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icon

This is to certify that the
thesis entitled

PARTICLE-BASED FLOW CYTOMETRY ASSAY TO DETECT
ANTI-ANGIOTENSIN ll TYPE I AND TYPE ll RECEPTOR
ANTIBODIES

presented by

MARIANE SETYABUDI

has been accepted towards fulfillment
of the requirements for the

Master of science degree in Clinical Laboratory Sciences

 

 

MWofessor's Signature

John A. Gerlach, Ph.D., D(ABHI)

 

December 11, 2008

MSU is an Affirmative Action/Equal Opportunity Employer

.....—.-.—.-a-u-u--u-c-—-—.-c—-o—-u-.-.---u¢-.—--.-.-

PARTICLE-BASED FLOW CYTOMETRY ASSAY TO DETECT ANTI-
ANGIOTENSIN II TYPE I AND TYPE II RECEPTOR ANTIBODIES

By

Mariane Sctyabudi

A THESIS

Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of

MASTERS OF SCIENCE

Clinical Laboratory Sciences

2008

ABSTRACT

PARTICLE-BASED FLOW CYTOMETRY ASSAY TO DETECT ANTI-
ANGIOTENSIN II TYPE I AND TYPE II RECEPTOR ANTIBODIES

By

Mariane Setya'budi

One of the major problems in renal transplantation is organ rejection which can
result in allografi loss. To prevent organ rejection, one needs to confirm that the kidney
from the donor is compatible with the recipient’s immune system. Currently, three major
steps are performed to prevent allograft rejection: Human Leukocyte Antigen (HLA)
typing, antibody screening, and compatibility testing. Antibody mediated allografi
rejection can be caused by HLA antibodies and non-HLA antibodies. Currently
screening for non-HLA antibodies is not included in renal transplantation.

One example of a non-HLA antibody that can cause allografi rejection is an anti-
angiotensin II receptor type 1 antibody (anti-ATI). A functional bioassay and enzyme-
linked immunosorbent assay (ELISA) have been developed to detect anti-AT] and anti-
angiotensin II receptor type 2 antibody (anti-ATz). Using the same principle as in the
ELISA, a microbead immunoassay using a flow cytometry based instrument was
developed to detect anti-AT; and anti-AT; antibodies.

The microbead immunoassay was not successfially developed due to the lack of
known positive controls for anti-AT] and anti-AT; antibodies. Another possible issue that
binding of the short peptide sequences to the microspheres changed their structure such

that immunogenicity was lost.

ACKNOWLEDGEMENT

Thank you to Dr. John Gerlach for all his time spent guiding me through this
research project. I am very grateful for all that he has done for me. Furthermore, I would
like to thank my committee members Dr. David Thome and Dr.Greg Fink for their time
and support throughout this research. I would like to thank Sue Fomey, Ann Gobeski,
and Ari Mankey for their help in the laboratory during this research. I would like to
thank Dr. Fisher for the human samples for this research. I would also like to thank
Dr.Kathy Doi g, Dr. Kathy Hoag, and Lindsy Hengesbach for their support throughout
this research. Finally, I would like to thank my family for their encouragement and

patience. These people made this degree possible.

iii

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................. vi
LIST OF FIGURES ........................................................................................................... ix
INTRODUCTION .............................................................................................................. I
BACKGROUND AND SIGNIFICANCE .......................................................................... 3
Renal transplantation ....................................................................................................... 3
Renal transplantation rejection and treatment ................................................................. 5
Renin angiotensin aldosterone system ............................................................................ 8
Angiotensin II receptors .................................................................................................. 8
Anti-AT; antibody ........................................................................................................... 9
Microbead immunoassay ............................................................................................... 1 1
Microsphere coupling .................................................................................................... 12
Luminex instrument ...................................................................................................... 14
Project Goal ................................................................................................................... 15
Materials ........................................................................................................................ l6
Microspheres ............................................................................................................. 1 6
Pepndes ...................................................................................................................... 16
Primary Antibodies .................................................................................................... 17
Secondary Antibodies ................................................................................................ 17
METHODS ....................................................................................................................... 19
General methods ............................................................................................................ l9
Peptide reconstitution ................................................................................................ l9
Peptide coupling with microspheres .......................................................................... 20
Microspheres modification with Adipic Acid Dihydrazide (AD‘H) .......................... 22
Peptide coupling to ADH-modified microspheres .................................................... 23
Determination of microspheres concentration ........................................................... 23
Blocking of microspheres with Bovine Serum Albumin (BSA) ............................... 24
Microbead immunoassay ........................................................................................... 24
Method modifications ................................................................................................... 25
Microbead immunoassay using vacuum manifold .................................................... 25
Microbead immunoassay using centrifugation .......................................................... 25
Microbead immunoassay by varying incubation conditions and secondary antibody
concentrations ............................................................................................................ 26
Microbead immunoassay by varying peptide concentrations #1 .............................. 26
Microbead immunoassay by varying peptide concentrations #2 .............................. 27
Microbead immunoassay by varying peptide concentrations and primary antibody
concentrations ............................................................................................................ 27
Microbead immunoassay by varying primary and secondary antibody concentrations
................................................................................................................................... 28
Rat sera screening using microbead immunoassay by centrifugation method .......... 28
Human sera screening using microbead immunoassay by centrifugation method... 29

Determination of peptide coupling efficiency ............................................................... 29

Enzyme-Linked Immunosorbent Assay (ELISA) ......................................................... 32
Dot Blot with non-fat dry milk as blocking agent ......................................................... 34
Dot Blot with Polyethylene Glycol (PEG) as Blocking Agent ..................................... 35
Data analysis ................................................................................................................. 35
RESULTS ......................................................................................................................... 37
Microbead immunoassay using centrifugation ............................................................. 38
Microbead immunoassay by varying incubation conditions and secondary antibody
concentrations ................................................................................................................ 39
Microbead immunoassay by varying peptide concentrations #1 .................................. 42
Microbead immunoassay by varying peptide concentrations #2 .................................. 42
Microbead immunoassay by varying peptide concentrations and primary antibody
concentrations ................................................................................................................ 44
Microbead immunoassay by varying primary antibody source for anti-ATg ................ 44
Rat sera screening using microbead immunoassay by centrifugation method .............. 46
Human sera using microbead immunoassay by centrifugation method ........................ 53
ELISA ............................................................................................................................ 6O
Dot Blot with non-fat dry milk as blocking agent ......................................................... 62
Dot Blot with PEG milk as blocking agent ................................................................... 64
DISCUSSION ................................................................................................................... 70
Microbead immunoassay ............................................................................................... 70
Microbead immunoassay using vacuum manifold .................................................... 70
Microbead immunoassay using centrifugation .......................................................... 71
Microbead immunoassay by varying incubation conditions and secondary antibody
concentrations ............................................................................................................ 7 l
Microbead immunoassay by varying peptide concentration #1 and #2 .................... 72
Microbead immunoassay by varying peptide concentrations and primary antibody
concentrations ............................................................................................................ 72
Microbead immunoassay by varying primary and secondary antibody concentrations
................................................................................................................................... 73
Problem solving ............................................................................................................. 73
Rat sera screening using the microbead immunoassay by centrifugation ................. 74
Human sera screening using microbead immunoassay by centrifugation ................. 75
Determination of peptide coupling efficiency ............................................................... 76
ELISA ............................................................................................................................ 77
Dot Blot with PEG as blocking agent ........................................................................... 77
CONCLUSION ................................................................................................................. 79
RECOMMENDATION .................................................................................................... 80
APPENDIX ....................................................................................................................... 81

LIST OF TABLES

Table 1. Angiotensin II receptor subtype function. ........................................................... 9
Table 2. Peptide sequences properties. ............................................................................ 16
Table 3. Primary antibodies. ............................................................................................ 17
Table 4. Secondary antibodies. ........................................................................................ 18
Table 5. Peptide sequences and corresponding microsphere group. ............................... 19
Table 6. Peptide sequence reconstitution. ........................................................................ 20
Table 7. Antibodies in microbead immunoassay (vacuum manifold). ............................ 25
Table 8. Antibodies in microbead immunoassay (centrifugation). .................................. 26

Table 9. Antibodies in microbead immunoassay (modification: incubation, secondary

anfibody) .......................................................................................................................... 26
Table 10. Antibodies in microbead immunoassay (modification: ATg secondary
antibody) ........................................................................................................................... 28
Table 11. ELISA plate map ............................................................................................. 33
Table 12. Median fluorescent intensity of microbead immunoassay (vacuum manifold).
........................................................................................................................................... 37
Table 13. Trimmed median fluorescent intensity of microbead immunoassay (vacuum
manifold). .......................................................................................................................... 37

Table 14. Mean fluorescent intensity ofmicrobead immunoassay (vacuum manifold). 38

Table 15. Trimmed mean fluorescent intensity of microbead immunoassay (vacuum
manifold). .......................................................................................................................... 38

Table 16. Fluorescent intensity for microbead immunoassay (centrifugation). .............. 39

Table 17. Median fluorescent intensity for microbead immunoassay (modification:
incubation, secondary antibody). ...................................................................................... 40

Table 18. Trimmed median fluorescent intensity for microbead immunoassay
(modification: incubation, secondary antibody). .............................................................. 40

Table 19. Mean fluorescent intensity for microbead immunoassay (modification:
, incubation, secondary antibody). ...................................................................................... 41

vi

Table 20. Trimmed mean fluorescent intensity microbead immunoassay (modification:
incubation, secondary antibody). ...................................................................................... 41

Table 21. Fluorescent intensity for microbead immunoassay (modification: peptide
concentrations). ................................................................................................................. 4”

a...

Table 22. Fluorescent intensity for microbead immunoassay (modification: peptide
concentrations, microsphere groups). ............................................................................... 43

Table 23. Fluorescent intensity for microbead immunoassay (modification: peptide
concentrations, primary antibody). ................................................................................... 44

Table 24. Fluorescent intensity for microbead immunoassay (modification: primary and

secondary antibody). ......................................................................................................... 45
Table 25. Median fluorescent intensity of rat sera screening. ......................................... 46
Table 26. Trimmed median fluorescent intensity of rat sera screening. .......................... 47
Table 27. Mean fluorescent intensity of rat sera screening. ............................................ 48
Table 28. Trimmed mean fluorescent intensity of rat sera screening. ............................. 49
Table 29. Median fluorescent intensity of rat sera screening (ADH microspheres) ........ 50

Table 30. Trimmed median fluorescent intensity of rat sera screening (ADH
microspheres). ................................................................................................................... 5 1

Table 31. Mean fluorescent intensity of rat sera screening (ADH microspheres). ........... 52

Table 32. Trimmed mean fluorescent intensity of rat sera screening (ADH microspheres).

........................................................................................................................................... 53
Table 33. Median fluorescent intensity of human sera screening. ................................... 54
Table 34. Trimmed median fluorescence intensity of human sera screening. ................. 54
Table 35. Mean fluorescence intensity of human sera screening. ................................... 55
Table 36. Trimmed mean fluorescence intensity of human sera screening. .................... 55

Table 37. Median fluorescent intensity of human sera screening (ADH microspheres). 56

Table 38. Trimmed median fluorescence intensity of human sera screening (ADH
microspheres). ................................................................................................................... 57

Table 39. Mean fluorescence intensity ofhuman sera screening (ADH microspheres). 57

vii

Table 40. Trimmed mean fluorescence intensity of human sera screening (ADH

microspheres). ................................................................................................................... 58
Table 41. Peptide absorbance at 230 nm. ........................................................................ 59
Table 42. Peptide absorbance at 260 nm. ........................................................................ 59
Table 43. Peptide absorbance at 280 nm. ........................................................................ 60
Table 44. Peptide absorbance at 320 nm. ........................................................................ 60
Table 45. ELISA result .................................................................................................... 61
Table 46. Peptide concentration based on absorbance at 230 nm ..................................... 76
Table 47. IVIICI'OSPIICI‘CS catalog number .......................................................................... 82
Table 48. Antibodies catalog number .............................................................................. 82
Table 49. Reagents catalog number ................................................................................. 83
Table 50. Consumables .................................................................................................... 83

viii

LIST OF FIGURES

Figure l: Microspheres classification. ............................................................................. 12
Figure 2. C arboxylated microsphere activation by EDC and Sulfo-NHS. ...................... 13
Figure 3. Microbead immunoassay principle ................................................................... 14
Figure 4. Microspheres concentration formula. ............................................................... 23
Figure 5. Dot blot map. .................................................................................................... 34
Figure 6. Dot blot map with 3% non-fat dry milk. ......................................................... 63
Figure 7. Dot Blot with 3% non-fat dry milk (without vacuum method). ........................ 63
Figure 8. Dot Blot with 3% non-fat dry milk (with vacuum method). ............................. 64
Figure 9. Dot blot map with PEG. ................................................................................... 66
Figure 10. Dot Blot with 1% PEG as a blocking solution (without vacuum method) ..... 66

Figure I I. Dot Blot with 3.5% PEG as a blocking solution (without vacuum method). 66

Figure 12. Dot Blot with 10% PEG as a blocking solution (without vacuum method)... 67

Figure 13. Dot Blot with 1% PEG as a blocking solution (with vacuum method). ......... 67
Figure 14. Dot Blot with 3.5% PEG as a blocking solution (with vacuum method). ...... 68
Figure 15. Dot Blot with 10% PEG as a blocking solution (with vacuum method). ....... 69
Figure 16. Beer’s Law formula ........................................................................................ 76

INTRODUCTION

In 2005, more than 485,000 Americans were treated for end stage renal disease
(ESRD). Diseases listed to cause ESRD were diabetes (36.9 percent (%)), high blood
pressure (24.2%), glomerulonephritis (16.2%), cystic kidney (4.6%), other urologic
(2.8%), and other unknown causes (17.9%) [I ]. Renal transplantation is one of the major
treatments for ESRD. One of the major problems in renal transplantation is organ
rejection which can result in allograft loss. To prevent organ rejection, one needs to
confirm that the kidney from the donor is compatible with the recipient’s immune system.
Currently, three major steps are performed to confirm the compatibility between the
donor and the recipient: Human Leukocyte Antigen (HLA) typing, antibody screening,
and compatibility testing. Antibody screening is performed to detect antibodies in the
recipient’s serum that react with HLA antigens to aid in donor selection. The
compatibility testing is performed to detect preformed antibodies in the recipient that are
reactive against the donor. Since 1970, a crossmatch is required prior to a renal
transplantation [2].

About one-third of acute rejections are antibody-mediated [3]. Antibody-
mediated rejection can be caused by HLA antibodies and non-HLA antibodies. Rejection
due to HLA antibodies can be minimized by matching HLA antigens, antibody screening
and compatibility testing. Currently, screening for non-HLA antibodies is not included in
renal transplantation. Some examples of non-HLA antibodies that can cause allograft
rejections are anti-phospholipid antibodies, anti-vimentin antibodies, antibodies specific
to endothelial antigens. and anti-angiotensin II receptor type I (anti-ATI) antibodies [4-

6].

The goal of this research is to develop a screening method to detect anti-
angiotensin II receptor type 1 (anti-ATI) and anti-angiotensin II receptor type 2 (anti-

ATz) antibodies using multiplex immunoassay.

Ix.)

BACKGROUND AND SIGNIFICANCE
Renal transplantation

Renal transplantation is one of the treatments for ESRD. To prevent allograft
rejection of the renal transplant, one needs to confirm that the organ from the donor is
compatible with the recipient’s immune system. Three steps are performed to confirm
the compatibility between the donor and the recipient: HLA typing, antibody screening,
and compatibility testing.

