What are the Components of an ELISA?
An enzyme-linked immunosorbent assay (ELISA) is an immunology technique used to detect an antigen or an antibody in a sample. An ELISA may also be used to quantify an antigen, antibody, protein, enzyme, or hormone in a sample.
The principle of an ELISA uses procedures to detect analytes recognized by an antibody in biological specimens. The procedures follow a basic principle including:
- Coating: coating solution is used to adsorb protein to plate surface
- Blocking: binding sites are blocked using a buffer designed to reduce non-specific binding and matrix interferences
- Detection Antibody: conjugated protein binds to analyte if present
- Substrate Addition: Colorimetric substrate added to wells catalyzed by enzyme
- Analysis: ELISA reader analyzes data
Surmodics™ IVD provides assay developers the critical components needed to build robust and reliable ELISA assays. Our complete line of dried protein stabilizers and blockers, sample/assay diluents, in-solution protein stabilizers, TMB substrates, and more are available for ELISA test development. Each of our immunoassay components help ensure accurate and reliable results when developing immunoassays.
The standard components in an ELISA kit include antibodies, antigens, dried protein stabilizers and blockers, wash buffers, in-solution protein stabilizers, substrates, stop solutions and sample/assay diluents if needed. Surmodics IVD acts as a one-stop-shop for assay developers to purchase individual components of an ELISA when building ELISA kits. Our extensive portfolio of ELISA products helps overcome technical issues and help assay developers in the continued optimization of their ELISA technology.
Visit the link below to learn more about Surmodics' ELISA components.
ELISA Buffers
Standard Buffers
The ELISA process uses several different buffers. There is a buffer for coating, blocking, stabilization, washing, and even sample or antibody dilution. Surmodics IVD offers an extensive selection of unique ELISA buffers.
Visit our ELISA Assay Solutions & Protocol page to review our line of ELISA buffers.
Coating Buffers
Coating is the beginning step in nearly every ELISA process. Coating requires suitably diluted antibodies or antigens to be incubated until completely adsorbed to the surface of a well. Adsorption occurs passively as hydrophobic interactions occur between amino acids on an antibody used for coating a surface. Coating buffers depend on time, temperature, and pH, and the concentration of a coated protein. The coating buffer maximizes adsorption to the plate, optimizing interactions for the next step in the immunoassay.
Visit the link below to learn more about our coating buffer.
Blocking / Stabilization Buffers
Blocking buffers are critical for preventing non-specific binding from the sample matrix and downstream assay components to a microwell plate surface. Blocking is a compromise between finding the desired sensitivity and reduced background. Surmodics IVD’s dried protein stabilizers and blockers are the industry’s gold standard for increasing assay sensitivity and reducing background noise. We offer three products at this step, StabilCoat™, StabilGuard™ and StabilBlock™.
Moreover, StabilCoat, StabilGuard and StabilBlock dried protein stabilizers and blockers preserve the conformation and activity of dried proteins coated on a wide range of surfaces, keeping antibodies and antigens at peak performance for long durations. At the same time, the blocking mechanisms in these reagents reduce non-specific binding of interfering proteins to maximize assay sensitivity. These ELISA blocking reagents are the industry gold standard for stability and blocking efficacy. The result is immediate improvement of assay performance in a one-step process for streamlined manufacturing.
Visit the link below to learn more about our ELISA bocking buffers.
Assay/Sample Diluents
Surmodics IVD’s ELISA blocking reagents also include sample or assay diluents that help to reduce matrix interferences in the assay. Surmodics™ Assay Diluent and MatrixGuard™ Diluent are the gold standard for reducing false positives in your assay. These formulations provide two options to use when different methods may be needed to block matrix interferences, while maintaining the signal of the assay.
For IVD kit manufacturers requiring a strong, consistent blocking diluent across a variety of assays, MatrixGuard Diluent provides unsurpassed blocking whereby matrix interferences are effectively blocked while the intended assay signal is maintained. Unlike other diluents that either are marginally effective at blocking matrix interferences or alternatively block out true assay signal, MatrixGuard Diluent achieves the goal of maximum blocking of matrix interferences, while simultaneously allowing signal to be maintained.
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Wash Buffers
Wash steps are often repeated between each step, helping to remove unbound materials given ELISA’s use surfaces that bind for separation. ELISA wash buffers were developed as a high performing washing solutions to be used in a variety of versatile ELISA formats.
Surmodics IVD’s BioFX™ Tris Buffered Saline (TBS) Wash Solution‐10X Concentrate contains non‐ionic surfactant, which does not interfere with assay reactants and reduces non‐specific binding.
The ELISA wash buffer available from Surmodics IVD may be used with antibody-sandwich ELISA’s and Direct ELISA applications
Visit the link below to learn more about our ELISA wash buffer.
