Preparation of antibody against alpha- toxin in guinea pigs and detection of yeast display vaccine / 中国医师进修杂志

ABSTRACT

Objective To prepare anti-serum against α-toxin in guinea pigs and purify anti-rHla IgG and identify it by SDS-PAGE observe the expression of Hla on the surface of yeast cells by ELISA and immunofluorescence confocal microscopy. Methods Guinea pigs were immunized with recombinant α-toxins to obtain anti-α-toxin serum and total IgG which contains anti-rHla IgG. The purity of IgG was Identified and evaluated with non-reducing SDS-PAGE. The expression of Hla was evaluated with whole cell ELISA and immunofluorescence confocal. Results The anti-α-toxin antibody of guinea pig was successfully obtained. Anti-α-toxin antibodies was captured by the protein A on the pre-packed column. The concentration of IgG in the unpurified serum was low, the α-rHla content in the flow-out peak was even lower, and the elution fraction contains IgG (α-rHla) with purity of about 85%. Through ELISA, transformant-1 and transformant-2 was identified to be positive with rHla on the surface, compared with that in the negative control. After stain with the purified anti-α-toxin IgG, the 1 yeast transformant showed green fluorescence under immunofluorescence confocal microscope. By contrast, without inducing, the 1 yeast transformant was negative with fluorescence. Conclusions The results show that Staphylococcus aureus Hla is successfully displayed on the surface of yeast, and this research could be further applied in the identification of clinical samples or pre-clinical research. This study provides two reliable methods for the development of yeast-display vaccine.