HLA is the major histocompatibility complex (MHC) located on chromosome 6
in humans. The MHC encodes proteins called MHC molecules or HLA. There are two
class of MHC: class I and class 11. Three major molecules for MHC class I are: HLA-A,
HLA-B, and HLA-C. Three major molecules for MHC class II are: HLA-DR, HLA-DQ,
and HLA-DP. MHC class I presents antigens to clusters of differentiation (CD) 8 T cell,
and MHC class 11 presents antigens to CD4 T cells [7].

HLA typing is done by microlymphocytotoxicity test. This test is performed in a
special microtest plate in which reagent serum is incubated with isolated lymphocytes.
Exogenous complement is added to the mixture and incubated to allow antigen and
antibody binding. Complement activation leads to cell death. Eosin dye is added to
show lysed lymphocytes. The lysed lymphocytes will take the eosin dye and appear dark
under a phase contrast microscope. An estimation of lysed cell count is determined using
a phase contrast microscope. Fonnalin is added as a fixative and to prevent non-specific
uptake of the eosin dye by the cells [8].

Antibody screening is performed to detect antibodies in the recipient’s serum that
react with the HLA antigens present on the donor lymphocytes. Antibody screening

assays are similar to the HLA typing, where lymphocytes from an individual whose HLA

types are known are incubated with the recipient’s serum [8, 9]. Assays using flow
cytometric technology with a panel of microbeads have been utilized for antibody
screening. This is done using a mixture of eight microbead groups that have a unique
fluorescent property essentially assigning an address. Each microbead group is coated
with purified HLA antigens from a single cell line. Recipient sera are added and HLA
antibody binds. Bound recipient antibodies are detected with anti-human globulin. Based
on the fluorescent signal on the surface of the microbeads and the microbead group
identification, HLA specificities can be determined. The use of microbeads coupled with
a single cell line of purified HLA antigens allow for a standardized quantity of antigen
present in the assay [10]. In a study comparing the microbead assay and the cytotoxicity
test, the microbead assay showed a higher sensitivity in the ability to detect H LA
antibodies at a higher serum dilution. The study was done on 1,421 sera from patients
who are waiting for a kidney transplant or following a kidney transplantation. The
microbead array method was able to detect anti-HLA antibodies on 18% of the patients
who are waiting for kidney transplant compared to 7% when using the complement
dependent cytotoxicity (CDC) method. In addition, the microbead array method was not
as labor intensive as the CDC method. Another advantage of using the microbead array
method is the ability to determine the HLA antibody specificities [1 1].

The last step to confirm the compatibility between the donor and the recipient is
the compatibility testing. The compatibility testing is performed to detect pro-existing
antibodies in the recipient against the donor [8]. The compatibility testing is performed
with the CDC method using donor lymphocytes that are incubated with the recipient’s

serum. The presence of cytotoxic antibodies is determined by looking at the level of

donor cell lysis. If cytotoxic antibodies are determined to be present, renal
transplantation is not performed. Compatibility testing using flow cytometric technology
can also been used. A titration study on compatibility testing using flow cytometric
technology showed a 30 to 250 fold of greater sensitivity [12].

However, there are patients who have allograft rejection without H LA antibodies.
A study showed that ten years after renal transplantation, 1 1% of rejection episodes did
not result in the development of anti-HLA antibodies. These patients were screened for
anti-HLA antibodies before and after renal transplantation and remains negative. Non-
HLA antibody mediated rejection could be a secondary effect from other cellular events
[13]. Another study showed that 20% of kidney transplantations performed failed and no
anti-HLA antibodies were found post—transplantation. The antibody specificity is not yet
resolved but preliminary studies indicate that the antibodies produced were against other
antigens [14]. In addition, in some cases of renal transplantation between HLA identical
siblings there was still a need for treatment with immunosuppressive drugs to prevent
rejection. These studies suggest that graft rejection can be caused by non-HLA
antibodies. Since screening for non-H LA antibodies is not included in pro-renal
transplantation, one of the causes for failure in kidney transplantation may be due to non-

HLA antibodies [15].

Renal transplantation rejection and treatment

There are three types of allograft rejection: hyperacute rejection, acute rejection,
and chronic rejection. Hyperacute rejection occurs within hours of transplantation and
the transplanted kidney must be removed. Hyperacute rejection is caused by preformed

lymphocytotoxic antibodies in the recipient serum directed towards donor antigens ] 16].

These antibodies should be identified in the antibody screen assay or the compatibility
testing [17]. Acute rejection occurs between 4 and 30 days oftransplant [16]. About
one—third of acute rejection episodes are antibody-rrrediated [3]. Antibody-mediated
rejection can be caused by HLA antibodies and non—HLA antibodies. Chronic rejection
occurs more than 3 months after transplantation. In the beginning, chronic rejection may
be caused by humoral immune responses since complement, immunoglobulins, and anti-
endothelial cell antibodies have been identified in the rejected kidney [18]. However,
further testing in later stages of chronic rejection showed the presence of antibody against
donor antigens in the recipient serum are decreased or absent [19]. A study using the rat
kidney allograft model showed a large numbers of macrophages. In addition, adhesion
molecules such as intercellular adhesion molecule 1 (ICAM-1) and vascular adhesion
molecule 1 (VCAM-l) were found on the glomerular capillary endothelium and other
renal structures [20]. These adhesion molecules are usually not present in nomral renal
tissue. This finding indicates that the T cell mediated immune response may be involved
in later stages of chronic rejection. Patients with chronic rejection eventually return to
dialysis therapy [16].

Rejection due to HLA antibodies can be minimized by matching HLA antigens
and modifying the recipients’ immune system [21]. Options to prevent and to treat
allograft rejection include: a short course of high dose corticosteroids, cytotoxic drugs
such as azathioprine, cyclophosphamide and mycophenolatc mofetil, or fungal and
bacterial derivative such as cyclosporin A, tacrolimus and sirolimus [22, 23].

A short course ofhigh dose corticosteroid is used in the treatment ofacute cellular

rejection. The effect of corticosteroid is a shift in the leukocyte population where the

6

number and preportion of neutrophils are increased and T lymphocytes are decreased
[24]. In addition, steroids also inhibit antigen stimulated proliferation ofT cells by
inhibiting interleukin 2 (IL-2). IL-2 is required for the initiation ofdeoxyribonucleic acid
(DNA) synthesis and proliferation ofT cells, which is product of cellular immune
response in acute cellular rejection [25].

The main effect ofcytotoxic drugs such as azathioprine is to interfere with DNA
synthesis. Azathioprine is a prodrug that releases 6-mercaptopurine. 6-mercaptopurine is
a tissue inhibitor of metalloproteinase which converted to thioguanine nucleotides.
Thioguanine derivatives may inhibit purine synthesis which is required for DNA
synthesis [3]. Mycophenolate mofetil also inhibit DNA synthesis by blocking de novo
synthesis of guanosine monophosphate, a nucleotide. C yclophospharnide is metabolized
to phosphoramide mustard which alkyl ates DNA and inhibiting DNA replication [22].

Fungal and bacterial derivatives such as cyclosporin A, tacrolimus and sirolimus
are immunosuppressive agents that interfere with T cell signaling. Cyclosporin A and
tacrolimus block T cell proliferation by inhibiting phosphatase activity of calcium
activated enzyme calciuncurin. Activation of calciuncurin allows for clonal expansion of
activated T cells. When the enzyme is inhibited, T cells proliferation is inhibited [22].
Sirolirnus act by inhibiting IL-2 driven T cell proliferation [3].

Therapies with anti-lyrnphocyte antibodies can also be used. Some examples are
anti-CD 20 which target B cell surface marker CD20 or anti-CD3 which target the T cell
surface marker CD3. Removal of preformed or newly developed antibodies can be done
by plasma exchange and intravenous immunoglobulin [26]. Plasma exchange has been

used to prevent and reverse antibody-mediated rejection [27].

Renin angiotensin aldosterone system

The renin angiotensin aldosterone system (RAAS) regulates renal activity,
maintains optimal salt and water balance, and controls tissue growth in the kidney.
Renin is an enzyme released by the renal juxtaglomerular cells located in the wall of
afferent arteriole. Renin synthesis is influenced by changes in blood pressure,
angiotensin II, and the plasma sodium and potassium levels in the body. Renin cleaves
the precusor angiotensinogen, which is synthesized by the liver, and forms angiotensin I.
Angiotensin l is converted to angiotensin II by angiotensin converting enzyme (ACE).
ACE is found in the endothelial cells of the lung, vascular endothelium, and cell
membranes of the kidneys, heart, and brain. Angiotensin II binds to angiotensin II
receptors found in the vascular endothelium, adrenal, kidney, liver and brain. Binding of
the angiotensin II to the angiotensin II receptors initiated the intracellular signaling
pathways that mediate vasoconstrictions and aldosterone release. Aldosterone is
synthesized by the adrenal gland. Aldosterone binds to the aldosterone receptors in the

kidney for reabsorption of water and sodium [28].

A ngiotensin 11 receptors

There are two subtypes of angiotensin 11 receptors: type 1 (ATI) and type 2
(AT3). AT] are located in the vasculature, kidney, adrenal gland, heart, liver, and brain.
ATg are present mainly in the fetus, but in adults they are located in adrenal medulla,
uterus, ovary, vascular endothelium, and brain [29-32]. AT] and AT; are polypeptides
containing approximately 360 amino acids that span the cell membrane seven times [33-
35]. The gene for AT. is located on chromosome 3, and for AT3 is on the X chromosome

[36, 37]. Both receptors are similar in structure, but each has different functions. In

general, AT2 receptor activation opposes functions mediated by the AT. receptor (Table
I) [38].

Table 1. Angiotensin II receptor subtype function.
(Adapted from Brewster and Perazella, 2004).

 

 

AT. function AT2 function

0 Systemic and renal vasoconstriction 0 Systemic and renal vasodilation
0 Increased renal sodium reabsorption o Decreased renal sodium
0 Activation of inflammatory reabsorption

cytokines o Decreased inflammation
0 Vascular smooth muscle growth 0 Decreased mitogenesis
0 Oxidative stress 0 Decreased myocyte hyperthrophy
o Endothelial dysfunction 0 Decreased cardiac fibrosis
0 Increased plasminogen activator

inhibitor 1 activity and thrombosis

 

A nti-A T. antibody

A study was performed on kidney transplant recipients who had allograft
rejection, but no anti-HLA antibodies. The study was done over 4 years (2000-2004) and
included 278 kidney transplantations, and 1 19 allograft rejections required treatment.

F mm 1 l9 allograft rejections, 23 were refractory to steroids. Further testing showed that
9 of 23 (39.1%) were due to anti-HLA antibodies, 10 of 23 (43.5%) were due to anti-AT.
antibodies, and 4 of23 (17.4%) were not due to anti-HLA antibodies or anti-AT.
antibodies. The method used to confinn the presence of anti-AT. antibodies was a
functional bioassay using spontaneously beating cultured neonatal-rat cardiomyocytes
that express several G-protein coupled receptors, including AT. receptors. The patient
sera were added, resulting in a linear increase of cellular activity. To confirm the AT.
receptor mediated response, irnmunoglobulin (IgG) stimulated cells (from paticnt’s) were
treated with short peptides corresponding to the sequence of the second extracellular loop

ofthe AT. receptors. The peptides inhibited the activity from IgG stimulated cells. This

9

inhibition continued that the I gG that bound to the receptors were anti-AT. antibodies
[39].

Another study observed that patients with hypertension may have autoantibodies
against AT.. Sera from patients with hypertension were screened for autoantibodies
against G-protein coupled cardiovascular receptors such as AT. and AT;. The study
included 14 patients with malignant essential hypertension (MEH), 12 patients with
malignant secondary hypertension (MSH), 11 patients with renovascular non-malignant
hypertension (RVH), and 35 subjects that were nonnotcnsive healthy blood donors
(control). From this population, anti-AT. autoantibodies were detected in 14% of MEH,
33% of MSH, 18% of RVH, and 14% ofthe control population. The presence of anti-
AT. autoantibodies suggests that it may be involved in the pathogenesis of malignant
hypertension. The method used in this study was an enzyme-linked immunosorbent
assay (ELISA). Peptide sequences from the AT. and ATg receptors were used to coat the
microtiter plate. The same peptide sequence used in anti-AT. antibody study, additional
peptide sequences from different parts of AT. and AT3 receptors were also used. Human
sera were added and incubated. To detect the presence of the antibody anti-human IgG
were added. Antibodies detected in the positive ELISA were purified and confinned

using the functional bioassay. The specificity was anti-AT. antibody [40].

10

.«llicrobead imnzzmoassay

Using the principle ofthc ELISA described above, my project was to design a
microbead immunoassay to allow an easier detection of anti-AT. and anti—ATZ
antibodies. The microbead immunoassay uses carboxylated polystyrene microspheres
from Luminex. The microspheres size is 5.6 pm. These microspheres are internally dyed
with red and infrared fluorophores. The concentrations of red and infrared dyes are
different for each group of microspheres. Each different group of microsphere is
assigned a specific number that corresponds to a different address based on the intensity
of the two dyes. There are up to 100 different microsphere groups available. A different
address will demonstrate different output signals during detection. A group of
microspheres with the same address will be called a microsphere group. Detection of the
different addresses of microsphere groups is done by the a classification laser in the
Luminex instrument. The classification laser excites the red and the infrared dyes inside
the microspheres and the intensities of the excitation emission are measured by a
detector. The classification laser wavelength is 635 nanometer (nm). Classification of
different microsphere group using forward and side scatter on the Luminex instrument is

in Figure 1.

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Figure 1: Microspheres classification.
(Adapted from Luminex Corporation. Austin, TX). Classification of 100 Luminex
microspheres based on red and infrared dye combination using forward and side scatter
on the Luminex instrument.
Microsphere coupling

On the surface of each microsphere carboxyl groups are present that can be
covalently coupled with the peptide sequences. The covalent bond will occur between
the carboxyl group of the microsphere and the N-terminus of the peptide. Coupling of
the peptide is done using 1-l€thy|-3-[3-dimethylaminopr0pyl] carboiimide hydrochloride

(EDC) and N—hydroxysulfosuceinimide (sulfo-NHS). The EDC will react with the

carboxyl group on the microsphere, forming an amine-reactive O-acylisourea
intermediate. The intermediate may react with an amine on the peptide sequence,
yielding a conjugate of the two molecules joined by a stable amide bond. The addition of
sulfo-NHS stabilizes the amine-reactive intermediate by converting it to an anime-

reactive sulfo-NHS ester (Figure 2) [41, 42].

0 cr—
\+/ |
k NH @/NH2 OK N/@ Stable
_ amide bond
0
Carboxylate N/
Molecule Regenerated
OOHK carboxyl group
\Cl ( Y

 

 

EDC Unstable reactive NH\ O§ISI /O-
o—aclysourea ester
< :)-— O
NH
[I o o ___i k /@
|| 0 k N N
N 0 § Isl / O _ O / H
r Stable
o amide bond
N
CH / Q) = Microspheres
Sulfo—NHS @ = Peptide

 

 

 

Figure 2. Carboxylated microsphere activation by EDC and Sulfo-NHS.
(Adapted from EDC package insert).

The carboxylated microspheres are coupled with a peptide sequence. Each
peptide sequence is coupled with one group of microspheres. After the different peptides
are coupled with a different microsphere group, the microspheres are blocked with

bovine serum albumin (BSA) to prevent non-specific binding. After the blocking step.

primary antibody which is the analyte is added (commercial control or patient’s sera).
After the addition of primary antibody, the microspheres are washed to remove all of the
unbound antibodies. To determine binding of the primary antibody to the peptide
sequence on the microspheres, the appropriate secondary antibody to match the primary
antibody is added. The secondary antibody is conjugated with phycoerythrin (PE) to
allow detection by the reporter laser in the Luminex instrument. The reporter laser
wavelength is 532 nm. The emission of the PE is detected by a photomultiplier tube with

detection bandwidth of 565-585 nm.

