A procedure for improving the specificity of enzyme-linked immunosorbent assays (ELISA) was devised, based on addition of antigen-specific or non-immune immunoglobulins to the citrated plasma sample and defining the difference in assay response between these two mixtures as the antigen-specific part of the response. When applied to measurement of tissue plasminogen activator (t-PA; EC 3.4.21.31) antigen in plasma, this procedure resulted in elimination of the overestimates obtained in a large proportion (10-20%) of patients' samples when assayed according to the conventional ELISA technique. Basal t-PA concentrations in plasma were found to be highly age-dependent, normal values being about 3 micrograms/L for adults near 30 years of age and about 10 micrograms/L for those over 60. Patients with gallbladder stone disease had increased mass concentrations of t-PA in plasma, even when corrected for the age effect; patients with multi-infarct dementia did not.
Immunization of a goat with partially reduced and S-carboxymethylated plasmin B-chain-alpha 2-antiplasmin complex resulted in a large population of antibodies with rather high specificity towards the complex. These antibodies do not react with plasminogen or plasmin in complex with other inhibitors than alpha 2-antiplasmin. However, they react fully with native alpha 2-antiplasmin, but a 200-fold higher concentration, as compared to plasmin-alpha 2-antiplasmin complex, is needed to obtain a similar displacement curve in a double-antibody radioimmunoassay. The results indicate a conformational change in the vicinity of the reactive site in alpha 2-antiplasmin, as a result of complex formation with plasmin. A method for determination of plasmin-alpha 2-antiplasmin complex in plasma has been elaborated using the described radioimmunoassay. About 1.5 mg plasmin-alpha 2-antiplasmin complex/l can be detected, which equals the condition when about 1% of the alpha 2-antiplasmin in plasma is in complex with plasmin. In normal individuals plasmin-alpha 2-antiplasmin complex could be detected only rarely. However, patient with acute processes, as evidenced by high fibrinogen levels, surgical patients postoperatively or patients with malignancy have often detectable levels.
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