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ID 49999
file
creator
KOHNO, Nobuoki
AKIYAMA, Mitoshi
KYOIZUMI, Seishi
TANABE, Masaru
OYAMA, Tetsu
YAMAKIDO, Michio
subject
RI-ELISA
Monoclonal antibody
KL-3 antibody
Soluble antigen
NDC
Medical sciences
abstract
A novel screening method was established to select new monoclonal antibodies which react with unknown antigenic determinants on molecules bearing antigen determinants reactive with established monoclonal antibodies. This new method is a sandwich assay termed "reversed indirect-enzyme linked immunosorbent assay" (RI-ELISA). Goat antimouse immunoglobulin antibodies are used as the primary immobilized antibody in this assay. They allow the non-purified monoclonal antibodies contained in hybridoma culture supernatants to bind to the microtest plate for enzyme immunoassay (EIA plate) much more efficiently than in the usual sandwich assay where the non-purified monoclonal antibodies are adsorbed directly to the polystyrene surface. The antigen solution is then reacted with the monoclonal antibodies and thereafter enzyme labeled monoclonal antibody with known specificity is added. Therefore, if the hybridoma culture supernatant contains monoclonal antibodies which were bound to the EIA plate and react with antigenic determinants on the soluble molecules which have antigen determinants recognized by the enzyme labeled antibody, the enzyme labeled antibodies will bind to induce an enzymatic reaction. The most important technical consideration in the RI-ELISA is the inhibition of direct binding of the enzyme labeled monoclonal antibodies to free sites remaining in the immobilized goat anti-mouse immunoglobulins antibodies. This problem could be effectively overcome by using normal mouse serum as blocking substance. These studies indicate that the RI-ELISA may be a useful screening method for selecting new monoclonal antibodies which react with unknown antigenic determinants on soluble molecules.
journal title
Hiroshima Journal of Medical Sciences
volume
Volume 36
issue
Issue 4
start page
319
end page
323
date of issued
1987-12
publisher
Hiroshima University Medical Press
issn
0018-2052
ncid
pubmed id
language
eng
nii type
Departmental Bulletin Paper
HU type
Departmental Bulletin Papers
DCMI type
text
format
application/pdf
text version
publisher
department
Graduate School of Biomedical Science
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