R&D Systems has over 25 years of experience designing, testing, and optimizing immunoassay kits to ensure the highest level of performance in analyte quantification. We currently offer more than 400 Quantikine®, Quantikine HS®, Quantikine IVD®, QuantiGlo®, Fluorokine® E, and Parameter™ ELISA Kits for numerous different analytes and species, including human, mouse, rat, canine, primate, and porcine. Choosing quality reagents that will lead to results you can trust is one of the most critical aspects of scientific research. How do you know if you are choosing a quality product? One measure of product quality is the frequency of citations in the scientific literature. R&D Systems ELISAs are referenced more than any other ELISA manufacturer and we are honored by the trust that so many have placed in our products.
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R&D Systems Is the Most Referenced ELISA Manufacturer. A literature survey was conducted to determine the number of citations referencing the use of R&D Systems ELISA Kits compared to the number referencing ELISAs manufactured by other companies. The survey included 860 manuscripts that were published in one of 44 high impact journals from several different general research areas, including immunology, signal transduction, development, neuroscience, bone/endocrinology, and hematology. A total of 433 ELISA citations referencing immunoassays from 66 different vendors were identified in the survey. |
R&D Systems is Committed to Producing High Quality ELISA Products
Making a Quality ELISA
Producing a quality ELISA strongly depends on optimization during development. R&D Systems ELISA Kits are required to meet stringent manufacturing and quality control standards to ensure that they provide the highest levels of performance and consistency. Quantikine Kits are complete, fully validated, ready-to-run immunoassay kits that are designed to measure proteins in a number of complex sample types. These assays are based on the two-site sandwich immunoassay principle in which two highly specific antibodies are used to detect a target analyte. Multiple steps are taken during development to ensure that Quantikine ELISA Kits will provide superior performance without the need for further assay optimization by the customer. These include:
- Careful selection of antibody pairs for optimal performance
- Automated microplate coating with precision of less than 10% coefficient of variation
- Cross-reactivity and interference testing with a panel of related factors
- Formulation of diluents that alleviate interferences due to matrix phenomena and heterophilic antibody interactions
- Correlation to NIBSC/WHO Standards when available
- Performance testing with all validated sample types
Evaluating the Performance of ELISA Kits
Quantikine ELISA Kits provide our customers with the precision, specificity, accuracy, and sensitivity that they expect due to rigorous validation testing. This testing includes:
- Intra- and inter-assay precision
- Analysis of natural samples in each of the validated matrices, such as serum, plasma, cell culture supernatets, and more
- Interference by blood components
- Linearity
- Recovery
- Component and kit stability
- Edge effect and assay drift
These tests are performed over many months by several technicians to ensure that the assay will be reproducible both well-to-well and lot-to-lot. Data obtained from performance testing on validation batches of our kits are provided in the product data sheets.
The validation process results in a comprehensive data packet that is reviewed by quality assurance personnel, who ensure that the test results meet established guidelines. These guidelines require that:
- Controls meet established specifications
- Non-specific binding, low standard, and high standard signals meet the established specifications
- Sensitivity, determined by assaying multiple replicates of the zero standard, falls within the established specifications
- Standards match master calibrators within the established specifications
The following sections outline the variables that may affect the outcome of your ELISA experiments and how these variables are addressed during the development of R&D Systems Quantikine ELISA Kits. By carefully considering these variables before the product is released, our scientists ensure that Quantikine Kits will provide you with reliable, reproducible results without the need for further assay optimization.
Quality Antibodies Provide the Foundation for a High Performance ELISA
Our ELISA kits are developed using highly purified antibodies. For our sandwich ELISA kits, including the Quantikine, Quantikine HS, Quantikine IVD, and QuantiGlo Kits, testing is performed on several different monoclonal and polyclonal antibodies to determine which combination optimally couples for use in analyte detection. Selected antibodies are carefully titered to ensure that the concentrations chosen will give the best possible results for the assay. During development, the capture antibody is coated onto the microplate in several different concentrations to determine the concentration that offers the most binding and the best precision. Different concentrations of the detection antibody are subsequently tested to determine the concentration at which the detection antibody optimally pairs with the capture antibody to give the best signal-to-noise ratio.
