基因工程抗体功能修饰及其在农业食品安全中的应用策略

doi:10.3864/j.issn.0578-1752.2025.02.011

摘要/Abstract

摘要：

基因工程抗体是抗体人工定向设计的巨大飞跃，其以重组抗原结合片段、单链抗体、纳米抗体等形式，广泛渗透应用到农业和食品安全中的各个领域；相关创新探索研究还在竞先推进，发展极为迅速。本文总结了当前基因工程抗体主要衍生形式及其依托的噬菌体、酵母、细菌、核糖体、哺乳动物细胞等抗体库搭载展示平台和相应配套的抗原特异性抗体靶向筛选体系；分析它们借助定点突变、链置换、易错PCR、DNA改组以及同源或异源抗体功能片段甚至与其他功能蛋白融合等策略，在体外开展亲和力成熟乃至提升环境胁迫稳定性等特性功能修饰的关键技术特点；概述它们采用昆虫和动物细胞、植物组织、酵母、大肠杆菌以及其他微生物等表达体系制备相应抗体蛋白状况和潜在优化策略。着重梳理基因工程抗体在产地环境危害物、农兽药投入品、真菌毒素、食源性致病微生物及其有毒代谢物、食源性过敏原等农业食品安全危害物免疫分析上的应用研究现状；同时结合笔者团队近年以Ab2β抗独特型抗体具备模拟抗原结构乃至生物活性功能的特性为理论依据，在模拟Bt Cry毒素结构及抗虫功能的Ab2β抗独特型基因工程抗体和模拟万古霉素抗金黄色葡萄球菌功能的Ab2β抗独特型基因工程抗体等方面创新研发的系列最新成果和相关研究经验，进一步探讨了基因工程抗体在农业食品安全危害物绿色检测和绿色防控创新应用策略研究上的未来发展动向和可行捷径，为基因工程抗体在农业食品安全乃至营养品质评估等领域相关应用研究提供最新最全面的具有参考价值的文献资料和潜在启发思路。

关键词: 基因工程抗体, 抗体亲和成熟, 抗体蛋白表达, 免疫检测, 抗独特型抗体, 杀虫抗体, 杀菌抗体

Abstract:

Genetically engineered antibodies (GEAbs) represent a giant leap forward in the artificial directed design of antibodies. They exist in forms such as recombinant antigen binding fragment, single chain variable fragment and nanobodies, etc., and have been widely used in various fields of agricultural and food safety. Relevant innovation exploration and research are still ongoing, and their development is extremely rapid. This paper summarized the main derivative forms of GEAbs, the carrying platforms of phage, yeast, ribosomal and mammalian cells on which they rely, and the corresponding antigen-specific antibody targeting screening system. The key technical characteristics of their characteristic functional modifications, such as affinity maturation and enhancement of environmental stress stability, were analyzed through strategies, such as site-directed mutagenesis, chain-shuffling, error-prone PCR, DNA shuffling, homologous or heterologous antibody functional fragments and functional protein fusion. The preparation of corresponding antibody proteins using the expression systems of insect and animal cells, plant tissues, yeast, Escherichia coli (E. coli) and other microorganisms and the potential optimization strategies were summarized too. The application and research status of GEAbs in the immunoassay of agricultural and food safety hazards, such as environmental hazards of origin growing area, pesticides and veterinary drugs inputs, mycotoxins, foodborne pathogenic microorganisms and their toxic metabolites, foodborne allergens were reviewed. Combined with the latest achievements and research experience in the innovative research and development of the simulants for simulating Bt Cry toxin structure and even anti-insect function of Ab2β anti-idiotypic GEAbs and for simulating vancomycin anti-Staphylococcus aureus (S. aureus) function of Ab2β anti-idiotypic GEAbs from author’s team, which is based on the theoretical basis that Ab2β anti-idiotypic antibody, could simulate the structure of antigen and even the function of biological activity. The future development trend and feasible shortcut of GEAbs in green detection and green prevention and control of agricultural and food safety hazards were further discussed. This paper was expected to provide the latest and comprehensive literature with reference value and potential inspiration for the application of GEAbs in the fields of agricultural, food safety and nutrition quality assessment.

Key words: genetically engineered antibody, antibody affinity maturation, expression of antibody protein, immunoassay, anti- idiotype antibody, insecticidal antibody, bactericidal antibody

徐重新, 沈建兴, 金嘉凤, 何鑫, 谢雅晶, 张霄, 朱庆, 刘媛, 刘贤金. 基因工程抗体功能修饰及其在农业食品安全中的应用策略[J]. 中国农业科学, 2025, 58(2): 355-386.

XU ChongXin, SHEN JianXing, JIN JiaFeng, HE Xin, XIE YaJing, ZHANG Xiao, ZHU Qing, LIU Yuan, LIU XianJin. Functional Modification of Genetically Engineered Antibodies and Their Application Strategies in Agriculture and Food Safety[J]. Scientia Agricultura Sinica, 2025, 58(2): 355-386.