Antigens & Antibodies
Surmodics IVD is the exclusive North American representative of DIARECT™ antigens and antibodies. We offer a comprehensive panel of autoimmune disease, infectious disease, allergy antigens and antibodies that provide outstanding assay performance for in vitro diagnostics applications.
DIARECT recombinant and native antigens have a proven track record for quality and sensitivity in ELISA, Western Blot and lateral flow applications. The lot-to-lot consistency of DIARECT antigens reduces development time and increases productivity of immunoassay developers.
Interaction between antibodies and antigens can be described in three different ways:
Specificity - specificity indicates whether an antibody binds to a unique epitope from a single antigen in a single species or whether it will bind to similar epitopes present on multiple molecules from several different species.
Affinity - affinity is the strength of binding that an antibody has on an individual epitope. Affinity also determines how quickly the binding process occurs and how long it will last.
Avidity - avidity accounts for the total stability of an antibody-antigen interaction on a solid phase.
Visit the link below to learn more about our antigens and antibodies.
In-Solution Protein Stabilizers
Surmodics IVD’s in-solution protein stabilizers (conjugate stabilizers) offer assay developers the gold standard for stability of protein conjugates at working strength concentration for ELISA/EIA, ELISpot, RIA and immunoblot/western blot applications. Our StabilZyme™ HRP, StabilZyme Protein-Free and StabilZyme NOBLE stabilizers provide the assay developer a complete set of tools to stabilize different HRP conjugates for long term, in-solution stability.
There are several technical advantages when using our in-solution protein stabilizers. When looking at their protein stabilizing capability, these reagents extend the shelf life of antibodies, antigens and other proteins at working strength concentrations and offer retained activity of enzymes in solution for up to two years.
In addition, our StabilZyme in-solution protein stabilizers offer outstanding assay compatibility. Furthermore, we offer multiple formulations with different stabilizing mechanisms to satisfy different enzyme/protein requirements. This includes Protein-free and BSA-free formulations to satisfy the background requirements for specific assay systems. In addition, these reagents stabilize enzymes and control materials in a variety of assays including ELISA/EIA, ELISpot, RIA, immunoblots and microarrays applications.
Visit the link below to learn more about our in-solution protein stabilizers.
Protein & Conjugate Stabilizers
Shop Protein & Conjugate Stabilizers
ELISA Substrates
The signal generation step of immunoassay systems is critical to obtaining an accurate measurement of the target protein. Surmodics’ portfolio of BioFX colorimetric and chemiluminescent substrates offers the stability, low background and sensitivity needed to meet the demands of assay manufacturers. A range of substrate sensitivities are available from Surmodics for ELISA, Western Blot, ELISpot and immunohistochemistry applications.
Detection limit, dynamic range, and reproducibility are cornerstones in the development of a successful immunoassay application. During the optimization process, antibody/antigen systems are carefully chosen to provide the specificity and sensitivity of the desired analyte measurement. However, the choice of enzyme/substrate can also have a substantial effect on achieving the above-mentioned parameters and require equal attention for optimal selection.
Visit the link below to learn more about our ELISA Substrates.
Substrate Selection
During assay development it is important to consider your substrate selection carefully and to have an understanding of your assay’s requirements. Several key elements can be important such as dynamic range, kinetic rate, and assay timing.
Dynamic range is especially important when developing an assay that requires quantitation of a large range of analyte concentrations. Kinetic rate is also an important criterion when finding the right substrate for your ELISA. As such, Surmodics IVD offers several TMB substrates at varying kinetic rates to ensure we have an option suitable for each unique assay in question.
In addition, the timing of your assay is important when considering factors such as dynamic range, reproducibility and detection limit. Assay timing can be optimized by choosing a TMB substrate with the kinetic rate that allows your assay to be performed within the desired timeframe.
Key assay parameters, such as dynamic range, kinetic rate and assay timing are important factors to consider when evaluating various TMB substrates. Each TMB substrate impacts these parameters differently. Choosing the best substrate for your assay will allow the assay developer to create a robust assay with superior performance.
Visit the link below to learn more about our recommendations for ELISA substrate selection.
White Paper: Substrate Selection in Immunoassay Development
Stop Solutions
The TMB stop solution is critical in application since it allows the substrate to undergo a complete reaction. The use of the stop solution on the enzyme-substrate in simple terms is for bringing about a change of color. The shift from blue to yellow is vital for the reactants to form the ultimate product for the process to stop. The yellow indicates that there is no further reaction taking place, hence the formation of the final product. The stop solution also changes the pH of the substrate to bring out the color change and make it optimal for analyzing the results of ELISA’s.
