中国新药杂志

H因子结合蛋白单克隆抗体的制备及ELISA法含量检测初探

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苏桂民 ¹^,² , 冀颖 ¹^,² , 纪国存 ¹^,² , 龙静 ¹^,² , 郭通 ¹^,² , 申卫家 ¹^,² , 张宇 ¹^,² , 朱卫华 ¹^,² , 杜琳 ¹^,²

中国新药杂志 | 生物医药前沿 2021,30(10): 921-928

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中国新药杂志 |生物医药前沿 2021 , 30 (10) : 921 -928

H因子结合蛋白单克隆抗体的制备及ELISA法含量检测初探

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苏桂民¹^,², 冀颖¹^,², 纪国存¹^,², 龙静¹^,², 郭通¹^,², 申卫家¹^,², 张宇¹^,², 朱卫华¹^,², 杜琳¹^,²

作者信息

¹北京市细菌性疫苗工程技术研究中心，北京　100176

²北京智飞绿竹生物制药有限公司，北京　100176

作者简介:

苏桂民，男，硕士研究生，生物制药高级工程师，主要从事疫苗研发工作。联系电话：（010）67872389，E-mail：suguimin@zhifeishengwu.com。

Preparation of monoclonal antibodies against fHBP and its application in determination of antigen content by ELISA

Gui-min SU¹^,², Ying JI¹^,², Guo-cun JI¹^,², Jing LONG¹^,², Tong GUO¹^,², Wei-jia SHEN¹^,², Yu ZHANG¹^,², Wei-hua ZHU¹^,², Lin DU¹^,²

Affiliations

¹Beijing Bacterial Vaccine Engineering Research Centre, Beijing 100176, China

²Beijing Zhifei Lvzhu Biopharmaceutical Co., Ltd., Beijing 100176, China

出版时间: 2021-05-30

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摘要

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目的：

制备B群脑膜炎球菌H因子结合蛋白A（fHBP A）和H因子结合蛋白B（fHBP B）亚家族的单克隆抗体，建立检测fHBP抗原含量的ELISA方法。

方法：

重组表达并纯化fHBP A（His）和fHBP B（His）标签蛋白免疫小鼠，将小鼠脾细胞与SP2/0细胞融合，筛选3株稳定分泌抗fHBP单克隆抗体的细胞株A，B和AB。以抗AB为一抗，辣根过氧化物酶标记抗A、抗B为二抗，分别建立检测fHBP A和fHBP B的双抗夹心ELISA方法。

结果：

3种单抗效价均在4.4×10⁵~8.8×10⁵，Western Blot及ELISA显示3种抗体均有结合特异性，且单抗AB和A，B对应不同的抗原识别位点，相互独立。双抗夹心ELISA方法灵敏度达到10 ng·mL^-1，变异系数<10%。

结论：

采用单克隆抗体检测fHBP抗原的ELISA方法特异性和适用性良好，为进一步建立快速灵敏检测和鉴定fHBP抗原的方法奠定了基础。

关键词

H因子结合蛋白 / 单克隆抗体 / ELISA / B群脑膜炎球菌

Abstract

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Objective:

To obtain monoclonal antibodies of the factor H binding protein (fHBP) A and B subfamily proteins in meningococcal group B, and establish an ELISA detection method which can provide a new way for quantitation and identification of fHBP antigen.

Methods:

Mice were immunized with purified histidine-labeled recombinant fHBP (His), the spleen cells of mouse were fused with SP2/0 cells. 3 cell strains with stable secretion of anti-fHBP monoclonal antibodies were acquired, respectively named A, B and AB. To establish a double-antibody sandwich ELISA method for detecting recombinant fHBP antigen, AB was coated as primary antibodies, A and B were labeled by horseradish peroxidase (HRP) as the second antibody.

Results:

The GMTs valences of A, B and AB antibodies produced in mouse ascites were all between 4.4~8.8×10⁵, and Western Blot showed that the 3 antibodies could specifically bind the corresponding recombinant fHBP antigens. The direct ELISA test also demonstrated that the 3 monoclonal antibodies merely reacted with proteins containing fHBP A or fHBP B through different recognition sites on antigens, independent of each other. The double-antibody sandwich ELISA method had good specificity and applicability, with sensitivity up to 10 ng·mL^-1 and coefficient variation of less than 10%.

Conclusion:

The double antibody sandwich ELISA method based on monoclonal antibody has good specificity and applicability, which laid a foundation to further establish a sensitive and rapid method for detection and identification of fHBP antigen.

Key words

fHBP / monoclonal antibody / ELISA / meningococcal group B

引用本文

苏桂民, 冀颖, 纪国存, 龙静, 郭通, 申卫家, 张宇, 朱卫华, 杜琳. H因子结合蛋白单克隆抗体的制备及ELISA法含量检测初探. 中国新药杂志, 2021 , 30 (10) : 921 -928 .

Gui-min SU, Ying JI, Guo-cun JI, Jing LONG, Tong GUO, Wei-jia SHEN, Yu ZHANG, Wei-hua ZHU, Lin DU. Preparation of monoclonal antibodies against fHBP and its application in determination of antigen content by ELISA[J]. Chinese Journal of New Drugs, 2021 , 30 (10) : 921 -928 .

