Help & FAQs: ELISAs

  • Can a partial Quantikine® ELISA plate be used?
  • The Quantikine® ELISA plates have removable strips of wells. Unused wells may be removed from the plate, returned to the foil pouch containing the desiccant pack, and stored at 2-8° C for up to one month.
  • Can ELISA Kits be used with tissue homogenates (or other non-validated sample types)?
  • Unfortunately, R&D Systems has not routinely validated tissue homogenates as a sample type for ELISA kits. This does not mean that ELISA kits are not suitable for other sample types than we have tested; it means further investigation is needed. One will need to perform a spike and recovery study to determine if an unvalidated sample type will work with a particular kit. To perform a spike and recovery experiment, one should divide a sample into two aliquots. In one of the aliquots, the user should spike in a known amount of the kit standard. A dilution series is performed comparing the spiked versus the unspiked sample. Generally, samples with expected recovery and linearity between 80-120% are considered acceptable. This method can be used to validate any sample type that has not been evaluated by R&D Systems. For a more detailed spike and recovery protocol, please contact Technical Service.Note: Acceptable ranges should be determined individually by each laboratory. Additionally, Technical Service can help determine if a buffer component is not compatible with a given ELISA kit. Please see the Citations tab on the product webpage for peer-reviewed papers utilizing a wide range of sample types.
  • Can I extend the standard curve (in either direction)?
  • R&D Systems cannot support kit results outside the stated range under any circumstances. A specific range was chosen because of confidence in the reproducibility of the assay.
  • Can recombinant proteins sold as stand-alone reagents be used as standards in the DuoSet® ELISA Development Kits?
  • These products are not calibrated for use in an ELISA system, and must be calibrated versus an existing mass calibrated standard. Standards provided in ELISA kits have been calibrated to a master calibrator.
  • Do you have information on which VEGF isforms your Human VEGF Quantikine® ELISA kit (Catalog # DVE00) recognizes?
  • The DVE00 kit detects the most abundant isoforms of Human VEGF A - specifically VEGF121 and VEGF165. However, the ability of the kit to detect VEGF189 has not been studied at this time.
  • How are cell lysates prepared for use in Quantikine® ELISA kits?
  • For those Quantikine® ELISAs where cell or tissue lysate is a validated sample type, sample preparation instructions for lysate are included in the product insert.  Components in lysate and lysis buffer can impact immunoreactivity, so if lysate is not a validated sample type, care must be taken in sample preparation and validation.   
  • How is an ELISpot assay different from an ELISA assay?
  • Both assays employ the sandwich ELISA format utilizing capture and detection antibodies. The traditional ELISA is designed measure the concentration of analyte in biological samples such as serum, plasma, and conditioned medium. In contrast, the ELISpot assay provides information about the relative number of cells secreting the specific analyte. In an ELISpot assay the cells are added to the plate and the immobilized capture antibody in the immediate vicinity of the cell binds the secreted protein of interest.The sites of cytokine secretion are visualized as spots in the bottom of the well.
  • How many samples can be assayed in a Parameter™ ELISA Kit?
  • The Parameter ELISA Kits will generally run a 7-point standard curve, non-specific binding wells, and 39 samples in duplicate. This may vary slightly by kit so please refer to each datasheet for details.
  • I am having a problem with high variability between sample duplicates. What could be happening?
  • The two main reasons for high variability in an assay is related to pipetting & wash technique. For tips on improving variability in your ELISA, please see https://www.rndsystems.com/resources/troubleshooting-guide-elisa-development  
  • I used your recombinant protein as a control in the corresponding ELISA kit. Why am I seeing discrepancy in mass values?
  • First, a large dilution is required to place the recombinant protein on the standard curve range. Typically this is a dilution from μg/mL to pg/mL. Any dilution step can introduce inaccuracy and the larger the dilution step the greater the potential for error. Any pipetting error or mis-calibrated pipet can result in apparent over- or under-recovery.Second, R&D Systems immunoassays have been developed to measure a level of protein captured by one antibody and detected by a second antibody. This measurement is calibrated to standards established when the kit was initially developed. The protein determination of these initial standards became the Master Calibrators to which all new standards are formulated. This provides R&D Systems immunoassay kits with consistency between manufacturing lots. In general, we would expect +/- 25% recovery of the amount stated on the vial when using the Quantikine® ELISA to determine a protein concentration. There may be slight differences in the immunologically recognizable mass between lots of protein, so the apparent concentration provided on the vial may vary from lot-to-lot when measured in the ELISA. If you are using proteins to make controls, it is better to value assign the mass based on measurement in ELISA and not use the mass on the vial when setting control levels.
  • If 540 nm or 570 nm wavelength correction is not available, what other wavelengths could work?
  • It has been suggested in literature that wavelength correction between 540-690 nm is suitable for TMB substrates.
  • The ELISA protocol does not list a plate shaker, is one needed?
  • If a shaker is required, it will be listed under the materials required section of the datasheet and information on the speed and orbit will be listed within the protocol. If the protocol does not call for a plate shaker, one is not needed and all incubations should follow the datasheet instructions.  Using a shaker where not required changes the immuno-kinetics of the assay and frequently impact the assay performance in a negative manner.
  • What is a competitive ELISA?
  • In the competitive immunoassay approach, also termed "labeled analyte technique", there exists a competition between the endogenous unlabeled antigen and an exogenous labeled antigen for a limited amount of antibody binding sites. Therefore, a decreasing signal indicates higher concentrations of the analyte being measured.
  • What is a sandwich ELISA?
  • A sandwich ELISA uses an immobilized capture antibody specific for the analyte of interest in a sample. After the analyte is bound to the immobilized antibody, a labeled secondary antibody specific for the analyte is used for detection. The analyte is "sandwiched" between the two antibodies. The sandwich ELISA is extremely sensitive, and the values obtained are quantitative when compared with a standard curve.
  • What is platelet poor plasma?
  • Platelet poor plasma is plasma devoid of platelets. To remove platelets, two centrifugation steps are required.  First, anticoagulated whole blood is centrifuged for 15 minutes (4 °C) at 1000 x g within 30 minutes of collection to ensure minimal platelet activation. Plasma should be carefully removed without pulling platelets from the top most layer of the packed blood cells. An additional high speed centrifugation step of the plasma at 10,000 x g for 10 minutes at 2 - 8 °C is then recommended to achieve platelet poor plasma.  Transfer platelet poor plasma to a fresh tube after final centrifugation step.
  • What is R&D Systems doing to reduce its use of packaging from non-renewable resources? R&D Systems should consider re-using styrofoam boxes that are in good condition after delivery.
  • Environmental stewardship is important to R&D Systems and its employees. R&D Systems regularly explores "green" options. First and foremost, the energy expended to ship back the styrofoam box is more detrimental to the environment than having the facility re-use or recycle it. Our stance is to encourage our customers to implement a recycling program locally and we can identify a recycling center for styrofoam nearest your facility if necessary. At R&D Systems we have chosen to reduce the use of styrofoam as much as possible by: Doing extensive stability testing in order to determine which products can be shipped with minimal packaging at ambient temperatures, Converting the use of non-recyclable packaging materials to recyclable plastics, cardboard, or biodegradable materials, and Continuing to investigate alternatives to dry ice shipments, the use of re-usable containers, and gel packs that allow for smaller styrofoam containers. Employees were key to initiating a recycling program at our facilities. Internally, we recycle paper, plastic, cardboard, aluminum, glass, and styrofoam.
  • What is the half life of a cytokine in serum/plasma/CCM for ELISA?
  • R&D Systems does not determine the half life of cytokines in natural samples such as serum, plasma, or cell culture supernates. Our general recommendation is to assay the sample immediately or aliquot into single use volumes and store these samples frozen. We recommend avoiding repeat freeze-thaw cycles with these samples to avoid protein degradation.
  • What sample types may be measured in R&D Systems' ELISA kits?
  • Typically the R&D Systems ELISA kits are validated for sera, two types of plasma, and cell culture supernate. However, the samples validated in an ELISA can vary from product to product. The product datasheet and ptoduct-specific web page states all sample types that have been validated for use with the ELISA kit. These are the only samples for which we can support the claims. References may exist for other sample types. See the "Citations" tab on the product-specific webpage for any published references citing the use of the kit with an alternate sample type. Unclaimed sample types should be validated by the customer.
  • Why are my wells green after adding the stop solution?
  • This happens when the substrate in the well does not completely mix with the stop solution. After addition of the stop solution, tap the plate gently or place on a shaker until the mixture in the wells turns yellow.
  • Why do I need to use a 4-PL curve fit for generating my standard curve?
  • R&D Systems develops and QCs most of our Quantikine® ELISA Kits using a 4-parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph.  The data may be linearized by plotting the log of the concentrations versus the log of the O.D., and the best fit line can be determined by linear regression. This procedure will produce an adequate but less precise fit of the data.
  • Why doesn't the assay range extend to the stated sensitivity?
  • Sensitivity is the lowest measurable value that is statistically not equal to zero. It is calculated based on the signal of the background and the inherent variability of the assay. It is commonly determined by taking the mean O.D. plus two standard deviations from 20 zero replicates. This value is converted into analyte concentration from the standard curve. The low standard is the lowest possible point at which R&D Systems feels confident that the value is in the linear portion of the standard curve and, therefore, quantifiable.
  • Why is there a brown precipitate in my wells after addition of the stop solution?
  • This is due to incomplete washing after the HRP-labeled detection antibody (or streptavidin-HRP) incubation. When HRP is present during the substrate and subsequent stop solution additions, an orange-brown or brown precipitate is observed. This may be remedied by the addition of a 30 second soak on each wash step followed by complete removal of all liquid in the wells.
  • Why is there a sample dilution necessary in this ELISA Kit?
  • There are primarily two reasons for dilutions. In some assays most samples read above the standard curve, thus requiring a dilution for analyte levels to fall within the range of the assay. A second reason for dilution is to limit interference due to factors in complex matrices.
  • Why must I use polypropylene tubes for standard dilutions in certain assays?
  • Certain proteins or analytes will bind to glass and polystyrene, but do not readily bind to the polypropylene tubes.
  • Will a Logit-Log curve fit work for ELISA data analysis?
  • R&D Systems develops and QCs most of our ELISA Kits using a 4-parameter logistic (4-PL) curve-fit, which is an extension of the Logit-Log curve fit equation.
  • Will R&D Systems' Quantikine ELISAs produce sample values similar to those produced in other vendors' ELISAs?
  • R&D Systems Quantikine ELISA standards are calibrated against an internal "Master Calibrator" to ensure consistency of the standard curve and sample values from lot-to-lot over the lifetime of a product. In addition to determining the standard's protein concentration based on A280 or Coomassie, each new lot of standard is compared against the Master Calibrator to ensure consistent immunoreactivity with the ELISA's antibodies. Tri-level controls (measuring the low, mid, and high range of the standard curve) and natural samples are run in each new lot (and compared to a previous lot) to verify consistent results are obtained.The challenge when comparing immunoassay results from different vendors is that there is no single Master Calibrator for each analyte that is used by all vendors. Calibration, coupled with other variables (such as antibodies, detection methods, buffers, and cross-reactivity/interference) can lead to differences in sample values from different vendors’ assays. If available, the Quantikine ELISA may have been tested against NIBSC/WHO standard material, and the  can be used as a general conversion factor against other immunoassays that have been tested against the same standard.
  • Won't addition of assay diluent cause a further dilution of a sample?
  • Since the assay diluent is added to all wells, standards and specimens are treated equally. Therefore, sample concentration can be read from the standard curve without adjusting for this dilution.
  • Your shaker protocol tells me to use 500 rpm. This is too fast for my shaker. Is this speed correct?
  • This is 500 rpm with a 0.12 orbit. If the plate shaker has a larger orbit, then 500 rpm will be too fast. In this case calibrate the speed to approximate R&D Systems recommendation. To do this, take a spare 96-well microplate and use wash buffer to load the amount of liquid that will be in the well for incubation on the shaker. Cover with a plate sealer and adjust your shaker speed so there is a fairly vigorous swirling almost to the top of the well. You do NOT want any splashing on the plate sealer or foaming of liquid in the sample. R&D Systems recommends the ThermoFisher Model # 4625 microtiter plater shaker.
  • Can fetal calf serum (FCS) be used instead of heat-inactivated goat serum for diluting the detection antibody?
  • The use of heat-inactivated goat serum is important to keep background low and improve the signal-to-noise ratio of the assay. The use of FCS is not recommended for this purpose.