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图/表 5

图1

表1

基因工程抗体常见功能修饰策略及相应代表性研究实例"

功能修饰及策略 Functional modification and strategies		供试对象 Experimental subject	修饰区位 Modified region or site	实施效果 Implementation effect	参考文献 Reference
亲和力修饰Affinity modification	定点突变 Site-directed mutagenesis	anti-TpoR rFab	V_H-CDR3 “DRKLGGSDYW→DRKLGGLDYW”、V_L-CDR3 “QQSNSFPWTF→ QRSNSPPYTF”	目标突变体的亲和力提高了20.68倍 The affinity of the target mutant was increased 20.68-fold	[58]
		anti-PD-L1 scFv	V_H-CDR3 “TKWELVDPYDY→SKWELVDPYAY”	目标突变体的亲和力提高了9.8倍 The affinity of the target mutant was increased 9.8-fold	[59]
		anti-sarafloxacin scFv	V_H-CDR3 “Y99H”	目标突变体的亲和力提高了7倍 The affinity of the target mutant was increased 7-fold	[95]
		anti-Bt Cry sdAb	V_H-CDR3 “N105L、R106L、V107Y、R114K”	目标突变体的抗原识别灵敏度提高了1.34-2.7倍 The antigen recognition sensitivity of target mutant was increased 1.34-2.7-fold.	[55]
		anti-cortisol scFv	V_H-FR1 “¹QVQLQQPGAE¹⁰→¹QVQLQQDPGAE¹⁰”	目标突变体的亲和力提高了31倍 The affinity of the target mutant was increased 31-fold	[66]
		anti-amantadine scFv	V_L-CDR3 “G107F”	目标突变体的亲和力提高了3.9倍 The affinity of the target mutant was increased 3.9-fold	[56]
		anti-ciprofloxacin scFv	V_L-CDR1 “V160S”	目标突变体的亲和力提高了16.6倍 The affinity of the target mutant was increased 16.6-fold	[61]
		anti-estradiol-17β scFv	V_L-CDR1 “I29V”	目标突变体的亲和力提高了5倍 The affinity of the target mutant was increased 5-fold	[62]
		anti-MC-LR scFv	V_L-CDR1 “ATWNMAY→ARYYWYGAFDI”	目标突变体的亲和力提高了27.8倍 The affinity of the target mutant was increased 27.8-fold	[63]
		anti-MC-LR scFv	V_L-CDR3 “F91Y”	目标突变体的亲和力提高了63.3倍 The affinity of the target mutant was increased 63.3-fold	[57]
		anti-amoxicillin scFv	V_L-CDR3 “S95E”	目标突变体的亲和力提高了6倍 The affinity of the target mutant was increased 6-fold	[64]
		anti-dengue virus scFv	V_H-CDR1 “D31L”、V_H-CDR3 “Y105W”、 V_L-CDR3 “S227W”	目标突变体的亲和力提高了~100倍 The affinity of the target mutant was increased ~100-fold	[96]
		anti-HBV scFv	V_L-CDR3 “Y96S”、V_H-CDR3 “D98S”	目标突变体的亲和力和热稳定性显著提高 The affinity and thermal stability of the target mutant were significantly improved	[97]
		anti-quinalphos Nbs	CDR1 “R29W”	目标突变体的抗原识别灵敏度提高了25倍 The antigen recognition sensitivity of target mutant was increased 25-fold	[32]
		anti-deoxynivalenol Nbs	CDR3 “T102Y”	目标突变体的亲和力提高了3.2倍 The affinity of the target mutant was increased 3.2-fold	[60]
		anti-aflatoxin B₁ Nbs	CDR3 “S102D”	目标突变体的抗原识别灵敏度提高了3.3倍 The antigen recognition sensitivity of target mutant was improved 3.3-fold	[98]
		anti-ochratoxin A Nbs	FR1 “G53Q”、CDR2 “S102D”	目标突变体的亲和力提高了1.36倍 The affinity of the target mutant was increased 1.36-fold	[65]
		anti-syn Nbs	FR3 “N77D”	目标突变体的亲和力提高了48.5倍 The affinity of the target mutant was increased 48.5-fold	[67]
	链置换 Chain- shuffling	anti-aflatoxin B₁ scFv	重链置换重组V_H-V_L Heavy chain replacement recombination V_H-V_L	目标突变体的抗原识别灵敏度提高了7.5倍 The antigen recognition sensitivity of target mutant was increased 7.5-fold	[69]
		anti-HBV scFv	重链置换重组V_H-V_L Heavy chain replacement recombination V_H-V_L	目标突变体的亲和力提高了6.5倍 The affinity of the target mutant was increased 6.5-fold	[99]
		anti-halofuginone scFv	轻链置换重组V_H-V_L Light chain replacement recombination V_H-V_L	目标突变体的抗原识别灵敏度提高了185倍 The antigen recognition sensitivity of target mutant was increased 185-fold	[70]
		anti-BoNT scFv	轻链置换重组V_H-V_L Light chain replacement recombination V_H-V_L	目标突变体的亲和力提高了77倍 The affinity of the target mutant was increased 77-fold	[100]
