Monoclonal Antibody Protocols

260 Pages·1995·14.92 MB·English

by  William C. Davis

CHAPTER 1
Methods of Immunization
to Enhance the Immune Response
to Specific Antigens InVivo
in Preparation for Fusions Yielding
Monoclonal Antibodies
Jon A Rudbach, John L. Cantrell,
and J. II Ulrich
1. Introduction
The first step in preparing useful monoclonal antibodies (MAbs) is to
immunize an animal with an appropriate “vaccine.” Animal and vaccine
are both emphasized in the preceding sentence because this chapter
describes how to generate satisfactory MAbs by maximizing interactions
between the two. The term vaccine was used purposefully to connote
that not only antigens of interest may be contained in the immunizing
product, but carriers and adjuvants may also be included. These latter
components can influence greatly the success of obtaining useful hybri-
domas, which produce antibodies of the desired specificity and quality.
Immunization protocols for obtaining only murine MAbs are covered
herein. Although cross-species hybridizations can be made, they usually
involve very specialized techniques. Moreover, the lessons that can be
derived from mouse immunization protocols can, in general, be extrapo-
lated to other species as well.
When considering which mouse strains to immunize, even though
some initial advantage may be obtained by selecting a strain other than a
Balb/c, there is an overriding consideration that must be taken into
From. Methods m Molecular Bology, Vol. 45’ Monoclonal Antrbody Protocols
Edlted by W C Davis Humana Press Inc., Totowa, NJ
1
2 Rudbach, Cantrell, and Ulrich
account. The usual mouse myeloma used for fusion IS a HAT-sensitive
variant of the Balbk-derived MOPC-2 1 myeloma. The fusion product of
the myeloma with the antibody-producing spleen cells will express both
Balbk antigens (from the MOPC-21) and those of the donor strain that
provided the spleen cells. Therefore, any production of ascites as a source
of MAb must be performed in a histocompatible mouse strain. This is
easiest if the spleen cell donor is a syngeneic Balbk mouse. If the spleen
cell donor is an inbred strain other than Balbk, then the F, progeny of a
Balbk-“spleen cell donor” cross, which contains both sets of histocompat-
ibility antigens, must be used to grow the hybridoma for ascites production.
With these genetic restrictions, hybridomas generated from spleen cells
donatedb y outbred mice would be allogeneic and precluded from growth m
any recipient. One way around this problem would be to generateM Abs
only from cell-culture fluids, thus avoiding the histocompatibility problem.
However, this usually resultsi n lower yields of antibodies. Therefore, most
investigators find it easier to manipulate the immunological responses of
Balbk mice with adjuvants and/or carriers rather than reverting to the
use of other inbred mouse strains for immunization.
An antigen is a molecule that, when introduced into an appropriate
animal, will stimulate an immunological (antibody) response in that ani-
mal. In basic terms, an epitope is the minimal chemical configuration in
an antigen that can be immunologically recognized as uniquely specific
by the immune system. Inasmuch as a controlling reason for generating
MAbs, instead of polyclonal antiserum, is to obtain a high degree of
specificity, immunization with an epitopically restricted antigenic mate-
rial is usually preferable to the use of a crude antigen. Such antigens with
restricted diversity can be obtained by blocking nondesired epitopes, by
chemically conjugating purified chemical groupings to a carrier, or by syn-
thesizing/cloning epitopically pure antigens. However, regardless of the
antigenic material used in the vaccine, the final selection of specificity
will be made during screening of the hybridoma supernatant fluids for
antibody. The nature of the ligand attached to the solid support is of prime
importance at this point.
