Quantification of Platelet-Associated IgG with Competitive Solid-Phase Enzyme Immunoassay

Abstract

In order to measure platelet-associated IgG (PAIgG), we devised a solidphase enzyme immunoassay employing a competitive binding of peroxidase-conjugated anti-IgG antiserum between platelets and polystyrene tubes coated with IgG. The amounts of peroxidase bound to the tubes were measured in a spectrophotometer by an enzymatic reaction. This method is highly sensitive, reproducible and can be carried out more simply. The PAIgG values of normal controls averaged 21.6 ± 6.6 (SD) ng/l0^τ platelets. 27 (93%) of 29 patients with idiopathic thrombocytopenic purpura (ITP), who had a platelet count of less than 15×10⁴/μl, had PAIgG values greater than those of controls by 2 SD and averaged 205.5 ± 323 ng. There was a significant inverse correlation between platelet count and PAIgG value of ITP patients. The PAIgG values of patients with aplastic anemia were within normal range.

An unclassified platelet function disorder associated with bleeding tendency.

“…Intracellular platelet /l-TG and PF4 were determined after ultrasonication of 1 ml PRP (50>< 104/,ul platelets) at 0°C for 30 sec in a 150 watt ultrasonic homogenizes at a wave amplitude 7 kHz. PAIgG was measured by competitive enzyme immunoassay (Tsubakio et al 1981). Glycoprotein of platelet membrane was stained following electrophoresis on SDS polyacrylamide gels (SDS-PAGE ).…”

In vitro Platelet Phagocytosis in Idiopathic Thrombocytopenic Purpura

“…The separation of platelet-rich plasma (PRP) from each patient and normal donor was based on a previously described method [3]. PRP was centri fuged at 1,200 g for 10 min, the cell buttons were sus pended in a small amount of RCD solution [9]; ap proximately 50 gC\ of radioactive chromium (5lCr) was added per sample, followed by 30-min incuba tion at room temperature.…”

“…Quantitative assays of platelet-associat ed IgG (PAIgG), including our competi tive solid-phase enzyme immunoassay [3], have been reported and the results indicat ed that in ITP, increased PAIgG correlat ed inversely with both platelet survival [4] and the level of thrombocytopenia [3][4][5][6][7][8]. We thought the PAIgG assay to be the most valuable method for detecting plate let autoantibody in ITP and investigated the relationship between the results ob tained by the direct assay and PAIgG val ues in ITP patients.…”

SLE Thrombocytopenia: From Peripheral Platelet Destruction to Central Hemopoietic Defect

“…However, accumulated evidence suggested that normal platelets have two IgG pools, one located on platelet surface (100 IgG molecules) and one intracellular pool (20,000 IgG molecules) [34,35]. Older methods also measured the IgG ofgranules, leading to falsely elevated results for surface IgG [36][37][38]. Surface immunoglobulin was later measured with I131-labeled monoclonal antibody for all IgG subclasses or labeled staphylococcal protein A [39][40][41].…”