		anti-MC-LR scFv	轻链置换重组V_H-V_HH Light chain replacement recombination V_H-V_HH	目标突变体的抗原结合活性显著提高 The antigen-binding activity of target mutant was significantly improved	[5]
		anti-Bt Cry toxin idiotypic scFv	双轻链置换重组V_L-V_L Double light chain replacement recombination V_L-V_L	目标突变体的亲和力提高了1.37倍 The affinity of the target mutant was increased 1.37-fold	[6]
		anti-Cry toxin scFv	双轻链置换重组V_L-V_L Double light chain replacement recombination V_L-V_L	目标突变体的抗原结合活性提高了1.44倍 The antigen-binding activity of target mutant was increased 1.44-fold	[101]
	易错PCR Error-prone PCR	anti-streptavidin rFab	V_L-CDR1 “H34R”、V_L-CDR3 “Y96H”	目标突变体的亲和力提高了10.7倍 The affinity of the target mutant was increased 10.7-fold	[73]
		anti-TCR scFv	V_H-CDR2 “Q50L”、V_L-FR1 “D1N”、V_L-CDR2 “Q53L”、V_L-FR3 “S65N”	目标突变体的亲和力提高了100倍 The affinity of the target mutant was increased 100-fold	[75]
		anti-aflatoxin B1 scFv	V_H-FR1 “T28P”、V_H-FR3 “A94V”、V_H-RF4 “T110A”、V_L-FR2 “E40V”、V_L-CDR2 “A57V”	目标突变体的亲和力提高了9倍 The affinity of the target mutant was increased 9-fold	[76]
		anti-CD22 scFv	V_L-CDR1 “N34A”	目标突变体的亲和力提高了10倍 The affinity of the target mutant was increased 10-fold	[102]
		anti-CIT scFv	V_H-CDR3 “T100P”、V_L-FR1 “M151T”	目标突变体的亲和力提高了13.25倍 The affinity of the target mutant was increased 13.25-fold	[25]
		anti-cortisol scFv	V_H“S21P” & linker “S8P”、V_L“K27R”	目标突变体的亲和力提高了63倍 The affinity of the target mutant was increased 63-fold	[77]
		anti-estradiol-17β scFv	V_H-FR1 “K19R”、V_H-CDR2 “Y57F”、V_H-RF3 “S88P、E89G”、V_H-CDR3 “L111Q”、V_L-CDR1 “Q166R、I168V”、V_L-FR2 “L175M”、V_L-CDR2 “H189N”、V_L-FR3 “S202G、S216G”	目标突变体的亲和力提高了151倍 The affinity of the target mutant was increased 151-fold	[103]
		anti-SAL scFv	V_H-CDR3 “T101S、F103Y”、V_L- CDR3 “S216N、V218T、T221L”	目标突变体的亲和力提高了~25倍 The affinity of the target mutant was increased ~25-fold	[74]
		anti-UreC Nbs	FR2 “N23K、P29R、R34Q”、CDR2 “F42I、F43S”、FR3 “N54D、G58R、N64D、F71Y、V84M”、FR4 “R109Q、R118Q、A119P”	目标突变体的抗原结合活性提高了1.5倍 The antigen-binding activity of target mutant was increased 1.5-fold	[104]
		anti-ClfA Nbs	FR1 “R19Y”、CDR1 “A24G”、CDR2 “G50A”、FR3 “D61V、K75R”	目标突变体的抗原结合活性提高了~80倍 The antigen-binding activity of target mutant was increased ~80-fold	[105]
	DNA改组 DNA shuffling	anti-fusarium scFv	V_H-FR1 “V23A” & V_H-CDR1 “D30G”、V_L-FR3 “G209S”	目标突变体的抗原结合活性提高了15倍 The antigen-binding activity of target mutant was increased 15-fold	[106]
		anti-ProGRP scFv	V_L-FR1 “P12A”、V_L-CDR1 “T36A”、V_H-CDR1 “V162M、S165P”、V_H- CDR3 “D238N”、V_H-FR3 “T248I”	目标突变体的亲和力提高了9倍 The affinity of the target mutant was increased 9-fold	[79]
		anti-SARS-CoV scFv	V_H-CDR2 “S52G、N57S”、V_H-FR3 “T91A”、V_H-FR4 “S117N”、V_L- CDR1 “S167N”、V_L-CDR2 “S189N”、V_L-FR3 “S202N”、V_L-FR4 “T239A”	目标突变体的亲和力提高了270倍 The affinity of the target mutant was increased 270-fold	[80]
	其他策略 Other strategies	anti-Salmonella Nbs	V_HH→V_HH-linker-V_HH	目标二价抗体复合物的抗原识别灵敏度提高了7.5倍 The antigen recognition sensitivity of the target bivalent-antibody complex was increased 7.5-fold	[81]
		anti-SARS-COV2 Nbs	Bivalent-V_HH recombinant fused by SpyTag/SpyCatcher protien	目标二价抗体复合物的亲和力提高了56倍 The affinity of the targeted bivalent-antibody complex was increased 56-fold	[107]
		anti-HER3 scFv	Tri-scFv recombinant fused by SpyTag/SpyCatcher protien	目标三价抗体复合物的亲和力提高了12倍 The affinity of the targeted trivalent-antibody complex was increased 12-fold	[82]
