Development of a new antibody to the human inhibin/activin betaB subunit and its application to improved inhibin B ELISAs.
Inhibin B has emerged as a clinically useful analyte for studies of reproductive function in both men and women. The antibody to the betaB subunit (C5) used in current commercially available assays (DSL and OBI) was raised in this laboratory to a synthetic peptide from the betaB subunit. These assays require pre-treatment of samples with hydrogen peroxide to oxidise two methionines in the epitope to the sulfoxide for full immunoreactivity. It was also claimed that this antibody cross-reacted significantly with inhibin A leading to a 0.5% cross-reaction of inhibin A in the current generation of immunoassays. Both of the above immunoassays required overnight incubation with sample. The development of improved antibodies to the betaB subunit has proved difficult due to the conservation of the betaB subunit between species. We describe the development of new monoclonal antibodies to the betaB subunit, by immunisation of mice with recombinant X. laevis activin B using the RIMMS method of immunisation. The result has been the development of highly specific antibodies in a short time period. One of these antibodies 46A/F is shown to be a highly effective capture antibody in a human inhibin B ELISA, without any sample pre-treatment. The results of the validation of an improved inhibin B assay using 46A/F as the capture antibody are shown, with comparison to one of the commercially available inhibin B assays. Overall, the inhibin B assay is simplified and the performance improved by using this new antibody 46A/F. It was further shown that the cross-reaction of inhibin A in both the OBI and DSL inhibin B ELISAs is ten fold less than previously reported. This can be attributed to the poor quality of recombinant inhibin B available for use as standard in 1996. Although the present generation of inhibin B assays has proved adequate to enable the physiological function of inhibin to be determined and novel clinical applications found, the simplification of the assay made possible by the improved antibody should make possible a new generation of more rapid, sensitive, convenient and robust tools for routine use.
Ludlow
Helen
H
Centre for Proteins and Peptides, School of Life Sciences, Oxford Brookes University, Headington, Oxford, OX3 0BP, UK. hludlow@brookes.ac.uk
Muttukrishna
Shanthi
S
Hyvönen
Marko
M
Groome
Nigel P
NP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D028322:Inhibin-beta Subunits / Q000097:blood* / Q000276:immunology; D007265:Inhibins / Q000276:immunology; D008297:Male; D051379:Mice; D008875:Middle Aged; D017698:Postmenopause / Q000097:blood; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D015203:Reproducibility of Results; D029867:Xenopus Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; C422402:INHBB protein, human; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins; D029867:Xenopus Proteins; C094377:inhibin-alpha subunit; D007265:Inhibins; D028322:Inhibin-beta Subunits