Abstract
A rabbit phage display library of single chain Fv (scFv) antibodies was constructed to screen specific scFv antibodies against melamine. Rabbits were immunized with melamine immunogens prepared via glutaraldehyde method. Two recombinant scFv antibodies with high specificity for melamine, termed A9 and B4, were isolated via library panning. SDS-PAGE analysis showed that the soluble A9-scFv and B4-scFv antibodies were expressed successfully and the scFvs showed good sensitivity with an IC50 of 12.64 and 10.32 μM to melamine, respectively. The cross-reactivity of 2 scFv antibodies with cyanuric acid and chloromycetin were below 1%. The high similarity of 2 scFv nucleotide sequences indicated that they may originate from 1 B cell clone that had undergone somatic diversification. As a consequence, the conjugation synthesis was successful and the selected scFvs could be used for sensitive detection of melamine in foods and feeds by immunoassay.
Similar content being viewed by others
References
Andersen WC, Turnipseed SB, Karbiwnyk CM, Clark SB, Madson MR, Gieseker CM, Miller RA, Rummel NG, Reimschuessel R. Determination and confirmation of melamine residues in catfish, trout, tilapia, salmon, and shrimp by liquid chromatography with tandem mass spectrometry. J. Agr. Food Chem. 56: 4340–4347 (2008)
Garber EAE. Detection of melamine using commercial enzymelinked immunosorbent assay technology. J. Food Protect. 71: 590–594 (2008)
Ehling S, Tefera S, Ho IP. High-performance liquid chromatographic method for the simultaneous detection of the adulteration of cereal flowers with melamine and related triazine by-broducts ammeline, ammelide, and cyanuric acid. Food Addit. Contam. 24: 1319–1325 (2007)
Scheepers ML, Meier RJ, Markwort L, Gelan JM, Vanderzande DJ, Kip BJ. Determination of free melamine content in melamineformaldehyde resins by Raman spectroscopy. Vibra. Spectrosc. 9: 139–146 (1995)
Yokley RA, Mayer LC, Rezaaiyan R, Manuli ME, Cheung MW. Analytical method for the determination of cyromazine and melamine residues in soil using LC-UV and GC-MSD. J. Agr. Food Chem. 48: 3352–3358 (2000)
Zhu XZ, Wang Sh, Liu Q, Xu Q, Xu S, Chen H. Determination of residues of cyromazine and its metabolite, melamine, in animal-derived food by gas chromatography-mass spectrometry with derivatization. J. Agr. Food Chem. 57: 11075–11080 (2009)
Filigenzi MS, Tor ER, Poppenga RH, Poppenga RH, Aston LA, Puschner B. The determination of melamine in muscle tissue by liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Sp. 21: 4027–4032 (2007)
Tran BN, Okoniewski R, Storm R, Jansing R, Aldous KM. Use of methanol for the efficient extraction and analysis of melamine and cyanuric acid residues in dairy products and pet foods. J. Agr. Food Chem. 58: 101–107 (2010)
Chan CP, Cheung YC, Renneberg R, Seydack M. New trends in immunoassays. Adv. Biochem. Eng. Biot. 109: 123–154 (2008)
Marks JD, Hoogenboom HR, Griffiths AD, Winter G. Molecular evolution of proteins on filamentous phage. J. Biol. Chem. 267: 16007–16010 (1992)
Marks C, Marks JD. Phage libraries — A new route to clinically useful antibodies. New Eng. J. Med. 335: 730–733 (1996)
Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR. Making antibodies by phage display technology. Annu. Rev. Immunol. 12: 433–455 (1994)
Sidhu SS, Li B, Chen Y, Fellouse FA, Eigenbrot C, Fuh G. Phagedisplayed antibody libraries of synthetic heavy chain complementarity determining regions. J. Mol. Biol. 338: 299–310 (2004)
Avrameas S. Coupling of enzymes to proteins with glutaraldehyde: Use of the conjugates for the detection of antigens and antibodies. Immunochemistry 6: 43–48 (1969)
Barbass III CF, Burton DR, Scott JK, Silverman GJ. Phage Display: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, NY, USA. protocol 9. 7 (2001)
Rader C, Barbas III CF. Phage display of combinatorial antibody libraries. Curr. Opin. Biotech. 8: 503–508 (1997)
Miller KD, Weaver-Feldhaus J, Gray SA. Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. Protein Expres. Purif. 42: 255–267 (2005)
Friguet B, Chaffotte AF, Djavadi-Ohaniance L, Goldberg ME. Measurements of the true affinity constant in solution of antigenantibody complexes by enzyme-linked immunosorbent assay. J. Immunol. Methods 77: 305–319 (1985)
Yuan QP, Clarke JR, Zhou HR, Linz JE, Pestka JJ, Hart LP. Molecular cloning, expression, and characterization of a functional single-chain Fv antibody to the mycotoxin zearalenone. Appl. Environ. Microb. 63: 263–269 (1997)
Roucairol C, Azoulay S, Nevers MC, Créminon C, Grassi J, Burger A, Duvalet D. Development of a competitive immunoassay for efavirenz: Hapten design and validation studies. Anal. Chim. Acta 589: 142–149 (2007)
Chalfie M. Green fluorescent protein. Photochem. Photobiol. 62: 651–656 (1995)
Kasai S, Horie T, Mizuma T, Awazu S. Fluorescence energy transfer study of the relationship between the lone tryptophan residue and drug binding sites in human serum albumin. J. Pharm. Sci. 76: 387–392 (1987)
Tout NL, Yau KYF, Trevors JT, Lee H, Hall JC. Synthesis of ligandspecific phage-display scFv against the herbicide picloram by direct cloning from hyperimmunized mouse. J. Agr. Food Chem. 49: 3628–3637 (2001)
Lei H, Shen Y, Song L, Yang J, Chevallier OP, Haughey SA, Wang H, Sun Y, Elliottet CT. Hapten synthesis and antibody production for the development of a melamine immunoassay. Anal. Chim. Acta 665: 84–90 (2010)
Yin W, Liu J, Zhang T, Li W, Liu W, Meng M, He F, Wan Y, Feng C, Wang S, Lu X, Xi R. Preparation of monoclonal antibody for melamine and development of an indirect competitive ELISA for melamine detection in raw milk, milk powder, and animal feeds. J. Agr. Food Chem. 58: 8152–8157 (2010)
Tomlinson IM, Cox JPL, Gherardi E, Lesk AM, Chothia C. The structural repertoire of the human VK domain. EMBO J. 14: 4628–4638 (1995)
Al-Lazikani B, Lesk AM, Chothia C. Standard conformations for the canonical structures of immunoglobulins. J. Mol. Biol. 273: 927–948 (1997)
Rader C, Ritter G, Nathan S, Elia M, Gout I, Jungbluth AA, Cohen LS, Welt S, Old LJ, Barbas III CF. The rabbit antibody repertoire as a novel source for the generation of therapeutic human antibodies. J. Biol. Chem. 275: 13668–13676 (2000)
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Huang, A., Lu, T. Production of single-chain Fv antibodies against melamine from a rabbit phage display library for the development of a melamine immunoassay. Food Sci Biotechnol 20, 1699–1704 (2011). https://doi.org/10.1007/s10068-011-0234-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10068-011-0234-z