Development Of The Amplified Luminescent Proximity Homogeneous Immunoassay For Detection Of Human Epididymis Protein 4 And Cancer Antigen 125

After cervical and uterine cancer, the incidence of ovarian cancer ranks third in gynecological tumors, while the death rate is the first. Ovarian cancer is a serious threat to women’s health, and its occurrence and development is relatively hidden. due to ovarian cancer early asymptomatic, lack of effective screening, clinical go up 80% of the patients with ovarian cancer diagnosis is period, the 5 year survival rate is only 20% to 30%, while 5 year survival rate for patients with early ovarian cancer is 70% to 90%. Early ovarian cancer prognosis is excellent, but only 10 to 30% of the survival for later period. Therefore, early detection of ovarian cancer patients is very important. High sensitivity and high specificity of ovarian tumor markers helps early detection of ovarian cancer, will improve the survival rate of ovarian cancer, and also help in the treatment of disease monitoring, efficacy evaluation and prognosis.Human epididymis secretory protein 4 (HE4) is a new potential markers of ovarian cancer, has good prospects in the early diagnosis and disease monitoring for ovarian cancer. HE4 belong whey acidic 4-disulfide Center (WFDC) family of proteins, full-length 11.78kb. HE4 gene was earliest found in human epididymal epithelium. HE4 gene associated with ovarian cancer by cDNA microarray analysis. This new serum marker in patients with benign tumors and normal is were very low, but in the serum of ovarian cancer patients is in high levels. And a growing number of research evidence, HE4 has a good prospect in the early diagnosis and disease monitoring for ovarian cancer. Cancer antigen 125 (CA125) was extracted in ovarian serous cystadenocarcinoma, and is a glycoprotein antigen with a relative molecular mass of 2×105～10×105. CA125 highly expressed in epithelial ovarian cancer cells, and secreted into the blood. CA125 is currently the most widely used clinical tumor marker for ovarian cancer, mainly used for diagnosis of ovarian cancer, after treatment efficacy of indicators and monitoring of the disease. Although the majority of patients with ovarian cancer serum CA125 levels increased, but less than 50% of these patients were in the early stage of the disease increased. Although CA125 has high sensitivity, but many patients with benign gynecological diseases are significantly elevated serum CA125 and can not distinguish the increase of CA125 in physiological conditions (menstrual period, pregnancy) and some benign disease (endometriosis, ovarian cysts and accompanied by ascites of cirrhosis of the liver). Study confirmed, HE4 expression in ovarian cancer tissues was significantly higher than in normal ovarian tissue, compared with the markers used in the past, HE4 has a high sensitivity in the diagnosis of ovarian cancer, especially in stage I ovarian cancer diagnosis aspects. The combined detection of serum levels of HE4 and CA125 obtained higher sensitivity than the single use of any one of the two markers.Labeling immunoassay is a progressive and comprehensive discipline and is the mainstream core technology of modern immunoassay. Based on the principle of labeling immunology, labeling immunoassay is characterized by high sensitivity and specificity, which has been widely used in medicine research and clinical examinations. In recent years, the development of new theories and new techniques has enormously improved the techniques in labeling immunoassay. The most widely used immunolabeling techniques are fluorescence immunoassay (FIA), enzymatic immunoassay (EIA), radio immunoassay (RIA), colloidal gold immunochromatographic assay (GICA), chemiluminescence immunoassay (CLIA), and time-resolved fluoroimmunoassay (TRFIA). Although all of these assays possess a certain degree of sensitivity and specificity, they have their inherent limitations in the labeling reagents involved. Therefore, it is necessary to develop a new labeling immunoassay to overcome these limitations. The amplified luminescent proximity homogenous immunoassay (AlphaLISA) technology employs luminescent oxygen channeling immunoassay and relies on PerkinElmer’s exclusive AlphaScreen(?). The new immunolabeling technique is developed for quantitatity in the recent year and provides a solution to the obstacle of the traditional labeling immunoassay. This technique does not require of washing-away of the free labels. In addition, amplified luminescent proximity homogenous assay could overcome other limitations from EIA, CLIA and TRFIA. As a homogenous assay, amplified luminescent proximity homogenous assay emerg the advantages of the laser technology, nano-scale microspheres and the half lifetime of singlet oxygen. Therefore, it carried out with high throughput, high sensitivity, no radio contamination, good repetition, simple process, amenability to automation and so on. It has been widely used in biomedical studies and represents the development of the analytical techniques.This thesis focuses on the development of the amplified luminescent proximity homogeneous immunoassay for detection of human epididymis protein 4 and cancer antigen 125, and to explore its feasibility for clinical testing.Method:In this thesis, amplified luminescent proximity homogenous assay kits were developed for detecting human epididymis protein 