Preparation of subunit and immunology characteristics of monoclonal antibody M26-32 against pan species malaria parasite
TAO Zhi-yong1,XU Sui2,XIA Hui1,GU Ya-ping2,LIU Yao-bao2,FANG Qiang1,GAO Qi2
1. Department of Microbiology and Parasitology, Bengbu Medical College,Anhui Key Laboratory of Infection and Immunity, Bengbu 233030, China; 2. Jiangsu Institute of Parasitic Diseases, Key Laboratory of Parasitic Disease Control and Prevention, Ministry of Health, Jiangsu Provincial Key Laboratory of Parasite Molecular Biology, Wuxi 214064, China
Abstract：The pentamer IgM monoclonal antibody M26-32 was modified into subunit and taken advantage of immunogold labeling of natural and modified M26-32 antibodies for detection of malaria antigens. After thawing and cultivation, M26-32 hybridoma was inoculated in BALB/c mice intraperitoneally and the ascitic fluid was collected. M26-32 was purified using HiTrap IgM Hp column, and the IgM antibody was lysed into IgM subunit with L-cysteine. After verification of titers by IFA, natural and L-cysteine lysed M26-32 antibodies were labeled by immunogold, and tested with both Plasmodium falciparum and Plasmodium vivax soluble antigens. A total of 20 mice were used and about 80 mL ascitic fluid was collected. Purified M26-32 antibody was obtained by using HiTrap IgM HP column. Native PAGE showed that L-cysteine could lyse the pentamer IgM into IgM subunit. IFA results showed that no titer difference between two types of M26-32 monoclonal antibody. After labeling with immunogold, both two types M26-32 could recognize 1∶100 diluted Plasmodium falciparum antigens and 1∶10 diluted Plasmodium vivax antigens. A method for modifying pentamer IgM M26-32 monoclonal antibody into IgM subunit by using L-cysteine have been proved, and a method for labeling both types M26-32 with immunogold have been established, providing useful information for designing malaria RDT based on M26-32 monoclonal antibody.
陶志勇，徐岁，夏惠，顾亚萍，刘耀宝，方强，高琪. 抗疟原虫单抗M26-32IgM的单体化及其免疫学特性研究[J]. 中国人兽共患病学报, 2014, 30(6): 551-555.
TAO Zhi-yong,XU Sui,XIA Hui,GU Ya-ping,LIU Yao-bao,FANG Qiang,GAO Qi. Preparation of subunit and immunology characteristics of monoclonal antibody M26-32 against pan species malaria parasite. Chinese Journal of Zoonoses, 2014, 30(6): 551-555.
[1]Li WL, Liu EX, Fan RG, et al. Characterization of Plasmodium yoelii antigens by monoclonal antibodies. I. Common antigens between erythrocytic stages of Plasmodium yoelii and human malaria parasites[J]. Chin J Parasitol Parasit Dis, 1984, 2(2): 83-87. (in Chinese) 李文渌,刘尔翔,樊汝恭,等.用单克隆抗体分析约氏疟原虫抗原的研究 I 约氏疟原虫红内期与人疟原虫的共同抗原[J].中国寄生虫学与寄生虫病杂志,1984,2(2):83-87. [2]Li WL, Liu EX,Fan RG, et al. Characterization of three monoclonal antibodys against Plasmodium falciparum[J]. Chin J Cell Mol Immunol, 1985, 1(4): 35-36. (in Chinese) 李文渌,刘尔翔,樊汝恭,等.三株抗恶性疟McAb的鉴定[J].细胞与分子免疫学杂志,1985, 1(4):35-36. [3]Wu LJ, Liu EX, Li WL, et al. Localization of the antigen in erythrogytic stages of Plasmodium yoelii by immuno-electron microscopy[J]. Chin J Parasitol Parasit Dis, 1992, 10(1): 30-32. (in Chinese) 吴莉菊, 刘尔翔, 李文渌, 等. 约氏疟原虫红内期抗原定位的免疫电镜研究[J]. 中国寄生虫学与寄生虫病杂志,1992, 10(1):30-32. [4]Gao Q, Yang ZY, Zhang WQ. Study on detecting blood stage P. falciparum antigen by using sandwiched monoclonal antibody-ELISA test[J]. Chin J Schistosomiasis Ctrl, 1990, 2(2): 43-45. (in Chinese) 高琪, 杨仲炎, 张炜琪. 用单克隆抗体-酶联免疫吸附试验双抗体夹心法检测人体疟原虫抗原的研究[J]. 中国血吸虫病防治杂志,1990, 2(2):43-45. [5]Gao Q, Yang CX, Zhang SY, et al. Detection of blood stage antigens of Plasmodium vivax by sandwich ELISA using pan-species monoclonal antibodies and polyclonal antibodies[J]. Southeast Asian J Trop Med Public Health, 1991, 22(3): 393-396.[6]Wang LQ, Gao Q, He W, et al. Analysis on subtypes and isoelectric points of effective immunoglobulins of monoclonal antibody M26-32 from ascitic fluid[J]. Chin J Parasit Dis Ctrl, 1997, 10(2): 84-86. (in Chinese) 王丽琴, 高琪, 何伟, 等. 单克隆抗体M26-32腹水有效免疫球蛋白的亚类及等电点的分析[J]. 中国寄生虫病防治杂志,1997, 10(2):84-86. [7]Andrew SM, Titus JA. Fragmentation of immunoglobulin M[M]. London: John Wiley and Sons, 2001:3-4. [8]Wang DQ, Tang LH, Gu ZC, et al. Application of the indirect fluorescent antibody assay in the study of malaria infection in the Yangtze River Three Gorges Reservoir, China[J]. Malar J, 2009, 8: 199. [9]Hu SF, Tao ZY, Xia H. Immunoreactivity comparison between Plasmodium vivax antigen and P. falciparum antigen[J]. Chin J Zoonoses, 2009, 25(10): 988-990. (in Chinese) 胡守锋, 陶志勇, 夏惠. 间日疟原虫和恶性疟原虫可溶性抗原免疫反应性比较[J].中国人兽共患病学报,2009,25(10):988-990. [10]Messeter L, Brodin T, Chester MA, et al. Mouse monoclonal antibodies with anti-A, anti-B and anti-A, B specificities, some superior to human polyclonal ABO reagents[J]. Vox Sang, 1984, 46(4): 185-194. [11]Yukawa N, Matsuda H, Seo Y, et al. Peroxidase labeling of IgMs fragment of ABO blood group specific mouse monoclonal IgM[J]. Bioconjug Chem, 1994, 5(3): 273-277. [12]Wang LQ, Gao Q, He W, et al. Comparison of three methods for purification of monoclonal antibody M26-32[J]. Chin J Schistosomiasis Ctrl, 1997, 9(2): 122. (in Chinese) 王丽琴, 高琪, 何伟, 等. 3种提纯M26-32单抗方法的比较[J]. 中国血吸虫病防治杂志,1997, 9(2):122. [13]Liu X, Song HX. Development and application of thiophilic adsorption chromatography for antibody purification[J]. Chin J Chrom, 2006, 24(1): 88-92. (in Chinese) 刘玄,宋宏新.嗜硫色谱纯化抗体技术的研究进展[J].色谱,2006,24(1):88-92. [14]Porath J, Maisano F, Belew M. Thiophilic adsorption-a new method for protein fractionation[J]. FEBS Lett, 1985, 185(2): 306-310.
2014, Vol. 30 
