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Detection of Canine Immunohematologic Reactions by Gel
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DETECTION OF CANINE IMMUNOHEMATOLOGIC REACTIONS BY GEL
IMMUNOCHROMATOGRAPHY USING IMMUNOGLOBULIN BINDING
PROTEINS
By

Elizabeth Ann Madej

A THESIS

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

MASTER OF SCIENCE
Medical Technology Program

2004

ABSTRACT
DETECTION OF CANINE IMMUNOHEMATOLOGIC REACTIONS BY GEL
IMMUNOCHROMATOGRAPHY USING lMMUNOGLOBULlN-BINDING
PROTEINS AND ANTI-CANINE GLOBULIN
By

Elizabeth Ann Madej

Detection of erythrocyte sensitization using a gel column can be beneficial
to veterinary immunohematology. Compatibility testing is recommended for
canine transfusion, but the traditional test tube method can often be difficult to
interpret and prone to variation depending on the skill of the individual performing
test. A gel method can ease interpretation and reduce variation. Agarose gels
were purchased containing bacterial proteins A, G, and L, and an anti-canine
globulin gel was created by adhering whole molecule rabbit anti-canine globulin
to a Cyanogen bromide activated agarose gel. Aliquots of each gel, with
erythrocytes sensitized with antibodies to dog erythrocyte antigens (DEA) 1.1,
1.X, 3,5, 4, 5, and 7, were centrifuged at 1200 gravities for 10 minutes. The
results were compared to those obtained by the traditional test tube method. Gel
columns with anti-canine globulin and columns without immunoglobulin binding
protein proved to be effective for detecting cells sensitized to DEAs 1.1, 4, and 5,
and in some cases, better than the test tube method. This method provides easy

interpretation, which is helpful for canine pre-transfusion testing.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................... v
LIST OF FIGURES ................................................................................ vi
LIST OF ABBREVIATIONS .................................................................... viii
INTRODUCTION ................................................................................... 1
LITERATURE REVIEW ........................................................................... 3
Transfusion history ........................................................................ 3
Basic immunohematology ............................................................... 3
Current practices in veterinary and human immunohematology .............. 5
DEA system ................................................................................. 8
Gel ........................................................................................... 1O
lmmunoglobulin binding proteins and anti-canine globulin .................... 12
MATERIALS AND METHODS ................................................................ 15
Anti-sera and cells ................................................................... .----15
Making a 3% erythrocyte suspension .............................................. 16
Gel preparation ........................................................................... 16
Grading gel reactions ................................................................... 18
Determining optimal microcentrifuge conditions ................................. 18
Screening the gels using anti-DEA 1.1 sensitized erythrocytes ............. 19
Screening the gels using anti-DEA 1.X sensitized erythrocytes ............. 20
Screening the gels using anti-DEA 3,5 sensitized erythrocytes ............. 21
Screening the gels using anti-DEA 4 sensitized erythrocytes ................ 22
Screening the gels using anti-DEA 5 sensitized erythrocytes ................ 23
Screening the gels using anti-DEA 7 sensitized erythrocytes ................ 24
RESULTS .......................................................................................... 26
Optimal microcentrifuge conditions ................................................. 26
Screening the gels using anti-DEA 1.1 sensitized erythrocytes ............. 26
Screening the gels using anti-DEA 1.X sensitized erythrocytes ............. 29
Screening the gels using anti-DEA 3,5 sensitized erythrocytes ............. 32
Screening the gels using anti-DEA 4 sensitized erythrocytes ................ 35
Screening the gels using anti-DEA 5 sensitized erythrocytes ................ 38
Screening the gels using anti-DEA 7 sensitized erythrocytes ................ 41
DISCUSSION ...................................................................................... 45
FUTURE PROSPECTIVE ...................................................................... 49

iii

TABLE OF CONTENTS (CONTINUED)

BIBLIOGRAPHY .................................................................................. 50

iv

LIST OF TABLES

Table 1 - Antigenic profiles ................................................................... 15
Table 2 - Results of centrifuge optimization .............................................. 26

Table 3 - Results for screening effectiveness for anti-DEA 1.1
sensitized cells .................................................................. 29

Table 4 - Results for screening effectiveness for anti-DEA 1.x
sensitized cells .................................................................. 32

Table 5 - Results for screening effectiveness for anti-DEA 3.5
sensitized cells .................................................................. 35

Table 6 - Results for screening effectiveness for anti-DEA 4
sensitized cells .................................................................. 38

Table 7 -' Results for screening effectiveness for anti-DEA 5
sensitized cells .................................................................. 41

Table 8 - Results for screening effectiveness for anti-DEA 7
sensitized cells ................................................................... 44

LIST OF FIGURES

Figure 1 - Interpretation guide for grading gels ........................................... 11
Figure 2 - Grading system for gel ............................................................. 18
Figure 3 - Protein A gels after the cells were reacted with anti-DEA 1.1........27
Figure 4 - Protein G gels after the cells were reacted with anti-DEA 1.1 .......... 27
Figure 5 - Protein L gels after the cells were reacted with anti-DEA 1.1 ........... 28
Figure 6 - ACG gels afteT the cells were reacted with anti-DEA 1.1 ................ 28
Figure 7 - Protein A gels after the cells were reacted with anti-DEA 1.x .......... 30
Figure 8 - Protein G gels after the cells were reacted with anti-DEA 1.X .......... 30
Figure 9 - Protein L gels after the cells were reacted with anti-DEA 1.X .......... 31

Figure 10 - ACG gels after the cells were reacted with anti-DEA 1.X ............... 31

Figure 11 - Protein A gels after the cells were reacted with anti-DEA 3,5 ......... 33
Figure 12 - Protein G gels after the cells were reacted with anti-DEA 3,5 ......... 33
Figure 13 - Protein L gels after the cells were reacted with anti-DEA 3,5 .......... 34

Figure 14 - ACG gels after the cells were reacted with anti-DEA 3,5 ................ 34
Figure 15 - Protein A gels after the cells were reacted with anti-DEA 4 ............ 36

Figure 16 - Protein G gels after the cells were reacted with anti-DEA 4 ............ 36
Figure 17 - Protein L gels after the cells were reacted with anti-DEA 4 ............. 37
Figure 18 - ACG gels after the cells were reacted with anti-DEA 4 .................. 37
Figure 19 - Protein A gels after the cells were reacted with anti-DEA 5 ............ 39
Figure 20 - Protein G gels after the cells were reacted with anti-DEA 5 ............ 39
Figure 21 - Protein L gels after the cells were reacted with anti-DEA 5 ............ 40

vi

LIST OF FIGURES (CONTINUED)

Figure 22 - ACG gels after the cells were reacted with anti-DEA 5 .................. 40
Figure 23 - Protein A gels after the cells were reacted with anti-DEA 7 ............ 42
Figure 24 - Protein G gels after the cells were reacted with anti-DEA 7 ............ 42
Figure 25 - Protein L gels after the cells were reacted with anti-DEA 7 ............ 43
Figure 26 - ACG gels after the cells were reacted with anti-DEA 7 .................. 43

Figure 27 - Protein A gel with anti-DEA 7 and anti-DEA 4 sensitized cells ........ 44

vii

LIST OF ABBREVIATIONS

Anti-Canine IgG ................................................................................ ACG
Dog Erythrocyte Antigen ..................................................................... DEA
lmmunoglobulin A ............................................................................... lgA
lmmunoglobulin G .............................................................................. IgG
lmmunoglobulin M .............................................................................. lgM
Institutional Animal Care and Use Committee ...................................... IACUC
Midwest Animal Blood Services ......................................................... MABS
Registered Trademark ............................................................................ ®
United States Department of Agriculture .............................................. USDA

viii

Introduction

Traditionally, test tube methods are used to detect agglutination for human
compatibility testing and typing; however, gel methods have gained popularity
due to their sensitivity, ease of performance and interpretation, and stability of
results. In commercially available human immunochromatographic gel cards,
immunoglobulin binding proteins that can bind to sensitized human erythrocytes
are adhered to gel particles (like Sepharose®). It is known that these
immunoglobulin binding proteins can bind to canine immunoglobulins. An
immunochromatographic method for detecting canine immunohematologic
reactions could prove to be beneficial.