 

.A at
‘41.-..- k, ‘25
fit, :3 are:

 

Polystyrene

microspheres WW” Primary antibody
fluorophores CADIUIC (anti lgG-PE)
ntcchde:

Peptide

Secondary antibody

(positive control:
commercial sera,
negative control: wash
l)trffi?r)

 

 

 

Figure 3. Microbead immunoassay principle.

(Figure adapted and modified from Liquichip® Application Handbook February 2006).
Polystyrene microspheres are coupled with peptide sequences from AT. and AT:
receptors. Detection of anti-AT. or anti-AT; antibody is done by adding secondary
antibody with PE.

Luminex instrument

The Luminex instrument is an instrument based on flow cytometry technology.
In the Luminex instrument there are two fluidie paths. The first fluidic path includes a
syringe driven mechanism that controls the sample intake from the sample container to

the cuvette. The advantage of this mechanism is the instrument requires small sample

14

volume. Sample is acquired using the sample probe from a 96-well microplate and
injected into the cuvette. From the cuvette the sample is purged with sheath buffer by the
second fluidics path which is driven by positive air pressure and supplies sheath fluid.
The second path removes residual sample to minimize carry over. The sample is
introduced to the optics path at a reduced rate to ensure that each microsphere is analyzed

individually [43].

Project Goal

The goal of this research is to develop a screening method for anti-AT. and anti-
AT2 antibodies using microbead immunoassay which utilize a flow cytometry based
instrument such as the Luminex that is commonly available in histocompatibility
laboratories. The microbead immunoassay will utilize the peptide sequences from the
functional bioassay and the ELISA method. The population of interest for this research is

renal transplant recipients.

15

MATERIALS

Materials

All reagents and supplies described, unless otherwise stated were purchased from
Michigan State University Stores (East Lansing, MI)

M icrosplzeres

 

xMAP® Multi-Analyte COOH Microspheres were purchased from Luminex
Corporation (Austin, TX). Six microsphere groups were selected based on the
manufacturer’s recommendation. The microsphere groups selected were: 033, 034, 035,
036, 037, and 038. Catalog numbers for the microspheres can be found in the appendix.
On the surface of each microsphere, carboxyl groups were present for covalent coupling
with a peptide sequence that is part of either the AT. or the AT2 receptor. The
concentration of each microsphere set was 1.25 x 107 microspheres/milliliter (mL).
Peptides

Peptides were synthesized by the Macromolecular Structure, Sequencing and
Synthesis Facility at Michigan State University (East Lansing, MI). The peptide
sequences were from Dragun et al. (2005) and F u et al. (1999). The peptide sequences
are parts of the AT. and AT; receptors. A list of the peptide sequences and its properties
are in Table 2.
Table 2. Peptide sequences properties.

Each letter in the peptide sequence represents a single amino acid abbreviation.
Underlined sequences used in both study (Adapted from Dragun et al. and Fuel al..)

 

 

 

 

 

 

 

Peptide sequence MW Reference _ _
IHRNVFFIENTNITVCAFHYESQNS'I‘L 3198.5 AT. receptor -_
AYEIQKNKPRNDD 1590.7 AT. receptor

ENTNIT 690.7 AT. receptor _ #
AFHYESQ 880.9 AT. receptor
ACLSSLPTFYFRDVRTIEYLGVNACI 2952.4 ATg receptgrm fl_

 

 

 

 

l6

Primary Antibodies

Primary antibodies used in the microbead immunoassay, ELISA, and Dot Blot are
listed in Table 3. Catalog numbers for the antibodies are listed in the appendix. These
commercial antibodies were used as positive controls.

For screening, 158 samples of human sera and 17 rat sera were used. The human
sera consist of renal transplant and preeclampsia patients, and the rat sera were
hypertension rat sera. All human samples and rat sera were handled in accordance with
the University Committee on Research Involving Human and Animal Subjects.

Table 3. Primary antibodies.

 

 

 

 

 

 

 

 

 

Primary antibody Source Stock
Concentration

Mouse anti human angiotensin 11 type 1 USBio 0.7 mg/mL

receptor (AT.) (Swampscott, MA) g

Rabbit anti human angiotensin 11 type 2 USBio l mg/‘mL

receptor (AT3) (Swampscott, MA)

Sheep polyclonal to angiotensin type 1 Abcam 1 mg/mL

receptor (AT.) (Cambridge, MA)

Rabbit polyclonal to angiotensin 11 type 2 Abcam 1 mg/mL

receptor (AT3) _ (Cambridge, MA)

 

Secondary Antibodies

 

Secondary antibodies used in the microbead immunoassay, ELISA, and Dot Blot
are listed in Table 4. Catalog numbers for the antibodies are listed in the appendix.
Antibodies with phycoerythrin (PE) were used in the microbead immunoassay. and

antibodies with alkaline phosphatase (AP) were used in the ELISA and Dot Blot.

l7

Table 4. Secondary antibodies.

(PE: phycoerythrin; AP=alkaline phosphatase).

 

 

 

 

 

 

 

 

 

 

 

 

(Cambridge, MA)

 

Product Source Concentration

Goat anti mouse IgG (PE) USBio 0.2 mg/mL
(Swampscott, MA)

Donkey anti rabbit IgG (PE) USBio 0.5 mg/mL
(Swampscott, MA)

Donkey polyclonal to sheep IgG (PE) Abcam 0.50 mg/mL
(Cambridge, MA)

Rabbit polyclonal to sheep IgG (AP) Abcam 1.00 mg/mL
(Cambridge, MA)

Donkey polyclonal to rabbit IgG (PE) Abcam 0.5 mg/mL
(Cambridge, MA)

Goat polyclonal to rabbit IgG (AP) Abcam 1 mg/mL
(Cambridge, MA)

Sheep polyclonal to rat IgG (AP) Abcam 1.00 mg/mL
(Cambridge, MA)

Goat polyclonal to rat IgG (PE) Abcam 0.5 mg/mL
(Cambridge, MA)

Goat polyclonal to human IgG (PE) Abcam 0.5 mg/mL

 

18

 

METHODS
General met/rods

Peptide reconstitution

 

Peptide reconstitution method was recommended by the Macromolecular
Structure, Sequencing and Synthesis Facility at Michigan State University (East Lansing,
MI). Peptide reconstitution was done in 1.5 mL polypropylene microcentri fuge tubes.

List of each peptide sequence coupled with the different microsphere group are in Table

 

 

 

 

 

 

 

5.

Table 5. Peptide sequences and corresponding microsphere grou' .
Peptide sequence Peptide ID Microsphere

group

IHRNVFFIENTNITVCAFHYESQNSTL AT.-033 033
AYEIQKNKPRNDD AT.-034 034
ENTNIT AT.-035 035
AFHYESQ AT.-036 036
ACLSSLPTFYFRDVRTIEYLGVNACI AT3-037 037
None None 038

 

 

 

 

 

A stock solution of each peptide sequences was prepared from the lyophilized
peptide. Based on the hydrophobic properties of the peptide sequences, peptide AT.~033
and peptide AT2-037 were dissolved in dimethyl sulfoxide (DMSO) (Si gma-Aldrieh, St.
Louis, MO) before the addition of distilled water (ngO) while peptide AT.—034, AT.-
035, and AT.-036 were dissolved in dHZO only. Table 6 lists the specific amount used to
make the stock solution of each peptide sequences. Peptide solutions were aliquoted and

stored at -20°C.

Table 6. Peptide sequence reconstitution.

 

 

 

 

 

 

 

Peptide ID Peptide DMSO ngO Final
(mg) (mL) (mL) Concentration
AT.-033 4 1 3 1 mg/mL
AT.-034 l 0 1 1 mgmL
AT.-035 1 0 1 1 mg/mL
AT.-036 1 0 1 1 mg/mL
AT3-037 1.5 0.5 1 1 mg/mL

 

 

 

 

 

 

Peptide coupling with microspheres

The peptide coupling method was adapted from the Luminex Corporation
(Austin, TX). Peptide coupling was done in 1 mL polypropylene microcentrifuge tubes
(Art Robbins, Sunnyvale, CA).

Microsphere stock solutions were resuspendcd by vortexing for 10 seconds (sec)
followed by sonication for 10 sec. The stock concentration of the microspheres was 1.25
x 107 microspheres/mL. 200 microliter (uL) of each microsphere group was aliquoted
into a separate 1 mL polypropylene microcentrifuge tube resulting in working
concentration of 2.5 x 106 microspheres/mL. The microspheres were washed by adding
100 uL of ngO, vortexing for 10 sec, sonicating for 10 sec and centrifugation for 8.000
x gravity (g) for two minutes (min). The supernatant was aspirated using a micropipette
and discarded after the wash step. The microspheres were resuspended with 80 pl. of
sodium phosphate monobasic buffer [0.1 molar (M) sodium phosphate monobasic buffer
(NaHzPO4) pH 6.2] (Sigma-Aldrich, St. Louis, MO) followed by vortexing for 10 sec and
sonication for 10 sec. 10 uL of 50 mg/mL sulfo-N-hydroxysulfosuccinirnide (Sulfo-
NHS) (Pierce, Rockford, IL) followed by 10 uL of 50 mg/mL (1-ethyl-3-[3-
dimethylaminopropyl]carbodiirnide hydrochloride (EDC) (Pierce, Rockford. II.) were

added to the microspheres. The microspheres were mixed gently by vortexing for 10 see.

The microspheres were incubated at room temperature for 20 min. During incubation,
the microspheres were vortexed at 10 min interval for 10 sec and covered with aluminum
foil to prevent photo bleaching.

After incubation, the microspheres were washed twice with 2[N-
Morpholino]ethanesulfoic acid (MES) buffer [0.05 M 2[N-Morpholino]ethanesulfoic acid
(MES) pH 5.0] (MES buffer #1) (Sigrna-Aldrich, St. Louis, MO). The microspheres
were washed by adding 250 uL of MES buffer #1 and centrifugation at 8,000 x g for two
min. The supernatant was aspirated by a micropipette and discarded after each wash.

The microspheres were resuspended in 100 pL of MES buffer #1. A specific amount of
each peptide concentration was added to each microsphere set followed by the addition of
MES buffer #1 for a total volume of 500 uL. Peptide concentrations used were 125, 100,
50, 25 and 5 pg. For microspheres group 038 only 500 uL of MES buffer #1 were added.
Microsphere group 038 serves as a blank. The microspheres were incubated at room
temperature for two hours on a rotator. During incubation the microspheres were covered
with aluminum foil to prevent photo bleaching.

The microspheres were washed by adding 500 uL of Phosphate Buffer Solution-
Tween (PBS-TBN) [PBS 0.138 M NaCI, 0.0027 M KC1, pH 7.4, 0.02% Tween 0.05%
sodium azide], and centrifugation for 8,000 x g for two min. The supernatant was
aspirated by a micropipette and discarded after each wash. Two more washes were
performed by adding 1000 uL of PBS-TBN, and centrifugation for 8,000 x g for two min.
The supernatant was aspirated and discarded after each wash. The microspheres were

resuspended in 1000 uL of PBS-TBN and stored at 2-8°C in the dark.

Microspheres modification with Adipic Acid Dihydrazide (ADH)

The microsperes modification with ADH method was adapted from Luminex
Corporation (Austin, TX). This method was performed to minimize binding issue
between the antibody of interest and the peptide sequences, especially with the shorter
peptide sequences. The ADH creates a space between the microsphere and the peptide so
that immunogenicity of the peptide was not hindered by its binding to the microsphere.

Microsphere stock solutions were resuspended by vortexing for 10 sec and
sonication for 10 see. 40 uL of each microsphere group was aliquoted into separate 1.0
mL polypropylene microcentrifuge tube resulting in working microsphere concentration
of 500,000 microspheres. 160uL of ngO were added into each microsphere group. The
microspheres were centrifuged at 1,000 x g for 2 min. The supernatant was aspirated and
discarded. 1 mL of MES buffer [0.1 M MES pH 6.0] (MES buffer #2) was added. The
microspheres were vortexed for 10 sec and centrifuged at 1,000 x g for 2 min. The
supernatant was aspirated and discarded. The microspheres were resuspended in 1 mL of
35 mg/mL ADH (Sigma-Aldrich, St. Louis, MO) and 200 uL of 200 mg/mL EDC. The
microspheres were incubated at room temperature for one hour in the dark on a rotator.
After incubation, the microspheres were washed three times with 1 mL of MES buffer
[0.1 M MES buffer pH 4.5] (MES buffer #3) and centrifuged at 8,000 x g for 2 min. The

ADH-modified microspheres were stored in the dark at 2-8°C.

Ix)
Ix.)

Peptide coupling to ADH-modified microspheres

After modification of microspheres with ADH, the microspheres were washed
with 1 mL of MES buffer #2. Wash step was performed by centrifugation of the
microspheres at 8,000 x g for 2 min, aspiration of the supernatant, and mix by vortex and
sonication for 10 see each. A specific amount of peptide concentration was added
followed by the addition of MES buffer #2 for a total volume of 500 uL. Peptide amount
used were 125, 100, 50, 25 and 5 pg. After the addition of the peptide solution and MES
buffer, 50 uL of 200 mg/mL EDC were added. The microspheres were incubated at
room temperature on a rotator for two hours in the dark. After incubation, the
microspheres were centrifuged at 8,000 x g for 2 min. The supernatant was aspirated and
discarded. The microspheres were resuspended in 1 mL of phosphate buffer (PBS) two

washes. After the last wash, the microspheres were resuspended in 1 mL of PBS buffer.

Determination ofmicrosplzeres concentration

 

The microspheres concentration was calculated after coupling the microspheres
with the peptide. Figure 4 lists the formula used to calculate the microsphere

concentration.

 

Microspheres/uL = average x 0.1

Average = the average number of microspheres from 8 large squares on both
sides of the hemocytometer.

 

 

 

Figure 4. Microspheres concentration formula.

F or each assay, a working peptide coupled microsphere solution was prepared for
each microspheres group. The concentration required for a working solution was 5000

microspheres for each microsphere group in 50 uL solution.

Blocking ofmicrospheres with Bovine Serum Albumin (BSA)

 

A working peptide coupled microsphere solution was prepared for each
microsphere group. PBS with 1% weight/volume (w/v) BSA (PBS 1% BSA buffer)
(Sigma-Aldrich, St. Louis, MO) was used as blocking solution for the microspheres. The
blocking step was performed by the addition of 100 uL PBS 1% BSA buffer into the
working microspheres solution followed by centrifugation at 8,000 x g for 2 min. The

microspheres were resuspended in 50 pL of PBS 1% BSA buffer.

Microbead immunoassay

 

After the microspheres were blocked with PBS 1% BSA, 50 pL of primary
antibody was added. PBS 1% BSA buffer were used to perform dilutions needed for
primary antibody. After primary antibody was added, the microspheres were incubated at
room temperature for 60 min on a plate shaker at 120 radius per minute (rpm) in the dark.
After incubation, the microspheres were washed twice using 100 uL of PBS 1% BSA
buffer. The microspheres were resuspended with 50 uL PBS 1% BSA and mixed. After
the last wash, 50 uL of secondary antibody was added and the microspheres were
incubated at room temperature for 30 min on a plate shaker at 120 rpm in the dark.
Following incubation, the microspheres were washed twice using 100 pl. of PBS 1%
BSA buffer. The microspheres were transferred to a 96-well microplate for detection on

the Luminex instrument (Austin, TX).

