摘要
甲胺磷(O,S-二甲基硫代磷酰胺)是一种剧毒的有机磷类农药,由于较好的杀虫效果往往被违禁使用,危害严重,有必要进一步对其加强监测。免疫分析法具有灵敏、简便、快速等特点,在农药残留检测中具有突出的优势。目前虽有制备甲胺磷多克隆抗体和单克隆抗体的报道,但其抗体制备需进行动物免疫及昂贵的细胞培养设备,周期长,成本高。第三代抗体—重组抗体的出现为快速、低成本制备小分子化学农药抗体提供了新的途径。为此,本论文开展了抗甲胺磷噬菌体单链抗体库的构建、筛选及抗甲胺磷单链抗体的表达与特性研究,主要研究内容及结果如下：
1 Balb/c小鼠的免疫及抗血清特性初步分析
利用活泼酯法将合成的甲胺磷半抗原β-(O,S-二甲基磷酰)氨基丙酸(HM3)与牛血清白蛋白(BSA)和卵清白蛋白(OVA)偶联分别制备免疫原HM3-BSA和包被原HM3-OVA。将HM3-BSA按高(H)、中(M)、低(L)3个不同剂量组分别免疫Balb/c小鼠。抗血清分析结果表明,经过8次近6个月时间的免疫在高(H)剂量组中获得了1只良好免疫效果的H3号Balb/c小鼠。
2抗体可变区基因的获得及抗甲胺磷噬菌体单链抗体库的构建
取H3号Balb/c免疫小鼠的脾细胞提取信使RNA(mRNA),通过反转录PCR (RT-PCR)扩增出了大小约340bp和320bp的抗体重(VH)、轻链(VL)可变区基因片段。纯化回收的VH与VL基因在接近等摩尔浓度下,首先采用含有Linker接头片段的引物利用重叠延伸拼接法(SOE-PCR)将VH与VL基因片段组装成单链抗体(ScFv)基因片段,再用带有限制性内切酶位点的引物(5’端SfiⅠ,3’端NotⅠ)扩增含内切酶位点的ScFv基因,获得了大小约750bp的目的片段,纯化回收并分别用SfiⅠ和NotⅠ酶切消化后,与经SfiⅠ、NotⅠ双酶切过的噬菌粒载体pCANTAB5E连接,电转化大肠杆菌E.coli TG1,建成了库容约9.8×105的抗甲胺磷噬菌体单链抗体库。经对随机转化子质粒的PCR及HindⅢ、EcoRⅠ双酶切鉴定,该抗体库重组率高。BstNⅠ酶切图谱显示该噬菌体单链抗体库具有良好的多样性。
3抗甲胺磷噬菌体单链抗体库的筛选和鉴定
借助辅助噬菌体M13K07的超感染,扩建了滴度约1.75×1013pfu/mL的抗甲胺磷噬菌体-ScFv表面展示文库。通过降低抗原包被浓度和增强洗脱力度的方法,以包被原HM3-OVA对展示库进行了五轮“吸附、洗脱、扩增”的富集筛选,结果显示,从第三轮开始,噬菌体回收率、多克隆噬菌体ELISA的OD值及阳性率均呈现上升趋势。从第5轮筛选后得到的细菌集落中分批随机挑选大量单克隆,经单克隆噬菌体ELISA及竞争ELISA进一步筛选鉴定,获得了6个特异性较强的抗甲胺磷噬菌体单链抗体阳性克隆,基因序列分析及Blast数据库检索表明所得6个阳性克隆的序列互不相同,均符合鼠源单链可变区抗体基因的结构和特征。
4抗甲胺磷单链抗体在大肠杆菌中的可溶性表达及性质鉴定
将6个甲胺磷噬菌体阳性克隆提取噬菌粒分别转化琥珀终止非抑制型大肠杆菌E.coli HB2151, IPTG诱导过夜小量制备各克隆的可溶性单链抗体。收集细菌沉淀及培养上清,采用渗透休克法提取菌体细胞周质中的可溶性单链抗体,并对培养上清进行超滤浓缩。SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示,周质腔提取物及浓缩上清在分子量约30KD处明显出现一增粗条带,与单链抗体的预期分子量相符,而阴性对照菌中没有该条带。间接ELISA结果显示,6个克隆的细菌培养上清和周质腔提取物中的抗体均能与包被抗原反应,说明分泌型单链抗体得到了正确的折叠和表达。间接竞争ELISA初步分析6个克隆新鲜制备的周质腔提取物对游离甲胺磷的竞争抑制性,结果显示,6个克隆的表达产物对游离甲胺磷均具有不同程度的抑制作用,其中克隆Met-93的周质腔提取物抑制中浓度I50值最高(887.66μg/mL),克隆Met-28D4的周质腔提取物I50值最低(146.74μg/mL)。
以抗体表达的最优化条件对Met-28D4阳性克隆株进行摇瓶发酵(1L)大量制备纯化单链抗体,抗体产量约0.98 mg/L培养基。以纯化单链抗体为免疫试剂,初步建立了甲胺磷竞争ELISA分析法。该ELISA标准曲线的线性范围为1～500μg/mL, I50为118.69μg/mL,检出限120为3.36μg/mL;在该线性范围内,标准曲线的批内和批间变异系数分别为2.3%和4.8%,稻谷和小白菜样品中添加甲胺磷后的平均回收率分别为83.8%和87.0%。交叉反应结果显示,本研究制备的纯化Met-28D4单链抗体仅与乙酰甲胺磷有部分交叉反应(4.9%),而与供试的其它5种有机磷农药(敌敌畏、乐果、甲拌磷、甲基对硫磷和水胺硫磷)的交叉反应率均小于0.1%,表明所制备的纯化单链抗体对甲胺磷的识别是高度特异的。抗体稳定性试验结果表明,在蛋白保护剂存在条件下,4℃保存下的纯化单链抗体其结合活性可维持约6-7周的时间,基本能满足短期内单链抗体研究的需要,但在大规模生产、应用等方面,就显得稳定性不够。
5抗甲胺磷单链抗体在巴斯德毕赤酵母中的表达及性质鉴定
在巴斯德毕赤酵母P. pastoris (X-33)中分泌表达Met-28D4-ScFv。设计引物从Met-28D4-pCANTAB5E-ScFv上扩增Met-28D4-ScFv,亚克隆至P. pastoris表达载体pPICZαC,获得酵母表达质粒pPICZαC-ScFv。表达质粒pPICZαC-ScFv线形化后,电转化P.pastoris (X-33)。随机挑选转化子菌落进行PCR鉴定,结果显示,转化子的酵母染色体中均整合有外源ScFv基因。在抗性梯度筛选多拷贝整合菌株试验中,共获得5个在2000μg/mL ZeocinTM的YPDS选择平板上正常生长的菌落,表型鉴定结果均为甲醇利用快速型(Mut+)。对5个多拷贝Mut+型转化菌进行甲醇诱导表达,SDS-PAGE结果显示,各菌株诱导48 h后在约30KD处均可见明显条带,72 h处表达量最大,各菌株表达量之间无明显差异；间接ELISA结果显示,各菌株诱导72h的表达产物具有较好的抗原结合活性。
选其中一个稳定高表达的X-33-Pp-Met-28D4-1菌株进行优化诱导表达试验,优化后的条件为：本实验室30℃、250 rpm的振荡培养条件下,菌体接种量OD600nm =1.5,培养基pH值6.5,每24 h补加甲醇至终浓度1.00%,诱导时间72 h。经过优化条件下的诱导表达,单链抗体的表达量达25 mg/L,比Invitrogen公司推荐的诱导表达条件下的表达量高5 mg/L。对优化条件下的诱导表达产物进行纯化并进行竞争ELISA初步试验,结果表明,酵母表达纯化抗甲胺磷单链抗体与游离甲胺磷具有较好的抗原结合特异性,其I50为93.52μg/mL,与大肠杆菌表达的单链抗体的性质基本接近；交叉反应结果显示,酵母表达纯化单链抗体与乙酰甲胺磷的交叉反应率为3.8%,与其它农药的交叉反应率均小于0.1%。稳定性试验表明,酵母表达纯化抗甲胺磷单链抗体的稳定性比大肠杆菌表达的单链抗体的稳定性有了一定的提高。
本论文研究结果为甲胺磷特异性抗体的大量制备奠定了一定的基础,对开发其它小分子化学农药的重组抗体具有重要的理论价值和实践意义。
Methamidophos (O,S-dimethyl phosphoramidothioate), an acutely toxic organophosphate, is still being used in violation of a ban because of its effective for the control of insect. Therefore, it is necessary to detect methamidophos residues for its threat. Immunoassay is being demonstrated as a sensitive, simple, and rapid alternative to traditional methods for pesticide analysis. There have been reported about polyclonal and monoclonal antibodies against methamidophos, but production of PAbs and Mabs is time-consuming and requires expensive instruments for culture cells, immunized-animals, and expensive costs. Recombinant antibodies, the third generation antibodies, can be produced cheaply and quickly through recombinant approaches. Here, we describe the construction, selection of phage-displayed single-chain antibody library against methamidophos and the expression, characterization of the single-chain antibodies.
