In order to generate an antibody against a small hapten molecule, the hapten is cross-linked with carrier protein to make it immunogenic. In this study, the hapten (Fumonisin B 1, FB 1) was coupled to ovalbumin (OVA) and bovine serum albumin (BSA), respectively by a short cross-linker reagent (glutaraldehyde, GA). To develop a technique for detecting the conjugation, the hapten-protein conjugates (FB 1-OVA and FB 1-BSA) were characterized thoroughly by ultraviolet (UV) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. The molecular weights of FB 1-BSA and FB 1-OVA were 74,355.301 Da and 48,009.212 Da, respectively determined by the method of MALDI-TOF-MS. The molecular coupling ratios were 11 and 5 in FB 1-BSA and FB 1-OVA, respectively. In this experiment, MALDI-TOF-MS was selected as the most efficient method to evaluate the cross-linking effect and calculate the molecular coupling ratio.
References
[1]
Reverberi, M.; Ricelli, A.; Zjalic, S.; Fabbri, A.A.; Fanelli, C. Natural functions of mycotoxins and control of their biosynthesis in fungi. Appl. Microbiol. Biotechnol 2010, 87, 899–911.
[2]
Stepien, L.; Koczyk, G.; Waskiewicz, A. FUM cluster divergence in fumonisins-producing Fusarium species. Fungal Biol 2011, 115, 112–123.
[3]
Bartok, T.; Tolgyesi, L.; Szekeres, A.; Varga, M.; Bartha, R.; Szecsi, A.; Bartok, M.; Mesterhazy, A. Detection and characterization of twenty-eight isomers of fumonisin B1 (FB1) mycotoxin in a solid rice culture infected with Fusarium verticillioides by reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight and ion trap mass spectrometry. Rapid Commun. Mass Spectrom 2010, 24, 35–42.
[4]
Draft Guidance for Industry: Fumonisin Levels in Human Foods and Animal Feeds; Availability; US Food and Drug Administration: Washington, DC, USA, 2002; Volume 65, p. 35945.
[5]
Gazzotti, T.; Zironi, E.; Lugoboni, B.; Barbarossa, A.; Piva, A.; Pagliuca, G. Analysis of fumonisins B1, B2 and their hydrolysed metabolites in pig liver by LC–MS/MS. Food Chem 2011, 125, 1379–1384.
[6]
Bernabucci, U.; Colavecchia, L.; Danieli, P.P.; Basirico, L.; Lacetera, N.; Nardone, A.; Ronchi, B. Aflatoxin B1 and fumonisin B1 affect the oxidative status of bovine peripheral blood mononuclear cells. Toxicol. In Vitro 2011, 25, 684–691.
[7]
Severino, L.; Russo, R.; Luongo, D.; de Luna, R.; Ciarcia, R.; Rossi, M. Immune effects of four Fusarium-toxins (FB1, ZEA, NIV, DON) on the proliferation of Jurkat cells and porcine lymphocytes: In vitro study. Vet. Res. Commun 2008, 32, S311–S313.
[8]
He, C.H.; Fan, Y.H.; Wang, Y.; Huang, C.Y.; Wang, X.C.; Zhang, H.B. The individual and combined effects of deoxynivalenol and aflatoxin B1 on primary hepatocytes of Cyprinus carpio. Int. J. Mol. Sci 2010, 11, 3760–3768.
[9]
Zhang, H.B.; He, C.H.; Fan, Y.H.; Liu, G.F. Isolation and identification of a strain of aspergillus tubingensis with deoxynivalenol biotransformation capability. Int. J. Mol. Sci 2008, 9, 2366–2375.
[10]
Yanhong, F.; Chenghua, H.; Guofang, L.; Haibin, Z. Optimization of the isolation and cultivation of Cyprinus carpio primary hepatocytes. Cytotechnology 2008, 58, 85–92.
[11]
de Oliveira Rocha, L.; Reis, G.M.; da Silva, V.N.; Braghini, R.; Teixeira, M.M.; Correa, B. Molecular characterization and fumonisin production by Fusarium verticillioides isolated from corn grains of different geographic origins in Brazil. Int. J. Food Microbiol 2011, 145, 9–21.
[12]
Firrao, G.; Torelli, E.; Gobbi, E.; Raranciuc, S.; Bianchi, G.; Locci, R. Prediction of milled maize fumonisin contamination by multispectral image analysis. J. Cereal Sci 2010, 52, 327–330.
[13]
Hussein, H.S.; Brasel, J.M. Toxicity, metabolism, and impact of mycotoxins on humans and animals. Toxicology 2001, 167, 101–134.
[14]
Zain, M.E. Impact of mycotoxins on humans and animals. J. Saudi Chem. Soc 2011, 15, 129–144.
