Objective: To establish a double antibody sandwich ELISA for detecting Csa2 protein in Candida albicans infection and evaluate its specificity and sensitivity.
Methods: A recombinant expression vector pPIC9K-Csa2 was constructed and transformed into Pichia pastoris GS115. A large-scale expression of recombinant Csa2 protein (rCsa2) was optimized using methanol, and the protein was purified in P. pastoris expression system. New Zealand Rabbits and guinea pigs were respectively immunized with the purified rCsa2 to prepare polyclonal antisera. The double antibody sandwich ELISA was established by choosing the optimal dilution of coating antisera and detecting antisera. Different concentrations of rCsa2 and culture supernatants of C. albicans collected at different time points were used to evaluate the sensitivity of detection. The specificity of the sandwich ELISA was evaluated by detecting culture supernatants of other three Candida spp, five Aspergillus spp, Cryptococcus neoformans and Penicillium marneffei.
Results: The rCsa2 protein was successfully expressed and purified. SDS-PAGE showed that its Mr was 13 300. Western blotting demonstrated that the protein bound to specific antibody. The sensitivity of the sandwich ELISA we established using the high-titer antisera was about 240 pg/mL of rCsa2, and could detect Csa2 protein in the culture supernatant of C. albicans when cultured for as early as 18 hours. There was no cross-reactivity between the culture supernatants of other 10 clinically important fungi and C. albicans.
Conclusion: The double antibody sandwich ELISA for detecting Csa2 protein has been established with good sensitivity and specificity. Csa2 protein could be used as a new diagnostic marker of C. albicans infection.