Antigen capture ELISA for platelet antibody detection: choice of conjugateinfluences assay result

The performance of an anti-IgG/A/M and two anti-IgG alkaline phosphatase (A P) conjugates in a commercial antigen capture ELISA (ACE) were compared for their ability to detect antibodies to human platelet antigens (HPAs) conta ined in 11 sera. Three anti-HPA-1a and one anti-HPA-3a were not detected by the anti-IgG/A/M conjugate, but both anti-IgG conjugates detected all HPA antibodies as a consequence of increased sensitivity in detecting specific antibody binding. This increase varied from 10% to 500%, depending on the s erum being tested. The increased sensitivity in some instances was also acc ompanied by an apparent increase in nonspecific binding, as measured by act ivity against irrelevant glycoproteins that should not have been recognized by the relevant HPA antibodies in the sera in question. Hence, anti-IgG co njugates would appear to be preferable for detecting platelet-reactive anti bodies in many clinical situations; however, the choice of anti-IgG conjuga te should be made judiciously (and appropriately validated), in order to av oid false positive results arising from increased nonspecific binding, whic h may otherwise be erroneously attributed to the presence of auto-antibodie s in the serum under investigation.