Robert J. Kinders

A very sensitive coupled luminescent assay for cytotoxicity and complement-mediated lysis

The demand for convenient and sensitive means of measuring cytotoxicity and complement-mediated killing is likely to be increased by the recent identification of Complement Factor H, an important regulatory protein of both the classical and alternate pathways of complement, as a tumor-associated antigen. Here we describe a simple luminometric assay capable of detecting the death of approximately 0.03 nucleated human-cell equivalent or approximately 1 rabbit-erythrocyte equivalent. The assay measures the release of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) from dead or damaged cells by coupling its enzymatic activity to production of ATP, which in turn is measured by well-known methods involving firefly luciferase. This is accomplished by means of a reaction series in which the activity of G3PDH is coupled with that of phosphoglycerate kinase, the next enzyme in the glycolytic pathway. As described, the assay uses inexpensive, commercially available reagents. This coupled assay was used to demonstrate that an anti-factor-H antibody is capable of enhancing complement-mediated killing of the Raji cancer cell line by > 1000%.

Interference in immunoassays by human anti-mouse antibodies

Mogensen and Moller [l] described interference in the Abbott CA 125 enzyme immunoassay (EIA) by putative human antimouse antibodies (HAMA).and demonstrated that this interference could be reduced by diluting the specimen with mouse serum. HAMA are predominantly IgGs that arise in response to immunizing doses of mouse monoclonal antibodies (Mabs) [2, 31. The high titres often found in HAMA sera should be distinguished from the low levels of anti-mouse activity resulting from heterophile antibodies, mostly IgM [4-81. Since HAMA are polyclonal and of complex specificity, many different types of interference can be observed in immunoassays with HAMAcontaining sera. The type of interference is dictated by the assay configuration (i.e. competitive vs. sandwich, one-step vs. twostep), the antibodies used (specificity, isotype and use as capture or probe reagent), the analyte and the subclass or isotype of the Mab used to immunize the patient. On injection of a patient with a Mab, HAMA are elicited that typically exhibit both anti-isotype and anti-idiotype specificities. The anti-isotype component may well bind to Mabs in an immunoassay and interfere, especially when the injected Mab and the Mabs used in the assay are of the same isotype. Elevated carcinoembryonic antigen (CEA) values were obtained in a patient who had been injected with mouse Mab B72.3 upon assay with the Abbott list 5863 product, which is a double monoclonal sandwich EIA in a two-step format (Table 1). That these increased values resulted from positive interference by HAMA was shown by reassay after removal of HAMA by either heat treatment or chromatography on immobilized protein A (patient 1, Table 2). Similar positive interferences in immunoassays have been reported for other commercially available immunoassays [2, 3, 9, lo]. These false positives, and presumably those described by Mogensen and Moller [ 11, result from HAMA bridging the probe and capture antibodies and were only partly corrected by diluting the specimen with mouse serum. Sandwich assay formats that we have evaluated and found to be most resistant to HAMA effects are double polyclonal assays or monoclonaYpolyclonal assays, in which the Mab is used as probe. Our CEA EIA One-Step (list 4439), which uses guineapig anti-CEA as capture antibody and a Mab as probe, yielded an appropriate value on the high titre HAMA specimen (Table 1). Efficient recovery of CEA (103%) added to this specimen demonstrated that false negatives were not produced. HAMA may also produce false negatives. For example, in immunoassays with mouse Mabs as capture antibodies and probes that are not mouse Mabs, HAMA binds to the solid phase Mab and sterically blocks capture of the antigen from the specimen, but does not recognize the probe. The addition of