Technique / Immunology / RIA radioimmunoassay

OBJECTIVE: To compare test characteristics of the Farr radioimmunoassay and an automated fluorescence immunoassay (ELIA dsDNA test) for the diagnosis of systemic lupus erythematosus (SLE). METHODS: A cross sectional study comprising 440 samples from 440 patients, sent to the laboratory over a three month period for anti-dsDNA testing. Chart review was performed, blinded for test results, to count for each patient the number of American College of Rheumatology criteria for the classification of SLE that were fulfilled. At least four criteria were met by 248 (56%) patients (SLE), one to three criteria by 77 (18%) (lupus-like disease, LLD), and no criterion by 115 (26%) (non-SLE/non-LLD). Results from serum samples from the non-SLE/non-LLD and SLE groups were used to calculate receiver operating characteristic curves. RESULTS: For the Farr assay, specificities of 95% and 99% corresponded to sensitivities of 72% and 56% respectively. For the ELIA dsDNA test these levels of specificity corresponded to sensitivities of 44% and 17% respectively. CONCLUSIONS: The Farr radioimmunoassay is superior to the ELIA dsDNA test for identifying patients with SLE.

For more than 25 years 12-S-hydroxyheptadecatrienoic acid (HHT) has been known to be a product of thromboxanesynthase (TX-Syn) when synthesized with thromboxane A2 (TXA2). Although there are some hints that HHT has anti-aggregatory effects, to date, it has neither been shown to have any specific pathological relevance nor is there much information about its physiological role. This review presents a summary of the physicochemical properties of HHT, its chemical synthesis, the impact of various biological systems on its enzymatic and non-enzymatic production and its physiological function and metabolization, as well as a survey of the most important methods for analyzing this unsaturated hydroxy-fatty acid. Due to the low antibody-raising potency expected in HHT, no immunological system for HHT quantification has been developed so far. In our report we present the development and validation of a sensitive and reliable, competitive radioimmunoassay (RIA) suitable for the quantitative determination of HHT. HHT was produced by an enhanced enzymatic method using platelet-rich plasma (PRP). With an effective and modified liquid-liquid and solid-phase extraction method we were able to produce highly purified HHT (97% purity by GC/MS) in sub-milligram ranges. These fractions were used for the synthesis of BSA-antigen-conjugates and for immunization of rabbits. The tritiated tracer was synthesized using prostaglandin H synthase for the production of prostaglandin H2 (PGH2) followed by an aqueous reaction with Fe(2+)-solution to rear-range PGH2 to HHT. The dynamic range of the assay was from 30-400 pg/tube, with a sensitivity of approximately 40 pg/tube. The evaluation of the assay was performed by a HPLC-RIA method as well as by correlation with a quantitative HPLC method and correlation with TXB2 concentrations in a blood coagulation study. The assay may be useful for the quantification of HHT in several tissues and body fluids under various physiological conditions and may also help to understand the possible physiological role of HHT in biological processes.