Synthesis of Ligand-Specific Phage-Display ScFv against the Herbicide Picloram by Direct Cloning from Hyperimmunized Mouse

Nl, Tout; Ky, Yau; Jt, Trevors; H, Lee; Jc, Hall

doi:10.1021/jf010136w

Cited by 39 publications

(40 citation statements)

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“…When affinities of RAbs and those of their parental MAbs have been compared in the literature, diverse results have been reported. While some authors reported that RAbs showed lower affinity than their parental MAb, others reported a comparable or even higher affinity of RAbs over their counterpart MAbs (Graham et al 1995;Kramer and Hock 2007;Nishi et al 2005;Tout et al 2001). Results obtained in this study and those reported in the literature point out that the binding site structure of scFvs differs somehow from that of their respective parental antibody molecules (MAb).…”

Section: Characterization Of Scfv Recombinant Antibodiessupporting

confidence: 39%

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Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit Juices

et al. 2013

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Abstract:A comparative study of the analytical performance of enzyme-linked immunosorbent assays (ELISAs), based on monoclonal and recombinant antibodies, for the determination of fungicide residues in fruit juices has been carried out. To this aim, three murine hybridoma cell lines secreting specific monoclonal antibodies against tetraconazole, thiabendazole, and imazalil were used as a source of immunoglobulin gene fragments for the production of single-chain variable fragment (scFv) and fusion scFv-pIII recombinant antibodies in Escherichia coli. Selected recombinant antibodies displayed cross-reactivity profiles very similar to those of the parent monoclonal antibodies. Imazalil and tetraconazole recombinant antibodies showed one order of magnitude lower affinity than their respective monoclonal antibodies, whereas the thiabendazole recombinant antibodies showed an affinity similar to that of their parent monoclonal antibody. On the other hand, scFv-pIII fusion fragments showed similar analytical properties as, and occasionally better than, scFv recombinant antibodies. Finally, ELISAs developed from each antibody type showed similar analytical performance when applied to the analysis of the target fungicides in fruit juices. Powered by Editorial Manager® and Preprint Manager® from Aries Systems Corporation AbstractA comparative study of the analytical performance of enzyme-linked immunosorbent assays (ELISAs), based on monoclonal and recombinant antibodies, for the determination of fungicide residues in fruit juices has been carried out. To this aim, three murine hybridoma cell lines secreting specific monoclonal antibodies against tetraconazole, thiabendazole, and imazalil were used as a source of immunoglobulin gene fragments for the production of single-chain variable fragment (scFv) and fusion scFv-pIII recombinant antibodies in Escherichia coli. Selected recombinant antibodies displayed cross-reactivity profiles very similar to those of the parent monoclonal antibodies. Imazalil and tetraconazole recombinant antibodies showed one order of magnitude lower affinity than their respective monoclonal antibodies, whereas the thiabendazole recombinant antibodies showed an affinity similar to that of their parent monoclonal antibody. On the other hand, scFv-pIII fusion fragments showed similar analytical properties as, and occasionally better than, scFv recombinant antibodies. Finally, ELISAs developed from each antibody type showed similar analytical performance when applied to the analysis of the target fungicides in fruit juices.

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Section: Characterization Of Scfv Recombinant Antibodiessupporting

confidence: 39%

“…In this sense, phage-displayed scFv analysis is a more laborious procedure because a phage rescue step is required. Moreover, the size and complexity of phage-displayed scFv may contribute to unspecific interactions that would mask the specific ones (Mersmann et al 1998;Tout et al 2001). …”

Section: Cloning and Expression Of Scfv And Scfv-piii Rab Fragmentsmentioning

confidence: 99%

Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit Juices

et al. 2013

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“…The assay sensitivity for the scFv antibodies is similar to those scFv obtained by Tout et al (2001) and Kramer (1998) for picloram and atrazine. The authors produced the scFv fragments using direct cloning of variable parts of antibody genes from immunized mice or employed recombinant phage antibody system from RPAS, Pharmacia.…”

Section: Discussionsupporting

confidence: 66%

Production of scFv recombinant fragments against 2,4-dichlorophenoxyacetic acid hapten using naďve phage library

Brichta¹,

Veselá²,

Fránek³

2003

Vet. Med. - Czech

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Three single chain variable fragment (scFv) antibodies against 2,4-dichlophenoxyacetic acid (2,4-D) herbicide were produced by the Griffin1.library. The selection of the scFv from the phage library was carried out by 2,4-D-protein coated tubes with different levels of hapten substitution in the conjugate. The scFv phage clones were isolated within the five round library panning and the antibodies were expressed in Escherichia coli HB2151. The recombinant products were purified by metal affinity chromatography yielding 200 g of pure scFv per 1 liter of bacterial culture. The antibody fragments provided steep curves in conventional indirect ELISA having the IC<sub>50</sub> values from 10.2 to 14.5 ng/ml established for 2,4-D standard. Interestingly enough, the recombinant ScFv E1 antibody exhibited 68% cross-reactivity with 2,4-dichlorphenol (2,4-D = 100%), and 38.0% with methylchlorophenoxyacetic acid (MCPA) whereas reaction with other phenoxyacetic compounds was low. Similar characteristics were obtained for other two recombinant products. Low stability for the isolated scFv antibodies was found in storage buffer even in the presence of stabilizers and protease inhibitors. Factors influencing stability of the recombinant antibodies are discussed.

