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This is to certify that the
thesis entitled

GENERATION OF A HUMAN AND/OR MURINE MONOCLONAL
ANTIBODY AGAINST HUMAN MAMMARY CARCINOMA

presented by

John Adam Gerlach

has been accepted towards fulfillment
of the requirements for

M.S. degfiein Clinical Laboratory
Science

Date NOV. 9, 1984

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GENERATION OF A HUMAN AND/OR MURINE MONOCLONAL
ANTIBODY AGAINST HUMAN MAMMARY CARCINOMA

By

John Adam Gerlach

A THESIS

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

MASTER OF SCIENCE

Department of Pathology

1984

ABSTRACT

GENERATION OF A HUMAN AND/OR MURINE MONOCLONAL
ANTIBODY AGAINST HUMAN MAMMARY CARCINOMA

By

John Adam Gerlach

An attempt to generate a monoclonal immunoglobulin directed against

human mammary carcinoma was made. The evidence supporting the generation
of such an immunoglobulin is that hosts develop immune responses to anti-
gens expressed by tumors. By using axillary lymph nodes from humans with
mammary carcinoma as the source of lymphocytes for fusion, the immunoglo-
bulin produced would be of human origin. Since human x murine fusions

are unstable, mice were immunized with tumor material and used to generate
a murine immunoglobulin directed against human mammary carcinoma. Immuno-
fluorescent and avidin—biotin immunoperoxidase methods were used for
screening of supernatants and immunized mouse sera. Neither a human nor

a murine monoclonal antibody was generated at the termination of this
project. Possible reasons for this were the instability of human x murine
fusions and the fact that only two tumors with corresponding lymph nodes

were used.

ACKNOWLEDGEMENTS

This project was possible, in part, because of funding from Butterworth

Hospital, Grand Rapids, Michigan.

ii

TABLE OF CONTENTS

Page
INTRODUCTIONOOOOOO00......0.00.0.0000....00...OOOOOOOOOOOOOOOOOOOOOOO 1

MATERIALS. 0 O O I O C O O I O O I O O I O O I O O O O O O O O I O O O O O O O O O O O O O O O O I O O O O O O O O I O O O O O 0
Tissue Material 0 O O O O O O O O O O O O O O O O O O O O O O O O O O 0 O O O O O O O O O O O O O O O O O O O O 0
Tumor Cell Lines 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L‘J-‘b

METHODS...OOOOOOOOOOOCOOOOOOOOOOOO0.... ....... OOOOOOOOOOOOOOOOOOOOOOO

Tumor Cell Line Maintenance.....................................
Human x Murine Fusion...........................................
Indirect Immunofluorescent Screening............................
Avidin-Biotin-Peroxidase Staining...............................
Enzyme-Linked Immunosorbant Assay (ELISA)

Screen for Human IgG Production................................
Immunization of Balb/C Mice with Tumor Material.................
Screening of Mouse Serum Using Avidin-Biotin

Peroxidase Staining............................................ 9
Murine x Murine Fusions......................................... 9
ELISA Screen for Murine Anti—Tumor Antibody Production..........10
Freezing of Hybridomas..........................................ll
Thawing of Frozen Cells.........................................12

\lO‘U‘IJ-‘b

CNN

RESULTS 0 O O O O O I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O I O O O O O O O O O O O O O O O O O O O O O O C O .13
Human x Murine FUSionS I O O O O O O O O O O O O O O O O O O O O O O O 0 O O O O I O O O O O O O O O O O 0 l3
Murine x Murine FUSionS O O O O O O 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 14

DISCUSSION 0 O O ...... O O O O O O O O O O O O O O O I O O O O O O O O O O O O O O O O O O O O O O O 0 O O O O O O O O O O 1-5

RECOMNDAT IONS O 0 O O O O O I O O O O O O O O O O O O 0 O O O O O O 0 O O O O O O O O O O O O O O O O O O O O O O O O O O 19

APPENDIX. 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 0 O O O O O O O .20

REFERENCESOOOOOOOO0.000......0....O0.0...0...0.0.0.0.000000000000000024

VITAOOOOOOOOOOOOOOOOO.0.0.0.0.0...O.0...IO0...0.0.00.00.00.000000000026

iii

LIST OF FIGURES

Figure Page
1. Avidin-biotin immunoperoxidase negative control................ 15

2. Avidin-biotin immunoperoxidase staining using normal
mouse Serumo.000......OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.... 16

3. Avidin-biotin immunoperoxidase staining using a 1:20
dilution pre-immune mouse serum................................ 17

4. Avidin-biotin immunoperoxidase staining using a 1:40
dilution of immune mouse serum.... ........................ ..... 18

iv

INTRODUCTION

Tumors are known to express antigens that are considered to be
"foreign" or "non-self" by the host (l,2,3,4,5). These antigens may be
divided into three categories: embryonic, viral related and tumor speci-
fic (2,5). It is thought, though unproven, that carcinoma of the breast
may be a tumor which is viral induced (2). Tumors of viral origin may
express a portion of the viral genome on the membranes of tumor cells,
thus producing the "foreign" or "non-self antigens.

