抗大片形吸虫Cat L1单克隆抗体的制备及其双抗体夹心ELISA检测方法的建立

doi:10.16431/j.cnki.1671-7236.2022.02.030

Abstract

Abstract: 【Objective】 This study was intend to obtain cathepsin L1(rFgCat L1) specific monoclonal antibody and construct the double antibody sandwich ELISA.【Method】 Five BALB/c mice were immunized with 1 mg/mL rFgCat L1 protein for four times.Mouse splenocytes were isolated and fused with SP2/0 cells to construct hybridoma cells.Strong positive hybridoma cell lines were screened, 1×10⁶ cells were injected intraperitoneally per mouse to prepare monoclonal antibodies.Antibody titer and antigenic epitope were detected using ELISA method, antibody subtype and specificity were identified using Western blotting method.The double antibody sandwich ELISA was constructed by combining the anti-rFgCat L1 polyclonal antibody, and its sensitivity and specificity were tested.The positive and negative critical value was screened by 20 negative sera with positive control, and the constructed double antibody sandwich ELISA was verified by 47 goat positive sera and 47 dairy cow positive sera.【Result】 After immunization, the antibody titers in serum of 4 mice were all more than 10⁴.After isolated mouse with the highest immune response spleen cells were fused with SP2/0 cells total of 8 of them were positive cell lines were obtained after selective culture.5D5 and 7G6 were identified as strong positive strains with stable antibody secretion.After multiple subcloning screens and subcultures, the antibodies secreted in the cell supernatant were stable, with titers of 2⁹ and 2¹⁰ respectively, with ascites titers of 10⁷ and 10⁸.Western blotting and antibody subtype identification kits identified that the two antibodies were IgG1 type and the light chain was kappa type, both of which could specifically bind FgESP.According to the same antigen site was recognized by the two kinds of antibodies, the antigen titer of the two monoclonal antibodies were comparied, 7G6 was used as the coating antibody, and anti-rFgCat L1 was used as the enzyme-labeled secondary antibody.The optimized condition of method was that 7G6 was coated at a concentration of 2 μg/mL, the dilution concentration of anti-rFgCat L1 polyclonal antibody was 25 μg/mL, the dilution of Don-HRP-conjugated was 1∶4 000, 5% skimmed milk powder was selected as the blocking solution and the color development time was 25 min.The method was proved that could recognize the lowest antigen concentration of 0.625 μg/mL, also could specifically recognize antigen of Fasciola fasciatus.The constructed sandwich ELISA method was used for antigen detection of 47 dairy cow positive serum and 47 goat positive serum infective samples kept in the laboratory and the positive antigen rate were 72.3% and 78.7%, respectively.【Conclusion】 Anti-rFgCat L1 monoclonal antibody was successfully prepared and the double-sheet sandwich ELISA method for fascioliasis was constructed, which provided a good theoretical basis and material basis for the development of low-cost and rapid diagnostic kits.

Key words: Fasciala gigantica; cathepsin L1; monoclonal antibody; double antibody sandwich ELISA

CLC Number:

R446.61

WANG Leming, WANG Yurui, ZENG Zixuan, RAO Guoshun, WU Zhengjiao, JIN Weikun, WANG Dongying. Preparation of Cathepsin L1 Monoclonal Antibody Against Fasclala gigantica and Establishment of Double Antibody Sandwich ELISA[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 677-686.

