| Ovarian cancer has been recognized as one of the most common women cancer and the first cause of cancer-related deaths of women.Intern ational Agency for Research on Cancer data show that in2008,the incidence is estimated to include6.3new cases per100,000and3.8deaths per100,000annually in the word.The incidence in Chinese cancer registration areas was7.95per100,000,and the mortality was3.44per100,000in2009.Due to some patients are often asymptomatic with early ovarian cancer and this often results in late diagnosis.More im por tantly,Early stage ovarian cancer has an excellent prognosis if trea ted,while advanced stage is associated with a poor survival rate of only10-30%in approximately70%of patients who was diagnosed. Therefore, there is a pressing clinical need for a test that finds a high sensitivity but also a high specificity for malignancies,especially for early detection.Cancer antigen125(CA125) is the most widely used currently available tumour marker in ovarian cancer. CA125is a marker of epithelial ovarian cancer and endometrial cancer, which as a glycoprote from the surface of non viscous type ovarian cancer cells,with the molecular weight of200kD. CA125is a bro ad-spectrum tumor marker,which was also seen in pancreatic cancer, lung cancer and other gynecological tumor.The sensitivity and specificity of CA125are far from ideal though its levels are raised in approximately80%of all epithelial ovarian cancers (EOC). Therefore, CA125can’t be used as a unique parameter in the prediction of malignancy. So a new cancer biomarkers to replace or complement CA125is urgently needed.Human epididymis secretory protein4(HE4) is one of the most promising novel ovarian cancer biomarkers and is of good prospect of application as candidate early detection markers and disease monitoring. It is also known as WFDC2because it contains two whey acid protein (WAP) domains and a "four disulphide bond core" made up of eight cysteine residues.The HE4gene resides on human chromosome20q12-13.The corresponding gene codes for a13kD protein. In its mature glycosylated form the protein has a molecular weight of approximately20-25kD and consists of a single peptide chain containing two WFDC domains[20].The study from domestic and overseas about HE4showed that,HE4is expressed at a very low level while high expressed both in tissue and serum of ovarian cancer under normal physiological conditions.Hellstrom elevated HE4protein levels in serum from patients with ovarian tumors, demonstrating a similar sensitivity to CA125, but higher specificity for malignant tumors as compared to benign disease.Increasingly evidence shows that, HE4has a good prospect of application in the early diagnosis and disease monitoring of ovarian.Furthermore, a series of papers showed that used a combination of CA125and HE4to predict the presence of malignant ovarian tumour in patient is a more accurate than either alone.When CA125was combined with HE4,the prediction rate was higher then either alone which showed a sensitivity for detecting malignant disease of76.4%at a specificity of95%.Labeled immunoassay is a comprehensive discipline which integrates with basic medicine, experimental techniques and clinical application. The detetion based on various tracer technique with highly sensitive combining with Immunology highly specific antigen-antibody reaction.In recent years, the development of new theories and new techniques has enormously improved the techniques in labeling immunoassay. Labeled immunoassay including radio immunoassay (RIA) and non radio immunoassay,such as fluorescence immunoassay (FIA),colloidal gold imm unochromatographic assay (GICA),enzymatic immunoassay (EIA), chemi lum inescence immunoassay (CLIA), and time-resolved fluoroimmunoassay (TRFIA). Time-resolved fluorescence immunoassay (TRFIA) is a sensitive quantitative immunoassay technology by using rare earth ion to label antigens or antibodies. Due to the unique fluorescence properties of lanthanide rare earth ions, the technique has advantages of high signal to noise ratio, high sensitivity (10-18mol/well), easy labeling process, long storage time, no radioactive contamination, good repeatability, wide range of detection, short operation process, not sensitive to natural fluorescence interference,multiple labelling and very wide range of applications.TRFIA has higher clinical application value compared with ELISA and RIA, which is widely used in the market for clinical detection of various antigens and antibodies.This thesis focuses on the development of Timed-resolved fluoroimmunoassay (TRFIA) for detection of Human epididymis secretory protein4and a dual-label time-resolved fluoroimmunoassay for the detection of Human epididymis secretory protein4(HE4) and cancer antigen125, and to explore their feasibility for clinical testing. Method:Ⅰ. Development of Time-Resolved Fluoroimmunoassay Kit for detection of Human epididymis protein4(HE4) in serum.1. Prepare standards and controls with HE4antigen, The concentration of standards were:Opmol/L、20pmol/L、50pmol/L、200pmol/L、500pmol/L、1000pmol/L.The standards prepared above were lyophilized according to1mL aliquot