1 Immunization of Balb/c mice and analysis of characterization of antiserum
The hapten of HM3 (3-(methoxy(methylthio)phosphorylamino)propanoic acid), was conjugated with proteins (BSA and OVA) using the active ester method. Balb/c mice were immunized in three different dose of an immunogen HM3-BSA. Identification of antiserum shows that H3 Balb/c mouse whose serum possessed high titers and the ability of recognition free methamidophos were obtained in the high dose group after 8 times immunization in 6 months.
2 Cloning and construction of phage-display scFv library against methamidophos
mRNA was extracted from the splenocyte of H3 Balb/c mouse and used to amplified VH and VL genes by RT-PCR. The predominant PCR products were of the expected sizes for VH(340bp) and VL(320bp) fragments. The approximately mol VH and VL genes which have been purified were successfully fused with a linker DNA (Gly4Ser)3 by splicing overlap extension PCR (SOE-PCR). The ScFv genes about 750bp generated by PCR using the primers that contain the slice sites of restriction endonuclease. After digested with Sfi I and Not I, the ScFv genes were ligated into the phagemid vector pCANTAB5E and subsequently transformed into the competent E.coli TG1 cells. The phage-display library consisted of 9.8×105 clones. The scFv genes of randomly picked clones were checked by PCR and digestion with HindⅢand EcoRⅠ, and it was found that all contained inserts of the expected size (750 bp). The phage-display library exhibited high diversity as judged by the BstNⅠrestriction pattern.
3 Selection and identification of specific phage-scFv antibodies against methamidophos from phage-display scFv library
The phage-displayed scFv antibody library against methamidophos containing 1.75×1013pfu/mL was constructed by infection with the helper phage M13K07. Then the library was biopanned 5 rounds through the stringency of selection by using the decreased concentration of coating conjugate HM3-OVA and the increased washing times. The phage recovery, the OD of polyclonal phage-ELISA, and the positive rate of clones were gradually increased from rounds 3 to 5. A great number of clones randomly picked from the 5th were further studied by monoclonal phage-ELISA and CI-ELISA, and 6 positive clones with the highest specificity for methamidophos were obtained. Sequence analysis of the 6 clones indicated that all VH and VL genes were homologous with the variable region of mouse antibody, and all sequences had different amino acid substitutions.
4 Expression and characterization of soluble scFv antibodies expressed in E. coli
Recombinant phages of the above 6 positive clones were used to infect E. coli HB2151 for expression of soluble scFv antibodies. The infected E. coli HB2151 were induced overnight by IPTG to express soluble scFv antibodies in small amounts. Both supernatants and pellets were collected. Periplasmic scFv was extracted by osmotic shock and culture supernatants were concentrated by Tangential flow filtration. Approximately 30KD scFv protein, which was consistent with expected molecular weight, could be detected both in the periplasmic extract sample and in the concentrated supernatants compared to negative control in SDS-PAGE analysis. Indirect ELISA also showed that soluble scFv expressed both in supernatant and in periplasmic had a good binding activity with the coating antigen. Data of CI-ELISA showed that soluble scFv of periplasmic extract of the 6 clones could compete with free methamidophos in various degrees, of which the Met-93 clone and the Met-28D4 clone possessed the highest I50 values (887.66μg/mL) and the lowest I50 values (146.74μg/mL) respectively.
Under the optimum conditions, expression scale-up with the Met-28D4 clone was accomplished by increasing the culture volume (1L). The products were purified and the scFv yield was 0.98 mg/L. Based on the purified scFv, CI-ELISA were constructed for methamidophos. The linear range, the I50 values, and the limit of quantification (I20) of CI-ELISA were 1～500μg/mL,118.69μg/mL, and 3.36μg/mL, respectively. In the linear range of CI-ELISA, the intra-assay CV is 2.3%, the inter-assay CV is 4.8%, the recovery rate from rice and cabbage are 83.8% and 87.0% respectively. The Cross-reactivity with other organophosphate pesticides (dichlorvos, dimethoate, phorate, parathionmethyl, isocarbophos) was below 0.1% except that acephate had a low cross-reactivity of about 4.9%, which showed that the soluble scFv antibodies were highly specific to methamidophos. The stability test of scFv antibodies showed that the binding activity of purified scFv retained only 6 weeks when stored at 4℃in the presence of protein stabilizers. The short-term stability of scFv antibodies may meet the demands of study on the characterization of scFv antibodies, but they are often limited in the practical applications.
5 Construction, production, and characterization of recombinant scFv antibodies against methamidophos expressed in Pichia pastoris
Pichia pastoris (x-33) was used to express soluble scFv antibodies of the Met-28D4-ScFv. The primers used for the PCR were synthesized according to antibody gene-specific primers and the cloning sites of the Pichia expression vector pPICZa C. The specific scFv gene was amplified from the Met-28D4-pCANTAB5E-ScFv and then subcloned into the expression vector pPICZa C. The resulting plasmid, pPICZa C-scFv, was linearized and transformed into Pichia pastoris (X-33). Transformants picked randomly were checked by PCR, and it was found that all had integrated target gene. Five clones, which were resistant to the high concentration of 2000μg/mL Zeocin and determined as Mut+ phenotype were obtained in the selection of transformants containing multiple copies of target genes. Then, the 5 transformants were cultured and induced with methanol. Approximately 30KD scFv protein, which was consistent with expected molecular weight, could be detected in the culture supernatants at 48 h in SDS-PAGE analysis. The amount of scFv protein produced by these 5 transformants was similar, and the expression of scFv was highest at 72 h after induction. The bioactivity of Pichia produced scFv was confirmed by indirect ELISA, which also suggested that the bioactivity of scFv in the culture supernatant reached the peak in 72h.
The clone named X-33-Pp-Met-28D4-1, which showed steady and strong expression was selected for further optimization for high-level protein production. In our lab culture conditions (30℃,250 rpm), the optimum process parameters for the expression of scFv produced with X-33-Pp-Met-28D4-1 are (a) an induction time of 72 h, (b) a pH of 6.5, (c) an inoculum density of OD6oonm=1.5, and (d) a methanol concentration of 1.0%. The scFv produced with X-33-Pp-Met-28D4-1 under the optimal conditions was purified and the yield was 25 mg/L, which is higher than that from unoptimized conditions based on recommendations from Invitrogen (20 mg/L). The bioactivity of X-33-Pp-Met-28D4-derived scFv antibodies was confirmed by CI-ELISA. The I50 values for methamidophos in assays was 93.52μg/mL, which indicate that the quality of the P. pastoris-derived scFv is comparable to that of parental E. coli-scFv. The Cross-reactivity of purified X-33-Pp-Met-28D4-derived scFv with other organophosphate pesticides (dichlorvos, dimethoate, phorate, parathionmethyl, isocarbophos) was below 0.1% except that acephate had a low cross-reactivity of about 3.8%. It was found that the stability of P. pastoris-derived scFv was better than that of E. coli-scFv.
The results generated in this study could be the foundation for large-scale production of specific antibodies against methamidophos, which would bring great significance in theorethics and practical for the development of recombinarit antibodies of other low-molecular-weight chemical pesticides.