		anti-aflatoxin B₁ scFv	scFv fused to alkaline phosphatase (ALP)	目标scFv-ALP复合物的抗原识别灵敏度提高了3倍 The antigen recognition sensitivity of the target scFv-ALP complex was increased 3-fold	[9]
		anti-HIV-1 Nbs	V_HH fused to ligand-tailored SH3 domain	目标V_HH-SH3融合复合物的亲和力提高了28倍 The affinity of the target V_HH-SH3 fusion complex was increased 28-fold	[10]
		anti-hCG Nbs	V_HH fused to PMMA-binding peptide	目标V_HH-PMMA-tag融合复合物的抗原结合活性提高了10倍 The antigen-binding activity of the target V_HH-PMMA- tag fusion complex was increased 10-fold	[108]
稳定性修饰Stability modification	热稳定性 Thermal stability	anti-adalimumab rFab	V_H“G10C/P210C” & VL “P40C/ E165C”	目标突变体的热稳定耐受性提高了6.5 ℃ The thermal stability tolerance of the target mutant was improved 6.5 ℃	[11]
稳定性修饰Stability modification	热稳定性 Thermal stability	anti-MS2 rFab	V_L-FR2 “F36Y、R46L”、 V_L-FR3 “Y87F”	目标突变体的热稳定耐受性提高了8.4 ℃ The thermal stability tolerance of the target mutant was improved 8.4 ℃	[109]
		anti-interleukin- 17A rFab	V_L-CDR1 “S30G”、V_L-CDR2 “D53A”、V_L-CDR3 “D92V” & V_H-CDR1 “V61P、S65G”、V_H-CDR3 “E97D”	目标突变体的热稳定耐受性提高了10.8 ℃ The thermal stability tolerance of the target mutant was improved 10.8 ℃	[110]
		anti-aflatoxin B₁ scFv	V_H“G44HC”、V_L“Q100C”	目标突变体的热稳定性显著增强 The thermal stability of the target mutant was significantly enhanced	[83]
		anti-CXCL13 scFv	V_L-CDR3 “SSYTYYDTYV→ASATLLDTYV”	目标突变体的热稳定耐受性提高了5 ℃ The thermal stability tolerance of the target mutant was improved 5 ℃	[111]
		anti-cTnI scFv	V_H-CDR3 “SSYQCSGDYC→SSYQASGDYA”	目标突变体的热稳定性显著增强 The thermal stability of the target mutant was significantly enhanced	[112]
		anti-EGFR scFv	V_H-V_L (lambda framework)→ V_H-V_L (kappa framework)	目标突变体的热稳性显著增强 The thermal stability of the target mutant was significantly enhanced	[84]
		anti-carbaryl Nbs	V_HH→V_HH-linker-V_HH	目标二价抗体复合物的热稳定性和亲和力显著提高 The thermal stability and affinity of the target bivalent antibody complex were significantly improved	[85]
		anti-hCG Nbs	CDR2 “N52S”、FR3 “N74S、N84T”	目标突变体的热稳性提高了~50% The thermal stability tolerance of the target mutant was improved ~50%	[86]
		anti-HAS Nbs	CDR1 “G32D”、CDR2 “R50L”、FR3 “G78A”、FR4 “S133R”	目标突变体的热稳定耐受性提高了12 ℃ The thermal stability tolerance of the target mutant was improved 12 ℃	[113]
		anti-TNF-α Nbs	CDR2 “E50Q、T53W”	目标突变体的热稳定耐受性提高了7.4 ℃且亲和力提高了3.86倍 The thermal stability tolerance of the target mutant was increased 7.4 ℃ and the affinity was increased 3.86- fold	[87]
		anti-α-synuclein peptide Nbs	FR3 “S96F”	目标突变体的热稳定耐受性提高了7.4 ℃且亲和力提高了7倍 The thermal stability tolerance of the target mutant was increased 7.4 ℃ and the affinity was increased 7-fold	[89]
		anti-lysozyme Nbs	FR3 “A79I”	目标突变体的热稳定耐受性提高了5 ℃ The thermal stability tolerance of the target mutant was increased 5 ℃	[114]
		anti-CD47 Nbs	CDR3 “G107W、T108H、S109V、F110A”	目标突变体的热稳定耐受性提高了7.36 ℃且亲和力提高了87.4倍 The thermal stability tolerance of the target mutant was increased 7.36 ℃ and the affinity was increased 87.4-fold	[88]
	pH稳定性 pH stability	anti-SAG1 scFv	V_H-14 sites/V_L-4 sites (突变位点较多，未细列 There are many mutation sites, not detailed list)	目标突变体的碱性pH耐受性增强了21% The alkaline pH tolerance of target mutant was enhanced 21%	[12]
	pH稳定性 pH stability	anti-alpaca-Fab Nbs	CDR1 “S30N”、FR2 “Q39E”、 CDR2 “H56Y”	目标突变体的碱性pH耐受性增强了~37%且亲和力提高了10倍 The alkaline pH tolerance of target mutant was enhanced ~37% and the affinity was increased 10-fold	[90]

