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Lab Protocols and Recipes

The protocols described below are for general application. Any product specific protocol supercedes these general recommendations.

Direct ELISA
is suitable for the detection of protein-based antigens and can be performed when the desired antibody is available in a conjugated form.

Indirect ELISA
is required if the primary antibody is not conjugated. With Indirect ELISA, a conjugated secondary antibody targets the specific isotype of the primary antibody. Both Direct and Indirect ELISA begin with the same steps.

Sandwich ELISA
measures the amount of antigen between two layers of antibodies. The antigens to be measured must contain at least two antigenic sites, capable of binding to antibody, since at least two antibodies act in the sandwich. Sandwich assays are restricted to the quantitation of multivalent antigens such as proteins or polysaccharides. Sandwich ELISAs for quantitation of antigens are especially valuable when the concentration of antigens is low and/or they are contained in high concentrations of contaminating protein.

ELISA Troubleshooting FAQ

Direct ELISA Protocol

A. Materials required:

   1. Primary antibody conjugated to biotin or HRP.

   2. Coating Buffer: Antigen standard is typically diluted in 50 mM sodium carbonate, pH 9.5.

   3. Wash Buffer: Typical wash solutions include 100 mM PBS or 100 mM Tris-buffered saline at neutral pH (7.4) with a detergent added such as Tween 20 or NP-40 at 0.05% to 0.1% (PBS-T).

   4. Blocking Buffer: The blocking buffer is also used as the diluent buffer and is composed of PBS-T plus a blocking agent. The most frequently used blocking agents are 5% BSA, or 5% non-fat dry milk.

   5. Primary Antibody Buffer: Primary antibody should be diluted in blocking buffer to prevent non-specific binding. Dilute antibody to the appropriate concentration based on recommended dilution.

   6. 96-well microtiter plate or 8-well strips.

   7. Color developing system (e.g., TMPT, OPD, enzyme-linked avidin).


B. Antigen Application

1. Add 100ul antigen solution and serial dilutions of calibrator standard (if available) diluted in coating buffer to appropriate numbers of microtiter wells.

2. Incubate at 4°C overnight (or 2 hours at RT) in a humid environment, i.e, covered by a glass plate or in a sealed box with a dampened paper towel inside.

3. Empty microtiter wells and invert plate to tap out excess liquid onto a clean tissue.


C. Blocking Step

4. Add 200-300ul of blocking solution to each well.

5. Incubate 10-15 min at RT, empty plate and tap out excess fluid onto a clean tissue.


D. Primary Antibody Incubation

6. Add 100ul of primary antibody solution diluted in blocking buffer to each well.

7. Incubate 1-2 hours at RT or 4 hours at 4°C with gentle agitation (on a rocker plate, for example).

8. Invert plate and tap out excess liquid onto a clean tissue.


E. Color Development

9. Develop wells according to the choice of conjugate on the primary antibody.


Indirect ELISA Protocol

In the absence of a pre-conjugated primary antibody, the introduction of a secondary detection antibody is needed. The conjugated secondary is directed specifically at the host species of the primary antibody and its specific isotype (e.g., mouse IgG1, goat IgM, rabbit IgG, chicken IgY, etc.).


A. Materials required:

1. primary antibody

2. Conjugated secondary antibody (HRP or biotin, etc.)

3. Colorimetric substrate (such as TMB, OPD, enzyme-linked avidin)

4. Coating buffer: 50 mM sodium carbonate, pH 9.5

5. 1X PBS (Phosphate buffered saline): 8.0g sodium chloride, 1.3g dibasic sodium phosphate, 0.2g monobasic sodium phosphate in 1.0 liter distilled water, pH 7.4.

6. 1X PBS-T (Phosphate buffered saline-Tween 20 solution (PBS-T): PBS containing 0.05% Tween-20.

7. Non-fat dry milk.

8. Blocking buffer: PBS-T, 5% non-fat dry milk..

9. ELISA 96 well microtiter plate or 8 well strips.

10. 2N HCl


B. Antigen Application

1. Add 100ul antigen solution and serial dilutions of calibrator standard (if available) diluted in coating buffer to appropriate numbers of microtiter wells.

2. Incubate at 4°C overnight (or 2 hours at RT) in a humid environment, i.e, covered by a glass plate or in a sealed box with a dampened paper towel inside.

