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Sandwich ELISA Protocol

This step-by-step protocol provides everything you need to carry out an ELISA assay. Written by our PhD qualified expert antibody team, this ELISA protocol includes advice for planning your ELISA assays, carrying them out safely, analyzing your results, as well as recipes for all the solutions you will need, and a troubleshooting guide.

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1. Introduction to ELISAs

The Enzyme-Linked Immunosorbent Assay (ELISA) is a powerful antibody-based technique for detecting and measuring a specific analyte, such as a protein, within a complex liquid sample. It can be used qualitatively to simply confirm if a target is present, or quantitatively to determine its exact concentration. A key advantage of ELISA over methods like a Western blot (WB) or immunohistochemistry (IHC) is its ability to provide precise quantitative data for a large number of samples efficiently. It is commonly used to measure biomarkers, hormones, and proteins like cytokines and chemokines.

 

2. Key Decisions

2.1 Types of ELISA

There are four main types of ELISA: Direct, Indirect, Sandwich and Competitive.

2.1.1 Direct

  • In a direct ELISA, the antigen is immobilized in the well of an ELISA plate. The antigen is then detected by an antibody directly conjugated to an enzyme such as HRP.
 
  • Due to the fewer steps required, direct ELISAs are much faster than other ELISA techniques and less prone to error due to fewer reagents being used. However, the disadvantages of direct detection methods include higher background noise in comparison to indirect ELISA due to non-specific immobilization of the antigen. Direct ELISA is also less flexible due to the requirement of a specific conjugated primary antibody needed for each target protein. Furthermore, as no secondary antibody is used, there is no signal amplification and therefore reduced assay sensitivity.

2.1.2 Indirect

  • The indirect ELISA method involves a two-step detection method, where the antigen is immobilized onto the ELISA plate and labeled with an unlabeled primary antibody which binds the specific antigen. Then an enzyme conjugated secondary antibody directed against the host species of the primary antibody is applied.
 
  • The indirect ELISA method has a higher sensitivity compared to direct methods due to the use of more than one labeled secondary antibody that can bind the primary antibody. It also provides greater flexibility as different primary antibodies can be used with a single labeled secondary antibody. Disadvantages of indirect ELISAs include the possibility of cross-reactivity of secondary antibody to the adsorbed antigen which may increase background noise. Indirect ELISAs also require longer protocols due to the additional incubation time of the secondary antibody required.

2.1.3 Sandwich

  • A sandwich ELISA features the use of matched antibody pairs, known as capture and detection antibodies, which recognize the same target analyte but bind to different epitopes. Using the sandwich analogy, the antigen is the “filling” whilst the two antibodies are seen as the “bread” capturing the filling. The capture antibody is used to coat the ELISA plate and binds the antigen which can then be detected in a direct or indirect ELISA configuration. 
 
  • To visualize and quantify the reaction, the detection antibody is either labelled directly or a secondary step included in the assay. Addition of a soluble substrate is then used, and the resulting color is directly proportional to amount of analyte present in the sample solution.
 
  • The key advantage of the sandwich ELISA is its high sensitivity in comparison to direct and indirect ELISAs. Sandwich ELISAs also have a high specificity due to the use of two antibodies to detect the antigen. The sandwich method also offers flexibility due to the ability to use either direct or indirect methods. The disadvantages of the sandwich method include time required to optimize the antibody pair to avoid cross-reactivity between capture-detection antibodies, if a standardized ELISA kit or tested antibody pair is not available.

2.1.4 Competitive

  • A competition/inhibition ELISA, also known as a blocking ELISA, is the more complex of the ELISA techniques. The competitive ELISA is commonly used to measure the concentration of an antigen in a sample by detecting interference in an expected signal output. The sample antigen or antibody “competes” with a reference for binding to a limited amount of labeled antibody or antigen, respectively. The higher the sample antigen concentration, the weaker the output signal, so the signal output inversely correlated with amount of antigen in the sample.
 

2.2 Colorimetric vs Fluorescent ELISA

  • Traditionally ELISAs use colorimetric systems for signal detection, where a colored compound is produced following catalysis of a substrate by an enzyme (e.g. HRP or AP). The benefit of using a colorimetric assay being able to use the relatively common and cheap plate readers that are widely available in most labs. The most widely used colorimetric ELISA substrate is TMB which is available in multiple strenths as either HB9992 - TMB ELISA Substrate (standard) or HB8566 - TMB ELISA Substrate (high sensitivity)
 
  • Fluorescent ELISAs work by direct labelling of an antibody with a fluorophore excited by a specific wavelength of light, which then emits light at a slightly longer wavelength. Fluorescence offers the ability to carry out multiplex arrays (probing for multiple antigens at the same time).
 