表1

表2

基因工程抗体主要表达策略及相应代表性研究实例"

表达策略Expression strategy			供试对象 Experimental subject	蛋白表达量 Protein expression level	参考文献 Reference
宿主Host		载体Vector	供试对象 Experimental subject	蛋白表达量 Protein expression level	参考文献 Reference
大肠杆菌表达系统 E. coli expression systems	E. coli BL21-DE3	pRSFDuet phoA-STII	anti-IGF1R rFab	6.3 mg∙L^-1*	[13]
	E. coli BL21-DE3	pLK04	synthetic human rFab	10 mg∙L^-1*	[116]
	E. coli BL21-DE3	pLac-DsbA/C	anti-MMP-14 rFab	30 mg∙L^-1*	[134]
	E. coli-CyDisCo	pET23	anti-Herceptin rFab	42 mg∙L^-1*	[118]
	E. coli FA113	pFAB1	anti-NhaA rFab	30 mg∙L^-1*	[117]
	E. coli BL21-pLysS	pET22b	anti-EGFRvIII scFv	133.33 mg∙L^-1	[135]
	E. coli Rosetta	pET22b	anti-CRP scFv	3.0 g∙L^-1*	[122]
	E. coli Rosetta	pET23a	anti-influenza PB2 scFv	20 mg∙L^-1*	[136]
	E. coli BL21-DE3	pET28a	anti-HER2 scFv	30 mg∙L^-1*	[119]
	E. coli BL21-DE3	pSAR-2	anti-HIV scFv	0.8 g∙L^-1*	[121]
	E. coli HB2151	pIT2 phagemid	anti-MC-LR scFv	2.56 mg∙L^-1*	[41]
	E. coli BL21-DE3	pET26b	anti-Van idiotypic sdAb	3.01 mg∙L^-1	[3]
	E. coli BL21-DE3	pET26b	anti-MC-LR Nbs	1.27 mg∙L^-1*	[115]
	E. coli Rosetta	pET25b	anti-DON Nbs	40 mg∙L^-1*	[137]
	E. coli BL21-DE3	pET45b	anti-α-Amylase Nbs	70 mg∙L^-1*	[138]
酵母表达系统 Yeast expression systems	Saccharomyces cerevisiae	pYE	anti-HIV-1 scFv	0.5 mg∙L^-1*	[14]
	Saccharomyces cerevisiae	pUR4548	anti-hapten Nbs	100 mg∙L^-1*	[123]
	Pichia pastoris	pPink-αHC	anti-ranibizumab rFab	30 mg∙L^-1*	[124]
		pPICZαA	anti-MC-LR scFv	44.6 mg∙L^-1*	[125]
		pPICZαA	anti-αIIbβ3 TEG4 scFv	30 mg∙L^-1*	[139]
		pTHI11-synMsn4	anti-gelsolin Nbs	8 g∙L^-1*	[126]
昆虫及动物细胞表达系统 Insect and animal cell expression systems	HEK293 cells	pJK7	anti-HPep rFab	5 mg∙L^-1*	[140]
	HEK293 cells	pcDNA	scFv	22 mg∙L^-1*	[141]
	CHO cells	pBIC-PS	anti-AIS rFab	1 g∙L^-1*	[15]
	Sf9 cells	pIB/V5	anti-VcHK scFv	-	[142]
		pFast-Bac	anti-AFB1 scFv	-	[143]
		pFast-Bac	anti-parathion-ethyl scFv	-	[144]
	Trichoplusia ni BTI-Tn5B1 cells	pIHAneo::pXINSECT	anti-bovine RNaseA rFab	120 mg∙L^-1*	[145]
	Bombyx mori BmN cells	pENTR11::pDEST8/v BmNPV/T3 bacmid	anti-SARS-CoV scFv	2.34 g∙L^-1*	[127]
植物表达系统 Plant expression systems	Tobacco	pENTR4®:: pK7WG2D	anti-venom scFv	~62 mg∙kg^-1**	[16]
		pBI-Ω	anti-TNC scFv	50—100 mg∙kg^-1 **	[146]
		pBI121	anti-HER2 Nbs	-	[147]
	Arabidopsis thaliana	pPhasBar	anti-TNF-α scFv	0.634 g∙kg^-1 **	[128]
	Arabidopsis thaliana	pPphas-GBP	anti-F4+ETEC Nbs	44.3 g∙kg^-1 **	[148]
	Rice	pZH2B	anti-human norovirus Nbs	4.63 g∙kg^-1 **	[129]
	Spirodela punctata	pRT100::pCAMBIA1304	anti-TNFα scFv	126 mg∙kg^-1 **	[130]
	Chlamydomonas reinhardtii	pBS	anti-αCD22 scFv	~61.5 mg∙kg^-1 **	[131]
其他表达系统 Other expression systems	Corynebacterium glutamicum	pPKStrast	anti-HER2 rFab	57.6 mg∙L^-1 *	[17]
	Bifidobacterium longum	pESH100	anti-exotoxin-A Nbs	1 mg∙L^-1 *	[18]
	Bacillus subtilis	pRBBm117	anti-lysozyme scFv	130 mg∙L^-1 *	[132]
	Brevibacillus choshinensis	pBIC	anti-trastuzumab rFab	145 mg∙L^-1 *	[149]
	Brevibacillus choshinensis	pBIC	anti-IZUMO1 Nbs	3 g∙L^-1 *	[150]
	Aspergillus niger	pAnGlaA	anti-DEC205 scFv	54 mg∙L^-1 *	[151]
	Aspergillus oryzae	phlACB	Nbs	0.61 g∙L^-1 *	[133]

表2

图2

表3

基于基因工程抗体的免疫分析策略在农业食品安全危害物监测中应用及代表性实例"

