| Heparin binding protein(HBP)is a novel inflammatory marker,which is significantly associated with a variety of infectious diseases.Therefore,HBP has been used as a diagnostic biomarker for a variety of diseases clinically.As a result,the establishment of a convenient,rapid,specific and sensitive HBP detection method is of great significance to improve the diagnostic efficiency of infectious diseases,and the high-affinity HBP antibody determines the sensitivity and accuracy of the HBP detection.Hence,in this study,monoclonal antibodies with high affinity were prepared based on hybridoma technology,and a fluorescence quantitative immunochromatography and a magnetic particles chemiluminescence immunoassay for HBP were established with monoclonal antibodies.The main research results are as follows:(1)BALB/C mice were immunized subcutaneously with HBP antigen for 4 times,after immunization,the antiserum titer of the mice was measured via ELISA,which reached1:1×105.7 hybridoma cell lines that stably secreted anti-HBP monoclonal antibodies were obtained by continuous subclonal screening and culture.The titer of ascites reached1:1×106.After purifying by affinity purification by magnetic separation,7 antibodies were obtained and the titer of antibodies by ELISA were 1:1×105 mg/m L.SDS-PAGE results showed that the antibody purity reached 90%.In addition,the affinity of the antibodies were less than 10-9 m,indicating their high affinity.(2)Preliminarily screen the HBP paired antibodies by molecular interaction instrument,and confirm that 22C5 is the coating antibody and 30G11 is the labeled antibody by fluorescence immunochromatography.Optimized the process of fluorescent immune layer by T/C value.The optimized process is as follows:The concentration of sheep-anti-rabbit Ig G on line C was 0.5 mg/m L,the width of spraying film on line T was 1.0 mm,the concentration of HBP-coated antibody was 2 mg/m L,the concentration of HBP labeled-antibody was 0.4mg/m L,the latex dilution ratio of immunofluorescent microbeads was 20%,the reaction time was 20 min,and the sample dilution ratio was 1:1.The performance was evaluated by the developed HBP fluorescence quantitative immunochromatographic kit.The results dispalyed the detection limit of the system was 3.37ng/m L,and the detection range was 3.37-1440 ng/m L.The recoveries at 7.5,30,120 ng/m L were 98.85%,98.18% and 98.81%,respectively.The coefficients of variation of 10 repeats at three concentration were 2.97%,3.80% and 5.13%,while the intra-batch and inter-batch differences CV were all less than 5%,indicating high accuracy and precision.There was no cross with inflammatory analogs such as procalcitonin,C-reactive protein and serum amyloid A,and the addition of plasma interferers such as bilirubin,triglyceride,hemoglobin and total protein had no interference in the detection,indicating that the assay has a good specificity.(3)HBP magnetic particle chemiluminescence detetion was constructed by using 22C5 as m Ab-MPs and 30G11 as AP-m Abs.The optimum reaction parameters were as follows: the reaction mode was one-step method,the dilution ratio of m Ab-MPs was 1:50,the dilution ratio of AP-m Abs was 1:200,the incubation time was 5 min,and the sample volume was 5 μL.The performance of the HBP chemiluminescence detection method was evaluated.The detection limit of the method was 4.74ng/m L,and the detection range was 4.74～1440ng/m L.The recoveries of samples at 7.5,30,120 ng/m L were 100.78%,99.03% and 98.58%,respectively,while the coefficient of variation of 10 replicates at three concentration points were 1.54%,1.92% and 2.49% and the intra-batch and inter-batch CVs were all less than 5%,indicating high accuracy and precision.There was no cross with inflammatory analogs such as procalcitonin,C-reactive protein and serum amyloid A,and the addition of plasma interferers such as bilirubin,triglyceride,hemoglobin and total protein had no interference in the detection,indicating that the assay has a good specificity.Compared with the two HBP detection,fluorescence immunochromatography kits require simple equipment,so it is more suitable for screening in a small area such as small hospitals or community hospitals.Magnetic particle chemiluminescence kits require large instruments,shorter reaction time and small sample size,which make them more suitable for large-scale screening. |