参考文献

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参考文献引证文献

排序方式：

[1]

邵祝军, 徐丽, 高源, 等. 中国流行性脑脊髓膜炎流行菌群变化趋势分析[J]. 中国计划免疫, 2007, 13(6): 541-544.

[2]

李军宏, 吴丹, 温宁, 等. 2015—2019年中国流行性脑脊髓膜炎血清群分布特征[J]. 中国疫苗和免疫, 2020, 26(3): 241-244.

[3]

VESIKARI T, ESPOSITO S, PRYMULA R, et al. Immunogenicity and safety of an investigational multicomponent, recombinant, meningococcal serogroup B vaccine (4CMenB) administered concomitantly with routine infant and child vaccinations: results of two randomised trials[J]. Lancet, 2013, 381(9869): 825-835.

[4]

MARTIN NG, SNAPE MD. A multicomponent serogroup B meningococcal vaccine is licensed for use in Europe: what do we know, and what are we yet to learn?[J]. Expert Rev Vaccines, 2013, 12(8): 837-858.

[5]

BEERNINK PT, LEIPUS A, GRANOFF DM. Rapid genetic grouping of factor h-binding protein (genome-derived neisserial antigen 1870), a promising group B meningococcal vaccine candidate[J]. Clin Vaccine Immunol, 2006, 13(7): 758-763.

[6]

SEIB KL, SCARSELLI M, COMANDUCCI M, et al. Neisseria meningitidis factor H-binding protein fHbp: a key virulence factor and vaccine antigen[J]. Expert Rev Vaccines, 2015, 14(6): 841-859.

[7]

GIUNTINI S, REASON DC, GRANOFF DM. Complement-mediated bactericidal activity of anti-factor H binding protein monoclonal antibodies against the meningococcus relies upon blocking factor H binding[J]. Infect Immun, 2011, 79(9): 3751-3759.

[8]

付昱, 朱喜梅. 新型隐球菌脑膜炎患者的抗真菌治疗分析与药学监护[J]. 今日药学, 2020, 30(8): 569-572.

[9]

GANDHI A, BALMER P, YORK LJ. Characteristics of a new meningococcal serogroup B vaccine, bivalent rLP2086 (MenB-FHbp; Trumenba^®)[J]. Postgrad Med, 2016, 128(6): 548-556.

[10]

MURPHY E, ANDREW L, LEE KL, et al. Sequence diversity of the factor H binding protein vaccine candidate in epidemiologically relevant strains of serogroup B Neisseria meningitidis[J]. J Infect Dis, 2009, 200(3): 379-389.

[11]

MASIGNANI V, COMANDUCCI M, GIULIANI MM, et al. Vaccination against Neisseria meningitidis using three variants of the lipoprotein GNA1870[J]. J Exp Med, 2003, 197(6): 789-799.

[12]

刘建东, 张静飞, 郑海发. B群流行性脑脊髓膜炎疫苗的研究进展[J]. 中国生物制品学杂志, 2019, 32(4): 472-475.

[13]

MADICO G, WELSCH JA, LEWIS LA, et al. The meningococcal vaccine candidate GNA1870 binds the complement regulatory protein factor H and enhances serum resistance[J]. J Immunol, 2006, 177(1): 501-510.

[14]

COSTA I, PAJON R, GRANOFF DM. Human factor H (FH) impairs protective meningococcal anti-FHbp antibody responses and the antibodies enhance FH binding[J]. mBio, 2014, 5(5): e01625-e01614.

[15]

ZLOTNICK GW, JONES TR, LIBERATOR P, et al. The discovery and development of a novel vaccine to protect against Neisseria meningitidis Serogroup B Disease[J]. Hum Vaccin Immunother, 2015, 11(1): 5-13.

[16]

FLETCHER LD, BERNFIELD L, BARNIAK V, et al. Vaccine potential of the Neisseria meningitidis 2086 lipoprotein[J]. Infect Immun, 2004, 72(4): 2088-2100.

[17]

VU DM, PAJON R, REASON DC, et al. A broadly cross-reactive monoclonal antibody against an epitope on the n-Terminus of meningococcal fHbp[J]. Sci Rep, 2012, 2: 341.

[18]

GIULIANI MM, SANTINI L, BRUNELLI B, et al. The region comprising amino acids 100 to 255 of Neisseria meningitidis lipoprotein GNA 1870 elicits bactericidal antibodies[J]. Infect Immun, 2005, 73(2): 1151-1160.

[19]

吴根鹏, 袁萍, 王月红, 等. 脑膜炎球菌表面蛋白fHBP与PorA的融合表达及其免疫原性[J]. 中国生物制品学杂志, 2016, 29(2): 124-128.

[20]

孙智锋, 杜念兴, 徐为燕. 兔出血症病毒单克隆抗体的抗原识别位点分析[J]. 南京农业大学学报, 1997, 20(1): 71-75.

2021年第30卷第10期

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首发时间：2026-10-28
出版时间：2021-05-30

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¹北京市细菌性疫苗工程技术研究中心，北京　100176

²北京智飞绿竹生物制药有限公司，北京　100176

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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