ELISA Assay, Principle, Protocols, Methods & Kits
ELISA Assays Principles, Protocols, Methods & Kits
At Assay Genie we have created a comprehensive guide outlining everything you need to know about ELISA assays!
Key Takeaways
- Versatile Research Tool: ELISA assays are crucial for detecting and quantifying proteins, hormones, and antibodies in biological samples.
- Different ELISA Formats: Multiple ELISA types like Sandwich, Competitive, and Indirect cater to a range of experimental requirements.
- Accurate and Sensitive Detection: ELISA provides high precision and sensitivity, essential for reliable measurement of target molecules.
- Applicable Across Various Sample Types: Effective for complex samples including serum, plasma, and urine.
- Essential in Multiple Scientific Fields: Due to its adaptability, ELISA is invaluable in areas like immunology and diagnostics.
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*Author: Seán Mac Fhearraigh PhD
What is an ELISA Assay?
ELISA (Enzyme-Linked Immunosorbent Assay) is an antibody based technique to measure levels of proteins, peptides, hormones or chemicals in biological samples.
In a sandwich ELISA assay a capture antibody is immobilised onto the surface of a 96 well plate, this is followed by the addition of a sample which contains the analyte of interest and a complex between the antibody and sample is formed.
Following an incubation step the well is washed using a wash buffer to remove unbound analyte. Detection occurs following the addition of a detection antibody which is conjugated to enzyme. Following incubation another wash step is carried out to removed excess antibody and unspecific bound proteins.
Substrate is then added and a colorimetric change occurs. The amount of analyte in the sample correlates to the increase intensity in colour change. Finally stop solution is added. Once the protocol is complete, samples are then analysed on an ELISA plate reader and a software program is used to plot and calculate results.
Types of ELISA Assays
There are 6 main types of ELISA assays that researchers use in their day to day studies, the most common being the Sandwich & Competitive ELISA assays, followed by ELISpot and Indirect ELISA assays. An outline of each ELISA type is provided below.
| ELISA Type | Description |
Sandwich ELISA Assay | Sandwich ELISA assays are the most common form of ELISA and are named after the formation of the capture and detection antibody sandwiching the analyte in between for detection. As mentioned above, Sandwich ELISA assays have their capture antibody immobilized onto a polystyrene ELISA plate. The sample is then incubated in the well of the ELISA plate, followed by a wash step. A enzyme linked detection antibody is then added followed by a further incubation and finally the addition of a substrate and stop solution to measure analyte levels. |
Competitive ELISA Assay | Competitive ELISA assays are mostly used to detect hormones. In a competitive ELISA assay the analyte of interest in immobilized onto the polystyrene ELISA plate. The competitive ELISA is named after the process the analyte in the sample competing with the immobilized analyte on the plate for the capture antibody. Therefore increased amounts of analyte in a sample will result in decreased signal and is inverse to the graph that you see in a Sandwich ELISA assay. |
ELISpot ELISA Assay | An ELISpot assay is commonly used for vaccine development, veterinary research, monocytes/macrophages/dendritic cells characterization. The principle of an ELISpot is similar to a sandwich ELISA assay, whereby a plate is coated with capture antibodies. Cells are then incubated in the ELISA plate for up to 3 hours, this can depend on application. Cytokines produced by the cells are then bound by the capture antibody immobilized onto the ELISA plate. Cells are then washed from the ELISA plate and a detection antibody is added, followed by a substrate and stop solution to allow for the detection of cytokines. |
FluoroSpot ELISA Assay | A FluoroSpot ELISA assay is identical to an ELISpot ELISA, however, instead of using a enzyme-linked detection antibody, the detection antibody is conjugated with a fluorophore to allow for detection and analysis. |
Indirect ELISA Assay | In an indirect ELISA assay the analyte of interest is bound by the primary capture antibody. A secondary antibody is then used to bind the primary antibody. This is similar to the method of a Western Blot assay. |
Direct ELISA Assay | The direct ELISA technique is a assay, whereby, an enzyme-labelled antibody is used to bind to an analyte in a solution. Once bound, the enzyme-labelled antibody can react with a substrate to provide a colour change, allowing for the quantification of the analyte. This removes the need for a secondary detection antibody as the capture antibody carries out both functions. |
Multiplex ELISA Assay | A multiplex ELISA assay allows researchers to analyse multiple analytes in a single sample. This is very useful when researchers are looking to measure multiple cytokines in a sample simultaneously. Multiplex ELISA assays can be carried out through multiple formats, including flow cytometry, plate based multiplex or using PVDF or Nitrocellulose membranes. Learn More! |
CLIA Assay | CLIA assays are similar in principle to a sandwich assay, however, instead of using a chromogenic substrate for the detection of samples, CLIA assays are chemiluminescent based. In a CLIA assay, the detection antibody converts a substrate to light. The amount of photons generated are proportional to the amount of analyte in a sample. To measure the amount of sample in an assay Luminescence is measured in Relative Light Units (RLU) by a luminometer. CLIA assays are typically used for the detection of low concentration analytes (limit of detection = zeptomole 10-21 mol). |
In-Cell ELISA | An In-Cell ELISA is an indirect ELISA technique and is performed using cells that have been plated and cultured overnight onto a polystyrene ELISA plate. Cells are then fixed, permeabilized and blocked. Target proteins are then detected using a primary antibody which is either enzyme conjugated or fluorescently tagged. Fluorescently tagged antibodies allow for the detection by either fluorescent plate reader or microscope, whereas the enzyme conjugated secondary antibodies would allow for detection by a plate reader. |
Sandwich & Competive ELISA Breakdown
While there are a number of different ELISA assay types the two most common are the Sandwich and Competitive ELISA. The key differences between these two approaches are outlined in detail below!