It has been demonstrated that the host immune system responds to
these tumor antigens in the form of cellular and humoral immune
responses (1,2,4). This concept of "immune response to tumors" is
supported by the evidence that animals who have had a tumor excised and
the same tumor re-introduced will remain tumor free (2,5). Further sup-
port is offered by the observation that resistance to a tumor induced
in one animal can be transferred to a different animal and enable it to
reject a tumor with the same markers (2,5). Stimulation of an immune
response by specified tumor antigens results in a decreased incidence of
tumors when oncogenic viruses are injected. A good examine of this is
seen in the effectiveness of Feline Leukemia Virus Vaccine (2,4,5). If
these concepts are applied to a human model, people with mammary carcinoma
should have an anti—tumor antibody.

There is evidence that these concepts can be applied to human models.
Histologically, a tumor lesion classically has inflammatory cells (in-

cluding plasma cells) associated with it. The lymph nodes proximal to

this tumor characteristically show the same involvement (2,5). Lymph

nodes proximal to a tumor lesion are exposed to products of that tumor due
to the drainage of lymph from that area. This lymph will possess the tumor
antigens expressed by that particular tumor due to secretion of the anti—
gen if it is a hormone or enzyme or by shedding of the surface antigens

due to membrane blebbing or cell death. If an immune reSponse is pre-
sent, the B—lymphocytes in the proximal lymph node will be stimulated by
these antigens.

In the mid-1970's, Kohler and Milstein pioneered the process of hy-
bridoma production providing unlimited supplies of monoclonal antibodies
(6,7). This process involves the fusion of B-lymphocytes with myeloma
cells resulting in the "hybrid" of the two cells referred to as a hybrid-
oma. The resultant hybridomas secrete the antibody of choice in culture
as long as they are maintained. This original work was a murine x murine
fusion.

Some investigators have had success in performing human x murine fu-
sions (7,8,9,lO). Several of these investigators have applied the con-
cepts of immune response to produce monoclonal antibodies against tumor-
associated antigens (9,10). Other investigators have also used a murine
x murine fusion to produce anti-tumor monoclonal antibodies (7). This
has involved immunization of the mouse with the tumor cells or tissue
homogenate prior to fusion to elicit the desired immune response. The
spleen of the animal was the source of the B-lymphocytes for fusion.

The production of a monoclonal antibody to a human mammary carcinoma
could have possible diagnostic or therapeutic applications (7,11,12,13,l4,
15,16). A highly specific antibody could be used for screening for early

detection of mammary carcinoma, as an imaging tag for tumor definition or

to locate possible metastases in radiologic diagnostic work if appro—
priately tagged. Much investigation is going on to see if Ehrlich's
"Magic Bullet" concept is realistic. This involves the delivery of anti-
tumor agents linked to anti—tumor antibody directly and only to the tumor
mass.

This project's premise was to use the concept of immune response to
tumor antigens and the monoclonal technology available to produce a
hybridoma that secretes an anti-mammary carcinoma. The use of human
axillary lymph nodes proximal to a tumor lesion would result in the pro-
duction of an antibody of human origin. Lymphocytes from this node were
fused with murine myeloma cells. Because of the instability of human x
murine fusions, mice were also immunized with tumor material and murine
x murine fusions performed (7). Hybridoma supernatants and mouse sera
were screened for immunoglobulin production against the immunizing tumor
using indirect immunofluorescent and immunoperoxidase assays. These tech—
niques were chosen over other methods for their sensitivity and safety

over other methods (17,18,19,20,21,22,23,24).

MATERIALS

Tissue Material:

 

Portions of two different tumors with corresponding lymph nodes were
used for this project. The first tumor (C.C.) received was described as
a massive poorly differentiated infiltrative ductal carcinoma of the
breast with extensive invasion of the axillary nodes. The second tumor
(E.G.) was described as a well differentiated carcinoma of the breast

without axillary node invasion.

Tumor Cell Lines:

 

Two myeloma cell lines were used in this project. The line used for
fusion was CRL-1580. It is non-secreting and produces stable fusions.
The other line that was used, CCL-130(MDPC-31C), secretes IgG. Both of

these cell lines were obtained from the Americal Type Culture Collection.

METHODS

Tumor Cell Line Maintenance:

 

The CRL—1580 myeloma cells were cultured in a humidified 5% C02 en-
vironment at 37C. They were split one-to-ten every two to three days
for maintenance of the cell line using Bull's Medium. In preparation for
fusion, the cells were split one-to—two twice to have the cells in log

phase growth.