Cell-Based Fluorometric ELISAs

  • How does R&D Systems recommend normalizing the data obtained using the Cell-Based ELISAs?
  • R&D Systems® Cell-Based ELISAs permit the simultaneous detection of two proteins in the same microplate well without the need for lysate preparation. Kits come in two formats: phospho-protein kits contain antibodies to measure both the phosphorylated and the total protein, while total protein kits contain antibodies to the protein of interest and a second housekeeping protein. Both formats allow for the normalization of the target protein in different wells. To normalize the data obtained in the Cell-Based ELISA, R&D Systems recommends the following procedure:At the end of the assay, the ELISA plate is read at 450 nm and at 600 nm.Take the average of the control wells and subtract from all wells read at each wavelength (subtract the average control reading at 450 nm from all readings at 450 nm and the average control reading at 600 nm from all readings at 600 nm). Use these numbers to continue the normalization.Normalize the 450 nm reading by setting one of the readings to 1 and normalizing the rest accordingly. For example, if the 450 nm readings are 58386 and 55357, 61201 and 53658 for 2 sets of duplicates, choose 55357 as 1 and then 58386/55357=1.055 so the 58386 reading becomes 1.055 normalized. Also, 61201/55357=1.106 and 53658/55357=0.969.Use these normalized 450 nm numbers to normalize the 600 nm numbers. For example, if corresponding 600 nm values were 709 and 612, 6426 and 6045 for the 2 sets of duplicates, then take 709/1.055= 672, 612/1=612; 6426/1.106= 5810 and 6045-0.969=6236. Now take the average of the 650 nm readings for each duplicate: (672+612)/2=642+-30; 5810+6236)/2=6024+-214. Use these values for further data analysis.Note: Separate treatments do not need separate normalizations for total protein.