Method modifications
Microbead immunoassay using vacuum manifold

The microbead immunoassay method was perfonned using vacuum manifold.
This experiment was performed using microsphere group 033 which was coupled with
125 pg of AT.-033 peptide, and microsphere group 038 which was not coupled with any
peptide. Microsphere group 038 serves as a blank. Each microsphere group was
aliquoted into a separate reaction wells. The volume used for primary and secondary
antibody was 50 pL. The primary and secondary antibody combinations and
concentrations used in this experiment are listed in Table 7.
Table 7. Antibodies in microbead immunoassay (vacuum manifold).

Primary and secondary antibody concentration used in the microbead immunoassay using
vacuum manifold. Volume used for each antibody was 50 pL.

 

 

 

 

 

Primary Antibody Mouse anti human AT. Mouse anti human AT.
(Concentration) (70 pg/mL) (294 pg/mL)
Secondary Antibody Anti mouse IgG- PE Anti mouse IgG- PE
(Concentration) (10 pg/mL) (4 pg/mL) _____

 

 

M icrobead immunoassay using centri/ugation

 

This method modification was performed due to insufficient washing observed
during the vacuum manifold method. The microbead immunoassay method was
performed using the centrifugation method for the washing step. The wash step was
performed with centrifugation at 8,000 x g for 2 min and aspiration of the supernatant
using a micropipette. This experiment was performed using microsphere group 033, 034.
035, 036 that were coupled with 125 pg of corresponding peptide, and microsphere group
038 as a blank. Each microsphere group was aliquoted into a separate reaction tube. The
volume used for primary and secondary antibody was 50 pL. The primary and secondary

antibody combinations and concentrations used in this experiment are listed in Table 8.

25

Table 8. Antibodies in microbead immunoassay (centrifugation).
Primary and secondary antibody concentration used in the microbead immunoassay using
centrifugation. Volume used for each antibody was 50 pL.

 

 

 

 

Primary Antibody Mouse anti human AT.
(Concentration) (420 pg/mL)
Secondary Antibody Anti mouse IgG- PE
(Concentration) (4 pg/mL)

 

 

Microbead immunoassay by varying incubation conditions and secondary antibody
concentrations

 

 

The microbead immunoassay method was performed using the centrifugation
method for the wash steps. This experiment was performed to optimize the assay by
varying primary and secondary antibody concentration combination. This experiment
was performed using microsphere group 033 that was coupled with 125 pg of AT.-033
peptide, and microsphere group 038 as a blank. During this experiment, each
microsphere group was aliquoted into a separate reaction tube. This assay was perfonned
under two different incubation conditions: room temperature and 37°C. The volume
used for primary and secondary antibody was 50 pL. The primary and secondary
antibody combinations and concentrations used in this experiment are listed in Table 9.
Table 9. Antibodies in microbead immunoassay (modification: incubation, secondary

antibody).
Two sets of assay was performed with room temperature incubation and 37°C.

 

 

 

Primary Antibody Mouse anti human AT. T Mouse anti human AT.
(Concentration) (700 pg/mL) (700 pg/mL) _‘
Secondary Antibody Anti mouse IgG- PE Anti mouse IgG- PE
(Concentration) (4 pg/mL) (40 pg/mL)

 

 

 

 

 

Microbead immunoassay by varying peptide concentrations #1

The microbead immunoassay method was performed using the centrifugation
method for the wash steps. This experiment was performed to optimize the assay. This

experiment was performed using microsphere group 033 that was coupled with 100, 50,

25, and 5 pg of AT.-033 peptide, and microsphere group 038 as a blank. During this
experiment, each microsphere group was aliquoted into a separate reaction tube. Primary
antibody used was 50 pL (70 pg/mL) of mouse anti human AT. (USBio, Swampscott,
MA). Secondary antibody used was 50 pL (4 pg/mL) of anti mouse IgG- PE (USBio,

Swampscott, MA).

Microbead immunoassay by varying peptide concentrations #2

Microbead immunoassay method was performed using the centrifugation method
for the wash steps. This experiment was performed using microsphere group 033, 034,
035, and 036 that was coupled with 100, 50, 25, and 5 pg of corresponding peptide, and
microsphere group 038 as a blank. During this experiment, each microsphere group was
aliquoted into a separate reaction tube. Primary antibody used was 50 pL (4 pg/mL) of
sheep polyclonal to angiotensin type 1 receptor (Abcam, Cambridge, MA). Secondary

antibody used was 50 pL (5 pg/mL) of anti rabbit IgG- PE (Abcam, Cambridge, MA).

Microbead immunoassay by varying peptide concentrations and primary antibody
concentrations

 

The microbead immunoassay method was performed using the centrifugation
method for the wash steps. This experiment was performed to optimize the assay by
varying the peptide, the primary antibody and secondary antibody concentration. This
experiment was performed using microsphere group 033 that was coupled with 100, 50,
25, and 5 pg of AT.-033 peptide, and microsphere group 038 as a blank. During this
experiment, each microsphere group was aliquoted into a separate reaction tube. Primary

antibody used was 50 pL (70 pg/mL and 140 pg/mL) ofrnouse anti human AT. (USBio,

Swampscott, MA). Secondary antibody used was 50 pL (4 pg/mL) of anti mouse IgG-

PE (USBio, Swampscott, MA).

Microbead immunoassay by varying primary and secondary antibody concentrations
The microbead immunoassay method was performed using the centrifugation
method for the wash steps. This experiment was performed to optimize the assay by
varying primary and secondary antibody combination. This experiment was performed
using microsphere group 033, 034, 035, 036, and 037 that was coupled with 125 pg of
the corresponding peptide, and microsphere group 038 as a blank. During this
experiment, each microsphere group was aliquoted into a separate reaction tube. The
volume used for primary and secondary antibody was 50 pL. The primary and secondary
antibody combinations and concentrations used in this experiment are listed in Table 10.

Table 10. Antibodies in microbead immunoassay (modification: ATz secondary

 

 

 

 

 

 

 

anfibody)
Anti-AT.
Primary Antibody Sheep polyclonal to angiotensin type 1 receptor (AT.)
(Concentration) (2 pg/mL)
Secondary Antibody Donkey polyclonal to sheep IgG-PE
(Concentration) (5 pg/mL)
Anti-AT;
Primary Antibody Rabbit anti human Donkey anti rabbit IgG—
(Concentration) angiotensin [I type receptor PE
(0.1 pg/mL) (USBio) (2.5 pg/mL)
Secondary Antibody Rabbit polyclonal to Donkey polyclonal to
(Concentration) angiotensin II type 2 rabbit IgG-PE
receptor (2.5 pg/mL)
(0.1 pg/mL) (Abcam)

 

 

 

 

Rat sera screening usingmicrobead immunoassay by centri/iigation met/rod

 

Microbead immunoassay method was performed using the centrifugation method.

In this experiment, different microsphere groups are combined prior the addition of

primary antibody. Since known positive control for the anti-AT. or anti-ATz was not
available, screening of hypertension rat sera was performed. This population was used
because it was described previously that patients with hypertension may have
autoantibodies against anti-AT. [3 9]. This experiment was performed with the peptide
coupled microspheres and ADH modified peptide coupled microspheres. Peptide
concentrations used was 125 pg. 50 pL of rat sera was used as primary antibody.
Secondary antibody used was 50 pL of 4 pg/mL of goat polyclonal to rat IgG-PE

(Abcam, Cambridge, MA).

Human sera screening using microbead immunoassay by centrifugation met/rod
Microbead immunoassay method was performed using the centrifugation method.
In this experiment, different microsphere groups are combined prior the addition of
primary antibody. Since known positive control for the anti-AT. or anti-ATg was not
available, screening of human sera from renal transplant recipient and preeclampsia
patients was performed. This population was used because it was described previously
that these patients may have the antibody of interest [27, 42]. This experiment was
performed with the peptide coupled microspheres and ADH modified peptide coupled
microspheres. Peptide concentrations used was 125 pg. 50 pL of human sera was used
as primary antibody. Secondary antibody used was 50 pL (4 pg/mL) of goat polyclonal

to human IgG-PE (Abcam, Cambridge, MA).

Determination ofpeptide coupling efficiency
Determination of peptide coupling efficiency was perfonned by measuring the

absorbance of the supernatant of the microspheres at 260 nanometer (nm). 280 nm, 320

29

nm, and 230 nm using a spectrophotometer (GeneQuant, Amersham Biosciences,
Pittsburgh, PA). This method was performed to ensure peptide binding to the
microspheres. This procedure was performed using the peptide coupling with
microspheres method with some modifications.

Microsphere stock solutions were resuspended by vortexing for 10 sec followed
by sonication for 10 sec. The stock concentration of the microspheres was 1.25 x 107
microspheres/mL. 5 pL of each microsphere group was aliquoted into a separate 1 mL
polypropylene microcentrifuge tube resulting in working concentration of 62,500
microspheres/mL. 395 pL of ngO were added. The microspheres were washed by
adding 100 pL of dHZO, vortexing for 10 sec, sonicating for 10 sec and centrifugation for
8,000 x g for two min. The supernatant was aspirated using a micropipette and discarded
after the wash step. The microspheres were resuspended with 80 pL of sodium
phosphate monobasic buffer followed by vortexing for 10 sec and sonication for 10 sec.
10 pL of 50 mg/mL Sulfo-NHS followed by 10 pL of 50 mg/mL EDC were added to
the microspheres. The microspheres were mixed gently by vortexing for 10 sec. The
microspheres were incubated at room temperature for 20 min. During incubation the
microspheres were vortexed at ten min interval for 10 sec and covered with aluminum
foil to prevent photo bleaching.

Following incubation, the microspheres were washed twice with MES buffer #1.
The microspheres were washed by adding 250 pL of MES buffer #1 and centrifugation at
8,000 x g for two min. The supernatant was aspirated by a micropipette and discarded
after each wash. The microspheres were resuspendcd in 100 pL of MES buffer .41. 125

pg of peptide were added, followed by the addition of MES buffer #1 for a total volume

30

of 500 pL . For microspheres group 038 only 500 pL of MES buffer #1 were added.
Microsphere group 038 serves as a blank. 200 pL of the supernatant was aspirated and
an absorbance was taken. This step was repeated at 0, 2, 4, 20 and 24 hours. At the end
of each reading, the supernatant was retumed to each tube and mixed by vortexing for 10
sec. The spectrophotometer was zeroed with MES buffer #1.

This experiment was also performed on ADH-modified microspheres.

31

E nzyme-Linked Immunosorbent Assay (ELISA)

The ELISA method was performed to check whether the commercial antibodies
for the positive control specificity matched the specificity of the peptide sequences
without the changing the peptide conformation due to the covalent binding to the
microsphere. A plate map of the ELISA is in Table 1 1. A 96-well plate was coated with
50 pL of 20 pg and 10 pg of peptide solution. The peptide was reconstituted with three
different pH: 5.5, 7.0, and 9.6. The plate was incubated at room temperature ovemight.
After incubation, the peptide solutions were removed and the wells were washed with
200 pL of PBS three times. 200 pL of 3% w/v non-fat dry milk diluted in PBS was
added as a blocking reagent. The plate was incubated at room temperature for one hour.
After incubation, the plate was washed with PBS three times. 100 pL of primary antibody
was added into the appropriate wells with the appropriate dilution. The plate was
incubated at room temperature for one hour. After incubation the plate was washed with
PBS three times. 100 pL of secondary antibody was added into the wells with the
appropriate dilution. The plate was incubated at room temperature for 30 min. After
incubation, the plate was washed with PBS three times. An ELISA substrate solution [20
mL of 0.1 M Glycine buffer 1 mM Mng 1 mM ZnClg pH 10.4 with 1 tablet of p-
nitrophenyl phosphate( pNPP)] (Sigma-Aldrich, St. Louis, MO) was prepared. Each well
was developed by adding 100 pL of ELISA substrate solution. The absorbance ofthc
microplate was read on a plate reader at 405 nm. The absorbance from the positive

control was compared with the negative control.

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33

Dot Blot wit/z non—fat dry milk as blocking agent

The dot blot method was performed to check whether the commercial antibodies
for the positive control specificity matched the specificity of the peptide sequences
without changing the conformation of the peptide from binding to the microplate or the
microsphere. A nylon membrane (Biorad, Hercules,CA) was cut into 7 centimeter (cm)
x 1 cm for each assay. The membrane was divided into 7 squares. Peptide solutions with
concentration of 20 pg, 5 pL of primary antibody (positive control), and 5 pL of BSA

(negative control) were added to the membrane. A general map for the dot blot is in

 

 

 

 

 

 

 

 

 

 

 

Figure 5.
Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8
Peptide Peptide Peptide Peptide Peptide BSA Anti-AT 1 Anti-AT)
AT. ~033 AT. -034 AT. —O35 AT. -036 AT2-037

 

 

 

 

 

Figure 5. Dot blot map.

Two methods used to apply peptide solutions onto the membrane: with vacuum
manifold and without vacuum manifold. The membranes were air dried overnight at
room temperature. The membranes were blocked with 3% non-fat dry milk-PBS solution
in a 15 mL conical tube on a rotator and incubated at room temperature for one hour.

The membranes were washed twice with 0.05% Tween-Phosphate Bigger Solution
(Tween-PBS) [PBS 0.138 M NaCl, 0.0027 KCI, pH 7.4, 0.05% Tween] and air dried for
30 minutes. The membranes were washed with 0.3% Tween-PB [PBS 0.138 M NaCI.
0.0027 KCI, pH 7.4, 0.03% Tween] and air dried for 15 minutes. Solutions with the
appropriate primary antibodies were prepared in a 15 mL conical tube. The membranes
were incubated with the primary antibody solutions and incubated on a rotator for 30 min

at room temperature. After incubation, the membranes were washed with 0,050.1. Tween-

34

PBS three times and PBS three times. The membranes were air dried for 30 minutes.
Solutions with the appropriate secondary antibodies were prepared in a 15 mL conical
tube. The membranes were incubated with the secondary antibody solutions and
incubated on a rotator for 30 min at room temperature. After incubation, the membranes
were washed with 0.05% Tween-PBS three times, followed by 0.05 M Sodium
bicarbonate (NaHCO3) buffer [0.05 M NaHC03 pH 9.5] (Sigma Aldrich, St. Louis,

MO). A BCIP/N BTFM substrate system (Sigma Aldrich, St. Louis, MO) was prepared by
' mixing 1 mL of BC IP/N BTTM substrate in 9 mL of NaHCO3 buffer. The membranes
were incubated in the liquid substrate system for 10 min and rinsed with water to stop the

reaction. The membranes were air dried.

Dot Blot with Polyethylene Glycol (PEG) as Blocking Agent

The experiment for Dot Blot was repeated with Polyethylene glycol (Sigma
Aldrich, St. Louis, MO) as blocking agent. PEG was used as an altemative blocking
agent to eliminate any issue with the large molecular weight property of BSA which may

interfere with peptide binding. PEG concentrations used were 1%, 3.5%, and 1094..

Data analysis

To determine the presence of anti-AT. or anti-AT: antibody, fluorescence
intensity between the microsphere groups which were coupled with the peptide such as
033, 034, 035, 036, and 037 was compared with the microsphere group 038 which was
not coupled with any peptide sequences and serve as a blank. For each run four data sets
were analyzed four ways: median fluorescence intensity, trimmed median fluorescence

intensity, mean fluorescence intensity, and trimmed mean fluorescence intensity. The

35

data analysis was calculated by the instrument based on 100 microspheres for each
microsphere group. The median was calculated by arranging the values from 100
microspheres from lowest to highest value and looking at the 50‘h value. The mean was
calculated by adding all the values from 100 microspheres and divide that by 100.
Trimmed median or trimmed mean were determined by removing the lower and upper
five percent of the extreme values.