引文
1. Abad A., Montoya A. Production of Monoclonal Antibodies for Carbaryl from a Hapten Preserving the Carbamate Group [J]. J. Agric. Food Chem,1994,42:1818-1823
2. Abad A, Moreno M J, Montoya A. Hapten Synthesis and Production of Monoclonal Antibodies to the N-Methylcarbamate Pesticide Methiocarb [J]. J. Agric. Food Chem,1998,46:2417-2426
3. Abad A, Moreno M J, Pelegry R, et al Determination of carbaryl, carbofuran and methiocarb in cucumbers and strawberries by monoclonal enzyme immunoassays and high-performance liquid chromatography with fluorescence detection:An analytical comparison [J]. J. Chromatogr. A,1999, 833:3-12
4. Adams G P. Improving the tumor specificity and retention of antibody-based molecules [J]. In Vivo, 1998,12:121-134
5. Ahn K C, Watanabe T, Gee S J, et al Hapten and antibody production for a sensitive immunoassay determinining a human urinary metabolite of the pyrethroid insecticide permethrin [J]. J. Agric. Food Chem,2004,52:4583-4594
6. Alcocer M J, Doyen C, Lee H A, et al Functional scFv antibody sequences against the organophosphorus pesticide chlorpyrifos [J]. J Agric Food Chem,2000,48:335-337 7. Alfredsson G, Branzell C, Granelli K, et al Simple and rapid screening and confirmation of tetracyclines in honey and egg by a dipstick test and LC-MS/MS [J]. Anal. Chim. Acta,2005,529: 47-51
8. Barbara S, Delmulle, Sarah M D, et al Development of an immunoassay-based lateral flow dipstick for the rapid detection of aflatoxin B1 in pig Feed [J]. J. Agric. Food Chem,2005,53:3364-3368
9. Barbas C F, Bain J D, Hoekstra D M, et al Semisynthetic combinatorial antibody libraries:a chemical solution to the diversity problem [J]. Proc Natl Acad Sci USA,1992,89(10):4457-4461
10. Barbas C F, Kang A S, Lerner RA, et al Assembly of combinatonal antibodylibraries on phage surfaces; the gene E1 site [J]. Proc Natl Acad Sci USA,1991,88(18):7978-7982
11. Bell C W, Roberts V A, Scholthof K-BG, et al. (1995) Recombinant antibodies to diuron A model for the phenylurea combining site. In:Nelson J.O., Karu A.E., Wong R.B.(eds) Immunoanalysis of agrochemicals:emerging technology [M]. ACS Symposium Series 586, Washington,DC, pp 50-71
12. Belleville E, Dufva M, Aamand J, et al Quantitative microarray pesticide analysis [J]. J. Immuno. Method,2004,286:219-229
13. Binz H K, Pluckthun A. Engineered proteins as specific binding reagents [J]. Curr. Opini. Biotechnol,2005,16:459-469
14. Blake D A, Pavlov A R, Yu H, et al Antibodies and antibody-based assays for hexavalent uranium [J]. Anal. Chim. Acta,2001,444:3-11
15. Bless N M, Smith D, Charlton J, et al Protective effects of an aptamer inhibitor of neutrophil elastase in lung inflammatory injury [J]. Current Biology,1997,7(11):877-880
16. Brichta J, Vesela H, Franek M. Production of scFv recombinant fragments against 2,4-dichlorophenoxyacetic acid hapten using naive phage library [J]. Vet Med,2003,48:237-247
17. Brimfield A A, Lenz D E, Graham C, et al Mouse monoclonal antibodies against paraoxon: potential reagents for immunoassay with constant immunochemical characteristics [J]. J. Agric. Food Chem,1985,33(6):1237-1242
18. Brocks B, Rode H J, Klein M A. TNF receptor antagonistic scFv, which is not secreted in mammalian cells, is expressed as a soluble mono- and bivalent scFv derivative in insect cells [J]. Immunotechnology,1997,3(3):173-184
19. Bucknall S, Silverlight J, Coldham N, et al Antibodies to the quinolones and fluoroquinolones for the development of generic and specific immunoassays for detection of these residues in animal products [J]. Food Additives and Contaminant,2003,20(3):221-228
20. Burton D R. Monoclonal antibody from combinatorial libraries [J]. Acc Chem Res,1993,26:405
21. Byrne F R, Grant S D, Porter A J, et al Cloning, expression and characterisation of a single-chain antibody specific for the herbicide atrazine [J]. Food Agric Immunol,1996,8:19-29
22. Cabilly S, Riggs A D. Immunoglobulin transcripts and molecular history of a hybridoma that produces antibody to carcinoembryonic antigen [J]. Gene,1985,40:157-61
23. Carlos F, Barbas Ⅲ. Selection of human anti-hapten antibodies from semisynthetic libraries [J]. Gene,1993,137:57-62
24. Carroll W L, Mendel E, Levy S. Hybridoma fusion cell lines contain an aberrant Kappa transcript [J]. Mol Immunol,1988,25:991-995
25. Centeno E R, Johnson W J, Sehon A H. Antibodies to two common pesticides, DDT and malathion [J]. Int. Arch. Allergy,1970,37:1-13
26. Chambers S J, Wyatt G M, Garrett S D, et al Alteration of the binding characteristics of a recombinant scFv anti-parathion antibody-2. Computer modelling of hapten docking and correlation with ELISA binding [J]. Food Agric Immunol,1999,11:219-228
27. Charlton K, Harris W J, Porter A J. The isolation of super-sensitive anti-hapten antibodies from combinatorial antibody libraries derived from sheep [J]. Biosens. Bioelectron,2001,16:639-646
28. Chester K A, Begent R H, Robson L, et al Phage libraries for generation of clinically useful antibodies [J]. Lancet,1994,343(8895):455-456
29. Chiu Y W, Chen R, Li Q X, et al Derivation and properties of recombinant Fab antibodies to coplanar polychlorinated biphenyls [J]. J Agric Food Chem,2000,48(6):2614-2624
30. Chowdhury P S, Pastan I. Improving antibody affinity by mimicking somatic hypermutation in vitro [J]. Nature Biotechnology,1999,17:568-572
31. Clackson T., Hoogenboom H.R., Griffiths A.D., et al. Making antibody fragments using Phage display libraries. Nature,1991,352(633):624-628
32. Clackson T, Well J A. In vitro selection from protein and peptide libraries [J]. Trends Biotechnol, 1994,(12):173-184
33. Clark S L, Remcho V T. Aptamers as analytical reagents [J]. Electrophoresis,2002,23:1335-1340
34. Conrad U, Fiedler U. Compartment-specific accumulation of recombinant immunoglobulins in plant cells:an essential tool for antibody production and immunomudulation of physiological functions and pathogen activity [J]. Plant Mol Biol,1998,38(1-2):101-109
35. Denardo S J, Denardo G L, Denardo D G Antibody phage libraries for the next generation of tumor targeting radio immuntherapeutics [J]. Clinical Cancer Research,1999,5(suppl): 3213s-3218s
36. Eldin P, Pauza M E, Heida Y. High level secretion of two antibody single chain Fv fragments by Pichia pastoris [J]. J Immunol Methods.1997,201:67-75
37. Ellinglon A D, Szostak J W. In vitro selection of RNA molecules that bind specific ligands [J]. Nature,1990,346:818-822
38. Emanuel P, Obrien T. Directing antigen specificity towards botulinum neurotoxin with combination phage display library [J]. Journal of lmmunological Methods,1996,193:189-197
39. Ferrieres L, Francez-Charlot A, Gouzy J, et al FixJregulated genes evolved through promoter duplication in Sinorhizobium meliloti [J]. Microbiology,2004,150(7):2335-2345
40. Fischer R, Drossard J, Emans N. Towards molecular farming in the future:Pichia pastoris-based production of single-chain antibody fragments [J]. Biotechnol Appl Biochem,1999,30:117-120
41. Franek M, Diblikova I, Cernoch I, et al Broad-Specificity Immunoassays for Sulfonamide Detection:Immunochemical Strategy for Generic Antibodies and Competitors [J]. Anal. Chem, 2006,78:1559-1567
42. Freyre F M, Vazquez J E, Ayala M. Very high expression of an anti-carcinoembryonic antigen single chain Fv antibody fragment in the yeast Pchia pastoris [J]. Journol of Biotechnology,2000, 76:157-163
43. Fuchs P, Dubel S, Breitling F, et al Recombinant human monoclonal antibodies. Basic principles of the immune system transferred to E. coli [J]. Cell Biophys,1992,21:81-91
44. Fuh G, Sidhu S S. Efficient phage display of polypeptides fused to the carboxy-terminus of the M13 gene-3 minor coat protein [J]. FEBS Lett,2000,480(1-2):231-234