There is a paradigm in immunology that the first antibody developed
after immunization is usually more specific than that produced later in
the immune response. On the other hand, a later antibody may have more
of the desired properties of affinity, class, and subclass (1). These con-
siderations, as well as the desirability of generating sufficient numbers
Immunization to Enhance Immune Response 3
of antibody-secreting cells to yield a quantitatively satisfactory fusion
run, require a well-designed immunization protocol. It is thought that
high-affinity antibody-producing cells can be selected by using minimal
(suboptimal) amounts of antigen (I). In order to use this approach and
not compromise some of the practical aspects of the procedure, immuno-
logical adjuvants can be employed. Appropriate adjuvants can be selected
that will increase the number of antibody-forming cells and also can
direct the response to yield a qualitatively desirable antibody. Antigens,
which are weakly immunogenic because they are functional molecules,
related to tissue antigens of the mice, denatured or lack appropriate physi-
cochemical properties, or too small, can have their immunogenicity
increased through the use of adjuvants. Furthermore, conjugation of
antigens to carriers, with or without coadministration of adjuvants, can
turn marginally immunogenic materials into useful antigens (2). Use of
carriers and procedures for conjugating them have been described else-
where (3), and are not covered in this chapter.
Adjuvants are materials that are not (usually) themselves immunogenic,
but which can be used in conjunction with antigens to alter an immune
response quantitatively and/or qualitatively (4). Although many types of
adjuvants are available, only those with proven utility for generating cells
useful for MAb production in mice are covered. These are commercially
available and do not require extensive preparation or manipulation. Com-
plete Freund’s Adjuvant (CFA) is a potent adjuvant that has been used
successfully for decades. It can be used with weakly antigenic materials
and has a reputation for stimulating the production of large amounts of
high-quality antibody (5). CFA, however, suffers from its toxicity. It has
a history of inducing necrotic lesions in animals even after a single use.
Moreover, many animal care committees have banned the use of CFA in
their facilities.
An alternative to CFA is the Ribi Adjuvant System (RAS), which has
gained wide acceptance both by immunologists and animal care commit-
tees (6). RAS is a ready-to-use product, two forms of which are
recommended for use in mice to generate cells suitable for fusions lead-
ing to MAb production. One of these contains synthetic trehalose
dicorynomycolate (S-TDCM) in a form that can be readily formulated
into an oil-in-water emulsion. The second form contains monophosphoryl
lipid A (MLA) as a second immunostimulant, in addition to the S-TDCM.
The choice of which one to use is somewhat empirical, but can be
Rudbach, Cantrell, and Ulrich
directed by the nature of the antigen and the quality of the antibodies
desired. Our experience has shown that the use of S-TDCM only as
the adjuvant produces predominantly IgGr isotype MAbs. The use of
S-TDCM + MLA increases the probability of a fusion yielding MAbs
of the IgG2 isotype.
2. Materials
2.1. Preparing Vaccine
1, Antigen: Prepareo r obtain antigen of choice.
2. Adjuvant RAS (see Section 3.2. for details).
3. Phosphate-buffered salme (PBS): 0.15M NaCl and O.OlM
NaH2P04-Na2HP04, pH 7.4.
4. Mouse: Use female mice (see Note 1).
2.2. Collecting Blood and Serum
1, Dry ice or a CO2 tank and regulator.
2. Cotton, 500~mL beaker (or other container that can be covered).
3. Solution of sodium heparin (1500 U/mL) in saline.
4. Pasteur pipets.
5. Razor blade (single-edged).
6. 1 mL Tuberculin syringes and 1/2-m., 27-gage needles.
7. 70% Alcohol.
8. Microcentrifuge and tubes.
3. Methods
3.1. Antigen Preparation
1. Antigens soluble in PBS: Solubilize selected antigen in sterile PBS,
ideally at a concentration per milliliter of about 50 times the amount to be
administered. For example, tf the antigen dose per injection for a mouse is
100 pg, then a stock solution of (50 x 100 or) 5000 pg/mL is desirable,
Because a mouse dose will be contained in 0.2 mL, this solution will be a
lo-fold concentrate. Store the PBS-soluble antigen preparation under con-
ditions deemed appropriate for the material (-7O”C, 4OC, and so
forth). This recommendation for preparation of an antigen solution is
ideal, but is not absolutely necessary.
2. Antigens soluble m detergent: Sometimes detergents are necessary to solu-
bilize very hydrophobic proteins. When possible, solubrlize antigen in
detergenta t a concentrations uch that when the solution is diluted to an
antigen concentration of five times a dose expected to be given to a mouse,
the detergent concentration should be 0.2% or less.
