Preparation of Monoclonal Antibody against P65 Protein of Mycoplasma hyopneumoniae

P65 protein, the major immunodominant protein of Mycoplasma hyopneu-moniae （Mhp） exhibiting no cross-reaction with other mycoplasmas, is general y used as a target protein for Mhp detection. In this study, BALB/c mice were immunized with prokaryotical y expressed P65 recombinant protein to prepare monoclonal anti-body. After screening with Mhp whole-cel protein and P65 protein, a specific hy-bridoma cel line, 3G12, was obtained by ELISA. Identification results indicated that the antibody secreted by 3G12 hybridoma cel s could react with P65 protein and Mhp whole-cel protein. According to indirect ELISA assay, 3G12 cel culture super-natant possessed a titer of 1∶12 800 against P65 protein and 1∶3 200 against Mhp whole-cel protein; 3G12 ascites possessed a titer of above 1∶4 000 000 against P65 protein and above 1∶20 000 against Mhp 168 whole-cel protein. After long-term in vitro culture and continuous freezing-thawing, 3G12 cel line could stably secrete antibodies. A monoclonal antibody against P65 protein and Mhp whole-cel protein was successful y obtained in the present study, which provided basis for further in-vestigating the pathogenic mechanism of Mhp and establishing diagnostic methods of Mycoplasmal pneumonia of swine （MPS）.

Reduction rate of monoclonal protein as a useful prognostic factor in standard-risk group of newly diagnosed multiple myeloma

BACKGROUND Multiple myeloma(MM)is a common hematologic malignancy that originates from a malignant clone of plasma cells.Solitary plasmacytoma,history of diabetes,and platelet count are considered as prognostic factors for MM.But some patients are still associated with much worse outcomes without any prognostic predictors.This study aimed to observe the reduction rate of monoclonal protein(M protein)after the first and fourth chemotherapy cycles,which is considered as a new prognostic factor for progression-free survival(PFS)in standard-risk group of newly diagnosed MM patients.AIM To investigate the reduction rate of M protein after first and fourth cycle chemotherapy as a useful prognostic factor.METHODS A total of 316 patients diagnosed with MM for the first time between 2010 and 2019 at the Lishui Municipal Central Hospital were included.All patients were diagnosed according to the National Comprehensive Cancer Network(NCCN)2020.V1 diagnostic criteria.The risk assessment was performed by the Mayo Stratification for Macroglobulinemia and Risk-Adapted Therapy guidelines.After diagnosis,164 patients were evaluated and underwent treatment with four to eight courses of continuous induction chemotherapy.The patients with no response after induction treatment were administered additional therapy following the NCCN 2020.V1 criteria.The following baseline data from the patients were collected:Gender,age at diagnosis,Durie-Salmon stage,glutamicpyruvic transaminase,glutamic-oxaloacetic transaminase,catabolite activator protein,albumin/globulin ratio,lactate dehydrogenase,translocation(t)(6;14),t(11;14),maintenance regimen,total cholesterol(TC),triglyceride,and phosphorous.All baseline data and the reduction rate of M protein after each chemotherapy cycle from the first to fourth were assessed by univariate analysis.The factors influencing the overall survival and PFS were then assessed by multivariate analysis.We found the first cycle(C1)reduction rate and the fourth cycle(C4)reduction rate as predictors of PFS.Then,PFS was compared between patients with a C1 reduction rate of M protein of≥25%vs<25%and≥50%vs<50%,and between patients with a C4 reduction rate of≥25%vs<25%,≥50%vs<50%,and≥75%vs<75%.RESULTS Multivariate analysis revealed age[hazard ratio(HR):1.059,95%confidence interval(95%CI):1.033-1.085,P≤0.001],International Staging System stage(HR:2.136,95%CI:1.500-3.041,P≤0.001),autotransplantion(HR:0.201,95%CI:0.069-0.583,P=0.019),TC(HR:0.689,95%CI:0.533-0.891,P=0.019),C1 reduction rate(HR:0474,95%CI:0.293-0.767,P=0.019),and C4 reduction rate(HR:0.254,95%CI:0.139-0.463,P=0.019)as predictors of PFS.The Kaplan-Meier survival analysis and the log-rank tests revealed that a higher reduction rate of M protein after first cycle(≥50%)and fourth cycle(≥75%)chemotherapy was associated with a longer PFS than the lower one.CONCLUSION Higher reduction rates of M protein after the first and fourth chemotherapy cycles can act as advantageous prognostic factors for PFS in standard-risk group of MM patients during initial diagnosis.

Preparation of monoclonal antibody to P53 and its clinical application

Objective：The aim of this study was to prepare monoclonal antibody against P53, a kind of tumor suppressor protein,and use the antibody initial y in clinical immunoassay. Methods：Monoclonal antibody was prepared and identified via the classic protocol of monoclonal antibody preparation. Identified monoclonal antibodies were purified by af inity chro-matography. Antibody titer was determined by enzyme linked immunosorbent assay （ELISA）. The specific binding activity of antibody was detected by Western blotting and immunohistochemistry. Results：Three strains of monoclonal antibodies named 1P15, 2P37 and 3P40 were obtained and purified by af inity chromatography. The purity of antibodies was higher than 90%. The titers of antibodies were more than 1：6000. Western blot and immunohistochemistry assay showed that the specific antibody can combine with endogenous P53 protein in the tumor celllines and determine the expression of P53 in tumor tis-sue. Conclusion：Three strains of monoclonal antibodies with high af inity to P53 were successful y established, which can be used for detecting the expression of P53 in tumor cells or tissue.

Expression and purification of Ctomp2aa167-aa434 and preparation of its mAb

Chlamydia trachomatis outer membrane protein 2 （Ctomp2） is a major immunogen in chlamydial infections and a highly genus-conserved structural protein of all Chlamydia species. To purify the protein and to prepare monoclonal antibodies （mAbs） against it, the recombinant protein was induced by IPTG, which was confirmed by SDS-PAGE and purified by means of a Ni^2＋ -charged resin colunm. The denatured protein was refolded in the GSH-GSSH buffer gradually and identified by Westem blotting. Then the BALB/c mice were immunized with the recombinant protein to prepare the mAb against Ctomp2. The obtained mAbs were characterized. Genital specimens were tested with indirect ELISA mostly made of the mAb and cell culture in 84 patients with genital symptoms. The results showed that high-level expression of the recombinant protein was achieved, which existed as inclusion body and amounted to 38 % of total bacterium protein. A mAb against Ctomp2 was obtained. It belongs to IgG 2b. The titers were as high as 1：40 000. The Western blotting showed that the mAb only reacted with the recombinant protein. It had no crossing reactions against E. coli, N. gonorhoea, M. hominis, U. urealyticum and M. penetrans. It had high specifity. In comparison with gold standard test-ceil culture, the sensitivities, specificities, positive predictive values and negative predictive values of indirect ELISA were 95.24%, 100%, 100% and 98.44%, respectively. The above-mentioned research work contributed not only to the further study of the structure and function of this protein, but also to the establishment of the method for its clinical application, for it had not been reported before.