ELISA Kits and Reagents
We offer a wide range of products to help you develop and run high quality ELISAs. We explain how our product range can help you optimize each step of your ELISA.
What is an ELISA?
An ELISA (Enzyme-Linked ImmunoSorbent Assay) is an antibody-based immunoassay used to detect and (usually) quantitate soluble analytes (typically proteins) in a plate-based format.
Types of ELISA
ELISAs can be formatted in several ways depending on the specific requirements of the experiment.
Please see our Back-to-Basics: What is an Immunoassay? and Immunoassay Walk-Through guides and our webinars, Benefits of Sample and Assay Diluents for ELISAs and Antibody-Sandwich ELISA: Solutions to simplify your development process for details of the different ELISA formats and tips on how to optimize the various steps.
To learn about what drives antibody-antigen interactions, take a look at Free Energy Basis Supporting Antibody-Antigen Bond Formation.
Two of the most common ELISA formats are outlined below.
Antigen-Down ELISA
| The microplate well is coated with the antigen of interest which subsequently binds antibodies (target antibodies) to the antigen that are present in the sample (e.g. serum or plasma). After a wash step to remove unbound components in the sample, bound target antibody is detected using an enzyme-labelled, species-specific antibody (e.g. HRP Goat Anti-Human IgG Fc could be used to detect bound IgG when using human serum as the sample). |
| After another wash step, to remove unbound enzyme-conjugated detection antibody, the bound detection antibody is measured using a suitable substrate. The signal strength is proportional to the amount of target antibody in the sample. |
Antibody-Sandwich ELISA
| A “capture” antibody (coated antibody) is adsorbed into the surface of a microplate well. The antigen of interest in the sample is captured by the “capture” antibody. After a wash step to remove unbound components from the sample, an enzyme-conjugated antibody (the “up” anti-antigen antibody) that recognizes a different epitope on the target antigen is added and binds to the target antigen. After another wash step, to remove unbound enzyme-conjugated antibody, the bound “up” detection antibody is quantitated using a suitable chromogenic substrate. |
| The signal strength is proportional to the amount of target antigen in the sample. Unless already validated (e.g. by the supplier), the ability of the “capture” and “up” antibodies to work together as a matched pair will need to be confirmed as part of the assay development. |
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| Alternatively, the “up” antibody can be biotinylated and detected using an HRP-streptavidin conjugate, in which case both capture and “up” antibodies can be from the same species. However, the hydrophobicity of the biotin tags can markedly increase non-specific binding. |
Developing an ELISA
We offer a range of reagents to develop and run high quality ELISAs (see our ELISA kits and reagents).
If you are new to developing ELISAs, or simply like the convenience, we have an Antigen-Down ELISA Development Kit and an Antibody-Sandwich ELISA Development Kit which contain the key reagents and detailed instructions to guide you through the assay development process.
In the sections below, we look at the key assay development steps and highlight the benefits of our high-quality ELISA reagents
Typical ELISA Workflows
Step 1: Binding antibody or antigen to microplate well & blocking the plate
| For ELISAs utilizing a colorimetric readout, clear polystyrene microwell plates with high binding affinity are used. We supply Costar® 96-Well EIA/RIA Stripwell Plates and Immulon® 2 HB 96-Well Microtiter EIA Plates. See Plates and Accessories. |
To optimize the binding of antigen (Antigen-Down ELISA) or capture antibody (Antibody-Sandwich ELISA) to plates, we have developed specific Antigen and Antibody Coating Buffers. These buffers enhance adsorption of the antigen and help stabilize the structure of the immobilized antigen or antibody. They enhance the specificity of the assay and help extend the shelf-life of the coated plates by preventing degradation / denaturation of the immobilized proteins during plate storage.
After coating the plate wells with antigen or antibody, it is important to block those sites on the plate wells that are not already coated with antigen / capture antibody; failure to do so will result in extremely high background signal due to sample components non-specifically binding to the unblocked sites. Blockers also stabilize the ‘sticky’ constant region of antibodies, which is advantageous for long-term storage of plates.
We offer a range of blocking buffers optimized to meet different needs.
For standard antibody-sandwich and antigen-down ELISAs, our General Block is a good starting point.
