Preparation And Application Of Monoclonal Antibodies Via Cytokine-assisted Immunization And Affinity-based Immunoassay Sensitivity Correlation Study

As an important component of immunoassay,the quality of antibodies directly determines the performance of the assay.For some target analytes with low immunogenicity or even without immunogenicity,it is difficult to obtain high quality target antibodies via traditional methods.At the same time,the influence of the affinity between the antigen and the antibody on the sensitivity of the immunoassay method cannot be overlooked.Therefore,it is of great significance to develop high-quality antibodies and to clarify the influence of their affinity on the sensitivity of immunoassay methods.In this study,the monoclonal antibodies were prepared successfully using cytokines as molecular adjuvants in both DNA immunization and subcutaneous immunization strategies.Further,we analyzed the specific epitope information recognized by the four antibodies and the influence of the epitope amino acid on the affinity of the antigen-antibody immune reaction.By analyzing the influence of competitive antigens with different affinities on the sensitivity of competitive immunoassay,the effect of the difference in affinity that the competitive antigen and the the antigen to be detected in the sample to the sensitivity of the competitive immunoassay has been clarified.Finally,the anti-mullerian hormone and copeptin fully automated chemiluminescence immuneassay were developed using the prepared high affinity monoclonal antibody,and it can be used for the detection of clinical samples.The main research results are as follows:?1?The pCAGGS-mFlt3L,pCAGGS-mGM-CSF and pCAGGS-mCCL20 recombinant vectors were successfully constructed via ligating the mFlt3L,mGM-CSF and CCL20 gene fragments to the vector pCAGGS.It was also confirmed that the recombinant vector can efficiently express the target protein in 293F cells.?2?High-affinity antibodies against AMH were successfully prepared by cytokine-assisted DNA immunization.AMH chemiluminescence detection method was successfully constructed using two high-affinity monoclonal antibodies with the value of K_D less than 4 nM.This analytical method showed high sensitivity and specificity for AMH.The detection range was 0.125²0 ng mL^-1,and the detection limit was 0.099 ng mL^-1.The incubation time was only 30 min,while the commercial ELISA kit took 3 hrs.It was used for the detection of AMH in clinical serum samples,and the results were highly consistent with the standard AMH detection ELISA kit.?3?After Cytokine-assisted immunization,the positive rate of hybridoma was significantly improved.Eight stably secreting antibody hybridomas were obtained after one hybridoma preparation and subcloning screening,while only 4 strains were prepared by subcutaneous of twice.So this strategy significantly improves the efficiency of antibody development.After purity and titer testing,the purity can reach 90%and the titers were greater than 1:10⁵.Prepared antibodies can specifically recognize the natural CPP and were used for immunoassay development.After affinity characterization,it was found that the affinity of the antibodies prepared by molecular adjuvant assisted immunization was much higher than that of the antibodies prepared by subcutaneous immunoprecipitation,and the highest affinity antibody had a K_D value of 10^-10 M.?4?After bioinformatics software analysis and peptide scanning,the epitope sequences of four high-affinity antibodies were finally determined,all were located between the 152-161amino acid sequence?APEPFEPAQP?.After alanine mutation scanning and molecular dynamics analysis,it was found that the key amino acids of the four antibodies were mainly proline?P?,phenylalanine?F?and glutamine?Q?.The position of the amino acid sequence containing more hydrophobic amino acids in the antigen is likely to form an epitope.?5?By studying the influence of the affinity change between the competitive antigen and the antibody on the sensitivity of the detection system in both the immunochromatographic and indirect competitive ELISA detection systems,it was found that the limit of detection?LOD?was significantly decreased after moderately reducing the affinity of the competitive antigen to the antibody.The most decrease was 9.5%of the original LOD in the immunochromatographic system,and in the indirect competitive ELISA system,it was 12.1%,the lowest LOD was 0.09 nmol L^-11 after reducing the affinity.It is indicated that a modest reduction in the affinity between the competitive antigen and the antibody can improve the sensitivity of the competitive immunoassay methodology.?6?The fully automated CLIA for CPP detection was successfully constructed using a high affinity anti-CPP antibody as capture antibody and magnetic particles as solid phase.The detection range was 1.2¹250 pmol L^-1 and the LOD was only 6.25 pmol L^-1,which meets the requirement for clinical applications?<18.9 pmol L-1?.Significantly,the whole incubation process could be completed in 30 min as compared to about 4.5 hr for the control ELISA kit.This method was also applied in the detection of CPP in clinical serum specimens and the results were significantly consistent with other commercially available CPP detection ELISA kits.Hence,our assay could provide an alternative platform for the rapid and accurate detection of CPP in clinical samples.