The CCL—l30 myeloma cells were cultured using COL-130 Medium in a
humidified 5% C02 37C environment. They were split one-to-four every
two to three days to maintain the cell line. Some flasks of cells were
allowed to grow until the media turned acid. The supernatant was then

collected and frozen for future use.

Human X Murine Fusion:

 

Lymphocytes from the draining regional lymph node were obtained by
perfusion of the node with Dulbecco's medium with 20% fetal calf serum
using a 25 gauge needle. The cells collected were then pelleted and the
supernatant was discarded. The pellet was resuspended with a NHucl—EDTA
solution to lyse any red blood cells present. The cells were then washed
three times with the Dulbecco's fetal calf serum medium. The rest of the
lymph node was ground with a tissue homogenator. The resultant homogenate
was processed in the same manner and used for subsequent fusions.

The suspension of human mixed lymphocytes and a suspension of CRL-1580
cells were then washed separately twice with Dulbecco's without fetal
calf serum. A manual cell count on each suspension was performed using
a hemocytometer. A mixture of human mixed lymphocytes and CRL-1580
myeloma cells was made using a ratio of 10:1 respectively. This mixture
was then washed once more using Dulbecco's without fetal calf serum. To
the resultant pellet, 0.2 ml of 30% polyethylene glycol (MW 1000) was
added and incubated at 37C for two minutes. This was then centrifuged for
ten minutes at 1000 g. Once pelleted, 5 ml of serum free Dulbecco's was
added while gently agitating the mixture. This was followed by the addi-
tion of 5 ml of Dulbecco's with 20% fetal calf serum. The suspension was

then centrifuged and washed twice with Dulbecco's with 20% fetal calf

serum. Resuspension of the pellet after the final wash with 24 ml of
hybridoma (HY) medium preceded the aliquoting of 1 ml fractions into

a 24 well tissue culture tray. Hybridoma medium contains 62% Dulbecco's,
20% fetal calf serum, 10% NCTC 109, 1% non-essential amino acids,

1% HEPES buffer, 1% sodium pyruvate, 1% bovine insulin, 1% thymidine,

1% oxaloacetic acid, 1% hypoxanthine, 1% glutamine and 1% penicillin and
streptomycin. All cultures were grown in a humidified 37C incubator with
5% C02.

The next day, each well was fed 0.5 m1 HY medium with 0.4 uM amino-
pterin (IXHAT). On the second day, each well was fed 0.5 m1 HY medium
with 0.8 uM aminopterin (2XHAT). Cultures were then fed 0.5 m1 HY medium
weekly. Cultures were periodically examined microscopically using an
inverted phase microscope to determine if there were hybridoma formation.

Supernatants of the cultures were collected at 21 and 35 days post-
fusion to be used in immunologic assay techinques to determine if there

were production of anti-tumor antibodies.

Indirect Immunofluorescent Screening:

 

An immunofluorescent technique was done using 4p frozen sections of
the appropriate tumor as a source of antigen. The sections were air dried
for ten minutes and then oven dried for one hour. Sections were rinsed
for ten minutes in phosphate buffered saline (PBS), overlaid with hybrid—
oma supernatants and incubated at 37C for one-half hour. Three 15-minute
rinses with PBS were performed prior to overlaying the sections with goat
anti-human IgG fluorescein tagged antibody. This was incubated for one-

half hour and followed with three 15-minute rinses with PBS. Sections

were then air dried and coverslipped using a 90% glycerol 10% PBS mount—
ing medium. The sections were then examined under a fluorescent micro-

scOpe to determine if any of the supernatants had anti—tumor activity.

Avidin-Biotin—Peroxidase Staining:

 

An avidin—biotin-peroxidase technique was used (Vectastain ABC kit).
Four micron frozen sections of the appropriate tumor were used as a
source of antigen. This technique used ten minute rinses between steps
and incubation periods of 20 minutes with each reagent. An initial
blocking of endogenous peroxidase activity was done using a 0.3% hydrogen
peroxide (H202) in methanol solution. Blocking of endogenous binding
of other components in the system was done by incubation with a 0.01%
avidin solution followed by a 0.01% biotin solution. A final blocking
of endogenous goat globulin binding was performed using a 2% normal goat
serum in PBS solution. The culture supernatants were then overlaid
followed by a biotinylated goat anti-human IgG antibody. A solution of
avidin-biotin-peroxidase complex was then overlaid. A chromogenic sub-
strate consisting of equal volumes of 1 mg/ml diaminobenzidine in
TRIS/HCI buffer and 0.03% H202 in distilled water was used. The sections
were then counterstained with Ehrlich's Hematoxylin and coverslipped with
a permanent mounting media. The presence of a microscopically visible

brown/black deposit indicated a "positive" or reactive staining pattern.