45. Galve R, Nichkova M, Camps F, et al Development and evaluation of an immunoassay for biological monitoring chlorophenols in urine as potential indicators of occupational exposure. Anal. Chem,2002,74:468-478
46. Garrard L J, Yang M, Connell M P, et al Fab assembly and enrichment in a monovalent phage display system [J]. Bio/Technology,1991,9:1373-1377
47. Garrett S D, Appleford D J A, Wyatt G M, et al Morgan M.R.A. Production of recombinant anti-parathion antibody (scFv); Stability in methanolic food extracts and comparison to an anti-parathion monoclonal antibody [J]. J. Agri. Food Chem,1997,45:4183-4189
48. George A J, Jamar F, Tai M S. Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site:technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide [J]. Proc Natl Acad Sci USA,1995,92: 8358-8362
49. Gerdes M, Meusel M, Spener F. Development of a displacement immunoassay by exploiting cross-reactivity of a monoclonal antibody [J]. Anal Biochem,1997,252:198-204
50. Glass T R, Ohmura N, Morita K, et al. Improving an immunoassay response to related polychlorinated biphenyl analytes by mixing antibodies. Anal. Chem,2006,78:7240-7247
51. Graham B M, Porter A J, Harris W J. Cloning, expression and characterization of a single-chain antibody fragment to the herbicide paraquat [J]. J Chem Technol Biotechnol,1995,63(3):279-289
52. Gram H, Marconi L A, Barbas C F, et al. In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library. Proc. Natl. Acad. Sci.USA,1992,89: 3576-3580
53. Greenman J, Jones E, Wright M D. The use of intracellular single chain antibody fragments to inhibit specifically the expression of cell surface molecules [J]. J Immunol Meth,1996,194(2): 169-180
54. Griffiths A D, Malmqvist M, Marks J D, et al Human anti-self antibodies with high specificity from phage display libraries [J]. EMBO,1993,12(2):725-734
55. Gupta A, Onda M, Pastan I, et al High-density functional display of proteins on bacteriophage lambda. J Mol Biol,2003,334 (2):241-254
56. Hammock B D, Mumma R O.1980. Potential of immunochemical technology for pesticide residue analysis. Pp.321-352 in Recent Advances in Pesticide Analytical Methodology [M]. Eds:Harvey J. Jr., Zweig G. pp:321-352. ACS Symposium Series, ACS Publications
57. Han Z, Xiong C, Mori T, et al Discovery of a stable dimeric mutant of cyanovirin-N (CV-N) from a T7 phage-displayed CV-N mutant library [J]. Biochem Biophys Res Commun,2002,292(4): 1036-1043
58. Hawkins R, Russell S, Winter G. Selection of phage antibodies by binding affinity. Mimicking affinity maturation [J]. Journal of Molecular Biology,1992,226:889-896
59. Hennecke F., Krebber C., Pliickthun A. Non-repetitive single-chain Fv linkers selectively-infective 1 page(SIP) technology [J]. Prot Eng,1998,19:405-410
60. Herrmann S, Leshem B, Lobel L, et al T7 phage display of Ep15 peptide for the detection of WNV IgG [J]. Journal of Virological Methods,2007,141:133-140
61. Hoess R H. Bacteriophage lambda as a vehicle for peptide and protein display [J]. Curr Pharm Biotechnol,2002,3(1):23-28
62. Hoogenboom H, Griffiths A, Johnson K. Multi-subunit proteins on the surfaces of filamentous phage:methodologies for displaying antibody (Fab) heavy and light chains [J]. Nucleic Acids Res, 1991,1:4133-4137
63. Hoogenboom H R, Chames P. Natural and designer binding sites made by phage display technology [J]. Immunol Today,2000,21:371-381
64. Houshmand H, Forman G, Magnusson G, et al Use of bacteriophage T7 displayed peptides for determination of monoclonal antibody specificity and biosensor analysis of the binding reaction [J]. Anal Biochem,1999,15:268(2):363-370
65. Huse W D, Sastry L, Iverson S A, et al Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda [J]. Science,1989,246(4935):1275-1281
66. Huston J S, Levnson D, Mudget-hunter M. Protein engineering of antibody binding site:recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli [J]. Proc. Natl. Acad. Sci.USA,1988,85 (12):879-5883
67. Jang M S, Lee S J, Xue X, et al Production and characterization of monoclonal antibodies to a generic hapten for class-specific determination of organophosphorus pesticides [J]. Bull. Korean Chem Soc,2002,23(8):1116-1120
68. Jayasena S. Aptamers:an emerging class of molecules that rival antibodies in diagnostics [J]. Clin. Chem,1999,45:1628-1650.
69. Johnson K S, Chiswell D J. Human antibody engineering [J]. Current Opin Strut Biol,1993,3: 564-571
70. Jourdan S W, Scutellaro A M, Fleeker J R, et al Determination of Carbofuran in Water and Soil by a Rapid Magnetic Particle-based ELISA [J]. J. Agric. Food Chem,1995,43:2784-2788
71. Kabat E A. Sequences of proteins of Immonological Interest [M]. (U.S.Dept. HealthHuman Services,Washington DC),1991; P1229
72. Karu A E, Scholthof K B G, Zhang G, et al Recombinant antibodies to small analytes and prospects for deriving them from synthetic combinatorial libraries [J]. Food agric. Immun,1994,6:277-286
73. Kettleborough C A, Ansell K H, Allen R W, et al Isolation of tumor cell-specific single-chain Fv from immunized mice using phage-antibody libraries and the reconstruction of whole antibodies from these antibody fragments [J]. Eur J Immunol,1994 Apr,24(4):952-958
74. Kohler G, Milstein C. Continuous culture of fused cells secreting antibody of predefined specificity [J]. Nature,1975,256:495-497
75. Kramer K. Evolutionary Affinity and Selectivity Optimization of a Pesticide-Selective Antibody Utilizing a Hapten-Selective Immunoglobulin Repertoire [J]. Environ Sci Technol,2002,36: 4892-4898
76. Kramer K. Synthesis of a group-selective antibody library against haptens [J]. J. Immuno. Methods, 2002,266:211-222
77. Kramer K, Hock B. Recombinant antibodies for environmental analysis [J]. Anal. Bioanal. Chem, 2003,377:417-426
78. Krebber A, Bomhauser S, Burmester J. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system [J]. Journal of Immunological Methods,1997,201:35-55
79. Kretzschmar T, Aoustin L, Zingel O. High-level expression in insect cells and purification of secreted monomeric single-chain Fv antibodies [J]. J Immunol Methods,1996,195(1-2):93-101
80. Krizan P. Recent progress in ring imaging Cherenkov counters with vacuum-based photon detectors [J]. Nuclear Instruments and Methods in Physics Research Section A:Accelerators, Spectrometers, Detectors and Associated Equipment,2003,502(1):28-35.
81. Kruif J, Boel E, Logtenberg T. Selection and application of human single chain Fv antibody fragments from a Semi-synthetic phage antibody display library with designed CDR3 regions [J]. Mol Biol,1995,248(1):97-105
82. Kusharyoto W, Pleiss J, Bachmann T T, et al Mapping of a hapten-binding site:molecular modeling and site-directed mutagenesis study of an anti-atrazine antibody [J]. Protein Eng,2002, 15:233-241
83. Larrick J. W. Bio/Technology,1989,71:934
84. Lee H-J, Shan G, Watanabe T, et al Enzyme-linked immunosorbent assay for the pyrethroid deltamethrin [J]. J. Agric. Food Chem,2002,50:5526-5532
85. Lee H-J, Shan G, Ahn K, et al Development of an enzyme-linked immunosorbent assay for the pyrethroid cypermethrin [J]. J. Agric. Food Chem,2004,52:1039-1043
86. Lehmann D, Sodoyer R, Leterme S. Characterization of BoHV-1 gE envelope glycoprotein mimotones obtained by phage display [J]. Veterinary Microbiology,2004,104:1-17
87. Lemeulle C, Chardes T, Montavon C. Anti-digoxin scFv fragments expressed in bacteria and in insect cells have different antigen binding properties [J]. FEBS Lett,1998,423(2):159-166
88. Levy R, Molineux I J, Iverson B L, et al Isolation of traps-acting genes that enhance soluble expression of scFv antibodies in the E. coli cytoplasm by lambda phage display [J]. Journal of Immunological Methods,2007,321:164-173