免疫分析策略 Immunoassay strategy		检测对象 Testing objects*		检测实效Detection effectiveness		参考文献 Reference
免疫分析策略 Immunoassay strategy		检测对象 Testing objects*		灵敏度 LOD	线性范围 LDR	参考文献 Reference
酶联免疫分析ELISA	IC-ELISA	重金属 Heavy metal	铀 Uranium (scFv)	2.2 nM	-	[19]
		农药 Pesticides	有机磷类农药 Organophosphorus pesticides (scFv)	4.1-17.6 ng∙mL^-1	-	[20]
			甲基对硫磷 Parathion-methyl (scFv)	0.9 ng∙mL^-1	-	[201]
			拟除虫菊酯Pyrethroid (scFv)	0.05 μg∙mL^-1	0.15—2.64 μg∙mL^-1	[157]
			胺甲萘Carbaryl (bispecific-Nbs)	0.8 ng∙mL^-1	2.1—270.9 ng∙mL^-1	[85]
			呋喃丹Carbofuran (Nbs)	0.65 ng∙mL^-1	1.44—30.39 ng∙mL^-1	[202]
			百草敌Dicamba (Nbs)	-	0.11—8.01μg∙mL^-1	[203]
			Bt Cry1F (scFv)	0.18 ng∙mL^-1	0.92—107.36 ng∙mL^-1	[42]
		兽药 Veterinary drugs	氯霉素Chloramphenicol (scFv)	1.11 ng∙mL^-1	-	[21]
			庆大霉素Gentamicin (scFv)	0.147 ng∙mL^-1	0.14—204.16 ng∙mL^-1	[204]
			环丙沙星Ciprofloxacin (scFv)	-	5.68—201.55 ng∙mL^-1	[61]
			沙氟沙星Sarafloxacin (scFv)	0.3 ng∙mL^-1	-	[95]
			恩诺沙星Enrofloxacin (Nbs)	0.975 ng∙mL^-1	-	[205]
			泰乐菌素Tylosin (scFv)	7.1 μg∙kg^-	-	[206]
			19-去甲睾酮 19-nortestosterone (Nbs)	0.1 ng∙mL^-1	-	[159]
		真菌毒素 Fungal-toxins	赭曲霉 Ochratoxin A (Nbs** & mAb)	0.001μg∙mL^-1	0.027—0.653μg∙mL^-1	[22]
			黄曲霉毒素 Aflatoxins(Nbs** & mAb)	0.015 ng∙mL^-1	0.018—0.079 ng∙mL^-1	[207]
			黄曲霉毒素Aflatoxins (rFab)	12 ng∙mL^-1	-	[152]
			呕吐毒素 Deoxynivalenol (Nbs** & mAb)	1.16 ng∙mL^-1	2.18—62.25 ng∙mL^-1	[137]
			呕吐毒素 Deoxynivalenol (scFv** & mAb)	<77.7 ng∙mL^-1	-	[208]
			稻曲病毒素Ustilaginoidins (Nbs)	-	1.17—32.13μg∙mL^-1	[209]
			青霉菌毒素Citreoviridin (scFv)	14.7 ng∙mL^-1	25—562 ng∙mL^-1	[25]
			桔霉毒素Citrinin (Nbs** & mAb)	-	5—300 ng∙mL^-1	[210]
		食源性致病微生物Food-borne pathogenic microorganisms	猪2型环状病毒 PCV2 (Nbs)	0.05μg∙mL^-1	-	[160]
		其他危害物 Other hazards	蘑菇毒伞肽Amatoxins (scFv)	1.91 ng∙mL^-1	-	[26]
			海豚毒素Citreoviridin (scFv)	14.7 ng∙mL^-1	25—562 ng∙mL^-1	[25]
			微囊藻毒素 MC-LR (Nbs** & mAb)	0.8 ng∙mL^-1	1.2—6.9 ng∙mL^-1	[27]
			膝沟藻毒Gonyautoxin (rFab)	0.7 ng∙mL^-1	-	[211]
			杏仁过敏原 Almond allergens (scFv)	40 ng∙mL^-1	-	[154]
	DAS-ELISA	农药 Pesticides	Bt Cry1Ab (scFv & pAbs)	0.008μg∙mL^-1	0.018—6.230μg∙mL^-1	[158]
		农药 Pesticides	Bt Cry1Ac (Nbs & Nbs)	5 ng∙mL^-1	10—1000 ng∙mL^-1	[212]
		真菌毒素 Fungal-toxins	交链孢霉毒素TeA Alternaria TeA (sdAb & sdAb)	0.08 ng∙mL^-1	0.26—25.90 ng∙mL^-1	[213]
		食源性致病微生物及其代谢毒素 Food-borne pathogenic microorganisms and their metabolizing toxins	金黄色葡萄球菌 S. aureus (Nbs & Nbs)	1.4×10⁵ CFU∙mL^-1	10⁴—10⁵CFU∙mL^-1	[23]
			沙门氏菌 Salmonella (Nbs & Nbs)	4.23-9.15×10³ CFU∙mL^-1	10³—10⁷CFU∙mL^-1	[161]
			李斯特菌 Listeria monocytogenes (Nbs & mAb)	1×10⁴ CFU∙mL^-1	-	[214]