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“…A maioria dos trabalhos envolve a utilização de camundongos, pois esses animais apresentam diversas vantagens sobre os demais, entre elas, as facilidades de obtenção, manutenção e manuseio, a vasta disponibilidade de animais de linhagem pura, destinados a diferentes estudos imunológicos, e a resposta imuno-humoral, cuja evolução é bastante semelhante à do homem 19,53 . Contudo, dependendo do tipo de composto a ser testado, nem sempre o uso de animais de pequeno porte, como camundongos 54,55 e coelhos 55,56 , pode levar a uma imunoresposta com a sensibilidade esperada, o que obriga o pesquisador a mudar de animal. Deste modo, ovelhas, porcos, bois, cavalos e camelos têm sido também utilizados em investigações específicas 53,[57][58][59][60] .…”

Section: Produção Dos Anticorpos (Imunização)unclassified

Métodos imunoquímicos para análise de contaminantes ambientais: conceitos, estado da arte e perspectivas

Nunes¹

2005

Quím. Nova

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Recebido em 6/2/04; aceito em 20/10/04; publicado na web em 28/2/05 IMMUNOCHEMICAL METHODS FOR ANALYSIS OF ENVIRONMENTAL CONTAMINANTS: CONCEPTS, STATE OF THE ART AND PERPECTIVES. Immunoassay techniques provide simple, powerful and inexpensive methods for analysis of environmental contaminants. However, the acceptance of immunoassays is dependent on the clear demonstration of quality and validity compared to more traditional techniques. In this review, primarily, the understanding and the fundamentals of immunoassay methods are given in order to make good use of immunoassays, especially of EIA tests. Special attention is given to the concepts related to the enzymelinked immunosorbent assay (ELISA) formats, such as inhibition concentration at 50% (IC 50 ), detection limit (LOD), cross-reactivity (CR %). It is also explained why some immunoassays are quantitative methods whereas others can only be used as screening methods. A list of main commercial kits for detection of priority pollutants is given in order to help analysts. Others formats, such as flowinjection immunoassay analysis (FIIA), immunoassay chromatography and immunosensors are also cited.Keywords: immunoassays; environmental contaminants; monitoring. INTRODUÇÃOA origem dos métodos imunológicos situa-se no final da déca-da de 50, quando Yalow e Berson 1 descreveram o primeiro ensaio imunológico para detectar insulina humana, o que lhes rendeu o Prêmio Nobel em 1977. A partir de então, as técnicas imunoquí-micas foram desenvolvidas como métodos de análise de macromoléculas (enzimas, hormônios), e posteriormente para moléculas orgânicas pequenas (drogas, fármacos), tendo seu maior êxito no campo da química clínica 2-4 . Em meados da década de 70, foi desenvolvido o primeiro ensaio biológico para pesticidas (aldrin e dieldrin) 5 . A este ensaio, seguiram-se outros, também baseados na marcação radioativa, chamados ensaios radioimunológicos ("radioimmunoassay, RIA"). Os ensaios imunoenzimáticos ("enzyme immunoassay, EIA") surgiram na década de 60 para identificação e localização de antígenos em preparações histológicas 2,6,7 , tendo sido a sua utilidade reconhecida posteriormente em outros campos distintos [8][9][10][11][12][13] . No início dos anos 80, começou-se a discutir as possibilidades que estes tipos de métodos analíticos podiam oferecer na detecção de contaminantes, tanto em amostras ambientais como de alimentos 12,14-16 , o que fomentou a sua aplicação no campo da química ambiental. A partir desse momento, começou-se a utilizar a marcação enzimática, que proporcionava menores riscos e apresentava sensibilidade similar aos métodos convencionais de análise 12,[15][16][17][18][19] . Inúmeras vantagens podem ser obtidas com a utilização dos métodos imunoquímicos e, paulatinamente, vão sendo disponibilizadas informações sobre diversos aspectos analíticos, tais como i) interpretação dos dados obtidos no desenvolvimento dos métodos; ii) estratégias utilizadas para a garantia da qualidade dos procedimentos operacionais; iii) análise dos dados e das informa...

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Synthesis of Ligand-Specific Phage-Display ScFv against the Herbicide Picloram by Direct Cloning from Hyperimmunized Mouse

Cited by 39 publications

References 46 publications

Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit Juices

Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit Juices

Production of scFv recombinant fragments against 2,4-dichlorophenoxyacetic acid hapten using naďve phage library

Métodos imunoquímicos para análise de contaminantes ambientais: conceitos, estado da arte e perspectivas

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