Establishment And Evaluation Of The Magnetic Particle-based Chemiluminescence Immunoassay For Pepsinogen I

Pepsinogen I（PGI）is an important marker of gastric mucosal inflammation,and its level monitoring plays an indispensable role in the evaluation of gastric function.It is of great significance to establish a rapid,specific and sensitive detection method for the prevention,control and treatment of gastric diseases.In this study,a chemiluminescence method for detecting PGI was established based on magnetic particles.The research work and results were as follows:1.Screening of antibody raw materials.Firstly,the optimal antibody materials were screened by antibody valence.Results showed that the titers of 7 antibody strains（B1,B2,C2,D1,D2,E1 and E2）could reach 1×10^-6 mg/mL.Secondly,antibody pairing screening was carried out.The results showed that 8 pairs of antibodies with high signal-noise ratio were screened preliminarily by chessboard titration based on chemiluminescence platform.Finally,clinical samples were used to screen the optimal antibody pairs.Results showed that when E1was used as the coated antibody and E2 as the labeled antibody,the correlation between the test results and the control kit was the highest（R²=0.9810）.This antibody pairs were finally selected to establish and optimize the chemiluminescence platform.2.Establishment of magnetic particle chemiluminescence detection method of PGI.Magnetic particles were coated with antibodies,Tosyl magnetic particles were selected as magnetic particles.The size distribution of magnetic particles was uniform and the dispersion was good before and after coated with antibodies.When the concentration of antibody was 200μg/mL,the stability of the immune magnetic particles was the best,and the hydration particle size was the smallest.The optimal coating process was as follows:the coating temperature was37℃,the coating buffer pH was 7.5,the coating time was 20 h,and the sealant was 1%BSA.When alkaline phosphatase was used to label another antibody,the ratio of alkaline phosphatase to antibody was 5:1,the ratio of cross-linking agent to antibody was 10:1,the pH of labeling buffer was 7.5,the signal-to-noise ratio was the highest.The buffer fluid system was optimized,in which the magnetic particle antibody diluent was Tris buffer,pH=7.5,the thermal acceleration stability was the best at 37℃,and the bias was-2.98%.When the alkaline phosphatase antibody diluent was MES buffer solution,pH=7.5,and the stabilizer was 4%glycine,the thermal acceleration stability was the best at 37℃,and the bias was-5.56%.The reaction system was optimized.The optimal reaction parameters were as follows:reaction mode was one-step double antibody layer,dilution ratio of magnetic particle antibody was 1:25,dilution ratio of alkaline phosphatase antibody was 1:1000,incubation time was 5 min,sample size was 20μL.3.Performance evaluation of PGI chemiluminescence detection method.The linear range of the method was 0～200 ng/mL（R²=0.9991）and the sensitivity was 0.048 ng/mL.The method had wide linear range and high sensitivity.The intra-analysis coefficient of variation were 3.26%and 2.89%,and inter-analysis coefficient of variation were 4.83%and 3.39%,respectively.The CV_S of variation were all less than 5%,indicating good precision.The average recovery was96.81%with good accuracy.In the high-concentration dose test,the nonlinearity of the luminescence values appeared at 400 ng/mL and significantly decreased at 1600 ng/mL,but these dose levels were far beyond the clinical dynamic range and did not affect clinical application.The addition of bilirubin 0.22 mg/mL,triglyceride 33 mg/mL,hemoglobin 5mg/mL and total protein 0.22 mg/mL did not interfere with the detection of PGI,and there was no cross reaction with PGII.The method had strong anti-interference ability and good specificity.The kit was placed at 37℃ for 1,4 and 7 days with good stability.It was predicted that the kit could be stored for 12 months at 2～8℃ with high stability of the method.4.Clinical application evaluation of PGI chemiluminescence detection method.The correlation between the method and Abbott direct chemiluminescence method was analyzed using batch clinical samples.The regression equation was y=1.008x+0.272,and the correlation coefficient R²=0.9981.The correlation between the two methods was good.Bland-Altman consistency analysis showed that the consistency between the two methods reached 95.84%,and there was no difference between the two methods.For different sample types,the test results of EDTA-K₂ plasma,heparin sodium plasma and heparin lithium plasma showed good correlation with serum detection without significant difference.Therefore,this method has strong clinical applicability.The preservation conditions of samples were explored.Results showed that serum samples could not be stored for more than 7 days at 2～8℃,and could be stored for 21 days at-20℃ and-80℃.The freeze-thaw frequency could be controlled within 3times to meet clinical testing requirements.In conclusion,the magnetic particle chemiluminescence analysis method constructed in this study can be used for rapid clinical detection of PGI,and provide a powerful tool for large-scale screening of gastric function health.