89. Li Q, Shivachandra S B, Leppla S H, et al Bacteriophage T4 Capsid:A Unique Platform for Efficient Surface Assembly of Macromolecular Complexes [J]. J Mol Biol,2006,363:577-588
90. Li Y, Cockburn W, Kilpatrick J, et al Selection of rabbit single-chain Fv fragment against the herbicide atrazine using a new phage display system [J]. Food Agric Immunol,1999,11:5-17
91. Li Y, William C, John B, et al High Affinity ScFvs from a Single Rabbit Immunized with Multiple Haptens [J]. Biochemical and Biophysical Research Communications,2000,268:398-404
92. Liu G, Riechers S L, Timchalk C, et al Sequential injection/electrochemical immunoassay for quantifying the pesticide metabolite 3,5,6-trichloro-2-pyridinol [J]. Electrochem. Communi,2005, 7:1463-1470
93. Longstaff M, Newell C A, Boonstra B, et al Expression and characterization of single-chain antibody fragments produced in transgenic plants against the organic herbicides atrazine and paraquat [J]. Biochim Biophys Acta,1998,1381(2):147-160
94. Lu J, Sloan S R. An alternating selection strategy for cloning phage display antibodies [J] Immunol Methods,1999,228(1-2):109-119
95. Luo D, Geng M, Noujaim T, et al An engineered bivalent single-chain antibody fragment that increases antibody binding activity [J]. Biochemistry,1997,121(5):831-834
96. Luzi E, Minunni M, Tombelli S, et al New trends in affinity sensing:aptamers for ligand binding [J]. Trends Anal. Chem,2003,22:810-818
97. Mahony J O, Nolan K, Smyth M R, et al Molecularly imprinted polymers—potential and challenges in analytical chemistry [J]. Anal. Chim. Acta,2005,534:31-39
98. Mak S K, Shan G, Lee H J, et al Development of a class selective immunoassay for the type II pyrethroid insecticides [J]. Anal. Chim. Acta,2005,534:109-120
99. Malmborg A C, Duenas W, Ohlint M, et al Selection of binders from phage displayed antibody libraries using the BIAcore biosensor [J]. J Immunol Methods,1996,198(1):51-57
100. Marks J D, Griffiths A D, Malmqvist M. By-passing immunization:building high affinity human antibodies by chain shuffling [J]. Biotechnology,1992,10(7):779-783
101. Marks J D, Hoogenboom H R, Bonnert T.P., et al. By-passing immunization human antibodies from V-gene libraries displayed on phage [J]. Mol Biol,1991,222(3):581-597
102. Martin Dufva M, Jesper Petersen J, Michael Stoltenborg M, et al Detection of mutations using microarrays of poly(C)10-poly(T)10 modiWed DNA probes immobilized on agarose Wlms [J]. Anal. Biochem,2006,352:188-197
103. Maruyama I N, Manryama H I, Brenner S, et al Lambda foo:a lambda phage vector for the expression of foreign proteins [J]. Proc Natl Acad Sci USA,1994,91:8273-8277
104. Moreno M J, Abad A, Montoya A. Production of Monoclonal Antibodies to the N-Methylcarbamate Pesticide Propoxur [J]. J. Agric. Food Chem,2001,49:2-78
105. Morozova V S, Levashova A I, Ermin S A. Determination of Pesticides by Enzyme Immunoassay [J]. Journal of Analytical Chemistry,2005,60(3):202-217
106. Nicholls P J, Johnson V G, Blanford M D, et al An improved method of generating single-chain antibodies from hybridomas [J]. J Immunol Methods,1993,165:81
107. Nieba L, Honegger A, Krebber C, et al Disrupting the hydrophobic patches at the antibody variable/constant domain interface:improved in vivo folding and physical characterization of an engineered scFv fragment [J]. Protein Engineering,1997,10:435-444
108. Ochi A.Transfer of a cloned immunoglobulin light-chain gene to mutant hybridoma cells restores specific antibody production [J]. Nature,1983,302-340
109. Ohage E, Steipe B. Intrabody construction and expression. I. The critical role of VL domain stability [J]. Journal of Molecular Biology,1999,291:1119-1128
110. Oiwode T F, Haggerty J E, Katz R, et al Synthetic compound libraries displayed on the surface of encoded bacteriophage [J]. Chem Biol,2003,10(9):847-858
111. Orlandi R, Gussow DH, Jones PT, et al Cloning immunoglobulin variable domains for expression by the polymerase chain reaction [J]. Proc Natl Acad SCi USA,1989,86:3833-3838
112. Patel D J, Suri A K. Structure, recognition and discrimination in RNA aptamer complexes with cofactors, amino acids, drugs and aminoglycoside antibiotics [J]. Rev. Mol. Biotechnol,2000,74: 39-60
113. Pereira S, van Belle P, Eleder D, et al Combinatorial antibodyies against human malignt melanoma [J]. Immunology,1997,16(1):11-15
114. Persic L, Righi M, Roberts A. Targeting vectors for intra cellular immunization [J]. Gene,1997, 87(1):1-8
115. Powers D B, Amersdorfer P, Poul M. Expression of single-chain Fv-Fc fusions in Pichia pastoris [J]. J Immunol Methods,2001,251:123-135
116. Putalun W, Tanaka H, Shoyama Y. Rapid detection of glycyrrhizin by immunochromatographic assay [J]. Phytochem. Anal,2005,16:370-374
117. Rau D, Kramer K, Hock B. Cloning, functional expression and kinetic characterization of pesticide-selective Fab fragment variants derived by molecular evolution of variable antibody genes [J]. Anal Bioanal Chem,2002,372(2):261-267
118. Reiter Y, Wright A F, Tonge D W. Recombinant single-chain and disulfide-stabilized Fv-immunotoxins that cause complete regression of a human colon cancer xenograft in nude mice [J]. Int J Cancer,1996,67(1):113-123
119. Salvatore G, Beers R, Margulies I, et al Improved cytotoxic activity toward cell lines and fresh leukemia cells of a mutant anti-CD22 immunotoxin obtained by antibody phage display [J]. Clin Cancer Res,2002,8:995-1002
120. Schlehuber S, Beste G, Skerra A. A novel type of receptor protein, based on the lipocalin scaffold, with specificity for digoxigenin [J]. J. Mol..Biol,2000,297:1105-1120
121. Shan G, Leeman W R, Stoutamire D W, et al. Enzyme-linked immunosorbent assay for the pyrethroid permethrin [J]. J. Agric. Food Chem,2000,48(9):4032-4040
122. Shan G, Stoutamire D W, Wengatz I, et al Development of an immunoassay for the pyrethroid insecticide esfenvalerate [J]. J. Agric. Food Chem,1999,47(5):2145-2155
123. Sheets M D, Amersdorfer P, Finnem R. Efficient construction of a large nonimmune phage antibody library:the production of high-affinity human single-chain antibodies to protein antigens [J]. Proc Natl Acad Sci USA,1998,95:6157-6162.
124. Shi X, Karkut T, Chamankhah M. Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris [J]. Protein Expr Purif,2003 Apr,28(2):321-330
125. Sidhu S S, Weiss G A, Wells J A, et al High copy display of large proteins on phage for functional selections [J]. Mol Biol,2000,296(2):487-495
126. Skerra A. Bacterial expression of immunoglobul in fragments [J]. Curr Opin Immunol,1993,5: 256-262
127. Skerra A, Pluckthum A. Assembly of a functional immunoglobul in Fv fragments in Escherichia Coli [J]. Science,1988,240:1038-1041
128. Spada S, Honegger A, Pliickthun A. Reproducing the natural evolution of protein structural features with the selectively-in-fective phage(SIP)technology [J]. Mol Bio,1998,283:395-407
129. Strachan G, Grant S D, Learmonth D, et al Binding characterization of anti-atrazine monoclonal antibodies and their fragments synthesised in bacteria and plants [J]. Biosens Bioelectron,1998,13: 665-673
130. Strohal R, Kroemer G, Wick, G, et al Complete variable region sequence of a nonfunctionally rearranged kappa light chain transcribed in the non-secretor P3X63-Ag8.653 myeloma cell line [J]. Nucleic Acids Res,1987,15:2771
131. Su Y C, Lim K P, Nathan S. Bacterial expression of the scFv fragment of a recombinant antibody specific for Burkholderia pseudomallei exotoxin [J]. J Biochem Mol Biol,2003,36(5):493-498
132. Tang Y, Jiang N, Parakkh C. Selection of linkers for a catalytic single chain antibody using phage display technology [J]. Journal of Biological Chemistry,1996,271:15628-15687
