Preparation And Clinical Application Of Elderberry Lectin Tumor Detection Reagent

In this paper,we used the principle of specific binding of elderberry agglutinin to abnormal glycoproteins of Siaα-2,6Gal structure to prepare an elderberry agglutinin tumor detection reagent and establish an assay for preliminary clinical application.The extraction process of elderberry agglutinin was optimized by single-factor test and Box-Behnken design response surface method.The optimal extraction process of elderberry agglutinin was established as follows:Tris-HCl buffer was used as the extractant,the materialliquid ratio was 1:16,the pH was 8.1,the extraction time was 2.1h,and the extraction temperature was 4℃.For further separation and purification,ammonium sulfate graded precipitation and anion exchange chromatography were used.The highest hemagglutination titer of the precipitated protein was obtained when the supernatant was 80%saturated ammonium sulfate solution;the eluent contained 0.2 mol/L NaCl solution when the column was passed,and elderberry agglutinin could be eluted down.The extraction and purification process is simple and lays the foundation for subsequent production.For the preparation of biotinylated elderberry agglutinin in the process of tumor detection reagent preparation,the luminescence values were tested for different amounts of biotin and different linking reaction times.The conditions were optimal when the biotin dosage was 0.5 mg and the linking reaction time was 6 h.The calibration materials were screened,and the luminescence test and linearity comparison method were used.The screening results showed that fetal bovine serum had the highest luminescence and linearity values.The assay conditions were optimized for optimal assay performance.The optimal ratio of biotinylated elderberry agglutinin to horseradish peroxidase was established at 1:1,and the optimal sample dilution of 10,000× was established,with the sample content stable within 2 days.We successfully prepared the elderberry agglutinin tumor detection reagent and established a feasible detection method,which is simple and convenient to operate,with few steps,stable process and good feasibility.The performance evaluation of the tumor detection reagents was performed mainly in terms of precision,correctness,blank limit,linearity,and anti-interference.The coefficients of variation of the precision of the assay reagents were all less than 15%,the recoveries of the correctness experiments were all between 85%and 105%,the blank limit was 207 U/mL,the linearity was greater than 0.99,and the measurement results were not affected by interfering substances when 500 mg/dl hemoglobin,40 mg/dl bilirubin,and 1000 mg/dl triglyceride were added.The precision,correctness,linearity and blank detection limit of the prepared tumor detection reagents were in accordance with the quality standard.In a clinical study of the established assay kit detection method,the highest level of abnormal glycoconjugate glycoprotein with Siaα-2,6Gal structure in the serum of early lung cancer patients was 13188±4124 U/mL when measured in healthy population and ten clinically common cancer patients;in a ROC curve analysis of the population of early lung cancer tumor patients,the area under the curve was 0.915 and Comparing the sensitivity and specificity of the elder tomb agglutinin assay prepared in this paper with that of the TAP assay,it was found that the assay and the detection method developed in this paper had high sensitivity and specificity for lung cancer,which laid a preliminary foundation for the application of the elder tomb agglutinin tumor detection reagent for lung cancer in the clinic.