For each data set, the fluorescent intensity of microsphere group ()38 with the
analyte (03 8-positive) is compared with the fluorescent intensity of microsphere group
033, 034, 035, 036, or 037 with the analyte (positive control). The expected difference

between 038-positive and the positive control was around 1000 fold.

36

RESULTS

Microbead immunoassay using vacuum manifold

The results from microbead assay using vacuum manifold are listed in tables 12-
15. Table 12 contains the median fluorescence intensity. Table 13 contains the trimmed
median fluorescence intensity. Table 14 contains the mean fluorescence intensity. Table
15 contains the trimmed mean fluorescence intensity. The amount of peptide solution
used to conjugate with the microspheres was 125 pL (1000 pg/mL).

Table 12. Median fluorescent intensity of microbead immunoassay (vacuum manifold).
Primary antibody used 50 pL (70 pg/mL and 294 pg/mL) (USBio). Secondary antibody

 

 

 

 

 

 

 

 

 

 

 

used 50 pL (10 pg/mL and 5 g/mL) (USBio).
Sample Primary Secondary Median Primary Secondary Median
Antibody Antibody Antibody Antibody
(rig/mL) (its/mL) (its/mL) (rig/ml.)
033—AT. (AT.) 70 10 7 294 5 11
033-AT. (BSA) 0 2 H 0 2
038 (AT.) 70 10 4 294 5 5
038 (BSA) 0 2 0 2

 

 

 

 

 

 

 

Table 13. Trimmed median fluorescent intensity of microbead immunoassay (vacuum

manifold).

Primary antibody used 50 pL (70 pg/mL and 294 pg/mL). Secondary antibody used 50

pL (10 pg/mL and 5 pg/mL).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Primary Secondary Trimmed Primary Secondary Trimmed

Antibody Antibody Median Antibody Antibody Median
(118/le (rig/mL) (148/le (118/le

033-AT. (AT.) 70 10 7 294 5 l l

033-AT. 0 2 O 2

(BSA) I

038 (AT.) 70 10 4 h 294 5 5

038 (BSA) 0 2 |] 0 2

 

37

 

 

Table 14. Mean fluorescent intensity of microbead immunoassay (vacuum manifold).
Primary antibody used 50 pL (70 pg/mL and 294 pg/mL) (USBio). Secondary antibody

used SOpL (10 pg/mL and 5 p,

g/mL) (USBio).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Primary Secondary Mean Primary Secondary Mean
Antibody Antibody Antibody Antibody
(rig/mm (its/mu (rig/mL) (Jig/”114)
033-AT. (AT.) 70 10 26.40 294 5 225.15
033-AT. 0 3.00 0 4.30
(BSA)
038 (AT.) 70 10 7.17 294 5 20.49
038 (BSA) 0 2.36 I] 0 2.84

 

 

Table 15. Trimmed mean fluorescent intensity of microbead immunoassay (vacuum

manifold).

Primary antibody used 50 pL (70 pg/mL and 294 pg/mL) (USBio). Secondary antibody

used 50 pL (10 p

g/mL and 5 pg/mL) (USBio).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Primary Secondary Trimmed Primary Secondary Trimmed

Antibody Antibody Mean Antibody Antibody Mean
(rig/mL) (rig/mL) (pg/mL) (ug/mL)

033-AT. (AT.) 70 10 8.56 294 5 15.10

033-AT. 0 2.39 O 2.93

(BSA)

038 (AT.) 70 10 4.65 294 5 5.90

038 (BSA) 0 2.13 ]| 0 2.59

 

 

 

Microbead immunoassay using centrifugation

The median, trimmed median, mean and trimmed mean from microbead assay

using centrifugation for the washing steps are listed in Table 16. The amount of peptide

solution used to conjugate with the microspheres was 125 pL (1000 pg/mL).

The

amount of primary antibody used was 50 pL (420 pg/mL) of anti—AT.. The amount of

secondary antibody used was 50 pL (4 pg/mL).

38

 

Table 16. Fluorescent intensity for microbead immunoassay (centrifugation).
Primary antibody used was 50 pL (420 pg/mL) of anti-AT. (USBio). Secondary
antibody used was 50 pL (4 pg/mL) (USBio).

 

 

 

 

 

 

 

 

 

 

 

 

Sample Median Trimmed Mean Trimmed Mean
Median
033-AT1 (AT1) ll 11 252.31 31.09
033-AT. (BSA) 3 3 9.93 4.57
034-AT. (AT1) 11 11 159.75 30.25
034-AT. (BSA) 2 2 2.82 2.30
035-AT. (AT1) 8 8 64.47 20.92
035-AT. (BSA) 2 2 3.03 2.16
036-AT. (AT1) 9 9 140.59 22.74
036-AT. (BSA) 10 10 14.55 11.29
038 (AT1) 5 5 36.45 7.89
038 (BSA) 2 2 3.17 2.60

 

 

 

 

 

 

Microbead immunoassay by varying incubation conditions and secondary antibody
concentrations

The results from microbead immunoassay experiment that vary the incubation
conditions along with secondary antibody concentrations are listed in tables 17-20.
contains the median fluorescence intensity. Table 18 contains the trimmed median
fluorescence intensity. Table 19 contains the mean fluorescence intensity. Table 20
contains the trimmed mean fluorescence intensity. The amount of peptide solution used

to conjugate with the microspheres was 125 pL (1000 pg/mL).

39

Table 17. Median fluorescent intensity for microbead immunoassay (modification:
incubation, secondary antibody).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Secondary antibody used was 50 pL (4 pg/ml and 40 pg/mL) (USBio).
Sample Primary Secondary Median Primary Secondary Median
Antibody Antibody Antibody Antibody
(rig/mL) (rue/mm f, (vamL) (ug/mL)
Room temperature incubation
033-AT. (AT.) 700 4 9 700 40 200
033-AT. (BSA) 0 3 0 17
038 (AT.) 700 41.5 700 247
038 (BSA) 0 2 0 5
37 °C incubation
033-AT. (AT.) 700 4 24 700 40 287.5
033-AT. (BSA) 0 4 0 2_2__fi
038 (AT.) 700 12 700 94
038 (BSA) 0 1 0 3
Table 18. Trimmed median fluorescent intensity for microbead immunoassay
(modification: incubation, secondary antibody).
Secondary antibody used was 50 pL (4 pg/ml and 40 pg/mL) (USBio).
Sample Primary Secondary Trimmed Primary Secondary Trimmed
Antibody Antibody Median Antibody Antibody Median
(118/le (rug/mu (pg/mL) (rig/mL)
Room temperature incubation
033-AT. (AT.) 700 4 9 700 40 200
033-AT. 0 3 0 17
(BSA)
038 (AT.) 700 41.5 700 247
038 (BSA) 0 2 0 5
37 °C incubation
033-AT. (AT.) 700 4 24 700 40 287.5
O33-AT. 0 4 0 22
(BSA)
038 (AT.) 700 12 700 93
038 (BSA) 0 1 0 3 __]

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

40

Table 19. Mean fluorescent intensity for microbead immunoassay (modification:
incubation, secondary antibody).

Secondary antibody used was 50 pL (4 p

g/ml and 40 pg/mL) (USBio).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Primary Secondary Mean Primary Secondary Mean
Antibody Antibody Antibody Antibody
(rig/mL) (rig/mL) .. (rig/mL) (rig/mu
Room temperature incubation
033-AT. 700 4 341.36 700 40 1500.75
(AT1)
033-AT. 0 6.05 0 110.61
(BSA)
038 (AT.) 700 728.63 700 2395.27.8_
038 (BSA) 0 7.29 0 29.77
37 °C incubation
033-AT. 700 4 369.55 700 40 1601.52
(AT1)
033-AT. 0 15.19 0 126.41
(BSA)
038 (AT.) 700 275.40 700 380.06
038 (BSA) 0 2.67 0 92.87

 

 

 

 

 

 

Table 20. Trimmed mean fluorescent intensity microbead immunoassay (modification:
incubation, secondary antibody).
Secondary antibody used was 50 pL Mpg/ml and 40 pg/mL)(USBio)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Primary Secondary Trimmed Primary Secondary Trimmed

Antibody Antibody Mean Antibody Antibody Mean
(rig/ml.) 018/le (rig/mu (rig/m1.)

Room temperature incubation ____fi

O33-AT. 700 4 59.43 700 40 641.21

(AT1)

033-AT. 0 3.48 0 26.83

(BSA)

038 (AT.) 700 322.29 700 2220644

038 (BSA) 0 2.66 0 7.66

37 °C incubation fl

033-AT. 700 4 52.8 700 40 735.84

(AT.) _,_____

O33-AT. O 6.43 0 57.71

(BSA)

038 (AT.) 700 20.65 700 _l___3_l.5_4

038 (BSA) 0 2 28 O 64".:21

 

 

 

 

4l

Microbead immunoassay by varying peptide concentrations #1

The median, trimmed median, mean and trimmed mean from microbead
immunoassay by varying peptide concentrations #1 are listed in Table 21 . The peptide
solution concentrations used for coupling were 100, 50, 25 and 5 pL (1000 pg/mL). The
amount of primary antibody used was 50 pL (70 pg/mL) anti-AT. (USBio). The amount
of secondary antibody used was 50 pL (4 pg/mL) (USBio).
Table 21. Fluorescent intensity for microbead immunoassay (modification: peptide

concentrations).
Peptide solution concentrations used for coupling were 100, 50, 25 and 5 pL (1000

pg/mL).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Peptide Median Trimmed Mean Trimmed
(pg) Median Mean
033-AT. (AT.) 100 4 4 10.12 5.10
033-AT. (BSA) 2 2 4.43 3.21
033-AT. (AT.) 50 4 4 28.25 6.23
033-AT. (BSA) 3 3 4.75 3.47
033-AT. (AT.) 25 4 4 68.42 6.68
033-AT. (BSA) 3 3 5.29 3.60
033-AT. (AT.) 5 4 4 196.37 9.37
033-AT. (BSA) 2 2 3.93 3.27
038 (AT.) 0 4 4 147.46 6.00
038 (BSA) 2 2 3.1 l 2.46

 

 

 

 

 

 

 

 

Microbead immunoassay by varying peptide concentrations #2

The median, trimmed median, mean and trimmed mean for microbead
immunoassay by varying peptide concentrations #2 are listed in Table 22. The peptide
solution concentrations used for coupling were 100, 50, 25 and 5 pg. The amount of
primary antibody used was 50 pL (4 pg/mL) anti-AT.. The amount of secondary

antibody used was 50 pL (5 pg/mL).

Table 22. Fluorescent intensity for microbead immunoassay (modification: peptide
concentrations, microsphere groups).
Peptide solution concentrations used for coupling were 100, 50, 25 and 5 pL (1000

pg/mL). Microsphere group 033, 034, 035, 036, 037 and 038 were used.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Peptide Median Trimmed Mean Trimmed

(pg) Median Mean
033—AT. (AT.) 100 7 7 19.21 9.78
033-AT. (BSA) 10 10 20.64 14.61
033-AT. (AT.) 50 7 7 11.61 8.22
033-AT. (BSA) 10 10 23.16 14.36
033-AT. (AT.) 25 10 10 83.81 12.77
033-AT. (BSA) 8 8 17.28 11.22
033-AT. (AT.) 5 6 6 23.62 8.28
033-AT. (BSA) 8 8 16.75 10.39
O34-AT. (AT.) 100 13 13 186.42 18.93
034-AT. (BSA) 6 6 16.31 8.82
034-AT. (AT.) 50 7 7 19.85 9.60
034-AT. (BSA) 5 5 13.82 8.24
034-AT. (AT.) 25 7 7 60.96 8.20
034-AT. (BSA) 5 5 9.78 8.02
034-AT. (AT.) 5 9 9 28.63 10.92
034-AT. (BSA) 6 6 10.61 8.62
035-AT. (AT.) 100 11 11 39.98 13.17
035-AT. (BSA) 6 6 9.82 8.00
035-AT. (AT.) 50 10 10 30.88 13.07
035-AT. (BSA) 6 6 10.05 8.17
035-AT. (AT.) 25 12 12 66.68 17.16
035-AT. (BSA) 7 7 10.82 8.96
035-AT. (AT.) 5 l3 13 127.46 15.90
035-AT. (BSA) 6 6 9.88 8.46
036-AT. (AT.) 100 9 9 52.74 11.30
036-AT. (BSA) 10 10 21.29 14.73
036-AT. (AT.) 50 7 7 25.28 8.05
036-AT. (BSA) 5 5 8.10 7.02
036-AT. (AT.) 25 9 9 19.25 10.64
036-AT. (BSA) 6 6 10.41 8.72
036-AT. (AT.) 5 15 15 189.50 24.96
036-AT. (BSA) 6 6 12.60 8.76
038 (AT.) 0 8 8 33.42 9.87
038 (BSA) 6 6 l 1.24 8.49

 

43

 

Microbead immunoassay by varying peptide concentrations and primary antibody

concentrations

The median, trimmed median, mean and trimmed mean for microbead

immunoassay by varying peptide concentrations and primary antibody concentrations are

listed in Table 23. The peptide solution concentration used for coupling was 100, 50, 25

and 5 pL (1000 pg/mL). The amount of primary antibody used was 50 pL (70 p g/mL

and 140 pg/mL) of anti- AT. (USBio). The amount of secondary antibody used was 50

pL (4 pg/mL) (USBio).

Table 23. Fluorescent intensity for microbead immunoassay (modification: peptide
concentrations, primary antibody).
Primary antibody used was 50 pL (70 pg/mL and 140 pg/mL). The peptide amount for
coupling were 100, 50, 25, and 5 pL (1000 pg/mL).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Peptide Primary Median Trimmed Mean Trimmed
(pg) Antibody Median M6311
(rug/mu
033-AT. (AT.) 100 70 4 4 31.81 4.93
033-AT. (AT.) 140 5 5 19.64 8.02
033-AT. (BSA) 0 3 3 5.64 4.46
033-AT. (AT.) 50 70 4 4 38.99 6.16 -
033-AT. (AT.) 140 4 4 60.56 6:2__I____ _1
033-AT. (BSA) 0 3 3 8.08 4.36
033-AT. (AT.) 125 70 4 4 28.37 5.79
033-AT. (AT.) 140 5 5 150.93 7.58
033-AT. (BSA) 0 3 3 6.04 4.91
033-AT. (AT.) 5 70 4 4 74.92 5.97
033-AT. (AT.) 140 4 4 81.46 5.77
033-AT. (BSA) 0 3 3 4.90 3.94
038 (AT.) 0 70 7 7 187.09 22.48”»
038 (AT.) 140 4 4 178.75 6.63 ___
038 (BSA) 0 2 2 3.10 2.51

 

 

 

Microbead immunoassay by varying primary antibody source/er anti-A T3

The median, trimmed median, mean and trimmed mean from microbead

immunoassay by varying secondary antibody concentrations are listed in Table 24. The

44

peptide solution concentration used for coupling was 125 pL (1000 pg’mL). For anti-
AT., primary antibody used was 50 pL (2 prymL) of anti-AT. (USBio). For anti-AT;
primary antibody used was 50 pL (0.1 pg/mL) of anti-AT; by two different
manufacturers: USBio and Abcam. Secondary antibody used was 50 pL (4 pg/mL).
Table 24. Fluorescent intensity for microbead immunoassay (modification: primary and
secondary antibody).