Enzyme—Linked Immunosorbant Assay(ELISA) Screen for Human IgG Production:

An enzyme-linked immunosorbant assay was also used to determine if

the cultures were producing any immunoglobulin (IgG). This was a

8

”modification of the above avidin—biotin—immunoperoxidase staining pro-
cedure. To facilitate the binding of any IgG antibodies in the cul-
ture supernatants to the polyvinyl chloride (PVC) microtiter plates,
they were incubated for 4 hours in an alkaline pH buffer (50 mM bicar-
bonate buffer, pH 9.6) solution. Any "non-Specific" binding sites

on the plastic wells were blocked by the use of 0.1% crystalline bovine
serum albumin solution. A further block using 2% normal goat serum
was used. Incubation of the biotinylated goat anti-human igG was
followed by the addition of the avidin—biotin-peroxidase complex. The
chromogenic substrate used in this assay consisted of equal volumes of
0.8 mg/ml o-phenylenediamine dihydrochloride in 100 mM citrate/phos-
phate buffer solution and 0.03% H202 in distilled water solution. The
color was allowed to develop and the absorbances for each well were ob-

tained using a BIOTEK microtiter plate reader.

Immunization of Balb/C Mice with Tumor Material:

 

Pre-immune serum was obtained by bleeding the mice prior to in-
jection of tumor material. Blood was collected by anesthetizing the
mice with ether and then using capillary tubes to rupture and collect
the blood from the median occipital vein. The serum was collected after
centrifugation and then heat inactivated for 30 minutes at 56C. This
was stored at -20C until used.

These same Balb/C mice were then injected intraperitoneally with
either free tumor cells (10,000 cells/injection) or a homogenate of
tumor tissue (250 ug of homogenate/injection) every 2 days for a total
of 3 injections. Seven days after the last injection post-immune serum

was collected and processed in the same manner as the pre-immune serum.

Screening of Mouse Serum Using Avidin-Biotin Peroxidase Staining:

Dilutions of 1:10, 1:20 and 1:40 in PBS of both pre- and post-immune
mouse sera were used to avoid the inherent "false positive" non-speci—
fic binding of mouse immunoglobulins. The paired dilutions of pre- and
post-immune mouse sera were used with the avidin-biotin-peroxidase
method described earlier with modifications. Four micron frozen sec—
tions of tumor were used as the antigen source. A 2% normal horse serum
to block endogenous binding of horse globulins and a biotinylated
horse anti—mouse IgG were used. Mice whose post-immune serum gave a
positive staining pattern at a higher dilution than their pre—immune
serum were considered to have mounted an immune response to the tumor

with which they were injected.

Murine X MUrine Fusions:

 

Mice that were considered to have an immune response were sacrificed
by dislocation of their cervical vertebrae. Their spleens were removed
by dissection and ground with a tissue homogenator to remove the lympho-

cytes needed for fusion. The homogenate obtained was washed with NHuC1

to remove red blood cells and processed in the same manner as the
mixed lymphocytes in the human x murine fusion technique described
earlier. The myeloma cell line used for fusion was CRL-1580.

The fusion protocol used was the same as that used in the human x
mouse fusions. The only deviation was in the use of polyethylene
glycol (MW4000) for a portion of the mixed lymphocyte-myeloma mixture
in an attempt to increase hybridoma production. This portion of the

pellet had the MW 4000 polyethylene glycol added and was allowed to

10

incubate at room temperature for five minutes and then slowly diluted
with 50 ml of Dulbecco's medium while gently agitating. From this
point, the fusion was carried out in the same manner for both portions
of the mixed lymphocyte-myeloma suspensions.

Feeding of 1XHAT, 2XHAT and weekly HY feedings were the same as
in the human x murine fusion. All cultures were incubated at 37C with
5% C02 and examined microscopically for hybridoma formation weekly.

Supernatants were harvested at 3 and 5 weeks post-fusion for screening.

ELISA Screen for Murine Anti-Tumor Antibody Production:

 

Microtiter trays were prepared with antigen by plating the antigen
material, free cells (1000/well) or tissue homogenate (10 ug/well), in
a 50 mM bicarbonate solution, pH 9.6, and incubated at 4C for 16-24
hours. Trays were prepared with tumor material (cells or homogenate)
and normal breast material (cells or homogenate). This was done in
order to differentiate non-specific antibody attachment from anti-
tumor antibody attachment. Supernatants were run in parallel on trays
with tumor material and on trays with normal breast material. Delta
Absorbances (AABS) between the paired trays were used to calculate a
mean (i) and standard deviation (SD). A supernatant that gave a AABS
greater than the i + 28D was considered significant and further screen-
ing would be performed.