Immunization to Enhance Immune Response 5
3. Immobilized antigen: Another type of antigen preparation that is frequently
encountered is a band cut from a polyacrylamide gel electrophoresis
(PAGE) gel. The slice of gel should be reduced to the smallest particles
practical by suspendmg it in a small amount of salme and expressing it
repeatedly through successively smaller hypodermic needles, beginning
with an 18-gage and finishing with a 27-gage needle. This suspension can
be treated as an antigen solution and prepared with the adjuvant as
described in Section 3.2.
It is recommended that the antigen under consideration be incorpo-
rated into the emulsion at a concentration range of 50-250 pg/mL of
saline. However, weak immunogens can be used at concentrations of up
to 1.0 mg/rnL. If the amount of antigen available is very limited, the
lower limit is the amount recommended. In these latter cases, experience
has shown that it is better to give multiple doses of small amounts of
antigen rather than to administer all of it in a single dose; this should be
considered when deciding on formulations of a precious antigen.
3.2. Vaccine Preparation with RAS
The RAS is available as an oil concentrate, which only requires recon-
stitution with a solution of antigen. Vaccines are formulated with RAS
adjuvants as follows:
1. Each vial of lyophilized adjuvant emulsion contains 0.5 mg of each
immunostimulant, 40 pL of oil (Squalene) and 4 uL of Tween-80. Vials
should be stored at 2-8°C until used.
2. Prior to reconstituting the emulsion, place the vial in a water bath
at 40-45”C for 5-10 mm (alternatively, the vial can be warmed in a beaker
of hot tap water for 5-10 min).
3. Reconstitute each vial with 2.0 mL of sterile PBS containing the desired
amount of antigen as follows:
a. Inject the antigen-PBS solution (2 mL) directly into the veal through
the rubber stopper, using a syringe fitted with a 20- or 21-gage needle
(leave the cap seal in place).
b. Vortex the vial vigorously for 2-3 mm to form emulsion, with rubber
stopper in place.
4. The final vaccine will contain 50 ug of each adjuvant/O.:! mL (a mouse
dose). The final emulsion also contains 2% oil (Squalene) and 0.2%
Tween-80.
If the entire contents of the vial will not be used initially, reconstitute
to 1 mL with saline, and mix aliquots 1: 1 with antigen in saline imrnedi-
6 Rudbach, Cantrell, and Ulrich
ately before use. Unused emulsion can be stored at 4°C (for up to 60 d) or
lyophilized. Do not store frozen. Prior to animal inoculation, warm the
vial to 37”C, and vortex briefly.
3.3. Immunization Protocol
When using a vaccine prepared with a RAS emulsion, it is recom-
mended to inject mice with 0.2 rrL ip or SC( 0.1 mL in each of two SC
sites). Our experience suggests the SCr oute is the preferred route. A
minimum protocol for immunizing mice to generate cells for preparing
hybridomas is as follows: immunize on d 0, boost on d 21, take a trial
bleeding on d 26; if the antibody titers are satisfactory, boost on d 35
with antigen only, intravenously, and remove the spleen to obtain cells
for fusion on d 38 (see Notes 3 and 4).
3.4. Collecting Sera
When screening mice for antibody responses during an immunization
regimen, in anticipation of deciding when to take the spleen for fusion, it
is preferable to test the serum of the actual potential spleen donor, rather
than that of a companion animal immunized in parallel. This necessitates
repeated bleedings of a single mouse. Repeated bleedings are possible,
owing to the very small volumes of sera needed for assay (see Chapter
lo), if care is taken with handling of the mice. Some institutional animal
care committees stipulate that mouse bleedings be performed under car-
bon dioxide anesthesia as outlined:
1. Either place a small piece of dry ice beneath cotton m a beaker or fill the
covered beaker with CO, gas from a tank.