			霉菌Aspergillus (Nbs & pAbs)	1μg∙mL^-1	-	[215]
			葡萄球菌肠毒素 A Staphylococcal enterotoxin A (Nbs & Nbs)	0.43 ng∙mL^-1	0.5—512.0 ng∙mL^-1	[24]
			肉毒杆菌ε毒素 Clostridium botulinum ε-toxin (rFab & mAb)	100 ng∙mL^-1	-	[162]
			H7N2禽流感病毒 H7N2-AIV (Nbs & Nbs)	2.946 ng∙mL^-1	5—100 ng∙mL^-1	[216]
			锦鲤疱疹病毒 Koi herpesvirus (scFv & scFv)	5 ng∙μL^-1	-	[217]
			柑橘衰退病毒CTV (scFv & scFv)	0.01μg CP∙mL^-1	-	[218]
		其他危害物 Other hazards	微囊藻毒素MC-LR (sdAb & sdAb)	0.14 nM	0.14—10000 nM	[219]
		其他危害物 Other hazards	膝沟藻毒 Gonyautoxin (sdAb & sdAb)	0.5 ng∙mL^-1	0.1->1000 ng∙mL^-1	[211]
侧流向免疫分析LFIA	Biotin & SA-AuNPs-LFIA	农药Pesticides	腐霉利 Procymidone (Nbs & mAb)	0.88 ng∙mL^-1	-	[165]
	Au@Pt-LFIA		3-苯氧基苯甲酸 3-PBA (Nbs & mAb)	0.005 ng∙mL^-1	-	[167]
	TRFM-LFIA		噻虫啉Thiacloprid (scFv & mAb)	0.003 ng∙mL^-1	0.01-10 ng∙mL^-1	[169]
	eGFP-AcmA@BMC-LFIA	兽药 Veterinary drugs	氟甲砜霉素 Florfenicol (scFv&mAb)	0.21 pg∙mL^-1	-	[170]
	Colloidal AuNPs-LFIA	真菌毒素 Fungal-toxins	伏马毒素Fumonisin B₁ (scFv@mAb)	2.5 ng∙mL^-1	-	[29]
	Avi/SA@QDs-LFIA		黄曲霉毒素 Aflatoxin B₁(Nbs & mAb)	1.25 ng∙mL^-1	-	[168]
	GFP-LFIA		黄曲霉毒Aflatoxin B₁ (Nbs & mAb)	4.8 ng∙mL^-1	-	[220]
	AuCPNs@PDA- photothermal-LFIA	食源性致病微生物代谢毒 Food-borne pathogenic microorganisms and their metabolizing toxins	葡萄球菌肠毒素B Staphylococcal enterotoxin B (Nbs & mAb)	0.58 ng∙mL^-1	-	[166]
	Colloidal AuNPs-LFIA	其他危害物 Other hazards	毒伞肽Amatoxins (scFv & mAb)	4 ng∙mL^-1	-	[164]
	Au^MBA@AgNPs-CM/ SERS-LFIA	其他危害物 Other hazards	甲壳类过敏原原肌球蛋白 Crustacean allergen tropomyosin (Nbs & mAb)	0.01 μg∙mL^-1	-	[44]
荧光免疫分析 FLIA	ALP-dc-FEIA	农药 Pesticides	对硫磷Parathion (Nbs)	0.2 ng∙mL^-1	-	[175]
	Rhodamine based FLIA		吡虫啉Imidacloprid (rFab)	10 ng∙mL^-1	-	[172]
	IC-TRFIA		Cry1Ie (scFv)	0.04 ng∙mL^-1	0.08—6.44 ng∙mL^-1	[30]
	QDs-FLISA		Cry2A (Nbs & pAbs)	0.41 ng∙mL^-1	2.6—1000 ng∙mL^-1	[171]
	QDs-FLISA		Cry3Bb (Nbs & Nbs)	8.45 ng∙mL^-1	31.25—500 ng∙mL^-1	[221]
	GFP-FLIA		阿维菌素Avermectin (scFv)	1.07 ng∙mL^-1	-	[173]
	HCR & CRISPR/ Cas12a-FLIA	兽药 Veterinary drugs	氯霉素 Chloramphenicol (scFv)	3.31 pg∙mL^-1	3.81 pg∙mL^-1—1 μg∙mL^-1	[178]
	Nanoluciferase (Nluc)-FLIA	真菌毒素 Fungal-toxins	交链孢霉毒素TeA Alternaria-TeA(Nbs** & mAb)	0.7 ng∙mL^-1	-	[174]
	ALP-FLIA		黄曲霉毒素 Aflatoxins (Nbs** & mAb)		0.005—100 ng∙mL^-1	[222]
	Resonance energy transfer- FLIA		赭曲霉 Ochratoxin A/B (Nbs-mAb)	0.06/0.12 ng∙mL^-1	0.25—20 ng∙mL^-1	[177]
	MBs&OPD/DAP-FLIA	食源性致病微生物Food-borne pathogenic microorganisms	大肠杆菌 E. coli F17 (Nb s& Nbs)	1.8 CFU∙mL^-1	10¹—10⁹ CFU∙mL^-1	[176]
化学发光免疫分析CLIA	AS@HRP-IC-CLIA	农药 Pesticides	二嗪农Diazinon (Nbs)	0.03 ng∙mL^-1	0.12—25.96 ng∙mL^-1	[181]