Gel cards are also available for human erythrocyte antigen typing. These
cards also contain gel particles. However, they contain anti-sera to the specific
antigen instead of a bacterially derived immunoglobulin binding protein.

Compatibility testing in humans is performed for all patients receiving a
transfusion with the exception of extreme emergencies when time does not
permit.1 Its purpose is to detect any testing errors due to ABO (significant,
naturally occurring antibodies) incompatibility and to a lesser extent
incompatibilities due to unexpected antibodies (antibodies due to pregnancy or
previous transfusion).1 Compatibility testing in dogs, although recommended and
beneficial to the recipient 2, may not be as frequently performed primarily
because incompatibility reactions to single transfusions are rare, and multiple
transfusions are not as frequently performed in veterinary medicine so there is

less concern over acquired antibodies. There are also no required standards for

veterinary pre-transfusion testing. The consequences of transfusing an
incompatible unit greatly range in severity. A transfusion with an incompatible
unit may be as harmless as being asymptomatic or, may lead to something more
severe, like death in humans.3 Although less information is available on the
effects of transfusing incompatible blood in dogs, the effects have a potential for
being severe.2

This study compared a modified gel test method, in a microcentrifuge
tube, to a traditional test tube method for the detection of canine
immunohematologic reactions. The gel method is not currently used in detecting
canine erythrocyte sensitization, but it may lead to a less complex and more
consistent method for typing and determining compatibility of canine blood. Gels
with different bound proteins were compared to gel without protein and to the test

tube method to determine the effectiveness of a gel test method for dogs.

Literature Review

Transfusion History

Some of the initial accounts of transfusions involve dogs as donors and/or
recipients. These early transfusions were not for therapeutic purposes in dogs,
but experimental for the benefit of humans. The objective was to determine if
inter-species transfusions or intra-species transfusions were safe and perhaps
had healthful results. Karl Landsteiner’s discovery of the human ABO blood
group in 1900 led to early methods of pre-transfusion testing.‘ As both veterinary
and human transfusion medicine developed better equipment and techniques,
therapeutic transfusion for dogs became possible.‘ In the 1960’s and 1970’s,
canine blood groups were first described; however, typing reagents were limited
because few laboratories were interested in producing typing sera. The leading
producer and distributor of canine typing sera was Dr. Robert Bull’s laboratory at

Michigan State University, East Lansing, MI.4

Basic immunohematology

Immunohematology focuses on the detection of immune responses to
antigens on blood cells. These antigens comprise proteins, carbohydrates,
and/or lipids. Methods for detecting in vivo and in vitro immune reactions involve
detecting sensitization of blood cells, i.e., antibody binding to blood cells. The
focus of this discussion is on erythrocyte immunohematology.

For antibodies to react, certain conditions must be met. Antibodies,

frequently immunoglobulin M (lgM), formed against carbohydrate antigens tend

to form stronger bonds at lower temperatures; conversely, antibodies, typically
immunoglobulin G (lgG), to protein antigens tend to form stronger bonds at
higher temperatures.6 Conditions, other than temperature, such as pH, reaction
time, and ionic strength of the solution are equally important. 6 The optimal pH is
species and immunoglobulin class dependent. Antibodies may interact with
erythrocyte antigens and form a complex by binding antigens on more than one
cell. The visible lattice that is formed is referred to as agglutination.6 For
antibodies to be able to encounter the antigen, the correct ratio of antibody to
antigen is necessary; for this reason, packed erythrocytes are often diluted to 2-4
percent (%) solutions. Optimally, antigens will not be exposed to too many
antibodies making cross-linking less likely, or too few antibodies, leaving many
erythrocytes unsensitized or undersensitized and thus show decreased visible
agglutination.

Testing methods are usually designed to mimic body conditions, to
prevent reactions that are not clinically significant (they do not occur under body
conditions). Thus, testing is frequently performed at normal human body
temperature 37 degrees Celsius (°C). There are also instances when incubating
at room temperature or 4 °C is recommended. These conditions often favor the
reaction of particular antibodies (usually lgM) in vitro.6

Reactions are evaluated by visually checking for the presence or absence
of agglutination. Frequently, antibody or complement will be present on an
erythrocyte, but due to the antigen sites’ locations and density agglutination does

not occur.‘5 In such cases, sensitization is detected by adding a secondary

antibody. In human immunohematology, this antibody is anti-human globulin
(AHG), anti-C3d (a component of the complement system), or a combination.
This antibody, if it is AHG, will bind the primary antibody or, if it is anti-C3d,
complement (already bound to the cell), forming a bridge to other cells, aiding
visible agglutination.6 In canine immunohematology, the antibody is anti-canine
lgG (ACG). This will bind the canine lgG if present.

Of the five classes of antibodies, lgM and lgG are the most significant
because they are present in the serum in significant quantities 7, and are
frequently formed against erythrocyte antigens.

lgM exists as a pentamer containing ten antigen binding sites; however,
only about five are readily available. Because it is multivalent, lgM tends to form
more visible agglutinates (erythrocytes cross-linked with antibodies to form
clusters) when exposed to erythrocyte antigens. It is the initial antibody
produced in response to foreign antigen. lgM can also help activate
complement, which may lead to lysis instead of agglutination.7

lgG is the most prevalent antibody in the sera. It is present only as a
monomer.7 Most subclasses of human lgG can activate complement as well as
cross the placenta.7 lgG on the surface of foreign particles enhances

interactions with phagocytes.7

Current practices in veterinary and human immunohematology
Tube testing methods have traditionally been used in human

immunohematology. They involve in vitro sensitization of erythrocytes using a 2-

4% suspension of erythrocytes to which serum is added. The reaction is allowed
to take place within a test tube at optimal temperature. The temperature selected
is dependent on the antibody Class for the blood group being observed. The test
tube is centrifuged, gently shaken until the cell button detaches from the bottom
of the test tube, and the reaction is graded based on the degree of agglutination,
which is assessed visually using an optical aide. The veterinary field also uses
this method for pre-transfusion testing. A major flaw of this test is the variation
due to the quality of the performers technique. The more modern gel test
minimizes variation and, according to manufacturers, has the advantage of
increasing productivity, standardization, and compliance with regulations.8
Other sources agree, saying it offers the benefits of decreased labor, consistent
results, and a stable reaction which may be reviewed at a later time.9

In the human gel test, a gel is created with Protein A and/or G, bacterially
derived immunoglobulin binding proteins, adhered to the gel particles.10 The gel
is also available with AHG adhered to the gel particles. The gel, whether bound
with proteins A and G or AHG, is placed in wells (similar to microcentrifuge
tubes) in a card. A 0.8% red blood cell solution from the donor is incubated at
optimal temperature with serum or plasma of the recipient in the wells of the
card. Next, the card is centrifuged and interpreted using visual comparison to a
chart. The gel-adhered protein binds the Fc portion of immunoglobulins, trapping
agglutinates in the gel, making them easy to interpret. A 4+ reaction appears as
a population of cells at the top of the gel column. Negative reactions appear as a

delineated population of cells at the bottom of the reaction well. Any agglutinates

captured within the gel denote a positive reaction and are graded according to
how far down the gel they are captured.”

