Establishment Of Immunochromatographic Detection Of Human Mammoglobin In Peripheral Blood

BackgroundAs one of the most common malignant tumors in women,the incidence of breast cancer has increased year by year in recent years.At present,the use of biomarkers has become a method to assist in the diagnosis and treatment of breast cancer,in which serum biomarkers are more convenient than other types of biomarkers.human mammaglobin(hMAM)is a breast-specific member of the uterine globin gene family.Its low expression in breast epithelial cells and high expression in breast cancer has been proved to be related to early detection and metastasis of tumor.And some studies have shown that hMAM in blood can be used as an independent prognostic marker of breast cancer and has become one of the specific indicators for the detection of breast cancer micrometastasis.Therefore,detecting the content of hMAM in peripheral blood can provide a screening method for breast cancer patients,but the commonly used methods for detecting hMAM have some limitations,so there is an urgent need to establish a rapid and quantitative detection method of hMAM.ObjectiveIn this study,based on the principle of double antibody sandwich method,a time-resolved fluorescence immunochromatography method for quantitative detection of hMAM was established,which provides a fast,accurate and economical technique for breast cancer diagnosis and treatment monitoring.Methods1.Expression,purification and identification of hMAM recombinant protein:The recombinant BL21-pET32a-hMAM was induced and expressed,and the target protein was purified by ultrasonic disruption,solution washing,urea denaturation and dissolution,DEAE anion exchange chromatography,Ni2+affinity chromatography and dialysis renaturation.The purified protein was detected by SDS-PAGE electrophoresis and HPLC.2.Preparation of hMAM antibody:The identified target protein immunized rabbits many times to obtain antiserum with certain titer.After antigen affinity purification,HPLC detected its purity,and paired it with hMAM monoclonal antibody as coated antibody.3.Preliminary establishment of fluorescence chromatography detection method:Through the study of the amount of hMAM labeled antibody,hMAM coating antibody,EDC and NHS for microsphere activation,and the formula of microsphere protective solution,the optimal preparation process of fluorescence immunochromatography was determined,and the detection method of hMAM fluorescence chromatography was established.4.Performance evaluation of fluorescent immunochromatographic test strips:The linear range,precision,specificity,accuracy and stability of the fluorescence chromatography test strip prepared in this study were evaluated,and compared with the methodology of CUSABIO Human Mammaglobin ELISA kit.Results1.After induced by 0.5 mmol/L IPTG at 37℃ for 5 h,the recombinant strain was highly expressed.,The purification process of recombinant protein was established,and the recombinant protein with high purity was obtained.2.The antiserum with high titer was obtained by immunizing rabbits.After antigen affinity purification,the purity of the peak region of the eluent was higher,and it could be well matched with hMAM monoclonal labeled antibody as coated antibody.3.A preliminary method for the detection of hMAM by fluorescence chromatography was established.The optimum raw materials for fluorescence chromatography were selected,and the optimal formula of microsphere protective solution was optimized.when activating 50 μL microspheres,the optimum amount of 10 mg/mL EDC was 20 μL,the optimum amount of 10 mg/mL EDC was 20 μL,the optimum amount of antibody labeling was 20 μg,the optimum concentration of T-line coating was 1.0 mg/ml,and the concentration of C-line coating was 2.0 mg/mL.4.Performance evaluation of fluorescent immunochromatographic test strips:Based on the above optimal process,the linear range of the test strip is 0.313-20 ng/ml,and the correlation is good at this time;The variation coefficients within the batch of the test strips are less than 10%,and the coefficient of variation between batches is less than 15%,which meets the precision requirements;The recovery rates of high,medium and low value samples are all within 8 5%-115%,the accuracy meets the requirements.There was no obvious cross-reaction with CEA and CA153,and the specificity was good,and there was a good correlation with 46 clinical samples detected in parallel with ELISA kit.Conclusions1.In this study,the recombinant protein was purified by two-step purification method,the purity of the target protein was high,and the polyantibody was purified by antigen affinity chromatography.2.The composition of the microsphere protective solution was optimized to make the microsphere coupling process more uniform.hMAM monoclonal antibody was used as labelled antibody and hMAM polyantibody was used as coating antibody,which solved the problem of poor matching and high cost of antibodies.3.A time-resolved fluorescence immunochromatography method for quantitative detection of human mammoglobin was established,which provides a convenient,fast,accurate and accurate auxiliary detection method for breast cancer screening,treatment and prognosis detection.