DuoSet Ancillary Reagent Kits

DuoSet ELISA Development Systems

  • After they are coated with capture antibody, can DuoSet® plates be stored for later use?
  • When DuoSets are tested in-house, plates are coated overnight at 4°C and then assayed the next day. DuoSet performance has been optimized with freshly coated plates. If coated plates absolutely must be stored, this general protocol can be followed:Coat plates overnight (8-18 hours) with the capture antibody as per the protocol for the DuoSet kit.Wash plates.Block plates with 1% BSA, 5%sucrose, 0.05% Sodium Azide in PBS for 1 hour.Aspirate blocking buffer, dryplates under vacuum, and seal in a plastic bag with desiccant.  Store at 2-8°C.  The use of plates coated and stored in this way is not part of routine DuoSet quality control testing, so each lab will need to empirically determine if and how long stored plates will remain stable. 
  • Can I use serum/plasma samples in the DuoSet® ELISA Development System?
  • The DuoSet ELISA Development System is designed for customers who have sufficient expertise to develop a useful ELISA but do not require the performance characteristics of a Quantikine ELISA kit. The development of DuoSet ELISA kits include testing for appropriate antibody pairing, reagent stability, parallelism using naturally-derived protein (if available), cross-reactivity/interference of related proteins, and lot-to-lot variability. The diluent and assay protocol suggested by the DuoSet ELISA datasheet will be suitable for most cell culture supernatant samples. Sample types such as serum, plasma, and tissue homogenates need to be validated by the researcher prior to use in the DuoSet ELISA Development System. Buffer components, sample matrix, complement, heterophilic antibodies, and rheumatoid factor can impact the accuracy of ELISA results. It is important that the diluents selected for reconstitution and dilution of the standard reflect the environment of the samples being measured. The diluent suggested in the datasheet may be suitable for most cell culture supernatant samples. Spike/recovery and linearity experiments should be performed to determine whether sample values reported from unvalidated sample types are accurate. To obtain a copy of a suggested spike/recovery and linearity protocol, please contact our Technical Service department. It is beyond the scope of R&D Systems' intention to provide customers with advice in building reliable plasma and serum assays from DuoSet ELISA kits. It is also not our intention to provide advice on how to QC and develop assays for sample types other than conditioned media. Customers wanting to measure protein levels in complex mixtures, like serum or plasma, should consult a reference guide, such as The Immunoassay Handbook, edited by David Wild, Nature Publishing Group, copyright 2001, or consider our Quantikine® ELISA kits.
  • Can recombinant proteins sold as stand-alone reagents be used as standards in the DuoSet® ELISA Development Kits?
  • These products are not calibrated for use in an ELISA system, and must be calibrated versus an existing mass calibrated standard. Standards provided in ELISA kits have been calibrated to a master calibrator.
  • Can the standard supplied in the DuoSet® ELISA Development System be used in cell culture?
  • The standard supplied in a DuoSet ELISA are specially formulated and may not be suitable for use in cell culture. Additionally, specific activity has not been determined for the standards.
  • Can Tocris BSA (Cat # 5217) be used with your DuoSet ELISA kits?
  • Our DuoSet ELISA kits that use Bovine Serum Albumin (BSA) are validated using Reagent Diluent Concentrate 2, R&D Systems Catlaog # DY995. The use of high quality BSA for the Reagent Diluent and Block Buffer is crucial for the optimal performance of our Duoset ELISA assays. Whenever possible, we recommend that customers use Catlaog # DY995. Tocris BSA (Catalog # 5217) is high quality and protease free and may be acceptable, but we have not tested this BSA in the Duoset ELISA kits. 
  • Do any of the provided components in the Duoset kit contain Azide?
  • No azide is added to any of the components supplied in the DuoSet. Neither the capture nor the detection antibody contain any preservatives.
  • For DuoSet assays that require heat-inactivated goat serum, what manufacturer/catalog number is recommended?
  • R&D Systems offers Heat-Inactivated Normal Goat Serum as catalog # DY005, Reagent Additive 1.
  • I am getting very low or no color development in my DuoSet® ELISA Development System. What is wrong?
  • Many things can contribute to low or no color development including the following common reasons.BSA Grade: It is important to choose a high quality grade BSA that is fatty acid free and/or 98% pure. This reduces the amount of globulins, binding proteins, or other proteins that can interfere in the assay. R&D Systems recommends using the same BSA used in development of the DuoSet ELISA (Catalog # DY995;  Catalog # DY997), or Millipore Probumin® Diagnostic Grade BSA (Millipore; Catalog # 82-045).Refer to package insert for selection of the appropriate Diluent Concentrate or contact Technical Service.Reagent Temperature:  Assay reagents that are not at room temperature before use, or even an excessively cool room, can cause suppressed signal.Choice of Microplate: ELISA Grade Microplates Plates designated as "high-binding ELISA" plates are required. Use of plates other than ELISA grade (such as tissue culture grade) are not recommended because they may not bind a sufficient amount of the capture antibody. We offer the same 96-well clear plates used in the Quantikine® ELISA Kits (Catalog # DY990) and black plates found in QuantiGlo® ELISA Kits (Catalog # DY991).Treatment of Reagents: Inadequate time allowed for complete solublization (5-10 minutes with gentle agitation is suggested) or inappropriate storage of reagents can affect color development.
  • If 540 nm or 570 nm wavelength correction is not available, what other wavelengths could work?
  • It has been suggested in literature that wavelength correction between 540-690 nm is suitable for TMB substrates.
  • Is the same antibody pair used for the Quantikine and DuoSet kits for the same analyte?  
  • Sometimes the antibody pairs are the same and sometimes they are different. Quantikine and DuoSet ELISA kits are designed separately and for different sample types, so optimization and validation is not the same, and our lab will choose the antibodies they empirically determine perform best.It is important to consider that the antibody pairing is only one factor that affects the specificity of an ELISA. The conjugation of the detection antibody, the formulation of the diluents, the concentrations of the antibodies, and general assay conditions will also affect specificity of the pairing. We recommend referring to the "Specificity" section near the end of each kit booklet for details on our empirical results of specificity testing of the ELISA assay.
  • The ELISA protocol does not list a plate shaker, is one needed?
  • If a shaker is required, it will be listed under the materials required section of the datasheet and information on the speed and orbit will be listed within the protocol. If the protocol does not call for a plate shaker, one is not needed and all incubations should follow the datasheet instructions.  Using a shaker where not required changes the immuno-kinetics of the assay and frequently impact the assay performance in a negative manner.
  • What antibody concentrations should be used with a DuoSet ELISA?
  • The recommended antibody concentrations are indicated in the lot specific Certificate of Analysis shipped with the product.  A lot specific Certificate of Analysis can also be downloaded on the DuoSet ELISA product specific page.
  • What grade BSA is recommend for use in ELISA and ELISpot Development Systems?
  • The grade of the BSA used has been found to be a critical component for running a successful ELISA. BSA with a minimum purity of 98% is recommended. R&D Systems recommends using the same BSAs used in development of the DuoSet; Catalog # DY995 Reagent Diluent Concentrate 2, Catalog # DY997 Reagent Diluent Concentrate 1. Millipore Probumin® Diagnostic Grade BSA (Millipore, Catalog # 820451) is another example.
  • What is a DuoSet® ELISA Development System?
  • The DuoSet ELISA Development System has enough Capture Antibody, Detection Antibody, Protein Standard, and Streptavidin-HRP to create about fifteen 96-well plates. A sample protocol and reagents recommended for use with this system is supplied. Our DuoSet ELISA Development Systems are designed primarily for the high-throughput screening of cell culture supernatants, although researchers experienced in ELISA development have been able to adapt them for samples with complex matrices such as serum and plasma.
  • What is the difference between a DuoSet and a DuoSet Economy pack?
  • R&D Systems will occasionally offer two size options for our DuoSet product line. The smallest size DuoSet kit (catalog# DYxxxx) contains sufficient reagents for fifteen 96-well plates. The next size is the DuoSet Economy pack (catalog# DYxxxxE), which contains sufficient reagents for forty-five 96-well plates. R&D Systems also offers an economy pack size for our DuoSet IC kits. The economy size for this product line has sufficient reagents for fifteen 96-well plates. The DuoSet IC kits are generally offered in a two plate, five plate, or fifteen plate sizes. These can be distinguished by the following catalog codes: DYCxxxx-2, DYCxxxx-5, or DYCxxxxE respectively.
  • What is the differences between Quantikine Kits and Duoset Kits?  
  • Quantikine kits are fully optimized and validated for the sample types listed in each specific protocol booklet. Each kit is supplied ready to use with one pre-coated 96-well plate, a detection antibody directly conjugated to HRP and all other necessary reagents. These kits are ideal for researchers who want the convenience of a ready to use and optimized ELISA product.Duoset Kits, in contrast, are ELISA development kits. These contain only vials of the capture and detection antibody, the mass-value calibrated standard, and streptavidin-HRP. These kits are usually validated for cell culture supernatant samples only and require further development and validation for accurate measurement in more complex samples such as serum and plasma.The DuoSet Kits offer an economical, flexible alternative for the experienced ELISA user.
  • Which ELISA plate is recommended for DuoSet® ELISA Development Kits?
  • R&D Systems offers clear (Catalog # DY990) and black (Catalog # DY991) microtiter plates, and plate sealers (Catalog # DY992). For added convenience, EvenCoat™ goat anti-mouse IgG microplates are available (Catalog # CP001; Catalog # CP002). EvenCoat microplates are clear, pre-blocked, 96-well polystyrene microplates coated with goat-derived antibody specific for the Fc region of mouse IgG. These plates may be used as a solid support for most sandwich ELISAs utilizing a mouse IgG capture antibody and a non-mouse IgG detection antibody. Other applications include competitive ELISA, IgG isotyping, and hybridoma screening/selection. EvenCoat Microplates may also be used with most DuoSet® ELISA Development Kits. Check your DuoSet ELISA datasheet to see if it can be used with EvenCoat Microplates. If there is no indication that it has been tested, please contact our Technical Service Department for more information.  
  • Why does the capture antibody vial in the DuoSet Kit appear to be empty?
  • Although there is always a slight possibility that a vial was not properly dispensed, this is a very rare occurrence and it would be extremely unusual to have received an empty capture antibody vial. There are multiple reasons why you may not observe a lyophilized pellet in your product. The two most common reasons for this observation are:The lyophilized product has become dislodged from the bottom of the vial during shipping/handling and is dispersed on the vial wall or cap. Tapping the vial firmly on the bench or a quick spin in a centrifuge may bring the lyophilized product back to the bottom of the vial.The capture antibody is formulated with a excipient to help protect the antibody during the lyophilization process and improve its long-term stability.  Over time, this excipient can absorb moisture and the product appearance may change from a typical lyophilized appearance to a transparent, film like appearance. Our extensive testing over many years has shown that this phase shift does not affect the performance or stability of the product as long as care is taken to ensure the complete solubilization of the antibody prior to transferring to another vessel.
  • Will a Logit-Log curve fit work for ELISA data analysis?
  • R&D Systems develops and QCs most of our ELISA Kits using a 4-parameter logistic (4-PL) curve-fit, which is an extension of the Logit-Log curve fit equation.
  • Will Color Development Reagent B (TMB) work if it has a solid or precipitate present?
  • This reagent is very close to the saturation point and a slight precipitate may be evident from time to time. Warm the reagent as instructed in the product insert.  If a precipitate remains, feel free to proceed. Vials of TMB containing precipitate have been tested and found to perform as expected. Avoid aspiration of the solid during pipetting.
  • Can fetal calf serum (FCS) be used instead of heat-inactivated goat serum for diluting the detection antibody?
  • The use of heat-inactivated goat serum is important to keep background low and improve the signal-to-noise ratio of the assay. The use of FCS is not recommended for this purpose.
  • Is there a formulation of Reagent Diluent and Blocking Buffer that will work for all DuoSet® ELISA Development kits?
  • Each analyte and corresponding antibody pair has unique physical and chemical properties that may require different formulation of reagent diluents for optimal assay performance. R&D Systems offers multiple formulation of Reagents Diluents for the DuoSet ELISA such as Catalog # DY995, DY997, and DY004. Check the product datsheet for the recommended Reagent Diluent for a specific analyte. Note that use of alternative formulations, brands, or reagents could cause lower signal and unusual results.