Antigen typing can also be performed by modifying this method. A mixture
of gel and anti-sera is within the well of the card. The unreacted erythrocytes are
placed on top of the gel and centrifuged. During centrifugation, the cells will
react with the anti-sera, agglutinate, and be captured within the gel.” Unlike the
tube method, reacted erythrocytes do not need to be washed for this test since
there is a matrix above the gel responsible for keeping unbound (free) antibody
from traveling through the matrix and binding to the immunoglobulin binding
proteins, thus inhibiting the sensitized cells from being bound. This reduces the
time it takes to perform compatibility testing, antibody screening, or antigen
typing. This test is more standardized and more sensitive than its tube
counterpart.4 However, it can be more expensive than the tube method due to
the initial investment in special equipment to incubate and centrifuge the cards,
as well as the regular purchase of gel cards and their corresponding diluents.
The true cost of this method can only be determined by accounting for
technologist time, number of samples being tested, and the extent to which the
cards are being used.11

During this study, a simplified gel test became available to veterinary
Clinics. This newer test, available from Midwest Animal Blood Services
(Stockbridge, Ml), utilizes a gel matrix without immunoglobulin binding proteins in
a microcentrifuge tube. The sieving properties of the gel particles are

responsible for capturing agglutinates in a positive reaction.

Another advantage human immunohematology has over veterinary
immunohematology is the availability of relevant monoclonal antibodies.
Monoclonal antibodies, often from murine sources, are readily available against
many human erythrocyte antigens. These antibodies provide consistent
specificity for, and strong reaction to, their corresponding antigen.7'12 Due to the
very high specificity of monoclonal antibodies, multiple different ones are often
mixed together.7 This mixing allows for more than one class or subclass of
antibody to be detected. Monoclonal antibodies to dog erythrocyte antigen
(DEA) 1.1 ‘3 and 4 ‘2 have already been created. A card agglutination test has
been developed using monoclonal anti-DEA 1.1.13 Other monoclonal reagents,
A, B, D, and E, have also been developed in Japan. Reagent A is thought to be

equivalent to anti-DEA 3.“

DEA System

Nomenclature for canine blood groups has changed over the years. The
current system used in the United States was proposed in 1976.15 Canine blood
groups are based on the DEA nomenclature system. In this system, there are
seven clinically significant DEAs: DEA 1.1, 1.2, 1.3, 3, 4, 5, and 7. The chemical
structures of these antigens are not known. Exposure to these erythrocyte
antigens may lead to a transfusion reaction in antigen-negative dogs.”
Antibodies responsible for eliciting the transfusion reaction may be produced
innately without exposure to an antigen; these are termed expected antibodies.

Conversely, the antibodies may be produced following exposure to a foreign

antigen, usually from previous transfusions or pregnancy; these are termed
unexpected antibodies. Some antigen-negative dogs may produce naturally
occurring antibodies (anti-) to DEA 3, 5, and 7, but most canine blood groups are
not known to have naturally occurring antibodies. Dogs are known to become
sensitized by pregnancy or transfusion to DEA 1.1, 1.2, 1.3, 3, 4, 5, and 7.”' ‘7' ”
DEA 1.1 and 1.2 are known to cause acute hemolytic transfusion reactions.”

The blood group system DEA 1 contains DEAs 1.1, 1.2, 1.3, and null
alleles.” Inheritance is likely autosomal dOminant.19 Exposure of a previously
exposed DEA 1.1 negative dog to DEA 1.1 positive erythrocytes may lead to
rapid adverse hemolytic transfusion events.” This is also true of DEA 1.2;
however, it typically takes longer for erythrocyte removal.” Both DEA 1.1 and
1.2 are known to be proteins. Less is known about DEA 1.3 because it is the
most recently recognized antigen in this group. Immunization of a null dog with
DEA 1.3 leads to antibodies that react against DEAs 1.1, 1.2, and 1.3.20

Dog erythrocyte antigens 3 and 5 are low incidence antigens.” They
have been known to cause delayed hemolytic transfusion reactions.” In both the
DEA 3 and 5 systems, dogs can have the antigen or the null phenotype.”
Naturally occurring antibodies to DEA 3 occur in up to 20% of the DEA 3 antigen
negative dogs.17 Within 5 days after a transfusion of DEA 3 positive
erythrocytes, dogs with antibody to DEA 3 experience erythrocyte destruction.17
Ten percent of randomly sampled dogs in the United States have naturally

occurring antibody to DEA 5.17 Dogs producing this antibody, which have been

transfused with DEA 5 positive cells, experience erythrocyte destruction within 3
days.‘7

Dog erythrocyte antigen 4 is thought to be a protein of about 32-40 kDa.21
There are two phenotypes, DEA 4 and null.” The significance of antibodies
against DEA 4, a high incidence antigen, in transfusion is debatable.” Early
erythrocyte survival studies showed no cell loss or hemolysis.17 However,
naturally occurring antibodies have been identified.22

The DEA 7 system, also known as the Tr systems, has three phenotypes,
Tr, O, and null.” Dog erythrocyte antigen 7 negative dogs may have a naturally
occurring antibody that may cause a delayed non-hemolytic transfusion reaction.
The significance of this reaction is dependent on the recipient’s ability to
regenerate its own erythrocytes.” It has been suggested that this antigen is

adsorbed onto erythrocytes from the serum.23

Gel

The gel method for detecting immunohematologic reactions was originally
developed by LaPierre.” The method LaPierre developed starts with a card
similar in size to a credit card. Within this card, there are wells (Figure 1). The
top of these wells is wide and shallow; it also contains fluid to keep the gel
column from drying. The area on top is provided for reagents to incubate.” The
tube narrows and lengthens at the bottom (this area is the column). The column
is where the gel is, and where the reaction is observed. Specialized incubators

and centrifuges are provided for use with these commercial gel cards.”

10

ln commercial gel card methods, a 0.8% erythrocyte suspension and
serum (if applicable) are added directly to a well. 'The entire card is placed in the
incubator. After incubation, the card is placed in the centrifuge and centrifuged at
70 gravities (g) for 10 min. The reaction is then graded based on the location at
which the cells remain in the column. A 4+ reaction is when the cells remain at
the top of the gel. The reaction strength is considered weaker the further down
the column the cells settle.” A negative reaction is when there is a delineated

population of cells at the bottom of the column (Figure 1).24

II II I ii? I”
" .f .‘ fit . - ‘:* .2 ;
H'\, 'hi\ r’ii‘I (’zg‘s t/il\

i i l i 3'

.. Ll ....: I. - I. J

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

' '
. v
I.

Figure 1: Interpretation guide for grading gels

The gel within this method has two functions. The first way the gel
captures immunohematologic reactions is by the size of the erythrocyte
agglutinates. The beads of gel act as a sieve. Larger agglutinates cannot
migrate as far down the column as smaller agglutinates. The second way the gel
captures immunohematologic reactions is by capturing immunoglobulins on the
surface of erythrocytes using immunoglobulin binding proteins on the particles of

gel. Weak gravitational forces and a long centrifuge time aid in the ability for the

11

immunoglobulin binding proteins and the sensitized erythrocytes to react. The
cells attached to the gel are thus prevented from migrating to the bottom of the
column during centrifugation.”

