Establishment Of Dual Detection Method For Time-resolved Flurescence Immunochromatography Of HE4 And CA15-3

Background Breast cancer is one of the most common malignant tumors in women with high incidence,and tends of this disease to be younger.Early diagnosis,early detection and early treatment are of great significance to improve the survival rate of breast cancer patients.CA15-3 is a common tumor marker for breast cancer,but its sensitivity is not ideal.Compare to single detection,HE4 and CA15-3 dual detection have shown high sensitivity and accuracy.Electrochemiluminescence is major method for HE4 and CA15-3 detection,but the method requires expensive equipment and combin of matching reagents,and requires professional operation,it is not conducive to the promotion and use of grass-roots hospitals.There is an urgent need to establish a convenient,fast and accurate method to realize HE4,CA15-3 dual detection in grass-roots hospitals.Objective Establishment of dual detection method for time-resolved fluorescence immunochromatography of HE4 and CA15-3,in order to provide a technical method for the diagnosis,prognosis and prediction of recurrence and metastasis of breast cancer.Methods1.A series of laboratory reference materials for the dual detection of HE4,CA15-3 fluorescent immunochromatography were prepared by dilution of HE4,CA15-3 antigen in matrix serum and calibration by Roche electrochemiluminescence detection reagent.2.HE4,CA15-3 single detection method establishment and optimization of its components: by establishing a single detection method of HE4,CA15-3 fluorescent immunochromatography,pairing antibodies were screened.The pH value of boric acid buffer and the ultrasonic time after redissolution in the preparation of immune microspheres were used to optimize the composition.3.HE4,CA15-3 dual detection method establishment and optimization the dual detectiont test strip: Based on the single detection method of HE4,CA15-3 fluorescence immunochromatography,the dual detection method of HE4,CA15-3 was established.The raw materials were screened by studying microsphere pad and nitrocellulose filter membrane.To explore the sequence of the detection line,sample pad treatment solution,labeled antibody concentration and coated antibody concentration,optimal proportion of dilution ratio of immune microspheres were investigated,and the sample detection time.Comprehensive analysis and selection,optimization of dual detection methods.4.Evaluation of the performance of the HE4,CA15-3 dual detection test strip: The linear range,minimum detection limit,specificity,precision and accuracy of the HE4,CA15-3 dual test strip prepared in this study were compared with those of Roche electrochemical luminous method to evaluate the performance indexes of the test strip.Result1.A series of linear,precise and specific indoor reference materials for HE4,CA15-3 fluorescence immunochromatography were prepared.It can be kept stably for 6 months at-20? to meet the experimental requirements.2.Establishment of single detection method for HE4,CA15-3 fluorescence immunochromatography: Rabbit IgG polyclonal antibody(labeled)-33 mouse anti-rabbit IgG monoclonal antibody(coated)was selected as the quality control line pairing antibody.CLIA and TRFMIA were used to screen out PE(labeled)-PC(coated)was selected as the optimal pairing antibody of HE4,QF(labeled)-QE(coated)was selected as the optimal pairing antibody of CA15-3.The optimum pH values of boric acid buffer in the preparation of HE4 and CA15-3 immune microspheres were 8.0and 8.5,ultrasonic time was determined to be 5 min.after twice centrifugal redissolution.3.Establishment of dual detection method for HE4,CA15-3 fluorescence immunochromatography: The "Z80" glass fiber cotton was selected as microsphere pad;CN140(without backing)was chosen as the optimum nitrocellulose filter membrane.Determining CA15-3 as the line T1 and HE4 as the line T2,the content of T-20 in the sample pad treatment solution is 0.25%,When 10 ?L fluorescent micros pheres were labeled with HE4,CA15-3 monoclonal antibody and rabbit IgG polyclonal antibody,the optimum labeling amount was 40?g,30?g and 10?g,respectively.The optimum coating concentration of HE4,CA15-3,mouse anti-rabbit IgG monoclonal antibody was 3mg/mL,2mg/mL,2mg/mL.When preparing HE4,CA15-3 monoclonal antibody and rabbit IgG polyclonal antibody three kinds of immune microspheres,the optimum dilution ratio was 1:1:2,and the optimum detection time was 15 min.4.Evaluation of the performance of the HE4,CA15-3 dual detection test strip:Linear regression processing was performed using log(X)-log(Y)mathematical model,and linear equation was fitted to determine the linear range of HE4 was 15.6-1500 pmol/L,CA15-3 linear range is 7.8-500 U/mL.The minimum detection limit of dual detection test strip HE4 was14.33 pmol/L,and the minimum detection limit of CA15-3 was 5.00 U/mL,and there was no cross reaction between HE4 and CA15-3.Bilirubin(5mg/L),hemoglobin(0.1g/L),biotin(50ng/mL),CA12-5(150U/mL),CA19-9(150U/mL),CEA(25ng/mL)does not interfere with the HE4 and CA15-3 detection,the test strip has good specificity.In the intra assay and Inter assay CV(%)was in agreement with less than 10%,the precision was good,and the recovery rate is between 85% and 110%,the accuracy is good.Compared with the results of Roche electrochemiluminescence,the results showed that the two methods had a good correlation.Conclusions:1.A series of indoor reference materials for dual fluorescence immunochromatography detection of HE4 and CA15-3 were prepared.2.A time-resolved fluorescence immunochromatography dual detection method for HE4 and CA15-3 was established.