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

Abstract

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.

Slot blotting and flow cytometry: two efficient assays for platelet antibody screening among patients with platelet refractoriness

“…However, the choice of methods that can be successfully used in refractory patients continues to be a problem [10]. The MAIPA, as a reference method, is a very complicated and time- consuming approach for it to be used routinely [12,26,27]. Therefore, introducing a feasible, cost-effective and efficient screening method is imperative.…”

Approach of using established and new laboratory tests to more comprehensively investigate noninfectious and nonhemolytic transfusion reactions – along with the experience in Japan

“…developed a panel of cell lines that stably express specific HPAs and can be used in the MR‐MAIPA test . A rapid ELISA‐based test (PAKPLUS ® Gen‐probe, San Diego, CA, USA) and a Luminex ® beads‐based test (PAKLX ® Gen‐probe) for detection of HPA antibodies are now available as commercial tests, although reports have suggested reduced sensitivity compared with that of the MAIPA assay . A summary of these tests is presented in Table .…”

A new transfectant panel cell line‐based MoAb‐independent antigen capture assay system for detection of CD36 antibody

“…GTI PakPlus ® , an enzyme‐linked immunosorbent assay kit, and LIFECODES ® Pak Lx™, which is designed to work on the Luminex instrument, are now commercially available to detect HPA Abs and CD36 Abs. Although a couple of studies evaluated the validity of these kits focusing on HPA Abs, there is no such study focusing on CD36 Abs .…”