Direct quantitation of platelet-associated IgG by electroimmunoassay

Abstract

An electroimmunoassay was applied to the quantitation of platelet- associated IgG (PAIgG). Protein solubilized by Triton X100 from washed platelets was electrophoresed at pH 5.0 in agarose gels containing carbamoylated rabbit anti-human IgG (pI approximately equal to 5.0). Because the rabbit antibody is immobilized under these conditions, while PAIgG is freely mobile, rocket precipitates were produced, the heights of which were directly proportional to the amount of antigen (PAIgG) present. By this method, PAIgG for normal individuals was found to be 4.3 +/- 1.7 fg/platelet (mean +/- 2 SD; n = 35). Increased PAIgG levels (direct assay) were found in 27 of 29 patients with a diagnosis of clinically active idiopathic thrombocytopenic purpura (ITP), ranging from 10.5 to 101.5 fg/platelet. Moderately elevated PAIgG was found in 8 of 10 patients in an early stage of recovery from ITP (range 7.5–9.5 fg/platelet) and in 3 of 6 patients with apparent nonimmune thrombocytopenia (range 14.5–24.0 fg/platelet). Electroimmunoassay for PAIgG can be performed on patients with platelet counts as low as 2000/microliters, yields results in less than 24 hr, is highly reproducible, and appears to provide a useful tool for the evaluation of patients with immunologically mediated thrombocytopenia.

Progress and development of platelet antibody detection

“…In the 1970s the availability of radiolabelled or immune fluorescence labelled anti-human immunoglobulins of the IgG, IgM or IgA class led to the development of more sensitive and specific methods (phase II) e.g. radio immune assay (RIA), platelet immunofluorescence test (PIFT) and mixed passive haemagglutination assay (MPHA) [7][8][9][10][11][12][13][14][15][16]. With these assays, detection of antibody binding on donor platelets incubated with serum from the patient (indirect method) or directly on patient platelets (direct method) became possible.…”

Flow cytometry analysis of platelet antibodies

“…Many tests have been devised to measure the circulating antibody to platelets on the antibody bound to the surface membrane (platelet-associated immunoglobulin-PAIgGm PAIgM, PAIgA). Among these are the antiglobulin consumption test (complement lysis inhibition) (7); the release of various platelet factors such as platelet factor III, serotonin, and adenosine (8); %r release assay (9); ' "I staphylococcal protein A (10); immunofluorescence (11); enzyme-linked immunoassays (12); electroimmunoassay (13); nephelometry (14); and flow cytometry (15)(16)(17).…”

Comparison of the measurement of surface or total platelet‐associated IgG in the diagnosis of immune thrombocytopenia

“…The specificity of increased PAIgG for the diagnosis of ITP is low, but many clinicians measure platelet-associated immunoglobulins to assist in the diagnosis of ITP [3-61. Three general types of assays can be used: (a) direct binding assays that measure the binding of labelled antiserum to the target platelets [7]; (b) two-stage assays that measure available antiserum following incubation of this reagent with the test platelets [ 11 ; and (c) assays that measure platelet-associated IgG following solubilization of the platelets [8,9]. These latter techniques are used with increasing frequency because of their simplicity.…”