and then stored at4℃until used.2. Antibodies were diluted to3ug/ml with coating buffer and coated into microplate with96wells. The plates were concussed for two hours at37℃,then incubated at4℃overnight, closed for three hours at37℃, vacuum pumped, and stored at4℃.3. Preparation of antibodies labelled with Eu3+:Purified antibodies were mixed with europium labeling reagent at a mass ratio of5:1,oscillated at25℃overnight, and purified over gel chromatography column of SephadexTM G-50at the next day. At the same time collect effluent (1ml/tube).Measured absorbance at A280nm,and combined the peak tubes,plused protective agent of10%BSA,-20℃preservation.4. Determination of6F8as the coating antibody and H5as the labelling antibody by compered different antibody.5. Determination of optimal concentration of coating antibody and labelled antibody using chessboard titration method.6. Determination of reference value:451healthy women serum samples were assayed by the proposed method. The statistical software is used for the analysis of distribution of data.7. Evaluation of assay performance7.1Calibration curve:A linearized standard curve was obtained on a log-log plot with six concentrations including zero calibrator. 7.2The linearity range and sensitivity.The zero calibration was detected for10times repeatedly and the mean signal value and the standard deviation were calculated. The signal value, which was obtained by the mean signal value of zero calibrations plus double standard deviation, was substituted into the standard curve equation to acquire the corresponding concentration.This concentration was defined as assay sensitivity.7.3Hook effect:The different concentrations of the HE4antigen was de termined by the proposed method.7.4Accuracy:Serial double dilutions of HE4antigen by Negative Serum as samples were determinated. Calibrated the ratio of real concentration to its theoretical value.7.5Precision:The quality control Ⅰ,Ⅱ,Ⅲ were measured repeatedly. Each mean concentration, standard deviation and the coefficients of variation were calculated.7.6Specificity:A certain amount of interference substances were detected. The ratio of measured and original were calculation.7.7Interference:The interferent effect of hemolysis, lipemia, and bilirubinemia was assessed by adding hemoglobin, bilirubin and triglyceride to the Quality Control Ⅰ and Quality Control Ⅲ.7.8Stability:Reagent performance was evaluated after stored at37℃for7days.8. Comparison:The samples were determined simultaneously by the proposed method and control reagents. The data was analysed, by linear correlation.Ⅱ.Development of a dual-label time-resolved fluoroimmunoassay for the detection of Human epididymis secretory protein4(HE4) and cancer antigen125(CA125)1. Prepare standards and controls with HE4antigen and CA125, the concentration of standards were:0pmol/L/0U/mL,20pmol/L/28.4U/mL,50pmol/L/53U/mL,200pmol/L/101.6U/mL,500pmol/L/445.9U/mL,1000pmol/L/815U/mL.The concentration of Quality Control Ⅰ,Ⅱ and Ⅲ were35pmol/L/16U/mL,110pmol/L/41U/mL and350pmol/L/265U/mL.The standards prepared above were lyophilized according to1mL aliquot and then stored at4℃until used.2. Anti-HE4McAb (6F8) and Anti-CA125McAb (M5807) were diluted with coating buffer and coated into microplate with96wells together. The plates were concussed for two hours at37℃,then incubated at4℃overnight, closed for three hours at37℃, vacuum pumped, and stored at4℃.3. Preparation of antibodies labelled with Eu3+:Purified antibodies were mixed with europium labeling reagent at a mass ratio of5:1,oscillated at25℃overnight, and purified over gel chromatography column of SephadexTM G-50at the next day. At the same time collect effluent (1ml/tube).Measured absorbance at A280nm,and combined the peak tubes,plused protective agent of10%BSA,-20℃preservation.4. Preparation of antibodies labelled with Sm3+:Purified antibodies were mixed with samarium labeling reagent at a mass ratio of2:1,oscillated at25℃overnight, and purified over gel chromatography column of SephadexTM G-50at the next day. At the same time collect effluent (1ml/tube).Measured fluorescence value,and combined the peak tubes,plused protective agent of10%BSA,-20℃preservation.5. To compared the line,the accuracy of blood values,the fluorescence values between HE4and CA125both labelled with Eu3+and Sm3+.6. Determination of optimal concentration of Anti-CA125McAb (M5807) and labelled Anti-CA125McAb (M5814) using chessboard titration method.7. Screening of proper sample volume (25μL、30μL、35μL、40μL、50μL). 