scholarly journals Heavy-chain subclass of round antiplatelet IgG in autoimmune thrombocytopenic purpura

Blood ◽  
1980 ◽  
Vol 56 (1) ◽  
pp. 84-87
Author(s):  
K Hymes ◽  
PH Schur ◽  
S Karpatkin
Keyword(s):  
Heavy Chain ◽  
Solid Phase ◽  
Protein A ◽  
Normal Serum ◽  
Staphylococcal Protein A ◽  
Antiplatelet Antibody ◽  
Thrombocytopenic Purpura ◽  
Autoimmune Thrombocytopenic Purpura ◽  
Solid Phase Radioimmunoassay ◽  
Monospecific Antisera

The gamma heavy-chain subclass of bound antiplatelet antibody was examined in six patients with autoimmune thrombocytopenic purpura (ATP) by a solid-phase radioimmunoassay. Monospecific antisera for gamma G1, gamma G2, gamma G3, and gamma G4 subclasses were employed in a “sandwich” technique, utilizing the binding of 126I-staphylococcal protein A. We have previously reported that serum antiplatelet antibody was restricted to be gamma G3 subclass in ATP. In contrast, all 4 IgG subclasses were found bound to platelets of ATP patients in the same distribution as that present in normal serum. It is suggested that the differences noted between serum antiplatelet IgG and platelet-bound IgG may represent different mechanisms of platelet injury.

scholarly journals Heavy-chain subclass of round antiplatelet IgG in autoimmune thrombocytopenic purpura

Blood ◽  
1980 ◽  
Vol 56 (1) ◽  
pp. 84-87 ◽  
Author(s):  
K Hymes ◽  
PH Schur ◽  
S Karpatkin
Keyword(s):  
Heavy Chain ◽  
Solid Phase ◽  
Protein A ◽  
Normal Serum ◽  
Staphylococcal Protein A ◽  
Antiplatelet Antibody ◽  
Thrombocytopenic Purpura ◽  
Autoimmune Thrombocytopenic Purpura ◽  
Solid Phase Radioimmunoassay ◽  
Monospecific Antisera

Abstract The gamma heavy-chain subclass of bound antiplatelet antibody was examined in six patients with autoimmune thrombocytopenic purpura (ATP) by a solid-phase radioimmunoassay. Monospecific antisera for gamma G1, gamma G2, gamma G3, and gamma G4 subclasses were employed in a “sandwich” technique, utilizing the binding of 126I-staphylococcal protein A. We have previously reported that serum antiplatelet antibody was restricted to be gamma G3 subclass in ATP. In contrast, all 4 IgG subclasses were found bound to platelets of ATP patients in the same distribution as that present in normal serum. It is suggested that the differences noted between serum antiplatelet IgG and platelet-bound IgG may represent different mechanisms of platelet injury.

scholarly journals Drug-dependent and non-drug-dependent antiplatelet antibody in drug- induced immunologic thrombocytopenic purpura

Blood ◽  
1985 ◽  
Vol 66 (2) ◽  
pp. 306-311 ◽  
Author(s):  
W Lerner ◽  
R Caruso ◽  
D Faig ◽  
S Karpatkin
Keyword(s):  
Solid Phase ◽  
Protein A ◽  
Staphylococcal Protein A ◽  
Antiplatelet Antibody ◽  
Drug Induced ◽  
Thrombocytopenic Purpura ◽  
Autoimmune Thrombocytopenic Purpura ◽  
History Of ◽  
Solid Phase Radioimmunoassay ◽  
Drug Dependent