133. Tanha J, Dubuc G. Narang S. A., et al. Selection by phage display of Iiama conventional V(H) H properties [J]. Immunol Methods,2002,263 (2):97-109
134. Tavladoraki P, Benvenuto E, Trinca S, et al Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack [J]. Nature,1993,366(6454):469-72
135. Tout N L, Yau K Y F, Trevors J T, et al Synthesis of ligand-specific phage-display scFv against the herbicide picloram by direct cloning from hyperimmunized mouse [J]. J Agric Food Chem, 2001,49(8):3628-3637
136. Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment [J]. Science,1990, 249(4968):505-510
137. Turner AP F. Biosensors sense and sensitivity [J]. Science,2000,290(5495):1315-1317
138. Turner D J, Riter M A, George A J. Importance of the linker in expression of single-chain Fv antibody fragments:optimization of peptide sequence using phage display technology [J]. Journal of Immunological Methods,1997,205:43-54
139. Verheijen R, Osswald I K, Dietrich R, et al Development of a one step strip test for the detection of (Dihydro)streptomycin residues in raw milk [J]. Food and Agri. Immunol,2000,12:31-40
140. Vuyisich M, Beal P A. Controlling Protein Activity with Ligand-Regulated RNA Aptamers [J]. Chemistry & Biology,2002,9(8):907-913
141. Ward V K, Schneider P G, Kreissig S B, et al Cloning, sequencing. and expression of the Fab fragment of a monoclonal monoclonal antibody to the herbicide atrazine [J]. Protein Eng,1993,6: 981-988
142. Weetal H W, Rogers K G. A simple assay for 2,4-dichlorophenoxyacetic acid using coated test-strips [J]. Anal. Lett,2002,35(8):1341-1348
143. Weiss G A, Lowman H B. Anticalins versus antibodies:made-to-order binding proteins for small molecules [J]. Chemistry & Biology,2000,7:177-184
144. Wels W, Beerli R, Hellmann P. EGF receptor and P185erbB-2-specific single chain antibody toxins differ in their cell-killing activity on tumor cells expressing both receptor proteins [J]. Int J Cancer,1995,60:137-142
145. Wengatz I, Stoutamire D W, Gee S J, et al Development of an enzyme-linked immunosorbent assay for the detection of the pyrethroid insecticide fenpropathrin [J]. J. Agric. Food Chem,1998, 46(6):2211-2221
146. Willuda J, Honegger A, Waibel R. High thermal stability is essential for tumor targeting of antibody fragments:engineering of a humanized anti-epithelial glycoprotein-2 (epithelial cell adhesion molecule) single-chain Fv fragment [J]. Cancer Research,1999,59:5758-576
147. Wing K D, Hammock B D. Stereoselectivity of a radioimmunoassay for the insecticide S-bioallethrin [J]. Experientia,1979,35:1619-1620
148. Wing K D, Hammock B D, Wustner D A. Development of an S-bioallethrin specific antibody [J]. J. Agric. Food Chem,1978,26:1328-1333
149. Winter G, Griffiths A D, Hawkins R E. Making antibodies by phage display technology [J]. Annu Rev Immunol,1994, (12):433-455
150. Winthrop M D, DeNardo S J, DeNardo G L. Development of a hyper immune anti-MUC-1 single chain antibody fragments phage display library for targeting breast cancer [J]. Clinical Cancer Research,1999,5(10 Suppl):3088s-3094s
151. Withoff S, Helfrich W. Bi-specific antibody therapy for the treatment of cancer [J]. Curr Opin Mol Ther,2001,3(1):53-62
152. Wood C R, Boss M A, Kenten J H. The synthesis and in vivo assembly of functional antibodies in yeast [J]. Nature,1985,314:446-449
153. Wyatt G M, Garrett S D, Lee H A, et al Alteration of the Binding Characteristics of a Recombinant scFv Anti-parathion Antibody—Ⅰ. Mutagenesis Targeted at the VH CDR3 Domain [J]. Food Agric.Immunol,1999,11:207-218 154. You K M, Lee S H, Im A, et al Aptamers as functional nucleic acids:in vitro selection and biotechnological applications [J]. Biotechnol. Bioprocess. Eng,2003,8:64-75 155. Zdanov A, Li Y, Bundle D R 1. Structure of a single-chain antibody variable domain (Fv) fragment complexed with a carbohydrate antigene at 1.7-Aresolution [J]. PNAS,1994,91(14):6423-6427 156. Zhang G P, Wang X N, Yang J F, et al Development of an immunochromatographic lateral flow test strip for detection of β-adrenergic agonist Clenbuterol residues [J]. J. Immunol. Methods,2006, 312:27-33
157.万泽生,王海涛,姜绍淳,等.人抗甲肝抗体重链可变区基因的序列分析[J].第三军医大学学报,1997,19(5)：418-420
158.万泽生,王海涛,姜绍淳,等.用PCR扩增的人抗体基因构建人源性噬菌体抗体库[J].中国免疫学杂志,1996,12(增刊)：107
159.吴海波,邵军军,常惠芸.免疫层析快速诊断试纸条在动物疾病诊断中的应用[J].中国动物检疫,2005,22(8)：43-45
160.张建琼,谢维,张雪萍,等.人源抗丙型肝炎病毒噬菌体抗体库的构建、筛选及表达[J].上海免疫杂志,2000,20(5)：304-307
161.张曙明,陈建明,王重庆,等.酶免疫分析在农药分析中应用的进展[J].农业环境与发展,1996,47(1)：13-17
162.曲建慧,成军,张玲霞,等.噬菌体展示技术筛选干扰素a启动子DNA结合蛋白[J].中华肝脏病杂志,2005,13(7)：520-523
163.王学,王海涛,陈万荣,等.人抗HBsAg噬菌体Fab段基因的序列分析及表达[J].军事医学院院刊,1997,21(1)：11-13
164.王弘,潘科,杨金易,等.抗克伦特罗单链抗体可溶性表达及特性鉴定[J].食品科学,2007,28(12)：332-335
165.王琰,刘群英,徐建军,等.人抗一HBsFab段基因的序列分析及表达[J].中华微生物杂志,1995,15(5)：304-307
166.秦丽莉,张春明.单链抗体研究进展及其在医学中的应用[J].国外医学免疫学分册,2005,29(6)
167.米歇尔·霍迪,肖恩·霍华德.免疫分析的发展趋势[J].检验医学,2006,21(增刊)：59-64
168.罗坤,金宁一,吴丛梅,等.重组鸡痘病毒与核酸疫苗联合免疫提高机体免疫功能研究[J].中国肿瘤生物治疗杂志,2001,8(1)：37-40
169.羿国香,赵静,李刚,等.氯嘧磺隆胶体金免疫检测试纸条的制备与应用[J].分析化学,2006,34(12)：1679-1682
170.范列英,韩焕兴,胡东平,等.人源性抗HBsAg单克隆抗体Fab片段的表达与纯化[J].中国免疫学杂志,1998,14(3)：169-171
171.董志伟,王琰.抗体工程[M].北京医科大学出版社,2001：125-126
172.蔡宁.抗Vitronectin兔单克隆抗体的制备与初步鉴定[M].浙江大学,2004：硕士学位论文.
173.谢忠平,崔萍芳,宋霞,等.利用HCV多表位复合抗原制备高效价抗体[J].中国生物制品学杂志,2005,18(4)：313-315
174.赵肃清,孙远明等.用多聚赖氨酸作载体制备抗甲胺磷抗体的研究[J].中国免疫学杂志,2002,18(4)：380
175.郑继平,刘向听,王令春,等.产肠毒素大肠杆菌混合载体疫苗的免疫原性评价[J].现代免疫学,2004,24(6)：482-485
176.钟彦伟,成军,刘妍,等.HCV非结构蛋白NS3人源单链抗体的筛选与鉴定[J].中华传染病杂志,2000,18：12-15
1. Alcocer M J, Doyen C, Lee H A, et al Functional scFv antibody sequences against the organophosphorus pesticide chlorpyrifos [J]. J Agric Food Chem,2000,48:335-337
2. Ames R,Tornetta M, Jones C, et al Isolation of neutralizing anti-Csa monoclonal antibodies from a filamentous phage monovalent Fab display library [J]. J Immunol,1994,152:4572-4581
3. Boado R J., Ji A, Pardridge W M. Cloning and expression in Pichia pastoris of a genetically engineered single chain antibody against the rat transferrin receptor [J]. J Drug Target,2000,8: 403-412
4. Brichta J, Vesela H, Franek M. Production of scFv recombinant fragments against 2,4-dichlorophenoxyacetic acid hapten using naive phage library [J]. Vet. Med,2003,48:237-247
5. Brierley R A, Davis G R, Holtz G C. Production of insulin-like growth factor-1 in methylotrophi yeast cells [P].1994, United States Patent.5:324-639
6. Brinkmann U, Chowdhuyr D, Roscoe D M, et al Phage display of disuflide-stabilized Fv fragment [J]. J. Immunol Methods,1995,182:41-52
7. Brun E M, Marta Garces-Garcia, Rosa Puchades, et al Enzyme-linked immunosorbent assay for the organophosphorus insecticide fenthion. Influence of hapten structure [J]. Journal of Immunological Methods,2004,295:21-35
8. Burrowes O J, Diamond G, Lee T C. Recombinant Expression of Pleurocidin cDNA Using the Pichia pastoris Expression System [J]. J Biomed Biotechnol,2005,4:374-384
9. Charlton K, Harris W J, Porter A J. The isolation of super-sensitive anti-hapten antibodies from combinatorial antibody libraries derived from sheep [J]. Biosens. Bioelectron,2001,16:639-646 10. Charlton K A., Moyle S, Porter A J R., et al Analysis of the diversity of a sheep antibody repertoire as revealed from a bacteriophage display library [J]. J Immunol,2000,164:6221-6229 11. Chowdhury P S, Chang K, Pastan I. Isolation of antimesothelin antibodies from a phage display library [J]. Mol Immuno,1997,84:9-20
12. Clackson T, Hoogenboom H R, Griffiths A D, et al Making antibody fragment using phage display libraries [J]. Nature,1991,352:624-628
13. Clare J J, Rayment F B, Ballantine S P, et al High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Biotechnology (N Y) [J].1991,9 (5):455-60.
