Evaluation of Antigen Preparation Methods for Polyclonal Antibody Production against Streptomyces spp.

Abstract

Aim: To determine the optimum antigen preparation method for producing specific polyclonal antibody specific for Streptomyces species. Study Design: Experimental study. Place and Duration of Study: Faculty of Science, Mahasarakham University, Mahasarakham Province, Thailand, between May 2011 and December 2011. Methodology: Two Streptomyces isolates were used for antisera production. The sonication method was chosen for antigen preparation. Antigen suspensions were emulsified with incomplete Freund’s adjuvant and 1 ml was injected into rabbit thigh muscle for the first, second and third immunization. The fourth and fifth immunizations were injected intravenously. Antibody titer, detection limit and specificity were measured using indirect-ELISA. Streptomyces antigen mixed with soil was investigated. Results: The 5 min sonication method gave a higher protein than other test methods, so this protocol was chosen for all subsequent work. The sonicated Streptomyces antigen was used for polyclonal antibody production. At week 7, the rabbit anti-Streptomyces sp. isolate STPR78 antibody and anti-Streptomyces sp. isolate STPR84 antibody gave maximum titers at 1:64000 and 1:32000, respectively. The detection limit of the anti-Streptomyces antibodies was 125 ng and 250 ng, respectively. Both anti-Streptomyces antibodies were also found to have good specificity. Minimal cross-reactivity was detected with antigens from other test bacteria. Regrettably however, neither antibody was capable of detecting Streptomyces spp. STPR78 or STPR84 in inoculated soil. Conclusion: A specific and high titer of polyclonal antibody was produced using sonicated antigen.