The gel column has been found to be more sensitive than the tube method
at detecting lgG in direct antiglobulin testing?5 Comparative testing between test
tube and gels for indirect globulin testing also shows a high degree of

sensitivity.26

lmmunoglobulin binding proteins and anti-canine globulin

The likely purpose of bacterial cell wall proteins A, G, and L is as virulence
factors. They adhere the bacteria to the host immunoglobulin’s Fc region,
coating the bacteria with these immunoglobulins. This enables the bacteria to
avoid phagocytes, which also utilize the Fc region.

Protein A (42 kilo-Daltons; kDa) 27, is a cell wall protein isolated from
Staphylococcus aureus. It has a wide range of possible uses due to the ability to
bind to lgG of most mammals.27 Protein A’s Fc receptors are specific for certain
subclasses of immunoglobulins. This leads to some limitations, but makes it a
good selective reagent.28 In dogs, protein A has been shown to bind some lgG,
lgM, and immunoglobulin A (lgA).28

Protein G (30 kDa) is a bacterial cell wall protein originally isolated from
Lancefield group G Streptococci. It has a strong affinity for lgG. This affinity is
mostly toward the Fc region of the lgG molecule. Protein G is able to bind to a

lesser degree to other classes of immunoglobulin due to recognition of Fab

12

domains.28 Protein G has been reported to bind approximately 100% of canine
lgG, 95% of lgA, and about 44% of IgIvI.29

Protein L is isolated from Peptostreptococcus magnus and is about 75
kDa.3° Protein L, like the other immunoglobulin-binding proteins, has multiple
copies of immunoglobulin-binding domains; however, protein L does not bind the
Fc region. Protein L interacts with the light chain (primarily kappa) 3‘; therefore, it
is not restricted by the class of immunoglobulin. It is known to bind the
immunoglobulins of many species. Studies of human polyclonal lgG showed
55% was bound to protein L. Twenty-two percent of polyclonal cat lgG was
bound to protein L32 Research also included other animals; mouse monoclonal
antibodies in particular were studied in more detail. Protein L bound strongly
(72% and 79% respectively) to monoclonal murine lgG3 kappa chains and lgG2b
kappa chains.32

Proteins A and G bind the FC region of lgG.28 About 82% 33 of canine lgG
and approximately 65% 33 of lgM can be bound by Protein A. Protein G has
been reported to bind 100% of canine lgG and 44% of lgM.29 The percentage of
polyclonal dog lgG bound to protein L was 20%.32 The affinity for these proteins
to canine immunoglobulins supports the idea that a similar gel test is feasible for
canine compatibility testing.

Whole molecule ACG from a rabbit source immunospecific for canine lgG
is available. This polyspecific reagent is commonly used as a Coombs’ phase

reagent for DEA 1. The human counterpart to ACG, anti-human globulin, is used

13

not only as a Coombs’ phase reagent,'but also in human gel cards as an

immunoglobulin binding protein.

14

Materials and Methods

Anti-sera and cells

The polyclonal anti-sera were from Michigan State University. The anti-
sera used were anti-DEA 1.1 (lot # 898227), anti-DEA 3,5 (lot # 1093AH), anti-
DEA 4 (lot Sara 02/27/02), anti-DEA 5 (lot # 51471800), and anti—DEA 7 (lot Dew
09/30/01). The erythrocytes, obtained from the canine colony at Midwest Animal
Blood Services Inc. (MABS; Stockbridge, MI), were as follows: Dog A (DEA 1.1"
and 5*), Dog B (DEA 1.2*, 4“, and 7*), Dog C (DEA 1.1”,3*, 4+ ,5+ , and 7*), Dog
D (DEA 1.3+ and 4*), Dog E (DEA 1.1+), Dog F (DEA 4+ and 5“). See Table 1 for
the complete antigen profiles. All antigen profiles were previously determined by
tube agglutination. All the dogs are covered under the Animal Use Form MABS
1-04 “Production of Antibody to Dog Erythrocyte Antigens”. The care and use
protocols were reviewed by the Institutional Animal Care and Use Committee
(IACUC) of MABS, United States Department of Agriculture (USDA) 13343.

Table 1. Antigenic profiles
“+” indicates antigen is present, and “0” indicates antigen is absent.

DEA1.1 DEA1.2 DEA1.3 DEA3 DEA4 DEA5 D 7
O 0 0 0 +

 

15

Making a 3% erythrocyte suspension

A 3% suspension was made because it provided enough cells so that the
cell layer could be visualized through the gel matrix. Approximately 500
microliters (pl) packed erythrocytes were transferred into a 12 X 75 millimeter
(mm) borosilicate glass test tube. This tube was filled with phosphate buffered
saline (PBS; 0.14 molar (M) NaCl, 0.003 M KCl, 0.01 M Na H2P04, 0.001 M
KHzPO4) and centrifuged in a centrifuge (lmmufuge ll; Baxter, Deerfield, IL) for 2
minutes (min) on high (1140 g). The PBS was vacuum aspirated leaving a cell
button. The tube was again filled with PBS and centrifuged for 2 min on high.
After vacuum aspiration, 75 pl of the erythrocyte button was transferred to a
glass tube containing 2.425 milliliters (ml) PBS. The cells were resuspended

using gentle end-over-end mixing until they were uniformly distributed.

Gel preparation

Streptococcal protein G adhered to lyophilized 4 % beaded agarose (45 to
165 micrometers (pm); P7700, Sigma, Saint Louis, MO) was stored in the freezer
prior to being swollen in 30 ml of water for 30 min. The resin was rinsed with
PBS and stored in storage buffer (PBS with 0.02% sodium azide) at 4 °C.

Lyophilized protein A adhered to 4% beaded agarose gel (45 to 165 um;
P9269, Sigma) was swollen in 30 ml Buffer A (0.02 M NaH2PO4, 0.15 M NaCI,
0.015 M sodium azide) for 30 min. The resin was rinsed five times with 10 ml

Buffer A and stored in storage buffer.

16

Protein L adhered to 4% beaded agarose gel (45 to 165 pm; P3351,
Sigma), was purchased in suspension and rinsed and stored in storage buffer at
4 °C.

Lyophilized, whole molecule, anti-canine lgG (D8650, Sigma) was coupled
to a cyanogen bromide activated 4% agarose (40-165 pm; 09210, Sigma) as
follows: First, the ACG was dissolved in coupling buffer (0.1 M NaHCO3, 0.5 M
NaCl, pH adjusted to 8.4). Each wash was removed by vacuum using a BiJchner
funnel with Whatman number 54 filter paper (VWR, West Chester, PA). The
cyanogen bromide activated agarose was swelled and washed ten times over a
period of 30 min using 20 ml of 1 millimolar (mM) HCI kept at 4 °C. The agarose
resin was washed ten times using a Btichner funnel and 7 ml of deionized water
per wash. The resin was rinsed once with 5 ml coupling buffer. The dissolved
ACG and agarose resin were mixed in a 50 ml conical vial for 2 hours (h) using a
tilting mixer at room temperature. The agarose resin, bound with ACG, was
filtered using a Bt'Ichner funnel, and washed with 5 ml coupling buffer. It was
blocked with 0.2 M glycine for 2 h on a tilting mixer at room temperature. After
blocking, the ACG agarose was subjected to five wash cycles, each consisting of
5 ml coupling buffer followed by 5 ml acetate buffer (0.015 M sodium acetate
trihydrate, 4.82 ml/L acetic acid, brought up to 1.0 liter (L) with 0.5 M NaCl). The
ACG agarose was stored at 4 °C in storage buffer.