Sandwich & Competitive ELISA Key Differences
What is a sandwich ELISA?
Sandwich ELISA (Enzyme-Linked ImmunoSorbent Assay) is a antibody based technique that allows researchers to quantify the amount of protein, hormone or analyte of interest in a sample. Capture and detection antibodies bind to non-overlapping epitopes on the protein to sandwich the protein, hence the name, Sandwich ELISA. Following the addition of the detection antibody, a chemical substrate is added (such as TMB) to produce a colorimetric signal that can be read by an ELISA plate reader.
Sandwich ELISA Video: An overview of a BDNF Sandwich ELISA principle and Method.
Capture Antibody & Detection Antibody
Sandwich ELISA Diagram of a detection and capture antibody: A schematic of a Sandwich ELISA, whereby the capture antibody and detection antibody have bound to the protein of interest.
The Sandwich ELISA is a common method used to measure the concentration of a particular protein in a sample. This assay relies on the binding of two antibodies to the protein of interest, forming a "sandwich" around the protein. The first antibody binds to the protein, and the second antibody (conjugated with an enzyme) binds to the first antibody. When an enzyme substrate is added, the Sandwich ELISA can measure the concentration of the protein in the sample by measuring the amount of enzyme activity.
The Sandwich ELISA is a quick and easy way to measure the concentration of a protein in a sample, making it a valuable tool for research and diagnostics. Additionally, this assay can be used to measure the concentration of proteins that are difficult to purify or otherwise quantify.
Capture antibodies are the key components of a sandwich ELISA, an assay used to detect and quantify proteins and other biomolecules in a sample. Capture antibodies bind to specific targets on the surface of the analyte molecule, capturing it for detection by a detection antibody.
Detection antibodies act as indicators of the presence or quantity of target proteins in a sample. They are often coupled to enzymes that can produce a detectable signal, such as light or color change, when they bind to their targets.
Antigen binding
The sandwich ELISA is a popular method for detecting antigens in samples. The principle behind this assay is that two antibodies are used to capture and detect the antigen of interest. The first antibody is used to capture the antigen from the sample, and the second antibody is used to detect the captured antigen. This technique is highly sensitive and can be used to detect very low levels of antigens in a sample. Sandwich ELISAs are often used to detect proteins, viruses, and other macromolecules in a variety of samples.
Sandwich vs Competitive ELISA
Sandwich ELISA and competitive ELISA are two popular methods for measuring the concentration of a specific analyte in a sample. Both methods use antibodies to bind to the analyte, but there are some key differences in the way that these procedures are carried out.
| ELISA Type | Description |
| Sandwich ELISA | Sandwich ELISA involves using two different antibodies, one of which is attached to a solid surface. The analyte is then added to the mixture and allowed to bind to the surface-bound antibody. A second antibody, which is conjugated to a detectable marker, is then added. This second antibody binds to the analyte, and the signal from the marker is used to determine the concentration of the analyte in the sample. |
| Competitive ELISA | Competitive ELISA, on the other hand, uses a single antibody that is conjugated to a detectable marker. The analyte and a known amount of "competing" analyte are added to the mixture. The competing analyte competes with the sample analyte for binding to the antibody. The signal from the marker is then used to determine the amount of analyte in the sample. |
So, what are the key differences between these two methods? Sandwich ELISA is more sensitive than competitive ELISA, meaning that it can detect lower concentrations of analyte. Sandwich ELISA is also less subject to interference from other substances in the sample, making it a more specific method. Competitive ELISA is faster and easier to carry out than Sandwich ELISA, making it a more convenient option in some situations.
Sandwich ELISA Antibodies
Antibodies used to create a Sandwich ELISA can be either polyclonal or monoclonal antibodies depending on the specificity, sensitivity and analyte being detected.
> Polyclonal antibodies
Polyclonal antibodies are often used to pull down as much analyte as possible in a sample. Polyclonal antibodies can bind to multiple facets of an epitope therefore, provide an increased capture opportunity for detecting proteins of interest.
> Monoclonal antibodies
Monoclonal antibodies allow researchers to pull down a single antigen. Therefore, allow researchers to distinguish between subtle differences in proteins. Monoclonal antibodies also provide increased consistency in data versus polyclonal antibodies.
Advantages of a Sandwich ELISA
| Advantage | Description |
| No sample purification required | Sandwich ELISA assays allow for the measure of proteins/analytes in complex samples without the need for purification. |
| High specificity | Since capture and detection antibodies are used, a Sandwich ELISA assay has increased sensitivity versus a direct or indirect ELISA assay. |
| Quantification | Versus other EIA methods such as a Western Blot, the Sandwich ELISA assay allows researchers to quantify the amount of protein in a sample. |
What is a Competitive ELISA?