  • Chemiluminescence enables the detection of sub-picogram protein concentrations and relies on the emission of light as a product of enzyme-mediated catalysis of a substrate. A commonly used substrate is luminol, which in the presence of HRP and H2O2 is oxidized to 3-aminophthalate. Unlike colorimetric assays, light is only present whilst the reaction is occurring. Once the substrate is exhausted, the signal stops. The assay must be measured using a luminometer or a plate reader with luminescent capabilities. Hello Bio offers a choice of two high quality ECL substrate kits that are compatible with ELISA assays: HB7090 - SuperBlot™ ECL Western Blotting Substrate Kit (Standard) and HB9308 - SuperBlot™ ECL Western Blotting Substrate Kit (High sensitivity)
 

2.3 Controls

 It is crucial to include the necessary controls when conducting any ELISA experiment to be confident of outcome validity. 

 
  • Negative control: Sample that does not contain analyte of interest i.e. contains only buffer.
  • Plate blank: Used for subtraction from other wells when reading the plate. Typically, 3-8 wells per assay. Blanks should include TMB and stop solution and be blocked and washed as all the other wells, but no reagent or sample added.
  • Other negative controls can be included as needed i.e. for cell culture supernatant you may wish to include cell culture medium alone.
  • Samples should be analyzed at minimum in duplicate.
 

2.4 One or Two Step Detection

 In an ELISA, either the detection antibody is directly labeled (one-step detection) or the detection antibody itself is detected in a second step (two-step detection). Two-step detection methods are considered more sensitive due to signal amplification.

  • One-step detection uses a HRP conjugated detection antibody. Some applications may benefit from this due to the slightly reduced sensitivity due to high abundance of analyte of interest in the sample.
  • Two-step detection uses a detection antibody conjugated with biotin, streptavidin is added which binds non-covalently to biotin molecules in a highly specific manner.

2.5 Choosing an enzyme and substrate pair

ELISAs are performed with colorimetric detection with selected ELISAs based on chemiluminescence or fluorescence. Chromogens are chemical compounds that are converted via a chemical reaction to produce a color. The use of these compounds in ELISA is popular due to the ability to see the result by eye and the requirement of commonly used photometers to perform readouts. The substrate chosen will depend on the type of enzyme used in the ELISA reaction:

 

 
  • Alkaline peroxidase (AP) is a mammalian enzyme found in two forms: tissue and intestinal. The AP reaction is slower than HRP but has a more linear activity. Also, in contrast to HRP, AP activity is not affected by the presence of antibacterial agents or sodium azide. If running an ELISA based on AP, it is recommended to use pNPP (p-Nitrophenyl Phosphate). This reaction typically takes longer than HRP-TMB, and it is recommended to take multiple readings at 30, 60 and 90 minutes. The plate should be incubated at room temperature away from direct light. pNPP produces a soluble yellow product which is measured at 405nm.

 

3. Equipment and Consumables

Equipment

Consumables
  • Rocker
  • Cold room or fridge
  • High binding ELISA plates
  • Multi-channel pipette
  • Plate reader (able to read absorbance 450nm)
  • Plate sealing strips

 

4. Protocol

This protocol is designed for colorimetric two step detection using a biotinylated detection antibody and streptavidin HRP.

4.1 Safety

Always follow local rules and read the full COSHH document for any chemical that you have not used previously. Always wear appropriate PPE such as a lab coat and gloves.

Specifically highlighted hazards:

  • The Stop Solution is a strongly acidic solution. When diluting concentrated acids always add acid to water and not the other way around. Work in a fume hood. Wear appropriate PPE when handling this reagent.

