THE manufacture of predefined specific antibodies by means of permanent tissue culture cell lines is of general interest. There are at present a considerable number of permanent cultures of myeloma cells1,2 and screening procedures have been used to reveal antibody activity in some of them. This, however, is not a satisfactory source of monoclonal antibodies of predefined specificity. We describe here the derivation of a number of tissue culture cell lines which secrete anti-sheep red blood cell (SRBC) antibodies. The cell lines are made by fusion of a mouse myeloma and mouse spleen cells from an immunised donor. To understand the expression and interactions of the Ig chains from the parental lines, fusion experiments between two known mouse myeloma lines were carried out.

Continuous cultures of fused cells secreting antibody of predefined specificity

Cell fusion techniques have been used to produce hybrids between myeloma cells and antibody-producing cells. The hybrid lines derived are permanently adapted to grow in tissue culture and are capable of inducing antibody-producing tumors in mice. Spleens from mice immunized against sheep red blood cells (SRBC) were fused to an 8-azaguanine-resistant clone (X63-Ag8) of MOPC 21 myeloma. Over 50% of the derived hybrid lines produce and secrete immunoglobulins different from the MOPC 21 myeloma. About 10% of the hybrid lines exhibit anti-SRBC activity. The high proportion of antibody-producing hybrids suggests that the fusion involves a restricted fraction of the spleen cell population, probably cells committed to antibody production. In order to avoid the presence of the MOPC 21 heavy chain in the specific hybrids, another myeloma cell line (NSI/1-Ag4-1) has been used. This is a nonsecreting variant of the MOPC 21 myeloma which does not express heavy chains. Three anti-SRBC (probably of the mu, gamma2b and gamma1 classes, respectively) and two anti-2,4,6-trinitrophenyl (of the mu class) antibody-producing hybrids have been repeatedly cloned. By random selection and by selection of specific clones according to their lytic activity (clone plaque selection), a number of different lines have been constructed. Such lines express different combinations of the four possible chains of each hybrid line: the myeloma gamma and K chains and the specific antibody heavy and light chains. In three cases (Sp1, Sp2 and Sp7) it is shown that only the specific H and L combination has activity and that the myeloma chains are unable to substitute for them. In most cases lines have been derived which no longer express the MOPC 21 chains but only the specific antibody chains.

Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion

A simple technique for detecting plaque-forming cells is described. It combines the sensitivity and improved optical conditions of the previously reported monolayer technique with the screening power and ease of quantification of the original agar-plate method.

Further improvements in the plaque technique for detecting single antibody-forming cells.

Publisher Summary This chapter discusses the strategies and procedures for the preparation of monoclonal antibodies (McAb). The derivation of permanent lines of hybrid cells, producing McAb, exhibiting certain desired properties, presents widely different degrees of difficulty. Desired properties include not only specific recognition of an antigen and other no less critical properties are the fine specificity of the antibody, avidity and kinetic parameters important for radioimmunoassays, cytotoxic properties necessary for direct complement-dependent lysis. The insoluble antigen and the antibody in the culture fluid are allowed to react. The free antibody is washed away. The amount of monoclonal antibody bound is measured directly or by binding of a second, labeled antibody capable of recognizing the first. Monoclonal antibodies can be easily labeled internally at high specific activity, using radioactive amino acid precursors. The choice of these is based on the efficiency of incorporation of labeled amino acids into secreted immunoglobulin in culture conditions. The hemagglutination assays are based on the ability of an antibody to agglutinate red cells, carrying the specific antigen. These assays have all the advantages in terms of extreme simplicity, speed, and direct visual reading of the results.

Preparation of monoclonal antibodies: strategies and procedures.

A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells

Distinct plaques, each of which is due to the release of hemolysin by a single antibody-forming cell, are revealed by complement after incubation, in an agar layer, of a mixture of sheep red cells and lymphoid cells from a rabbit immunized with sheep red cells.

http://science.sciencemag.org/content/sci/140/3565/405.1.full.pdf

Plaque Formation in Agar by Single Antibody-Producing Cells

Plaque Forming Cells: Methodology and Theory

DELAYED, or tuberculin-type, sensitivity occurs in the course of many bacterial infections. It is characterized by skin reactions appearing with 24–48 hr. after intracutaneous injections of bacteria or their soluble products. Like immunological reactions, delayed skin reactions are highly specific, but the products eliciting the reactions need not be antigenic per se. Circulating antibodies have not been demonstrated to play a part. Delayed sensitivity is not transferable passively by serum, but can be transferred with the cells of sensitized organisms.

Production of tuberculin sensitivity.

Delayed Hypersensitivity in Tuberculous Mice

/pdf/british-association-of-allergists-4nkss5tuq2.pdf

Albert A. Nordin

Papers

Plaque Formation in Agar by Single Antibody-Producing Cells

Plaque Forming Cells: Methodology and Theory

Production of tuberculin sensitivity.

Delayed Hypersensitivity in Tuberculous Mice

British Association of Allergists