Surmodics IVD offers several TMB stop solutions, including our most recently developed TMB stop solution, BioFX 450 nm Liquid Nova-Stop Solution for TMB Microwell Substrates (Product Code: NSTP). This TMB stop solution offers assay developers maximum performance, and minimal signal drift compared to sulfuric or hydrochloric acid. Absorbance is read in the yellow spectral range (450 nm) and can be read up to 3 hours after the TMB reaction has been stopped using the Nova-Stop Solution. In addition, this TMB stop solution offers assay developers a safer alternative as it is non-corrosive to both skin and eyes.
Visit the link below to learn more about our stop solutions.
ELISA Formats
There are three main types of ELISA kits, observed by looking at the ways antibodies are bonded to analytes.
Sandwich ELISA
Sandwich ELISA assays are the most commonly used assay test. In a sandwich ELISA assay, two specific antibodies sandwich an antigen or antibody of interest, known as matched antibody pairs. First a capture antibody is coated on a microplate. Target proteins bind to the captured antibody if present. A conjugated antibody binds to detect the target protein when added. Then an ELISA substrate is added, producing a signal proportional to the given analyte in a sample. Although these ELISA tests are highly specific assays, they often need additional optimization due to a risk for false positive results.
Direct ELISA
The Direct ELISA process is completed when an antigen is immobilized directly onto a plate. A patient sample is then added, if the antibody of interest is present in the sample, it will bind to the antigen on the surface. A detection antibody is then added to bind to the sample antibody. Then a substrate is added, producing a signal that is directly proportional to the amount of analyte concentration within a given sample. One difficulty with this type of assay can be coating the antigen so the binding site is recognized by the antibody.
Competitive ELISA
Competitive assays are typically used for small molecules. This ELISA is used when the target protein is too small to be used in a sandwich ELISA with two antibodies. A capture antibody is coated on a microplate. Instead of using a conjugated detection antibody, a conjugated antigen is used to compete for binding with the antigen present in the sample. The more antigen present in the sample, the less conjugated antigen will bind to the capture antibody. A substrate is then added, and the produced signal is inversely proportional to the amount of protein in the given sample.
Preparation and Handling
A specific protocol is recommended for standard sandwich ELISA’s. A standard protocol is completed using a 96-well plate for colorimetric detection:
- Coat Capture Antibody:
- Using your starting concentration, add 100 ul capture antibody to appropriate wells
- Starting Concentration Example: 240 μg/mL --> 1:240 --> 1.0 μg/mL in COAT (1X)
- Incubate 1 hour at Room Temperature
- Wash 3X with wash buffer
- Recommended reagents for this step:
- Blocking and Plate Stabilization:
- Add 150 μL of Dried Protein Stabilizer/Blocker to all wells on the plate
- Incubate 1 hour at Room Temperature
- Recommended reagents for this step:
- Continue and Finish Assay in Same Day:
- Aspirate plate or wash with wash buffer
- Proceed to step 5
- Recommended reagents for this step:
- BioFX Tris Buffered Saline Wash Solution - 10X Concentrate (Product Code: WSHW)
- Long Term Plate Stability and Storage:
- Aspirate blocker/stabilizer from wells and blot plate dry to remove residual liquid.
- Dry plates using methods optimized by end user.
- Recommended drying methods:
- Dry in a humidity-controlled chamber (less than 15% humidity) until dry (4-24 hours)
- Dry 2-4 hours in a sealed desiccated container
- Dry plates at 30-40C in a vacuum over for 4 hours
- Package plates in temperature sealed moisture proof pouch with desiccant
- Standards, Samples and Controls: (concentrations pre-determined by end user titration):
- Using your starting concentration, add 100 μL of standards, samples and controls to appropriate wells across plate
- Incubate at Room Temperature for 2 hours
- Wash 3X with wash buffer
- Recommended reagents for this step:
- Primary Antibody (concentration pre-determined by end user titration):
- Using your starting concentration, add 100 μL to all wells on plate
- Starting concentration example: 2.0 mg/mL --> 1:20,000 --> 100 ng/mL in StabilZyme™ Stabilizer
- Incubate 1 hour at Room Temperature
- Wash 3X with wash buffer
- Recommended reagents for this step:
- Secondary HRP/AP Conjugated Antibody (concentration pre-determined by end user titration)
- Using your starting concentration, add 100 μL to all wells on plate
- Example Starting Concentration: 2.0 mg/mL --> 1:20,000 --> 100 ng/mL in StabilZyme Conjugate Stabilizer
- Incubate 1 hour at Room Temperature
- Wash 3X with wash buffer
- Recommended reagents for this step:
- Detection:
- Add 100 μL of BioFX TMB Substrate to all wells
- Incubate for 5 - 30 min at Room Temperature, protect from light
- Add 100 μL of BioFX Liquid Stop Solution to stop plate
- Read plate @ 450 nm
- Recommended reagents for this step:
Please contact us here to connect with one of our R&D scientists directly to learn more about our ELISA protocol recommendations and request free samples for evaluation!
