Preparation,Characterization And Diagnostic Application Of HPV16 E7 Protein Monoclonal Antibodies

Background and Objective:Cervical cancer is the fourth most common cancer in women,being next to breast cancer and with approximately 570,000 cases of cervical cancer and 311,000 deaths from the disease in 2018.The persistent infection of high risk human papillomavirus?HR-HPV?is the direct cause of cervical intraepithelial neoplasia?CIN?and cervical cancer,among which HPV16is the highest?53%?.HPV16 E7 protein is the key molecule of HPV carcinogenesis.HPV16 E7protein is selectively expressed in tumor cells.During the development of cervical intraepithelial neoplasia?CIN?from CIN1 to CIN3,the expression of HPV16 E7 protein is increasingly obvious and the expression amount is increasingly high,with HPV16 E7 protein finally appearing in cervical exfoliated cells.Cytology examination?such as TCT?together with HR-HPV DNA detection can increase the detection rate of CIN3,cervical adenocarcinoma in situ and invasive cervical adenocarcinoma.At present,the clinical detection method of human papillomavirus?HPV?is mainly based on the PCR method.However,this method can only be employed to detect HPV DNA and HPV types rather than HPV E6 and E7 protein,causing low accuracy in predicting HPV-positive cancers such as cervical cancer.The purpose of the study is to conduct supplementary detection of HPV16 E7 oncoprotein for those women who test negative in cytological detection while positive in HR-HPV DNA detection.In other words,anti-HPV16 E7 protein monoclonal antibodies will be prepared and utilized to establish a chemiluminescence immunoassay for quantitative detection of HPV16 E7 protein at the protein level.The presence of HPV16 E7 protein indicates the existence of cervical cancer cells,which will be likely to be found in CIN2,CIN3 and even CIN1 stages of cervical intraepithelial neoplasia,having the effect of warning cervical cancer in the early stage.Methods:In this study,HPV16 E7 protein was prepared by using gene cloning technology.Anti-HPV16 E7 protein monoclonal antibodies were prepared by employing hybridoma technology.The characterizations of anti-HPV16 E7 protein monoclonal antibodies were identified and analyzed including monoclonal antibodies pairing,amino acid sequence and affinity determination of mAbs,identification and conservation analysis of the dominant linear B cell epitopes of mAbs,etc.The effectiveness,specificity and preliminary diagnostic value were explored of anti-HPV16 E7 protein monoclonal antibodies for qulitative detection of natural HPV16 E7 protein by employing immunochemistry,immunofluorescence,Western blot and other methods.The diagnostic application values were investigated of anti-HPV16 E7 protein monoclonal antibodies 79A11 and 69E2?79A11as a capture antibody and 69E2 as a detection antibody?for quantitative detection of HPV16 E7protein in ng levels in cervical exfoliated cells or tissues of patients with CIN and cervical cancer by employing the detection system of route double antibody sandwich ELISA based on HRP-detection antibody and TMB,and by employing the detection system of LSAB-lumino-dual-antibody sandwich ELISA.Results as follows:1.Cloning and identification of HPV16 E7 gene and purification of HPV16 E7proteinBy using the total DNA of CaSki cell as the template,PCR amplified about 322bp of HPV16E7 gene.The amino acid sequence of HPV16 E7 protein expressed by recombinant plasmid pET-28a?+?-HPV16 E7 was different from that of CaSki cell line at 28th amino acid?28F and 28L?and was identical with that of SiHa cell line.The molecular weight of HPV16 E7 protein expressed by engineering bacteriumpET-28a?+?