Precise quantitation of palgg: A new radiometric microtechnique

Abstract

We report the development of a radiometric assay for platelet-bound IgG that is both sensitive and quantitative. The assay utilized 96-well millititer plates incorporating a 0.2 microns filter membrane in the bottom. A 125I-labeled monoclonal antihuman IgG, as a secondary antibody, detected the platelet-bound human IgG. Since 5 x 10(6) platelets were used for each assay, tests for platelet-bound IgG can be performed on persons with severe thrombocytopenia. For the detection of circulating antiplatelet alloantibodies, as little as 10 microliters of platelet-free plasma per assay is required. Antiplatelet IgG was quantitated by using anti-PIA1 antibody that was purified with affinity and elution and DEAE chromatography. This purified antiplatelet antibody was labeled with 125I and was used to determine the binding ratio of secondary antibody to primary antibody. Under our standard conditions, this ratio was found to be stable at approximately 0.35 over the sensitivity range of the assay. The assay can detect approximately 200 molecules of human IgG per platelet (0.1 ng of secondary antibody bound per 5 x 10(6) platelets). It has a linear range from 0 to 7,000 molecules per platelet. Quantitation of anti-PIA1 binding for platelets stored for up to 6 months under refrigeration showed no change in number of PIA1 binding sites. Clinical studies showed that 18 of 19 ITP patients had an increased number of IgG molecules per platelet as did patients with malignancy and drug-induced immune thrombocytopenia. Patients who had received multiple platelet transfusions had antiplatelet antibody in their plasma. Normal amounts of PAIgG were observed in platelets and plasma of patients with nonimmune thrombocytopenia. The advantages of this method are that it is: 1) a more precise quantitation of PAIgG via direct measurement of binding ratio with PIA1 antibody; 2) performed with small amounts of platelets and plasma; 3) both sensitive and specific; and 4) reliably reproducible with both fresh and stored platelets.

Increased representation of the PTPN22 mutation in patients with immune thrombocytopenia

“…Indeed, normal O-blood group subjects have a consistently elevated VWFpp/VWF:Ag ratio, a sensitive index of increased clearance, and a shorter VWF survival after demopressin, again in keeping with increased VWF clearance [8]. This blood group-dependent effect on VWF levels has been demonstrated also in heterozygotes for VWF null alleles or R854Q mutations [10].…”

“…Genotyping of PTPN22 1858C/T SNP was performed by PCR‐restriction fragment length polymorphism (RFLP) [9]. A radiometric technique was used for quantitation of autologous platelet surface IgG [10]. Demographic data describing the patients’ age and race or ethnic background were obtained from the patients.…”

“…A radiometric technique was used for quantitation of autologous platelet surface IgG [10]. Demographic data describing the patientsÕ age and race or ethnic background were obtained from the patients.…”

MHC Class I–Associated Peptides Identified From Normal Platelets and From Individuals With Idiopathic Thrombocytopenic Purpura

“…Patients were diagnosed with ITP based on a high level of platelet-associated immunoglobulin G (PAIgG) in addition to a low platelet count, normal bone marrow, and no other etiology for thrombocytopenia [5]. PAIgG was used to detect antiplatelet autoantibodies by a radiometric micro technique [25]. This technique measures total IgG bound to the platelet surface, which includes both antiplatelet autoantibodies and nonspecifically bound IgG.…”