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Generation of human and rabbit recombinant antibodies for the detection of Zearalenone by phage display antibody technology.
Talanta. 2019 Aug 15; 201:397-405.T

Abstract

This article reports the identification, engineering and characterisation of recombinant single chain variable fragment (scFv) antibody against Zearalenone (ZEN), an oestrogenic mycotoxin, using phage display antibody technology. To increase the chance of obtaining clones that can bind to free toxin, the conjugated proteins of the target antigen, i.e. bovine serum albumin ZEN-BSA and ovalbumin ZEN-OVA, were switched during the biopanning. One phage-displayed scFv clone specific to free ZEN, designated yZEN2A8, could be isolated. The gene encoding the yZEN2A8 scFv was sub-cloned into the pET-21d (+) and pKP300 delta III vectors to generate the recombinant scFv and scFv-AP antibody formats, respectively. After ELISA optimisation by checkerboard titration, the sensitivities of the recombinant yZEN2A8 scFv antibody and scFv-AP fusion were improved approx. 2 and 60 folds, respectively. Competitive ELISA indicated that the median inhibition concentration (IC50) of recombinant yZEN2A8 scFv antibody and scFv-AP fusion after ELISA optimisation were 90 and 14 ng mL-1, with a limit of detection (LOD) of 20 and 2 ng mL-1, respectively. No cross-reactivity to other common mycotoxins was observed. Homology modelling illustrated specific binding of the recombinant antibody to ZEN and demonstrated the role of complementary determining regions (CDRs) of both the variable heavy and light chains in antibody-antigen interactions. Efficient application of scFv-AP for the detection of ZEN contamination in corns and wheat samples were investigated for the first time. The antibody in the form of scFv-AP can be used as a prototype for the development of a convenient reagent for the detection of ZEN contamination in various format, including biosensor-based.

Authors+Show Affiliations

Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand.Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand.Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand.Department of Biochemistry, Faculty of Science, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand.Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand. Electronic address: montarop@sut.ac.th.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31122440

Citation

Sompunga, Pensuda, et al. "Generation of Human and Rabbit Recombinant Antibodies for the Detection of Zearalenone By Phage Display Antibody Technology." Talanta, vol. 201, 2019, pp. 397-405.
Sompunga P, Pruksametanan N, Rangnoi K, et al. Generation of human and rabbit recombinant antibodies for the detection of Zearalenone by phage display antibody technology. Talanta. 2019;201:397-405.
Sompunga, P., Pruksametanan, N., Rangnoi, K., Choowongkomon, K., & Yamabhai, M. (2019). Generation of human and rabbit recombinant antibodies for the detection of Zearalenone by phage display antibody technology. Talanta, 201, 397-405. https://doi.org/10.1016/j.talanta.2019.04.034
Sompunga P, et al. Generation of Human and Rabbit Recombinant Antibodies for the Detection of Zearalenone By Phage Display Antibody Technology. Talanta. 2019 Aug 15;201:397-405. PubMed PMID: 31122440.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Generation of human and rabbit recombinant antibodies for the detection of Zearalenone by phage display antibody technology. AU - Sompunga,Pensuda, AU - Pruksametanan,Natcha, AU - Rangnoi,Kuntalee, AU - Choowongkomon,Kiattawee, AU - Yamabhai,Montarop, Y1 - 2019/04/14/ PY - 2019/01/25/received PY - 2019/04/11/revised PY - 2019/04/12/accepted PY - 2019/5/25/entrez PY - 2019/5/28/pubmed PY - 2019/6/18/medline KW - Alkaline phosphatase fusion KW - Competitive ELISA KW - Phage display KW - Recombinant antibody KW - Zearalenone KW - scFv SP - 397 EP - 405 JF - Talanta JO - Talanta VL - 201 N2 - This article reports the identification, engineering and characterisation of recombinant single chain variable fragment (scFv) antibody against Zearalenone (ZEN), an oestrogenic mycotoxin, using phage display antibody technology. To increase the chance of obtaining clones that can bind to free toxin, the conjugated proteins of the target antigen, i.e. bovine serum albumin ZEN-BSA and ovalbumin ZEN-OVA, were switched during the biopanning. One phage-displayed scFv clone specific to free ZEN, designated yZEN2A8, could be isolated. The gene encoding the yZEN2A8 scFv was sub-cloned into the pET-21d (+) and pKP300 delta III vectors to generate the recombinant scFv and scFv-AP antibody formats, respectively. After ELISA optimisation by checkerboard titration, the sensitivities of the recombinant yZEN2A8 scFv antibody and scFv-AP fusion were improved approx. 2 and 60 folds, respectively. Competitive ELISA indicated that the median inhibition concentration (IC50) of recombinant yZEN2A8 scFv antibody and scFv-AP fusion after ELISA optimisation were 90 and 14 ng mL-1, with a limit of detection (LOD) of 20 and 2 ng mL-1, respectively. No cross-reactivity to other common mycotoxins was observed. Homology modelling illustrated specific binding of the recombinant antibody to ZEN and demonstrated the role of complementary determining regions (CDRs) of both the variable heavy and light chains in antibody-antigen interactions. Efficient application of scFv-AP for the detection of ZEN contamination in corns and wheat samples were investigated for the first time. The antibody in the form of scFv-AP can be used as a prototype for the development of a convenient reagent for the detection of ZEN contamination in various format, including biosensor-based. SN - 1873-3573 UR - https://www.unboundmedicine.com/medline/citation/31122440/Generation_of_human_and_rabbit_recombinant_antibodies_for_the_detection_of_Zearalenone_by_phage_display_antibody_technology_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0039-9140(19)30423-0 DB - PRIME DP - Unbound Medicine ER -