Competitive ELISA assays are used by researchers determine the amount of analyte in their sample and changes in analyte level under different conditions. Competitive ELISA assays allow researchers to measure a range of metabolites, hormone and proteins in a sample efficiently, quantitatively & cheaply. Similar to a Sandwich ELISA, a competitive ELISA utilizes antibodies to determine the amount of analyte in a sample and uses an enzyme-linked secondary antibody to quantitatively measure the amount of analyte in a sample.
Figure 1: Schematic of a competitive ELISA principle, whereby the primary antibody has bound the analyte plated onto the ELISA plate. The enzyme-linked detection secondary antibody has bound the capture antibody.
The competitive ELISA assay is a great tool to measure key analytes including universal hormones & signalling molecules such as cortisol, T3, hydroxyproline, serotonin. As many researchers study these types of molecules, the competitive ELISA provides a cheap, effective and fast way to measure these analytes without the high costs of expensive machinery.
Competitive ELISA Advantages & Disadvantages
| Advantages | Disadvantages |
| Measure your analyte in complex samples: A key advantage that a competitive ELISA assay provides is it's ability to measure analytes of interest in complex biological samples. As the main step in a competitive ELISA is to mix the capture antibody with your sample, it allows for the formation of the capture-analyte complex effectively,. | Negative controls can give positive results: If wells are not blocked correctly, antigens or secondary antibodies may bind to the well non-specifically. |
| Signal is inversely related to the amount of antigen in a sample: Meaning that for samples with high concentrations of antigen, a weaker signal is generated and affecting the range of the assay. Dilution of samples may be required to measure samples in a significant range. |
Competitive ELISA standard curve
Figure 2: Competitive ELISA standard curve. The amount of analyte in a sample is inverse to absorbance values due to the binding of antigen with capture antibody in the complex formation step.
Competitive ELISA assays are based on the principle of the capture antibody competing for an antigen in a sample. Firstly, an unlabeled capture antibody is incubated with a sample containing the antigen of interest. This leads to the formation of an antibody-antigen complex. During this step, excessive antibody is added to the mixture, therefore free antibody is leftover after the incubation step.
Next step is the addition of the antibody-antigen complex to a an ELISA plate pre-coated with the antigen of interest (inhibitor antigen). The free antibody remaining from the initial complex forming step is then able to bind the inhibitor antigen on the plate, while the preformed antibody-antibody complexes are washed off during wash steps.
Next, an enzyme-linked detection antibody is added to the plate, which binds the primary antibody bound to the inhibitor antigen. Finally, a substrate is added and a colorimetric change occurs, whereby the intensity of signal is inversely correlated with the amount of antigen in the sample.
ELISA Sample Preparation
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
Serum | If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. |
Plasma | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit. |
Urine & Cerebrospinal Fluid | Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid. |
Cell culture supernatant | Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately. |
Cell lysates | Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
Tissue homogenates | The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C. |
Tissue lysates | Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
Breast Milk | Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles. |
ELISA Protocols
It is essential that the correct ELISA protocol is followed when conducting an ELISA in order to obtain accurate and precise results. At Assay Genie we have grouped our protocols together in one convenient place. Click a protocol to expand the information!
Sandwich ELISA Protocol
| Step | Procedure |
| 1. | Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells! |
| 2. | Aliquot 0.1ml standard solutions into the standard wells. |
| 3. | Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well. |
| 4. | Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids) into test sample wells. |
| 5. | Seal the plate with a cover and incubate at 37 °C for 90 min. |
| 6. | Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2. |
| 7. | Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall. |
| 8. | Seal the plate with a cover and incubate at 37°C for 60 min. |
| 9. | Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash. |
| 10. | Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min. |
| 11. | Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min. |
| 12. | Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 15-30 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious colour. |
| 13. | Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately. |
| 14. | Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution. |
Figure 3: Sandwich ELISA protocol for a pre-coated ELISA plate. Step by step schematic for the steps involved in a sandwich ELISA assay. Firstly, prepare standards, followed by the addition of samples to the ELISA plate & incubate. Once incubated, wash the plate followed by the addition of labelled antibody & incubate. Following incubation, wash the plate and add the SABC working solution. Wash the plate and add the TMB substrate, followed by an incubation. Finally add stop solution and measure.