If you require a protein-free blocker, we recommend our ELISA Blocking Buffer (formerly Synblock); this buffer is also recommended if you are having issues with high non-specific background signal. We recommend the use of Costar® 96-Well EIA/RIA Stripwell Plates with this blocker.
For antigen-down ELISAs designed to measure serological levels, we suggest our ELISA Blocking Buffer - Non-Mammalian-based (formerly Neptune Block). This blocker uses a non-mammalian protein extract and small molecule stabilizers to provide highly efficient blocking. The small size of the blocking reagents minimizes steric hindrance and prevents masking of small coated (peptide) antigens, thus maximizing their specific antigenic signal. The non-mammalian nature of this blocking buffer means that it is antigenically foreign to most mammalian immune systems. When detecting antigen-specific antibodies, this reduces the possibility of false positives due to endogenous antibodies in the sample reacting with blocking proteins. It is also good for antigen-sandwich assays with high background issues. It is a good option to reduce non-specific binding when using biotin / avidin-HRP signal amplification protocols.
If these blockers do not give satisfactory ELISA performance, we have other blockers in our range that may help resolve your ELISA issues.
Alternative Block ELISA Blocking Buffer is a unique protein-free, detergent-free ELISA blocking buffer. It contains a heterogeneous mixture of proprietary blockers and synthetic stabilizers that block the uncoated regions of the plate without the use of conventional cross-reactive protein additives or detergents.
Monster Block ELISA Blocking Buffer provides a high degree of blocking efficiency using a heterogeneous mixture of non-mammalian protein blocking agents. It is designed to address high background problems in antigen-down and sandwich immunoassays.
We also have Phosph-Free Block ELISA Blocking Buffer (Note: not compatible with Immulon II plates) which is a non-protein, phosphate-free blocking buffer for ELISAs using alkaline phosphatase detection, or for assays requiring ultra-sensitivity.
To aid assay optimization, we offer a Block Buffer Optimization Pack containing 100 mL each of General Block, ELISA Blocking Buffer – Non-Mammalian (formerly Neptune Block), ELISA Blocking Buffer (formerly Synblock), Alternative Block, and Monster Block.
While it is possible to prepare plates fresh, it makes sense to prepare batches of stable, dried plates so that you are always ready to run your ELISA. Take a look at: What is your advice for preparing stable dried ELISA plates?
Step 2: Adding the sample
The test sample is likely to be a complex biological matrix such as plasma, serum or tissue culture medium. It is important to consider interference from the sample matrix (the “matrix effect”) when developing your ELISA. This is the sum of all the non-target sample components that impact on the final ELISA reading. The matrix effect can lead to impaired analyte-antibody binding in the sample compared to the standard solution, which normally has a much less complex matrix.
Sample viscosity:
High sample viscosity interferes with antibody-antigen binding kinetics. If the sample matrix is substantially more viscous than the standard solutions, this can lead to a significant underestimation of sample target concentration. If the target concentration is sufficiently high, then simply diluting your sample >1:20 in the same diluent used to prepare the standard curve may be sufficient to overcome potential viscosity issues. If the assay sensitivity / analyte concentration is such that the sample cannot be extensively diluted, then steps must be taken to minimise matrix differences between the sample and the standard (see Sample Matrix Viscosity, Another Common Contributor to Signal Inhibition in Enzyme Immunoassays).
Complement inhibition of signal:
Complement binds to the Fc region of immunoglobulins and may block analyte-specific binding sites via steric hindrance. Inhibiting Complement C1 formation by using calcium chelators can eliminate this type of assay interference.
Other immune-based elements may also interfere with ELISA performance by interacting with antibody-sandwich ELISA antibodies leading to false negative or false positive readings (see Endogenous Heterophile and Human anti-animal Antibodies Mode of Action with Respect to ELISA Interference).
We have developed a range of Sample and Assay Diluents to help minimize matrix effects and optimize ELISA performance.
Sample Diluents:
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Sample diluents are used to dilute samples into the functional range of the assay and to create the standard curve. They are suitable for antibody-sandwich or antigen-down ELISAs. |
Protein-Free Sample Diluent is designed for the dilution of biological samples (e.g. serum, cell culture media) into the useful range of antibody-sandwich or antigen-down ELISA assays. It contains a heterogeneous mixture of proprietary molecules to reduce background noise associated with non-specific bridging of signal-generating conjugates to the plate well surface.
