Types of ELISA

There are 5 main ELISA types used to determine protein concentration:

  • Direct
  • Indirect
  • Sandwich
  • Competitive
  • ELISpot

Direct ELISA

The direct ELISA detection method uses a labelled primary antibody which reacts directly with the antigen on the protein of interest. Direct detection can be performed when the antigen of interest is directly immobilised on the plate or in capture assay format.

In a direct ELISA a conjugated primary antibody binds directly to the analyte

 Sample Direct ELISA Protocol

  1. Coat the antigen onto the microtiter plate as described below.
  2. Dilute the antigen to a final concentration of 20ug/ml in PBS. The concentration used may vary between assays and require optimisation.
  3. Coat the wells of the microtiter plate by pipetting 50ul of the antibody dilution into the wells of the plate.
  4. Ensure the samples contain the antigen at a concentration that is within the detection range of the antibody.
  5. Cover the plate with adhesive plastic and incubate for 2h at room temperature or 4°C overnight. The incubation time may vary depending on the assay and require optimization.
  6. Remove the antibody coat from the wells.
  7. Wash x2 with 200ul of PBS.
  8. Once the PBS wash is complete, empty the plate onto a paper towel to remove any remaining residual PBS.
  9. Block protein binding sites using blocking buffer (5% non-fat dry milk/PBS or BSA). Add 200ul of blocking buffer to each well and incubate for at least 2 h at room temperature or overnight at 4°
  10. Wash the plate x2 with 200ul of PBS.
  11. Add 100ul of antibody diluted in blocking buffer (use at optimal concentration as per manufacturer’s instructions).
  12. Cover the plate and incubate. 2 h is recommended however this may require optimisation depending on the strength of the signal and overnight incubation may be required.
  13. Wash the plate x4 with 200ul of PBS.
  14. Add 100ul of substrate solution to each well.
  15. Following sufficient colour development add the stop solution.
  16. Read the absorbance (OD, optical density) of each well with a plate reader.
  17. Prepare a standard curve with concentration on the X axis vs absorbance on the Y axis. Interpolate the concentration of the sample from this standard curve.

*Note – Antigen protein concentration should not be over 20ug/ml as this will cause saturation of binding sites. Ensure the concentration of the standards spans the most dynamic detection range of antibody binding. The concentration range may need to be optimized to obtain a suitable standard curve. Always run samples and standards in duplicate or triplicate.

Indirect ELISA

Indirect ELISA is performed in 2 steps, the binding of a primary antibody to the antigen followed by the binding of the labelled secondary antibody to the primary antibody. Indirect ELISA is one of the most popular forms of ELISA assay.

In an indirect ELISA the secondary antibody binds to the primary antibody similarly to a Western Blot

Sample Indirect ELISA Protocol

  1. Coat the antigen onto the microtiter plate as described below.
  2. Dilute the antigen to a final concentration of 20ug/ml in PBS. The concentration of antigen used may vary between assays and require optimisation.
  3. Coat the wells of the microtiter plate by pipetting 50ul of the antibody dilution into the wells of the plate.
  4. Ensure the samples contain the antigen at a concentration that is within the detection range of the antibody.
  5. Cover the plate and incubate for 2 h at room temperature or 4°C overnight. The incubation time may vary depending on the assay and require optimization.
  6. Remove the antibody coat from the wells and wash x2 with 200ul of PBS.
  7. Empty the plate onto a paper towel to remove any remaining residual PBS.
  8. Block protein binding sites using blocking buffer (5% non-fat dry milk/PBS or BSA). Add 200ul of blocking buffer to each well and incubate for at least 2 h at room temperature or overnight. The incubation time may vary depending on the assay and require optimization.
  9. Wash the plate x2 with 200ul of PBS.
  10. Add 100ul of diluted primary antibody to the plate.
  11. Cover the plate and incubate for at least 2 h at room temperature. This may require optimisation depending on the strength of the signal and overnight incubation may be required.
  12. Wash the plate x4 with 200ul of PBS.
  13. Add 100 µl of conjugated secondary antibody diluted in blocking buffer to the selected wells of the microtiter plate (The optimal concentration should be used as per manufacturers instructions).
  14. Cover the plate and incubate for 1-2 h at room temperature.
  15. Wash the plate x4 with 200ul of PBS.
  16. Add 100ul of substrate solution per well.
  17. Following sufficient colour development add 100ul of stop solution.
  18. Read the absorbance (OD, optical density) of each well with a plate reader.
  19. Prepare a standard curve with concentration on the X axis vs absorbance on the Y axis. Interpolate the concentration of the sample from this standard curve

*Note – Ensure the concentration of the standards spans the most dynamic detection range of antibody binding. The concentration range may need to be optimized to obtain a suitable standard curve. Always run samples and standards in duplicate or triplicate.