DuoSet ELISA Development Systems - 5/15 Plate Configuration

  • After they are coated with capture antibody, can DuoSet® plates be stored for later use?
  • When DuoSets are tested in-house, plates are coated overnight at 4°C and then assayed the next day. DuoSet performance has been optimized with freshly coated plates. If coated plates absolutely must be stored, this general protocol can be followed:Coat plates overnight (8-18 hours) with the capture antibody as per the protocol for the DuoSet kit.Wash plates.Block plates with 1% BSA, 5%sucrose, 0.05% Sodium Azide in PBS for 1 hour.Aspirate blocking buffer, dryplates under vacuum, and seal in a plastic bag with desiccant.  Store at 2-8°C.  The use of plates coated and stored in this way is not part of routine DuoSet quality control testing, so each lab will need to empirically determine if and how long stored plates will remain stable. 
  • Do any of the provided components in the Duoset kit contain Azide?
  • No azide is added to any of the components supplied in the DuoSet. Neither the capture nor the detection antibody contain any preservatives.

DuoSet IC (Intracellular) ELISA Development Systems

  • After they are coated with capture antibody, can DuoSet® plates be stored for later use?
  • When DuoSets are tested in-house, plates are coated overnight at 4°C and then assayed the next day. DuoSet performance has been optimized with freshly coated plates. If coated plates absolutely must be stored, this general protocol can be followed:Coat plates overnight (8-18 hours) with the capture antibody as per the protocol for the DuoSet kit.Wash plates.Block plates with 1% BSA, 5%sucrose, 0.05% Sodium Azide in PBS for 1 hour.Aspirate blocking buffer, dryplates under vacuum, and seal in a plastic bag with desiccant.  Store at 2-8°C.  The use of plates coated and stored in this way is not part of routine DuoSet quality control testing, so each lab will need to empirically determine if and how long stored plates will remain stable. 
  • Can Tocris BSA (Cat # 5217) be used with your DuoSet ELISA kits?
  • Our DuoSet ELISA kits that use Bovine Serum Albumin (BSA) are validated using Reagent Diluent Concentrate 2, R&D Systems Catlaog # DY995. The use of high quality BSA for the Reagent Diluent and Block Buffer is crucial for the optimal performance of our Duoset ELISA assays. Whenever possible, we recommend that customers use Catlaog # DY995. Tocris BSA (Catalog # 5217) is high quality and protease free and may be acceptable, but we have not tested this BSA in the Duoset ELISA kits. 
  • I am getting very low or no color development in my DuoSet® ELISA Development System. What is wrong?
  • Many things can contribute to low or no color development including the following common reasons.BSA Grade: It is important to choose a high quality grade BSA that is fatty acid free and/or 98% pure. This reduces the amount of globulins, binding proteins, or other proteins that can interfere in the assay. R&D Systems recommends using the same BSA used in development of the DuoSet ELISA (Catalog # DY995;  Catalog # DY997), or Millipore Probumin® Diagnostic Grade BSA (Millipore; Catalog # 82-045).Refer to package insert for selection of the appropriate Diluent Concentrate or contact Technical Service.Reagent Temperature:  Assay reagents that are not at room temperature before use, or even an excessively cool room, can cause suppressed signal.Choice of Microplate: ELISA Grade Microplates Plates designated as "high-binding ELISA" plates are required. Use of plates other than ELISA grade (such as tissue culture grade) are not recommended because they may not bind a sufficient amount of the capture antibody. We offer the same 96-well clear plates used in the Quantikine® ELISA Kits (Catalog # DY990) and black plates found in QuantiGlo® ELISA Kits (Catalog # DY991).Treatment of Reagents: Inadequate time allowed for complete solublization (5-10 minutes with gentle agitation is suggested) or inappropriate storage of reagents can affect color development.
  • If 540 nm or 570 nm wavelength correction is not available, what other wavelengths could work?
  • It has been suggested in literature that wavelength correction between 540-690 nm is suitable for TMB substrates.
  • The ELISA protocol does not list a plate shaker, is one needed?
  • If a shaker is required, it will be listed under the materials required section of the datasheet and information on the speed and orbit will be listed within the protocol. If the protocol does not call for a plate shaker, one is not needed and all incubations should follow the datasheet instructions.  Using a shaker where not required changes the immuno-kinetics of the assay and frequently impact the assay performance in a negative manner.
  • What grade BSA is recommend for use in ELISA and ELISpot Development Systems?
  • The grade of the BSA used has been found to be a critical component for running a successful ELISA. BSA with a minimum purity of 98% is recommended. R&D Systems recommends using the same BSAs used in development of the DuoSet; Catalog # DY995 Reagent Diluent Concentrate 2, Catalog # DY997 Reagent Diluent Concentrate 1. Millipore Probumin® Diagnostic Grade BSA (Millipore, Catalog # 820451) is another example.
  • What is a DuoSet® IC ELISA Development System?
  • The DuoSet IC (Intracellular) ELISA Development System contains the basic components required for the development of a sandwich ELISA (or activity assay) to measure intracellular proteins in cell lysates. Each kit has sufficient capture antibody, detection antibody, standard, and Streptavidin-HRP to create two, five, or fifteen 96-well plates. R&D Systems supplies a sample protocol and reagents recommended for use with this system.
  • What is the difference between a DuoSet and a DuoSet Economy pack?
  • R&D Systems will occasionally offer two size options for our DuoSet product line. The smallest size DuoSet kit (catalog# DYxxxx) contains sufficient reagents for fifteen 96-well plates. The next size is the DuoSet Economy pack (catalog# DYxxxxE), which contains sufficient reagents for forty-five 96-well plates. R&D Systems also offers an economy pack size for our DuoSet IC kits. The economy size for this product line has sufficient reagents for fifteen 96-well plates. The DuoSet IC kits are generally offered in a two plate, five plate, or fifteen plate sizes. These can be distinguished by the following catalog codes: DYCxxxx-2, DYCxxxx-5, or DYCxxxxE respectively.
  • Why does the capture antibody vial in the DuoSet Kit appear to be empty?
  • Although there is always a slight possibility that a vial was not properly dispensed, this is a very rare occurrence and it would be extremely unusual to have received an empty capture antibody vial. There are multiple reasons why you may not observe a lyophilized pellet in your product. The two most common reasons for this observation are:The lyophilized product has become dislodged from the bottom of the vial during shipping/handling and is dispersed on the vial wall or cap. Tapping the vial firmly on the bench or a quick spin in a centrifuge may bring the lyophilized product back to the bottom of the vial.The capture antibody is formulated with a excipient to help protect the antibody during the lyophilization process and improve its long-term stability.  Over time, this excipient can absorb moisture and the product appearance may change from a typical lyophilized appearance to a transparent, film like appearance. Our extensive testing over many years has shown that this phase shift does not affect the performance or stability of the product as long as care is taken to ensure the complete solubilization of the antibody prior to transferring to another vessel.
  • Will Color Development Reagent B (TMB) work if it has a solid or precipitate present?
  • This reagent is very close to the saturation point and a slight precipitate may be evident from time to time. Warm the reagent as instructed in the product insert.  If a precipitate remains, feel free to proceed. Vials of TMB containing precipitate have been tested and found to perform as expected. Avoid aspiration of the solid during pipetting.