The avidin—biotin-peroxidase procedure used with the culture super-
natants was similar to the avidin-biotin-peroxidase enzyme-linked immuno-
sorbant assay used for screening cultures for human IgG production des—
cribed earlier. Because the antigen source was tissue homogenate or

free cells, all wells were blocked with 0.3% H202, avidin, biotin and

11

normal horse serum solutions prior to the addition of culture supernatants.
Biotinylated horse anti-mouse IgG was used as a secondary antibody and
preceded the addition of the avidin-biotin-peroxidase complex. The
chromogenic substrate was the o-phenylenediamine dihydrochloride/H202
mixture described earlier. Once the color had developed, the superna-
tants were transferred to clean PVC trays.

Any supernatant giving a AABS value greater than 2+28D would be
tested by an assay similar to the avidin-biotin-peroxidase screen used
on pre- and post-immune mouse sera. The culture supernatant would be
run in parallel with the supernatant of CCL-130 (American Type Culture
Collection) mouse myeloma cells. COL-130 cells secrete immunoglobulin.
The protein concentration of the CCL-130 supernatant would be adjusted
to match that of the culture supernatant and run on 4p frozen sections
of the tumor using the avidinubiotin-peroxidase protocol. If the cul-
ture supernatant had a positive staining pattern and the CCL-l30 did
not, then the hybridoma was considered to be secreting an anti-tumor
antibody and would be sent to Butterworth Hospital in Grand Rapids,
Michigan for further evaluation of its clinical usefulness. If both the
culture and the CCL-l30 supernatants gave positive staining patterns, the
hybridoma was considered to produce a non-specific antibody and discarded.
The experimental protocol called for screening a library of soft tissue
tumor sections using the supernatants of value by the avidin-biotin-

peroxidase method.

Freezing of Hybridomas;

 

A semi-automated freezing system using liquid nitrogen was used.

The cells to be frozen were washed free of culture media and

12

resuspended in freezing medium at a density of 1,000,000 cells/ml. The
freezing medium was kept at 4C prior to the resuspension. One milliliter
aliquots of the suspensions were placed into tubes and kept in an ice
bath until placed in the 4-5C precooled chamber. Once the tubes were in
the sealed chamber, the instrument was placed in automatic mode and
cooled in stepwise intervals until the desired temperature was reached.
At this point, the tubes (with the cells) were removed and stored in

liquid nitrogen until needed.

Thawing of Frozen Cells:

 

Cells to be thawed were removed from the liquid nitrogen storage
tank and immediately placed in a 37C water bath. As soon as the 1 ml
aliquot within the tube was thawed, it was transferred to a tube with
9 ml of the medium in which the cells were grown. This mixture was
then centrifuged and the pellet resuspended with fresh medium. The
suspension was then placed into culture in a humidified 37C incubator

With 570 C02.

13

RESULTS

Human x Murine Fusion:

 

Three human x murine fusions were performed. Two of these fusions
used lymphocytes obtained by perfusion of lymph nodes from patients
identified as C.C. and E.G. The third fusion used lymphocytes obtained
from the homogenate of the C.C. lymph node.

Of the three human x murine fusions performed, only one hybridoma
was produced. It was seen on the thirteenth day following fusion of
C.C. lymphocytes obtained by perfusion. The hybridoma was a cluster of
sixteen well-rounded cells with defined margins. On the fourteenth day
following fusion, this hybridoma was no longer visible. The superna—
tants from this fusion were screened using the indirect immunofluores—
cent technique. Distinguishing a reactive fluorescent pattern from a
non-reactive pattern using this technique was very difficult due to the
amount of autofluorescence produced by the tumor tissue itself. The
immunofluorescent technique was not used for future screening of culture
supernatants because of its inherent problem. Screening of these and
the subsequent fusion culture supernatants using the avidin-biotin-
peroxidase method on 4p sections failed to yield any supernatants giving
a reactive staining pattern.

The only fusion that was screened for human IgG production was the
third and final human x murine fusion that used the lymphocytes obtained
from the homogenate of the C.C. lymph node. A mean absorbance of 0.071
with a standard deviation of 0.029 absorbance units was obtained. None
of the supernatants screened had an absorbance greater than the 0.129

absorbance units needed to indicate significant IgG production (x+28.D.).

l4

Murine x Murine Fusions:

 

Two sets of six mice were used for immunization. Three mice of
each set were injected with tumor material from C.C.; the other three
received tumor material from E.G. One mouse injected with C.C. tumor
material and one mouse injected with E.G. tumor material from each set
were chosen to be fused. A total of four murine x murine fusions were
accomplished.