DIY Sandwich ELISA Protocol
Coating Plate with Capture Antibody
| Step | Procedure |
| 1. | Add 100µl of diluted Capture Antibody to every well. |
| 2. | Cover with a plastic plate cover and incubate at 4°C overnight. |
| 3. | Remove the cover and wash the plate as follows: |
| 4. | Add 100µl of Blocking Buffer to every well. |
| 5. | Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) for 2 hours. |
| 6. | Remove the cover and wash the plate as follows: |
| 7. | For Immediate use of the plate(s) continue to next section. If you wish to store the coated and blocked plates for future use, bench dry each plate at room temperature (18 to 25°C) for 24 hours. Then store at 2-8°C in a sealed plastic bag with desiccant for up to 12 months. |
Protocol
| Step | Procedure |
| 1. | Prepare Standard curve. |
| 2. | Add 100µl of each Standard, Sample, zero (Standard Dilution Buffer) to appropriate wells in duplicate. |
| 3. | Add 50μl of diluted Detection Antibody into all wells. |
| 4. | Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) for 1 hour. |
| 5. | Remove the cover and wash the plate as follows: |
| 6. | Add 100μl of Streptavidin-HRP solution into all wells. |
| 7. | Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) for 30 minutes. |
| 8. | Repeat wash step 5. |
| 9. | Add 100μl of ready-to-use TMB Substrate Solution into all wells. |
| 10. | Incubate in the dark for 5-15 minutes* at room temperature. Avoid direct exposure to light by wrapping the plate in aluminium foil. |
| 11. | Add 100μl of Stop Reagent into all wells. |
| 12. | Read the absorbance value of each well (immediately after step 11) on a spectrophotometer using 450 nm as the primary wavelength and optionally 630 nm as the reference wave length (610 nm to 650 nm is acceptable). |
Schematic
Figure 4: Sandwich ELISA protocol for a development ELISA kit. Step by step schematic for the steps involved in a sandwich ELISA assay.
Competitive ELISA Protocol
The below protocol is a sample protocol for a competitive ELISA kits. Competitive ELISA kits allow for the detection and quantification of an analyte in a sample. This can be very useful when looking for increases in protein concentration, phosphorylation of proteins or decreases on protein concentrations.
| Step | Procedure |
| 1. | Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells. |
| 2. | Add Sample and Biotin-detection antibody: Add 50µL of Standard, Blank (Sample/Standard dilution buffer), or Sample per well. The blank well is added with Sample / Standard dilution buffer. Immediately add 50 µL of Biotin-detection antibody working solution to each well. Cover with the Plate sealer we provided. Gently tap the plate to ensure thorough mixing. Incubate for 45 minutes at 37°C. (Solutions are added to the bottom of micro ELISA plate well, avoid touching plate walls and foaming). |
| 3. | Wash: Aspirate each well and wash, repeating the process x3 according to instructions. |
| 4. | HRP-Streptavidin Conjugate (SABC): Add 100µL of SABC working solution to each well. Cover with a new Plate sealer. Incubate for 30 minutes at 37°C. |
| 5. | Wash: Repeat the aspiration/wash process for x5 times according to instructions. |
| 6. | TMB Substrate: Add 90µL of TMB Substrate to each well. Cover with a new plate sealer. Incubate for about 15-20 minutes at 37°C. Protect from light. The reaction time can be shortened or extended according to the actual color change, but not more than 30 minutes. When apparent gradient appears in standard wells, you can terminate the reaction. |
| 7. | Stop: Add 50µL of Stop Solution to each well. Wells will turn to yellow immediately. The adding order of stop solution should be as the same as the substrate solution. |
| 8. | OD Measurement: Determine the optical density (OD Value) of each well at once, using a microplate reader set to 450 nm. You should open the microplate reader ahead, preheat the instrument, and set the testing parameters. |
Schematic
Figure 5: Step by step schematic for the competitive ELISA protocol.
Direct ELISA Protocol
The direct ELISA technique is a assay, whereby, an enzyme-labelled antibody is used to bind to an analyte in a solution. Once bound, the enzyme-labelled antibody can react with a substrate to provide a colour change, allowing for the quantification of the analyte.
| Step | Procedure |
| 1. | Coat the microtiter plate with antigen/analyte. |
| 2. | Cover the plate and incubate overnight at 4°C. |
| 3. | Wash the plate three times with 300ul of wash buffer. |
| 4. | Add blocking buffer and incubate for 1 hr at 37°C. |
| 5. | Wash four times with 300ul of wash buffer. |
| 6. | Add samples and standards to the selected wells at the appropriate concentrations. |
| 7. | Incubate for 90 minutes at 37°C or overnight at 4°C. |
| 8. | Wash three times with 300ul of wash buffer. |
| 9. | Add the biotin-conjugated streptavidin in wash buffer. |
| 10. | Incubate for 1 hr at 37°C. |
| 11. | Wash three times with wash buffer. |
| 12. | Add the substrate solution to the selected wells. |
| 13. | Incubate at room temperature until the desired colour change is observed. |
| 14. | Add stop solution. |
| 15. | Read the absorbance values. |
Indirect ELISA Protoco
An indirect ELISA is similar to a Western Blot, whereby, a secondary antibody binds to a primary antibody.
| Step | Procedure |
| 1. | Coat the microtiter plate with antigen/analyte. |
| 2. | Cover the plate and incubate overnight at 4°C. |
| 3. | Wash three times with 300μl of wash buffer. |
| 4. | Add blocking buffer and incubate for 1 hr at 37°C. |
| 5. | Wash four times with 300μl of wash buffer. |
| 6. | Add samples and standards to the selected wells at the appropriate concentrations. |
| 7. | Incubate for 90 minutes at 37°C or overnight at 4°C. |
| 8. | Wash three times with 300μl of wash buffer. |
| 9. | Add the detection antibody in wash buffer to the selected wells. |
| 10. | Incubate for 1 hr at 37°C. |
| 11. | Wash three times with wash buffer. |
| 12. | Add the substrate solution to the selected wells. |
| 13. | Incubate at room temperature until the desired colour change is observed. |
| 14. | Add stop solution. |
| 15. | Read the absorbance values. |
Popular ELISA Kits
Immunoassay vs EIA
An immunoassay is an assay that uses antibodies to detect an antigen of interest. An EIA (Enzyme Immunoassay) is an assay that uses an enzyme conjugated to a detected antibody to allow for detection and quantification.