Abstract The mechanism of drug-dependent immunologic thrombocytopenic purpura (DITP) was investigated by studying the sera of four patients with classic DITP (two with quinidine-, one with acetaminophen-, and one with phenazopyridine-dependent antiplatelet antibody) using a solid- phase radioimmunoassay with 125I-staphylococcal protein A. Two forms of antiplatelet antibody could be demonstrated: one that required drug to bind to platelets and one that bound to platelets in the absence of drug. Drug-dependent antiplatelet antibody required the simultaneous addition of drug and the Fc domain of the drug-dependent IgG molecule for binding to platelets. It did not require serum complement or factor VIII-related antigen for binding to platelets. Drug-dependent binding of antibody to platelets was saturation-dependent. Non-drug-dependent antiplatelet antibody of two patients (one with quinidine-induced thrombocytopenia and the other with acetaminophen-induced thrombocytopenia) reacted with autologous platelets as well as with homologous platelets, indicating that they were autoantibodies. Both autoantibodies had disappeared when their sera were tested 23 and 138 days, respectively, after withdrawal of their initial positive sera. Non-drug-dependent antiplatelet antibody binding could be demonstrated with the F(ab')2 fragment of the purified IgG of the serum of the second patient with quinidine DITP, who did not have detectable alloantibodies against HLA. None of the four patients with non-drug- dependent antiplatelet antibody had a past or present history of autoimmune thrombocytopenic purpura.

scholarly journals Drug-dependent and non-drug-dependent antiplatelet antibody in drug- induced immunologic thrombocytopenic purpura

Blood ◽  
1985 ◽  
Vol 66 (2) ◽  
pp. 306-311
Author(s):  
W Lerner ◽  
R Caruso ◽  
D Faig ◽  
S Karpatkin
Keyword(s):  
Solid Phase ◽  
Protein A ◽  
Staphylococcal Protein A ◽  
Antiplatelet Antibody ◽  
Drug Induced ◽  
Thrombocytopenic Purpura ◽  
Autoimmune Thrombocytopenic Purpura ◽  
History Of ◽  
Solid Phase Radioimmunoassay ◽  
Drug Dependent

The mechanism of drug-dependent immunologic thrombocytopenic purpura (DITP) was investigated by studying the sera of four patients with classic DITP (two with quinidine-, one with acetaminophen-, and one with phenazopyridine-dependent antiplatelet antibody) using a solid- phase radioimmunoassay with 125I-staphylococcal protein A. Two forms of antiplatelet antibody could be demonstrated: one that required drug to bind to platelets and one that bound to platelets in the absence of drug. Drug-dependent antiplatelet antibody required the simultaneous addition of drug and the Fc domain of the drug-dependent IgG molecule for binding to platelets. It did not require serum complement or factor VIII-related antigen for binding to platelets. Drug-dependent binding of antibody to platelets was saturation-dependent. Non-drug-dependent antiplatelet antibody of two patients (one with quinidine-induced thrombocytopenia and the other with acetaminophen-induced thrombocytopenia) reacted with autologous platelets as well as with homologous platelets, indicating that they were autoantibodies. Both autoantibodies had disappeared when their sera were tested 23 and 138 days, respectively, after withdrawal of their initial positive sera. Non-drug-dependent antiplatelet antibody binding could be demonstrated with the F(ab')2 fragment of the purified IgG of the serum of the second patient with quinidine DITP, who did not have detectable alloantibodies against HLA. None of the four patients with non-drug- dependent antiplatelet antibody had a past or present history of autoimmune thrombocytopenic purpura.

scholarly journals Cumulative experience with a simplified solid-phase radioimmunoassay for the detection of bound antiplatelet IgG, serum auto-, allo-, and drug-dependent antibodies

Blood ◽  
1982 ◽  
Vol 60 (4) ◽  
pp. 807-813 ◽  
Author(s):  
D Faig ◽  
S Karpatkin
Keyword(s):  
Solid Phase ◽  
Protein A ◽  
Serum Antibody ◽  
Antibody Detection ◽  
Staphylococcal Protein A ◽  
Antiplatelet Antibody ◽  
Microtiter Plates ◽  
Antibody Titers ◽  
Solid Phase Radioimmunoassay ◽  
Drug Dependent