DuoSet IC (Intracellular) Phospho-site Specific ELISA Development Systems

  • Can the Phospho control in my DuoSet® IC (Intracellular) Phospho-specific ELISA kit be diluted to make a standard curve?
  • The Phospho control has not been validated for use as standard curve material and further dilutions may not yield linear signal development. The concentration recommended in the kit insert has been verified to function well as a positive control in order to compare treated to non-treated/control samples.
  • I am getting very low or no color development in my DuoSet® ELISA Development System. What is wrong?
  • Many things can contribute to low or no color development including the following common reasons.BSA Grade: It is important to choose a high quality grade BSA that is fatty acid free and/or 98% pure. This reduces the amount of globulins, binding proteins, or other proteins that can interfere in the assay. R&D Systems recommends using the same BSA used in development of the DuoSet ELISA (Catalog # DY995;  Catalog # DY997), or Millipore Probumin® Diagnostic Grade BSA (Millipore; Catalog # 82-045).Refer to package insert for selection of the appropriate Diluent Concentrate or contact Technical Service.Reagent Temperature:  Assay reagents that are not at room temperature before use, or even an excessively cool room, can cause suppressed signal.Choice of Microplate: ELISA Grade Microplates Plates designated as "high-binding ELISA" plates are required. Use of plates other than ELISA grade (such as tissue culture grade) are not recommended because they may not bind a sufficient amount of the capture antibody. We offer the same 96-well clear plates used in the Quantikine® ELISA Kits (Catalog # DY990) and black plates found in QuantiGlo® ELISA Kits (Catalog # DY991).Treatment of Reagents: Inadequate time allowed for complete solublization (5-10 minutes with gentle agitation is suggested) or inappropriate storage of reagents can affect color development.
  • What grade BSA is recommend for use in ELISA and ELISpot Development Systems?
  • The grade of the BSA used has been found to be a critical component for running a successful ELISA. BSA with a minimum purity of 98% is recommended. R&D Systems recommends using the same BSAs used in development of the DuoSet; Catalog # DY995 Reagent Diluent Concentrate 2, Catalog # DY997 Reagent Diluent Concentrate 1. Millipore Probumin® Diagnostic Grade BSA (Millipore, Catalog # 820451) is another example.
  • What is a DuoSet® IC ELISA Development System?
  • The DuoSet IC (Intracellular) ELISA Development System contains the basic components required for the development of a sandwich ELISA (or activity assay) to measure intracellular proteins in cell lysates. Each kit has sufficient capture antibody, detection antibody, standard, and Streptavidin-HRP to create two, five, or fifteen 96-well plates. R&D Systems supplies a sample protocol and reagents recommended for use with this system.
  • What is the difference between a DuoSet and a DuoSet Economy pack?
  • R&D Systems will occasionally offer two size options for our DuoSet product line. The smallest size DuoSet kit (catalog# DYxxxx) contains sufficient reagents for fifteen 96-well plates. The next size is the DuoSet Economy pack (catalog# DYxxxxE), which contains sufficient reagents for forty-five 96-well plates. R&D Systems also offers an economy pack size for our DuoSet IC kits. The economy size for this product line has sufficient reagents for fifteen 96-well plates. The DuoSet IC kits are generally offered in a two plate, five plate, or fifteen plate sizes. These can be distinguished by the following catalog codes: DYCxxxx-2, DYCxxxx-5, or DYCxxxxE respectively.
  • Why does the capture antibody vial in the DuoSet Kit appear to be empty?
  • Although there is always a slight possibility that a vial was not properly dispensed, this is a very rare occurrence and it would be extremely unusual to have received an empty capture antibody vial. There are multiple reasons why you may not observe a lyophilized pellet in your product. The two most common reasons for this observation are:The lyophilized product has become dislodged from the bottom of the vial during shipping/handling and is dispersed on the vial wall or cap. Tapping the vial firmly on the bench or a quick spin in a centrifuge may bring the lyophilized product back to the bottom of the vial.The capture antibody is formulated with a excipient to help protect the antibody during the lyophilization process and improve its long-term stability.  Over time, this excipient can absorb moisture and the product appearance may change from a typical lyophilized appearance to a transparent, film like appearance. Our extensive testing over many years has shown that this phase shift does not affect the performance or stability of the product as long as care is taken to ensure the complete solubilization of the antibody prior to transferring to another vessel.