Two examples of an EIA are a Western Blot and an ELISA. A Western Blot is an EIA that used Nitrocellulose or PVDF to immobolize proteins. This is followed by the addition of a primary antibody to bind the protein of interest, followed by the incubation with a enzyme conjugated-secondary antibody to detect your analyte of interest. In an ELISA assay, the capture antibody is immobilized onto a polystyrene plate, followed by incubation a sample which contains the analyte of interested, followed by detection with a detection antibody and colorimetric change using TMB as a substrate.
Figure 6: An example of detection using an EIA format such as Western Blot vs Sandwich ELISA format for target capture and analysis.
ELISA Assay Plates
ELISA assays are carried out in either 96 or 384 well polystyrene plates. ELISA plate maps, plate standards, plate readers and plate washing are all important considerations when running an ELISA assay. Below we have broken down each of these considerations further.
ELISA Plates
Proteins and antibodies can immobilize to the 96 well ELISA plate following incubation. In a Sandwich ELISA assay a capture antibody is immobilized to the ELISA plate. However, in a Competitive ELISA assay, the analyte of interest is bound to the ELISA plate. The ELISA plate is then blocked with a BSA blocking solution to prevent the binding of non-specific proteins during the ELISA assay.
Binding of antibodies or analytes to the plates allows for wash steps with a wash buffer and thus the removal of non-specifically binding analytes.
ELISA plates are flat bottomed to allow the plate reader to read the absorbance in an ELISA plate reader at 450nm.
Figure 7 : Cartoon of 96 well polystyrene ELISA plate
ELISA Plate Map
When carrying out an ELISA assay it is important to layout where you place your standards, blanks, controls and samples on the ELISA plate for analysis of your results. Each 96 well ELISA plate allows researchers to analyze up to 40 samples at once on a ELISA plate reader.
As most Sandwich & Competitive ELISA kits have a 7-point dilution of their standard, 14 wells are taken up by standards on the ELISA plate. In addition to standards, it is also necessary to have a blank control which is just sample/standard dilution buffer. Researchers may also use additional wells for positive, negative or spike recovery controls on the plate.
Figure 8: ELISA plate map for setting up an assay. Purple circles = Standards, Grey circles = Samples, White circles = Blanks.
ELISA Plate Standards
ELISA standards allows researchers to determine the amount of analyte they have in their sample by extrapolating absorbance readings of samples to known concentrations of standards, thus allowing researchers to quantitatively determine analyte amounts. In an ELISA kit, researchers are provided with a stock solution of standard which will be diluted by the sample/standard diltution buffer.
ELISA standards allows researchers to determine the amount of analyte they have in their sample by extrapolating absorbance readings of samples to known concentrations of standards, thus allowing researchers to quantitatively determine analyte amounts. In an ELISA kit, researchers are provided with a stock solution of standard which will be diluted by the sample/standard diltution buffer.
Figure 9: 7-point serial dilution of stock standard using standard dilution buffer.
Standard dilution guidelines
*Note: Refer to datasheet provided with the kit for comprehensive sample dilution guidelines.
Dilute each standard vial provided with 1ml (Volume will depend on kit) sample standard dilution buffer to create the Standard Stock Solution. Keep tube at room temperature for 10 min and mix thoroughly.
To create the standard series, label 6 microcentrifuge tubes and aliquot 300µl of the Sample/Standard dilution buffer into each tube. Add 300µl of the standard stock solution into 1st tube and mix thoroughly. Transfer 300µl from 1st tube to 2nd tube and mix thoroughly. Repeat this dilution process until the standard series is complete (see Figure 1 above for details).
ELISA Plate Readers
What is an ELISA microplate reader?
A microplate reader is an instrument used by researchers to detect and analyse a range of biochemical changes. An ELISA plate reader can read and analyze multiple wells at once at a range of wavelengths, the ELISA plate reader allows researchers to improve efficiency and save operational time and costs while automating a data analysis for a range of applications.
How does an ELISA plate reader work?
An ELISA plate reader works by detecting light at a specific wavelength. For the final steps in the ELISA assay, TMB (3,3',5,5'-Tetramethylbenzidine) is converted to a blue coloured solution by HRP and finally converted to a Yellow Coloured solution following the addition of stop solution. The amount of colour in the well is proportional to the amount of analyte that is in a sample.
Once the plate is placed in the ELISA plate reader the absorbance of each sample is read at 450nm to determine the amount of analyte in each well. Researchers can then plot the value of their samples vs known values of their standard curve to calculate the amount of analyte they have in each sample.
96 well ELISA plate readers
When carrying out an ELISA assay you will perform your assay in a 96 well plate to measure the amount of analyte in each sample. The formatting of your plate will consist of an 8 point standard curve and blanks in duplicate. This will take up 20 wells on your 96 well ELISA plate. This the researcher to measure 38 samples in duplicate on their 96 well ELISA plate.
