The Establishment Of ELISA Method For PAPP-A And Its Initial Clinical Application

Objective:Develop an enzyme linked immunoassay (ELISA) that coated biotinstreptavi-din for detecting pregnancy-associated plasma protein A (PAPP-A) in human serum. This method will be used to test the PAPP-A concentration of relative diseases, and estimate its clinical value to the diagnosis and therapy of the diseases.Methods:1. Purify the protein of PAPP-A: we collected adequate serum of full term pregnant women. After dialysis, we selected two effective chromatography media:Sephadex G-200 and anion exchanger. Based on the molecule structure and physicalchemical characteristic of PAPP-A. We used SDS-PAGE and immunoblotting techniques to verify the protein.2. Antibody labeling and purification:(1) Labelled mAb(10E1) against PAPP-A with HRP by improved NaIO4 method. And purify the conjugate by gel filtration chromatography of molecular sieve. (2) Labelled mAb(10E2) against PAPP-A with biotin. Removed the free biotin by dialysis to achieve the purpose of purification.3. Establishment of the method and selection of reacting conditions:We coated the plate with biotinylation bovin serum albumin (BBC) and streptavidin. The plate would react with biotinylation mAb10E2 against PAPP-A, and established the assay of indirect coating ELISA. Optimized the reacting conditions such as the best coating level, the best working concentration of labelled antibody, the reaction time and temperature by checkerboard titration.4. Estimation of the method:Estimated the sensitivity, reproducibility, accuracy and stability etc. of the method we have developed. And compared the associativity with the overseas same kind kit.5. The initial clinical application:(1) The concentration measurement of PAPP-A for normal pregnancy women in the first trimester (from 8 to 14 weeks). Discussed the effects of age, weight and pregnant weeks. (2) The concentration measurement of PAPP-A for the patients of acute coronary syndrome (ACS). Analyzed the disease associativity by using healthy adult serum as comparisons.Results:1. Purify the protein of PAPP-A:We established a series of purifier techniques of PAPP-A, and obtained a high pure protein of PAPP-A. The analysis of the final purified PAPP-A by SDS-PAGE showed a single band of 200KD, and immunoblott-ing techniques showed that the main purified protein was PAPP-A.2. Labelled antibody:After the gel filtration chromatography of sephadex G-200, the first peak we collected was purified conjugate. The concentration was 0.9g/L, and IgG was 0.31g/L.3. Establishment of the assay and reacting conditions:(1) The result of checkerboard titration showed that, the best coating levels of BBC and streptavidin were each for 1:3000(3.33μg/ml) and 1:4000(3.Oμg/ml). (2) Both the two labelled antibodies' working concentration were at 1:4000. (3) We obtained a series standard preparation of PAPP-A from diluting the purified antigen of PAPP-A, the concentrations of them each for 0,1,2.5,5,10,25,50μg/ml. The linear range of the test was 0-50μg/ml. (4) The best reaction temperature was 37℃for 30min.4. Estimation of the assay: The sensitivity of the assay we developed was 0.31μg/ml and its recovery rates all between 95% and 100%. The linearity, stability and specificity of the assay are good. The CVs inner the group all did not exceed 10%, and the CVs between groups all did not exceed 15%. The assay we developed was done the analysis associativity with enzyme immunoassay kit abroad, r=0.982, which indicated the two assays were correlated greatly.5. The initial clinical application:(1) The objects of this study were the 160 normal pregnancy women in first trimester (from 8 to 14 weeks), screened Down's Syndrome. The results showed that the Multiples of the Medium (MoM) of serum PAPP-A level in the normal pregnancy women were increased along with the pregnant weeks, this result was coherent with other reports. Furthermore, we also found that:The serum level of PAPP-A in the older pregnancy women (≥35 years) was significantly lower than in the younger pregnancy women; And the serum level of PAPP-A was decreased in the high weight pregnancy women. (2) After the detection of 50 serum samples of ACS patients, the results showed that we need a further study to improve the sensitivity.Conclusions:1. We established a series of purifier techniques of PAPP-A, and obtained a high pure protein of PAPP-A, which could provide a potential value for establishing an ELISA method to measure the serum concentration of PAPP-A.2. The ELISA assay we established for PAPP-A detection by coating the plate indirectly has high sensitivity, stability and specificity. Compared with enzyme immunoassay kit abroad, the two assays were correlated greatly.3. PAPP-A detection had significance to Down's Syndrome and for Acute Coronary Syndrome, we need a further study to improve the sensitivity.