Numerous hybridomas were visible microscopically 12-14 days post-
fusion in all four of the fusions performed. The supernatants of the
two C.C. fusions had AABS means of 0.001 and 0.002. Their respective
standard deviations were 0.042 and 0.022. None of the individual super—
natants tested had a AABS greater than x+23D and all were therefore con-
sidered insignificant. The supernatants of the two E.G. fusions were
also tested using the ELISA technique and had AABS means of 0.063 and
0.122. Their respective standard deviations were 0.045 and 0.036.

None of these supernatants had an individual AABS value that was greater
than i+23D. These fusions did not produce an antibody directed against
human mammary carcinoma.

The experimental protocol of this project called for the avidin-
biotin-immunoperoxidase screening of supernatants of value in parallel
with the CCL-130 supernatant. This portion of the protocol was never
actualized. No supernatants were sent to Butterworth Hospital in

Grand Rapids, Michigan for evaluation of its potential utility.

15

DISCUSSION

Much of the time and effort afforded this project involved perfec—
tion of the screening methods. During this project, it was found that
undiluted mouse sera overlaid on tissue sections bind non-specifically
leading to "false" positive results (see figures 1 and 2). Parallel
serial dilutions of pre— and post-immune serum can overcome this pro—
blem (see figures 3 and 4). It was also found that breast tissue has
endogenous avidin binding activity (24) and must be blocked with avidin
and biotin solutions to make it possible to use an avidin-biotin—
peroxidase staining procedure. While the production of a monoclonal
antibody directed against human mammary carcinoma was not actualized,
the screening methods established can be used for future projects of

similar intent.

 

Figure l. Avidin-biotin immunoperoxidase unegative control. This sec—
tion of tumor tissue was stained using the avidin—biotin immunoperoxi—
dase staining methodology. The addition of mouse serum was omitted
and replaced with phosphate buffered saline at that step in the pro—
cedure.

l6

 

Figure 2. Avidin-biotin immunoperoxidase staining using normal mouse
serum. This section of tumor tissue was stained using the avidin—
biotin immunoperoxidase staining methodology. Undiluted serum from a
mouse that was not immunized with tumor tissue was used as the source
of primary antibody. It was not expected to show deposition of a
brown/black pigment.

l7

 

Figure 3. Avidin-biotin immunoperoxidase staining using a 1:20 dilu—
tion of pre—immune mouse serum. This section of tissue was stained
using the avidin—biotin immunoperoxidase staining methodology. Serum
from a mouse not immunized with tumor tissue was used as the source

of primary antibody. This serum was diluted 1:20 in phosphate buffered
saline. There is no deposition of a brown/black pigment indicating

a non-reactive result.

18

 

Figure 4. Avidin-biotin immunoperoxidase staining using a 1:40 dilu-
tion of immune mouse serum. This section of tissue was stained using
the avidin-biotin immunoperoxidase staining methodology. Serum from
the same mouse as in figure 3 was collected after immunization with
tumor material and diluted 1:40 in phosphate buffered saline and used
as the source of primary antibody. The deposition of a brown/black
pigment indicates a reactive result.

l9

RECOMMENDATIONS

Given the opportunities of more time and more tumors with corres-
ponding lymph nodes, the following modifications of the procedures used
in this project may yield the monoclonal immunoglobulin sought. Pro-
curement of a non-secreting human myeloma cell line would be the most
significant change that could be made. The use of such a cell line
would enable an investigator to generate stable hybridomas using human
lymphocytes. Unfortunately, such a cell line is currently unavailable.
Fusion with rat myeloma cells (CRL—163l) may offer more numerous and
more stable fusion partners for human B-lymphocytes. Also, the use of
heteromyelomas (25) may also offer the same advantages over murine cell
lines. Heteromyelomas are the product of the fusion of mouse myeloma
cells and non-secreting human lymphocytes. It is thought that human
chromosomes would less likely be lost using these as fusion partners
and thus produce stable hybridomas. This variation, coupled with the
recent advances at Johns Hopkins University (26) using avidin labelled
antigen to select only the specific B-lymphocytes for electro-fusion
with biotinylated myeloma cells, may offer the key to the success of

this project. Only time, specimens and further investigation can answer

these questions.

APPENDIX

Reagents:

A. Media

1). Bull Media 83

10

0.45

l

l

2). Hybridoma (HY) Medium 62
l

l

10

20

0.45

l
1
3).

