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Method for determining the affinity of monoclonal antibody using non-competitive ELISA: a computer program.

IR@IMTECH: CSIR-Institute of Microbial Technology, Chandigarh


Field	Value

Title	Method for determining the affinity of monoclonal antibody using non-competitive ELISA: a computer program.

Creator	Raghava, G.P.S. Agrewala, J N

Subject	QR180 Immunology

Description	A simple and reliable method based upon law of mass action for calculating affinity of a monoclonal antibody using non-competitive ELISA, is described. In this method, the binding of an antibody (Ab) with an antigen (Ag) is measured by ELISA using serial dilutions of both antigen (coated on the plate) as well as antibody. When the OD measured after the antigen antibody interaction was plotted against the concentration of Ab, added to the wells, a hyperbolic curve was obtained. The OD, at any point of the curve, was considered as a direct reflection of the amount of antibody bound to the antigen. The OD-100 denotes the occupancy of maximum no. of epitopes available on the antigen molecules, accessible to the antigen. The concentration of antibody (Ab, Ab') at corresponding levels of antigen concentration (Ag, Ag'), presents the value obtained at OD-50. The [Ag] and [Ag'] are not the true antigen concentrations but are the measurement of antigen density on the plate. The affinity constant K(aff) was calculated by using the formula K(aff) = (n - 1)/2(n[Ab'] - [Ab]), derived from law of mass action, where n = [Ag]/[Ag']. A computer program to calculate the affinity of antibody to the antigen using method described in this manuscript has been developed and discussed.

Publisher	Marcel Dekker

Date	1994-05

Type	Article PeerReviewed

Identifier	Raghava, G.P.S. and Agrewala, J N (1994) Method for determining the affinity of monoclonal antibody using non-competitive ELISA: a computer program. Journal of immunoassay, 15 (2). pp. 115-28. ISSN 0197-1522

Relation	http://crdd.osdd.net/open/410/