Production of Monoclonal Antibodies Against Recombinant Human Interferon-Gamma: Screening of Hybridomas Without Purified Antigen

Abstract

A panel of mouse monoclonal antibodies (MAbs) against the recombinant human gamma interferon (HuIFN-γ) has been produced for the study of the structure-function relationships of this important lymphokine. Enzyme linked immunosorbant assay (ELISA) is the current method of choice to screen hybridomas for specific MAb production. The purity of the antigen used for screening dictates the specificity of the ELISA. As often is the case in many systems, adequately purified biologically active HuIFN-γ was not readily available for this purpose. A sandwich ELISA which allowed the use of unpurified HuIFN-γ for hybridoma screening was developed. A rabbit antiserum against the denatured HuIFN-γ purified by SDS-PAGE was prepared and the nonspecific binding activity was removed by adsorption to control cell proteins immobilized on Sepharose. The adsorbed immunoglobulin fraction was bound to the ELISA plate: (i) to trap HuIFN-γ specifically from the whole cell lysate, thus providing specificity for MAb detection, and (ii) to avoid direct adsorption of the HuIFN-γ to the ELISA plate because others have found that this prevented detection of neutralizing MAb. The sandwich ELISA detected both neutralizing and non-neutralizing MAbs with relatively low false positive reactions. This approach to the development of an ELISA method to screen hybridomas without purified antigen should be applicable to the production of MAbs to other proteins.