Development Of The Amplified Luminescent Proximity Homogeneolls Immunoassay For Detection Of Human Follicke-stimnlating Hormone And Luteinizing Hormone

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. In EIA, the enzymes used are often easily inactivated with the sensitivity lowered. On the other side the enzyme-labeling with large molecules often results in the steric hindrance effect on the labeled molecules and hence the sensitivity is lowered as well. In CLIA, lots of factors, especially the environment, affect its luminescence. Because of the extremely short life time of luminescence, the samples cannot be measured repetitively. In addition, due to the un-open reagent, it is expensive. In TRFIA, the procedures are complicated. The instrument and maintenance are costly. Moreover, isotopes in the environment and samples can cause the background-fluorescence. Besides, all of the assays have a common drawback:the separation of the labeled and free label by washing. 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, AlphaLISA could overcome other limitations from EIA, CLIA and TRFIA. As a homogenous assay, AlphaLISA 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.Human follicle-stimulating hormone (hFSH) is synthesized and secreted by gonadotrophs of the anterior pituitary, which is a single chain polypeptide hormone. It’s found that the level of hFSH is increased much in primary testicular failure, testicular seminoma, congenital testicular hypoplasia, congenital ovarian hypoplasia, primary amenorrhea, primary gonadal dysfunction, menopausal syndrome. However, it would decrease in women infertility, long-term using of contraceptives, large-scale dose of using sex hormones and so on. It’s very important for clinical diagnosis to determin the level of hFSH in serum. Human luteinizing hormone (hLH) is a glycoprotein which is secreted by the basophilic cells of the anterior pituitary with molecular weight about30,000daltons. hLH molecule consists of two non-covalently linked peptide chains (a and (3) composition, the a chain is similar to the human thyroid stimulating hormone (hTSH), human follicle-stimulating hormone (hFSH) and human chorionic gonadotropin (hCG). However, the β chain consists of distinct sequence and composition of amino acid, which makes it specific as the decision of the biological activity of hLH. In females, an acute rise or change of the level of hLH triggers ovulation and development of the corpus luteum. In males, hLH had also been called interstitial cell-stimulating hormone, it stimulates leydig cell production of testosterone. The level of hLH is changed by the ovulation cycle in healthy women with menstrual cycle, so the level of hLH in serum can be used to predict ovulation. In addition, the level of hLH is associated to many diseases directly and it’s also important for clinical diagnosis to determin the level of hLH in serum.This thesis focuses on the development of the amplified luminescent proximity homogeneous immunoassay for detection of human follicle-stimulating hormone and and luteinizing hormone, and to explore its feasibility for clinical testing.Method:In this thesis, AlphaLISA kits were developed for detecting hFSH and hLH using antibody-sandwich method.1. Calibrations:The calibrator series were carried out by diluting antigen in the buffer. The desired standard concentrations for hFSH were0,1,4,16,64and192U/L. The desired standard concentrations for hLH were0,0.55,2.2,11,66and135U/L.The standards prepared above were lyophilized according to1mL aliquot and then stored at4℃until used.2. Coupling of antibody to beads:0.2mg of antibody was added to microspincolumn, and was centrifuged for8min at9000rpm. Then, the antibody was washed six times using0.13mol/L sodium phosphate buffer (pH8.0). The solution was added to1mg acceptor beads with10μL of25mg/mL NaBH3CN and1.25μL of10%Tween-20and then incubated at37℃for48hours. The total volume of the reaction solution was200μL. To block nonconjugated sites, a fresh carboxy-methoxyl amine (CMO) solution (65mg/mL) was prepared in a0.8M NaOH.10μL of CMO solution was added to the reaction, and incubated for an hour at37℃. The reaction solution was centrifuged and the supernatant was removed. Then the bead pellet was re-suspended in200μL of0.1M Tris-HC1, pH8.0, centrifuged and washed. After the last centrifugation, the beads were re-suspended in storage buffer (200μL of PBS+0.05%Proclin-300as a preservative) at the concentration of5mg/mL3. Biotinylated antibody:1mg of antibody was added to microspincolumn, and was centrifuged for8min at9000rpm. Then, the antibody was washed six times using0.1mol/L carbonate buffer (pH9.5) containing0.1%NaN3repeatedly. NHS-D-Biotin was dissolved in DMSO immediately prior to use in dark place at a concentration of22mg/mL. Then a volume equal to10%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 for4hours. The unreacted NHS-D-Biotin was filtered with microspincolumn. The biotinylated antibody was stored (0.5mg/mL) at-20℃until use.4. Evaluation of assay performance4.1Accuracy:The ratio of real concentration of standards determined to concentration marked is between0.90and1.10, which were regarded as a criterion of the accuracy.4.2Calibration curve:A linearized standard curve was obtained on a log-log plot with six concentrations including zero calibrator.4.3The measurement range and the hook effect:The different concentrations of the hFSH antigen and the hLH antigen were determined by the proposed method.4.4Sensitivity:The zero calibration was detected for20times 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.4.5Analytical recovery:Spiking recovery was assessed by adding one of the calibrations to the control serum.10μl of the calibration was spiked into200μl of the control serum for the ratio of1:20. The evaluations were done by calculating the ratio between the calculated and the expected values.4.6Precision:The control serum was measured10times repeatedly. Each mean concentration and standard deviation was achieved. Thus, the coefficients of variation were calculated.4.7Specificity:A certain amount of disturbing substances were regarded as samples and were detected. The gained concentration was served as the analysis of specificity.4.8Interference: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.4.9Reference value:The healthy serum samples were determined by the proposed method. The statistical software is used for the analysis of distribution of data.4.10Comparison:The samples were determined simultaneously by the proposed method and control reagents. The data was analysed, including the Wilcoxon signed-rank test and the linear correlation.Result:The ratio of real concentration of standards determined to concentration marked is between0.90and1.10. The analytical sensitivity of the AlphaLISA kit for hFSH was0.09U/L. The measurement range was from0.09to192U/L. The intra-assay coefficients of variation were4.2%-7.1%and the inter-assay coefficients of variation were7.5%-8.3%. No cross reactivity was found with hLH, hTSH and hCG. The testing results were not subjected to interference by hemoglobin, triglycerides, or bilirubin. The result of the Wilcoxon signed-rank of100serum samples involved was Z=-0.825(P=0.409>0.05) and indicated that there was no statistical significance between hFSH-AlphaLISA assay and commercial SIEMENS Immulite FSH kit. The correlation coefficient was0.993.For hLH detection, the measurement range was from0.164to135U/L. The analytical sensitivity was0.164U/L. The intra-assay coefficients of variation were4.3%-6.1%and the inter-assay coefficients of variation were7.4%-8.1%.No cross-reactivity with hFSH, hTSH and hCG. The testing results were not subjected to interference by hemoglobin, triglycerides, or bilirubin. The result of the Wilcoxon signed-rank of158serum samples involved was Z=-1.295(P=0.195>0.05) and indicated that there is no statistical significance between hLH-AlphaLISA assay and commercial ECLIAhLH kit (Roche). The correlation coefficient was0.989.Conclusion:The results demonstrate that the AlphaLISA reagent for hFSH and hLH were developed and met the demand for clinical application and could be the substitute of the import products for basic research and clinical examinations.