Sandwich ELISA

A sandwich ELISA quantifies antigen expression sandwiched between two antibodies (the capture and detection antibody). The antigen under investigation must contain at least two antigenic epitopes to be capable of binding two antibodies.

Monoclonal or polyclonal antibodies can be used as detection/capture antibodies in a sandwich ELISA. Polyclonal antibodies are mainly used as capture antibodies to pull down as much antigen as possible. Monoclonal antibodies recognise a single epitope making then ideal for fine detection and quantification of small differences in antigen.

A sandwich ELISA is said to be 2-5 times more sensitive than direct or indirect ELISA.

Steps in a sandwich ELISA protocol. 1. Capture antibody is bound to the microtitre plate. 2. Analyte is incubated with the capture antibody and binds. 3. A second antibody against the analyte of interest conjugated to a alkaline phosphatase or horseradish peroxidase binds to the analyte. In the presence of TMB/pNPP the conjugate enzyme catalyzes the reaction resulting in the production of colour.

Sample Sandwich ELISA protocol

  1. Coat the microtiter plate with the capture antibody (our kits come pre-coated with your capture antibody of choice) at 1–10 μg/mL concentration in carbonate/bicarbonate buffer (pH 9.6). The concentration of antibody used may vary between assays and require optimisation.
  2. Cover the plate and incubate overnight.
  3. Remove the capture antibody solution.
  4. Wash x2 with 200ul of PBS.
  5. Once the PBS wash is complete, empty the plate onto a paper towel to remove any remaining residual PBS.
  6. Block protein binding sites using blocking buffer (5% non fat dry milk/PBS). Add 200ul of blocking buffer to each well and incubate for at least 2h at room temperature or overnight. The duration of incubation may vary between assays and require optimisation.
  7. Wash the plate x2 with 200ul of PBS.
  8. Add 100 μL of diluted samples to each well. Incubate for 90 min at 37°C.
  9. Remove samples and wash x2 with 200ul of PBS.
  10. Incubate with the detection antibody as outlined below.
  11. Add 100 μL of diluted detection antibody to each well. The concentration used may vary between assays and require optimisation.
  12. Cover the plate with adhesive plastic and incubate for 2 h at room temperature.
  13. Wash the plate x4 with 200ul of PBS.
  14. Add 100 μL of conjugated secondary antibody, diluted in blocking buffer immediately before use.
  15. Cover the plate with adhesive plastic and incubate for 1–2 h at room temperature.
  16. Following sufficient colour development add 100ul of stop solution.
  17. Read the absorbance (OD, optical density) of each well with a plate reader.
  18. Prepare a standard curve with concentration on the X axis vs absorbance on the Y axis. Interpolate the concentration of the sample from this standard curve.

*Note – Ensure the concentration of the standards spans the most dynamic detection range of antibody binding. The concentration range may need to be optimized to obtain a suitable standard curve. Always run samples and standards in duplicate or triplicate. Check that the detection antibody recognizes a different epitope on the target protein to the capture antibody. This prevents interference with antibody binding. Use a tested matched pair whenever possible.

Competitive ELISA

A competitive ELISA is carried out between 2 samples which compete for binding

to the antigen/analyte (already bound to the plate and blocked). Both samples contain the antibody of interest however one is enzyme conjugated (this is normally not the sample of interest) and the other remains unconjugated.

The samples are added to the plate and compete for binding to the antigen/analyte. The plate is then washed and a colourless substrate added, followed by a stop solution. Unlike indirect, direct and sandwich ELISA an inverse relationship with colour is noted here. A strong signal indicates that more

antibody was found in the enzyme conjugated sample than the sample of interest, indicating low expression levels. The reverse is also true with a weaker signal indicating a greater amount of antibody in your sample of interest.