8. Evaluation of assay performance8.1Calibration curve:The linearized standard curves of HE4-TRFIA and CA125-TRFIA were obtained on a log-log plot with six concentrations including zero calibrator.8.2The measurement range, sensitivity and Hook effect:The zero calibration(A) was detected for20times repeatedly and the mean signal value and the standard deviation were calculated. The assay sensitivity, which were obtained by the mean signal value of zero calibrations plus double standard deviation, was substituted into the standard curve equation to acquire the corresponding concentration.The different concentrations of the HE4antigen and CA125antigen were determined by the proposed method together.8.3Accuracy:Serial double dilutions of HE4antigen and CA125antigen by negative serum as samples were determinated. Calibrated the ratio of real concentration to its theoretical value.8.4Precision:The quality control serum Ⅰ、Ⅱ、Ⅲwere measured repeatedly. Each mean concentration and standard deviation were achieved. Thus, the coefficients of variation were calculated.8.5Specificity:A certain amount of interference substances were detected and cross-reactivity were calculated.8.6Interference:The interferent effect of hemolysis, lipemia, and bilirubinemia was assessed by adding hemoglobin, bilirubin and triglyceride to the serum samples of the serum sample.8.7Stability:Reagent performance was evaluated after stored at37℃for7days.9. Comparison:The samples were determined simultaneously by the proposed method and control reagents. The data was analysed, by linear correlation. Result:I. Development of Time-Resolved Fluoroimmunoassay Kit for detection of Human epididymis protein4(HE4) in serum.Anti-HE4McAb (6F8) was determined as coating material,Anti-HE4McAb (H5) was determined as labeling material by the screening of raw materials by TRFIA.Considering the background signal values and signal to noise ratio factor, choose3ug/ml as the optimal concentration of coated antibody, choose1:200as the optimal dilution of lablled antibody.451healthy women serums were assayed. The level of HE4over the age of50were significantly higher than those under the age of50by t test;96.9%samples had concentration value below90pmol/L; ROC curve showed that,when the concentrationwas at90pmol/L,the sensitivity was75%and the specificity was96.1%; So that the HE4concentration cut-off was set at90pmol/L.The sensitivity was0.73pmol/L.The measure range of developed kit was0.73-1000pmol/L.The correlation coefficient of the dose-response curve was0.9996within the linear range.No HOOK effect was found in samples with concentration below2000pmol/L. The recovery rate of dilution was between90%to113%.The intra and inter-assay coefficients of variation (CV) were4.6%-7.5%and8.3%-14.4%, respectively. There was no cross reactivity with Cancer Antigen15-3(CA15-3),Carcinoembryonic Antigen(CEA), Alpha-Fetoprotein (AFP), Hepatitis B virus surface Antigen (HBsAg),Cancer Antigen125(CA125),Cancer Antigen19-9(CA19-9),Cancer Antigen50(CA50) and Human Chorionic Gonadotropin(hCG). No interferences were detected from triglycerides, bilirubin and hemoglobin.The reagent showed good stability proved by performance evaluation after stored7days at37℃.Compared with Roche reagent kit,133samples were detected, correlation formula was y=0.794x+12.255, R2=0.976, P<0.001, the result reflected that this reagent can meet the requirements of clinical detection very well. II.Development of a dual-label time-resolved fluoroimmunoassay for the detection of Human epididymis secretory protein4(HE4) and cancer antigen125(CA125)Anti-HE4McAb (H5) labelled with Eu3+and Anti-CA125McAb (M5814) labelled with Sm3+,based on comparing the line,the accuracy of blood values,the fluorescence values between HE4and CA125both labelled with Eu3+and Sm3+.Considering the background signal values and signal to noise ratio factor, choose3ug/ml as the optimal concentration of Anti-CA125McAb (M5807), choose1:50as the optimal dilution of lablled antibody of Anti-CA125McAb (M5814). Detection limits of HE4and CA125were0.476pmol/L and1.6U/mL, respectively.The measure range of developed kit were0.476-1000pmol/L for HE4and1.6-815U/mL for CA125.The correlation coefficient of the dose-response curve of HE4and CA125were0.9994and1.0000, respectively, within the linear range. The recovery rate of dilution were between93.5%to107.3%for HE4and97.9%to105.0%for CA125.The Intra-and inter-assay coefficients of variation of HE4-TRFIA were3.02%-8.81%and0.65%-8.57%and for CA125-TRFIA were2.60%-9.27%and5.24%-7.88%, respectively. There were no cross reactivity with Cancer Antigen15-3(CA15-3), Carcinoembryonic Antigen(CEA), Alpha-Fetoprotein (AFP), Hepatitis B virus surface Antigen (HBsAg),Cancer Antigen19-9(CA19-9),Cancer Antigen50(CA50) and Human Chorionic Gonadotropin(hCG). No interferences were detected from triglycerides, bilirubin and hemoglobin. The reagent showed good stability proved by performance evaluation after stored7days at37℃.Compared with Roche reagent kit,147samples were detected, correlation formula of HE4was y=0.9756x-0.1956, R2=0.905, P<0.001, and153samples were detected, correlation formula of CA125was y=0.9953x-10.070, R2=0.986, P<0.001, the result reflected that this reagent can meet the requirements of clinical detection very well.Conclusion:These results demonstrated that the HE4-TRFIA reagent and HE4and CA125-TRFIA reagent developed in this study had good performance, including accuracy, sensitivity, precision, specificity, etc., which could meet the requirements of clinical application. The donor beads coated with streptavidin showed similar performance with the imported products, and are expected to be used in production by further optimization. |
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