Measuring antibody titer in antiserum or culture medium with ELISA
1, Dilute the antigen to a final concentration of 1 µg/ml (peptide antigen) or 10 µg/ml (protein antigen) in PBS. Coat the wells of a PVC 96-well plate with the antigen by pipetting 100 µl of the antigen dilution.
2, Cover the plate with an adhesive plastic and incubate at 4°C overnight.
3, Remove the coating solution and wash the plate three times by filling the wells with 100 µl PBS. The solutions or washes are removed by vacuum aspirator. The remaining drops are removed by patting the plate on a paper towel.
1, Block the remaining protein-binding sites in the coated wells by adding 100 µl blocking buffer, which contains 3% BSA, and 0.1% Tween-20 in PBS, per well. (Tween-20 is vital to reduce non-specific binding.)
2, Cover the plate with an adhesive plastic and incubate for 30min at room temperature.
3, Dilute antiserum (1:1,000-1:1,000,000) or cell culture medium (1:2-1:10) into blocking buffer.
4, Add 100 µl of diluted primary antibody to each well and incubate for 30min-1hr at room temperature with gentle shaking. (Incubation for overnight at room temperature or 4°C is not recommended, because long-term incubation will cause antibodies to bind non-specifically to the PVC plastic well instead of the antigen, therefore generating false positive results.)
5, Wash the plate three times with blocking buffer.
6, Dilute secondary antibody, HRP conjugated (1:1,000) into blocking buffer.
7, Add 100 µl of diluted secondary antibody to each well and incubate for 30min-1hr at room temperature with gentle shaking.
8, Wash the plate three times with blocking buffer.
HRP substrate
TMB (3,3',5,5'-tetramethylbenzidine) and H2O2
1, Remove the blocking buffer and wash the plate three times by filling the wells with 100 µl PBS. The solutions or washes are removed by vacuum aspirator. The remaining drops are removed by patting the plate on a paper towel.
2, Add 100 µl of the substrate solution to each well and incubate for <5min at room temperature (protected from light).
3, After sufficient color development, take a picture of the plate with iPhone.
Figure legend
2 independent peptide antigens of the CCB79 protein were used to immunize rats (3 animals per antigen). One antigen elicited immune response achieving antibody titer of 1,000,000, whereas the other antigen elicited antibody titer of 10,000. Procedure: 96-well plate was coated with peptide at 1 µg/ml in PBS, followed by incubation with rat antiserum for 30min and anti-rat-HRP for 30min. HRP substrate (blue color) develops within 5 min. No acid was added. Image was taken with iPhone.
Figure legend
Hybridoma cells made from a rat immunized with a peptide antigen of CCB79 protein were tested for antibody binding affinity in cell culture medium. Procedure: 96-well plate was coated with peptide at 1 µg/ml in PBS, followed by incubation with hybridoma supernatant for 30min and anti-rat-HRP for 30min. HRP substrate (blue color) develops within 5 min. No acid was added. Image was taken with iPhone.