Development Of Diagnostic Reagents For Free β-hCG And PAPPA Detection Using Amplified Luminescent Proximity Homogeneous Assay (AlphaLISA)

Down syndrome, also called innate clumsy, is the most common in fetus with an occurrence rate of about1in600to1in800(Korenberg et al.1994; Shaw et al.,2008), according to a general statistics, there were about27thousands infants borning with Down syndrome every year, that to say, one case Down syndrome was borned every twenty minutes average, take about one tenth in intelligent dysplasia. Down syndrome is a most common chromosome inherit disease cased by chromosome defect in number twenty one, and the mainly feature were seriously innate intelligent dysplasia、special facial features and usually occurs with differently congenital malformation. Presently there is no method to cure Down syndrome completely, so patients can add tremendous financial burden and mental pressure to the family. The opinion of good prenatal and postnatal care was accepted by most of people these years, so it is necessary to perfect the prenatal diagnosis, also screening for Down syndrome has become to hotspot. Now how can discover Down syndrome in antenatal to prevent and reduce its birth is the important task in improving the health of newborns in prenatal disgnosis in our country.About ten years ago, maternal serum screening for Down syndrome have been acknowledged in all over the word when using free β-human chorionic gonadotrophin(Free β-hCG), a-fetoprotein (AFP) and unconjugated estriol (uE3) in second-trimester. But if prenatal screening for Down syndrome in second-trimester, it need induced labour, so first-trimester cause people’s attention for its advantage in reducing sprit and body pain for induced labour. Later Wald found combination detection of free β-human chorionic gonadotrophin(Free β-hCG) and pregnancy-associated plasma protein-A(PAPPA) in first-trimester has a equal detection rate with multiple detection in second-trimester when synthetical analysed seven serum maternal including Free β-hCG and PAPPA. Then with the development of high resolution ultrasound technology, ultrasound is used more and more common in prenatal screening and prenatal diagnosis. Bianchi DW discovered that maternal serum pregnancy-associated plasma protein-A (PAPPA) and free β-human chorionic gonadotrophin(Free β-hCG) combination with sonographic nuchal translucency (NT) measurement had a detection rate of86.3percent, it was regarded as the most common and effect coalition screening methods in early trimester of pregnancy, also stand for the trend of prenatal screening for Down syndrome.At present, the methods used in prenatal screening in all hospitals at home and abroad are labeling immune analysis technology. This technique is characterized by high sensitivity and specificity, which has been widely used in basic medicine research and clinical examinations. In recent years, the most widely used immunolabeling techniques are enzymatic immunoassay (EIA), chemiluminescence immunoassay (CLIA) and time-resolved fluoroimmunoassay (TRFIA). But all of these assays have their inherent limitations in the labeling agent used. In EIA, the enzymes used are often easily inactivated to lower the sensitivity, and enzyme labeling of large molecules often results in alterations in the spatial structure of the labeled molecules to restrict the improvement of sensitivity. In CLIA, lots of factors affect its luminescence, especially the environment. Because of the extremely short life time of luminescence, the samples cannot be repetitively measured. In addition, due to the un-open reagent, it is expensive. In TRFIA, the procedures are complicated and need relatively long react time for physical absorption, multiple and maintenance are costly. Moreover, isotopes in the environment and samples can generate 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 the limitations. In recent years, the development of new theories and new techniques has enormously improved the techniques in labeling immunoassay. The amplified luminescent proximity homogenous assay (AlphaLISA) technology employs luminescent oxygen channeling immunoassay and relies on PerkinElmer’s exclusive AlphaScreen(?). The new and quantitative immunolabeling technique which emerges in the recent year provides a solution to the obstacle of the traditional labeling immunoassay. This technique does not require washing away of the free label. In addition, AlphaLISA could overcome other limitations in EIA, CLIA and TRFIA. As a homogenous assay, AlphaLISA combined with the laser technology, nano-scale microspheres and the half lifetime of singlet oxygen is characterized by 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 trend of the analytical techniques.This thesis used this new immunoassay technology, combined antibody-sandwich method and btiotin-even bad affinity element amplifier system to manufacture the quantify detection reagent for Free β-hCG and PAPPA in maternal serum, and evaluated the each