ELISA Immunoassay Detailed Protocol

ELISA Immunoassay Detailed Protocol

Enzyme-linked immunosorbent Assay (ELISA) is a biochemical method used to detect the presence and concentration of antibodies or antigens in a sample.

Materials needed:

– ELISA Plates

– Target antigen or antibody

– Non-reactive protein (e.g., BSA or casein)

– Primary and secondary antibodies

– Substrate for the enzyme (e.g., TMB)

– Buffer solutions

– Wash, Blocking, and Incubation buffer

– Stop solution (sulfuric acid)

– Plate reader

Image credits: Elsevier

Detailed Protocol:

1. Coating: Coat the ELISA plates with your target antigen/antibody and incubate overnight at 4°C. The concentration should ideally be determined empirically for each antigen/antibody used.

2. Blocking: After incubation, discard the coating solution. Block nonspecific binding sites by adding 200 – 300 µl of blocking buffer (e.g., 5% non-fat milk/PBS or BSA/PBS) to each well and incubating the plate at room temperature for 1-2 hours.

3. Washing: The plate should then be washed with wash buffer (e.g., PBS + Tween 20) to remove unbound proteins.

4. Primary Antibody Addition: Add a primary antibody of appropriate dilution to each well and incubate for 1–2 hours at room temperature or overnight at 4°C.

5. Washing: Wash the plate again to remove any unbound primary antibodies.

6. Secondary Antibody Addition: Add an enzyme-linked secondary antibody to each well. This antibody should be specific to the primary antibody used. Incubate for 1–2 hours at room temperature, followed by repeated washings to remove unbound secondary antibodies.

7. Substrate Addition: The enzyme on the secondary antibody will react with the substrate to produce a color change (blue with TMB). Add the appropriate enzyme substrate to each well and incubate in the dark for a time, depending on the substrate and enzyme used.

8. Stop Solution Addition: When the appropriate color has developed, add a stop solution to halt the reaction. This is usually an acid (e.g., sulfuric acid or phosphoric acid) for colorimetric assays.

9. Read the Plate: Using an ELISA plate reader, determine each well’s optical density (OD) within 30 minutes of adding the stop solution. The absorbance is directly proportional to the concentration of the antigen in the sample. Read the plate at a wavelength appropriate to your specific substrate (commonly at 450nm).

Remember that this is a general outline for a standard “indirect” ELISA and should be adjusted according to your specific needs. The times, temperatures, and concentrations will depend on the specific kit or reagents being used. Always follow the manufacturer’s instructions or your lab’s protocol.

Chemical Recipes

Stop solution

(1) Take sulfuric acid (95%-98%) 27.2ml and dissolve it in distilled water or deionized water.

(2) Further dilute to 1000 mL.

(3) The final concentration is around 2M.

Since the stop solution only stops the biochemical reaction, 0.5–2.0M sulfuric acid can all work. Don’t worry if your stop solution is not so accurately prepared.

After stopping the reaction, the whole ELISA plate will turn yellow instead of blue. Then, read the ELISA plate with an ELISA reader at 450nm.

TMB substrate

Note: Buy 3,3′,5,5′-tetramethylbenzidine (TMB) solution directly from the company or prepare in the lab by following

A simple way to prepare a stock solution of TMB is to weigh 10 mg of TMB and dissolve it in 10 mL of DMSO. To prepare a working substrate-chromogen solution for ELISA, dilute 1 mL of TMB-DMSO stock solution with 9 mL of phosphate-citrate buffer pH 5.2 and add 2 microliters of hydrogen peroxide

Buffer Preparation for ELISA


NaCl: 80g

KCl: 2g

Na2HPO4: 14.4g

KH2PO4: 2.4g

ddH2O: 800ml

Dissolve well.

Adjust pH to 7.4.

Bring up the volume to 1 L with ddH2O

Sterilize by autoclaving

1X Wash Buffer

10X PBS: 100ml

ddH2O: 900ml

Tween-20: 50ul

1X Blocking Buffer

10X PBS: 10ml

ddH2O: 90ml

BSA: 1g

Dissolve well

Stop Solution

0.5M H2SO4

Dr AF Saeed

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