Competitive ELISA protocol

Competitive ELISA Sample protocol

  1. Coat the microtiter plate well with 100ul of the antigen solution. A concentration of between 1-10 ug/ml should be used. This may require optimisation and can vary between assays.
  2. Cover the plate and incubate overnight at 4°
  3. Wash the plate x3 times with 200ul of wash buffer.
  4. Add 150ul of blocking buffer and block the plate for 1 h at 37°
  5. Wash x4 times with 200ul of wash buffer.
  6. Prepare the antibody-antigen mixture by adding 50ul of antigen to 50ul of antibody. It is recommended to use a range of antigen concentrations.
  7. Incubate this mix for 1h at 37°
  8. Add 100ul of this mix to each well of the microtiter plate and incubate for 1h at 37°
  9. Wash x3 times with 200ul of wash buffer.
  10. Add 100ul of enzyme-conjugated secondary antibody ( dilution used will require optimisation) to each well. Incubate for 1 hr at 37°
  11. Wash x3 times with 200ul of wash buffer.
  12. Add 100ul of substrate solution to each well and incubate at room temperature (in the dark if light sensitive) for 30 minutes or until the desired colour change is required.
  13. Read the absorbance (OD, optical density) of each well with a plate reader.
  14. Prepare a standard curve with concentration on the X axis vs absorbance on the Y axis. Interpolate the concentration of the sample from this standard curve.

ELISPOT

ELISPOT, or enzyme linked immunospot is a technique used primarily for the detection of secreted proteins such as growth factors and cytokines.  ELISPOT assays are performed using a precoated 96-well plate, precoated with the antibody specific for the secreted protein of interest on a PVDF membrane. The sample containing cells are added to the plate and stimulated to produce protein which will bind its specific antibody.

Following this a detection antibody is added which binds the bound protein and can be detected by either by enzymatic reaction or through the use of fluorescent tag. The plate can then be analysed by manually counting the coloured spots or through the use of a specialised plate reader. Each cell secreting the protein of interest appears as a spot of colour/fluorescence and therefore provides a quantitative method for evaluating protein secretion.

Sample ELISPOT Protocol

  1. Prepare a PVDF membrane in a 96-well plate incubate in 35% ethanol for 30s.
  2. Wash thoroughly to remove any residual ethanol from the plate.
  3. Coat with PBS diluted capture antibody and incubate overnight at 4°
  4. Add approximately 0.5–1 µg per well of antibody. This concentration may require optimisation and can vary between assays.
  5. Empty the wells and wash with 200ul of PBS.
  6. Add 100ul of 2% dry milk solution to each well to prevent non-specific binding. Block for up to 1 h. This time may require optimisation and can vary between assays.
  7. Wash the plate x3 with 200ul of PBS and leave to dry.
  8. Prepare PBMCs.
  9. Add the cells at the required concentration to the wells. This may require optimisation and may vary depending on the targets.
  10. Culture overnight at 37°
  11. Wash away the cells and any unbound cytokine by incubating with PBS 0.1% TWEEN 20 for 10 min.
  12. Dilute the conjugated detection antibody in PBS 1% BSA and add to the wells. The concentration may vary between assays and require optimisation.
  13. Incubate for 1–2 h at room temperature. The incubation may vary between assays and require optimisation.
  14. Wash plate x3 times with PBS 0.1% TWEEN 20 to remove non-specific detection antibody binding.
  15. Add the conjugated detection antibody, dilute in 1% BSA.
  16. Incubate for 1–2 h at room temperature. The incubation time may vary between assays and require optimisation.
  17. Wash plate x3 times with PBS 0.1% TWEEN 20 to remove non-specific detection antibody binding.
  18. Add the enzyme substrate solution to each well.
  19. For enzymatic detection protocols, the base should be taken off the bottom of the plate to enable thorough washing of the membrane before adding substrate/chromogen.
  20. After replacing the base and adding the substrate, carefully monitor spot formation. Stop the reaction by gently washing the plate with PBS 0.1% TWEEN
  21. 20 once development appears to slow. Take the base off the plates and wash both sides of the membrane with distilled water to stop the spot formation.
  22. Dry the plates and allow the membranes to dry at room temperature.
  23. Punch the membranes out of the wells onto a sticky plastic sheet. This step will depend on your reader’s requirements. Consult the reader manual.
  24. Measure the sheet and analyse the membrane circles