 

4.2 Sandwich ELISA Protocol

  1. Coat a 96-well high-binding ELISA microplate with 100µl/well capture antibody – incubate overnight at 4°C. 
    1. When coating the capture antibody onto the ELISA plate, recommended concentrations are 0.5-4µg/ml but this will need optimisation
    2. There are a range of different coating buffers that are used which differ in their effectiveness depending on what protein is being coated: 
      1. Carbonate-bicarbonate buffer (0.1M, pH 9.6) is the most popular coating buffer. The higher pH helps solubility of many proteins and peptides and ensures most are protonated with an overall negative charge which aids binding to the positively charged plate.
      2. 0.1M carbonate buffer (pH8 - 8.2) is another effective coating buffer using less basic conditions than the standard carbonate-bicarbonate buffer.
      3. PBS / TBS are also widely used as they keep the protein at a physiological pH but are often less efficient at coating.
    3. It’s important to ensure the plate used facilitates the binding of high amounts of protein. For ELISAs it is recommended to use flat-bottomed plates usually made of polystyrene which enables the binding of proteins via hydrophobic bonding. Our protocol is designed for 96-well plates, with 100µl being sufficient per well for reagents and 200-300µl per well used for each wash step. While it is possible to coat plates at room temperature with a few hours incubation it is highly recommended to incubate overnight at 4°C.

 

  1. Block plate with 200µl/well blocking buffer (2% BSA in PBST) – incubate for 1 hour at RT. 
    1. Blocking the plate ensures free binding sites in the well are saturated, eliminating the possibility of non-specific binding. Different protein blockers can be used, however bovine serum albumin (BSA) is the most popular and well documented choice for blocking.

 

  1. Wash 3 x times 300µl/well wash buffer (PBST or TBST). 
  1. In the ELISA procedure, assay reagents are added in excess to ensure saturation. To avoid unspecific binding and carryover, it is important to sufficiently wash the plate between steps. This can be done manually or by using an automated washer. Wash buffers are composed of PBS and a detergent such as Tween-20 (0.02%). Volumes used in wash steps are recommended to be higher than those used when adding reagents as this ensures no residual reagent is bound to the walls of the wells. After washing excess buffer is removed by tapping the plate on absorbent paper. Be careful to move straight onto the next step, not allowing the plate to dry!

 

  1. Add 100µl/well sample or standard diluted in assay diluent. Incubate for 2 hours at RT or overnight at 4°C.
  1. All samples and standards should be diluted a minimum of 1:2 in the appropriate ELISA dilution buffer.  The composition of buffer used with vary depending on sample type i.e. for cell culture supernatant it is recommended to use PBS with 0.05% Tween-20 and 0.1% BSA. 
  2. Following reconstitution of the standard, prepare serial dilutions of the standard in the same buffer used for sample dilutions. Dilutions used should cover the standard range and be prepared no longer than 30 minutes before use. 
  1. Wash 3 x times 300µl/well wash buffer (PBST or TBST). 

 

  1. Add 100µl/well biotinylated detection antibody diluted to recommended concentration with blocking buffer. Incubate 1 hour at room temperature.
  1. If no recommended concentration is available this will need optimising with a good concentration range being 0.1 - 3µg/ml. 

 

  1. Wash 3 x times in 300µl/well wash buffer (PBST or TBST). 

 

  1. Add 100µl/well streptavidin HRP-conjugate. Incubate 30 mins at room temperature.
  1. This may need optimising depending on the ELISA with there being a wide range of dilutions commonly used from 1:1000 all the way to 1:50,000. 

 

  1. Wash 3 x times in 300µl/well wash buffer (PBST or TBST). 

 

  1. Develop with 100µl/well detection reagent (keep out of direct light).
  1. For TMB incubate for 15 minutes at room temperature before then stopping the reaction using 100µl/well stop buffer (0.16M H2SO4)
  2. For pNPP incubate for up to 90 minutes at room temperature and take multiple readings at 30, 60 and 90 minutes. 

 

  1. Use a plate reader to measure absorbance.
  1. For TMB measure at 450nm
  2. For pNPP measure at 405nm

 

5. ELISA Data Analysis

The readout provided by the plate reader provides optical density (OD) values for each well. These can be used to compare samples of unknown concentrations to the known concentration of serially diluted standards. Firstly, subtract the OD of the reference wavelength measurement from each well (some plate reader software makes this subtraction for you if appropriate parameters set). The plate blank can also be subtracted from all values before analysis (again, certain plate reader software may do this for you as well). 

5.1 Quantitative or Qualitative?

  • ELISA assays enable researchers to achieve a quantitative measure of protein abundance present in a sample. This is calculated by comparison of optical density (OD) values of a sample to that of a standard curve, whereby the sample protein concentration is interpolated.
 
