Preparation Of Maackia Amurensis Lectin Ⅱ Tumor Detection Reagent And Its Clinical Application

Cancer is one of the most dreaded diseases in the 21st century,and its incidence and lethality are still further increasing,and now cancer has become the main cause of death from diseases in China.Nowadays,domestic cancer diagnosis methods are relatively single,mainly imaging tests,which are costly and complicated,with low success rate and radiological hazards in detecting early tumors.The study of abnormal glycoproteins as early diagnostic markers of tumors has become a hot topic,and lectins can specifically bind glycoproteins and have been widely used in tumor diagnosis and prognosis.In this thesis,an in vitro diagnostic reagent was prepared and evaluated by chemiluminescence using the property of recognition of glycoprotein terminal sialic acid Siaα-2,3Gal by Korean Sophora lectin Ⅱ(Maackia Amurensis Lectin Ⅱ,MAL Ⅱ).The main contents are as follows:(1)In the study of the extraction and isolation and purification process of Korean Sophora agglutinin Ⅱ,MAL Ⅱ was extracted from Korean Sophora as raw material using single-factor test and star point design effect surface method,and its preliminary isolation and purification were carried out.It was determined that the highest specific viability of Korean acacia lectin was achieved under the conditions of extraction time of 6 h,material-liquid ratio of 1:12 and pH of 8.0.The crude extract was precipitated by 40%-60%ammonium sulfate gradation and subjected to DEAE-52 anion exchange chromatography and dextran gel chromatography;MALII was successfully separated using a specific affinity sorbent fetal globulin agarose 4B gel column.The extraction and purification process is simple and the resulting product is pure and can be used for production.(2)In the study of the preparation process of Korean Sophora agglutinin Ⅱ tumor detection reagent,biotin was combined with lectin,and the optimal dosage of biotin was 0.5 mg and the optimal reaction time was 6 h by single factor.;the optimal amount of luminescent substrate HRP was determined as 100 μL and the reaction kinetics was analyzed;fetal bovine serum was screened as the calibrator,and the calibrator and quality control were lyophilized with fixed values;The concentrations of lectin and HRP-SA were optimized to be 1 μg/mL and 2μg/mL,respectively,and the dilution of specimens was 3000 and the specimens were stable within two days;the tumor detection reagents were successfully prepared and the important parameters of the reagents were optimized to achieve the best detection effect.(3)In the performance evaluation determination of Korean Sophora agglutinin Ⅱ tumor detection reagent,precision,correctness,blank limit,linear interval and anti-interference tests were performed,and the test results were:repeatability and precision CV were less than 15%;correctness(recovery)was within 85%～115%;blank limit was established at 185.04 μg/mL;validated linear interval was good with correlation coefficient greater than 0.99;the endogenous interfering substances bilirubin,hemoglobin,triglyceride and celiac particles had no effect on the test results.In conclusion,the performance of the kit prepared in this experiment is stable and meets the requirements of in vitro diagnostic reagents,which is expected to be applied in production after further adjustment.(4)In the preliminary clinical application of Korean Sophora agglutinin Ⅱ tumor detection reagent,ten common clinical cancer species and healthy population serum samples were measured,and the reference interval<4900 U/mL was determined for healthy individuals;ROC curve analysis yielded a sensitivity of 83.3%,specificity of 86.3%,AUC=0.90(95%confidence interval 0.87～0.93)for ovarian cancer group,P<0.001,and the diagnostic values of the tests were all statistically significant;and the methodological comparisons with commercially available similar products showed good correlation and consistency,high accuracy,and can be applied to clinical auxiliary diagnosis.In conclusion,the assay established in this study not only has high sensitivity and accuracy of measurement value,but also the reagent performance meets the research requirements,so it has a good market prospect in the auxiliary diagnosis of malignant tumors.