There are a range of ELISA plate readers on the market that will allow you to measure your ELISA assay including SPECTROstar Nano from BMG LABTECH, plus any plate reader from Bio-Rad, Thermo, Tecan, Molecular Devices or Biotek. Each platform comes with it’s own dedicated plate readers software to allow researchers interpret their data.
| Company | Platereader |
BMG Labtech | SPECTROSTAR Nano |
Biotek | PowerWave HT Microplate Spectrophotometer |
BYONOY | ABSORBANCE 96 PLATE READER |
Bio-Rad | xMarkâ„¢ Microplate Absorbance Spectrophotometer |
Plate Reader Settings to Measure ELISA
Depending on the ELISA plate reader that you have, settings may vary. However, the following settings may apply when reading your sample.
| Type | Settings |
Optic Settings | Absorbance spectrum, Endpoint test |
General Setting | Number of flashes: 45 |
ELISA Plate Washing
| ELISA Type | Description |
TMB Reaction | In order to measure output of an ELISA experiment numerous substrates can be used to detect the final antibody-antigen bound complex. Colorimetric, chemiluminescent & fluorescent based assays are the three main technologies. Commonly ELISA refers to the colorimetric based measurement of samples, whereby TMB (3,3’,5,5’-Tetramethylbenzidine) acts as a hydrogen donor for the reduction hydrogen peroxide to water in the presence of horse radish peroxidase. This reaction results in the production of a diimine which changes the colour of the solution to blue. This colour change to blue can be read on a spectrophotometer at 370 & 650 nm. To stop this reaction sulphuric acids is added to the sample and results in a colour change to yellow. The ELISA plate can then be analyzed using a spectrophotometer and read at 450 nm. |
ELISA wash steps | A key step in the ELISA protocol is the wash steps. Washing steps are critical in order to reduce background signal, which can be due to unbound, conjugated antibody resulting in the increase in ratio of signal to noise. Therefore washing steps ensure that only high fidelity binding interactions occur between antigen and antibody. When inefficient washing occurs, this can result in high background levels, which can obstruct the acquisition of data, result in high variation between samples, ultimately resulting in poor results. |
ELISA washing volumes & parameters | The volume of wash buffer added to the ELISA plate is key to ensure effective washing of your plate. Depending on whether you use an ELISA plate washer or multichannel pipette, pay attention to the volume required when washing your plate. Washing buffer volume should be higher than the coating buffer volume added to each well. For instance, if your plate was coated with 100µL of coating buffer, you will need to added a higher volume of wash buffer to ensure complete washing of the well such as 200 – 350 µL. |
ELISA plate wash cycles | After wash volume, the number of wash cycles are important to increase the removal of background but also to prevent the unneccessary washing of bound antigen-analyte. If samples are over-washed, this may reduce the signal strength and make it difficult to measure and analyze your data. Depending on your postion in the protocol 1-3-5 wash steps maybe required. In general, pre-coated plates require fewer wash steps than DIY coated plates, however, this may depend on the manufacturer and the technology used to read the ELISA plate. |
Aspiration of buffers during ELISA plate washing – manual | Aspiration of buffers during an ELISA protocol is key to remove residual buffer, unwanted complexes or antibody. When using a multichannel pipettte, place fresh pipettes tips on the multichannel between steps. When aspirating buffer, place the multichannel at a titled angle into the well, while being careful not to touch the side or the bottom of the wells. Following removal of buffer, you can turn the plate upside and tap the plate on paper towels to remove residual buffer. Do not let the plate dry between wash steps and buffer steps. |
ELISA Sensitivity & Range
An ELISA assay allows researchers to determine the amount of analyte they have in their sample (serum, plasma, supernatant, milk, urine) within a defined range using a set of known standards. When carrying out an ELISA assay a known concentration of analyte is use as a benchmark of the amount of analyte in a sample these are called the standards.
During an ELISA assay, a stock of the standard is provided, usually in ng/mL or pg/mL amount, this stock is then diluted 6-7 fold to provide a range of known concentrations of an analyte in a volume. When plotting values a standard curve is created and unknown concentrations of samples are calculated versus these values.
Detection Strategies for ELISA
The detection step in an ELISA is last step to measure the amount of analyte that you have in your sample. The signal generated during the detection step is proportional to the amount of an. There are three options for the detection of analytes in an ELISA; radioactive (Radioimmunoassay) or fluorescent tags or using a chromogenic substrate.
Chromogenic is the most popular and most widely used technique for ELISA detection and involves the horse radish peroxidase (HRP) substrate TMB (3, 3’, 5, 5’-tetramethylbenzidine) which yields a blue colour when oxidized and turns to yellow following the addition of sulphuric acid. Which allows samples to be read at 450nm on an ELISA plate reader.
Other detections methods such as chemiluminescence can be used based on HRP using Luminol as a substrate which emits light at 425 nm.