  • ELISAs can also be used to more simply provide qualitative data, indicating whether the antigen of interest is present in the sample or not. This is achieved by comparison of the sample to a negative control (blank) and a positive control expressing the antigen (ideally present at a low level to provide a lower threshold for antigen detection).
 
  • ELISAs can also be “semi-quantitative” where the analyte concentration is compared to that of a positive control. 

5.2 Standard Curves?

  • The standard curve in an ELISA assay is used to determine the concentration of a chosen analyte in unknown samples. This is achieved through analysis of known amounts of purified protein serially diluted and analyzed alongside the samples of unknown concentration. The standard curve is generated by plotting the absorbance values of standard wells of known concentrations.
 
  • The standard curve plot is generated by averaging the standard replicates for each concentration then subtracting the absorbance values for the blank control. These values are then plotted, and an appropriate curve fitted. The absorbance values of the unknown samples can then be compared to the standard curve to interpolate the protein concentration.
 
  • A standard curve can simply be made by drawing straight lines between points of each concentration; however, this method will not provide an estimate between the standard data points and would not provide an accurate value of the true curve. Therefore, generally plotting a standard curve involves modeling of the standard data to generate a line equation which can be used to predict concentrations of the samples. How this model is fitted is very important to the accuracy of the results of an ELISA assay.
 
  • The simplest model to fit is a linear regression, which uses the linear range of an assay. The goodness of fit, which is how well the model describes the data, is determined by the R2 value, where R2 value > 0.99 is considered a very good fit.
 
  • To generate a standard curve, plot the log10 of the standard concentration against the log10 of the OD. Some plate reader software feature more advanced model fitting options that allow automatic generation of the standard curve, in this case it is recommended to choose a curve with a 4- or 5-parameter fit.  
 
  • When quantifying, don’t forget to adjust the determined sample concentrations by multiplying by the dilution factor. If the OD value of the sample falls outside of the standard range, it is advised to repeat the assay and dilute the sample at a higher dilution factor. 

6. Reagents

  • 0.1M Carbonate - Bicarbonate buffer pH9.6 - Add 5.76g/L NaHCO3 and 3.33g/L Na2CO3 then check that the pH is 9.6 and adjust if necessary.
 
  • 0.1M Carbonate buffer pH8.2 - Add 8.4g/L NaHCO3 and check that the pH is 8-8.3
 
  • 10x PBS pH7.4 - Add 80g/L NaCl, 2g/L KCl, 14.4g/L Na2HPO4, 2.4g/L KH2PO4 and adjust pH to 7.4. Alternatively PBS is available as HB5330 - PBS (100 Tablets).
 
  • PBST (0.02% Tween-20) - Add 100ml/L 10x PBS then add 0.2ml/L Tween-20 (for accurate pipetting it is easier to make a 10% Tween-20 stock then add 2ml/L of 10% Tween-20). Alternatively PBST is available with 0.1% Tween-20 as HB8088 - PBS buffer with Tween 20 (20x)
 
  • 10x TBS pH7.4 - Add 80g/L NaCl and 24.2g/L Tris base then adjust pH to 7.4. Alternatively TBS is available as HB7121 - TBS (25x) (pH 7.4)
 
  • TBST (0.02% Tween-20) - Add 100ml/L 10x TBS then add 0.2ml/L Tween-20 (for accurate pipetting it is easier to make a 10% Tween-20 stock then add 2ml/L of 10% Tween-20). Alternatively TBST is available with 0.1% Tween-20 as HB6971 - TBS-T with Tween 20 (20x) 
 
  • Blocking Buffer (2% BSA in PBST / TBST) - Add 2g/100ml BSA to either TBST or PBST depending on what buffer the ELISA is being run in.

