Influence of affinity of antibodies upon their detection by liquid phase radiobinding assay and solid phase enzyme linked immunosorbent assay. Demonstration using monoclonal antibodies raised against rDNA human proinsulin

Hybridomas producing proinsulin antibodies were cloned by limiting dilution of cell cultures obtained by fusion of splenocytes of immunized mice with immortal myeloma cells. Some proinsulin monoclonal antibodies crossreacted with labelled insulin but none did with labelled C-peptide indicating that...

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Bibliographic Details
Published in:Diabetologia 1991-07, Vol.34 (7), p.463-468
Main Authors: SODOYEZ, J. C, KOCH, M, LEMAIRE, I, SODOYEZ-GOFFAUX, F, RAPAILLE, A, FRANCOIS-GERARD, C, SONDAG, D
Format: Article
Language:English
Subjects:
Animals
Antibodies, Monoclonal - analysis
Antibody Affinity
Antigen-Antibody Complex
Antigen-antibody reactions, antigen-antibody complexes, antibody-complement and others. Study of affinity. Antigen presentation
Biological and medical sciences
DNA, Recombinant
Enzyme-Linked Immunosorbent Assay
Fundamental and applied biological sciences. Psychology
Fundamental immunology
Humans
Immunoglobulin G - analysis
Iodine Radioisotopes
Kinetics
Mice
Mice, Inbred BALB C - immunology
Mice, Inbred Strains - immunology
Molecular immunology
Proinsulin - immunology
Radioligand Assay
Recombinant Proteins - immunology
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Summary:Hybridomas producing proinsulin antibodies were cloned by limiting dilution of cell cultures obtained by fusion of splenocytes of immunized mice with immortal myeloma cells. Some proinsulin monoclonal antibodies crossreacted with labelled insulin but none did with labelled C-peptide indicating that the involved epitopes were at one of the insulin/C-peptide junctions or included in the insulin moiety. Hybridoma supernatants were assayed for IgG concentration by a solid phase assay and for ligand binding by a radiobinding assay and an enzyme linked immunosorbent assay. The half-life of immune complexes formed with radioligand was measured and, as expected, correlated with affinity as measured by the method of Scatchard. Antibody titres determined by enzyme linked immunosorbent assay did not correlate to those measured by radiobinding assay. IgG concentration correlated to enzyme linked immunosorbent assay titres but not to radiobinding assay titres. Finally, a significant correlation was found between radiobinding assay titre and the product of enzyme linked immunosorbent assay titre by the period of immune complexes. It is concluded that, except for very low affinity antibodies, enzyme linked immunosorbent assay is a capacity assay whereas radiobinding assay is influenced by both antibody concentration and affinity. The former assay is thus best suited to detecting low affinity antibodies whereas the latter is more efficient in the presence of low levels of high affinity antibodies.
ISSN:0012-186X
1432-0428
DOI:10.1007/BF00403281