| Breast cancer is one of the most common malignant tumors among women.According to the latest survey,breast cancer ranks first in the incidence and mortality of malignant tumors among women in the world,posing a serious threat to women’s lives and health.The initial symptoms of breast cancer are not obvious,and most of the patients have advanced to the middle and advanced stage by the time they visit the doctor.Therefore,it is of great significance to develop a detection reagent to improve the early diagnosis of breast cancer and to monitor the curative effect during the treatment of the patients.Carbohydrate antigen 15-3(CA15-3)is currently recognized as a specific tumor marker in the diagnosis of breast cancer.The purpose of this project is to develop a detection kit of CA15-3 based on the Light Initiated Chemiluminescence method,and improve the performance of domestic detection of CA15-3 reagent by utilizing the characteristics of homogeneous phase,wash-free,rapid and high sensitivity of the Light Initiated Chemiluminescence,so as to reach the international first-class level,achieve import substitution,and reduce the cost expenditure of the domestic medical industry.First of all,based on light initiated chemiluminescence platform confirmed CA15-3 test kit,double antibody sandwich principle of development,then purchase for reagent from well-known raw material company to develop a pair of CA15-3 monoclonal antibody and antigen of CA15-3,will the matching antibody cross-linked respectively to luminous particles and biotin as a kit reagent 1 and reagent 2 components inside,antigen respectively diluted into six different concentration points as a calibration 1 to 6 respectively,And two high and low concentration points were used as the precision sample 1 and precision sample 2 in the kit.Through the optimization of the preparation process of the reaction system,reagent concentration,sample addition,reaction time of the kit,and other conditions,we finally determined that the buffer used for the cross-linking of the kit luminescent particles was CB buffer,with a cross-linking mass ratio of 10:0.1,NaHCO3 buffer for biotin cross-linking,and a cross-linking molecular ratio of 1:20.In addition,the final reagent 1 was used at a concentration of 50 μ g/mL,reagent 2 was used at a concentration of 1.5 μ g/mL,the sample was added at a volume of 10 μL,and the reaction time was 20 minutes.After determining the above conditions,we established a kit traceability system,and used Roche’s CA15-3 detection kit to calibrate the working calibration products prepared by us to determine the concentration of each working calibration product point.In addition,an internal set of measurement procedures is selected to coordinate with the working calibration products to assign values to the product calibration products of the kit.The above traceability system is used to ensure the reliability of the measured value of the kit in the later stage.Finally,we used clinical samples for verification.There were a total of 111 clinical samples used in this trial,and 5 outlier samples appeared,accounting for 4.5%,which exceeded the acceptable range of 2.5%as required by general requirements.Statistical analysis was performed after the exclusion of 5 outliers:the regression equation of two groups of data Passing-Bablok was y=-1.205+1.028x,and each parameter met the corresponding requirements.Bland-altman analysis results of the two groups of data showed that 5 points were distributed outside the range of ±1.96SD,accounting for 4.7%of the total cases,which was less than the 5%deviation requirement.The Kappa value of the two methods was analyzed,and the Kappa value was finally calculated as 0.89,which proved that the two methods had good consistency.Then,through further analysis of the above outliers,we found that the measured values of the 5 samples were uniformly low compared with the reference system,and we speculated that the current detection system was missing compared with the reference system.Therefore,we consider adding lectin to the existing system to fill in the leakage.We chose the peanut agglutinin with two different types of wheat germ agglutinin lectin,through to the two kinds of lectins and CA15-3 antibody reactivity test,determine the two lectins were not CA15-3 antibodies with nonspecific adsorption,another through two kinds of lectin and the reactivity of CA15-3 antigen test,select strong reactivity of wheat germ agglutinin for further research.After the above conditions were determined,wheat embryo lectin was cross-linked with biotin,and it was finally determined that the leakage effect of wheat embryo lectin on 5 outliers was significantly better than that of cross-linked with biotin.Finally,after adding the luminescent particle crosslinked wheat embryo lectin,we retested the original 111 clinical samples,and a total of 2 outliers appeared,accounting for 1.8%,meeting the acceptable range of 2.5%of the general requirements.Statistical analysis was carried out after the exclusion of 2 outliers:the regression equation of two groups of data Passing-Bablok was y=-1.244+1.043x,and each parameter met the corresponding requirements.Bland-altman analysis results of the two groups of data showed that 5 points were distributed outside the range of ±1.96SD,accounting for 4.7%of the total cases,which was less than the 5%deviation requirement.The Kappa value of the two methods was analyzed,and the Kappa value was finally calculated as 0.93,which proved that the two methods had good consistency.Finally,through the above process optimization,we confirmed that the CA15-3 detection kit with lectin reaction system developed on the Light Initiated Chemiluminescence Assay platform was highly similar to the correlation and consistency of imported reagents in the detection of clinical samples. |
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