performance and indicator of the reagent, then compared with the similar kit from foreign and evaluated their possibility in clinical diagnosis. At the same time, we detected the concentration of Free β-hCG and PAPPA in serum in different gestational weeks from Southern China, established the median values of different gestational weeks. This can supply the evidence for confirming the normal reference range.Method:In this study, using antibody-sandwich method AlphaLISA kits detecting for Free (3-hCG and PAPPA were developed.1. Free (3-hCG and PAPPA calibrations:For calibration purposes, the calibrator series were carried out by diluting antigen in the buffer. The desired standard concentrations for Free β-hCG were0,3,10,30,100and200ng/mL. The desired standard concentrations for PAPPA were0,10,40,200,800and2000mU/L.The standards prepared above were lyophilized according to0.5mL 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 pH8.0repeatedly. 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-methoxy lamine (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-HCl, 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/mL.3. 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 (1mg/mL) at-20℃until use.4. Evaluation of assay performance4.1Calibration curve:A linearized standard curve was obtained on a log-log plot with six concentrations including zero calibrator.4.2Sensitivity: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, was substituted into the standard curve equation to acquire the corresponding concentration. This concentration was defined as assay sensitivity.4.3Analytical recovery. Take two serum samples, whose concentration have been tested, then dilute them with dilution buffer according to different rates. The evaluations were done by calculating the ratio between the calculated and the expected values.4.4Precision:The control serum was measured10times repeatedly. Each mean concentration and standard deviation was achieved. Thus, the coefficients of variation were calculated.4.5Specificity:A certain amount of disturbing substances were regarded as samples and were detected. The gained concentration was served as the analysis of specificity.4.6Interference: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.4.7Reference value:The healthy materal serum samples were determined by the proposed method. The statistical software is used for the analysis of distribution of data.4.8Comparison:The samples were determined simultaneously by the proposed method and DELFIA kits (PerkinElmer). The data was analysed, including the Wilcoxon signed-rank test and the linear correlation.Result:This study manufactured the diagnosis reagent for Free β-hCG and PAPPA in maternal serum for prenatal screening in first-trimester, and evaluated the each performance and indicator of the reagent, including accuracy、sensitivity、analytical recovery、specificity、precision, also comparison with Free β-hCG DELFIA kits (PerkinElmer), then estimated the possibility of its using in clinical diagnosis. The result showed that under the optimum condition, for Free β-hCG, the detection limit was0.11ng/ml and the analytical range was0.11-200ng/ml; Analytical recovery was98%-108%; The intra-assay coefficients of variation were1.32%-4.50%and the inter-assay coefficients of variation were4.69%-4.95%; There was no cross-reaction to LH, FSH, HCG and TSH, showing that antibody has a good specificity;4168maternal serum samples of8-20weeks were tested using reagernt made by ourself, median values during different weeks and the reference value were established; The result of the Wilcoxon signed-rank of188serum samples involved was Z=-0.842(P=0.412>0.05) and there is no statistical significance between Free β-hCG-AlphaLISA assay and commercial Free β-hCG DELFIA kits (PerkinElmer). The correlation coefficient was0.989. The correlation equation was AlphaLISA=1.045TRFIA+1.580, P<0.001.For PAPPA, the measurement range was from0to10000mU/L. The analytical sensitivity was1.365mU/L; The intra-assay coefficients of variation were5.61%-6.51%and the inter-assay coefficients of variation were2.07%-4.82%; No cross-reactivity with AFP、RPL、HPL and HCG was found. The testing results were not subjected to interference by hemoglobin, triglycerides, or bilirubin;853maternal serum samples of9-13weeks were tested using reagernt made by ourself, median values during different weeks and the reference value were established; The result of the Wilcoxon signed-rank of165serum samples involved was Z=-0.197(P=0.844>0.05) and there is no statistical significance between PAPPA-AlphaLISA assay and commercial PAPPA DELFIA kits (PerkinElmer). The correlation coefficient was0.987, The correlation equation was AlphaLISA=0.976TRFIA+46.808, P<0.001.Conclusion:The results demonstrate that the AlphaLISA reagent for Free β-hCG and PAPPA we made meet the demand for clinical application and could be the substitute of the import products for basic research and clinical examinations for prenatal screening in first-trimester.