A) 1XHAT

20

ml

ml

ml

ml

ml

ml

ml

ml

ml

m1

ml

ml

m1

ml

ml

m1

ml

ml

ml

Appendix 1

Dulbecco's with NaHC03
HEPES Buffer

Non-essential Amino Acids
Fetal Calf Serum

Na Pyruvate

Penicillin and Streptomycin
Glutamine

Dulbecco's with NaHC03
HEPE's Buffer

Non-essential Amino Acids
NCTC 109

Fetal Calf Serum

Na Pyruvate

Bovine Insulin

Thymidine

Oxaloacetic Acid
Hypoxanthine

Penicillin and Streptomycin

Glutamine

Hypoxanthine/Aminopterin/Thymidine (HAT) Medium

99 ml HY Media

4).

5).

l)

2)

3)

4)

c.
1)

2)

21

1 ml 40 uM aminopterin
B) 2XHAT 98 m1 HY Media

2 ml 40 uM aminOpterin

CCL-130 Medium 75 m1 Leivowitz Medium
1 ml HEPE's Buffer
1 m1 Non-essential Amino Acids
20 m1 Fetal Calf Serum
0.45 ml Na Pyruvate
1 m1 Glutamine
1 ml Penicillin and Streptomycin
Freezing Medium 80 m1 RPMI Medium with Antibiotics
10 ml Fetal Calf Serum
10 ml Dimethylsulfoxide (DMSO)
Fusion Reagents:
Dulbecco's with Fetal Calf 80 ml Dulbecco's
20 m1 Fetal Calf Serum
Dulbecco's serum free 99 ml Dulbecco's
1 ml HEPE'S Buffer
30% Polyethylene Glycol 3 ml Polyethylene glycol
MW 1000
7 ml Dulbecco's serum free
NHuCl—EDTA 0.17 M
Immunofluorescent screening:
Phosphate Buffered Saline (PBS) pH 7.4
Goat anti-Human IgG Fluorescein Conjugated Cappel Laboratory

Centrifuged to remove unconjugated fluorescein and diluted 1/100

in PBS

3)

l)

2)

3)
4)

5)

6)

7)

l)
2)
3)
4)

5)

22

Coverslipping Medium 90% Glycerol

10% PBS
Avidin—Biotin—Peroxidase (Immunoperoxidase)
Staining on Frozen Sections:
Phosphate Buffered Saline pH 7.4
0.03% H202 0.1 ml 30% H202

9.9 m1 Methanol

0.01% Avidin 1 mg of Avidin dissolved in 10 ml PBS
0.01% Biotin 1 mg of Biotin dissolved in 10 m1 PBS
Vectastain ABC Kit Vector Laboratories

A) For Human IgG Detection PK-4003
B) For Mouse IgG Detection PK—4002

Chromogenic substrate 5 mg diaminobenzidine tetrahydro—
chloride

5 ml TRIS/HCl buffer pH
10 pl 30% H202
5 ml deionized distilled H2O

Counterstain: Erhlich's Hematoxylin

Enzyme—Linked Immunosorbant Assay (ELISA) screen for human
IgG production:

Phosphate Buffered Saline pH 7.4

Alkaline Buffer 50 mM Bicarbonate pH 9.6

0.1% Crystalline Bovine Serum Albumin

Vectastain ABC Kit PK-4003
Chromogenic Subtrate 8 mg o—phenylenediamine dihydro—
chloride

10 ml 100 mM citrate/phosphate buffer

10 pl 30% H202

1)
2)

3)

4)
5)
6)

7)

Phosphate Buffered Saline

Alkaline Buffer

0.03% H202

0.01% Avidin
0.01% Biotin
Vectastain ABC Kit

Chromogenic Substrate

23

10 ml deionized distilled H2O

. ELISA Screen for murine anti-tumor antibody production:

pH 7.4
50 mM Bicarbonate pH 9.6
0.1 ml 30% H202
9.9 ml Methanol
1 mg Avidin dissolved in 10 m1 PBS
1 mg Biotin dissolved in 10 ml PBS
PK—4002

8 mg o-phenylenediamine dihydro-
chloride

10 ml 100 mM citrate/phosphate
buffer

10 pl 30% H202

10 m1 deionized distilled H20

REFERENCES

10.

ll.

12.

24

REFERENCES

Simpson, E.: Antigens associated with H-2, embryos and tumors.
Nature vol 306 22/29 December, 1983.

Hokama, Y. and Nakamura, R.: Immunology and Immunopathology,
Little, Brown and Company, Boston, MA., 1982.

 

Bakerman, 8.: Tumor Markers: Types and applications. Chem
Cues, March 1984.

Wheelock, E. F. and Robinson, M.K.: Biology of disease, endo-
genous control of the neoplastic process. Lab Invest vol 48
no 2, 1983.

Eisen, H.: Immunology, Harper and Row Publishers, Philadelphia,
PA, 1980.

 

Kohler, G. and Milstein, C.: Continuous cultures of fused cells
secreting antibody of predefined specificity. Nature vol 256
August, 1975.