Primary antibody used was 50 pL (2 pg/mL) for anti—AT. (USBio); 50 pL (0.1 pg/mL) of
anti-AT; from USBio and Abcam. Secondary antibody for anti-AT. used was 50 pL (5

pg/mL); Secondary antibody for anti-ATg antibody used was 50 LL (2.5 pg/mL).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Primary Median Trimmed Mean Trimmed
Antibody Median Mean
(vs/mL)
033-AT. (AT.) 2 4 4 14.59 4.92
033-AT. (BSA) 0 5 5 10.58 6.58
034-AT. (AT.) 2 4 4 6.08 4.91
034-AT. (BSA) 0 3 3 3.89 3'4..(3__-
035-AT. (AT.) 2 4 4 6.08 4.56 #
035—AT. (BSA) 0 3 3 5.04 4.37_
036-AT. (AT.) 2 4 4 16.25 4.71 __J
036-AT. (BSA) 0 3 3 4.84 3.89
037-AT2 (AT2)
(USBio) 0.1 4 4 5.67 4.83
037-AT2 (AT2)
(Abcam) 0.1 4 4 5.84 5.12
037-AT2 (BSA)
(USbio) O 4 4 5.75 4.93
037-AT2 (BSA)
(Abcam) 0 4 4 5.40 492 _fl
038 (AT.) 2 4 4 7.07 4.63
038 (AT2) (USBio)
0.1 5 5 6.05 5.35 _
038 (AT2) (Abcam) l
0.1 3 3 4.41 3.90
038 (BSA) 0 2 2 2.94 2.67”__é
038 (BSA) (USBio)
0 3 3 6.96 3.65
038 (BSA) (Abcam)
0 2.5 2.5 3.19 2.92

 

 

 

45

Rat sera screening using microbead immunoassay by centrifugation method

The results from rat sera screening using microbead immunoassay by
centrifugation method are listed in tables 25-28. Table 25 contains the median
fluorescence intensity. Table 26 contains the trimmed median fluorescence intensity.
Table 27 contains the mean fluorescence intensity. Table 28 contains the trimmed mean
fluorescence intensity. In this experiment, different microsphere groups were combined
prior the addition of primary antibody. The peptide solution concentrations used to
couple with the microspheres was 125 pL (1000 pg/mL). The amount of rat sera used
was 50 pL of undiluted rat sera. The amount of secondary antibody used was 50 pL (4
meL) (Abcam).
Table 25. Median fluorescent intensity of rat sera screening.

The rat sera screening was performed using the microbead immunoassay by
centrifugation method. Rat sera used was 50 pL, and secondary antibody used was 50

M (4 rte/mu.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Microsphere group
AT.-O33 AT.—034 AT.-035 AT.-036 AT3-037 038
Negative 12.5 8 8 ll 20 ____ 5 _
1 206.5 11 8 9 21.5 9_
2 21 10 9 9 20 81
3 52.5 12 12 13 17 13
4 24 10 10 7 4 9
5 11 6 8 8 11.5 8_
6 35 11 10 8 22 13
7 21 8 9 9 27 9.5
8 41 12 10 9 24 9
9 224 9 7 6 9 6
10 376.5 18 18 16 54 _ 2‘04
11 36 9 9.5 8 8.5 _7__4
12 25 8 8 8 19 ~_ __7_’
13 32 10 10 8 14.5 7
14 35 21 20 20 42 18"
15 43 7 9 10 25 9
16 126 25 25 21 22 27;
17 21 6 9 7 1 . -8.

 

 

 

46

Table 26. Trimmed median fluorescent intensity of rat sera screening.
The rat sera screening was performed using the microbead immunoassay by
centrifugation method. Rat sera used was 50 pL, and secondary antibody used was 50

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

11L (4 vs/mL).
Sample Microsphere group

AT.-033 AT.-034 AT.-035 AT.-036 AT2-037 038

Negative 12.5 8 8 1 1 20 5
1 206.5 11 8 9 21.5 9

2 21 10 9 9 20 8

3 52.5 12 12 I3 17 13

4 24 10 10 7 4 9

5 11 6 8 8 11.5 8

6 35 11 10 8 22 13

7 21 8 9 9 27 9.5

8 41 12 10 9 24 9

9 224 9 7 6 9 6

10 376.5 18 18 16 54 20

11 36 9 9.5 8 8.5 7
12 25 8 8 8 19 7
13 32 10 10 8 14.5 7
14 35 21 20 20 42 18
15 43 7 9 10 25 9
16 126 25 25 21 22 27
17 21 6 9 7 l 8

 

 

 

 

 

 

 

 

47

 

Table 27. Mean fluorescent intensity of rat sera screening.
The rat sera screening was performed using the microbead immunoassay by
centrifugation method. Rat sera used was 50 pL, and secondary antibody used was 50

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

pL (4 pg/mL).
Sample Microsphere group
AT.—033 AT .—034 AT.-035 AT.—036 AT3-037 038
Negative 40.62 18.22 18.38 18.39 45.78 16.15
1 701.02 46.14 51.16 20.90 145.66 27.07
2 117.05 22.17 18.80 27.14 88.10 36.91
3 142.20 23.26 26.52 20.97 46.72 24.10
4 60.35 17.29 18.01 20.24 14.03 20.78
5 36.07 13.75 22.47 14.75 17.29 24.25
6 129.47 22.96 21.29 20.18 99.03 26.32
7 120.96 20.19 24.97 22.90 96.44 24.29
8 170.15 24.18 28.68 27.87 88.50 19.40
9 599.77 12.97 12.90 13.74 235.97 41.01
10 1326.80 68.96 83.19 31.07 874.78 63,152.,
11 132.61 16.33 22.01 37.41 39.67 17.141]
12 62.83 17.14 33.32 21.89 54.59 19.4_1__4
13 96.62 19.78 25.50 25.00 35.46 33.27
14 95.23 28.45 31.19 29.10 88.07 31.00
15 137.27 23.80 21.51 19.97 33.84 25.17
16 341.45 30.96 34.23 36.00 170.34 45.36
17 59.08 16.55 19.00 18.39 57.70 19.64

 

 

 

 

 

 

 

 

48

 

Table 28. Trimmed mean fluorescent intensity of rat sera screening.

The rat sera screening was performed using the microbead immunoassay by
centrifugation method. Rat sera used was 50 pL, and secondary antibody used was 50

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

L (4 pg/mL).
Sample Microsphere group
AT.-033 AT.-034 AT.-035 AT.-036 AT3-037 03g

Negative 23.89 13.59 15.38 15.16 31.17 12.16
1 478.28 16.22 15.01 15.17 66.76 15.36
2 59.56 15.40 14.09 14.94 51.81 13.37
3 93.11 16.94 18.54 17.48 30.45 18.03
4 42.14 14.17 14.27 13.35 12.70 14.01
5 28.60 11.47 12.61 12.27 17.29 12.55
6 72.44 16.72 16.16 15.55 62.90 17.65
7 40.49 15.36 15.54 15.09 71.89 15.41
8 95.61 18.37 16.86 16.61 43.24 16.77
9 416.66 10.88 10.49 10.31 155.18 9.47
10 843.83 23.73 25.09 22.85 580.76 29.55
11 62.97 13.94 14.13 14.37 25.32 13.02
12 43.07 14.37 14.01 14.46 39.77 13.87
13 57.85 15.68 15.82 15.67 22.64 15.19
14 66.75 23.40 24.09 24.06 66.46 24.13
15 99.03 14.29 14.93 15.90 26.87 15.51
16 235.00 26.81 27.94 24.57 101.88 29.70
17 42.89 13.12 14.29 14.31 35.78 14.86

 

 

 

 

 

 

 

 

 

The results from rat sera screening using microbead immunoassay by

centrifugation method and ADH modified microspheres are listed in tables 29-32. Table

29 contains the median fluorescence intensity. Table 30 contains the trimmed median

fluorescence intensity. Table 31 contains the mean fluorescence intensity. Table 32

contains the trimmed mean fluorescence intensity. In this experiment, different

microsphere groups were combined prior the addition of primary antibody. The peptide

solution concentrations used to couple with the ADH modified microspheres was 125 pL

(1000 pg/mL). The amount of rat sera used was 50 pL of undiluted rat sera. The amount

of secondary antibody used was 50 pL (4 pg/m L) (Abcam). Due to limited amount of

sample, sample #9 and #1 1 were not used for this experiment.

49

Table 29. Median fluorescent intensity of rat sera screening (ADH microspheres).
The rat sera screening was performed using the microbead immunoassay by
centrifugation method and ADH modified microspheres. Rat sera used was 50 pL, and
secondary antibody used was 50 pL (4 pg/mL).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Microsphere group
AT.-033 AT. -034 AT.-035 AT.-036 AT..-037 038
Negative 14 7.5 8 6 12 12
1 8.5 11 5.5 8 10 8
2 6 4.5 5.5 7 7 6.5%
3 9 5.5 11 7 10 10
4 10 9 10 8.5 6 10
5 8.5 9 7 4.5 11 9
6 7.5 10 8 16 8 13.5
7 7 5 8 11.5 8 __ ______1_()__
8 14 11 6 8 10 7.5
10 13.5 10 4 12 15 10
12 10 8 9 8 9.5 8.5
13 8 8 9 9 4 9
14 22 27.5 28 29 30 27
15 10 8 8 14.5 10 12
16 26 29 25 22.5 26.5 31
17 12.5 9 9 7.5 8 8

 

 

 

50

Table 30. Trimmed median fluorescent intensity of rat sera screening (ADH

microspheres).

The rat sera screening was performed using the microbead immunoassay by
centrifugation method and ADH modified microspheres. Rat sera used was 50 pL, and
secondary antibody used was 50 pL (4 me).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Microsphere group
AT. -033 AT.-034 AT.—035 AT.-036 AT2-037 038
Negative 14 7.5 8 6 12 124
l 8.5 11 5.5 8 10 8
2 6 4.5 5.5 7 7 6.5
3 9 5.5 11 7 10 10
4 10 9 10 8.5 6 10
5 8.5 9 7 4.5 11 9
6 7.5 10 8 16 8 13.5
7 7 5 8 11.5 8 10
8 14 11 6 8 10 7.5
10 13.5 10 4 12 15 10
12 10 8 9 8 9.5 8.5
13 8 8 9 9 4 9
14 22 27.5 28 29 30 27
15 10 8 8 14.5 10 12
16 26 29 25 22.5 26.5 31
17 12.5 9 9 7.5 8 8

 

 

 

51

Table 31. Mean fluorescent intensity of rat sera screening (ADH microspheres).

The rat sera screening was performed using the microbead immunoassay by
centrifugation method and ADH modified microspheres. Rat sera used was 50 pL, and
secondary antibody used was 50 pL (4 pg/mL).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Microsphere group
AT.-033 AT.-034 AT.-035 AT.-036 AT3-037 03L
Negative 19.13 10.22 12.01 11.79 15.29 17.76
1 13.21 14.23 12.22 13.06 13.91 12.22
2 20.33 11.14 12.04 12.60 12.67 15.19
3 15.16 12.72 14.60 13.78 15.64 15.13
4 21.69 16.82 15.74 14.09 14.39 15.28
5 15.72 12.42 11.97 11.39 16.11 17.22
6 13.28 14.58 23.89 21.31 17.89 24.91
7 13.08 11.78 13.33 14.38 13.58 14.29
8 16.90 13.78 13.43 15.53 19.47 12.40
10 19.99 16.39 12.82 12.90 16.48 13.57
12 14.87 21.96 12.73 21.66 14.70 13.77
13 16.67 15.94 14.17 14.08 12.04 14.39
14 26.71 29.71 31.30 31.41 33.63 29.46
15 14.04 14.29 13.20 19.80 19.72 16.25
16 27.57 29.22 29.34 24.73 31.79 33.23
17 26.63 12.56 13.40 14.02 11.36 14.29

 

52

 

Table 32. Trimmed mean fluorescent intensity of rat sera screening (ADH microspheres).
The rat sera screening was performed using the microbead immunoassay by
centrifugation method and ADH modified microspheres. Rat sera used was 50 pL, and
secondary antibody used was 50 pL (4 pg/mL).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Microsphere group
AT.-033 AT.-O34 AT.-035 AT.-036 AT2-037 038
Negative 16.37 9.19 10.39 10.05 14.32 13.16
1 11.03 12.83 10.21 11.44 11.82 10.10
2 11.98 9.44 10.63 11.36 10.92 11.14
3 13.16 11.01 13.36 11.76 14.14 13.97
4 14.41 13.91 14.46 12.43 11.23 13.67
5 12.92 10.97 10.61 9.45 14.50 12.43
6 11.48 12.69 11.92 18.92 13.97 16.25“
7 10.76 10.06 11.91 13.35 10.21 12.78
8 14.76 12.22 11.89 13.82 15.26 11.21
10 15.76 15.03 11.09 11.95 15.22 12.50
12 12.19 12.73 11.29 10.97 13.05 11.78
13 12.23 10.90 10.96 12.80 8.88 12.55
14 24.49 28.30 29.50 30.54 31.91 28.27
15 12.60 12.73 11.86 15.62 13.59 14.87
16 25.82 28.47 28.12 23.57 28.04 32.15
17 16.47 11.55 11.47 12.23 10.00 12.36

 

 

 

Human sera using microbead immunoassay by centrifugation method

The results from human sera screening are listed in tables 33-36. There were 158

samples of human sera used. The results listed here are representative of 158 of the

human samples. The microbead immunoassay by centrifugation method was used. Table

33 contains the median fluorescence intensity. Table 34 contains the trimmed median

fluorescence intensity. Table 35 contains the mean fluorescence intensity. Table 36

contains the trimmed mean fluorescence intensity. In this experiment, different
microsphere groups were combined prior the addition of primary antibody. The peptide
solution concentrations used to couple with the microspheres was 125 pL (1000 pg/mL).
The amount of human sera used was 50 pL. The amount of secondary antibody used was

50 pL (4 pg/mL) (Abcam).

53

Table 33. Median fluorescent intensity of human sera screening.
The human sera screening was performed using the microbead immunoassay by

centrifugation method. Human sera used was 50 pL, and secondary antibody used was

 

 

 

 

 

 

 

 

 

 

 

 

 

50 pL (4 tug/m1.) (Abcam).
Sample Microsphere group
AT.-033 AT.-034 AT.-035 AT.-036 AT3-037 038

Negative 5 3 2 3 2 4
1 5 4 3 3 2 3
2 6 4 4 2 4 3
3 3 3 3 4 6 4
4 4 3 2 4 6 5
5 5 3 4 4 5.5 3
6 5 3 2 3 4 4
7 5 3 3 4 5.5 32
8 5 3 3 4 3 4
9 6 3 3 4 4 4
10 5 5 2 5 5 3

 

 

 

 

 

 

 

 

 

Table 34. Trimmed median fluorescence intensity of human sera screening.

The human sera screening was performed using the microbead immunoassay by

centrifugation method. Human sera used was 50 pL, and secondary antibody used was

50 pL (4 pg/mL) (Abcam).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Microsphere group
AT.-033 AT.-034 AT.-035 AT.-036 AT2-037 038
Negative 5 3 2 3 2 4
1 5 4 3 3 2 3
2 6 4 4 2 4 3
3 3 3 3 4 6 4
4 4 3 2 4 6 5
5 5 3 4 4 5.5 3
6 5 3 2 3 4 4
7 5 3 3 4 5.5 3
8 5 3 3 4 3 4
9 6 3 3 4 4 4
10 5 5 2 5 5 3

 

 

54

Table 35. Mean fluorescence intensity of human sera screening.

