Preparation Of Monoclonal Antibodies Against Anti-müllerian Hormone (AMH) And Development Of Biosensor Detection Methods For AMH

Anti-Mullerian hormone（AMH）is the main marker reflecting ovarian reserve function and evaluating fertility.The detection of AMH can be used to identify embryonic sex,reflecting the ovarian reserve function,diagnosis of premature ovarian failure,polycystic ovary syndrome,assessment of male infertility,and assisted reproduction.Compared with other fertility markers,AMH concentration is relatively stable,not limited by specific testing time,and is not easy to be affected by external factors（such as hormones,drugs,menstrual cycle and pregnancy）.It can decrease with age to about 25 years,and is often used as an important indicator of clinical testing.AMH detection technology has developed rapidly,from early immunocytochemical analysis and radioimmunoassay technology to enzyme-linked immunosorbent analysis（ELISA）,and has developed to automatic chemical light detection technology.Immunocytochemical analysis can only be qualitative,not quantitative;radioimmunoassay has the risk of environmental pollution.ELISA method is simple and suitable for large serological investigation and has great application prospect,but repeated washing requires limited detection sensitivity;reagents and instruments required for chemical lighting detection are expensive and professional operation.Therefore,it is of great significance to explore the efficient,specific and inexpensive detection methods for the detection of AMH.The existing immunological detection methods of AMH are all based on the reaction of antigen antibody.At present,the quality of AMH antigens and antibodies produced in China is uneven,and the import price is very expensive.The preparation of high price,strong specificity and low cost of AMH antigen antibodies can provide effective reagents for the detection of AMH.This paper aims to prepare AMH antigens and antibodies with strong specificity and high activity as raw materials to develop a high sensitivity electrochemical immunosensor based on CMWCNTs/NH₂-Si O₂for the detection of AMH and provide a new detection method for AMH diagnosis.The research contents of this paper are described as follows:1.AMH antigen preparationHuman AMH gene sequences retrieved from NCBI Gene Bank（accession number:NM＿000479.5）were analyzed and optimized,and the Ndel and Xhol restriction sites were introduced at both ends of the AMH gene sequence to chemically synthesize AMH gene.It was fused to the p ET 28（+）vector to construct a p ET28a（+）-AMH recombinant plasmid,and then the recombinant plasmid was introduced into E.coli BL21 for induced expression.After that,the induction conditions were optimized to perform the mass expression of AMH under optimal conditions.AMH protein was purified using nickel ion affinity chromatography column,and a series of biological characteristics（purity,concentration,specificity,reactogenicity）were identified.2.AMH monoclonal antibody preparationMix the AMH recombinant protein prepared above with an equal volume adjuvant,emulsify it thoroughly,and use it as an immunogen to immunize BALB/c mice using long-term immunization.Take the spleen of mice after 4 immunizations and 1 booster immunization,prepare spleen cell suspension,and use PEG1500 to fuse the above cell suspension with SP2/0 myeloma cells.Repeat screening of the supernatant of fusion cells and subcloned cells using indirect ELISA.Expand the cultivation of stable secreting monoclonal cell lines,inject them into the abdominal cavity of BALB/c mice to induce ascites monoclonal antibodies,purify the ascites using Protein A column,and identify the biological characteristics of the purified AMH antibodies.3.developing a highly sensitive electrochemical immunosensor based on CMWCNTs/NH₂-Si O₂modified SPE.Si O₂nanospheres were prepared by the sol gel method,and APTES was added to combine with the Si-O-Si bond to obtain surface aminated silica;Activating the aminated silica nanospheres with EDC/NHS crosslinking agent to form stable NHS esters with carboxylated nanotubes,and mixing with CMWCNTs by ultrasonic method to obtain composite materials;SEM electron microscopy was used to characterize its surface scanning and spatial element distribution;Then CMWCNTs were fixed on the surface of the screen electrode by physical action,and the amide bond was used to bind Si O₂nanoparticle,and the peptide bond was used to covalently bind AMH recombinant protein.After modification,the electrode material was electrochemically characterized by CV cyclic voltammetry,which proved that the material was successfully modified on the electrode surface;And evaluate the sensitivity,repeatability,specificity,and clinical applicability of the constructed sensor.4.Establish a rapid detection method of AMH based on