3. Empty microtiter wells and invert plate to tap out excess liquid onto a clean tissue.


C. Blocking Step

4. Add 200-300ul of blocking solution to each well.

5. Incubate 1-2 hours at RT, empty plate and tap out excess fluid onto a clean tissue.

6. Wash wells three times with PBS-T.

D. Primary antibody Incubation

7. Add 100ul of primary antibody solution diluted in blocking buffer to each well.

8. Incubate 1-2 hours at RT (or 4 hours at 4°C) with gentle agitation (on a rocker plate, for example).

9. Invert plate and tap out excess liquid onto a clean tissue.

10. Wash wells three times with PBS-T.

E. Secondary antibody incubation

11. Add 100ul of secondary antibody diluted in blocking buffer to each well.

12. Incubate 1-2 hours at RT with gentle agitation.

13. Invert plate and tap out excess liquid onto a clean tissue.

F. Wash Microtiter Wells

14. Fill each well with Wash solution (PBS-T), agitate 5 min at RT.

15. Invert plate to empty and tap out residual fluid onto a clean tissue.

16. Repeat wash step 3 times.

G. Color development

17. Add TMB Substrate to each well. Seal with tape and incubate the plate for 15-30 minutes at RT.

18. Add STOP Solution (2N hydrochloric acid or 2N sulfuric acid) to each well. Shake gently for a few seconds. NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.

19. Read absorbance at 450 nm with microplate reader within 30 minutes after adding STOP Solution.


Sandwich ELISA Protocol

A. Materials Required

1. Primary antibody

2. Conjugated (e.g., HRP, biotin) secondary antibody directed against antigen of interest.

3. Colorimetric substrate (such as TMB, OPD, enzyme-linked avidin)

4. Coating buffer: 50 mM sodium carbonate, pH 9.5

5. 1X PBS (Phosphate buffered saline): 8.0g sodium chloride, 1.3g dibasic sodium phosphate, 0.2g monobasic sodium phosphate in 1.0 liter distilled water, pH 7.4.

6. Wash buffer: 1X PBS-T (Phosphate buffered saline-Tween 20 solution (PBS-T): PBS containing 0.1% Tween-20.

7. Non-fat dry milk.

8. Blocking buffer: PBS-T, 5% non-fat dry milk..

9. ELISA 96 well microtiter plate or 8 well strips.

10. 2N HCl


B. Coating the Primary antibody to the plate

1. Dilute the capture antibody in 0.1M Bicarbonate buffer, pH 9.2. Add 100ul to each well of the microtiter plate.

2. Cover the antibody coated plate with plastic wrap and incubate at 4°C overnight in a moist box containing a wet paper towel or at RT in a humid chamber for two hours.

3. Empty the plate, and wash 3x with PBS.


C. Blocking step

4. Block the unoccupied sites with 100ul of blocking buffer containing 100mM phosphate buffer, pH 7.2, 1% BSA, 0.5% Tween-20 for 30 min at RT.

5. Empty the plate and wash three times with wash buffer (100mM phosphate buffer, 150mM NaCl, 0.2% BSA and 0.1% Tween 20).


D. Adding standards and samples

6. Dilute the antigen solution in antigen buffer (100mM phosphate buffer, 150mM NaCl). Add to the plate in a volume of 100ul per well. Incubate the plate at RT for 45-60 min.

7. Empty the plate again. Wash three times with wash buffer.


E. Secondary antibody incubation

8. Dilute the enzyme-labeled antibody appropriately in blocking buffer. Add 100ul to each well and incubate at RT for 30 min.

9. Empty the plate again. Wash three times with wash buffer.


F. Color development

10. Add color development system (e.g., TMPD, OPD) and measure absorbance at appropriate wavelength.



ELISA Troubleshooting for low absorbance values

A.Target protein not expressed in sample used/ Low level of target protein expression in sample used:
Check the expression profile of the target protein to ensure it will be expressed in your samples. If there is low level of target protein present, increase the amount of sample used, or you may need to change to a more sensitive assay. Ensure that the positive control is within the detection range of the assay.


B. Insufficient antibody:
Check that the recommended amount of antibody is being used. The concentration of antibody may need to be increased for optimization of results.


C. Substrate solutions not fresh or combined incorrectly:
Prepare the substrate solutions immediately before use. Ensure the stock solutions are in date and have been stored correctly, and are being used at the correct concentration. Be sure the reagents are used as directed at the correct concentration.


D. Reagents not fresh or not at the correct pH:
Ensure reagents have been prepared correctly and are not past their expiration date.


E. Incubation time not long enough:
Ensure you are incubating the antibody for the recommended amount of time, if time is suggested. An increased incubation time may be required to optimize the signal.


F. Incubation temperature too low:
Antibodies will have optimum binding activity at the correct temperature. Ensure the incubations are carried out at the correct temperature. Incubation temperature may require some optimization. Ensure all reagents are at RT before proceeding.


G. Stop solution not added:
Addition of stop solution increases the intensity of color reaction and stabilizes the final color reaction.