The human sera screening was performed using the microbead immunoassay by
centrifugation method. Human sera used was 50 pL, and secondary antibody used was
50 pL (4 pg/mL) (Abcam).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Microsphere group
AT.-033 AT.-034 AT.-035 AT.-036 AT3-037 038

Negative 10.33 5.74 4.36 4.84 11.00 5.48
l 10.68 5.34 4.61 4.99 5.88 4.99
2 8.60 5.73 4.86 4.39 6.24 4.57
3 9.34 5.80 5.22 5.69 7.95 6.48
4 8.06 5.62 4.92 6.56 8.31 7.30
5 9.52 5.85 6.80 6.61 8.53 11.12
6 13.42 6.16 6.01 5.76 9.82 6.64
7 8.18 5.94 6.94 7.27 9.08 6.31
8 8.76 5.02 5.31 5.58 9.61 7.02
9 10.66 6.43 6.41 5.90 6.21 7.40
10 8.21 5.78 5.23 12.48 7.42 5.94

 

 

 

 

 

 

 

Table 36. Trimmed mean fluorescence intensity of human sera screening.

The human sera screening was performed using the microbead immunoassay by
centrifugation method. Human sera used was 50 pL, and secondary antibody used was
50 pL (4 pg/mL) (Abcam).

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Microsphere group . ___ _g
AT.-033 AT.-034 AT.-035 AT.-036 AT3-037_+ 2-93312.
Negative 5.54 4.40 3.68 4.37 4.13 4'8].
l 6.21 4.86 4.01 4.44 3.78 4.42%

2 6.82 5.07 4.34 3.81 5.40 3.88

3 5.80 4.52 4.62 4.87 6.47 5.32

4 6.56 5.01 4.29 5.58 6.78 6.45

5 7.27 5.18 6.07 5.62 7.16 4.95

6 6.64 5.50 5.25 5.01 7.02 5.94

7 6.49 5.33 6.09 5.36 6.60 5.67

8 5.68 4.60 4.75 5.08 5.24 4.94

9 7.02 5.63 4.92 5.19 5.29 5.46

10 6.50 5.15 4.65 5.95 6.70 5.21

 

 

 

 

 

 

 

 

 

55

The results from human sera screening using the ADH modified microspheres are
listed in tables 37—40. The microbead immunoassay by centrifugation method was used.
Table 37 contains the median fluorescence intensity. Table 38 contains the trimmed
median fluorescence intensity. Table 39 contains the mean fluorescence intensity. Table
40 contains the trimmed mean fluorescence intensity. In this experiment, different
microsphere groups were combined prior the addition of primary antibody. The peptide
solution concentrations used to couple with the ADH modified microspheres was 125 p1.
(1000 pg/mL). The amount of human sera used was 50 pL. The amount of secondary
antibody used was 50 pL (4 pg/mL) (Abcam).

Table 37. Median fluorescent intensity of human sera screening (ADH microspheres).
The human sera screening was performed using the microbead immunoassay by

centrifugation method and ADH modified microspheres. Human sera used was 50 pL,
and secondary antibody used was 50 pL (4 pg/mL) (Abcam).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Microsphere group
AT.-O33 AT.-034 AT.-035 AT.-036 AT3-037 038

Negative 16 25 5 13 45 18__
1 4 4 4 4 5 3 l
2 4 5 3 4 5 5
3 5 4 4 4 4 4 _l
4 3 4 3 4 1 4 j
5 4 3 4 4 2 _4_,
6 3 3 3 2 3.5 ___ _3__
7 4 4 2.5 4 5+ _____ _5
8 4 3 2 4 3 __ _5:
9 6 4 4 4 4 6 j
10 4 4 3 4 4 4

 

 

 

 

 

 

 

 

56

Table 38. Trimmed median fluorescence intensity of human sera screening (ADH

microspheres).

The human sera screening was performed using the microbead immunoassay by
centrifugation method and ADH modified microspheres. Human sera used was 50 pL,
and secondary antibody used was 50 pL (4 pg/mL) (Abcam).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Microsphere group

AT. -033 AT. -034 AT.-035 AT.-036 AT3-03 7 ()3_8__
Negative 16 25 5 13 45 1_8_ j
1 4 4 4 4 5 2.-

2 4 5 3 4 5 5

3 5 4 4 4 4 4

4 3 4 3 4 1 4

5 4 3 4 4 2 4

6 3 3 3 2 3.5 3

7 4 4 2.5 4 5 5
8 4 3 2 4 3 3 l
9 6 4 4 4 4 6 l

10 4 4 3 4 4 4

 

 

 

Table 39. Mean fluorescence intensity of human sera screening (ADH microspheres).
The human sera screening was performed using the microbead immunoassay by
centrifugation method and ADH modified microspheres. Human sera used was 50 pL.
and secondary antibody used was 50 pL (4 pg/mL) (Abcam).

 

‘_—1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

57

Sample Microspheregrog)
AT.-033 AT.-034 AT.-035 AT.-036 AT3-037 038- g
Negative 25.22 30.17 5.36 13.61 56.44 32.519.
1 4.98 5.09 4.54 4.88 10.23 3262..
2 6.41 5.81 4.66 5.70 6.84 5.85
3 9.92 4.45 4.79 7.26 5.54 4.83
4 4.43 6.12 4.21 4.42 4.33 5.14
5 5.82 5.21 5.55 5.38 6.81 5.54
6 4.81 4.68 4.50 4.30 5.15 -_6.__5_7~;
7 5.03 4.94 4.91 4.36 5.68 5.38%
8 4.48 4.01 3.79 4.89 4.82 4.55”,
9 12.07 6.53 6.14 6.44 7.45 8.14
44-9—4 4M 1
10 5.13 6.54 5.51 4.131] _ 5 479_L __ 4.74

 

 

Table 40. Trimmed mean fluorescence intensity of human sera screening (ADH

microspheres).

The human sera screening was performed using the microbead immunoassay by

centrifugation method and ADH modified microspheres. Human sera used was 50 pL,
and secondary antibody used was 50 pL (4 pg/mL) (Abcam).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Microsphere group
AT.-033 AT.-034 AT.-035 AT.-O36 AT..-037 038

Negative 17.77 27.27 5.13 13.20 51.73 18.76
1 4.26 4.54 4.21 4.39 5.03 4.19
2 5.68 5.13 4.09 5.16 5.91 5.38
3 5.13 4.08 4.31 4.72 4.92 4.50
4 3.88 5.03 3.86 4.03 3.77 4.56
5 5.31 4.82 4.96 5.05 3.96 5.02
6 4.39 4.03 4.10 3.88 4.37 4.01
7 4.70 4.47 3.85 4.15 5.32 5.13
8 3.99 3.69 3.44 4.60 4.40 3.83
9 8.30 5.63 5.56 5.37 5.69 6.36
10 4.57 4.62 3.66 4.20 4.22 4.39

 

In addition to microbead immunoassay experiments, other experiments such as

determination of peptide coupling efficiency, the ELISA and the dot blot were perfomed.

These experiments were performed to aid in the development of the microbead

immunoassay.

58

 

Determination of peptide coupling efficiency

The results from the determination of peptide coupling efficiency by measuring

the absorbance at 230 nm overtime are listed in Table 41. Initial peptide solution

concentration used for coupling was 125 pL (1000pg/mL).

Table 41. Peptide absorbance at 230 nm.

Initial peptide solution concentration was 125 pL (1000pg/mL).

 

 

 

 

 

 

 

 

 

 

 

 

 

Peptide ID Absorbance at 230 nm 2
0 hours 2 hours 4 hours 20 hours 24 hours

AT.- 033 1.756 1.698 1.683 1.633 1.613
AT.- 034 0.959 0.910 0.892 0.882 0880
AT.- 035 0.183 0.167 0.154 0.139 0.129
AT.- 036 1.403 1.274 1.261 1.255 1.239
AT;- 037 0.909 0.772 0.768 0.763 0.758 _.-
Blank 0.066 0.093 0.067 0.059 0.057
(MES buffer)

 

 

 

The results from the determination of peptide coupling efficiency by measuring

the absorbance at 260 nm overtime are listed in Table 42.

Table 42. Peptide absorbance at 260 nm.

Initial peptide solution concentration was 125 pL (1000pg/mL).

 

 

 

 

 

 

 

 

 

 

 

 

 

Peptide ID Absorbance at 260 nm

0 hours 2 hours 4 hours 20 hours 24 hours
AT.- 033 0.049 0.059 0.053 0.051 0.018
AT.- 034 0.095 0.096 0.084 0.082 0.079
AT.- 035 0.011 0.024 0.011 0.000 0.002
AT.- 036 0.208 0.195 0.183 0.177 _ 0.163 _
AT;- 037 0.077 0.045 0.042 0.047 __ 9,942 _7
Blank 0.030 0.061 0.041 0.034 0.031
(MES buffer) _g

 

 

 

The results from the determination of peptide coupling efficiency by measuring

the absorbance at 280 nm overtime are listed in Table 43.

59

Table 43. Peptide absorbance at 280 nm.
Initial peptide amount was 125 pL (1000pg/mL).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Peptide ID Absorbance at 280 nm
0 hours 2 hours 4 hours 20 hours 24 hours

AT.- 033 0.036 0.046 0.041 0.038 0.005
AT.- 034 0.173 0.165 0.155 0.151 0.151
AT.- 035 0.007 0.019 0.007 -0.004 0.001
AT.- 036 0.255 0.237 0.225 0.221 0.202
AT2- 037 0.074 0.039 0.036 0.040 0.040
Blank 0.021 0.050 0.030 0.023 0.018
(MES buffer)

 

The results from the determination of peptide coupling efficiency by measuring

the absorbance at 320 nm overtime are listed in Table 44.

Table 44. Peptide absorbance at 320 nm.

Initial peptide amount was 125 pL (1000pg/mL).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Peptide ID Absorbance at 320 nm 5 p l
0 hours 2 hours 4 hours 20 hours 24 hours
AT.- 033 0.009 0.019 0.013 0.020 -0.007
AT.- 034 -0.009 0.000 -0.01 1 -0.009 -0.006
AT.— 035 0.005 0.004 -0.008 -0.014 -0.014
AT.- 036 0.010 0.019 0.006 0.005 -0.01 1
AT2- 037 0.020 0.001 -0.001 0.006 0.002
Blank -0.009 0.10 -0.006 -0.1 1 -0.016
(MES buffer)
ELISA

The absorbance reading at 405 nm from the ELISA is in Table 45.

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61

Dot Blot with non-fat dry milk as blocking agent

This method used non- fat dry milk as blocking agent. A general map for this dot
blot is in Figure 6. The result for dot blot without vacuum method is in Figure 7 and the
dot blot with the vacuum method is in Figure 8. The peptide concentration solution used
was 10 uL (1000 ug/mL). The amount of BSA solution used was 20 uL (20 ug/mL).
The amount of anti-AT] and anti-AT; amount used were 5 uL (pg/mL). The amount of
primary antibody used for AT. was 10 mL (2 ug/mL) and for AT; was 10 mL (0.1
ug/mL). The amount of secondary antibody used for anti-IgG-ATl—AP was 10 mL (1

ug/ml), and for anti-IgG-ATg-AP was 10 mL (1 ug/mL).

62

 

 

 

 

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8
Peptide Peptide Peptide Peptide Peptide BSA Anti- Anti-ATZ
AT1-033 AT1—034 AT1-035 A'l‘l-036 AT2-037 A'l‘l

 

 

 

 

 

 

 

 

 

 

 

Figure 6. Dot blot map with 3% non-fat dry milk.

 

 

Dot 81m res-m lO/‘linO? ms Court?)

Anti-AT]
, > , positive
033 034 035 036 037 BSA Anti—AT] Anti-A'l‘g

Anti-AT,
negative

Anti-ATz
positive

Anti-All
negative

 

 

 

Figure 7. Dot Blot with 3% non-fat dry milk (without vacuum method).

Primary antibody used for AT. was 10 mL (2 ug/mL) and for AT2 was 10 m1, (0.]
ug/mL). Secondary antibody for anti-IgG-ATl-AP was 10 mL (1 ug/ml). and for anti-
IgG-ATz-AP was 10 mL (1 ug/mL).

63

 

not 8101 0“! Mia? MS {p.393

033 034 035 036 037 BSA Anti-AT. Anti-AT;

    

 

 

 

 

 

Figure 8. Dot Blot with 3% non-fat dry milk (with vacuum method).
Primary antibody used for ATI was 10 mL (2 ug/mL) and for AT2 was 10 mL (0.1
ug/mli). Secondary antibody for anti-IgG-ATl—AP was 10 mL (1 ug/ml), and for anti-
lgG-ATz—AP was 10 mL (1 ug/mL) (handwritten).
Dot Blot with PEG milk as blocking agent

This method used PEG as blocking agent. PEG concentrations used were 1%.
3.5%, and 10%. Figure 9 is the map for the dot blot with PEG as blocking agent. The
dot blot without vacuum method result is in figures 10-12 and the dot blot with the

vacuum method is in figures 13-15. Figure 10 is the dot blot result without vacuum

method with 1% PEG as blocking agent. Figure 1 1 is the dot blot result without vacuum

64

method with 3.5% PEG as blocking agent. Figure 12 is the dot blot result without
vacuum method with 10% PEG as blocking agent. Figure 13 is the dot blot result
without vacuum method with 1% PEG as blocking agent. Figure 14 is the dot blot result
without vacuum method with 3.5% PEG as blocking agent. Figure 15 is the dot blot
result without vacuum method with 10% PEG as blocking agent.

The peptide solution concentration used was 10 uL (1000 ug/mL). The amount
of BSA solution used was 20 uL (20 ug/mL). The amount of anti-AT] and anti-AT;
amount used were 5 uL (pg/mL). The amount of primary antibody used for AT. was 10
mL (2 ug/mL) and for AT; was 10 mL (0.1 ug/mL). The amount of secondary antibody
for anti-IgG-ATl-AP used was 10 mL (1 ug/ml), and for anti-lgG-ATg-AP was 10 mL (1

tie/mid).

65

 

Lane

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

l 2 3 4 5 6 7 8 9
blank Peptide Peptide Peptide Peptide blank BSA blank Anti-AT]
AT1-033 AT1-034 AT1-035 AT1-036
Figure 9. Dot blot map with PEG.
O33 O34 O35 O36 BSA Anti-AT!

 

Anti-AT. positive

Anti-AT. negative

 

Figure 10. Dot Blot with 1% PEG as a blocking solution (without vacuum method).

 

 

O33 O34 035

036

BSA

Anti-AT]

Anti-AT. positive

Anti-AT. negative

 

Figure 1 1. Dot Blot with 3.5% PEG as a blocking solution (without vacuum method).

66

 

 

 

 

O33 034 035 036

 

BSA Anti-AT]

Anti-AT. positive

Anti-AT] negative

 

 

Figure 12. Dot Blot with 10% PEG as a blocking solution (without vacuum method).

 

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67

 

 

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i» .,
‘ 1
.‘1 e.
. 1
5'. . . ~ :.
A 4s~_vu.-.n.'-r......_.....; Q33.— , ......__, w. M 1,0 . ._._ . 3. _ _. ‘ .
" - ~, -fl,“. ‘ --.~. -\ ~ . -. e..L—_..t. : .4..-.....—....~..~.~.~~ , < - -.-—.1.1--r-ul.-.-. or.- ---.. . . ‘ uvuo-‘—_n.'-: !

 

 

 

Figure 15. Dot Blot with 10% PEG as a blocking solution (with vacuum method)
(handwritten).

69

DISCUSSION

The goal of this research was to develop a screening method for anti-AT; and

anti-ATg antibodies using microbead immunoassay.

