The Development Of Rheumatoid Factor Kit

Rheumatoid Arthritis (RA) is a common rheumatic disease, the prevalence rate of it is about 0.4% to 1.0%, if the disease can not be diagnosed or treated in time, it often cause joint deformities, which will seriously affect the ability to work and daily life, after one to three years. Increasing its rate of early diagnosis is the doctors concern. Rheumatoid factor (RF) is one of the most important bases for the diagnosis of rheumatoid arthritis, the fast and accurate detection of RF is very valuable to the diagnosis and prognosis of RA. There are kinds of methods to measure RF: latex agglutination, ELISA (enzyme linked immunosorbent assay), imnunoturbidimetry(nephelometryand and tronsmisson turbidimetry and immunolatex turbimetry). Latex agglutination of RF is a classical method, but it's only a semi-quantitative determination, and can only through IgM-RF. The last two methods can carry out quantitative determination of the RF, ELISA method can determine the RF and its subtypes. According to the recent development of automation equipment, scattering turbidity and transmission method has been used a lot in clinical diagnosis. Most of the current reagents are imported, they are very expensive.The theme of this research is to study the latex Turbidimetry volume of RF, and develop the domestic intellectual property rights RF kit for clinical diagnosis of low-cost, high value clinical diagnostic reagentsThe modest size of PEG, uniform particles of polystyrene microspheres,were chosen and pre-treated for the preparation of working microsphere. The human immunoglobulin IgG was exstracted and purified by ammonium sulfate precipitation and affinity chromatography. The immuno-mirospheres were obtained by cross linking the IgG to working microspheres. The serials of concentration of PEG6000 were added into the antigen-antibody reaction system, and the optimal concentration of accelerating agent(PEG6000) was determined. The optimal reaction time, reaction temperature,the quantity of application of sample and test wave length were determined by orthogonal experiment. the standard curve was made and the linearity range was determined. The coefficient of variability of inter-batch and intra-batch were checked by repeatability test, and the systematic error was checked by recovery test. The reference range was established. The sensitivity and specificity of the reagents were test by testing of clinical samples.Highly purified human immunoglobulin IgG was obtained successfully. The desired immuno-microspheres were successfully prepared. The optimal concentration of PEG6000 was 3.8%, and the optimal concentration of latex was 0.3%; the optimized quantity of application of sample was 10μl,the detection wave length was 500nm, the total reaction time was 15 minutes. The coefficient of variability of intra-batches and inter-batches was 1.80-4.33% and 7.53-14.14%, respectively. The recovery rate was 94.2-106.5%.Detection sensitivity was 81.5% and specificity was 69.0%, youden index was 50.5%. The lowest detective sensitivity was 3IU/ml,and the assured stable experiment reagent time was 12 monthes at 4℃refrigerator.The latex Turbidimetry rheumatoid factor detecting kits was successfully developed, which providing an alternative selection for the dection of rheumatoidfactor in clinical laboratory.