14. Clare J J, Romanos M A. Expression of cloned genes in the yeasts Saccharomyces cerevisiae and Pichia pastoris [J]. Methods Mol Cell Biol,1995,5:319-329
15. Clare J J, Romanos M A, Rayment F B, et al Production of mouse epidermal growth factor in yeast:high-level secretion using Pichia pastoris strains containing multiple gene copies [J]. Gene, 1991,105(2):205-12
16. Cregg J M, Vedvick T S, Raschke W C. Recent advances in the expression of foreign genes in Pichia pastoris [J]. Biotechnology(N Y),1993,11(8):905-910
17. Cwirla S E, Peter E A, Barrett R.W, et al peptides on phage:A vast library of peptides for identifying ligands [J]. Proc. Natl. Acad. Sci. USA,1990,87:6378-6382
18. David R H, James M C. PichiaProtocols [M]. [S.l.] Humana Press Inc.1998
19. Dedman J R, Marcia A K, Hsiao C C, et al Selection of targeted biological modifiers from a bacteriophage library of random peptides. J Biol Chem,1993,268:23025-23030
20. Di Paolo C, Willuda J, Kubetzko S, et al A recombinant immunotoxin manized epithelial cell adhesion molecple-specific single-chain antibody Fragment has potent and selective anti-tumor activity [J]. Cin Cancer Res,2003,9(7):2837-2848
21. Eldin P, Pauza M E, Hieda Y, et al High-level secretion of two antibody single chain Fv fragments by Pichia pastoris [J]. J Immunol Methods,1997,201:67-75
22. Ellington A D, Szostak J W. In vitro selection of RNA molecules that bind specific ligands [J]. Nature,1990,346:1104
23. Freia J. Detection of pesticides residuces by enzyme immunoassay [J]. Pestic. Sci,1989,26: 303-307
24. Freyre F M, Vazquez J E, Ayala M, et al Very high expression of an anti-carcinoembryonic antigen single chain Fv antibody fragment in the yeast Pichia pastoris [J]. J Biotechnol,2000,76:157-163
25. Gallop M, Barrett R, Dower W. Applications of combinatorial technonologies to drug discovery Background and peptide combinatorial libraries [J]. Journal of Medicinal chemistry,1994,37: 1223
26. Garrett S D, Appleford D J A, Wyatt G M, et al Production of recombinant anti-parathion antibody (scFv); Stability in methanolic food extracts and comparison to an anti-parathion monoclonal antibody [J]. J. Agri. Food Chem,1997,45:4183-4189
27. Gellissen G, Kunze G, Gaillardin C, et al New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and dimorphic Arxula adeninivorans and Yarrowia lipolytica-A comparison [J]. FEMS Yeast Res,2005,5:1079-1096
28. Graham B M, Porter A J, Harris W J. Cloning, expression and characterization of a single-chain antibody fragment to the herbicide paraquat [J]. J Chem Technol Biotechnol,1995,63(3):279-289
29. Grant R A, Lin T C, Webster R E. Structure of filamentous bacteriophage:isolation, characterization, and localization of the minor coat proteins and orientation of the DNA [J]. Prog Clin Biol Res,1981,64:413-428
30. Griffiths A D, Duncan A R. Strategies for selection of antibodies by phage display [J].Curr Opin Biotechnol,1998,9:102-108
31. Gurkan C, Symeonides S N, Ellar D J. High-level production in Pichia pastoris of an anti-p185HER-2 scFv using an alternative secretion expression vector [J]. Biotechnol Appl Biochem,2004,39:115-122
32. Hans J, de Haard, Nicole, et al A Large Non-immunized Human Fab Fragment Phage Library That Permits Rapid Isolation and Kinetic Analysis of High Affinity Antibodies [J]. J Biol Chem,1999, 26:18218-18230
33. Hellwig S, Emde F, Raven N P G, et al Analysis of. single-chain antibody production in Pichia pastoris using on-line methanol control in fed-batch and mixed-feed fermentations [J]. Biotechnol Bioeng,2001,74:344-352
34. Henderikx P, Kandilogiannaki M, Petrarca C, et al Human single-chain Fv antibodies MUC1 core peptide selected from phage display libraries recognize unique epitopes and predominantly bind adenocarcinoma [J]. Cancer Res,1998,58 (4):4324-4332
35. Hoogenboom H R. Designing and optimizing library selection strategies for generating high-affinity anti-bodies [J]. TIB TECH,1997,15(2):62-69
36. Ito H., Fukuda Y, Murata K, et al Transformation of intact yeast cells treated with alkalications [J]. J Bactoriol,1983,153(1):163
37. Johnson G,Wu T T. The Kabat database and a bioinformatics example [J]. Met. Mol. Biol,2004, 248:11-25
38. Jung F, Meyer H D, Hamm R T. Development of a sensitive Enzyme-linked Immunosor bent Aassay for the Fungicide Fenpropimorph [J]. J Agric Food Chem,1989,37:1183-1187
39. Kalidas C, Joshi L, Batt C. Characterization of glycosylated variants of β-Lactoglobulin expression in Pichia pastoris [J]. Protein Eng,2001,14(3):201-207
40. Kay B K, Winter J, McCafferty J. Phage display of Peptides and Proteins [J]. Academic Press. 1996
41. Kramer K. Synthesis of a group-selective antibody library against haptens [J]. J. Immuno. Methods, 2002,266:209-220
42. Kramer K, Fiedler M, Skerra A, et al A generic strategy for subcloning antibody variable regions from the scFv phage display vector pCANTAB 5 E into pASK85 permits the economical production of Fab fragments and leads to improved recombinant immunoglobulin stability. Biosens [J]. Bioelectron,2002,17:305-313
43. Kramer K, Hock B. Recombinant antibodies for environmental analysis [J]. Anal. Bioanal. Chem, 2003,377:417-426
44. Kretzschmar T, von Ruden T. Antibody discovery:phage display [J]. Curr Opin Biotechnol,2002, 13(6):598-602
45. Lange S, Schmitt J, Schmid R D. High-level expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris [J]. J Immunol Methods.2001,255: 103-114
46. Lee J K, Ahn K C, Stoutamire D W, et al Development of an enzyme-linked immunosorbent assay for the detection of the organophosphorus insecticide acephate [J]. J Agric Food Chem,2003,51: 3695-3703
47. Li Y, Cockburn W, Kilpatrick J, et al Selection of rabbit single-chain Fv fragment against the herbicide atrazine using a new phage display system. [J] Food Agric Immunol,1999,11:5-17
48. Li Y, Cockburn W, Kilpatrick J B. et al High affinity ScFvs from a single rabbit immunized with multiple haptens [J]. Biochem. Biophys. Res. Com,2000,268:398-404
49. Longstaff M, Newell C A, Boonstra B, et al Expression and characterization of single-chain antibody fragments produced in transgenic plants against the organic herbicides atrazine and paraquat [J]. Biochim Biophys Acta,1998,1381(2):147-160
50. Mansur M, Cabello C, Hernandez L, et al Multiple gene copy number enhances insul in precursor secretion in the yeast Pichia pastoris [J]. Biotechnol Lett,2005,27(5):339-45
51. Marco M P, Gee S J, Hammock B D. Immunochemical techniques for environmental analysis. II. Antibody production and immunoassay development [J]. Trends Anal. Chem,1995,14(8):415-425
52. Mark J D, Hoogenboom H R, Bonnert T.P., et al. By-passing immunization. Human antibodies from Ⅴ-gene libraries displayed on phage [J]. J Mol Biol,1991,222:81-597
53. McElhiney J, Drever M, Lawton L A. Rapid isolation of a single-chain antibody against the cyanobacterial toxin microcystin-LR by phage display and its use in the immunoaffinity concentration of microcystins from water [J]. Appl Environ Micro biol,2002 Nov,68(11):5288-95
54. Michael D. Sheets, Peter Amersdorfer, Ricarda Finnern, et al Efficient construction of a large nonimmune phage antibody library:The production of high-affinity human single-chain antibodies to protein antigens [J]. Cell Biology,1998,11:6157-6162
55. Miller K D, Weaver-Feldhaus J, Gray S A, et al Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli [J]. Protein Expr Purif,2005,42:255-267
56. Monod Y M, Guidicelli V, Chaume D, et al IMGT/JunctionAnalysis; the first tool for the analysis of immunoglobulin and T cell receptor complex V-J and V-D-J junctions [J]. Bioinformatics,2004, 20:1379-1385
57. Nathan S, Li H, Mohamed R, et al Phage display of recombinant antibodies toward Burkholderia pseudomallei exotoxin [J]. J Biochem Mol Biol Biophys,2002 Feb,6(1):45-53
58. Orlandi R, Gussow D H, Jones P T, et al. Cloning immunoglobulin variable domains for expression by the polymerase chain reaction [J]. Proc Natl Acad Sci USA,1989,86:3833
59. Reiter Y, Brinkmann U, Webber K O, et al Engineering interchain disulfide bonds into conserved framework regions of Fv fragments:improved biochemical characteristics of recombinant immunotoxins containing disulfide-stabilized Fv [J]. Protein Eng,1994,7:697-704
60. Rigano R, Profumo E, Siracusano A. New perspectives in the immunology of Echinococcus granulosus infection [J]. Parassitologia,1997,39(4):275-277
61. Rodi D J, Makowski L. Phage-display technology-fording a needle in a vast molecular haystack [J]. Curr Opin Hiotechnol,1999,10:87-93
62. Sanchez L, Ayala M, Freyre F, et al Higt cytoplasmic expressin in E.coli, purification, and in vitro refolding of a single chain Fv antibody fragment against the hepatitis B surface antigen [J]. Journal of Biotechnology,1999,72:13-20
63. Scorer C A, Clare J J, McCombie W R., et al Rapid selection using 6418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression [J]. Biotechnology (N Y), 1994,12(2):181-4
64. Shenlan M, Changshou G, Chih-Hung L, et al Phage-display library selection of high-affinity human single-chain antibodies to tumor-associated carbohydrate antigens sialyl Lewisx and Lewisx [J]. Medical Sciences,1999,12:6953-6958.
