Multiplexed immunoassay for detection of rheumatoid factors by FIDISTM technology.

Our objective was to (i) compare FIDIS Rheuma, a new multiplexed immunoassay designed for simultaneous detection of IgM class rheumatoid factors (RF) directed against Fc determinants of IgG from humans and animals, with agglutination and ELISA (conventional methods) and (ii) evaluate the clinical sensitivity and specificity of biological markers for rheumatoid arthritis (RA). To do this, FIDIS technology was employed using the Luminex system. It consists of distinct color-coded microsphere sets, a flow cytometer, and digital signal processing hardware and software. Agglutination and ELISA tests were performed with commercial kits. The study included 134 samples from RA patients and 105 from healthy blood donors. For human specificity, we compared FIDIS with latex agglutination and ELISA. Relative sensitivities were 98.9% and 88.5% and specificities were 90.2% and 94.6%, respectively. For animal specificity, we compared FIDIS with Waaler-Rose and ELISA. The results were 84.9% and 71.9% for the sensitivities and 97.5% and 98.4% for the specificities, respectively. Detection of IgG anti-CCP by ELISA and IgG antikeratin by immunofluorescence was also determined in order to compare their clinical sensitivity and specificity with IgM-RF, according to the method used. The results were: IgG anti-CCP 72.3%, 97.2%; IgG antikeratin 36.6%, 100%; latex agglutination 66.4%, 97.2%; Waaler-Rose 55.9%, 96.3%; FIDIS human 73.9%, 92.1%; FIDIS animal 49.2%, 97.2%; ELISA human 93.2%, 95.5%; and ELISA animal 74.6%, 91.3%. The results showed the efficiency of FIDIS with analytical performance equivalent to the conventional methods, but having the advantage of giving quantitative results (IU/mL).

Production of polyclonal antibodies towards the immunodetection of insecticide phosalone.

Hapten synthesis for the production of specific insecticide phosalone polyclonal antibodies was carried out starting from an intermediate of the phosalone synthesis, 6-chloro-2-benzoxazolone 1. Two haptens containing different spacers have been prepared: N-5-carboxypentyl-6-chloro-2-benzoxazolone 7 and N-(2-oxo-3-aza-5-carboxypentyl)-6-chloro-2-benzoxazolone 12. Each of these two haptens conjugated to bovine serum albumine (BSA) was used to immunize four rabbits. Immunoassays of phosalone were performed with ELISA using solid-phase bound hapten thyroglobulin conjugate and horseradish peroxidase labelled goat antirabbit IgG. The more sensitive response was observed when the antiserum obtained from the rabbit immunized with the hapten-BSA conjugate containing the N-2-oxo-3-aza-5-carboxypentyl spacer was in competition with the same hapten coupled to thyroglobulin. An identical response was obtained under the same conditions when using benzoxazolone instead of phosalone as competitor, showing a good recognition of the specific aromatic part of the organophosphate insecticide phosalone. Reduction of coating conjugate concentration (from 2 to 0.05 micrograms/well) and also the use of heterologous coating protein instead of homologous did improve the sensitivity, resulting in a concentration of phosalone required to inhibit binding by 50% of 2 mg/l and a detection limit of 0.02 mg/l.