Preparation of the plain gel was identical to the ACG gel except no ACG

was added to the gel. The plain gel was also stored at 4 °C in storage buffer.

17

Prepared gels were transferred to 0.2 ml PCR tubes (#501-PCR; Dot
Scientific, Burton, MI), 200 pl per tube, and centrifuged at 1200 g for 10 min to

compact the gel prior to use.

Grading gel reactions

The interpretation scheme for grading human gel cards was used as a
guide to grading the gel reactions. The shape of the wells in the card and the
shape of the PCR tube differ; however, a strong positive reaction (4+) resulted in
all the cells remaining at the top of the gel, 3+ reactions showed cells near the
top of the gel, 2+ reactions resulted in cells throughout the gel, 1+ reactions had
cells within the lower half of the gel, and a negative reaction (0) resulted in a

pellet at the bottom of the gel. The reaction grading is demonstrated in Figure 2.

 

Figure 2. Grading system for gel
The full spectrum of reaction strengths is shown from strongest, 4+ agglutination
on the far left, to 0 (no agglutination) on the far right.

Determining optimal microcentrifuge conditions
For every run, two 3% erythrocyte suspensions were made. The first

suspension was made using cells known to express the antigen DEA 4. The

18

second suspension consisted of cells without DEA 4. Six reaction test tubes
were set up. The first two tubes (S+) contained 50 pl anti-DEA 4 with 50 pl DEA
4 positive cells, the second two (W+) had 50 pl diluted (1/80) anti-DEA 4 with 50
pl DEA 4 positive cells, and the third two (N) had 50 pl anti-DEA 4 with DEA 4
negative cells. All six tubes were incubated at 4 °C for 30 min. One tube of 8+,
W+, and N were centrifuged in a centrifuge (lmmufuge II; Baxter, Deerfield, IL) on
high for 15 seconds (3). They were interpreted for agglutination visually using
gentle shaking and observation in a mirror. The cells in the remaining three
tubes were washed three times using PBS. After the last wash, the washed cells
from reaction tubes were transferred to a corresponding protein A gel tube. The
gel tubes were centrifuged in a microcentrifuge (Micro-centrifuge 59A; Fisher,
Pittsburgh, PA).

The time and force with which the gel tubes were centrifuged differed for
each run. The combinations of conditions included 900 g for 15 min, 1050 g for
10 min, 1050 g for 15 min, and 1200 g for 10 min. Once optimal conditions were
determined, two more runs were performed for confirmation, and cells reacted
with more dilute sera (1/160 and 1/240) were also centrifuged through gel

columns to show the full spectrum of reaction strength.

Screening the gels using anti-DEA 1.1 sensitized erythrocytes
Fifty microliters of a 3% suspension of dog A erythrocytes were added to
each of six test tubes labeled “pos 1.1”. The same amount of a 3% suspension

of dog B cells was added to five other test tubes labeled “neg 1.1”. Into all of the

19

tubes, 50 pl of anti-DEA 1.1 was added. The tubes were incubated at 37 °C for
15 min. After incubation one “pos 1.1” tube and one “neg 1.1” tube were
centrifuged for 15 s at 1140 9. They were gently shaken and visually interpreted
using a viewing aide (Agglutination Viewer model 5384; Clay Adams,
Parsippany, NJ). All the tubes were filled with PBS and centrifuged at 1140 g for
1 min. The supernatant was discarded after the wash. This wash step was
completed two more times. When discarding the supernatant for the last wash,
excess fluid was removed‘using absorbent paper (Kimwipe, Kimberly-Clark,
Roswell, GA) for the two tubes that were previously visually interpreted. To
these two tubes, 50 pl Coombs’ reagent (D7407; Sigma) was added, the tubes
were centrifuged at 1140 g for 15 s, and interpreted visually. All other tubes were
transferred to gels using glass Pasteur pipets. Each gel type (A, G, L, and ACG)
had two tubes, a positive and a negative. These gels were centrifuged in a
microcentrifuge for 10 min at 1200 g. The gels were then interpreted visually
without an aide.

The entire process was completed two more times with five tubes each
time. The plain tubes, one positive and one negative, were tested in the same

manner as the other gels, but were only tested once.

Screening the gels using anti-DEA 1.X sensitized erythrocytes
Fifty microliters of a 3% suspension of dog B erythrocytes were added to
each of six test tubes labeled “pos 1.X”. The same amount of a 3% suspension

of dog F cells was added to five other test tubes labeled “neg 1.X”. Into all of the

20

tubes, 50 pl of anti-DEA 1.X was added. The tubes were incubated at 37 °C for
15 min. After incubation one “pos 1.X” tube and one “neg 1.X” tube were
centrifuged for 15 s at 1140 g in a centrifuge. They were gently shaken and
visually interpreted using a viewing aide. All the tubes were filled with PBS and
centrifuged at 1140 g for 1 min. The supernatant was discarded after the wash.
This wash step was completed two more times. When discarding the .
supernatant for the last wash, excess fluid was removed using absorbent paper
(Kimwipe, Kimberly-Clark, Roswell, GA) fer the two tubes that were previously
visually interpreted. To these two tubes 50 pl Coombs reagent (D7407; Sigma)
was added, the tubes were centrifuged at 1140 g for 15 s, and interpreted
visually. All other tubes were transferred to gels. Each gel type (A, G, L, and
ACG) had two tubes, a positive and a negative. These gels were centrifuged in a
microcentrifuge for 10 min at 1200 g. The gels were then interpreted visually
without an aide.

The entire process was completed one more time with five tubes due to
the lack of available sera. The plain tubes, one positive and one negative, were
tested in the same manner as the other gels, but were only tested once.

Unsensitized, washed dog B erythrocytes were centrifuged in an ACG gel

tube to test for reactivity of the erythrocytes with the ACG gel.
Screening the gels using anti-DEA 3, 5 sensitized erythrocytes

Fifty microliters of a 3% suspension of dog C erythrocytes were added to

each of six test tubes labeled “pos 3,5”. The same amount of a 3% suspension

21

of dog D cells was added to five other test tubes labeled “neg 3,5”. Into all of the
tubes, 50 pl of anti-DEA 3,5 was added. The tubes were incubated at 4 °C for 30
min. After incubation one “pos 3,5” tube and one “neg 3,5” tube were centrifuged
for 15 s at 1140 g in a centrifuge. They were gently shaken and visually
interpreted using a viewing aide. The remaining tubes were filled with PBS and
centrifuged at 1140 g for 1 min. The supernatant was discarded after the wash.
This wash step was completed two more times. The tubes were transferred to
gels. Each gel type (A, G, L, and ACG) had two tubes, a positive and a negative.
These gels were centrifuged in a microcentrifuge for 10 min at 1200 g. The gels
were then interpreted visually without an aide.

The entire process was completed two more times with five tubes The
plain tubes, one positive and one negative, were tested in the same manner as
the other gels, but were only tested once.

One additional run was performed entirely (including centrifugation steps)

at 4 °C to test for strict cold reactivity.