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Antibody Pairs Are Carefully Selected for Quantikine ELISA Development. Quantikine ELISAs are based on a two-site sandwich principle in which two highly specific antibodies are used to detect the target analyte. Quantikine Kits provide a 96-well microplate pre-coated with a capture antibody specific for the analyte of interest. Upon incubation with experimental samples, standards, or controls, the target analyte is captured by this antibody. A conjugated detection antibody that binds to a different epitope on the target analyte is used to complete the sandwich. A substrate solution is subsequently added to produce a signal that is proportional to the amount of analyte bound. |
ELISA Precision & Reproducibility: Providing Confidence in Your Results
Immunoassay precision is defined as the reproducibility of results within and between assays. This characteristic of an immunoassay is extremely important in order to: 1) provide assurance that the results obtained throughout a study are accurate and reproducible from one experiment to the next and 2) decide if two results are the same or different. Precision is measured as a coefficient of variation (CV) from the mean value. Two types of precision should be considered, intra-assay precision and inter-assay precision. Intra-assay precision is the reproducibility between wells within an assay. This allows the researcher to run multiple replicates of the same sample on one plate and obtain similar results. Inter-assay precision is the reproducibility between assays. Inter-assay precision guarantees that the results obtained will be reproducible using multiple kits over a period of time. R&D Systems Quantikine Immunoassays typically have CV values less than 10% across the standard curve for both intra- and inter-assay precision. These low CV values allow the researcher to perform repeated assays and be confident that the results are consistent throughout the study.
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| Quantikine ELISA Kits Are Tested for Stability and Reproducibility. A. Three samples with different concentrations of IL-6 (colored lines) were assayed using the same lot of the Human IL-6 Quantikine ELISA Kit (Catalog # D6050B) over a 15 month period. B. Three samples with different IL-6 concentrations (colored lines) were assayed using four different lots of the Human IL-6 Quantikine ELISA Kit (Catalog # D6050B) over a 12 month period. |
ELISA Specificity: Measuring Only the Analyte of Interest
Immunoassay specificity can be compromised by antibody cross-reactivity and interference. Cross-reactivity occurs when a molecule other than the analyte of interest is bound by both antibodies leading to a false positive result. Interference occurs when other substances in the sample matrix modify the antigen-antibody interaction, preventing an assay from recognizing its designated analyte.
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The Use of Quality Antibodies Is the First Step in the Development of a Reliable ELISA. Antibodies play a crucial role in the development of a high performance ELISA. Antibody pairs used for R&D Systems ELISAs are selected to ensure high signal, low background, and the best possible sensitivity. Antibodies are tested for specificity, cross-reactivity with molecules other than the target analyte, and interference with matrix components. |
These problems are mitigated by proper development and testing. False positive results may be due to matrix effects that only diligent validation and quality control measures can identify. This being the case, an ELISA cannot be judged solely based on whether or not it produces a signal, until that signal is confirmed to be produced by the analyte of interest. In most cases, this can be accomplished by assaying the linearity of dilution.
R&D Systems carefully selects antibodies and optimizes coating buffers, conjugate buffers, and assay diluents to eliminate matrix effects. To gauge the specificity of an assay, factors related to the analyte are tested for cross-reactivity and interference. The members of the panel and the results of this testing are reported in our product data sheets.
Binding differences may occur between natural and recombinant samples due to conformational changes of the antigen after it is bound to the capture antibody. These conformational changes may affect the binding of the detection antibody. Quantikine Kits are optimized so that the antibodies recognize both recombinant and natural antigen with equal efficacy.
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False Positive ELISA Signals Can Be Identified by Assaying the Linearity of Dilution. Serial dilutions of a cell culture supernate were assayed for natural linearity using two different TIMP-2 ELISA Kits. Diluted samples measured using the Human TIMP-2 Quantikine Kit (Catalog # DTM200) gave recovery results between 105-108% of the neat sample, supporting the linearity claim of the kit. In contrast, the target analyte was not detectable beyond the first dilution in samples measured with the second kit, indicating that the assay was producing a false positive signal. ND=Not detectable. *Samples were diluted prior to the assay as directed in the product data sheet. |
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Interference Testing of the Human TNF-alpha Quantikine ELISA Kit. TNF-alpha, at concentrations of 125-1000 pg/mL, was measured in the presence or absence of soluble TNF receptors (sTNF RI or sTNF RII) using the Human TNF-alpha Quantikine ELISA Kit (Catalog # DTA00D). The results demonstrate that the presence of the soluble TNF receptors at concentrations up to 1000 ng/mL does not affect the TNF-alpha concentration determined using the Quantikine ELISA Kit. |
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Quantikine ELISA Kits Are Developed to Detect Natural and Recombinant Proteins. A serum sample containing activated human TGF-beta1 was serially diluted (blue line) and compared to the TGF-beta1 standard (red line). Results show that the Human TGF-beta1 Quantikine ELISA Kit (Catalog # DB100C) measures recombinant and natural TGF-beta1 with equal effectiveness. |
ELISA Recovery Experiments
Complex sample matrices, such as serum and plasma, may contain interfering factors that affect the ability of the assay to accurately quantify the target analyte. Recovery experiments are used to determine if assays are affected by interfering factors. Low, medium, and high concentrations of analyte are spiked into all validated sample types and then analyzed for recovery. The results are expressed as a percentage of analyte recovered and are reported in each product data sheet. Our criteria require that recoveries are between 80-120% across the concentration range of the assay, demonstrating no quantifiable matrix interference for each sample type. If interfering factors are found, R&D Systems formulates diluents that minimize their effects.