Pre-coated ELISA & DIY ELISA
At Assay Genie we provide a range of ready to use pre-coated ELISA kits and ELISA development kits for the detection of cytokines, chemokines, hormones, signalling proteins and a 1000’s of other biomarkers.
| Features | DIY ELISA | Pre-coated ELISA | Multiplex ELISA |
Read out | HRP-TMB | HRP-TMB | PE/APC |
Incubation time | 2-3 hours | 2-3 hours | 2 hours |
Sensitivity | pg/ml | pg/ml | pg/ml |
Number of targets per well | 1 | 1 | 24 |
Instrumentation required | ELISA plate reader | ELISA plate reader | Flow Cytometer |
High Sensitivity PharmaGenie ELISA Kits
PharmaGenie ELISA Kits from Assay Genie are high quality ELISA Kits designed to meet the needs of pharma and biotech research. Focussing on high quality monoclonal antibody pairs & reagents that have been been validated according ISO 9001:2000 quality systems, PharmaGenie ELISA Kits are excellent assays to help discover our future.
- Accuracy and reliability are guaranteed as all our reagents have been validated according ISO 9001:2000 quality systems
- Recognises both Natural and Recombinant antigen Specificity
- No cross reactivity with other human cytokines tested
- Standard Calibration to NIBSC
- Development ELISA Kits (Antibody Pairs)
- Antibody pairs allow research
Development ELISA kits (antibody pairs)
Development ELISA kits allow researchers to create their own ELISA plates. Development ELISA kits come with antibody pairs (matched capture & detection antibodies) and buffers. This allows researchers to coat and plate their own ELISA kits. ELISA Genie provide a range of high quality development ELISA kits called SuperSet ELISA kits.
- Highly optimised monoclonal antibody pair.
- High quality purified recombinant protein to generate consistent standard curves.
- Reagents have been validated according ISO 9001:2000 quality systems.
- Natural and Recombinant antigen Specificity.
- No cross reactivity with other cytokines tested.
- Standard Calibration to NIBSC.
- ELISA Kits developed with pharmaceutical and biotech research sectors in mind.
- Different Kits Sizes for Efficient Research!
Antibody Types for ELISA
ELISA kits are made using either Monoclonal or Polyclonal antibodies for the detection and capture antibodies. Monoclonals provide the advantage of recognizing a single epitope, therefore provide a accurate analysis of a particular antigen. However, polyclonals have the advantage of capturing increased amounts of antigen. Lately, recombinant monoclonal antibodies have been used to create ELISA kits, thus providing increased specificity and consistency.
The Assay Genie PharmaGenie range of ELISA kits are made using high quality monoclonal antibodies for key cytokine targets including IL-1 beta, IFN-gamma, IL-2, IL-4, IL-6, CXCL8/IL-8 and many more targets.
ELISA Buffers & Recipes
What is a Blocking Buffer?
In order to prevent non-specific binding of proteins to an ELISA assay plate, blocking buffers are used to coat a plate. The binding capacity of a ELISA plate is higher than the amount of protein coated (Capture Antibody/Antigen) coated onto the plate. Therefore the remaining area must be blocked to prevent non-specific binding of antibodies or other proteins during subsequent incubation steps. Therefore a blocking buffer is used using a protein that will not be bound or form complexes with other proteins or detection antibodies in subsequent steps. Therefore, the blocking buffer increases ELISA sensitivity as it prevents binding of non-specifics proteins, reduces background noise and therefore increases signal-to-noise ratio.
During ELISA development several different blocking buffers must be tested to optimize the assay and improve signal to noise ratio. Factors that can influence non-specific binding of proteins is the formation of protein:protein interactions. When optimizing a block buffer, the key is to optimize for an increase in signal to noise ratio, this is found by using a control well without the addition of the sample with target analyte.
When optimizing blocking buffer it is also important to not use excessive concentrations of blocker, which can inhibit antibody-antigen interactions or potentially inhibit enzyme activity, thus reducing the signal to noise ratio. When optimizing for blocking, a few buffers may be tested in order to optimize for the best signal.
| Recipe for Blocking Buffer |
| Phosphate buffered saline (PBS) |
| 1% BSA |
Coating Buffer
An ELISA coating buffer is used to immobilize proteins/analytes or antibodies on microtitre plates. Key factors in immobilization of analytes/antibodies on to microtitre plates can be the pH of the coating buffer. Selecting a coating buffer between pH 7.4 and pH 9.6 can have an affect on the steric structure of protein/antibody/analyte binding and thus affect their immobilization. Testing of coating buffers can help increase mobility and performance of immobilized antibodies. In SuperSet Development ELISA kits for one plate, add 100µl of Capture Antibody into 10ml of Coating Buffer.
| Coating Buffer Recipe (1L) | Ingredient Amounts |
| Naâ‚‚COâ‚‚ | 1.5g |
| NaHCO₃ | 2.93g |
| Distilled Water | 1L (pH 9.6) |
TMB
In the presence of HRP (Horseradish peroxidase) conjugated enzymes, TMB and peroxide react to produce a blue bypproduct which has a maximum absorbance at 605 nm. The colour intensity produced by HRP activity is proportional to the levels of analyte in the ELISA assay. Following TMB (3,3’,5,5’ – tetramethylbenzidine) incubation a stop solution of 0.16M sulfuric acid is added to halt the reaction. The amount of stop solution added should be equal to the amount of TMB substrate added to each well of the ELISA plate (typically 50 – 100 uL per well). Following addition of sulfuric acid stop solution. The color changes from blue to yellow, which stabilizes the color development and allows the accurate measurement of intensity at 450nm using a spectrophotometer.
