Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis.

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.

FUSION of myeloma cells which grow in tissue culture with spleen cells from an immunised mouse provides a general method for obtaining cell lines (hybridomas) which make antibody of the desired specificity1–3. Hybrids derived from these myelomas make the immunoglobulin (Ig) heavy and light chains of the myeloma parent as well as the antigen-specific heavy and light chains of the spleen cell parent. In conditions in which the two heavy and two light chains associate randomly, a hybridoma would make 10 distinct Ig molecules, and the specific antibody would comprise only 1/16 of the total Ig4,5. To obtain hybridomas making only the specific antibodies requires a tumour cell fusion partner that itself makes no Ig but which can nevertheless be fused with spleen cells to obtain hybrids secreting only the specific antibody. We report here the identification of such a cell line, Sp2/0-Ag14.

A better cell line for making hybridomas secreting specific antibodies

Production of monoclonal antibodies: strategy and tactics.

A hybridoma clone which secretes a macrophage (MΦ)-specific monoclonal antibody, F4/80, was produced by fusing spleen cells from a rat hyperimmunized with cultured thioglycollate-induced mouse peritoneal MΦ with a mouse myeloma, NS1. Binding of antibody to primary cells and cell lines was detected by radioimmune indirect binding assay, autoradiography or fluorescence-activated cell sorter analysis. F4/80 binds to mouse MΦ from the peritoneal cavity or other sources, blood monocytes, MΦ derived from bone marrow precursors in culture and MΦ-like cell lines, but not to other cells, including polymorphonuclear leukocytes, lymphocytes or fibroblasts. F4/80 does not bind to MΦ via Fc receptors, is not cytotoxic and is of the rat IgG2b subclass. Since F4/80 binds to all MΦ defined by adherence, morphology and immune phagocytosis, it provides a new marker to define the MΦ in the mouse. Large differences in expression of antigen F4/80 were found, depending on intraperitoneal stimulation, time in culture and stage of maturation. Immunoprecipitation experiments demonstrated that the antigen F4/80 is part of a component of Mr 160000 which is synthesized by the MΦ and, at least in part, exposed on the cell surface.

F4/80, a monoclonal antibody directed specifically against the mouse macrophage

FUSION between myeloma cells and spleen cells from immunised donors has been shown to be a successful method of deriving homogeneous anti-SRBC (anti-sheep red blood cell) and anti-TNP antibodies1,2. One of the most powerful features of this approach is that, by cloning, one may easily derive cell lines synthesising monoclonal antibodies despite using non-purified immunogens. The multiple components of a heterogeneous population of hybrid cells are resolved by cloning techniques. This feature makes the system a very powerful tool in the study of complex antigenic structures. The established cell lines offer the further advantage of unlimited permanent supply of material, and the possibility of worldwide standardisation.

Antibodies to major histocompatibility antigens produced by hybrid cell lines.

Eukaryotic structural gene mutations occurring spontaneously in a mouse myeloma cell line offer the opportunity to study somatic mutation in animal cells at the molecular level. Studies on the myeloma protein and on mRNA have enabled us to characterise four such mutants representing four different mutation mechanisms. The results may have some bearing on the origin of antibody diversity.

Molecular analysis of spontaneous somatic mutants

A rat myeloma cell line which does not express an immunoglobulin chain, YB2/3.0.Ag.20, is prepared from the cell line Y3-Ag 1.2.3 via a hybrid myeloma cell line. This cell line, and variants thereof prepared by passaging and/or cloning the line, may be fused with immunocyte cells from an animal sensitized to an immunogen to produce hybrid myeloma cell lines which provide a source of monoclonal antibodies to said immunogen.

Rat myeloma cell lines

The complete sequence of the mouse immunoglobulin kappa light chain MOPC 21 messenger RNA has been determined using a chain termination method and chemically synthesised deoxyoligonucleotides to initiate the synthesis of a DNA molecule complementary to the mRNA template. Five such oligonucleotide primers have been used for the sequence analysis of this messenger RNA. The approach is excellent for comparative studies of mouse k-chain mRNAs because they can be made on impure mRNA preparations. The MOPC 21 light chain mRNA is 943 nucleotides in length excluding the poly(A) region. An unexpected finding was that there are only three bases in the 5' non-coding region and its significance in terms of ribosome binding is discussed; 87 code for the precursor or leader sequence of the protein, 642 for the mature protein and 211 for the 3' non-coding region. The codons for the precursor region allows the previously undetermined amino acid sequence to be predicted. In common with other precursor regions a high proportion of the predicted amino acids are hydrophobic.

Cesar Milstein

Papers

Antibodies to major histocompatibility antigens produced by hybrid cell lines.

Molecular analysis of spontaneous somatic mutants

Rat myeloma cell lines

Complete sequence of an immunoglobulin mRNA using specific priming and the dideoxynucleotide method of RNA sequencing

Somatic cell genetics of antibody-secreting cells: studies of clonal diversification and analysis by cell fusion.