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Analysis of the Recovery of Dkk-1 Using the Quantikine ELISA Kit. The recovery of Dkk-1 spiked to various levels throughout the range of the assay was assessed for all validated sample types of the Human Dkk-1 Quantikine ELISA Kit (Catalog # DKK100B). *Samples were diluted prior to the assay as directed in the product data sheet. |
ELISA Samples: Linearity of Dilution
Dilutions should always derive the same final analyte concentration for a sample. This is known as assay linearity. Interfering factors can compromise assay linearity unless the assay is designed to overcome these effects. We generate a dilution series using kit diluents across the dynamic range of the assay for each validated sample type. The results are expressed as a percent observed from expected. Values between 80-120% show good assay linearity. Each product data sheet reports the mean and the range of percent linearity for all validated sample types.
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Linear Dilution to Assess ELISA Matrix Effects. A. Expected results from a linearity of dilution experiment when no interfering factors are present in the matrix. B. Potential results from the same experiment if interfering factors are present in the matrix. Factors in complex matrices can interfere with the analyte of interest. This effect may be revealed by unexpected linear dilution values. |
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| Assay Linearity Is An Important Measure of Immunoassay Accuracy. A. Spiked heparin plasma samples were serially diluted and assayed for human Thrombomodulin using two different ELISA kits. Samples measured with the Human Thrombomodulin/BDCA-3 Quantikine ELISA Kit (Catalog # DTHBD0) had recovery values between 90-110% of the neat sample and displayed acceptable assay linearity (gold line). In contrast, the percent recovery of diluted samples measured with the second kit had a range of 141-325% (burgundy line), suggesting that interfering factors were preventing accurate measurement of the target analyte. B. Spiked heparin plasma samples were serially diluted and assayed for human Tissue Factor Pathway Inhibitor (TFPI) to determine the accuracy of two different immunoassay kits. Samples measured with the Human TFPI Quantikine ELISA Kit (Catalog # DTFP10) showed acceptable recovery values (90-110% of the neat sample; red line), while those measured with the second kit (green line) did not (80-7% of the neat sample). |
ELISA Sensitivity: Measuring Proteins at the pg/mL Range
The minimum detectable dose is the lowest measurable value that is statistically different from zero. It is calculated by adding two standard deviations to the mean optical density value of several zero standard replicates and determining the corresponding analyte concentration from the standard curve. The better the sensitivity of an assay, the lower the useful working range (standard curve range) will be. Quantikine ELISAs are optimized to ensure high signal, low background, and the best sensitivity possible.
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| The Minimum Detectable Dose for Many Quantikine ELISA Kits Allows Proteins Present at the pg/mL Range to be Accurately Measured. A. Serum from 86 apparently healthy individuals was assayed using the Human IL-12/IL-23 p40 Quantikine ELISA Kit (Catalog # DP400). B. Serum from 41 apparently healthy individuals was assayed using the Human IL-6 Quantikine HS ELISA Kit (Catalog # HS600C). |
ELISA Calibration: Ensuring Consistency from One Lot to the Next
Each Quantikine ELISA Kit includes an immunoassay standard that is calibrated against highly purified material. R&D Systems assigns a mass value to a standard based on comparison to a master calibrator. These master calibrators are manufactured during the development of an ELISA and are used to maintain the consistency of kit standards. All future lots are compared to the master calibrator to ensure that no drift in sample values occurs.
Due to the fact that different mass value assignments are made for ELISA standards, sample values produced using one manufacturer’s kit may not be directly comparable to those obtained using another manufacturer’s kit. R&D Systems supplies a correlation to a WHO international reference material when available. This calibration allows a researcher to take the values obtained with a Quantikine ELISA Kit and compare them to values obtained with other assays (assuming that the other ELISA manufacturer provides this conversion factor as well).