Microbead immunoassay

Microbead immunoassay using vacuum manifold

 

The microbead immunoassay using vacuum manifold method was adapted from
the Luminex Corporation (Austin, TX). In this method, a commercial anti-AT] antibody
was used. Sample 033-AT. (AT.) was the positive control for microsphere group 033
coupled with peptide AT1-033. Sample 038 (AT1) was the blank for the assay. To
determine the presence of anti-AT; or anti-ATg the fluorescence intensity between 033-
AT. (AT.) and 038 (AT1) was compared. From the median fluorescence intensity
between the 033-ATI (AT.) and 038 (AT1) (Table 12), it showed that there was an
increase in the fluorescence intensity however the increase was lower than 1000 fold.
The trimmed median fluorescence intensity result (Table 13) was identical as the median
fluorescence intensity. The mean fluorescence intensity result (Table 14), showed a
higher increase in fluorescence intensity signal, however, when the outliers were taken
out, which was shown with the trimmed mean fluorescence intensity (Table 15), the
signal increase was still not as great as expected. One possible cause for the low signal
was that there was a low amount of primary antibody bound to the peptide on the
microsphere resulting in low amount of secondary antibody bound shown by the low
fluorescence intensity. To solve this problem, the amount ofthc primary antibody was
increased. When the amount of primary antibody was increased, the difference was still

not as great as expected.

70

One issue found while performing the microbead immunoassay with vacuum
manifold was the inconsistency of the aspiration of the wash buffer between wells. To
ensure that the microspheres were well equally washed, the washing step, where it was
aspirated with the vacuum manifold, was replaced with a centrifugation method where
the microspheres were centrifugated, and the supernatant aspirated manually using a
micropipette.

Microbead immunoassay using centrifugation

In this method, the amount of primary antibody was increased almost double from
the vacuum manifold method. The secondary antibody concentration used was slightly
lower than previous experiment. Similar results (Table 16) were produced in this
experiment; there was an increase in fluorescence intensity but not as great as expected.

Microbead immunoassay by varying incubation conditions and secondary antibody
concentrations

 

To optimize antigen antibody binding, two sets of incubation conditions were
performed: 37°C and room temperature. In addition, different combinations of
secondary antibody concentrations were used. By comparing the fluorescence intensity
between O33-AT1 positive and 038 positive (Tables 17-20) at room temperature
incubation, it showed that when the secondary antibody concentration was increased, a
higher background shown by the increase signal for 038 positive was produced. In
addition, the change in incubation condition to 37°C also produced a higher background.

A possible cause, discussed previously, could be insufficient antibody binding to
the peptide on the microsphere and resulting in a low signal. Due to the limit of primary
antibody concentration available, one can decrease the amount of the peptide bound to

the microspheres and therefore decrease the amount of primary antibody required.

71

Microbead immunoassay by varying peptide concentration #1 and #2

 

Experiments done up to this point were only using microsphere group 033 which
was coupled with peptide AT.-033. Peptide AT.-O33 was chosen because it was the
longer peptide and contains peptide AT.-035 and AT.-O36. The result from varying the
peptide concentration (Table 21) showed the same trend as previous results, where there
was a slight increase of signal for the positive control.

One possible cause was the specificity of the primary antibody used could be
directed to other parts of the AT. receptor. The AT. receptor consists of polypeptides
containing approximatey 360 amino acids that span the cell membrane 7 times [33-35].
The epitope for the antibody used for the experiments may not correspond to the peptide
sequence AT.-033. To address this, an experiment with the other peptide sequences was
performed. In addition, a different manufacturer for the primary and secondary antibody
was used. The result (Table 22) showed similar trend as previous result where there was
a slight increase of signal for the positive control. In addition, as the peptide
concentration decrease, the background signal for the negative control of each
microsphere group increased. This showed that when there was less peptide bound to the
microsphere, there was an increase in non-specific binding.

Microbead immunoassay by varying peptide concentrations and primary antibody
concentrations

 

 

To optimize antigen antibody binding, different peptide concentrations and
primary antibody concentrations were used to obtain the correct antigen antibody ratio.
The result in Table 23 showed that as the peptide concentration decreased. the
background signal was increased. This showed that there was an increase in non—specific

binding.

Microbead immunoassay by varying _primary and secondary antibody concentrations

 

This experiment was performed with other peptide sequences to address the
specificity issue. In addition, different combinations of primary and secondary antibody
concentrations were used. The result (Table 24) showed similar trend as previous results

where there was a slight increase of signal for the positive control.

Problem solving

The problem with the specificity of the commercial antibody used was not solved
by using the other peptide sequences. An attempt to obtain positive control used in the
ELISA and the functional bioassay method was made and failed. A search for anti-AT.
and anti-AT: commercial antibodies that have the specificity matching the peptide
sequences was done and the antibodies of interest were not found. The commercial
antibodies for AT. and AT2 receptor that were available were directed to other parts of
the receptor that were not used in the ELISA or the bioassay studies.

Since the search for a commercial primary antibody or a known positive primary
antibody failed, 17 samples of sera from hypertension rat were used with the microbead
assay method. This sample population was used because it was described previously that
patients with hypertension may have autoantibodies against anti-AT. [39].

Another problem with the antibody specificity issue could be caused by the
peptide sequences. When the peptide sequences were coupled to the microspheres, it
may change the immunogenicity of the peptide and result in no binding to the primary
antibody. In addition, the blocking step with BSA may also hide the peptide and
therefore inhibit binding to the primary antibody. This issue may occur with the shorter

peptide sequences AT.-035 and AT.-036. One solution suggested was to add an ADH

73

molecule to the microsphere prior to the addition of the peptide sequence. This method
was performed using the ADH modified microspheres and the peptide coupling to the
ADH modified microspheres. The next experiment with the rat sera screening was
performed with the microspheres coupled with the peptide and the ADH modified
microspheres coupled with the peptide.

Rat sera screening using the microbead immunoassay by centrifugation

 

Rat sera screening using the microbead immunoassay by centrifugation was
performed on 17 samples. By comparing the median fluorescence intensity (Table 25)
between the negative control and the sample, there were a few that showed a greater
increase in fluorescence intensity. The same trend was also observed in the trimmed
median, mean and trimmed mean results (Tables 26-28).

When the samples were screened using the ADH modified microspheres the
fluorescence intensity between the negative control and the sample were about the same
(tables 29-32). This showed that ADH modified microspheres did not work. Further
consultation on the ADH modified microspheres method with the manufacturer showed
that the method was performed incorrectly. By lowering the amount of microspheres
used, the amount of ADH and other reagents in the method should have been decreased
proportionally. Initially, the manufacturer did not recommend decreasing the ADH and
other reagents proportionally. However, further discussion with the manufacturer after
the project was completed suggests that the ADH and other reagents should have been

decreased proportionally.

Another possible solution was to screen human sera from renal transplantation

population. This population was used because the availability of human sera from the

74

MSU lmmunohematology and Serology Laboratory and the presence ofanti-AT.
antibody have been described previously [27]. In addition, human sera from
preeclampsia patients were used since the presence of anti-AT. autoantibody has been

described [42].

Human sera screening using microbead immunoassay b)" centrifugation

 

The human sera screening using the microbead immunoassay by centrifugation
was performed using the microsphere coupled with the peptide and the ADH modified
microspheres coupled with the peptide. 158 human sera were used in this experiment.
The median fluorescence intensity (Table 33) between the negative control and the
sample showed very small or no difference. The same trend was also observed in the
trimmed median, mean and trimmed mean results (Tables 34-36). This showed that
there was no antibody present or the assay was still not working.

When the samples were screened using the ADH modified microspheres the
fluorescence intensity between the negative control and the sample were about the same
(Tables 37-40). The same method was performed on this set of samples; therefore the

failed result could be due to incorrect method used.

One possibility discussed previously was regarding the amount ofthc peptide
sequence on the microsphere. To determine if the peptide coupling was successful, one
can measure the absorbance of the supernatant of the microsphere solution. As the
peptide bound with the microsphere, the amount of the peptide in the supernatant should

decrease.

75

Determination ofpeptide coupling efficiency

The absorbance was taken using four different wavelengths. Based on the

absorbance, the absorbance that showed the greatest decrease over time was used to

determine the concentration. From the results in tables 41 -44, the wavelength of choice

to determine the decrease in peptide concentration was 230 nm. Using the initial

concentration and the initial absorbance for each peptide sequence, the molar absorptivity

for each peptide sequence was calculated. The molar absorptivity was calculated based

on Beer’s Law (Figure 16).

Figure 16. Beer’s Law formula

 

 

A: absorbance
e = molar absorptivity
b = path length of the sample (1 cm)
c = concentration (M)

A=sbc

 

 

Using the molar absorptivity for each peptide sequence, the concentration of each

peptide over time was calculated and shown in Table 46.

Table 46. Peptide concentration based on absorbance at 230 nm

 

 

 

 

 

 

 

 

 

 

 

 

Peptide 1D Concentration (ug/mL)

0 hours 2 hours 4 hours 20 hours 24 mug:
AT.- 033 125 120.7 119.6 115.9 114.4 ,2
AT.- 034 125 118.2 115.6 1142 _ 1_13_.9
AT.- 035 125 108.2 94.5 1218“ _ 4r“ ___68.3__~_-
AT.- 036 125 112.9 111.7 111.2 109.7 .2
AT;— 037 125 104.7 104.1 103.4 l_02.7___

 

 

 

The result (Table 46) showed a decrease in peptide concentration over time which

indicates binding of the peptide to the microsphere. In addition, incubation past two

hours did not increase the efficiency ofthe peptide binding to the microsphere.

76

Another solution proposed to solve the antibody specificity issue was the change
in the conformation of the peptide during the coupling reaction to the microspheres.
Two methods were performed to overcome this problem: the ELISA method and the Dot
Blot method.

ELISA

The result (Table 45) showed small difference in absorbance between each wells.
This showed either no peptide binding to the microplate or the specificity of the primary

antibody used was incorrect.

Dot Blot wit/z non-fat dry milk as blocking agent

The dot blot result with non-fat dry milk with the vacuum method (Figure 8)
showed that there was no binding between the peptide and the primary antibody. One
possible problem with the vacuum method was the possibility of the peptide solution to
be filtered out fiom the membrane. To overcome this problem, another dot blot with
non-fat dry milk without the vacuum method was performed. The result (Figure 7) still
showed no binding between the peptide and the primary antibody.
Dot Blot with PEG as blocking agent

One issue with using non-fat dry milk as a blocking agent was that since it has a
large molecular weight, it may cover the peptide sequence bound to the membrane. To
address this problem, PEG which has lower molecular weight was used as the blocking
agent. This method was performed with the vacuum method (Figures 13-15) and the
result showed that there was no binding between the peptide and the primary antibody
used. In addition, the positive control (primary antibody and secondary antibody only)

color was less compare to the dot blot that used non-fat dry milk as blocking agent. This

77

indicates that PEG did not block as well and therefore there was an increase in non
specific binding. In the method without the vacuum (Figures 10-12), there was also no

binding between the peptide and the primary antibody.

78

CONCLUSION

The development of the microbead immunoassay to detect anti-AT. and anti-AT:
was not successfully developed. Two possible sources of the problem: the peptide
sequences and the primary antibody.

The peptide sequences were coupled with the microspheres. The peptide
efficiency experiment showed that there was peptide bound to the microsphere.
However, this may create an issue with the immunogenicity of the peptide to bind the
antibody of interest. An attempt to omit the coupling step that may hinder the peptide’s
immunogenicity was done by performing the ELISA method and the dot blot method.

Another factor was the primary antibody, which is a known positive control to the
anti-AT. or anti-ATg that will react with the peptide sequences used in this assay. The
peptide sequences used in this research were selected from the ELISA and the bioassay
method. The commercial antibodies available for purchase directed to ATI and AT;
receptors were directed to a different part of the receptor than the ELISA or the bioassay
studies. If a known positive controls were available, it is possible that anti-AT. or anti-
AT2 could be detected. In addition, performing the ADH modified microspheres
coupling method with the appropriate reagent amount may also solve the problem with

the short peptide sequences.

79

RECOMMENDATION

Major problem with the development of this assay was the availability of the
positive control for the anti-AT] and anti-AT; If a known positive control available, I
would recommend to do the dot blot assay to check the binding ability of the antibody to
the peptide sequences used in the assay.

The result from the rat sera screening showed that there were a few samples
showing a good separation between the positive and the negative control. With the
availability of more hypertension rat sera, I would recommend repeating the assay. In
addition, varying peptide concentrations, secondary antibody concentration, and rat sera

volume used may help in the development of this assay.

80

APPENDIX

81

Table 47. Microspheres catalog number

 

 

 

 

 

 

 

 

Microsphere ID Catalog #
033 L100—C033-01
034 L l 00-C034-01
035 L100-C035-01
036 L100-C036-01
037 L100-CO37-01
038 L 100-C038-01

 

 

 

Table 48. Antibodies catalog number

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Product Source Catalog #
Mouse anti human angiotensin 11 type 1 receptor USBio A2295-07
(AT1)

Goat anti mouse IgG (PE) USBio 11904-74X _4
Rabbit anti human angiotensin 11 type receptor (AT2) USBio A2295-075
Donkey anti rabbit IgG H&L (PE) USBio [1903-1313
Sheep polyclonal to angiotensin factor 1 receptor Abcam Ab31667
Donkey polyclonal to sheep IgG H&L (PE) Abcam Ab7009
Rabbit polyclonal to sheep IgG H&L (AP) Abcam Ab6748
Rabbit polyclonal to angiotensin 11 type 2 receptor Abcam Ab31210
Donkey polyclonal to rabbit IgG (PE) Abcam Ab7007
Goat polyclonal to rabbit IgG (AP) Abcam Ab6722
Sheep polyclonal to rat IgG H&L (AP) Abcam Ab6853
Goat polyclonal to rat IgG H&L (PE) Abcam Ab7010
Goat polyclonal to human IgG H&L (PE) Abcam Ab7006 _

 

 

 

 

Table 49. Reagents catalog number

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

83

 

 

Product Source Catalog #
ADH (Adipic acid dihydrazide) Sigma A0638
BCIP/NBT Liquid (Dot Blot substrate) Sigma B191 1
BSA (Bovine serum albumin) Sigma A3803-10g
DMSO (Dimethyl sulfoxide) Sigma D2650
Dry milk (non fat) Carnation
EDC (1-ethyl-3-[3- Pierce 22980
dimethylaminopropyl]carbodiimide
hydrochloride)
Glycine Sigma (37126
MgClg (Magnesium Chloride) JT Baker 2444-01
Sulfo-NHS (Sulfo-N- Pierce 24510
hydroxysulfosuccinimide)
MES (2[N-Motholinolethanesulfoic acid) Sigma M—2933
Tween-20 (CAS 9005-64-5) USB 20605
NaN3 (Sodium Azide) Sigma S2002
NaCl (Sodium chloride) JT Baker 3624-05
NaOH (Sodium hydroxide) JT Baker 3722-05
N8H2PO4 (Sodium phosphate monobasic, Sigma S-501 1-5006
anhydrous)
NaHCO3 (Sodium hydrogencarbonate) JT Baker 3506-05
NaOH (Sodium hydroxide) JT Baker 3722-05
KCl (Potassium chloride) Colombus 14673
Chem.
Industries
KH2P04 (Potassium phosphate) Sigma P5379-5006
PEG (Polyethylene glycol MW 1,500) Fluka 73034
pN PP (J-nitrophenyl phosphate) tablets Sigma N2765 __
ZnClg (Zinc chloride) Sigma Z4_8_75_ “___"
Table 50. Consumables
Product Source Catalog #
1.2 pm PVDF filter micro titer plates Millipore MABVN 12
50
1.5 ml tube Eppendorf 022364111”:
1.0 ml microeentrifuges tube Art Robbins 16674-2
Instruments
15 ml conical polypropylene tube Corning “#430053“ l
50 ml conical polypropylene tube Corning ___ _480290 _ _<
250 ml filter system, vacuum Nalgene 126;0_(_)45__ _!
96-wells, 250 pl, V-bottom microplate Whatman J_7701__1250 _ l

 

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