Establishment Of The Detection For Anti-mullerian Hormone Based On Magnetic Particle Chemiluminescence Assay And Its Application In Diagnosis Of Precocious Puberty Diseases

Anti-Mullerian hormone is one of the most effective markers for evaluating ovarian reserve function.It not only attracts the attention of clinicians in the field of female reproduction,but also in neonatal and child endocrine diseases.The detection of human serum AMH concentration has gradually become a popular item in medical laboratories.However,there is no uniform standard for AMH detection methods and reference intervals.So far,a variety of AMH test techniques have been available in foreign countries,but the research on AMH diagnostic reagents in China is still in the development stage.We have established a magnetic particle chemiluminescence detection method,which can detect AMH quickly,highly sensitively and specifically.Part 1 Establishment of magnetic particle chemiluminescence detection method for anti-Mullerian hormoneIn order to innovate the diagnostic methods of AMH in vitro and provide reference for clinical application,this study established the magnetic particles chemiluminescence method of AMH through the selection of reagents,the optimization of reaction system and detection process,the evaluation of analytical performance,the establishment of reference value and the study of stability.1 In terms of reagents,we finalized the AMH antigen 2 and AMH conjugates of Anti-AMH-AP(1.0μg/mL)and Anti-AMH-FITC(0.5Μg/mL)from Beijing Zecheng Biotechnology Co.,Ltd.2 In the reaction system and detection process,0.5%BSA was added to the Tris buffer of AMH anti-reagent.And the protein buffer BUF3(Tris-HCl buffer,150mmol/L,PH7.5,containing 0.5%BSA and 0.05%proclin300)was used to prepare the calibratorto ensure the stability of the reagents and reaction system.The sample loading mode was determined as 30μL sample+30μL anti-reagent A+30pL anti-reagent B 15 min+30μL magnetic particle reagent 5 min+300μL washing solution,3times+200μL substrate,and then tested on a luminescence detector.3 In the evaluation of analytical performance,we evaluated the precision,accuracy,specificity,blank detectionlimit,linear range,methodological comparison and hook effect.The intra-batches CV of quality control(QC)1 and QC 2 were 2.31%and 1.89%,the inter-batches CF were 3.79%and 6.64%,respectively;the accuracy was within±4%,and the recovery rate was 96%.The blank detection limit was 0.05ng/mL.100ng/mL inhibin A,100ng/mL activin A.and 500mIU/mL LH were added to the zero-content AMH sample,and the results were not higher than 0.07ng/mL.The specificity was better,and the specific detection requirements were met.The linear range was 0.07ng/mL-20ng/mL,and the correlation coefficient was r=0.999;hook effect was not observed when the AMH level of the sample tested as high as 800ng/mL;The results of the method and Roche electrochemiluminescence test were highly consistent.The linear regression equation Y=0.996Z+0.029,r=0.997,and the correlation was good.4 we analyzed 560 nor,al human samples,and determined the reference range of AMH by the percentile method.Healthy male:1.43ng/mL-11.6ng/mL;healthy female:20-24 years old(1.66ng/mL-9.49ng/mL);25-29 years old(1.18ng/mL-9.16ng/mL);30-34 years old(0.672ng/mL-7.55ng/mL);35-39 years old(0.777ng/mL-5.24ng/mL);40-44 years old(0.097ng/mL-2.96ng/mL);45-50 years old(0.046ng/mL-2.06ng/mL).5 Through the analysis of the accelerated stability and bottle opening stability ot the reagents,we found that the titer of anti-reagent decreased by about 30%after 9 days of accelerated destruction at 37 C and the calibratorto showed good stability and no obvious degradation after 8 hours of opening the bottle.The results indicate that the main performance indicators of the anti-Mullerian hormone magnetic particle chemiluminescence detection method meet the requirements of clinical testing with high sensitivity,high specificity and suit for clinical application.Part 2 Changes and significance of serum anti-Mullerian hormone levels in girls with central precocious pubertyTo evaluate ovarian reserve function in sirls around puberty and the diagnostic value of AMH in central precocious puberty(CPP),we determined the serum AMH levels of CPP girls aeed 5-8 years old and normal developing girls by the newly established method.The ovarian function of the two groups were compared,and the diagnostic efficacy of AMH for CPP was judged by receiver operating characteristic curve.We found that AMH levels were higher in girls with central precocious puberty than in normal girls(2.80ng/mL vs.2.10ng/mL)with the difference statistically significant(P<0.05)and the height and weight were significantly increased.The area under curve(AUC)was 0.681.When the Youden index reached the maximum,the optimal diagnostic level of AMH intercepted was 2.01ng/mL.Among them,the sensitivity reached 75%and the specificity was 63.33%.T he results showed that serunm AMH level has certain reference value for the diagnosis of CPP girls which provides a new idea for clinical evaluation of ovarian reserve and treatment plans.