Antigen capture ELISA for platelet antibody detection: choice of conjugate influences assay result.
The performance of an anti-IgG/A/M and two anti-IgG alkaline phosphatase (AP) conjugates in a commercial antigen capture ELISA (ACE) were compared for their ability to detect antibodies to human platelet antigens (HPAs) contained 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 serum being tested. The increased sensitivity in some instances was also accompanied by an apparent increase in nonspecific binding, as measured by activity against irrelevant glycoproteins that should not have been recognized by the relevant HPA antibodies in the sera in question. Hence, anti-IgG conjugates would appear to be preferable for detecting platelet-reactive antibodies in many clinical situations; however, the choice of anti-IgG conjugate should be made judiciously (and appropriately validated), in order to avoid false positive results arising from increased nonspecific binding, which may otherwise be erroneously attributed to the presence of auto-antibodies in the serum under investigation.
Lubenko
A
A
National Blood Service--Leeds Blood Centre, West Yorkshire, UK. anatole.lubenko@nbs.nhs.uk
Savage
J
J
D000328:Adult; D000906:Antibodies / Q000097:blood*; D000918:Antibody Specificity; D016824:Antigens, Human Platelet / Q000097:blood / Q000276:immunology; D001792:Blood Platelets / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards; D005188:False Negative Reactions; D005189:False Positive Reactions; D006023:Glycoproteins / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007231:Infant, Newborn; D011933:Reagent Kits, Diagnostic / Q000592:standards; D012680:Sensitivity and Specificity
D000906:Antibodies; D016824:Antigens, Human Platelet; D006023:Glycoproteins; D007074:Immunoglobulin G; D011933:Reagent Kits, Diagnostic