PNPP (p-nitrophenyl phosphate)
pNPP is a chromogenic substrate for alkaline phosphatases. pNPP for use with alkaline phosphate-conjugated antibodies. Alkaline phosphatase catalyzes the hydrolysis of pNPP to pNP. This results in the production of a yellow phenolate which has a maximal absorption at 405nm. pNNP is sensitive to light and thus should be protected.
Wash Buffer
The goal of an ELISA wash is to remove any signaling altering debris and preserve ELISA components. ELISA plates are washed prior to the addition of standards and samples, following the addition of detection antibody and following the addition of HRP conjugate antibody. Both Tris based and PBS based wash buffers can be used in ELISA protocols.
| Recipe for PBS Wash Buffer |
| Phosphate buffered saline (PBS) |
| 0.05% V/V TWEEN-20 |
| Recipe for Tris-based Wash Buffer (1L) |
| 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 |
| Recipe for Wash Solution (1L) |
| 1 L of TBS |
| 5 ml of 10% Tween 20 |
Analysing ELISA Data
Following the completion of your ELISA protocol the next step is to acquire and analyse the data using an ELISA plate reader.
When performing an ELISA it is recommended to run your samples in duplicate or triplicate to ensure statistical significance of your results. Further, positive controls such as standards, known positive controls and negative controls such as blanks or samples without the antigen of interest may be required.
- Negative controls: Samples with no presence of your analyte
- Positive controls: Samples with a known presence or quantity of your analyte
Depending on the type of ELISA used (qualitative, semi-quantitative or quantitative) data output will vary. Therefore you choose the specific ELISA you want to use based on the data that you want to analyse. Data is presented as a plot of optical density (OD) vs the log concentration of sample. Standards with known concentrations are used to generate a standard curve from which the concentration of an unknown analyte can be determined.
Coefficient of variation
The coefficient of variation helps identify any inconsistences and inaccuracies in the results. It is expressed as a percentage of variance to the mean. The larger the variance the greater the inconsistency and error. The coefficient variation (CV) is the ratio of the standard deviation σ to the mean µ:
Cv= σ/µ
A High CV can be attributed to some or all of the following:
- Pipetting inaccuracies
- Sample contamination with bacteria/fungi or other reagents
- Temperature variation- plates should be incubated at a stable temperature away from drafts.
- Drying out of wells – plates should be covered during all incubation steps
Calculating Results
Calculate using the following equation:
The relative O.D.450 = (the O.D.450 of each well) – (the O.D.450 of Zero well)
The standard curve can be plotted as the relative O.D.450 of each standard solution (Y) vs. the respective concentration of the standard solution (X). The concentration of the samples can be determined from the standard curve. It is recommended to use professional software such as curve expert 1.3.
Note: If the samples measured were diluted, multiply the dilution factor to the concentrations from
interpolation to obtain the concentration before dilution.
Horse Radish Peroxidase (HRP) & Alkaline Phospatase are the most widely used enzymes for the detection of analytes by Sandwich ELISA method and provide different options for researchers depending on the application.
| Component | Description |
P-Nitrophenyl-phosphate (pNPP) | pNPP is the ALP substrate. Following the addition of pNPP, incubate samples at room temperature for 10-30 mins. Stop the reaction by adding 0.75M NaOH and read samples at 405nm. |
Hydrogen Peroxide | Hydrogen peroxide is the substrate for HRP, which allows for a colour change during the reaction. |
TMB (3,3’,5,5’-tetramethylbenzidine) | TMB undergoes colour change following it's reduction of hydrogen peroxide in the presence of HRP. To quench the reaction, sulfuric acid is added and the reaction results in a colour change that can be read at 450nm by an ELISA plate reader. |
Hemolysis & ELISA
Why Does Hemolysis Effect an ELISA?
Hemolysis can affect an ELISA (enzyme-linked immunosorbent assay) because it can interfere with the accuracy of the results. Hemolysis is the breakdown of red blood cells, which releases hemoglobin into the sample. Hemoglobin can bind to the detection reagent used in ELISA, which may cause false positive results.
ELISA works by detecting the presence of a specific target molecule in a sample using antibodies that are specific to the target molecule. The antibodies are immobilized on a solid surface, such as a microplate, and the sample is added. If the target molecule is present in the sample, it will bind to the antibodies. Then, a detection reagent, which is also specific to the target molecule, is added. The detection reagent typically consists of an enzyme linked to a second antibody that recognizes a different part of the target molecule. If the target molecule is present, the detection reagent will bind to it, and the enzyme will catalyze a reaction that produces a detectable signal.
However, if hemoglobin or other substances released from hemolysis are present in the sample, they may bind to the detection reagent instead of the target molecule. This can result in a false positive signal, indicating the presence of the target molecule when it is not actually there. Additionally, hemolysis can cause the release of other substances, such as enzymes or cytokines, which can interfere with the ELISA reaction or cause non-specific binding.
Therefore, it is important to minimize hemolysis when collecting and handling samples for ELISA to ensure accurate results. If hemolysis is present, it may be necessary to repeat the assay with a new, non-hemolyzed sample, or to use a different detection method that is not affected by the interference caused by hemolysis.