-HPV16 E7-Rosetta?DE3?pLysS is about 18 kDa,and it has high expression,soluble expression and the secondary structure as?helix.The HPV16 E7 protein with a purity of more than 95%was obtained by nickel column affinity chromatography and DEAE column purification.2.Preparation of anti-HPV16 E7 protein monoclonal antibodiesHPV16 E7 protein was utilized to immunize the hind-leg foot pad and abdominal cavity of BALB/c mice 5 times.The titer of serum of immunized BALB/c mice before cell fusion was1:128000¹:256000.The fusion rates of mouse inguinal lymph node cells and spleen cells with SP2/0-Ag14 were both 100%.The main subtype of mAbs with high titer from lymph node cells was IgG2a,including 69E2,69D10,54D5,54F4 and 54G5,of which 69A6 was IgM,while the main subtype of monoclonal antibody with high titer from spleen cells was IgM,which was represented by mAb 79A11.The purity of IgG2a subtype monoclonal antibody 69E2 purified by two-step method-caprylic acid-ammonium sulfate precipitation and Protein G purification was over 95%and the antibody titer of 69E2 was the highest?0.19 ng?.The purity of IgM subtype monoclonal antibody 79A11 purified by four-step ammonium sulfate precipitation plus gel filtration chromatography plus IgM column plus gel filtration chromatography was more than 95%,the titer of mAb 79A11 was about 6.25ng.3.Characterization of anti-HPV16 E7 protein monoclonal antibodiesEight mAbs,namely 79A11,69E2,69A6,54D5,54F4,54G5,74F3 and 72E6,reacted with a human c-myc?HIS-c-Myc?containing histidine tag by Western blot without exposure band appearing,excluding the false-positive reaction between the eight mAbs and histidine tag.The 8mAbs were paired with 56 pairs,and there were 4 pairs representing strong positive signals:79A11and 69E2,79A11 and 69A6,79A11 and 74F3,79A11 and 54D5.MAb 79A11 was suitable for serving as capture antibody,while mAbs 69E2,69A6,54D5 and 74F3 were suitable for being detection antibody.Three monoclonal antibodies,79A11,69E2 and 69A6,were determined to be three new anti-HPV16 E7 protein mAbs.The amino acid sequences of heavy chain and light chain of 79A11 and 69E2 were quite different.The affinity between mAb 69E2 and HPV16 E7 protein was high?5.60E-9M?,which was because the binding between mAb 79A11 and HPV16 E7 protein was eluted by high salt,there was considered to be no specific binding between mAb 79A11 and HPV16 E7 protein.The specific epitopes of the three mAbs 79A11,69E2 and 69A6 were all conformational epitopes that were exposed,discontinuous and adjacent peptides HPV16 E7_49-66,HPV16 E7_73-853-85 and HPV16 E7_91-97.The amino acid sequences of the three exposed specific epitopes of three mAbs 79A11,69E2 and 69A6,HPV16 E7_49-66,HPV16 E7_73-853-85 and HPV16 E7_91-97,are highly homologous with those of HPV16 E7 protein of other 30 HPV16 strains downloaded from NCBI.There were two differences in the dominant linear B cell epitopes of three mAbs 79A1,69E2and 69A6.The first was that the results of indirect ELISA of the overlap peptides showed a strong signal in the reaction between mAb 69E2 and HPV16 E7_85-98?p<0.01?,whereas no positive signal was expressed between the two monoclonal antibodies 79A11 and 69A6 and HPV16 E7₈₅-₉₈.The second difference was that the results of indirect competitive ELISA of overlapping peptides at 10?mol/L showed that the inhibition rates of overlapping peptides HPV16 E7_49-669-66 inhibited by three mAbs 79A11,69E2 and 69A6 were 40.666%,94.28%and 16.28%respectively.The two differences suggested that the dominant linear B cell epitopes of these three mAbs were different.4.Diagnostic application of anti-HPV16 E7 protein monoclonal antibodies inimmunochemistry,immunofluorescence and Western blotEight mAbs,namely 79A11,69E2,69A6,54D5,54F4,54G5,74F3 and 72E6,reacted with CaSki cells by immunocytochemistry at the content as low as 7.8125 ng,all of which showed typical brownish granules in CaSki cells,while no brownish granules in HeLa cells appeared in reaction with HeLa cells.When three mAbs,namely 79A11,69E2 and 69A6,reacted with HPV16-positive cervical cancer tissues at 0.895?g/tablet,5?g/tablet and 1.84?g/tablet respectively,typical brownish yellow granules appeared,while no brownish yellow granules were showed in reaction with HPV18 positive cervical cancer tissues.All of the three mAbs 79A11,69E2,69A6 reacted with CaSki cells at 1?g/mL and showed green fluorescence,while no green fluorescence appeared in reaction with HeLa cells.Three mAbs 79A11,69E2 and 69A6 reacted with the total protein of CaSki cells by Western blot,generating typical exposure bands at 18 kDa,while no typical exposure bands were expressed in reaction with the total protein of HeLa cells.5.Diagnostic application of mAbs 79A11 and 69E2 for quantitative detection of HPV16 E7 protein by employing the detection system of double antibody