Cloning and Characterization of a Hybridoma Secreting a 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Specific Monoclonal Antibody and Recombinant F(ab)

Toxins

Heather Wanczyk⁵  Tolga Barker³  Debra Rood⁵  Daniel I. Zapata⁶  Amy R. Howell²  Stewart K. Richardson²  John Zinckgraf¹  Gregory P. Marusov⁴  Michael A. Lynes⁴ 

[1] Immucell, Portland, ME 04101, USA; E-Mail:;Department of Chemistry; University of Connecticut, Storrs, CT 06268, USA; E-Mails:;National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA; E-Mail:;Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06268, USA; E-Mails:;Department of Animal Science, University of Connecticut, Storrs, CT 06268, USA; E-Mails:;New York Medical College, New York, NY 10162, USA; E-Mail:

关键词: TSNAs;    F(ab);    NNK;    monoclonal antibody;    2A cleavage;   

DOI  :  10.3390/toxins5030568

来源: mdpi

【 摘 要 】

Smokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These potent carcinogens are formed during tobacco curing and as a result of direct nitrosation reactions that occur in the oral cavity. In the current work we describe the isolation and characterization of a hybridoma secreting a high-affinity, NNK-specific monoclonal antibody. A structurally-related benzoyl derivative was synthesized to facilitate coupling to NNK-carrier proteins, which were characterized for the presence of the N-nitroso group using the Griess reaction, and used to immunize BALB/c mice. Splenocytes from mice bearing NNK-specific antibodies were used to create hybridomas. Out of four, one was selected for subcloning and characterization. Approximately 99% of the monoclonal antibodies from this clone were competitively displaced from plate-bound NNKB conjugates in the presence of free NNK. The affinity of the monoclonal antibody to the NNKB conjugates was K_d = 2.93 nM as determined by surface plasmon resonance. Free nicotine was a poor competitor for the NNKB binding site. The heavy and light chain antibody F(ab) fragments were cloned, sequenced and inserted in tandem into an expression vector, with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab) with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers.

【 授权许可】

CC BY   
© 2013 by the authors; licensee MDPI, Basel, Switzerland.