A simple approach to improving sensitivity in a one-step monoclonal antibody-based ELISA for human IIbIIIa using multiple conjugates.
This paper describes an approach which can be used to increase both the speed and the sensitivity of a monoclonal antibody (MAb) based ELISA for human IIbIIIa by using a mixture of enzyme labeled MAbs (conjugates) and a one step incubation format (where analyte and conjugates were incubated simultaneously for 2 h in the MAb coated well). A simple competitive blocking method is described for mapping the relative epitopes of five monoclonal antibodies (MAbs) with minimum preparation of enzyme conjugate. This method permits the selection of three MAbs which recognize distinctly different epitopes, and are suitable for use in a one step ELISA format. Compared to the regular two step ELISA using two MAbs, this simple modification improves the assay sensitivity by three-fold and decreases the incubation time by 75%, while maintaining a high level of precision (2-15%). The assay is very specific and can accurately measure both recombinant and native IIbIIIa.
Rowland
A M
AM
Department of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, CA 94080.
Wooldridge
M A
MA
Rosenthal
K A
KA
Wong
W L
WL
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001667:Binding, Competitive; D003429:Cross Reactions; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes; D006801:Humans; D010980:Platelet Membrane Glycoproteins / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010980:Platelet Membrane Glycoproteins