Plasma Sample Diluent is designed for dilution of neat plasma and serum samples. As well as reducing non-specific IgG absorption onto coated and blocked plates, it is specifically formulated to solve the issue of unwanted clotting events in plasma and serum samples.
General Serum Diluent and General Serum Diluent, 2X are formulated with BSA to provide a stable, protein-friendly environment for dilution of serum, cell culture, ascites, urine, and other aqueous-based biological samples for evaluation within an ELISA format. They are an excellent generic matrix for diluting antigen standards within antibody-sandwich formats. They also provide an excellent serum dilution medium for antigen-down ELISAs assessing humoral immune responses.
Sample Diluent - Non-Mammalian-based (formerly Neptune Sample Diluent) is a non-mammalian protein-containing solution highly recommended for use with serum or plasma samples from mouse, porcine, bovine or rabbit in an antigen-down format. Also suitable for samples such as serum or cell culture media in antibody-sandwich and antigen-down ELISAs. Good for ‘sticky’ porcine IgG samples.
We also offer a Sample Diluent Optimization Pack, containing 100 mL each of General Sample Diluent, Plasma Sample Diluent, Sample Diluent - Non-Mammalian-based (formerly Neptune Sample Diluent) and Protein-Free Sample Diluent, ideal for rapidly and economically screening for the best Sample Diluent during assay development.
Assay Diluents:
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Assay Diluents are a second level of support to help overcome matrix effects. They are added to all assay wells (sample, standard and controls) to help minimize the difference in matrix complexity between the sample and the diluent used to generate the ELISA standard curve. Our Assay Diluents are formulated to inhibit clotting in plasma and serum samples. |
General Assay Diluent and General Assay Diluent, 2X are mammalian protein-based reagents designed to equalize sample and standard matrices for more accurate results. They reduce non-specific protein interaction with the plate and reduce complement and thrombin interference in serum and plasma samples. Formulated for testing serum, plasma, urine, and cell culture samples in all sandwich ELISA formats. Can General Assay Diluent be used as sample/standard diluent?
Antigen-Down Assay Diluent is designed for use with serum and plasma samples in antigen-down ELISAs. Proprietary additives reduce non-specific binding and minimize clotting potential and interference from complement and thrombin. It enhances specific signal without denaturing the plate-adsorbed antigen molecules.
Neptune Assay Diluent is a non-mammalian protein-based reagent formulated to equalize complex matrix differentials that are often encountered when evaluating serum and plasma samples in antibody-sandwich ELISA formats.
IgM-Reducing Assay Diluent is designed to address IgM-mediated conjugate bridging interference in problematic serum and plasma samples in antibody sandwich ELISA formats.
We also offer an Assay Diluent Optimization Pack, which contains 100 mL each of General Assay Diluent, IgM-Reducing Assay Diluent, Neptune Assay Diluent, and Antigen-Down Assay Diluent, ideal for rapidly and economically screening for the best Assay Diluent during assay development.
Step 3: Washing the plate
It is crucial to wash each assay well thoroughly to remove unbound sample components prior to adding the detection antibody. Failure to do so will result in high non-specific background signal.
ELISA Wash Buffer, 10X is an optimal formulation to wash ELISA plates between reagent addition steps to remove signal-altering debris yet preserve specifically bound assay components and so reduce background and increase signal. It is suitable for antibody-sandwich ELISAs and antigen-down ELISAs.
Step 4: Add detection antibody
We offer a range of HRP-based antibody conjugates for different ELISA set-ups.
HRP Goat Anti-Rabbit IgG Fc is antigen affinity-purified and recognizes normal rabbit IgG F(c) fragment. It is non-reactive with other rabbit serum proteins.
HRP Goat Anti-Mouse IgG Fc is antigen affinity-purified and recognizes normal mouse IgG F(c) fragment. It does not react with kappa or lambda light chains.
HRP Goat Anti-Human IgG Fc is antigen affinity-purified and recognizes normal human IgG F(c) fragment. It is specific for human IgG and is non-reactive with kappa or lambda light chains.
| If you want to generate your own HRP-antibody conjugates, we offer Conjugation-Ready HRP Maleimide. This reagent avoids both the inactivation of HRP seen with periodate oxidation methods and the aggregation and precipitation issues encountered with glutaraldehyde methods. |
To learn why HRP is such a popular ELISA detection enzyme, take a look at HRP Redox Reaction Driven TMB Color Development and our webinar Features of HRP which provide the Chemical Basis for its routine inclusion within Colorimetric ELISA assays
We also offer a range of enzyme conjugate stabilizers to improve conjugate stability and performance. Each is designed to meet specific ELISA needs. All are designed to reduce background signal.
