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The unexplained occurrence of thrombocytopenia in cases of Gramnegative sepsis in man led us, in the light of animal experiments indicating the blood platelet as the target cell for endotoxin, to examine the effect of Salmonella enteritidis lipopolysaccharide B on human platelets. Human platelets were separated from a coat of plasma proteins by Sepharose 2B filtration or by a combined procedure of albumin gradient and Sepharose 2B filtration. The action of endotoxin on human platelets resulted in membrane changes manifested by dose-dependent release of [3H]serotonin and adenine nucleotides. Cytoplasmic marker, lactic dehydrogenase, and lysosomal marker, beta glucuronidase, were retained indicating that the release reaction was selective. Release of [3H]serotonin was specific for endotoxin since other particulates, zymosan and erythrocyte stroma, were without effect. Endotoxin, added to gel-filtered human platelets, induced a significant evolution of platelet factor 3 procoagulant activity. Preincubation of endotoxin with a membrane-rich homogenate of human platelets inhibited its labilizing effect on human platelets thus suggesting an interaction between endotoxin and the platelet membrane itself. Other plausible factors in this interaction [fibrinogen, adenine nucleotides, thrombin, sialic acid residues, and IgG] were eliminated on the basis of a series of control experiments. From the negligible effect of aspirin and indomethacin, we may infer that the interaction of endotoxin with platelets does not depend on the platelet prostaglandin synthesis pathway. The direct interaction of endotoxin with the human platelet membrane comprises a new mechanism which may help to clarify the pathogenesis of vascular and haemostatic disorders accompanying bloodstream infections due to Gram-negative bacteria.
A radioimmunometric antibody-binding assay was developed with the use of 125I-labeled protein A of Staphylococcus aureus (SpA) for the evaluation of xenoantisera to human melanoma-associated antigens. Antisera were produced in New Zealand male albino rabbits by the injection of cultured human melanoma cells or soluble, partially purified melanoma-associated antigens isolated from these cells. Xenoantisera were rendered operationally specific for melanoma-associated antigens by absorption with human red cells and cultured lymphoblasts. The methodologic parameters and the quantitative relationships among xenoantisera, cultured melanoma target cells, and 125I-labeled SpA and their effect on the measurement of xenoantibody binding were critically evaluated. Data indicated the usefulness of the radioimmunometric assay in monitoring the efficacy of absorption and in characterizing the specificity of xenoantisera to melanoma-associated antigens. The radioimmunometric binding assay when modified and used as a binding inhibition assay was effective in the assessment of the serologic activity of soluble melanoma-associated antigens and thus may be used to monitor the progress of antigen purification.