Eliminating interference from heterophilic antibodies in a two-site immunoassay for creatine kinase MB by using F(ab')2 conjugate and polyclonal mouse IgG.

Heterophilic antibodies interfere in two-site immunoassays. Our purpose was to screen for the samples containing heterophilic antibodies that react with mouse immunoglobulins and to use them to determine how to eliminate interference in various two-site immunoassays being developed in our laboratory. Of approximately 2600 samples screened, 81 had heterophilic antibodies. When creatine kinase MB (CKMB) concentration was measured with intact antibody conjugate in these 81 samples, 18 (22%) samples had apparent CKMB values significantly greater than values measured with Hybritech's "Tandem -E CKMB immunoenzymetric" assay and Corning agarose-gel electrophoresis. Adding up to 133 mg/L of polymerized IgG or up to 1666 mg/L polyclonal mouse IgG in the assay did not eliminate the interference in all the samples. However, adding F(ab')2 conjugate plus 40 mg/L of polymerized IgG or 83 mg/L of polyclonal mouse IgG eliminated the interference in all the samples. This approach was also effective in eliminating the interference in 15 samples containing 4.7-165.2 mg/L of human antimouse antibody (HAMA). Combined use of F(ab')2 conjugate and polyclonal mouse IgG is recommended to eliminate interference from heterophilic antibodies that react with murine immunoglobulins or HAMA in two-site murine-antibody-based assays.