	HRP@luminol-DAS-CLIA		Bt Cry2A (pAbs-Nbs)	0.09 ng∙mL^-1	0.1—1000 ng∙mL^-1	[223]
	HRP@luminol-IC-CLIA		Bt Cry1Ab (mAb-Nbs**)	6.45 ng∙mL^-1	10.49—307.1 ng∙mL^-1.	[31]
	ALP@AMPPD-IC-CLIA	兽药 Veterinary drugs	庆大霉素Gentamicin (scFv)	0.38 ng∙mL^-1	0.546—3.428 ng∙mL^-1	[179]
	ALP@AMPPD-IC-CLIA		孔雀石绿Malachite green (scFv)	0.04 ng∙mL^-1	0.09—11.5 ng∙mL^-1	[182]
	HRP@luminol-IC-CLIA		诺氟沙星Norfloxacin (scFv)	0.105μg∙kg^-1	-	[224]
	ALP@CSPD-CLIA	真菌毒素 Fungal-toxins	伏马毒素 Fumonisin B₁(Nbs** & mAb)	0.12 ng∙mL^-1	0.93—7.73 ng∙mL^-1	[180]
	HRP@luminol-DAS-CLIA	食源性致病微生物及其代谢毒 Food-borne pathogenic microorganisms and their metabolizing toxins	克罗诺杆菌 Cronobacter sakazakii (Nbs & Nbs)	1.04×10⁴ CFU∙mL^-1	-	[183]
	HRP@luminol-DAS-CLIA		鼠伤寒沙门氏菌 Salmonella Typhimurium (Nbs & Nbs)	3.63×10³ CFU∙mL^-1	5.1×10³—1.2×10⁶ CFU∙mL^-1	[225]
	ALP@AMPPD-DAS-CLIA		葡萄球菌肠毒素-B Staphylococcal enterotoxin-B (Nbs & mAb)	1.44 ng∙mL^-1	3.12—50 ng∙mL^-1	[184]
(光)电化学免疫分析(P) ECLIA	PVA-CA-NFM@HRP-ECLIA	农药 Pesticides	对硫磷Parathion (Nbs)	2.26 pg∙mL^-1	0.01—100 pg∙mL^-1	[185]
	ATTO₅₂₀-PECLIA		喹硫磷Quinalphos (Nbs)	7 ng∙mL^-1	-	[32]
	EDC/NHS@HRP-ECLIA		Bt Cry1Ab (Nbs & Nbs)	0.07 ng∙mL^-1	0.1—1000 ng∙mL^-1	[186]
	GO-Th@GCE-ECLIA		Bt Cry1C (Nbs & Nbs)	3.2 pg∙mL^-1	0.01—100 pg∙mL^-1	[188]
	IMBs@HRP-ECLIA	真菌毒素 Fungal-toxins	呕吐毒素Deoxynivalenol (rFab)	63 ng∙mL^-1	100—4500 ng∙mL^-1	[187]
	SPCEs@HRP-ECLIA	真菌毒素 Fungal-toxins	黄曲霉毒素 Aflatoxin M₁ (Nbs** & mAb)	0.09 ng∙mL^-1	0.25—5 ng∙mL^-1	[226]
	HRP-ECLIA	其他危害物 Other hazards	丙烯酰胺Acrylamide (Nbs)	0.033μg∙mL^-1	0.39—50 μg∙mL^-1	[227]
	SA & ALP-ECLIA	其他危害物 Other hazards	花生过敏原Ara h1 Peanut allergen Ara h1 (Nbs)	0.86 ng∙mL^-1	4.5—55 ng∙mL^-1	[28]
表面等离子共振免疫分析 SPRIA	Sensor chip CM5-SPRIA	真菌毒素 Fungal-toxins	玉米赤霉烯酮 Zearalenone (scFv)	7.8 ng∙mL^-1	-	[228]
	AuNPs-SPRIA	食源性致病微生物Food-borne pathogenic microorganisms	副溶血弧菌 Vibrio parahaemolyticus (Nbs)	10³ CFU∙mL^-1	-	[189]
	2-carboxyethyl-SPRIA	食源性致病微生物Food-borne pathogenic microorganisms	肠炎沙门氏菌 Salmonella Enteritidis (rFab)	-	10³—10⁸ CFU∙mL^-1	[153]
	Sensor chip CM5-SPRIA	其他危害物 Other hazards	蘑菇毒伞肽 Amatoxins (scFv)	0.17 ng∙mL^-1	0.554—44.7 ng∙mL^-1	[26]
免疫PCR IM-PCR	IM-PCR	农药Pesticides	Bt Cry1Ac (Nbs)	0.1 pg∙mL^-1	0.001—100 pg∙mL^-1	[33]
	PD-IM-PCR	真菌毒素 Fungal-toxins	玉米赤霉烯酮 Zearalenone (Nbs** & mAb)	6.5 pg∙mL^-1	0.01—100 pg∙mL^-1	[190]
	rt-IM-PCR		桔霉毒素 Citrinin (Nbs** & mAb)	0.08 ng∙mL^-1	0.1—1000 ng∙mL^-1	[229]
	IM-PCR		赭曲霉毒素 Ochratoxin A (Nbs** & mAb)	4.17 pg∙mL^-1	0.01—1000 pg∙mL^-1	[191]

表3

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