The Complete Guide to Antibody Detection by Immunoenzyme Technique: Enzyme-linked Immunosorbent Assay (ELISA) for Soluble Antigen
The immunoenzyme technique is an immunological technique that combines the specificity of the antigen-antibody reaction and the efficient catalytic action of the enzyme on the substrate’s color reaction. Because of its specificity and high sensitivity, it has been widely used for screening and identification of monoclonal antibody.
Enzyme-linked Immunosorbent assay (ELISA) for Soluble Antigen
a. Dilute purified antigen to 1–20ug/ml with coating buffer.
b. Add the diluted purified antigen from last step to the coated wells at 50–100ul/hole. Maintain it overnight at 4 ℃or absorb for 2 hours at 37 ℃.
c. Discard the liquid inside the hole, while washing with washing solution for 3 times. Each time lasts for 3–5 minutes, and then pat dry.
d. Add 200 μl of blocking solution to each well. Maintain it overnight at 4 ° C or absorb for 2 hours at 37 ° C. This step can be omitted for some antigens.
e. Wash the plate from the last step with washing solution for 3 times. Maintain the coated plate at -20℃ or 4℃ for later use.
f. Add 50–100ul hybridoma cell supernatant remain untested into each hole. Meanwhile, while establishing positive control, negative control and blank control. Incubate for two hours at 37℃, then wash and tap dry.
g. Add 50–100ul enzyme-labeled secondary antibodies into each hole. Incubate for 1–2 hours at 37 ℃, then wash and pat dry.
h. Add 50–100ul fresh substrate solution into each hole, and incubate for 10–30 minutes at 37 ℃.
i. Terminate the reaction with 2mol/L H2SO4. Read the OD value on the enzyme-linked immuno reader.
j. Results analysis: If P/N ≧ 2.1 or P≧N + 3D, it is positive. If the negative control hole is colorless or close to colorless, while the positive control hole shows clear color, you can directly observe the results with naked eyes.
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