Kohler and Miltstein
Monoclonal Antibody Production
Immunization

The purpose of immunization is to induce the production of primed B cells which produce antibodies of defined specificity. Any molecule can be used if it is immunogenic. If it is smaller than 3000 Daltons, it must be linked to a carrier protein (e.g., Thyroglobulin or BSA).

Immunization is typically done with two doses: 1) an initial priming dose, and 2) a second booster dose. The boosts are given at 4 week intervals to allow the animal to rest. After the 'boosters' the antibody develops high titer and affinity. Test bleeds determine the quality of the reponse.

The molecule may contain only 1 epitope,

or it may have a number of antigenic determinants -

Each of which will induce the formation of its own specific antibody.


In most cases, an adjuvant is required; Freund's Adjuvant is commonly used. Freund's adjuvant is a water-in-oil emulsion used experimentally for stimulating a vigorous immune response to an antigen (that is in the aqueous phase). Complete Freund's adjuvant contains heat-killed tubercle bacilli; these are omitted from Freund's incomplete adjuvant.

Theoretically, any species could be used, but the mouse is the preferred recipient because of its convenience, availability and rapid breeding. The exact protocols for immunization of different animals vary. Other species that are commonly used are rabbits, rats, goats (used to produce large quantities) and donkeys (also used to produce large quantities).

Preparation of Spleen Cell Suspension

The spleen is removed once a good antibody response is obtained. The diagram below illustrates how the spleen tissue is made into a cell supension.


This diagram is kindly reproduced from a slide from Dr. Anne White.

Myeloma Cells

Myelomas are tumors of the bone marrow, or B-cell tumors. There are now numerous myeloma cells which have been used for the generation of hybridomas.

It is important that the myeloma cells come from the same strain. Interstrain or even interspecies hybrids are possible, but they are often unstable.

Non-secretor myeloma cells, i.e. those which do not secrete their own antibodies, are preferred for fusion. If a hybridoma is producing Ig chains for two differnt types of antibody (one from the myeloma cell and one from the primed B-cell) when the Ig chains randomly associate, only a small proportion will be entirely from the immunized animal. This small proportion of antibodies will be the only ones that will interact properly with the antigen that was used in the immunization.

Cell Fusion

The purpose of cell fusion is for the production of hybridomas which have the characteristics of immortality and the production of pure, homogeneous antibody.

The fusion of cells is completely random. There are three possible scenarios for a cell:

It does not fuse:


It fuses with a cell of the same type, i.e., it has homogeneous parent cells:


It fuses with a cell of a different type, i.e., it has heterogeneous parent cells:


Only a small percentage of cells will form hybridomas.

HAT Culture

HAT medium is a selective growth medium for animal tissue cells that contains Hypoxanthine, the folate antagonist Aminopterin (or amethopterin) and Thymine. It is used for selection of hybrid somatic cell lines, as in the production of monoclonal antibodies. In HAT medium, cells are forced to use these exogenous bases, via the salvage pathways, as their sole source of purines and pyrimidines. Parental cells lacking enzymes such as HGPRT or TK can be eliminated while hybrids grow.

HAT culture is performed to select successful hybridomas. After the cell fusion step there will be a mixture of cells present: unfused cells, fused cells which have homogeneous parents, and fused cells which have heterogeneous parents.


Separation of successful hybridomas from the other cells is accomplished by culturing the cells after the fusion step, in wells containing the HAT medium. After 7-10 days only successful hybridoma cells will have survived.

Cloning of Successful Hybridomas

Cloning is performed to obtain a cell line producing only a homogeneous antibody. Positive cultures can be cloned by plating one cell to each well.

Screening

Screening is performed to identify the successful hybridomas which are secreting antibody of a defined specificity. As previously mentioned, not all hybrids will secrete antibody. When hybridomas are formed, they are usually tetraploid. In this form they are unstable.

As the cell continues to divide, it will lose a number of chromosomes derived from one or both parents. This continues until the cell stabilizes. The loss is random, and there is a possibility that the cell will loose genes which are necessary for its survival. Alternatively, it could lose the chromosomes carrying the genes necessary for antibody production. Thus, screening is important to identify hybridoma cells which are Ig secretors.

A second reason to screen the hybridomas is to identify those which secrete the exclusive, unique Ig which is sought. Molecules with multiple antigenic determinants will produce multiple antibodies which recognize multiple haptens.

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