Abstract A simplified, sensitive, solid-phase radioimmunoassay employing 125I- staphylococcal protein A has been developed that is capable of detecting bound antiplatelet IgG as well as serum auto-, allo-, and drug-dependent antiplatelet antibodies. The simplified assay employs a ratio of test over control platelet counts per minute (cpm) for detection of positive results. All reagents are commercially available. The assay can be performed with as little as 10(6) washed platelets (10 microliters of whole blood) that have been stored for as long as 8 wk at 4 degrees C in microtiter plates. The assay time, employing stored platelets, is 4 hr. Bound platelet IgG is positive in 93% of 46 thrombocytopenic patients with autoimmune disease and correlates inversely with their platelet count, r = -0.65, p less than 0.001. The ability of this assay to detect serum antibody was studied with a rabbit anti-human platelet antibody capable of giving optimal immunoprecipitation with solubilized platelet membranes at a tier of 1:10. The present assay increases the sensitivity of antibody detection 256-fold to a titer of 1:2560. Human serum antiplatelet membrane antibody was positive in 2 of 2 patients with anti-PLA-1 antibody (titers of 1:256 and greater than 1:64); 7 of 12 multiply transfused patients who were refractory to platelet transfusion (2 had titers of greater than 1:256 and greater than 1:32); 5 of 19 patients with autoimmune thrombocytopenic purpura (2 had titers of 1:64 and 1:32); and 10 of 14 patients with clinical histories of drug-dependent antiplatelet antibody (2 had titers of 1:1280 for quinidine and 1:384 for phenazopyridine).

scholarly journals Cumulative experience with a simplified solid-phase radioimmunoassay for the detection of bound antiplatelet IgG, serum auto-, allo-, and drug-dependent antibodies

Blood ◽  
1982 ◽  
Vol 60 (4) ◽  
pp. 807-813
Author(s):  
D Faig ◽  
S Karpatkin
Keyword(s):  
Solid Phase ◽  
Protein A ◽  
Serum Antibody ◽  
Antibody Detection ◽  
Staphylococcal Protein A ◽  
Antiplatelet Antibody ◽  
Microtiter Plates ◽  
Antibody Titers ◽  
Solid Phase Radioimmunoassay ◽  
Drug Dependent

A simplified, sensitive, solid-phase radioimmunoassay employing 125I- staphylococcal protein A has been developed that is capable of detecting bound antiplatelet IgG as well as serum auto-, allo-, and drug-dependent antiplatelet antibodies. The simplified assay employs a ratio of test over control platelet counts per minute (cpm) for detection of positive results. All reagents are commercially available. The assay can be performed with as little as 10(6) washed platelets (10 microliters of whole blood) that have been stored for as long as 8 wk at 4 degrees C in microtiter plates. The assay time, employing stored platelets, is 4 hr. Bound platelet IgG is positive in 93% of 46 thrombocytopenic patients with autoimmune disease and correlates inversely with their platelet count, r = -0.65, p less than 0.001. The ability of this assay to detect serum antibody was studied with a rabbit anti-human platelet antibody capable of giving optimal immunoprecipitation with solubilized platelet membranes at a tier of 1:10. The present assay increases the sensitivity of antibody detection 256-fold to a titer of 1:2560. Human serum antiplatelet membrane antibody was positive in 2 of 2 patients with anti-PLA-1 antibody (titers of 1:256 and greater than 1:64); 7 of 12 multiply transfused patients who were refractory to platelet transfusion (2 had titers of greater than 1:256 and greater than 1:32); 5 of 19 patients with autoimmune thrombocytopenic purpura (2 had titers of 1:64 and 1:32); and 10 of 14 patients with clinical histories of drug-dependent antiplatelet antibody (2 had titers of 1:1280 for quinidine and 1:384 for phenazopyridine).

