General Questions Related to ELISA
1. How do I design a monoclonal antibody or Mab-based sandwich ELISA?
You need to find a pair of monoclonal antibodies with different subclass (IgG1, IgG2a, IgG2b, IgG3, or IgM). They can also differ by the light chains, i.e., one Ab with kappa chain and the other with lambda chain. Then, test them one as capture Ab and the other detection, and vice versa. Choose the combination that yields better signals.
2. How do I determine the antibody concentration in ascites or tissue culture supernatant?
Use a purified immunogloblin of the same subclass/subtype as a calibration standard (see our General Reagent section), run a generic version of ELISA or F-ELISA of your choice or our kits (XXXXX). The concentration in the ascites or tissue culture supernatant can be derived from the standard curve for the purified immunogloblin.
3. How do I lower the background in the F-ELISA?
The wash step after the incubation of enzyme conjugates is very critical for reducing the background. The suggested practices include tap dry the plates to remove any residual liquid and double the number of wash cycles for this wash step compare to other wash steps, such as wash twice with 3 washes in each wash program and tap dry the plates in between.
4. When serum samples are tested, how do I lower the background?
The following two methods in the step for serum sample dilution and/or incubation can be used to reduce the background: 1) Higher salt (NaCl, citrate buffers preferred); and/or 2)Inclusion of certain surfactants (such as polysorbate-20, polysorbate-80, or Triton X-100).
5. How is the sensitivity of F-ELISA compared to the OD version?
F-ELISA is generally 5-10 fold more sensitive than ELISAs using TMB or OPD as substrates