2. Place mouse in beaker until It is anesthetized.
3. Remove the mouse, and rapidly bleed by one of the followmg techniques:
a. Retro-orbital: Insert the tip of a Pasteur pipet, which has been “wetted”
with the heparin solution, into the retro-orbltal space, anterior to the
eye. Rotate gently to disrupt the vascular plexus, and collect by capil-
lary action about 100 FL of blood.
b. Cardiac: With the mouse on its back, wet the chest with alcohol, and
insert a 27-gage needle into the heart, between the ribs or under the
sternum, through the diaphragm, Collect 100 PL of blood mto the
“heparm-wetted” syringe.
c. Tall vein: With the corner of a new, alcohol-wiped razor blade, nick a
lateral vein, longitudinally, near the tip of the tad. Collect, by capdlary
action, 100 pL of blood into a “heparm-wetted” Pasteur pipet. Compress
Immunization to Enhance Immune Response 7
with dry cotton to stop the blood flow. Warming mice under a heat
lamp for a few minutes immediately before bleeding will increase blood
flow through the veins and speed the process of blood collection.
4. Express the blood into a nncrocentrifuge tube that contains 10 pL of the hep-
arm solution in its tip, vortex well, and centrifuge to separate the plasma.
5. Remove the plasma to a second microcentrifuge tube, seal, and store m a
freezer, if not tested immediately.
4. Notes
1. It is recommended that female mice be used for immunization. Male
Balb/c mice fight; many times the tails are so damaged that inJections and
bleedings are impaired.
2. If sufficient antigen is available, mice should be immuruzed to prepare a
pool of polyvalent antiserum. The enzyme immunoassay (EIA) assay
should be optimized with this antiserum pool (see Chapter 10). The short
time between successful screenmg of the culture supernatant fluids for
antibody after the fusion and reculturmg for clonmg or expansion gener-
ally is not sufficient for optimizmg the EIA assay.
3. With most antigens, a good antibody titer can be achieved after a single
booster injection. If, however, the serum antibody titer is too low, a second
booster injection, with adJuvant, should be given, and another test bleed-
ing taken to determine if satisfactory titers have been obtained.
4. In order to increase the chances of obtainmg a hybridoma that will yield
the desired quality of antibody, the immunization protocol should be
designed to yield the maxtmum number of antibody-forming cells from
the spleen. Experience has shown that a mouse with a higher serum anti-
body response yields splenic cells that result in proportionately greater
numbers of specific MAb-producing hybridomas. Therefore, the immuni-
zation protocol should be designed to maximize serum antrbody titers.
References
1. Davis, B D., Dulbecco, R , Eisen, H. N., Ginsberg, H. S., Wood, W. B.,
and McCarty, M (1973) Antibody formation, in Mzcrobiology, 2nd ed , Harper &
Row, New York, pp 484,485
2 Benjamini, E. and Leskowitz, S (1991) Immunology. A Short Course, 2nd ed.,
Wiley-Liss, New York, pp. 38-40.
3. Kabat, E A and Mayer, M. M (1961) Experimental Immunochemistry, 2nd ed ,
Thomas, Spnngfield, IL, pp 446-450,798-802,8 13-8 15
4 Hui, G S. N., Chang, S P , Gibson, H., Hashimoto, A., Hashno, C , Barr, P. J.,
and Kotam, S. (1991) Influence of adJuvants on the antibody specificity to the
Plasmodmm falciparum maJor merozoite surface protein, gp195 J Immunol 147,
39353941
Rudbach, Cantrell, and Ulrich
5. Kabat, E. A. and Mayer, M. M. (1961) Experimental Immunochemuty, 2nd ed.,
Thomas, Springfield, IL, pp. 309-310, 872
6 Rudbach, J. A., Johnson, D. A., and Ulrich, J T. (1995) Ribi adJuvants: chemistry,
biology and utility in vaccines for human and veterinary medicine, in Adjuvants.
Theory and Practical Applications (Stewart-Tull, D. E S , ed.), Wiley, New York,
pp. 287-313.