Parameter Colorimetric ELISA / Assay Kits

QuantiGlo Chemiluminescent Sandwich ELISAs

  • Are control sets available for Human Quantikine® ELISA kits?
  • R&D Systems offers tri-level control sets for the Human Quantikine ELISA Kits (colorimetric), Quantikine HS ELISA kits (high sensitivity), and QuantiGlo® ELISA kits (chemiluminescent). Catalog numbers and pricing for control sets can be found in the "Supplemental Products" tab on the product webpage.
  • Are Quantikine® ELISA Kit Wash Buffers interchangeable?
  • Wash buffer can be interchanged between like kits provided they have the same part number.Note: The Wash Buffer from a Quantikine ELISA Kit CANNOT be used in a Quantikine HS (High Sensitivity) ELISA Kit. The phosphate contained in the Quantikine ELISA Kit Wash buffer will interfere with the NADPH driven amplification of signal in the Quantikine HS ELISA kits. Additional bottles of Wash Buffer are available.
  • Can a partial Quantikine® ELISA plate be used?
  • The Quantikine® ELISA plates have removable strips of wells. Unused wells may be removed from the plate, returned to the foil pouch containing the desiccant pack, and stored at 2-8° C for up to one month.
  • Can I stop a Quantikine® ELISA kit or Parameter™ assay at any point, extend an incubation time, or change the suggested incubation temperature? 
  • R&D Systems has optimized the assays for both incubation times and temperatures. Each kit has only been validated for the protocol described in the kit datasheet. We cannot guarantee the performance of our kits when the protocol has been altered in any way. 
  • Can the reagents from Quantikine®, QuantiGlo®, Parameter™, or Surveyor™ IC ELISA Kits be interchanged?
  • Assay Diluent(s), Calibrator Diluent(s), and Substrate may be interchanged if they have the same part number AND lot number. R&D Systems does "whole kit QC" which means that we cannot support the use of reagents from other lots or sources being substituted into an assay. Plates and Conjugate cannot be interchanged under any circumstance.
  • How are ELISA kit results converted from mass to units?
  • For those analytes of which there is a WHO standard, an equation for converting to units is provided in the kit insert. Please note that these units are determined in bioassay, and are not mass calibrated.
  • How many samples can be assayed in a Human Quantikine® ELISA Kit?
  • Most Quantikine ELISA Kits will run the standard curve and 40 samples in duplicate. Please refer to the datasheet for details on each kit.
  • I don't have enough of the Calibrator Diluent in my Quantkine Kit for dilutions of my cell culture supernates. What should I do?
  • The kits are designed with enough calibrator diluent to ensure that the vast majority of samples fall within the indicated range of the assay. Should you find that there is not enough diluent provided in the kit to dilute your samples, you have at least two options. Option 1) Samples can be diluted in two steps. The initial dilution in culture medium and a final dilution, of at least 1:10, into the Calibrator Diluent provided in the kit. Option 2) For a nominal charge, you can purchase additional diluent provided the same lot included in the kit is still available. Contact Technical Service for more information.
  • Is it necessary to add the full volume of assay diluent stated in the protocol, or can additional sample volume be used instead?
  • The assay diluent is specially formulated and required for optimal performance of the ELISA, and the full volume of assay diluent stated in the kit booklet should be used.
  • Quantikine® ELISA kits often contain multiple diluents. Which diluent is used to dilute samples? What is the correct use of the assay diluent?
  • In general, the calibrator diluent should be used to make standard curve dilutions and any needed sample dilutions. If there are multiple calibrator diluents in a kit, the kit booklet will describe which one is correct for each sample type. Diluting both the standards and the samples with the same diluent allows them to be in a similar matrix while incubating in the plate.The assay diluent is only used at the beginning of the protocol where a specific amount is added to the plate prior to adding samples, standards, or controls. There is excess assay diluent provided for this step, and there is no other use for the assay diluent. The assay diluent allows for the introduction of components into the sample incubation and will prevent other substances present in the samples from interfering with the assay.
  • The Assay Diluent, RD1, supplied in my Quantikine® ELISA Kit looks like it contains a precipitate. Is this OK?
  • Due to saturating amounts of some buffer components, some of the RD1 Assay Diluents contain a light to heavy precipitate. In these instances, it is normal and it will be noted in the specific protocol booklet. If it is not noted in the protocol booklet, please contact Technical Service.
  • The RLU values observed in QuantiGlo ELISA and those published in the Typical Data section of the protocol booklet provided with the kit are significantly different. Why? 
  • RLU stands for Relative Light Unit. Luminometers typically do not yield a measurement directly in units of photons, thus the use of the term Relative. The RLU's observed between different luminometers (of different or the same manufacturer) can vary significantly and should not be used as an indicator of a successful assay. The example data in the kit booklets was collected using the Dynex MLX luminometer. As long as the standard curve still has an R2 value near 1, the assay should still be valid.
  • What are the shipping conditions for the optional ELISA kit controls?
  • The shipping method for the control depends on the target analyte.  Some Quantikine® ELISA controls ship at ambient temperature, some ship on cold packs, and some ship on dry ice. 
  • What is included in a Quantikine® ELISA Kit?
  • Quantikine ELISA Kits are a complete kit consisting of a precoated microplate, Conjugated Detection Antibody, Standard, Diluents, Substrate, Stop Solution, Wash Buffer, and plate sealers. They are fully validated ELISAs for the sample types listed in the specific datasheet. They have been exhaustively tested for superior quality.
  • What is the difference between the Quantikine® and QuantiGlo® ELISA Kits?
  • Quantikine ELISA kits are a colorimetric assay that require a standard microplate reader with the appropriate wavelength filters for reading and wavelength correction. The QuantiGlo ELISA Kits have a Luminol-based substrate that is read by the intensity of light emitted or relative light units (RLU). Therefore, the QuantiGlo kits must be read on a luminometer. In general, the QuantiGlo ELISA has a wider dynamic range than the standard Quantikine ELISA. Please see the datasheet for specifications.
  • Why is my wash buffer yellow?
  • R&D Systems wash buffers contain ProClin® 300. ProClin 300 is a preservative used in diagnostic and research reagents that is clear to pale yellow in color. The yellow color may become more pronounced over time and occurs more rapidly at room temperature than at 2-8 °C. This color change has no effect on efficacy of the preservative or the performance of the assay. 
  • Will a Logit-Log curve fit work for ELISA data analysis?
  • R&D Systems develops and QCs most of our ELISA Kits using a 4-parameter logistic (4-PL) curve-fit, which is an extension of the Logit-Log curve fit equation.
  • Won't addition of assay diluent cause a further dilution of a sample?
  • Since the assay diluent is added to all wells, standards and specimens are treated equally. Therefore, sample concentration can be read from the standard curve without adjusting for this dilution.
  • Will your Quantikine ELISA kit detect normal sample values of my target?
  • You will be able to quantify samples down to the lowest point on the standard curve. In some cases, the standard curve does go down low enough to detect normal samples. In other cases, normal samples have levels of the target that are just too low to detect. You can check the Sample Values section in your kit booklet to find out what kind of sample values we obtained from apparently healthy individuals. You may also want to check the literature to find out if there is an established normal range for your target. We also have Quantikine HS kits for some of the cytokines that are known to be very low in normal samples. Even in the high sensitivity (HS) kits, it is important to check the standard curve range and the sample values section to determine if the kit is likely to detect the target in your samples

