Glutaraldehyde fixation of target cells to plastic for ELISA assays of monoclonal anti-HLA antibodies produces artefacts

We found that an ELISA method for screening mouse monoclonal antibodies raised against antigens of the HLA system, that involves glutaraldehyde fixation of the target cells, produces both false negatives and false positives. The false negative results are due to destruction and/or modification of the antigenicity of some HLA-D region coded molecules; the false positives are mostly caused by non-specific adhesion of IgM, even at 1.625 μg/ml, to glutaraldehyde fixed cells. To a lesser extent there is nonspecific binding of IgG2a and IgG2b monoclonal antibodies particularly at concentrations above 12.5 μg/ml. These findings are unfortunate because the fixation of cells to the plates appeared to be a major technical advance as it removed the need for multiple centrifugation steps in the ELISA assay.