[15]
Wang, X.C.; Zhang, H.B.; Liu, H.M.; He, C.H.; Zhang, A.H.; Ma, J.R.; Ma, Y.N.; Wu, W.D.; Zheng, H. An immunoarray for the simultaneous detection of two mycotoxins, Ochratoxin A and Fumonisin B1. J. Food Saf 2011, 31, 408–416.
[16]
Zhang, H.B.; Zhang, A.H.; Ma, Y.N.; Feng, L.L.; Wang, Y.; He, C.H.; Wang, X.C. Development of a sensitive competitive indirect ELISA method for determination of Ochratoxin A levels in cereals originating from Nanjing, China. Food Control 2011, 22, 1723–1728.
[17]
Khayoon, W.S.; Saad, B.; Salleh, B.; Ismail, N.A.; Manaf, N.H.A.; Latiff, A.A. A reversed phase high performance liquid chromatography method for the determination of fumonisins B1 and B2 in food and feed using monolithic column and positive confirmation by liquid chromatography/tandem mass spectrometry. Anal. Chim. Acta 2010, 679, 91–97.
[18]
Shiu, C.M.; Wang, J.J.; Yu, F.Y. Sensitive enzyme-linked immunosorbent assay and rapid one-step immunochromatographic strip for fumonisin B1 in grain-based food and feed samples. J. Sci. Food Agric 2010, 90, 1020–1026.
[19]
Goodrow, M.H.; Harrison, R.O.; Hammock, B.D. Hapten synthesis, antibody development, and competitive-inhibition enzyme-immunoassay for S-triazine herbicides. J. Agric. Food Chem 1990, 38, 990–996.
[20]
Singh, K.V.; Kaur, J.; Varshney, G.C.; Raje, M.; Suri, C.R. Synthesis and characterization of hapten-protein conjugates for antibody production against small molecules. Bioconjug. Chem 2004, 15, 168–173.
[21]
Szurdoki, F.; Szekacs, A.; Le, H.M.; Hammock, B.D. Synthesis of haptens and protein conjugates for the development of immunoassays for the insect growth regulator fenoxycarb. J. Agric. Food Chem 2002, 50, 29–40.
Zhou, Y.; Li, Y.; Pan, F.; Liu, Z.; Wang, Z. Identification of tetrodotoxin antigens and a monoclonal antibody. Food Chem 2009, 112, 582–586.
[24]
Liu, H.; Gaza-Bulseco, G.; Chumsae, C.; Newby-Kew, A. Characterization of lower molecular weight artifact bands of recombinant monoclonal IgG1 antibodies on non-reducing SDS-PAGE. Biotechnol. Lett 2007, 29, 1611–1622.
[25]
Sang, G.; Luo, S.; Lin, J.; Qiu, L.; Chen, C.; Yang, H.; Xia, Y. Syntheses of haptens and hapten-protein conjugates for insecticide propoxur and cyhalothrin. Sci. China Chem 2010, 53, 1108–1113.
[26]
Hillekamp, F. Determination of the hapten density of immunoconjugates by MALDI-Mass Spectrometry. Anal. Lett 1992, 25, 1983–1977.
[27]
Kong, J.; Yu, S. Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim. Biophys. Sin (Shanghai) 2007, 39, 549–559.
[28]
Siegel, M.M. Protein and Peptide Analysis by Mass Spectrometry; Chapman, J.R., Ed.; Humana Press Inc: Totowa, NJ, USA, 1996; p. 211.
[29]
Keough, T.; Lacey, M.P.; Trakshel, G.M.; Asquith, T.N. The use of MALDI mass spectrometry to characterize synthetic protein conjugates. Int. J. Mass Spectrom 1997, 169, 201–215.
[30]
Adamczyk, M.; Gebler, J.C.; Mattingly, P.G. Characterization of protein-hapten conjugates. 2. Electrospray mass spectrometry of bovine serum albumin-hapten conjugates. Bioconjug. Chem 1996, 7, 475–481.
[31]
Gourlaouen, N.; Bolbach, G.; Florentin, D.; Gonnet, F.; Marquet, A. Characterization of protein-hapten conjugates by mass spectrometry. C. R. Acad. Sci. Ser. IIC 1998, 1, 35–40.
[32]
Yu, F.Y.; Chu, F.S. Production and characterization of antibodies against fumonisin B1. J. Food Protect 1996, 59, 992–997.
[33]
Quantity one 1-D analysis Software, version 4.6.9; Bio-Rad Laboratories, Inc.: Hercules, CA, USA, 2001.
[34]
Sambrook, J.; Russell, D.W. The Condensed Protocols from Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, USA, 2006.