65. Sheth H B, Sporns P. Development of a single ELISA for detection of Sulfanamides [J]. J Agric Food Chem.1991,39 (9):1696-1700
66. Shi X, Karkut T, Chamankhah M, et al Optimal conditions for the expression of a single-chain antibody (ScFv) gene in Pichia pastoris [J]. Protein Express. Purifi,2003,28:321-330
67. Smith G P. Filamentous fusion phage:novel expression vectors that dispay cloned antigens on the virion surface [J]. Science,1985,228:1315-1317
68. Sreekrishna K, Brankamp R G, Kropp K E, et al Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris [J]. Gene,1997,190:55-62
69. Strachan G, Grant S D, Learmonth D, et al Binding characterization of anti-atrazine monoclonal antibodies and their fragments synthesised in bacteria and plants [J]. Biosens Bioelectron,1998,13: 665-673
70. Szurdoki F, Bekheit H K M, Marco M P, et al Important factors in hapten design and enzyme-linked immunosorbent assay development. In:Kurtz D.A., Skerritt J.H., Stanker L. New Frontiers in Agrochemical Immunoassays, AOAC, Arlington, VA,1995, P.39
71. Tout N L, Yau K Y F, Trevors J T, et al Synthesis of ligand-specific phage-display scFv against the herbicide picloram by direct cloning from hyperimmunized mouse [J]. J. Agric. Food Chem, 2001,49:3628-3637
72. Vaughan T J, Williams Sc, Pritchard K, et al Human antibodies with subnanomolar affinities isolated from a large non-immunized phage display library [J]. Nat Biotechnol,1996,14:309-314
73. Wang Y C, Chen Z R, Chen Z W, et al Recombinant human heparin-binding neurite-promoting factor expressed with yeast stimulates neuritis out growth [J]. Chin Med J.2002,115:1352-1357
74. Wells J A, Clackson T. In vitro selection from protein and peptide libraries [J]. TIB TECH,1994, 12:173-183
75. Willuda J, Honegger A, Waibel R, et al High thermal stability is essential for tumor targeting of antibody fragments:engineering of a humanized anti-EGP-2 (Ep-CAM) scFv fragment [J]. Cancer Res,1999,59:5758-5767
76. Winter G, Griffiths A D. Making antibodies by phage display technology [J]. Ann Rev Immunol, 1994,12 (2):433
77. Worn A, Plueckthun A. Stability engineering of antibody single-chain Fv fragments [J]. J Mol Biol, 2001,305:989-1010
78. Wu S, Geoffrey J, Letchworth. High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiotheitol [J]. Drug discovery and genomic technologies,2004,36(1):152-157
79. Yazaki P J, Shively L, Clark C, et al Mammalian expression and hollow fiber bioreactor production of recombinant anti-CEA diabody and minibody for clinical applications [J]. J. Immunol. Method, 2001,253(1-2):195-208
80. Yuan Q, Hu W, Pestka J J. Expression of a functional antizearalenone single-chain Fv antibody in transgenic Arabidopsis plants [J]. Appl. Env. Microb,2000,66,3499-3505
81. Zhang Y D, Yang B L. In vivo optimizing of intracellular production of heterologous protein in Pichia pastoris by fluorescent scanning [J]. Anal Biochem,2006,357:232-239
82. Zhao S Q, Sun Y M, Zhang C Y, et al Studies on purification of methamidophos monoclonal antibodies and comparative immunoactivity of purified antibodies [J]. Biomed Environ Sci,2003, 16(2):119-125
83. Ziegler A, Cowan G H, Torrance L, et al Expression of functional recombinant antibody molecules in insect cell expression systems [J]. Protein Express. Purifi,2000,18:221-228
84.董国伟,刘贤进,王沫,等.间接竞争ELISA检测甲胺磷残留[J].华中农业大学学报,2001,20(5)：434-437
85.董国伟,王沫,刘贤进,等.兔抗甲胺磷多克隆抗体的制备[J].华中农业大学学报,2001,20(4)：340-343
86.杜桂鑫,于长明,毛春生,等.人噬菌体抗体库的构建和甲肝抗体的筛选[J].中华微生物学和免疫学杂志,2000,20：49-52
87.侯颖春,杨为松,白雪帆,等.人源性汉坦病毒噬菌体抗体基因的筛选、测序和表达[J].中华微生物学和免疫学杂志,1999,19：38-41
88.胡世界,罗素兰,张吉贞,等.巴斯德毕赤酵母表达系统及其高水平表达策略[J].生物技术,2007,17(6)：78-83
89.刘凤权,许志刚,王金生.定量测定甲胺磷残留的间接竞争ELISA的建立和初步应用[J].农业生物技术学报,1998,6(2)：140-146
90.刘庆慧,韩文军,黄捷,等.WSSV-VAP1基因克隆及在毕赤酵母中的表达[J].高技术通讯,2005,15(5)：67-70
91.吕海芹,张建琼.噬菌体抗体库技术研究进展[J].微生物学免疫学进展,2002,29(1)：90-93
92.毛春生,李季枝,宋宏彬.人源性抗HIV-1 V3环噬菌体抗体的基因克隆与序列分析[J].细胞与分子免疫学杂志,1999,15：178-181
93.萨姆布鲁克等.《分了克隆实验指南》[M].北京：科学出版社,1995年第二版
94.沈倍奋主编.分子文库[M].北京,科学出版社,2001
95.孙亚范,王海宽,向微,等.甲醇毕赤酵母Pichia methanolica的高效电转化方法[J].食品与发酵工业,2004,30(5)：35-39
96.孙远明,赵肃清,张春艳,等.Synthesis and identification of methamidophos artificial antigen [J].免疫学杂志,2002,18(3)：163-166
97.王弘,潘科,杨金易,等.抗克伦特罗单链抗体可溶性表达及特性鉴定[J].食品科学,2007,28(12)：332-335
98.王琐,化冰,刘群英,等.半合成抗体库的构建及鉴定[J].中华微生物学和免疫学杂志,1999,19：238-241.
99.王琐,王欲晓,朱迎春,等.将抗体库所获抗-HBs Fab转换为全长人IgG[J].中华微生物学和免疫学杂志,2000,20：41-44.
100.王志毅,刘祀,万泽生,等.抗-HBs的人源性噬菌体抗体库的构建[J].中华肝脏病杂志,1999,7(3)：159-161.
101.于长明,杜桂鑫,毛春生,等.人源抗HAV噬菌体抗体的筛选及基因序列分析[J].中华肝脏病杂志,1999,7(3)：162-164.
102.张奇.速灭威等化学农药免疫速测技术研究.南京农业大学,2004,硕士学位论文.
103.赵肃清,孙远明,乐学义,等.农药人工抗原合成的研究进展.农药,2002,41(3)：9-11.
104.赵肃清,孙远明,张春艳.甲胺磷单克隆抗体制备及鉴定.免疫学杂志,2003,19(2)：142-145.
105.赵肃清,孙远明等.用多聚赖氨酸作载体制备抗甲胺磷抗体的研究.免疫学杂志,2002,18(4)：380.
106.甄永苏,邵荣光主编.抗体工程药物.化学工业出版社.2002.