CHAPTER2
Methods of Immunization
to Enhance the Immune Response
to Specific Antigens In Vitro
Margaret E. SchelZing
1. Introduction
In vitro immunization involves the exposure of spleen cells to antigen
in tissue culture rather than the antigenic stimulation of spleen cells via
immunization of mice. The production of monoclonal antibodies (MAbs)
to highly conserved molecules, such as enzymes (1,2), is possible using
in vitro immunization. MAbs to such “self’-antigens often are not pos-
sible to make using traditional in vivo methods owing to immune sup-
pression or tolerance. Utilizing in vitro immunization, it is possible to
elicit the formation of MAbs in response to picogram quantities of anti-
gen (3-6). Although certain protocols (I, 7) indicate a minimum require-
ment of from 30-100 yg antigen for in vitro immunization, we have
found that the nanogram or picogram quantities of antigen available from
blotted polyacrylamide gels provide sufficient antigen for the prepara-
tion of MAbs by in vitro immunization (3,.5). Additional advantages of
in vitro immunization include shortening the immunization procedure
from the 5 or 6 wk required for in vivo immunization to 4 d, allowing
defined antigen concentrations, and controlling antigen degradation (3).
In vitro immunization is modulated by regulating the activation and
maturation of antigen-specific B-lymphocytes using growth/differentia-
tion factors. An extensive literature describes interactions between the
various lymphokines and cell types involved in the regulation of B-cell pro-
liferation and differentiation, but these interactions are not sufficiently
From Methods m Molecular Brology, Vol. 45. Monoclonal Antibody Protocols
Edtted by W. C Davis Humana Press Inc , Totowa, NJ
9
Schelling
defined to provide a complete overview. Factors that appear to increase the
immune response to specific antigens in vitro include interleukin-2
(IL-2). When IL-2 was included in the in vitro immunization, Pollock
and d’Apice (8) found that cultures produced a higher yield of hybrido-
mas producing MAbs of the desired specificity. Additionally, muramyl
dipeptide (MDP) has been reported to increase the yield of specific anti-
bodies in in vitro systems (8-10). The MDP effect on lymphocytes is
attributed (8) to the ability of MDP to stimulate interleukin- 1 (IL- 1) pro-
duction by monocytes/macrophages, which activates helper T-cells, and
its adjuvant effect on immunizations. MDP is not a polyclonal activator
of human lymphocytes, which may be important in limiting the number
of activated but irrelevant lymphocytes available for fusion following
antigen stimulation. The IL-2 effect is also possibly the result of its effect
on helper T-cells. Jacot-Guillarmod (II) reported the use of 10% condi-
tioned medium as a source of B-cell growth and differentiation factors.
This conditioned medium consisted of a 2-d-old supernatant from
human spleen cells cultured in the presence of pokeweed mitogen. The
activity of the conditioned medium was replaced by 20 p,g/niL MDP and
200 UlmL IL-2 (I I). &helling (3) reported the addition of dextran sulfate
to thymocyte-conditioned medium (TCM) (12,13) for increased specific
MAb formation for viral proteins. Martin et al. (14), however, reported
that the addition of specific and nonspecific cell activators such as Sta-
phylococcus aweus Cowan I strain cells, lipopolysaccharide, or dextran
sulfate, to the immunizing medium did not increase the in vitro secretion
of specific human antibodies to Haemophilus influenzae type B.
The number of specific MAbs produced is higher when no more than
2% fetal bovine serum (FBS) is used in the in vitro unmunization system
(3). Additionally, the number of specific MAbs produced is greater when
the addition of FBS is delayed until 24 h following the addition of anti-
gen to the in vitro immunization system, thus avoiding a competition
of the antigen with components of the FBS in the in vitro immunization
system for the production of MAbs.
In vitro immunization of B-lymphocytes frequently results in the pro-
duction of IgM MAbs. If IgG MAbs are preferred, it is possible to inject
mice prior to harvest of the spleen cells for in vitro immunization
according to in vivo technique immunization schedules. It has been
reported that sequential in vitro immunizations are possible, but given
the short-lived existence of spleen cells in culture, it is difficult to