Screening the gels using anti-DEA 4 sensitized erythrocytes

Fifty microliters of a 3% suspension of dog C erythrocytes were added to
each of six test tubes labeled “pos 4”. The same amount of a 3% suspension of
dog E cells was added to five other test tubes labeled “neg 4”. Into all of the
tubes, 50 pl of anti-DEA 4 was added. The tubes were incubated at 4 °C for 30
min. After incubation one “pos 4” tube and one “neg 4” tube were centrifuged for

15 s at 1140 g in a centrifuge. They were gently shaken and visually interpreted

22

using a viewing aide. The remaining tubes were filled with PBS and centrifuged
at 1140 g for 1 min. The supernatant was discarded after the wash. This wash
step was completed two more times. The tubes were transferred to gels. Each
gel type (A, G, L, and ACG) had two tubes, a positive and a negative. These
gels were centrifuged in a microcentrifuge for 10 min at 1200 g. The gels were
then interpreted visually without an aide.

The entire process was completed two more times with five tubes. The
plain tubes, one positive and one negative, were tested in the same manner as

the other gels, but were only tested once.

Screening the gels using anti-DEA 5 sensitized erythrocytes

Fifty microliters of a 3% suspension of dog C erythrocytes were added to
each of six test tubes labeled “pos 5”. The same amount of a 3% suspension of
dog E cells was added to five other test tubes labeled “neg 5”. Into all of the
tubes, 50 pl of anti-DEA 5 was added. The tubes were incubated at 4 °C for 30
min. After incubation one “pos 5” tube and one “neg 5” tube were centrifuged for
15 s at 1140 g in a centrifuge. They were gently shaken and visually interpreted
using a viewing aide. The remaining tubes were filled with PBS and centrifuged
at 1140 g for 1 min. The supernatant was discarded after the wash. This wash
step was completed two more times. The tubes were transferred to gels. Each
gel type (A, G, L, and ACG) had two tubes, a positive and a negative. These
gels were centrifuged in a microcentrifuge for 10 min 1200 g. The gels were then

interpreted visually without an aide.

23

The entire process was completed two more times with five tubes. The
plain tubes, one positive and one negative, were tested in the same manner as

the other gels, but were only tested once.

Screening the gels using anti-DEA 7 sensitized erythrocytes

Fifty microliters of a 3% suspension of dog C erythrocytes were added to
each of six test tubes labeled “pos 7”. The same amount of a 3% suspension of
dog D cells was added to 'five other test tubes labeled “neg 7". Into all of the
tubes, 50 pl of anti-DEA 7 was added. The tubes were incubated at 4 °C for 30
min. After incubation one “pos 7” tube and one “neg 7” tube were centrifuged for
15 s at 1140 g in a centrifuge. They were gently shaken and visually interpreted
using a viewing aide. The remaining tubes were filled with PBS and centrifuged
at 1140 g for 1 min. The supernatant was discarded after the wash. This wash
step was completed two more times. The tubes were transferred to gels. Each
gel type (A, G, L, and ACG) had two tubes, a positive and a negative. These
gels were centrifuged in a microcentrifuge for 10 min at 1200 g. The gels were
then interpreted visually without an aide.

The entire process was completed two more times with five tubes. The
plain tubes, one positive and one negative, were tested in the same manner as
the other gels, but were only tested once.

One additional run was performed entirely (including centrifugation steps)

at 4 °C to test for strict cold reactivity.

24

To ensure that the centrifuge and gels were working properly, a protein A
gel previously centrifuged for anti-DEA 7 was reused by centrifuging with anti-
DEA 4 sensitized cells.

To test for lgM activity, 450 pl of anti-DEA 7 serum with 50 pl 50 mM
dithiothreitol (DTT; D0632, Sigma), and 450 pl of anti-DEA 7 with 50 pl PBS were
incubated at 37 °C for 30 min. Fifty microliters of each was pipetted into separate
test tubes with 50 pl 3% erythrocyte suspension of dog C. A negative control
tube contained 50 pl anti-DEA 7 and 50 pl 3% dog D cells. All tubes were

incubated a 4 °C for 30 minutes, centrifuged for 15 s and graded with an aide.

25

Results

Optimal microcentrifuge conditions

Results of the microcentrifuge study as well as the dilutions tested to

demonstrate negative through 4+ reactions are shown in Table 2.

Table 2. Results of centrifuge optimization

Results scored on a scale of 0 (no agglutination) to 4+ (complete agglutination).
8+ = DEA 4 cells with undiluted anti-DEA 4, W+ = DEA 4 cells with 1/80 dilution

of anti-DEA 4, and N = DEA 4 negative cells with anti-DEA 4.

 

 

 

 

 

 

 

Test 900 g 1050 g 1050 g 1200 g 1200 g 1200 9

tube 15 min 10 min 15 min 10 min 10 min 10 min

(run #2) (run #3)
8+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
W+ 2+ 3+ 3+ 3+ 3+ 3+ 3+
N 0 1+ 1+ 0 0 0 0
1/160 dilution 1+ 2+
1/240 dilution w+ 1+

 

 

 

 

 

 

 

 

Screening the gels using anti-DEA 1.1 sensitized erythrocytes

Examples of gels A, G, L, and ACG are shown in Figure 3, Figure 4,

Figure 5, and Figure 6 respectively. The results of screening gels A, G, L, ACG,

control, and test tube using anti-DEA 1.1 sensitized erythrocytes are shown in

Table 3.

26

 

 

Figure 3. Protein A gels after the cells were reacted with anti-DEA 1.1
DEA1.1 positive cells are in the tube on the left, and DEA 1.1 negative cells are
on the right. Both sets of cells were incubated with anti-DEA 1.1 and washed
three times before being transferred to the gels and centrifuged at 1200 g for 10
mIn.

 

Figure.4. Protein G gels after the cells were reacted with anti-DEA 1.1
DEA1.1 positive cells are in the tube on the left, and DEA 1.1 negative cells are
on the right. Both sets of cells were incubated with anti-DEA 1.1 and washed
three times before being transferred to the gels and centrifuged at 1200 g for 10
min.

27

 

Figure 5. Protein L gels after the cells were reacted with anti-DEA 1.1
DEA1.1 positive cells are in the tube on the left, and DEA 1.1 negative cells are
on the right. Both sets of cells were incubated with anti-DEA 1.1 and washed
three times before being transferred to the gels and centrifuged at 1200 g for 10
min.

 

Figure 6. ACG gels after the cells were reacted with anti-DEA 1.1
DEA1.1 positive cells are in the tube on the left, and DEA 1.1 negative cells are
on the right. Both sets of cells were incubated with anti-DEA 1.1 and washed
three times before being transferred to the gels and centrifuged at 1200 g for 10
mIn.

28

Table 3. Results for screening effectiveness for anti-DEA 1.1 sensitized cells
Results scored on a scale of 0 (no agglutination) to 4+ (complete agglutination).
The term “positive” refers to the tube with the cells containing DEA 1.1 antigen.

“Negative” refers to the tube with cells that do not contain the antigen.

Run #1 Run #2 Run #3

Protein
A

Test tube

w/Coombs
w/Coombs
Plain Gel

 

Screening the gels using anti-DEA 1.X sensitized erythrocytes

Examples of gels A, G, L, and ACG are shown in Figure 7, Figure 8,
Figure 9, and Figure 10 respectively. The results of screening gels A, G, L, ACG,
plain, and test tube using anti-DEA 1.X sensitized erythrocytes are shown in

Table 4.

29

 

Figure 7. Protein A gels after the cells were reacted with anti-DEA 1.X
DEA1.X positive cells are in the tube on the left, and DEA 1.X negative cells are
on the right. Both sets of cells were incubated with anti-DEA 1.x and washed
three times before being transferred to the gels and centrifuged at 1200 g for 10
mm.

