Design and production of antibodies for the detection of Streptococcus uberis

• Published in: Enzyme and microbial technology 2017-01, Vol.96, p.135-142
• Main Authors: Mihklepp, K., Kivirand, K., Nikopensius, M., Peedel, D., Utt, M., Rinken, T.
• Format: Article
• Language: English
• Subjects: Adhesins, Bacterial - chemistry; Adhesins, Bacterial - genetics; Adhesins, Bacterial - immunology; Amino Acid Sequence; Animals; Anti-S. uberis antibodies; Antibodies, Bacterial - biosynthesis; Antibodies, Bacterial - metabolism; Antibody Specificity; Antigens, Bacterial - chemistry; Antigens, Bacterial - genetics; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - immunology; Bioinformatic analysis; Biosensing Techniques; Cattle; Female; In silico selection of antigenic peptides; Mastitis, Bovine - diagnosis; Mastitis, Bovine - immunology; Mastitis, Bovine - microbiology; Milk - microbiology; Peptides - chemistry; Peptides - genetics; Peptides - immunology; Protein Conformation, alpha-Helical; Rabbits; Rapid detection of S. uberis; Streptococcal Infections - diagnosis; Streptococcal Infections - immunology; Streptococcal Infections - veterinary; Streptococcus - genetics; Streptococcus - immunology; Streptococcus - isolation & purification
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2016.10.009

•In silico selection of antigenic sequences for production of anti-S. uberis antibodies.•Production of selected antigenic peptides and corresponding antibodies.•Purified antibodies bind specifically to S. uberis.•Application of anti-S. uberis antibodies for the detection of S. uberis. Streptococcus uberis (S. uberis) is an important environmental pathogen causing mastitis in dairy cattle. Mastitis or mammary gland infection is the most common disease in milking cows and cause significant economic burden to farmers due to the reduction of the amount of milk and its quality. The development of rapid analytical methods for the determination of mastitis-causing pathogens in milk is of utmost importance for the early identification of the causes of mastitis and the beginning of timely treatment of cows. Combining in silico bioinformatic analysis and solid phase peptide synthesis using Fmoc chemistry, we have made two different peptides to mimic the adhesion protein of S. uberis, which is promoting the attachment of bacteria to epithelial cells. After purification with RP-HPLC, the peptides were conjugated with a larger carrier protein (KLH) and used for immunization of rabbits to produce specific antibodies. The separation of anti-S. uberis antibodies from rabbit blood antisera was carried out with affinity chromatography, using the synthetic peptides as affinity ligands. The purified antibodies showed high affinity and specificity towards S. uberis and were used for rapid bio-recognition and identification of S. uberis with an immunobiosensing system.