Establishment Of A Method For Combined Detection Of Serum Amyloid A And C-reactive Protein For Fluorescent Immunochromatography

BackgroundThe infections of bacterial and viral are the most common infectious diseases,which poses a seriously threat to human health.These two infectious diseases have insidious onset and lack of specificities in clinical symptoms and signs,but have a significant difference in clinical medication.Therefore,it is particularly important to distinguish bacterial from viral infection by means of detection effectively before treatment.At present,C-reactive protein(CRP)and serum amyloid A(SAA)were selected as clinical identification indicators for identifying bacterial or viral infection in some large and medium-sized hospitals.Chemiluminescence and immunoturbidimetry are the main detection methods.However,the limitations of these two methods are not only in SAA and CRP could not be detected at the same time,but also have a difficult to be applied into community clinics in a large scale due to the needs of equipment and professional operation.Therefore,this study intends to establish a method for quantitative detection of serum SAA and CRP simultaneously by using time-resolved fluorescence immunochromatography,which aims to enable the institutions of the basic,community and large hospitals to do rapid bedside detection and small sample detection.ObjectiveThe aim of this study is to establish a quantitative detection method for simultaneous detection of CRP and SAA in serum by time-resolved fluorescence immunochromatography with the principle of double antibody sandwich method,which can meet the needs of rapid detection of bedside in grassroots or community medical institutions.Early screening of bacterial and viral infections and selection of drugs provide a convenient and accurate detection technique.Methods1.Screening of paired antibodies: Based on the fluorescence immunochromatographic technology platform established in our laboratory,the antibodies encapsulated by quality control line,SAA and CRP ligands were screened respectively.1.1 Screening of quality control line coated antibodiy:The rabbit IgG polyclonal antibody purified in laboratory was labeled by routine method.Four strains of mouse anti-rabbit IgG monoclonal antibodies(SKT 28,SKT 29,SKT 30 and SKT 31)were used as coatings for paired screening.The quality control line coated antibodiy was screened by observing the fluorescence intensity of C line.1.2 Screening of SAA paired antibody:Based on linear correlation,paired screening of three mouse anti-human SAA monoclonal antibodies numbered MS 2201,MS 2202 and MS 2203 was carried out by detecting a series of SAA indoor reference materials.1.3 Screening of CRP paired antibody:By detecting a series of CRP indoor reference materials and based on the linear correlation,three strains of mouse anti-human CRP monoclonal antibodies(MC 003,MC 004 and MC 005)were screened pairwise.2.Establishment of SAA fluorescence immunochromatographic assay method: The microsphere mats and coatings required for this study were determined by screening glass fiber cotton and nitrocellulose membranes;to determine the optimal preparation process of test strip by studying the optimum proportion of EDC and NHS,the optimum pH value of buffer in the process of microsphere labeling,the formulation of microsphere protective solution,the composition of sample diluent,and to investigate the linearity,accuracy,precision and other properties of test strip.3.Establishment of a method for combined detection of Serum amyloid A and C-reactive protein for Fluorescent Immunochromatography: The optimal mixing ratio of three markers(fluorescent microsphere-MS 2203,fluorescent microsphere-MC 004,fluorescent microsphere-rabbit IgG polyclonal antibody).The optimal labeling amount and coating amount of SAA and CRP paired antibodies were studied to determine the preparation process of fluorescence immunochromatographic strips for SAA and CRP,and preliminary establishment of the combined detection methods of SAA and CRP fluorescence immunochromatography.4.Performance evaluation of SAA and CRP fluorescence immunochromatography test strips: Linearity,precision,accuracy,stability and cross-reaction of SAA and CRP fluorescence immunochromatography test strips developed in this study were evaluated,and methodological comparisons were performed with Siemens BN ProSpec? specific protein analysis system.Results1.SKT 30 mouse anti-rabbit IgG monoclonal antibody was selected as quality control line coated antibody;MS 2203(label)and MS 2201(coating)as the SAA pairing antibody of this study;MC 004(label)and MC 003(coating)as CRP pairing antibody in this study.2.A fluorescent immunochromatographic method for quantitative detection of SAA in serum was established.The suitable microsphere pads and coating film were preliminarily screened out,and the optimum activator ratio and pH of the labeling buffer were determined,and the optimum formulation of microsphere protective solution with the best stability was selected.The test strips made by this process have the best linear correlation at 0.78~200 mg/L,and the intra-assay coefficient of variation is less than 10%,and the inter-assay coefficient of variation is less than 15%,which meets the precision requirements.The detection of high,medium and low value samples all meet the requirements of recovery rate of 85%~115%.SAA content in 80 clinical serum samples was detected simultaneously with the Siemens BN ProSpec? specific protein analysis system(scattering nephelometry)showed a good correlation between the two methods.3.A time-resolved fluorescence immunochromatographic method for simultaneous quantitative detection of SAA and CRP in serum was established.The optimum microsphere mixing ratio of three markers(fluorescent microsphere-MS 2203,fluorescent microsphere-MC 004 and fluorescent microsphere-rabbit IgG polyclonal antibody)was determined.The optimum labeling amount of SAA and CRP monoclonal antibodies was 40 ?g when 10 ?L microspheres were activated.On this basis,the optimum coating amount of SAA and CRP monoclonal antibodies was determined to be 2 mg/mL and 1.5 mg/mL,respectively,by detecting linear range and linear correlation.4.The performance of SAA and CRP fluorescence immunochromatography test strips was evaluated.The linear ranges of SAA and CRP were 0.78~200 mg/L,and the correlation was good.The intra-batch and inter-batch variation coefficients of SAA and CRP were less than 10% and 15% respectively,which met the precision requirements.The recoveries of SAA and CRP were between 85% and 115%,which met the recovery requirements.Common interfering substances(hemoglobin,triglyceride,bilirubin)in serum had no effect on the detection of SAA and CRP.The specificity of test strip was good,and there was no cross reaction and no interference when SAA and CRP were detected separately.They could be stored at 37? for 21 days with good stability.The results of methodological comparison with Siemens BN ProSpec? specific protein analysis system(scattering turbidimetry)showed that the two detection methods have good correlation in detecting SAA and CRP.Conclusions1.In this study,the formulation of microsphere protective solution was determined,the test strip can be stored stably for 21 days in accelerated damage test at 37?.2.Using of rabbit Ig G polyclonal antibody labeling and mouse anti-rabbit Ig G monoclonal antibody coating as quality control line pairing antibody,solved the difficult problem of simultaneous detection of two markers on the same test strip.3.A time-resolved fluorescence immunochromatographic method for combined detection of SAA and CRP was preliminarily established,which provided a sensitive,specific,convenient and accurate detection technology for early screening and auxiliary diagnosis of bacterial and viral infections,and also provided a reference for joint detection of other markers.