 

Figure 8. Protein G gels after the cells were reacted with anti-DEA 1.X
DEA1.X positive cells are in the tube on the left, and DEA 1.X negative cells are
on the right. Both sets of cells were incubated with anti-DEA 1.X and washed
three times before being transferred to the gels and centrifuged at 1200 g for 10
min.

30

 

Figure 9. Protein L gels after the cells were reacted with anti-DEA 1.X
DEA1.X positive cells are in the tube on the left, and DEA 1.X negative cells are
on the right. Both sets of cells were incubated with anti-DEA 1.X and washed
three times before being transferred to the gels and centrifuged at 1200 g for 10
mIn.

 

Figure 10. ACG gels after the cells were reacted with anti-DEA 1.X
DEA1.X positive cells are in the tube on the left, and DEA 1.x negative cells are
on the right. Both sets of cells were incubated with anti-DEA 1.X and washed
three times before being transferred to the gels and centrifuged at 1200 g for 10
min.

31

Table 4: Results for screening effectiveness for anti-DEA 1.x sensitized cells
Results scored on a scale of 0 (no agglutination) to 4+ (complete agglutination).
The term “positive” refers to the tube with the cells containing DEA 1.X antigen.
“Negative” refers to the tube with cells that do not contain the antigen.

run #1 run #2
Protein
A

Test tube

positive
w/Coombs

negafive
w/Coombs

Plain Gel

 

Screening the gels using anti-DEA 3, 5 sensitized erythrocytes

Examples of gels A, G, L, and ACG are shown in Figure 11, Figure 12,
Figure 13, and Figure 14 respectively. The results of screening gels A, G, L,
ACG, control, and test tube using anti-DEA 3,5 sensitized erythrocytes are

shown in Table 5.

32

 

Figure 11. Protein A gels after the cells were reacted with anti-DEA 3,5
DEA 3 positive cells are in the tube on the left, and DEA 3 negative cells are on
the right. Both sets of cells were incubated with anti-DEA 3,5 and washed three
times before being transferred to the gels and centrifuged at 1200 g for 10 min.

 

Figure 12. Protein G gels after the cells were reacted with anti-DEA 3,5
DEA 3 positive cells are in the tube on the left, and DEA 3 negative cells are on
the right. Both sets of cells were incubated with anti-DEA 3,5 and washed three
times before being transferred to the gels and centrifuged at 1200 g for 10 min.

33

 

Figure 13. Protein L gels after the cells were reacted with anti-DEA 3,5
DEA 3 positive cells are in the tube on the left, and DEA 3 negative cells are on
the right. Both sets of cells were incubated with anti-DEA 3,5 and washed three
times before being transferred to the gels and centrifuged at 1200 g for 10 min.

 

Figure 14. ACG gels after the cells were reacted with anti—DEA 3,5
DEA 3 positive cells are in the tube on the left, and DEA 3 negative cells are on
the right. Both sets of cells were incubated with anti-DEA 3,5 and washed three
times before being transferred to the gels and centrifuged at 1200 g for 10 min.

Table 5. Results for screening effectiveness for anti-DEA 3,5 sensitized cells
Results scored on a scale of 0 (no agglutination) to 4+ (complete agglutination).
The term “positive” refers to the tube with the cells containing DEA 3 antigen.
“Negative” refers to the tube with cells that do not contain the antigen.

run#1 run#2 run#3 4°C

Protein
.A

Testtube

 

Screening the gels using anti-DEA 4 sensitized erythrocytes

Examples of gels A, G, L, and ACG are shown in Figure 15, Figure 16,
Figure 17, and Figure 18 respectively. The results of screening gels A, G, L,
ACG, control, and test tube using anti-DEA 4 sensitized erythrocytes are shown

in Table 6.

35

 

Figure 15. Protein A gels after the cells were reacted with anti-DEA 4
DEA 4 positive cells are in the tube on the left, and DEA 4 negative cells are on
the right. Both sets of cells were incubated with anti—DEA 4 and washed three
times before being transferred to the gels and centrifuged at 1200 g for 10 min.

 

Figure 16. Protein G gels after the cells were reacted with anti-DEA 4
DEA 4 positive cells are in the tube on the left, and DEA 4 negative cells are on
the right. Both sets of cells were incubated with anti-DEA 4 and washed three
times before being transferred to the gels and centrifuged at 1200 g for 10 min.

36

 

Figure 17. Protein L gels after the cells were reacted with anti-DEA 4
DEA 4 positive cells are in the tube on the left, and DEA 4 negative cells are on
the right. Both sets of cells were incubated with anti-DEA 4 and washed three
times before being transferred to the gels and centrifuged at 1200 g for 10 min.

 

Figure 18. ACG gels after the cells were reacted with anti-DEA 4
DEA 4 positive cells are in the tube on the left, and DEA 4 negative cells are on
the right. Both sets of cells were incubated with anti-DEA 4 and washed three
times before being transferred to the gels and centrifuged at 1200 g for 10 min.

37

Table 6. Results for screening effectiveness for anti-DEA 4 sensitized cells
Results scored on a scale of 0 (no agglutination) to 4+ (complete agglutination).
The term “positive” refers to the tube with the cells containing DEA 4 antigen.
“Negative” refers to the tube with cells that do not contain the antigen.

run #1 run #2 run #3
Protein
A

Test tube

Plain Gel

 

Screening the gels using anti-DEA 5 sensitized erythrocytes

Examples of gels A, G, L, and ACG are shown in Figure 19, Figure 20,
Figure 21, and Figure 22 respectively. The results of screening gels A, G, L,
ACG, control, and test tube using anti-DEA 5 sensitized erythrocytes are shown

in Table 7.

38

 

Figure 19. Protein A gels after the cells were reacted with anti-DEA 5
DEA 5 positive cells are in the tube on the left, and DEA 5 negative cells are on
the right. Both sets of cells were incubated with anti-DEA 5 and washed three
times before being transferred to the gels and centrifuged at 1200 g for 10 min.

 

Figure 20. Protein G gels after the cells were reacted with anti-DEA 5
DEA 5 positive cells are in the tube on the left, and DEA 5 negative cells are on
the right. Both sets of cells were incubated with anti-DEA 5 and washed three
times before being transferred to the gels and centrifuged at 1200 g for 10 min.

39

 

Figure 21. Protein L gels after the cells were reacted with anti-DEA 5
DEA 5 positive cells are in the tube on the left, and DEA 5 negative cells are on
the right. Both sets of cells were incubated with anti-DEA 5 and washed three
times before being transferred to the gels and centrifuged at 1200 g for 10 min.

 

Figure 22. ACG gels after the cells were reacted with anti-DEA 5
DEA 5 positive cells are in the tube on the left, and DEA 5 negative cells are on
the right. Both sets of cells were incubated with anti-DEA 5 and washed three
times before being transferred to the gels and centrifuged at 1200 g for 10 min.

40

Table 7. Results for screening effectiveness for anti-DEA 5 sensitized cells
Results scored on a scale of 0 (no agglutination) to 4+ (complete agglutination).
The term “positive” refers to the tube with the cells containing DEA 5 antigen.
“Negative” refers to the tube with cells that do not contain the antigen.

run #1 run #2 run#3

Protein
A

Test tube

Plain Gel

 

Screening the gels using anti-DEA 7 sensitized erythrocytes

Examples of gels A, G, L, and ACG are shown in Figure 23, Figure 24,
Figure 25, and Figure 26 respectively. The results of screening gels A, G, L,
ACG, control, and test tube using anti-DEA 7 sensitized erythrocytes are shown
in Table 8. Both the D'l'l' treated and the PBS sera demonstrated a 3+ reaction.