A Solid Phase Radio-Immuno Assay for Platelet-Bound IgG Employing 125-I protein Extraction of 7S IgG and Identification of Its Subclasses

1979 ◽  
Author(s):  
K. Hymes ◽  
S. Shulman ◽  
P. Schur ◽  
S. Karpatkin
Keyword(s):  
Gradient Analysis ◽  
Protein Extraction ◽  
Solid Phase ◽  
Protein A ◽  
Igg Subclasses ◽  
Human Igg ◽  
Thrombocytopenic Purpura ◽  
Autoimmune Thrombocytopenic Purpura ◽  
Marker Proteins ◽  
Immuno Assay

Studies on bound anti-platelet Ab have not differentiated Ab from Ag-Ab complexes. A sensitive solid phase radio-immuno assay has been developed for IgG, which detects picogram IgG. Washed platelets are frozen, thawed, sonicated and centrifuged at 20,000 g. The supernatant is applied in serial dilution to a series of plastic wells, capable of adsorbing protein. After incubation, the wells are washed with 1% BSA in PBS, pH 7.2. Commercial rabbit anti-human IgG in 1% BSA-PBS (or specific anti-human IgG subclass) is then applied. Unbound anti-IgG is washed away and the wells treated with 125-I staphprotein A (10,000 cpm/well; 2x107 cpm/μgm). Unbound protein A is washed away and the wells assayed for radioactivity. Twenty-six of 28 thrombocytopenic patients (92%) with autoimmune thrombocytopenic purpura had IgG greater than 2 SD of controls and averaged autoimmune thrombocytopenic purpura had IgG greater than 2 SD of controls and averaged, 172±155 (SD) ng/106 platelets; 11 healthy controls, 12.3±4.9 ng; 5 ‘thrombocytopenic controls, ‘8.2±5.7 ng. The platelet count (X) correlated inversely with IgG/platelet (Y) according to the equation: log Y=-0.61 log X + 4.4; r=0.64. P<0.00l. Sucrose gradient analysis, employing 7S and 19S marker proteins, revealed platelet ‘extract IgG’ activity migrating as 7S protein, not as Ag-Ab complex. Three patients were tested for IgG subclasses: γG1, γG2, γG3 and γG4. These averaged 75±9.7, 13±6.1, 9.7±4.2 and 2.3±1.3% of total bound IgG. We conclude that a 7S immunoglobulin can be extracted from platelets which is IgG and contains all 4 IgG subclasses at a distribution found in normal plasma.

scholarly journals Experience with protein A-immunoadsorption in treatment-resistant adult immune thrombocytopenic purpura

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2237-2245 ◽  
Author(s):  
HW Snyder ◽  
SK Cochran ◽  
JP Balint ◽  
JH Bertram ◽  
A Mittelman ◽  
...  
Keyword(s):  
Side Effects ◽  
Immune Thrombocytopenic Purpura ◽  
Protein A ◽  
Staphylococcal Protein A ◽  
Treatment Resistant ◽  
Thrombocytopenic Purpura ◽  
Specific Serum ◽  
Clinical Responses ◽  
Acute Increase

Abstract Extracorporeal immunoadsorption of plasma to remove IgG and circulating immune complexes (CIC) was evaluated as a therapy for adults with treatment-resistant immune thrombocytopenic purpura (ITP). Seventy-two patients with initial platelet counts less than 50,000/microL who had failed at least two other therapies were studied. They received an average of six treatments of 0.25 to 2.0 L plasma per procedure over a 2- to 3-week period using columns of staphylococcal protein A-silica (PROSORBA immunoadsorption treatment columns; IMRE Corp, Seattle, WA). The treatments caused an acute increase in the platelet count to greater than 100,000/microL in 18 patients and to 50,000 to 100,000/microL in 15 patients. The median time to response was 2 weeks. Responses were transient (less than 1 month duration) in seven of those patients (10%), but no additional relapses were reported over a follow- up period of up to 26 months (mean of 8 months). Clinical responses were associated with significant decreases in specific serum platelet autoantibodies (including anti-glycoprotein IIb/IIIa), platelet- associated Ig, and CIC. Thirty percent of treatments were associated with transient mild to moderate side effects usually presenting as a hypersensitivity-type reaction. Continued administration of failed therapies for ITP, which always included low-dose corticosteroids (less than or equal to 30 mg/d), had no demonstrable influence on the effectiveness of immunoadsorption treatment but did depress the incidence and severity of side effects. The degree of effectiveness of protein A immunoadsorption therapy in patients with treatment-resistant ITP is promising and further controlled studies in this patient population are warranted.

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