4 and cancer antigen 125 using antibody-sandwich method.1. Coupling of antibody to beads:0.2 mg of antibody was added to microspincolumn, and was centrifuged for 8 min at 9000 rpm. Then, the antibody was washed six times using 0.13 mol/L sodium phosphate buffer (pH 8.0). The solution was added to 1 mg acceptor beads with 10μL of 25 mg/mL NaBH3CN and 1.25 μL of 10% Tween-20 and then incubated at 37 ℃ for 48 hours. The total volume of the reaction solution was 200μL. To block nonconjugated sites, a fresh carboxy-methoxyl amine (CMO) solution (65 mg/mL) was prepared in a 0.8 M NaOH.10μL of CMO solution was added to the reaction, and incubated for an hour at 37 ℃. The reaction solution was centrifuged and the supernatant was removed. Then the bead pellet was re-suspended in 200 μL of 0.1 M Tris-HCl, pH 8.0, centrifuged and washed. After the last centrifugation, the beads were re-suspended in storage buffer (200μL of PBS+0.05% Proclin-300 as a preservative) at the concentration of 5 mg/mL.2. Biotinylated antibody:0.5 mg of antibody was added to microspincolumn, and was centrifuged for 8 min at 9000 rpm. Then, the antibody was washed six times using 0.1 mol/L carbonate buffer (pH 9.5) containing 0.1% NaN3 repeatedly. NHS-D-Biotin was dissolved in DMSO immediately prior to use in dark place at a concentration of 20 mg/mL. Then a volume equal to 10% of the total volume of the antibody solution was added into the NHS-D-Biotin solution in portions to the antibody solution with gentle stirring, and incubated at room temperature for 4 hours. The unreacted NHS-D-Biotin was filtered with microspincolumn. The biotinylated antibody was stored at-20 ℃ until use (0.5 mg/mL).3. Calibrations:The calibrator series were carried out by diluting antigen in the buffer. The standards prepared above were lyophilized according to 1 mL aliquot and then stored at 4℃ until used.4. Evaluation of assay performance4.1 Calibration curve:A linearized standard curve was obtained on a log-log plot with six concentrations including zero calibrator.4.2 Hook effect:The different concentrations of the antigen were determined by the proposed method.4.3 Sensitivity:The zero calibration was detected for 20 times repeatedly and the mean signal value and the standard deviation were calculated. The signal value, which was obtain by the mean signal value of zero calibration plus double standard deviation and minus the background value, was substituted into the standard curve equation to acquire the corresponding concentration. This concentration was defined as assay sensitivity.4.4 Accuracy:The ratio of real concentration of standards determined to concentration marked is between 0.90 and 1.10, which were regarded as a criterion of the accuracy.4.5 Recovery:Spiking recovery was assessed by adding one of the calibrations to the control serum. 10μl of the calibration was spiked into 200 μl of the control serum for the ratio of 1:20. The evaluations were done by calculating the ratio between the calculated and the expected values.4.6 Precision:The control serum was measured 10 times repeatedly. Each mean concentration and standard deviation was achieved. Thus, the coefficients of variation were calculated.4.7 Specificity:A certain amount of disturbing substances were regarded as samples and were detected. The gained concentration was served as the analysis of specificity.4.8 Interference:The effect of hemolysis, lipemia, and bilirubinemia was assessed by adding hemolysate, bilirubin (unconjugated), triglyceride and sodium ascorbate to the serum samples of the patient volunteers.4.10 Comparison:The samples were determined simultaneously by the proposed method and control reagents. The data was analysed by linear correlation.Result:The ratio of real concentration of standards determined to concentration marked is between 0.90 and 1.10. The analytical sensitivity of the AlphaLISA kit for HE4 was 0.81 pMol/L. The intra-assay coefficients of variation were 4.3%-7.2% and the inter-assay coefficients of variation were 6.1%-7.7%. No cross reactivity was found with CA15-3, AFP, CEA and CA125. The testing results were not subjected to interference by hemoglobin, triglycerides, or bilirubin. Compared with the ECLA, the correlation coefficient of the developed immunoassay was 0.978 (P<0.0001).For CA125 detection, the analytical sensitivity was 0.43 U/mL. The intra-assay and the inter-assay coefficients of variation were all less than 10%. No cross-reactivity with CA15-3, AFP, CEA, HE4, CA19-9 and CA50. The testing results were not subjected to interference by hemoglobin, triglycerides, or bilirubin. Compared with the ECLA, the correlation coefficient of the developed immunoassay was 0.992(P<0.0001).Conclusion:The results demonstrate that the amplified luminescent proximity homogenous assay reagent for human epididymis protein 4 and cancer antigen 125 were developed and met the demand for clinical application and could be the substitute of the import products for basic research and clinical examinations. As reported, human epididymis secretory protein 4 combination with cancer antigen 125 demonstrated unique advantages when used in diagnosis of ovarian cancer, we look forward to use our epididymis secretory protein 4 and cancer antigen 125 amplified luminescent proximity homogenous assay reagent can be applied in the diagnosis, condition monitoring and curative effect evaluationof ovarian cancer.