ELISA Procedures

• Indirect ELISA
  
• Capture ELISA

Indirect ELISA

Reagents and Equipment

1. Phosphate buffered saline (PBS) tablet: 10 mM phosphate buffer, pH 7.4, 150 mM NaCl (Product No. P4417) and 0.1% sodium azide (Product No. S2002).
2. Carbonate-Bicarbonate buffer capsule, pH 9.6 (Product No. C3041).
3. Washing buffer (PBS-T): 10 mM phosphate buffer pH 7.4, 150 mM NaCl, 0.05% Tween 20 (Product No. P3563).
4. Monoclonal primary antibody.
5. Antibody controls: species- and isotype-matched, non-specific immunoglobulin (e.g., mouse myeloma proteins as a control for a mouse monoclonal primary antibody)
6. Alkaline phosphatase-conjugated secondary antibody or peroxidase-conjugated secondary antibody
7. Substrate for alkaline phosphatase conjugated secondary antibody (such as SIGMAFAST™ pNPP tablets, Product No. N1891) or substrate for peroxidase conjugated secondary antibody (such as SIGMAFAST™ OPD tablets, Product No. P9187).
8. Stopping reagent for alkaline phosphatase: 3 M NaOH (optional) or stopping reagent for peroxidase: 3 M HCl or 3 M H₂SO₄ (optional).
9. Microtiter plates
10. Microtiter plate reader equipped with a 405 nm or 450/492 nm filter (for pNPP or OPD, respectively).
    

Procedure

• Antigen Coating
  
• Primary Antibody Reaction
  
• Application of Secondary Antibody
  
• Substrate Preparation
  
• Development

Antigen Coating

1. Prepare an antigen solution at the appropriate concentration in carbonate-bicarbonate buffer or PBS.
2. Pipette 0.2 ml of the above solution to each well of the microtiter plate.
3. Incubate at 37 °C for 30 min., or incubate (covered) overnight at 4 °C.
4. Remove the coating solution. Wash three times with PBS-T.
   
   Note: If problems with non-specific binding occur, an additional blocking step (30 min. 5% BSA-PBS) may be required. For further information see: Vogt, R.F., et al., J. Immunol. Meth., 101, 43 (1987).

Primary Antibody Reaction

1. Dilute the monoclonal primary antibody in PBS-T. The optimal dilution should be determined using a titration assay.
2. Add 0.2 ml of the diluted monoclonal antibody to each well. The negative control should be species- and isotype-matched, non-specific immunoglobulin diluted in PBS-T.
3. Incubate at room temperature for 2 hours.
4. Wash as in step 4 of Antigen Coating.

Application of Secondary Antibody

1. Dilute the enzyme-conjugated secondary antibody in PBS-T. Add 0.2 ml of this solution to each well. The optimal dilution should be determined using a titration assay.
2. Incubate at room temperature for 2 hours.
3. Wash as in step 4 of Antigen Coating.

Substrate Preparation

1. During the last incubation and immediately before use, prepare the enzyme substrate or bring the premade liquid substrate to room temperature.

Development

1. Add 0.2 ml of the freshly prepared substrate to each well.
2. Color should develop in positive wells after 30 minutes (yellow or orange, for pNPP or OPD, respectively).
3. Absorbance may be read directly in a microplate reader (at 405 nm or 450 nm, for pNPP or OPD, respectively) or the reaction may be stopped with 50 µL per well of the appropriate stopping reagent and absorbance read later (at 405 nm or 492 nm, for pNPP or OPD, respectively).

Capture ELISA

Capture ELISA (also known as "sandwich" ELISA) is a sensitive assay to quantitate picogram to microgram quantities of substances (such as hormones, cell signaling chemicals, infectious disease antigens and cytokines.). This type of ELISA design may be needed rather than a direct or indirect ELISA for several reasons. The substance to be analyzed may be too dilute to bind to the polystyrene microtiter plate (such as a protein in a cell culture supernatant) or does not bind well to plastics (such as a small organic molecule). It may be mixed with too many other substances to bind well to the plate, two Sigma kits that describe or utilize capture ELISA are the TNF-alpha kit and the Mouse Isotyping Reagents kit (Product No. ISO2).

Reagents and Equipment

1. Phosphate buffered saline (PBS): 10 mM phosphate buffer, pH 7.4, 150 mM NaCl tablet (Product No. P4417) and 0.1% sodium azide (Product No. S2002)
2. Carbonate-Bicarbonate buffer capsule, pH 9.6 (Product No. C3041)
3. Washing buffer (PBS-T): 10 mM phosphate buffer pH 7.4, 150 mM NaCl, 0.05% Tween 20 (Product No. P3563)
4. Capture or coating antibody
5. Control antigen to be used for standard curve
6. Labeled detection antibody
7. Substrate. Typically the most sensitive substrate is used (such as TMB or OPD if using peroxidase as the enzyme label).
8. Stop solution (optional)
9. Microtiter plates
10. Microtiter plate reader
    

Procedure

• Coating of capture antibody
  
• Application of control and samples
  
• Application of detection antibody

Note: This is provided as a general protocol. Optimal dilutions for the capture antibody, samples, controls, and detecting antibodies as well as incubation times will need to be determined empirically and may require extensive titration. Ideally, one would use an enzyme-labeled detection antibody as described in this procedure. However, if the detection antibody is unlabeled, the secondary antibody cannot cross-react with either the coating antibody or the sample. The appropriate negative and positive controls should also be included.

Coating of Capture Antibody

1. Appropriately dilute the capture or coating antibody in carbonate-bicarbonate buffer or PBS. Capture antibodies are typically plated at 0.2 to 10 µg/ml. It is preferable to use affinity purified antibodies or at a minimum use an IgG fraction.
2. Pipette 0.2 ml of the diluted capture antibody to each well of a microtiter plate.
3. Incubate the plate (covered) for 1 hour at 37 °C.
4. Remove the coating solution. Wash the plate 3 times with washing buffer (PBS-T).

Application of Control and Samples

1. Add 0.2 ml of appropriately diluted samples and controls into the appropriate wells. Generally samples are diluted in PBS in the 10 ng-10 µg/well range (the more sensitive the assay, the less sample is required).
2. Incubate the plate at room temperature for 1 hour.
3. Remove the sample or control solution. Wash the plate 3 times with washing buffer (PBS-T).

Application of Detection Antibody

1. Dilute the enzyme-labeled detection antibody.
2. Pipette 0.2 ml of the appropriately diluted detection antibody to each well.
3. Incubate the plate at room temperature for 30 minutes.
4. Remove the detection antibody solution. Wash the plate 3 times with washing buffer (PBS-T).
5. Add the appropriate substrate solution.
6. Allow the plate to develop (typically 30 minutes) and add stop solution (optional).
7. Read the absorbance at the appropriate wavelength in a microplate reader.

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3. Crowther JR. Basic Immunology.1-34. https://doi.org/10.1385/0-89603-279-5:1

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5. 1996. Immunoassay. Elsevier.

6. Feren?ík M. 1993. Handbook of Immunochemistry. https://doi.org/10.1007/978-94-011-1552-0

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