Quantikine Colorimetric Sandwich ELISAs

  • Will your Quantikine ELISA kit be able to detect my target analyte in normal samples?
  • Samples which read within the standard curve are able to be quantified.  In some cases, normal samples have levels of the target analyte that are below the detection limit of the kit. Check the "Sample Values" section in the kit booklet to find the sample values R&D Systems obtained from apparently healthy individuals. It is also recommended to review literature to detirmine if there is an established normal range for your target.  There are also Quantikine High Sensitivity kits available for some cytokines, that are known to be present in very low amounts in normal samples. However, it is still important to check the standard curve range and the sample values section to determine if the kit is likely to detect the target in your samples, when using the High Sensitivity Quantikine kits.
  • Are control sets available for Human Quantikine® ELISA kits?
  • R&D Systems offers tri-level control sets for the Human Quantikine ELISA Kits (colorimetric), Quantikine HS ELISA kits (high sensitivity), and QuantiGlo® ELISA kits (chemiluminescent). Catalog numbers and pricing for control sets can be found in the "Supplemental Products" tab on the product webpage.
  • Are Quantikine® ELISA Kit Wash Buffers interchangeable?
  • Wash buffer can be interchanged between like kits provided they have the same part number.Note: The Wash Buffer from a Quantikine ELISA Kit CANNOT be used in a Quantikine HS (High Sensitivity) ELISA Kit. The phosphate contained in the Quantikine ELISA Kit Wash buffer will interfere with the NADPH driven amplification of signal in the Quantikine HS ELISA kits. Additional bottles of Wash Buffer are available.
  • Are there any suggestions on how to collect and handle non-validated sample types such as CSF?
  • We are not able to provide guidance for clinical sample collection.  Generally, we recommend looking to literature for published methodology and preparing samples in a manner that minimizes freeze-thaw cycles.
  • Can a partial Quantikine® ELISA plate be used?
  • The Quantikine® ELISA plates have removable strips of wells. Unused wells may be removed from the plate, returned to the foil pouch containing the desiccant pack, and stored at 2-8° C for up to one month.
  • Can I stop a Quantikine® ELISA kit or Parameter™ assay at any point, extend an incubation time, or change the suggested incubation temperature? 
  • R&D Systems has optimized the assays for both incubation times and temperatures. Each kit has only been validated for the protocol described in the kit datasheet. We cannot guarantee the performance of our kits when the protocol has been altered in any way. 
  • Can the reagents from Quantikine®, QuantiGlo®, Parameter™, or Surveyor™ IC ELISA Kits be interchanged?
  • Assay Diluent(s), Calibrator Diluent(s), and Substrate may be interchanged if they have the same part number AND lot number. R&D Systems does "whole kit QC" which means that we cannot support the use of reagents from other lots or sources being substituted into an assay. Plates and Conjugate cannot be interchanged under any circumstance.
  • Does the Human VEGF Quantikine® ELISA kit (Catalog # DVE00) detect porcine samples?
  • Our lab has not tested porcine samples in the kit, however many customers have published use of the kit with porcine samples.  Links to these references can be found in the citations tab by selecting for Porcine in the Species filter.
  • Does the Human VEGF Quantikine® ELISA kit (Catalog # DVE00) detect human VEGF121?
  • Yes, the kit detects VEGF121.  In specificity testing, 100% cross-reactivity was observed with recombinant human VEGF121. 
  • Does the Human VEGF Quantikine® ELISA kit (Catalog # DVE00) detect mouse VEGF?
  • No, the kit does not detect mouse VEGF. Specificity testing with recombinant mouse VEGF 120 and VEGF 164 showed no cross-reactivity. For the detection of mouse VEGF, please refer to the Mouse VEGF Quantikine® ELISA kit (Catalog # MMV00).
  • How are ELISA kit results converted from mass to units?
  • For those analytes of which there is a WHO standard, an equation for converting to units is provided in the kit insert. Please note that these units are determined in bioassay, and are not mass calibrated.
  • How many samples can be assayed in a Human Quantikine® ELISA Kit?
  • Most Quantikine ELISA Kits will run the standard curve and 40 samples in duplicate. Please refer to the datasheet for details on each kit.
  • How many samples can be assayed in a non-human Quantikine® ELISA Kit?
  • Each plate in the non-human Quantikine ELISA Kit will run the standard curve, control, and 39 samples in duplicate per plate. Some of these ELISA kits come with two plates. Please see the datasheet for details.
  • I don't have enough of the Calibrator Diluent in my Quantkine Kit for dilutions of my cell culture supernates. What should I do?
  • The kits are designed with enough calibrator diluent to ensure that the vast majority of samples fall within the indicated range of the assay. Should you find that there is not enough diluent provided in the kit to dilute your samples, you have at least two options. Option 1) Samples can be diluted in two steps. The initial dilution in culture medium and a final dilution, of at least 1:10, into the Calibrator Diluent provided in the kit. Option 2) For a nominal charge, you can purchase additional diluent provided the same lot included in the kit is still available. Contact Technical Service for more information.
  • Is it necessary to add the full volume of assay diluent stated in the protocol, or can additional sample volume be used instead?
  • The assay diluent is specially formulated and required for optimal performance of the ELISA, and the full volume of assay diluent stated in the kit booklet should be used.
  • Is the same antibody pair used for the Quantikine and DuoSet kits for the same analyte?  
  • Sometimes the antibody pairs are the same and sometimes they are different. Quantikine and DuoSet ELISA kits are designed separately and for different sample types, so optimization and validation is not the same, and our lab will choose the antibodies they empirically determine perform best.It is important to consider that the antibody pairing is only one factor that affects the specificity of an ELISA. The conjugation of the detection antibody, the formulation of the diluents, the concentrations of the antibodies, and general assay conditions will also affect specificity of the pairing. We recommend referring to the "Specificity" section near the end of each kit booklet for details on our empirical results of specificity testing of the ELISA assay.
  • Quantikine® ELISA kits often contain multiple diluents. Which diluent is used to dilute samples? What is the correct use of the assay diluent?
  • In general, the calibrator diluent should be used to make standard curve dilutions and any needed sample dilutions. If there are multiple calibrator diluents in a kit, the kit booklet will describe which one is correct for each sample type. Diluting both the standards and the samples with the same diluent allows them to be in a similar matrix while incubating in the plate.The assay diluent is only used at the beginning of the protocol where a specific amount is added to the plate prior to adding samples, standards, or controls. There is excess assay diluent provided for this step, and there is no other use for the assay diluent. The assay diluent allows for the introduction of components into the sample incubation and will prevent other substances present in the samples from interfering with the assay.
  • The Assay Diluent, RD1, supplied in my Quantikine® ELISA Kit looks like it contains a precipitate. Is this OK?
  • Due to saturating amounts of some buffer components, some of the RD1 Assay Diluents contain a light to heavy precipitate. In these instances, it is normal and it will be noted in the specific protocol booklet. If it is not noted in the protocol booklet, please contact Technical Service.
  • What are the shipping conditions for the optional ELISA kit controls?
  • The shipping method for the control depends on the target analyte.  Some Quantikine® ELISA controls ship at ambient temperature, some ship on cold packs, and some ship on dry ice. 
  • What is included in a Quantikine® ELISA Kit?
  • Quantikine ELISA Kits are a complete kit consisting of a precoated microplate, Conjugated Detection Antibody, Standard, Diluents, Substrate, Stop Solution, Wash Buffer, and plate sealers. They are fully validated ELISAs for the sample types listed in the specific datasheet. They have been exhaustively tested for superior quality.
  • What is R&D Systems recommendations for lysis buffer to evaluate tissue homogenate or cell lysate samples in an ELISA kit?  
  • R&D Systems provides Lysis or Extraction Buffers in select ELISA kits, if tissue homogenate or cell lysates are validated sample types.  In other ELISA kits, the additional buffer may be listed in the "Other Supplies Required" section in the product insert or general sample preparation instructions will be provided in the product insert's "Sample Values" section.
  • What is the difference between the Quantikine® and QuantiGlo® ELISA Kits?
  • Quantikine ELISA kits are a colorimetric assay that require a standard microplate reader with the appropriate wavelength filters for reading and wavelength correction. The QuantiGlo ELISA Kits have a Luminol-based substrate that is read by the intensity of light emitted or relative light units (RLU). Therefore, the QuantiGlo kits must be read on a luminometer. In general, the QuantiGlo ELISA has a wider dynamic range than the standard Quantikine ELISA. Please see the datasheet for specifications.
  • What is the difference between the traditional Quantikine® ELISA Kits and the High Sensitivity Quantikine HS ELISA Kits?
  • The High Sensitivity (HS) versions of Quantikine ELISA Kits are complete kits generally used when very low levels of the cytokine are expected. Quantikine HS Kits incorporate an amplification system designed to enhance the signal accompanying low analyte concentrations. It is strongly recommended to do a literature search to determine which kit range would best suit your needs.
  • What is the differences between Quantikine Kits and Duoset Kits?  
  • Quantikine kits are fully optimized and validated for the sample types listed in each specific protocol booklet. Each kit is supplied ready to use with one pre-coated 96-well plate, a detection antibody directly conjugated to HRP and all other necessary reagents. These kits are ideal for researchers who want the convenience of a ready to use and optimized ELISA product.Duoset Kits, in contrast, are ELISA development kits. These contain only vials of the capture and detection antibody, the mass-value calibrated standard, and streptavidin-HRP. These kits are usually validated for cell culture supernatant samples only and require further development and validation for accurate measurement in more complex samples such as serum and plasma.The DuoSet Kits offer an economical, flexible alternative for the experienced ELISA user.
  • What is the stability of supplemental ELISA kit controls (Quantikine, Quantikine HS, or QuantiGlo ELISA kits)?
  • Controls are assigned an expiration date of 3 months from date of receipt. They are to be used once and discarded. If the lyophilized controls are stored properly, it is possible that they will remain stable for an extended period of time, although we have not conducted extended stability testing. The controls have not been tested for stability after reconstitution.
  • Why don't we offer a kit specific for HMW adiponectin?
  • R&D Systems scientific staff have reviewed the literature regarding the selective measurement of different forms of human Adiponectin by using proteases to pre-treat samples. It is our conclusion that a protease treatment is so condition-dependent that the development of a kit capable of obtaining reproducible results would be problematic. However, we are currently exploring the possibility of developing an alternative method that doesn't rely on the use of proteases for measuring the HMW form of Adiponectin.
  • Why is my wash buffer yellow?
  • R&D Systems wash buffers contain ProClin® 300. ProClin 300 is a preservative used in diagnostic and research reagents that is clear to pale yellow in color. The yellow color may become more pronounced over time and occurs more rapidly at room temperature than at 2-8 °C. This color change has no effect on efficacy of the preservative or the performance of the assay. 
  • Will a Logit-Log curve fit work for ELISA data analysis?
  • R&D Systems develops and QCs most of our ELISA Kits using a 4-parameter logistic (4-PL) curve-fit, which is an extension of the Logit-Log curve fit equation.
  • Will Color Development Reagent B (TMB) work if it has a solid or precipitate present?
  • This reagent is very close to the saturation point and a slight precipitate may be evident from time to time. Warm the reagent as instructed in the product insert.  If a precipitate remains, feel free to proceed. Vials of TMB containing precipitate have been tested and found to perform as expected. Avoid aspiration of the solid during pipetting.
  • Won't addition of assay diluent cause a further dilution of a sample?
  • Since the assay diluent is added to all wells, standards and specimens are treated equally. Therefore, sample concentration can be read from the standard curve without adjusting for this dilution.
  • Will your Quantikine ELISA kit detect normal sample values of my target?
  • You will be able to quantify samples down to the lowest point on the standard curve. In some cases, the standard curve does go down low enough to detect normal samples. In other cases, normal samples have levels of the target that are just too low to detect. You can check the Sample Values section in your kit booklet to find out what kind of sample values we obtained from apparently healthy individuals. You may also want to check the literature to find out if there is an established normal range for your target. We also have Quantikine HS kits for some of the cytokines that are known to be very low in normal samples. Even in the high sensitivity (HS) kits, it is important to check the standard curve range and the sample values section to determine if the kit is likely to detect the target in your samples

