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Precise quantitation of palgg: A new radiometric microtechnique
Author(s) -
Schwartz Kenneth A.,
Gauger John A.,
Davis John M.
Publication year - 1990
Publication title -
american journal of hematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.456
H-Index - 105
eISSN - 1096-8652
pISSN - 0361-8609
DOI - 10.1002/ajh.2830330304
Subject(s) - platelet , antibody , chemistry , monoclonal antibody , microbiology and biotechnology , immunology , medicine , biology
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 μm filter membrane in the bottom. A 125 l‐labeled monoclonal antihuman IgG, as a secondary antibody, detected the platelet‐bound human IgG. Since 5 ± 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 μl of platelet‐free plasma per assay is required. Antiplatelet IgG was quantitated by using anti‐Pl A1 antibody that was purified with affinity and elution and DEAE chromatography. This purified antiplatelet antibody was labeled with 125 l 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 ± 10 6 platelets). It has a linear range from ) to 7,000 molecules per platelet. Quantitation of anti‐Pl A1 binding for platelets stored for up to 6 months under refrigeration showed no change in number of Pl A1 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 PAlgG 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 PAlgG via direct measurement of binding ratio with Pl A1 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.