Dual-radius Enhanced Latex Immunoturbidimetric Assay For The Quantitative Determination Of C-reactive Protein

Objective:C-reactive protein(CRP)is a marker of acute inflammation,which can increase rapidly within a few hours after the occurrence of inflammation.Its detection is of great significance for early diagnosis,identification,curative effect observation,and prognosis judgment of the disease.As one of the routine clinical testing items,diverse methods are currently developed for the CRP determination,however,there are still certain limitations in practical applications.Dual-radius enhanced latex immunoturbidimetric assay is a detection method based on the principle of mixing dual-radius immune microsphere.The large radius immune microsphere is used to increase the upper limit of detection,while the small radius immune microsphere is used to increase the detection sensitivity.It has been confirmed that the above method performs well in terms of stability,sensitivity,accuracy,repeatability,and anti-interference.This research aims to adjust a method for the quantitative detection of CRP,improve the analytical sensitivity and linear range of immunoreagents,verify detection capabilities,and provide a detection kit with good diagnostic performance.Methods:Covalently coupled latex microspheres with uniform texture and different radius were coated with CRP antibodies to obtain sensitized immune microsphere.Difference radius immune microspheres were screened respectively,and the best latex microsphere type,antibody concentration,cross-linking agent concentration,and immune microsphere concentration were determined through the results of the standard curve,linear fitting curve,and residual graph.According to the above screening results,detection kits were assembled to ensure that the final mixing system presented the best response state of immune microsphere with different radius,thus completing the quantitative detection of CRP.To improve the detection sensitivity and linear range,the double-radius immune microsphere reaction system was optimized,and the best sample diluent(R1),immune microsphere(R2),and sample volume were screened in this study.Automatic biochemical analyzer was used to evaluate the detection limit,precision,linear range,accuracy,anti-interference ability,and stability of the detection kit.Then,clinical serum samples were collected to compare the detection capabilities between commercial kits and self-developed kits and verify the detection capabilities of self-developed kits.Results:This study successfully established the dual-radius enhanced latex immunoturbidimetric assay.The best response of 70 nm latex microspheres was shown when the EDC/NHS was 1.5 mg/ml,the monoclonal antibody was 2.0 mg/ml,and the absorbance of the final sensitized immune microsphere was 1.5.In addition,the best response of 200 nm latex microspheres was shown when the EDC/NHS was 1.0 mg/ml,the polyclonal antibody was 1.4 mg/ml,and the final concentration of sensitized immune microsphere was 1.5.The method established in this study was applied in fully automatic biochemical analyzer,so we adjusted the detection parameters.After optimizing the conditions of the above mixing system,it was finally determined that the volume of R1 was 100μl,the volume of R2 was 100μl,and the sample volume was2μl.Then,performance analysis was carried out to evaluate the assembled kit.Finally,we got the limit of blank of 0.10 mg/L,the limit of quantification of 0.50 mg/L,the linear range of 0.50～320 mg/L,the recovery rate of 100.31%～106.13%,and the average recovery rate of 102.94%.All within-run,between-run,and within-laboratory coefficient of variation of samples with high,medium,and low concentrations were less than 10%.Besides,204μmol/L bilirubin,6 mmol/L triacylglycerol,10 g/L hemoglobin,and 32 mg/dl ascorbic acid without significant interference in blood samples.The stability of the reagent can be maintained for more than 14 days after opening the bottle for the first time and the storage stability can be maintained for more than 12 months.Self-developed detection kits and commercial detection kits were used to quantitatively detect 40 clinical blood samples at the same time,and the final linear fitting r~2 was 0.99.Therefore,we have reason to believe that the self-developed detection kit can replace commercial detection kits to a certain extent.Conclusions:In this study,we have successfully innovated a CRP detection kit based on dual-radius enhanced latex immunoturbidimetric assay,which has been verified to meet clinical requirements and can successfully assemble an automatic biochemical analyzer.