 

7. Troubleshooting

Problem

Potential cause

Suggested Solutions

High background

Insufficient washing

         Increase number of washes

         Add 30 second soak step in between washes

Too much streptavidin-HRP

Check dilution, titrate if needed

Insufficient blocking

         Increase blocking time

         Check blocking solution dilution calculation

Interfering substances in sample or standard

Run appropriate controls

 

Incubation time too long

Reduce incubation times

Buffers contaminated

Make up fresh buffers

No Signal

Reagents incorrectly prepared or added in incorrect order

         Repeat assay

         Check calculations, make up new buffers, standards etc

         Review protocol steps

Contamination of HRP with azide

Use fresh reagent

Insufficient antibody used

Increase antibody concentration

Standard expired (see signal in sample wells)

         Check standard handled appropriately according to directions

         Use new vial of standard

Capture antibody did not bind to plate

         Check correct plate used (must be ELISA-compatible plate, not a tissue culture plate)

         Dilute in PBS without additional protein

Buffers contaminated

Make fresh buffers

Too much signal – plate uniformly blue

Insufficient washing – unbound HRP remaining

         Increase number of washes

        Add 30 second soak step in between washes

Incubation time is too long

Follow recommended incubation times for each step

Overly concentrated streptavidin-HRP used

Check dilution, titrate is necessary

Reused plate sealers or reagent reservoirs. Resulting in presence of residual HRP, resulting in non-specific color change of TMB solution.

Use fresh plate sealer and reagent reservoir for each step.

Contaminated buffer – residual HRP

Make fresh buffers

Standard curve has poor discrimination between points/low or flat curve

Insufficient streptavidin-HRP

Check dilution, titrate if necessary

Capture antibody did not bind to plate

         Check correct plate used (must be ELISA-compatible plate, not a tissue culture plate)

         Dilute in PBS without additional protein

Insufficient detection antibody

Check dilution, titrate if necessary

Plate not developed long enough

Increase incubation time with substrate

Procedure carried out incorrectly

Check protocol, remove any modifications

Errors in calculation of standard curve dilutions

Check calculations, repeat standard curve with new dilutions

Poor duplicates

Insufficient washing

         Increase number of washes

         Add 30 second soak step in between washes

Uneven plate coating

         Check correct plate used (must be ELISA-compatible plate, not a tissue culture plate)

         Dilute in PBS without additional protein

        Check coating and blocking volumes, times and method of reagent addition. Check plate used

Reused plate sealer

        Use fresh plate sealer for each step

No plate sealer used

Use plate sealers

Buffers contaminated

Make fresh buffers

Poor assay to assay reproducibility

Insufficient washing

         Increase number of washes

         Add 30 second soak step in between washes

Variations in incubation time/temperature

         Carry out incubations according to recommended time and temperatures

         Avoid incubating plates where environmental conditions may vary

Variations in protocol

Adhere to same protocol steps from assay to assay

Plate sealer reused

Use fresh plate sealer for each step

Incorrect calculation of standard curve dilutions

         Check calculations, make new standard curve dilutions

         Use internal controls

Buffers contaminated

Make fresh buffers

No signal in sample wells

Sample matrix masking detection

Dilute samples minimum 1:2 in sample diluent, or do series of dilutions to look at recovery

Sample signal too high, standard curve looks fine

Samples contain target analyte in levels above assay range

Dilute samples and repeat assay

Low readings across plate

Incorrect wavelengths selected

Check filters/plate reader parameters

Insufficient development time

Increase development time

Coated plates used are too old

Coat fresh plates

Capture antibody did not bind to plate

          Check correct plate used (must be ELISA-compatible plate, not a tissue culture plate)

         Dilute in PBS without additional protein

Green color develops upon addition of stop solution when using streptavidin-HRP

Reagents not mixed sufficiently

Tap/shake plate

Edge effects

Uneven temperatures around plate area

         Avoid incubating plates in areas where environmental conditions may vary

         Use plate sealers

Drift

Interrupted assay set-up

Assay set-up should be continuous, have all reagents/standards prepared before commencing assay

Reagents not at room temperature

Ensure all reagents at room temperature before beginning assay

 

8. Further Reading

  • Lindstrom and Wager, 1978. IgG Autoantibody to Human Serum Albumin Studied by the ELISA-Technique. Scandanavian Journal of Immunology. doi: 10.1111/j.1365-3083.1978.tb00472.x
  • Kato et al., 1977. Use of Rabbit Antibody IgG Bound onto Plain and Aminoalkylsilyl Glass Surface for the Enzyme-Linked Sandwich Immunoassay. Journal of Biochemistry. doi: 10.1093/oxfordjournals.jbchem.a131678 
  • Yorde et al., 1976. Competitive enzyme-liked immunoassay with use of soluble enzyme/antibody immune complexes for labeling. I. Measurement of human choriogonadotropin. Clin Chem. PMID: 949847
  • Engvall and Perlmann., 1971. Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry. doi: 10.1016/0019-2791(71)90454-X