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Indirect ELISA Protocol

This protocol outlines the procedure for performing indirect ELISA methodology. Infomration includes required reagents, preparation of reagents and the procedure for all steps involved.

Materials:

  1. Antigen
  2. Primary Antibody
  3. HRP Conjugated Secondary Conjugate
  4. Coating Buffer, 0.05 M Carbonate-Bicarbonate, pH 9.6
  5. Wash Solution, 50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0
  6. Blocking (Postcoat) Solution, 50 mM Tris, 0.14 M NaCl, 1% BSA, pH 8.0
  7. Sample/Conjugate Diluent, 50 mM Tris, 0.14 M NaCl, 1% BSA, 0.05% Tween 20, pH 8.0
  8. Enzyme Substrate, TMB
  9. Stop Solution, 0.18 M H2SO4 or other appropriate solution
  10. High Protein Binding Microtiter Plate (i.e. Nunc C bottom Immunoplate 96 well, 446612)

Preparation of Materials:

Bethyl Laboratories ELISA Accessory Kit may be used or prepare the following reagents as specified below.

Coating Buffer

3.7 g Sodium Bicarbonate (NaHCO3)
0.64 g Sodium Carbonate (Na2CO3)
1 L of distilled water


Tris Buffered Saline (TBS)

6.06 g Tris Base
8.2 g NaCl
6.0 ml 6 M HCl
1 L of distilled water

pH should be 7.2 to 7.8, conductivity should be 14,000 to 16,000


Wash Solution

1 L of TBS
5 ml of 10% Tween 20


Blocking (Postcoat) Solution

1 L of TBS
10 g BSA


Sample/Conjugate Diluent

1 L of TBS
10 g of BSA
5 ml of 10% Tween 20


0.18 M H2SO4

Stock is 18 M
10 ml of Stock solution
1 L distilled water


Procedure:

Perform all steps at room temperature.

Coat with Antigen

  1. Determine the number of single wells needed. Standards, samples, blanks and/or controls should be analyzed in duplicate.
  2. Dilute the antigen in Coating Buffer to the desired concentration. For purified antigens 1 – 2 mcg/ml is sufficient. For non-purified antigens, optimal concentration needs to be determined. Transfer 100 mcl of diluted antigen to each well.
  3. Incubate coated plate for 60 minutes.
  4. After incubation, aspirate the Antigen solution from each well.
  5. Wash each well with Wash Solution as follows:
    1. Fill each well with Wash Solution
    2. Remove Wash Solution by aspiration
    3. Repeat for a total of 3 washes.

Blocking (Postcoat)

  1. Add 200 mcl of Blocking (Postcoat) Solution to each well.
  2. Incubate 30 minutes.
  3. After incubation, remove the Blocking (Postcoat) Solution and wash each well three times.

Primary Antibody

  1. Dilute primary antibody samples in Sample/Conjugate Diluent as per manufacturer’s recommendation or determine optimal concentration empirically.
  2. Add 100 ul of diluted primary antibody to each well.
  3. Incubate 60 minutes.
  4. After incubation, remove the Blocking (Postcoat) Solution and wash each well three times.

HRP-Conjugated Secondary Antibody

  1. Dilute the HRP Conjugate Secondary Antibody in Conjugate Diluent to the optimal concentration.
  2. Transfer 100 mcl to each well.
  3. Incubate 60 minutes.
  4. After incubation, remove HRP Conjugate and wash each well 5 times.

Enzyme Substrate Reaction

  1. Prepare the substrate solution according to the manufacturer's recommendation. TMB is highly recommended but OPD or ABTS can be used.
  2. Transfer 100 mcl of substrate solution to each well.
  3. Incubate plate 5 - 30 minutes.
  4. To stop the TMB reaction, apply 100 mcl of 0.18 M H2SO4 to each well. If using another substrate, use the stop solution recommended by manufacturer.
  5. Using a microtiter plate reader, read the plate at the appropriate wavelength for the substrate. (450 nm for TMB)

Notes: This procedure is suitable for alkaline phosphatase conjugated antibody. Use an appropriate substrate for alkaline phosphatase

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