sandwich ELISA based on HRP-69E2 and TMBThe optimized conditions of the detection system included:the optimal coating concentration of capture antibody 79A11 was 2?g/ml,the optimal concentration of detect antibody HRP-69E2 was 1?g/ml,the background of 5%skim milk for 2 h was low and the positive signal was strong.Double antibody sandwich ELISA showed that when the antigen HPV16 E7 protein was diluted with the method of equal ratio and equal difference?0,25,50,100,200,400,600,800,1000 ng/well?at ng levels,the determination coefficient R² of the straight-line fitting of reference curve was 0.9723.The abscissa was the two points with the antigen as 0 and the low concentration of 25 ng with the variation coefficient?CV?value lower than 10%,and the corresponding RLU value was the ordinate,so as to obtain the equation of calculation the limit of detection is Y=0.01122*X+0.6896 and the value of determination coefficient R²=0.9889,the RLU=3*0.6896=2.0688,the limit of detection=?2.0688-0.6896?/0.01122=122.9234ng.The CV value was less than 20%,therefore the limit of detection was also the limit of quantitation as 122.9234ng,generating the need of exploring a detection system with higher sensitivity.6.Diagnostic application of mAbs 79A11 and 69E2 for quantitative detection of HPV16 E7 proteinin by employing chemiluminescence Immunoassay based on LSAB-lumino-dual-antibody sandwich ELISAThe optimized conditions of chemiluminescent immunoassay included:the optimized coating concentration of capture antibody 79A11 was 2 ug/mL,0.25%BSA blocking solution was sealed for2 h,the antigen HPV16 E7 protein was diluted at ng level?0,25,50,100,200,400,600,800,1000ng/well?and reacted with the capture antibody at 37?for 2 h,the optimization working concentration of detection antibody biotin-69E2 and HRP-streptavidin were both 1 ug/ml,and luminol reaction for 10 min,etc.The reference curve was prepared with optimized conditions,in which regression equation of straight line fitting was Y=53.35*X+10294,and the determination coefficients R² of straight line fitting and double logarithm fitting were 0.9666 and 0.956respectively.The content of HPV16 E7 protein in 0 and low concentration of 25ng was taken as abscissa,the corresponding relative light units?RLU?value was taken as ordinate and the regression equation Y=363.1*X+3441 was obtained for calculation the limit of detection,R²=0.9823,RLU=3*3441=10324 with S/N=3,the limit of detection=?10324-3441?/363.1=18.9562ng.The CV value was less than 20%,therefore the limit of detection was also the limit of quantitation as18.9562ng,whose sensitivity was 6.48 folds as high as that of routine double antibody sandwich ELISA.The RLU values of the samples were brought into the equation Y=53.35*X+10294,and it was found that the content of HPV16 E7 protein in 20?g total protein of four normal cervical tissues and four cervical HPV52 positive cancer tissues were less than the limit of quantification of 18.9562ng;and that the content of HPV16 E7 protein in 20?g total protein of 12 HPV16 positive cervical cancer tissues fluctuated from 102.1743 to 438.2473ng,showing significant difference with that in normal cervical tissues and HPV52 positive cervical cancer tissues?p<0.01?.ConclusionIn this study,HPV16 E7 protein with a molecular weight of about 18 kDa,soluble expression and a secondary structure as?helix was prepared.Two new representative mAbs 79A11?IgM?and69E2?IgG2a?were obtained,whose specific epitopes were both conformational epitopes and whose locations were on two monomers of HPV16 E7 protein dimer respectively.MAbs 79A11 and 69E2had potential diagnostic value for qualitative detection of natural HPV16 E7 protein by employing immunochemistry,immunofluorescence and Western blot.MAbs 79A11 and 69E2 were used in chemiluminescence immunoassay based on LSAB luminol double antibody sandwich ELISA,the limit of quantitative detection of HPV16 E7 protein was 18.9562 ng.The detection rate of HPV16E7 protein by applying this chemiluminescent immunoassay in 12 cases HPV16 positive cervical cancer was 100%,which suggested that mAbs 79A11 and 69E2 had potential diagnostic value for quantitative detection of natural HPV16 E7 protein in ng levels by employing this chemiluminescent immunoassay.