HRP Conjugate Stabilizer, Mammalian, 1X is used to preserve concentrated stock conjugates, reconstitute lyophilized conjugates, and dilute antibody-HRP conjugates to their working concentration.
HRP Conjugate Stabilizer, Non-Mammalian-based, 1X (formerly Neptune HRP Conjugate Stabilizer) preserves the activity of concentrated and diluted IgG-HRP conjugates in traditional antigen-down or antibody sandwich ELISA formats. It uses hydrolyzed non-mammalian proteins instead of BSA.
Antigen-Down HRP Conjugate Stabilizer, 5X is a unique formulation with proprietary non-mammalian protein additives. It inhibits conjugate binding to non-specific serum proteins bound to the plate, reducing conjugate bridging on the plate surface and background noise. May be particularly useful when assessing low-positive humoral antibody responses.
HRP Conjugate Stock Stabilizer, 5X is designed for users who require an all-purpose HRP-conjugate diluent and stabilizer.
Alkaline Phosphatase Conjugate Stabilizer for stabilizing and diluting alkaline phosphatase conjugates. It minimizes non-specific interactions and reduces background.
For more details, please see our Conjugates and Stabilizers page.
Step 5: Washing the plate
It is crucial to wash the plate wells again to remove unbound detection antibody prior to adding the substrate and visualizing / quantitating the bound detection antibody. See Step 3 for details.
Step 6: Substrate addition / stopping the detection reaction
A suitable substrate is required for the enzyme-conjugated detection antibody to produce a colored reaction product that enables quantitation of the bound analyte.
Our substrates enhance the sensitivity of your ELISA, reduce background noise, and provide linearity for enhanced reproducibility.
HRP as the detection enzyme:
ABTS 1-Component HRP Microwell Substrate (SUBA) is designed to be used when the required detection level is in the µg-ng/mL range. Ideal for ELISAs where the test samples contain high concentrations of the target molecule or when it is advantageous to utilize lower sample dilution factors.
TMB 1-Component HRP Microwell Substrate (SUBT) is designed to be used when the required detection level is in the ng-pg/mL range.
TMB Slow Kinetic 1-Component HRP Microwell Substrate (SUBK) is designed for use where the test samples contain high levels (µg-ng/mL) of the target molecule, for assays with long incubation periods, and for assays that do not need a high level of sensitivity.
TMB Super Sensitive 1-Component HRP Microwell Substrate (SUBS) is our most sensitive TMB reagent. It is ideal for when very high dilutions of test samples (e.g. 1:10,000) are required; with samples exhibiting high steric hindrance; with low binding capacity antibodies; or when a short incubation time is required.
Alkaline Phosphatase as the detection enzyme:
Our pNPP 1-Component AP Microwell Substrate with Stabilizing Pellets (SUBP) is a stabilized formulation of pNPP substrate to extend the shelf life of the substrate on your benchtop to 2-3 days.
Stopping the detection reaction:
After an appropriate incubation period, the detection reaction should be terminated with a Stop Solution.
Stop Solution for TMB Substrates (STOPT) is a ready-to-use liquid acidic stop solution suitable for use with our TMB substrates. When the reaction is stopped using Stop Solution for TMB Substrates, the chromogen changes from blue to yellow. This product is suitable for all endpoint ELISAs using a TMB substrate for color development. Read absorbance at 450 nm.
Stop Solution for AP Substrates (STOPP) is designed for use with pNPP-based colorimetric substrates. It is suitable for all endpoint ELISAs using an alkaline phosphatase detection substrate. Read absorbance at 405-420 nm.
Step 7: Reading the microplate
Once the detection reaction has been terminated, the microplate should be read in a suitable plate reader to quantitate the results.
Step 8: Calculating your results
The concentration / amount of analyte in each sample is quantitated by reference to the analyte standard / calibration curve. Plate readers often contain software for constructing and reading standard curves. Otherwise, various commercial curve-plotting software packages are available.