The negative DTI’ control was negative.

41

 

Figure 23. Protein A gels after the cells were reacted with anti-DEA 7
DEA 7 positive cells are in the tube on the left, and DEA 7 negative cells are on
the right. Both sets of cells were incubated with anti-DEA 7 and washed three
times before being transferred to the gels and centrifuged at 1200 g for 10 min.

 

Figure 24. Protein G gels after the cells were reacted with anti-DEA 7
DEA 7 positive cells are in the tube on the left, and DEA 7 negative cells are on
the right. Both sets of cells were incubated with anti-DEA 7 and washed three
times before being transferred to the gels and centrifuged at 1200 g for 10 min.

42

 

Figure 25. Protein L gels after the cells were reacted with anti-DEA 7
DEA 7 positive cells are in the tube on the left, and DEA 7 negative cells are on
the right. Both sets of cells were incubated with anti-DEA 7 and washed three
times before being transferred to the gels and centrifuged at 1200 g for 10 min.

 

Figure 26. ACG gels after the cells were reacted with anti—DEA 7
DEA 7 positive cells are in the tube on the left, and DEA 7 negative cells are on
the right. Both sets of cells were incubated with anti-DEA 7 and washed three
times before being transferred to the gels and centrifuged at 1200 g for 10 min.

43

 

Figure 27: Protein A gel with anti-DEA 7 and anti-DEA 4 sensitized cells
The initial test, with anti-DEA 7 sensitized cells, is responsible for the cell
population at the bottom, and later test, with anti-DEA 4 sensitized cells, is
responsible for the cell population at the top.

Table 8. Results for screening effectiveness for anti-DEA 7 sensitized cells
Results scored on a scale of 0 (no agglutination) to 4+ (complete agglutination).
The term “positive" refers to the tube with the cells containing DEA 7 antigen.
“Negative" refers to the tube with cells that do not contain the antigen.

 

Discussion

The conditions chosen for the microcentrifuge, 1200 g for 10 min, were
chosen for two reasons. First, the positive reaction appeared positive and the
negative appeared negative. The second reason was that it was only a 10 min
centrifugation as opposed to 1050 g for 15 min, which also showed a clear
positive and negative. .

The ACG gel demonstrated the strongest reaction to erythrocytes
sensitized to anti-DEA 1.1 followed by the. control gel which was stronger than
the test tube. Gels A and L reacted, but were weaker than the control gel. Gel G
had no visible reaction to the sensitized cells (Table 3).

Anti-DEA 1.X sensitized cells reacted in the ACG positive and negative
gels. The other gels were ineffective (Table 4). The unexpected positive in the
ACG tube with DEA 1.x antigen negative erythrocytes is not due to the reaction
of dog A’s erythrocytes alone, since dog A cells unreacted with anti-sera resulted
in a negative reaction when centrifuged through the gel. There must be a
reaction occurring due to the sera, or that particular sera in combination with dog
A’s cells. If more sera were available, this could be tested by using cells from
another dog negative for DEA 1. The gel itself reacted as expected in screens
performed before and after this one; therefore it is not likely that the gel was
compromised.

None of the gels displayed any reaction to anti-DEA 3,5 sensitized cells.
The test tube showed a moderately positive reaction (Table 5). After performing

one run where the equipment and the reagents were all at 4 °C. the reactions

45

remained unchanged. This run was performed with the intent of attempting to
enhance the reaction, although anti-DEA 3,5 reaction is known to occur at room
temperature as well.

All positive gels maintained a strong positive reaction to anti-DEA 4
sensitized cells. The positive test tube was also very strong (Table 6). The
immunoglobulin binding proteins present in the gels were not necessary to
observe a strong reaction.

The ACG gel showed the strongest reaction to anti-DEA 5 sensitized cells.
Protein G and control gels showed moderate reactivity similar to the test tube
method. Gel L was slightly weaker than the test tube (Table 7). The reaction
strength displayed in the ACG gel suggest that not only sieving properties are
responsible for the strength, but the ACG is likely acting as an immunoglobulin
binding protein against the immunoglobulins present on the sensitized cells.

The test tube positive DEA 7 reaction was a consistent moderately strong
reaction, and the negative tubes were negative. In contrast, all gels were
unreactive to erythrocytes sensitized with anti-DEA 7 (Table 8). The protein A
gel that was previously run with anti-DEA 7 sensitized cells and rerun with anti-
DEA 4 sensitized cells, was used to test the function of the centrifuge and the
gels. This resulted in a strong positive reaction for anti-DEA 4 sensitized
erythrocytes, indicating that the gel was functional. Performing an entire run at 4
°C, including equipment and reagents, was done in an attempt to enhance any
cold reaction that may have been missed with the initial method. Enhancement

did not occur. The addition of D'l'l’ would have destroyed the ability of IgM to

46

agglutinate or activate complement.34 lmmunoglobulin M is not likely to be fully
responsible for the anti-DEA 7 activity since D'l'l' treatment did not alter the
reactivity. The lack of positive results in the gels may be due to the fact that
DEA 7 antigen may be adsorbed onto the cell similar to the human Lewis blood
group system,24 and therefore, may be removed from the cells during the gel
testing procedure.

It remains unclear why any of the protein gels would appear negative
while the plain gel was positive, but this occurred for only the anti-DEA 1.1 serum
and only comparing the plain gel to protein A, G, and L gels (not the ACG gel).
This suggests either a difference between the gels themselves or differences
resulting from gel handling and processing. Gel particles were sold as being of
the same size, and the method for attaching the proteins was the same for each.
However, the plain and ACG gels were processed in-house, and the other gels
were processed before purchase. Thus, differences in processing are possible.
It is also likely that the gels themselves came from different lots for the different
proteins. Perhaps there exists delicate balance between the narrowness of the
tube, which is important for protein interaction with immunoglobulins, and the
centrifuge conditions necessary for a sieving effect. A gel with a smaller range in
gel particle size may provide more consistent sieving properties.

The grading of the gel was more difficult than the human method for the
weaker reactions. This may be due to the shape of the PCR tube. Cells often
stay to the sides of the tube in the conical portion; therefore they may not be

experiencing the sieving effect in this portion of the tube. The human gel cards

47

have a long narrow column with parallel sides, while a PCR tube or
microcentrifuge tube has a shorter column and a conical shape with tapering
sides of the tube.

Since most DEA reactions were visible in the control gel and the ACG gel,
they are likely to be the best matrices to use for immunochromatographic
methods of detecting immunohematologic reactions in dogs. The control gel is
the more cost effective choice. The reactions are less strong than in the ACG
gel; however, they are easily discernable from a negative. The strength of the

reaction in the ACG gel is likely greater due to the protein (ACG)-lgG interaction.

48

Future prospective

Currently, a plain gel matrix seems to be the best gel option. ACG gel is
also very effective. Though more expensive, its reactions are sometimes
stronger than the plain gel. If a cost effective source of ACGs, particularly a
mixture of monoclonal antibodies, is developed, an ACG gel for compatibility
testing may prove to be more useful. To apply the immunoglobulin binding
protein gel to DEA typing, a monoclonal antibody for each significant DEA type,
that is also recognized by an immunoglobulin binding protein like protein A (the
most cost effective protein in this study), could theoretically provide consistent,
reliable results. If cost-effective monoclonal antibodies are developed, it may be
less expensive to create an immunochromatographic gel because less serum,
compared to the test tube method, would be necessary to achieve a positive

result.

49

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