Kennett, R. H., McKearn, T. J. and Bechtol, K. B.: Monoclonal
Antibodies Hybridomas: A New Dimension in Biological Analyses,

 

 

Plenum Press, New York and London, 1981.

Cute, R. J., Morrissey, D. M., Houghton, A.N., Beattie, Jr., E. J.,
Oettgen, H. F. and Old, L. J.: Generation of human monoclonal anti-
bodies reactive with cellular antigens. Proc Natl Acad Sci USA
vol 80 April, 1983.

Glassy, M. C., Handley, H. H., Lowe, H., 80101, R. E., Leonard, J.
and Royston, I.: A human monoclonal antibody reactive against a
human tumor-associated antigen. Fed Proc, 41:805, 1982.

Schlom, J., Wunderlich, D. and Teramoto, Y. A.: Generation of
human monoclonal antibodies reactive with human mammary carcinoma
cells. Proc Natl Acad Sci USA vol 77 no 11 November, 1980.

Collier, R. J. and Kaplan, D. A.: Immunotoxins. Sci Am vol 251
no 1 July, 1984.

Miller, R. A., Maloney, D. C., Warnke, R. and Levy, R.: Treatment
of B-cell lymphoma with monoclonal anti-idiotype antibody. N Eng
J Med vol 306 no 9 March, 1982.

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Gordon, D. 8.: Fast track for monoclonal antibodies. Med Lab Obs
July, 1983.

Oldham, R. K.: Current status of monoclonal antibodies in cancer
therapy. Clin Imm Newsletter vol 4 no 10 October, 1983.

Goldstein, G. and Sanders, M.: Monoclonal antibodies in clinical
medicine. Clin Imm Newsletter vol 4 no 6 June, 1983.

Reizenstein, P.: Immunological and biological response modification
in tumor treatment. Clin Imm Vnol Newsl vol 4 no 2 February, 1983.

Falini, B. and Taylor, C.: New developments in immunoperoxidase
techniques and their application. Arch Pathol Lab Med vol 107
March, 1983.

Lusheng, S. and Whiteside, T. L.: Immuno phenotyping of lymphoid
cells in tissues with monoclonal antibodies by avidin-biotin—
peroxidase techniques. Clin Imm Newsletter vol 4 no 3 March, 1983.

Chien, L.: Fluorescent antibody techniques. Diagnostic Procedures
For Viral And Ricketts Infection 4th ed.

 

 

Johnson, G. D. and Holbrow, E. J.: Immunology. Handbook of Diag—
nostic Clinical Immunology vol 1 Oxford, 1974.

 

 

DeLellis, R. A., Sternberger, L. A., Mann, R. B., Banks, P. M.
and Nakane, P. K.: Immunoperoxidase technics in diagnostic path-
ology. A workshop by Am Soc Clin Path May, 1979.

Taylor, C. R.: Immunoperoxidase techniques. Arch Pathol Lab
Med vol 102 March, 1978.

Giorno, R.: Characterization of mononuclear cells in cytocentri-
fuge and imprint preparations using monoclonal antibodies and an
avidin-biotin immunoperoxidase staining system. J Histochem and
Cytochem vol 31 no 11 1983.

Wood, G. S. and Warnke, R.: Suppression of endogenous avidin-binding
activity in tissues and its relevance to biotin-avidin detection
systems. J Histochem and Cytochem vol 29 no 10 1981.

Teng, N. N. H., Lam, K. S., Riera, F. C. and Kaplan, H. 8.: Con-
struction and testing of mouse-human heteromyelomas for human mono—
clonal antibody production. Proc Natl Acad Sci USA vol 80
December, 1983.

Klausner, A.: Hopkins lab improves hybridoma production. Biotechnol
September, 1984.

VITA

26

VITA

The author was born on June 11, 1954, in Detroit, Michigan. He
received his primary education in a parochial school in Romeo, Michigan.
He graduated from the public high school in Romeo, Michigan in June of
1972. The fall of that year he attended Michigan State University and
graduated with a Bachelor of Science degree in Medical Technology the
spring of 1976. The author spent one year of clinical internship at
the Washington Hospital Center, Washington, D.C. In the fall of 1977,
the author was certified by the American Society of Clinical Patholo-
gists as a Medical Technologist and began working at Community Hospital
in Battle Creek, Michigan. The author was appointed supervisor of the
transfusion service for this facility and remained there until the fall
of 1982. It was at this time that the author returned to Michigan
State University to pursue a Master of Science degree in Clinical
Laboratory Science. The author has been involved in continuing educa-
tion programs throughout the state for several years and has served on
committees for the American Red Cross (Lansing) and the Michigan Associa-
tion of Blood Banks. Beginning in January of 1985, this author will
begin the pursuit of a Doctoral degree in Pathology at Michigan State

University.

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