micro-nano fiber couplerThe biosensor of GO integrated helical micro-nano fiber coupler（DHMC）was constructed by covalently bonding graphene oxide（GO）on a 6μm diameter micro-nano fiber coupler Go was deposited on the surface of DHMC to provide an enhanced local evanescent light field and abundant binding sites for antibody molecules.AMH monoclonal antibody were modified on the sensor surface as target molecular recognition units,covalent binding to different concentrations of AMH antigen for immunological detection,such as specificity,clinical experiments.Result:1.The results of double enzyme digestion identification and sequencing showed that a significant gene band appeared at 1689 bp,indicating the successful construction of the p ET28a（+）-AMH recombinant plasmid.The optimal IPTG induction expression conditions for the optimized p ET28a（+）-AMH engineering strain are:30℃,0.8 mmol/L,and 10 hours.The purified AMH protein reached electrophoretic purity through SDS-PAGE identification,and the protein concentration measured by BCA identification was 0.7 mg/m L;The Western blot identification results showed a clear imprinting band at 62 k D,which was consistent with the expected results.After incubating with commercially available AMH antibodies,the ELISA test results showed a strong reaction,indicating good reactivity of AMH.2.During the process of preparing AMH monoclonal antibodies by cell fusion between mouse spleen cells and myeloma cells,the cell fusion rate was 100%.Six monoclonal cells stably secreting AMH antibodies were successfully cloned three times on the fused cells,named 5-G12,5-G11,5-C7,2-B12,1-H3,1-F5.Select two strains of 5-G12（subtype Ig G1）and 5-G11（subtype Ig G2b）with higher potency for expanded culture,prepare ascites,and purify them.SDS-PAGE electrophoresis identification showed clear bands at 25 k D and 50 k D,which were consistent with the light and heavy chain sizes of Ig G type antibodies.This indicates that the purified monoclonal antibody has high purity,and the BCA protein quantification concentration is 1.08mg/m L.The Western blot identification results showed that the prepared monoclonal antibody could specifically bind to the AMH recombinant protein,and the target band was consistent with expectations.The purified ascites titer measured by ELISA can reach 1:1024000.3.Successfully built an electrochemical immunosensor platform and established an AMH detection method.The detection range of AMH antigen was 1fg/m L-100 ng/m L by differential pulse voltammetry,the detection limit LOD was 0.15 fg/m L,and the corresponding linear equation R²=0.9807.Through four repetitive experiments,it can be seen that the current changes at the same concentration are small and the repeatability is good.DPV detection was performed on AMH and different serum markers（such as AFP,DCP,ST2）,and the results showed that only AMH samples caused a strong current effect,with a current change of up to 130μA.The sample without AMH markers showed no significant changes in current,indicating good specificity of the sensor.In addition,diluting the concentration of three negative clinical samples and six positive clinical samples by 50 times can detect biomarkers in serum samples,and the difference in current signal between the negative and positive groups is significant,with a current difference around 60μA,the results indicate that the sensor has good clinical performance.4.The constructed DHMC sensor is functionalized（such as hydroxylation,silanization,precipitated GO,activated carboxylic,fixed AMH monoclonal antigen,closed binding site）,monitored and analyzed by spectrometer.It can be seen that the spectrum is constantly shifted to the right with the increase of refractive index RI concentration.Sensitivity experiments showed that the spectrum leveled off at 50μg/m L,with a spectral sensitivity range of 200 fg/m L-50μg/m L and a detection limit lower than 200fg/m L.By selecting three groups of AMH clinical samples and AFP and DCP for specific clinical experiments,it can be seen that DHMC is well specific and has clinical application value.ConclusionIn this study,highly active AMH protein was successfully prepared by prokaruclear expression system,and AMH monoclonal antibody with high efficiency and specificity was prepared by cell fusion and subclonal screening.And constructed AMH high sensitivity electrochemical immune sensor based on CMWCNTs/NH2-Si O₂modified screen electrode and AMH biological sensor based on micro-nano optical fiber coupler,from the sensitivity,repeatability,specificity and clinical detection methods,the results of the results show that the two methods have high sensitivity,repeatability,strong specificity,can be used in clinical samples AMH detection,provides a new detection technology for AMH detection,has important clinical application prospects.