References

[1] MASCOMA S. Epidemiology of fascioliasis in human endemic areas[J]. Journal of Helminthology, 2005, 79(3): 207-216.
[2] WEBB C M, CABADA M M. Recent developments in the epidemiology, diagnosis, and treatment of Fasciola infection[J]. Current Opinion in Infectious Diseases, 2018, 5(31): 409-414.
[3] ANURACPREEDA P, SRIRAKAM T, PANDONLAN S, et al. Production and characterization of a monoclonal antibody against recombinant cathepsin L1 of Fasciola gigantica[J]. Experimental Parasitology, 2014, 136: 1-9.
[4] MEEMON K, SOBHON P. Juvenile-specific cathepsin proteases in Fasciola spp. : Their characteristics and vaccine efficacies[J]. Parasitology Research, 2015, 114(8): 2807-2813.
[5] 黄维义, 张为宇, 田锷. 大片吸虫分泌排泄抗原的分析[J]. 广西农业大学学报, 1997, 2: 36-40.
HUANG W Y, ZHANG W Y, TIAN E. Analysis of Fasciola gigantica excretory-secreting antigens[J]. Journal of Guangxi Agricaltural University, 1997, 2: 36-40. (in Chinese)
[6] ELSIBAEI M M, ALI N M, IBRAHIM A N. Evaluation of the diagnostic efficacy of Fasciola adult worm vomit for serodiagnosis of human fasciolosis[J]. Parasitology Research, 2013, 112(5): 1849-1855.
[7] ABDOLAHI K S, SARKARI B, MOSHFE A, et al, Production of monoclonal antibody against excretory-secretory antigen of Fasciola hepatica and evaluation of its efficacy in the diagnosis of fascioliasis[J]. Monoclonal Antibodies in Immunodiagnosis & Immunotherapy, 2017, 36(1): 8-14
[8] ANURACPREEDA P, CHAWENGKIRTTIKUL R, SOBHON P. Immunodiagnostic monoclonal antibody-based sandwich ELISA of fasciolosis by detection of Fasciola gigantica circulating fatty acid binding protein[J]. Parasitology, 2016, 143(11): 1369-1381.
[9] COLLINS P R, STACK C M, O'NEILL S M, et al. Cathepsin L1, the major protease involved in liver fluke (Fasciola hepatica) virulence propeptide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells[J]. Journal of Biological Chemistry, 2004, 279(17): 17038-17046.
[10] BERASAIN P, GONI F, MCGONIGLE S, et al. Proteinases secreted by Fasciola hepatica degrade extracelullar matrix and basement membrane components[J]. Journal of Parasitology, 1997, 83(1): 1-5.
[11] SMITH A M, DOWD A J, MCGONIGLE S, et al. Purification of a cathepsin L-like proteinase secreted by adult Fasciola hepatica[J]. Molecular and Biochemical Parasitology, 1993, 62(1): 1-8.
[12] CARMONA C, DOWD A J, SMITH A M, et al. Cathepsin L proteinase secreted by Fasciola hepatica in vitro prevents antibody-mediated eosinopHil attachment to newly excysted juveniles[J]. Molecular & Biochemical Parasitology, 1993, 62(62): 9-17.
[13] BRADY M T, O'NEILL S M, DALTON J P, et al. Fasciola hepatica suppresses a protective Th1 response against bordetella pertussis[J]. Infection & Immunity, 1999, 67(10): 5372-5378.
[14] O'NEILL S M, BRADY M T, CALLANAN J J, et al. Fasciola hepatica infection downregulates Th1 responses in mice[J]. Parasite Immunology, 2000(22): 147-155.
[15] O'NEILL S M, MILLS K H G, DALTON J P. Fasciola hepatica cathepsin L cysteine proteinase suppresses Bordetella pertussis-specific interferon-γ production in vivo[J]. Parasite Immunology, 2001, 23(10): 541-547.
[16] 王晓旭. 肝片吸虫病诊断抗原的筛选及间接ELISA方法的建立[D]. 大庆: 黑龙江八一农垦大学, 2019.
