Development of ELISA based on Bacillus anthracis capsule biosynthesis protein CapA for naturally acquired antibodies against anthrax
Fig 4
Comparison of PAD1-ELISA and CapA322-ELISA.
Checkerboard titration between CapA322 and (A) horse hyperimmunized anti-Bacillus anthracis serum (PC1) or (B) naive horse serum (NC1) in CapA322-ELISA. Each dilution of serum sample was tested in technical triplicate by CapA322-ELISA. The twofold serum dilution starts with a dilution of 1:100, and CapA322 dilution starts with 1.6 μg/well. From the result, we determined the optimal concentrations of antigen, antibody, and serum dilutions as follows: antigen, 0.8 μg/well; serum dilution, 1:100; second antibody dilution, 1:15,000. (C) Comparison of PAD1-ELISA and CapA322-ELISA on horse sera. Each serum samples of horse hyperimmunized anti-Bacillus anthracis (PC1), horse naturally infected with B. anthracis (PC2 and PC3), naive horses (NC1 and NC2), and horse vaccinated with Sterne34F2 strain (Vac_H1D21-Vac_H4D21) was analyzed in technical triplicate by PAD1-ELISA and CapA322-ELISA. (D) One-way ANOVA with Tukey’s multiple comparison test on relative OD values of positive (PC; n = 3), negative (NC; n = 2), and vaccinated (Vac; n = 3) groups.