Abstract: Elisa checkerboard titration plays an important role in saving detection time and cost. During establishing an Elisa method, selection of the best working concentration of antigen and antibody can rapidly find Elisa optimal conditions.
Keywords: Elisa Checkerboard Titration, Antibody Titration Elisa, Elisa Titration Test, Quantitative Elisa Test
1. 7 Steps for Elisa Checkerboard Titration
Take sandwich Elisa for antigen detection for example, working concentration selection procedures are specified below.
- Use coating buffer to dilute monoclonal antibody with different concentrations coated in each Elisa strip plate : 10ug/ml, 5.0ug/ml, 2.5ug/ml, 1.0ug/ml; 100ul/well coated at 4℃ overnight; Wash three times;
- Use 5% skimmed milk powder to block Elisa plate; Incubate for 1 hour at 37℃; Wash three times;
- Add 100ul blank control and standards in each coated well respectively; Incubate for 1 hour at 37℃; Wash three times;
- Add rabbit polyclonal antibody at dilution of 1:2000, 1:4000, 1:8000 and 1:16000 in each coated strip respectively(Figure 1); Incubate for 1 hour at 37℃; Wash three times;
- Add HRP-conjugated goat anti-rabbit IgG at recommended dilution in each coated strip respectively; Incubate for 30min at 37℃; Wash five times;
- Add TMB substrate; Stain for 10-15min in a dark place at 37℃; Use 2mol/L H2SO4 to stop the reaction; Read OD value on a microplate reader at 450nm wavelength;
- Ideal OD value: measured standards: about 2.0; negative control: below 0.1
Notes: It's suggested to serially dilute antibodies at the same original concentration. Errors caused by pipettes and manual operations can be minimized.
2. Antibody Titration Elisa
The best coating condition can be identified by a further checkerboard titration according to the initially determined concentration. Sample loading method is specified in Figure 1 below.
Figure 1. 1. strip 1-4 coats monoclonal antibody with different concentration marked as red; 2. add standards and blank control with diluted polyclonal antibody in columns; 3. Add secondary antibody at recommended dilution in the plate(S - Standards).
Seen from Figure 2, the combination of 1ug/ml and 1:4000 achieves the best effect.
Figure 2. Sample data for IL-6, range: 300pg/ml.
3. FineTest Elisa Kit
FineTest provides professional catalog Elisa kits and custom Elisa services. In future articles, how to develop a qualified Elisa kit will be introduced.
REFERENCES
Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction, PMID: 29553569.