WANG X X, Screening of diagnostic antigen for fascioliasis hepatica and establishment of indirect ELISA[D]. Daqing: Heilongjiang Bayi Agricultural University, 2019. (in Chinese)
[17] 王乐铭, 侯林静, 饶国顺, 等. 大片形吸虫CatL1蛋白的原核表达及其特异性多克隆抗体的制备[J]. 黑龙江畜牧兽医, 2020, 18: 68-71.
WANG L M, HOU L J, RAO G S, et al. Prokaryotic expression of Fasciola gigantica cathepsin L1 protein and preparation of its specific polyclonal antibody[J]. Heilongjiang Animal Science and Veterinary Medicine, 2020, 18: 68-71. (in Chinese)
[18] 万文徽. 单克隆抗体亲和常数的测定[J]. 细胞与分子免疫学杂志, 1993, 2: 72-75.
WAN W H. Determination of affinity constant of monoclonal antibody[J]. Journal Monoclonal Antibody, 1993, 2: 72-75. (in Chinese)
[19] FAFETINE J M, TIJHAAR E, PAWESKA J T, et al. Cloning and expression of Rift valley fever virus nucleocapsid (N) protein and evaluation of a N-protein based indirect ELISA for the detection of specific IgG and IgM antibodies in domestic ruminants[J]. Veterinary Microbiology, 2007, 121(1-2): 29-38.
[20] 任瑞敏, 王云龙, 张怡青, 等. 利用改良后的脾内免疫和半固体培养基法制备单克隆抗体[J]. 生物技术通报, 2013, 8: 166-169.
RENG R M, WANG Y L, ZHANG Y Q, et al. The improved intrasplenic immunization and semi solid medium to prepare monoclonal antibody[J]. Biotechnol Bulletin, 2013, 8: 166-169. (in Chinese)
[21] 吴文德. 罗非鱼无乳链球菌抗原抗体快速检测方法的研究[D]. 南宁: 广西大学, 2017.
WU W D. Study on the rapid detection method of Streptococcus agalactiae antigen and antibody from Tilapia[D]. Nanning: Guangxi University, 2017. (in Chinese)
[22] 刘乐, 廖旻晶, 徐叶, 等. 人巨细胞病毒gH蛋白表达及其单克隆抗体的制备[J]. 激光生物学报, 2020, 29(3): 260-267.
LIU L, LIAO M J, XU Y, et al. Expression of Human cytomegalovirus gH protein and preparation of its monoclonal antibody[J]. Acta Laser Biology Sinica, 2020, 29(3): 260-267. (in Chinese)
[23] 杨佰启, 温贵兰, 田浪, 等. 猪繁殖与呼吸综合征病毒BJ3毒株N蛋白的原核表达及其单克隆抗体的制备[J]. 中国兽医科学, 2020, 50(9): 1119-1126.
YANG B Q, WEN G L. TIAN L, et al. Prokaryotic expression of N protein of Porcine reproductive and respiratory syndrome virus BJ3 strain and preparation of its monoclonal antibody[J]. Chinese Veterinary Science, 2020, 50(9): 1119-1126. (in Chinese)
[24] 蒋佳伊, 张磊, 秦露, 等. 高灵敏度黄曲霉毒素B₁单克隆抗体的制备及其在中药材酸枣仁污染快速检测中的应用[J]. 中国中药杂志, 2020, 45(16): 3900-3907.
JIANG J Y, ZHANG L, QIN L, et al. Preparation of highly sensitive monoclonal antibody against aflatoxin B₁ and its application in rapid detection of contamination in Ziziphi Spinosae semen[J]. China Journal of Chinese Materia Medica, 2020, 45(16): 3900-3907. (in Chinese)
[25] 李雪, 魏志鹏, 赵明龙, 等. 大片形吸虫病早期诊断夹心ELISA方法的建立及应用[J]. 中国畜牧兽医, 2016, 43(4): 899-905.
LI X, WEI Z P, ZHAO M L, et al. Development and applpcation of sandwich ELISA for diagnosis of Fasciola gigantica early stage[J]. China Animal Husbandry & Veterinary Medicine, 2016, 43(4): 899-905. (in Chinese)
[26] 李超, 林祥梅, 梅琳, 等. 牛γ-干扰素单克隆抗体的制备及ELISA检测方法的建立[J]. 中国畜牧兽医, 2011, 38(1): 72-76.
LI C, LIN X M, MEI L, et al. Developement of monoclonal antibody against bovine interferon-γ and establishment of ELISA detection method[J]. China Animal Husbandry & Veterinary Medicine, 2011, 38(1): 72-76. (in Chinese)

Preparation of Cathepsin L1 Monoclonal Antibody Against Fasclala gigantica and Establishment of Double Antibody Sandwich ELISA

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