An isotype-specific spot-ELISA for the enumeration of antibody-secreting hybridomas and the determination of isotype switch variants. 1991

J M Coco-Martin, and P Koolwijk, and C A van der Velden-de Groot, and E C Beuvery
National Institute for Public Health and Environmental Protection (RIVM), Laboratory for Inactivated Viral Vaccines, Bilthoven, The Netherlands.

A solid-phase spot enzyme-linked immunosorbent assay (spot-ELISA) using rat monoclonal antibodies (MAbs) and an image-processing system is described. This isotype-specific spot-ELISA permits the enumeration of antibody-secreting cells irrespective of the specificity of the secreted antibodies. When used in combination with an ELISA, the antibody production per cell can also be evaluated. In addition, isotype switch variants, which arise spontaneously in antibody-producing cell lines, can be determined. This study compared four assays: three antigen-specific spot-ELISAs, using enzyme-conjugated polyclonal antibodies as well as rat MAbs; and an isotype-specific spot-ELISA using rat MAbs. There were no significant differences between these four spot-ELISA systems. For one tested cell line (alpha huIgA1/gamma 1), the number of antibody-secreting cells fluctuated between 60% and 95% during several passages. For the other tested cell line (alpha huIgA1/gamma 2b), the number of antibody-secreting cells decreased from 90% to 70% after several passages. The results of the spot-ELISA were in agreement with flow cytometric (FC) analysis of cytoplasmic IgG. This indicates that for these two cell lines, the synthesized IgG was also secreted into the culture fluid. Using the isotype-specific spot-ELISA, the switch frequency of five murine hybridomas (alpha huIgA1/gamma 1, alpha huIgA1/gamma 2b, alpha HRP, RIV6, MN12) was determined. The switch frequencies varied from 1/82,000 for the alpha HRP cell line to 1/660,000 for the alpha huIgA1/gamma 2b cell line.

UI MeSH Term Description Entries
D007132 Immunoglobulin Isotypes The classes of immunoglobulins found in any species of animal. In man there are nine classes that migrate in five different groups in electrophoresis; they each consist of two light and two heavy protein chains, and each group has distinguishing structural and functional properties. Antibody Class,Ig Isotype,Ig Isotypes,Immunoglobulin Class,Immunoglobulin Isotype,Antibody Classes,Immunoglobulin Classes,Class, Antibody,Class, Immunoglobulin,Classes, Antibody,Classes, Immunoglobulin,Isotype, Ig,Isotype, Immunoglobulin,Isotypes, Ig,Isotypes, Immunoglobulin
D004797 Enzyme-Linked Immunosorbent Assay An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed. ELISA,Assay, Enzyme-Linked Immunosorbent,Assays, Enzyme-Linked Immunosorbent,Enzyme Linked Immunosorbent Assay,Enzyme-Linked Immunosorbent Assays,Immunosorbent Assay, Enzyme-Linked,Immunosorbent Assays, Enzyme-Linked
D006825 Hybridomas Cells artificially created by fusion of activated lymphocytes with neoplastic cells. The resulting hybrid cells are cloned and produce pure MONOCLONAL ANTIBODIES or T-cell products, identical to those produced by the immunologically competent parent cell. Hybridoma
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D000911 Antibodies, Monoclonal Antibodies produced by a single clone of cells. Monoclonal Antibodies,Monoclonal Antibody,Antibody, Monoclonal
D000921 Antibody-Producing Cells Cells of the lymphoid series that can react with antigen to produce specific cell products called antibodies. Various cell subpopulations, often B-lymphocytes, can be defined, based on the different classes of immunoglobulins that they synthesize. Antibody-Producing Cell,Antibody-Secreting Cell,Antibody-Secreting Cells,Immunoglobulin-Producing Cells,Immunoglobulin-Secreting Cells,Antibody Producing Cell,Antibody Producing Cells,Antibody Secreting Cell,Antibody Secreting Cells,Cell, Antibody-Producing,Cell, Antibody-Secreting,Cell, Immunoglobulin-Producing,Cell, Immunoglobulin-Secreting,Cells, Antibody-Producing,Cells, Antibody-Secreting,Cells, Immunoglobulin-Producing,Cells, Immunoglobulin-Secreting,Immunoglobulin Producing Cells,Immunoglobulin Secreting Cells,Immunoglobulin-Producing Cell,Immunoglobulin-Secreting Cell
D051379 Mice The common name for the genus Mus. Mice, House,Mus,Mus musculus,Mice, Laboratory,Mouse,Mouse, House,Mouse, Laboratory,Mouse, Swiss,Mus domesticus,Mus musculus domesticus,Swiss Mice,House Mice,House Mouse,Laboratory Mice,Laboratory Mouse,Mice, Swiss,Swiss Mouse,domesticus, Mus musculus
D051381 Rats The common name for the genus Rattus. Rattus,Rats, Laboratory,Rats, Norway,Rattus norvegicus,Laboratory Rat,Laboratory Rats,Norway Rat,Norway Rats,Rat,Rat, Laboratory,Rat, Norway,norvegicus, Rattus

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