[PubMed] [Google Scholar] 40. of foods. To maintain the quality and safety of foods, polyclonal and monoclonal antibodies have been tested as a tool for quality control. They have been increasingly recognized for their value in the detection of microorganisms, contaminants, and toxins in food systems (26). In this decade, active monoclonal antibody fragments have been synthesized by microbial expression systems (3, 41). This technological breakthrough has facilitated industrial applications of antibodies at a more reasonable cost. Recently, the versatility of recombinant antibody fragments for use in clinical diagnoses (7, 44), protein purification (2, 6), or food pathogen binding (30) has been demonstrated. Mometasone furoate These antibodies are likely to further enhance applications of antibodies in investigations in applied and environmental microbiology, because they should be relatively inexpensive and HUP2 readily available. Recombinant antibodies contain the variable regions of the heavy- and light-chain domains that can associate into an antigen binding unit in vivo (41). The average size of a recombinant antibody is approximately 30,000 Da, which is only one-fifth of a normal immunoglobulin G (IgG) molecule. Mometasone furoate These antibodies have an affinity for antigen that is similar to or slightly lower than that of their parent monoclonal antibodies (3, 16, 41, 44). However, the heavy and light chains of the recombinant protein may dissociate and have limited stability at low protein concentrations (14), because the intermolecular disulfide bonds that linked the heavy and light chains of native Mometasone furoate antibody do not exist. To prevent this problem, a short linker peptide has been used (16) to connect the domains of the heavy- and light-chain variable regions and to form a single-chain antibody. This design allows recombinant antibody to fold into the correct conformation and increases its stability in many applications (14). Huston et al. (17) indicated that the peptide linker should span at least 3.5 nm between the two domains to maintain the correct conformation. This length is approximately that of a 10-amino-acid peptide; however, a peptide of 14 to 15 amino acids seems to be an optimal linker for single-chain antibody. The glycine-serine peptide linker (17) that contains 3 U of (Gly)4-Ser linkage is one popular choice for construction of single-chain antibody. In the mammalian immune system, the antibody-synthesizing B cells rearrange their immunoglobulin biosynthesis genes before they produce specific antibodies. The intron sequences are removed from the transcribed RNA and form the mature mRNA for antibody expression (15). Different strategies have been developed for cloning the immunoglobulin genes (16, 32). For example, reverse transcription-PCR (RT-PCR) allows direct Mometasone furoate cloning and synthesis of rearranged immunoglobulin variable-region genes. The mRNA isolated from hybridoma cells can be used as the template for cDNA synthesis, and the target cDNA that encodes the antibody gene can be amplified by PCR. Thus, selection and enrichment of immunoglobulin variable-region genes can be finished within a few hours. Strep tag is a 10-amino-acid peptide that binds to streptavidin through a biotin-streptavidin-like interaction (39). Strep tag-fused proteins can be recovered directly from cell lysates by single-step affinity chromatography with an immobilized streptavidin column (40) and can be detected directly by a streptavidin-conjugated enzyme system (39). In the present study, the genes encoding the variable regions of a monoclonal anti-spore IgG were cloned by RT-PCR and constructed into a fusion protein gene. The strep tag sequence was joined to the construct to form a bifunctional single-chain antibody gene. The RT-PCR cloning strategy, gene modification, vector construction, protein expression, and functional assays used are presented and discussed. MATERIALS AND METHODS Hybridomas, bacterial spores, and cells. Hybridoma cell lines that produce the monoclonal antibody against T spores were previously screened in our laboratory (35). The myeloma cell line X63-Ag8.653 (20) was used as the fusion partner. The hybridoma culture and native monoclonal antibody tissue culture supernatant were prepared by the College of Veterinary Medicine, North Carolina State University. JM109 {(rk?, mk+) BL21 (DE3), which carries the T7 RNA Mometasone furoate polymerase gene under the promoter control, was purchased from Novagen (Madison, Wis.). Competent cells used for gene transformation were prepared by the polyethylene glycol-dimethyl sulfoxide protocol described by Chung et al. (10). Vegetative cells of T were grown in Trypticase soy broth at 30C for 12 h. Spores of T were prepared on fortified nutrient agar sporulation medium.