The Establishment And Application Of The Fluorescence Quantitative Immunochromatography Method For Canine C-reactive Protein

C-reactive protein(CRP),a major acute-phase protein,is crucial to disease diagnosis and control.CRP can rise rapidly with high sensitivity during the early stages of inflammatory diseases,and thus making it a suitable systemic inflammatory biomaker of diseases of the canine.It is of great significance to develop a rapid and sensitive method for the diagnosis of diseases in pet canine.Hence,in this study we successfully established a fluorescence quantitative immunochromatographic assay of canine CRP(CCRP)based on the principles of immunochromatography.In this research,CCRP recombinant protein was used as the immunogen and BALB/C mice were immunized by subcutaneous injection for several times.The antiserum titer of mice after immunization reached 1:10~6.Using continuous subcloning and indirect ELISA,we finally selected two hybridoma cell lines from 87 cell lines secreting anti-ccrp monoclonal antibody,and named them as 2C5 and 2G8,respectively.The titer of ascites produced by intraperitoneal injection in mice was higher than 1:10~6.After purification of the monoclonal antibodies by method of ammonium sulfate precipitation,SDS-PAGE showed that the purity of prepared antibody was not significantly different from that of the control,and the titer could reach 1:1×10~5.Western-blot results showed two distinct specific binding to the CCRP antigen with good specificity.CCRP immunofluorescence microspheres were also prepared successfully in this study.The fluorescence spectrometer showed that the fluorescence intensity did not decrease significantly after the coupling of antibody with fluorescent microspheres,and the phenomenon of basic fluorescence quenching did not appear.The scanning electron microscope(SEM)showed that the fluorescence microspheres coupled with CCRP antibody didn't agglomerate and the morphology was stable and uniform.Dynamic light scattering(DLS)and Zeta potential were used to characterize the prepared CCRP immunofluorescence microspheres,and the results showed that the stability of CCRP immunofluorescence microspheres was improved under certain conditions.In our experiment,the final optimized conditions were coupling activation time of 20 min,EDC and 5 mg NHS,CCRP antibody labeling concentration of 600?g/mL,coupling time of 2 h,and the MES buffer pH was 6.0.Besides,the antibody pair was selected by fluorescent immunochromatography and the optimal test conditions were obtained by optimizing the process conditions of the test strip.The optimal strip parameters were:0.5 mg/mL of chicken IgY on line C,and 2 mg/mL of CCRP antibody on the T line.The distance between the labeled and coated antibody was 0.6cm and the volume of antibody sprayed on the T and C lines was 1.5?L/cm.The CCRP immunofluorescence microsphere dilution ratio was 15%and 1:200 for sheep anti-chicken IgY and drying of the membrane for 48 h.The dilution ratio of CCRP standard was 1:200 and the immune reaction time was 180 s.Finally,the CCRP fluorescence quantitative immunoassay kit was developed for performance evaluation and clinical application.The results showed a standard curve of y=0.909x2-0.5441x+1.0839(R~2=0.9993),the limit of detection(LODs)was 0.418 mg/L and the detection range was 0.418-600 mg/L.The average recoveries of the kit at 500 mg/L,250mg/L,and 5 mg/L were 100.21%,100.74%,and 99.79%,respectively,and the coefficient of variation were 0.60%,1.29%,and 5.46%,respectively.The coefficient of intra-assay variation was 0.68%-6.92%and the inter-assay was 0.89%-8.79%,indicating the high accuracy and precision of the kit.The interference test and analysis of accelerated damage stability showed no cross-reactivity with 9 kinds of interfering substances such as bilirubin,cholesterol and triglyceride,and no significant changes in T/C value after accelerated destruction was observed,which demonstated excellent specificity and stability.This method was applied in the detection of CCRP in clinical specimens,and the results were highly comparable to the commercially available I-CHROMA kit.Therefore,this study developed an alternative diagnostic method for the rapid and accurate detection of CCRP in clinical samples,which could be well applied to the prevention and monitoring in clinical treatment of canine diseases.