Quantification of anti cd-HAP antibody responses using ELISA in immunized BALB/c mice

Anti-cd-HAP IgG in the serum of different mouse groups from pre-immunization, days 10, 24, 38 (n = 9 mouse sera in each group in mentioned time points) and 180 (n = 4 mouse sera in each group) after primary immunization were evaluated using ELISA. Optimized antigen concentrations and the dilution of the primary and secondary antibodies were evaluated by a checkerboard cross-titration assay using different concentrations of antigen and antibody dilutions. The optimized concentration of cd-HAP (1000 ng/ml, 100 ng/well) was added into MaxiSorp flat-bottom 96-well ELISA plates (Jet Biofil, Guangzhou, China) and incubated at 4 °C overnight. After blocking, optimized dilution of serum samples (1:200) was added to the desired wells. After incubation and rinsing of the wells, a secondary HRP-conjugated anti-mouse IgG antibody at 1:25,000 dilution (Sigma-Aldrich Co., USA) was added. Anti-cd-HAP IgG was detected using TMB as a substrate. The reaction was stopped with 2N H₂SO₄, and the absorbance was measured using an ELISA microplate reader (BioTek, Winooski, VT, USA) at OD₄₅₀nm. The cut-off values were estimated from the average of the 20 NMS plus three standard deviations (SD). Further, the anti-cd-HAP IgG subclasses (IgG1, IgG2a, IgG2b and IgG3) were evaluated by the ELISA as described above; however, the secondary antibodies specific to mouse IgG1 and IgG3 at 1:2000 dilution, and IgG2a, and IgG2b at 1: 1000 dilution of antibodies were used (Sigma-Aldrich Co.). Subsequently, the plates were incubated with 1:10,000 dilution of anti-goat IgG HRP (Sigma-Aldrich Co., USA) at RT for 1 h. For analysis of the antibodies persistence, the levels of antibodies on day 180 (6 months after immunization) was measured and compared with day 38 (10 days after the second boost) using paired sample t-test.