Quantikine HS High Sensitivity Colorimetric Sandwich ELISAs

  • Will your Quantikine ELISA kit be able to detect my target analyte in normal samples?
  • Samples which read within the standard curve are able to be quantified.  In some cases, normal samples have levels of the target analyte that are below the detection limit of the kit. Check the "Sample Values" section in the kit booklet to find the sample values R&D Systems obtained from apparently healthy individuals. It is also recommended to review literature to detirmine if there is an established normal range for your target.  There are also Quantikine High Sensitivity kits available for some cytokines, that are known to be present in very low amounts in normal samples. However, it is still important to check the standard curve range and the sample values section to determine if the kit is likely to detect the target in your samples, when using the High Sensitivity Quantikine kits.
  • Are control sets available for Human Quantikine® ELISA kits?
  • R&D Systems offers tri-level control sets for the Human Quantikine ELISA Kits (colorimetric), Quantikine HS ELISA kits (high sensitivity), and QuantiGlo® ELISA kits (chemiluminescent). Catalog numbers and pricing for control sets can be found in the "Supplemental Products" tab on the product webpage.
  • Are Quantikine® ELISA Kit Wash Buffers interchangeable?
  • Wash buffer can be interchanged between like kits provided they have the same part number.Note: The Wash Buffer from a Quantikine ELISA Kit CANNOT be used in a Quantikine HS (High Sensitivity) ELISA Kit. The phosphate contained in the Quantikine ELISA Kit Wash buffer will interfere with the NADPH driven amplification of signal in the Quantikine HS ELISA kits. Additional bottles of Wash Buffer are available.
  • Can a partial Quantikine® ELISA plate be used?
  • The Quantikine® ELISA plates have removable strips of wells. Unused wells may be removed from the plate, returned to the foil pouch containing the desiccant pack, and stored at 2-8° C for up to one month.
  • Can I stop a Quantikine® ELISA kit or Parameter™ assay at any point, extend an incubation time, or change the suggested incubation temperature? 
  • R&D Systems has optimized the assays for both incubation times and temperatures. Each kit has only been validated for the protocol described in the kit datasheet. We cannot guarantee the performance of our kits when the protocol has been altered in any way. 
  • Can the reagents from Quantikine®, QuantiGlo®, Parameter™, or Surveyor™ IC ELISA Kits be interchanged?
  • Assay Diluent(s), Calibrator Diluent(s), and Substrate may be interchanged if they have the same part number AND lot number. R&D Systems does "whole kit QC" which means that we cannot support the use of reagents from other lots or sources being substituted into an assay. Plates and Conjugate cannot be interchanged under any circumstance.
  • How are ELISA kit results converted from mass to units?
  • For those analytes of which there is a WHO standard, an equation for converting to units is provided in the kit insert. Please note that these units are determined in bioassay, and are not mass calibrated.
  • How many samples can be assayed in a Human Quantikine® ELISA Kit?
  • Most Quantikine ELISA Kits will run the standard curve and 40 samples in duplicate. Please refer to the datasheet for details on each kit.
  • I don't have enough of the Calibrator Diluent in my Quantkine Kit for dilutions of my cell culture supernates. What should I do?
  • The kits are designed with enough calibrator diluent to ensure that the vast majority of samples fall within the indicated range of the assay. Should you find that there is not enough diluent provided in the kit to dilute your samples, you have at least two options. Option 1) Samples can be diluted in two steps. The initial dilution in culture medium and a final dilution, of at least 1:10, into the Calibrator Diluent provided in the kit. Option 2) For a nominal charge, you can purchase additional diluent provided the same lot included in the kit is still available. Contact Technical Service for more information.
  • Is it necessary to add the full volume of assay diluent stated in the protocol, or can additional sample volume be used instead?
  • The assay diluent is specially formulated and required for optimal performance of the ELISA, and the full volume of assay diluent stated in the kit booklet should be used.
  • Is the same antibody pair used for the Quantikine and DuoSet kits for the same analyte?  
  • Sometimes the antibody pairs are the same and sometimes they are different. Quantikine and DuoSet ELISA kits are designed separately and for different sample types, so optimization and validation is not the same, and our lab will choose the antibodies they empirically determine perform best.It is important to consider that the antibody pairing is only one factor that affects the specificity of an ELISA. The conjugation of the detection antibody, the formulation of the diluents, the concentrations of the antibodies, and general assay conditions will also affect specificity of the pairing. We recommend referring to the "Specificity" section near the end of each kit booklet for details on our empirical results of specificity testing of the ELISA assay.
  • Quantikine® ELISA kits often contain multiple diluents. Which diluent is used to dilute samples? What is the correct use of the assay diluent?
  • In general, the calibrator diluent should be used to make standard curve dilutions and any needed sample dilutions. If there are multiple calibrator diluents in a kit, the kit booklet will describe which one is correct for each sample type. Diluting both the standards and the samples with the same diluent allows them to be in a similar matrix while incubating in the plate.The assay diluent is only used at the beginning of the protocol where a specific amount is added to the plate prior to adding samples, standards, or controls. There is excess assay diluent provided for this step, and there is no other use for the assay diluent. The assay diluent allows for the introduction of components into the sample incubation and will prevent other substances present in the samples from interfering with the assay.
  • The Assay Diluent, RD1, supplied in my Quantikine® ELISA Kit looks like it contains a precipitate. Is this OK?
  • Due to saturating amounts of some buffer components, some of the RD1 Assay Diluents contain a light to heavy precipitate. In these instances, it is normal and it will be noted in the specific protocol booklet. If it is not noted in the protocol booklet, please contact Technical Service.
  • What are the shipping conditions for the optional ELISA kit controls?
  • The shipping method for the control depends on the target analyte.  Some Quantikine® ELISA controls ship at ambient temperature, some ship on cold packs, and some ship on dry ice. 
  • What is included in a Quantikine® ELISA Kit?
  • Quantikine ELISA Kits are a complete kit consisting of a precoated microplate, Conjugated Detection Antibody, Standard, Diluents, Substrate, Stop Solution, Wash Buffer, and plate sealers. They are fully validated ELISAs for the sample types listed in the specific datasheet. They have been exhaustively tested for superior quality.
  • What is the difference between the traditional Quantikine® ELISA Kits and the High Sensitivity Quantikine HS ELISA Kits?
  • The High Sensitivity (HS) versions of Quantikine ELISA Kits are complete kits generally used when very low levels of the cytokine are expected. Quantikine HS Kits incorporate an amplification system designed to enhance the signal accompanying low analyte concentrations. It is strongly recommended to do a literature search to determine which kit range would best suit your needs.
  • What is the differences between Quantikine Kits and Duoset Kits?  
  • Quantikine kits are fully optimized and validated for the sample types listed in each specific protocol booklet. Each kit is supplied ready to use with one pre-coated 96-well plate, a detection antibody directly conjugated to HRP and all other necessary reagents. These kits are ideal for researchers who want the convenience of a ready to use and optimized ELISA product.Duoset Kits, in contrast, are ELISA development kits. These contain only vials of the capture and detection antibody, the mass-value calibrated standard, and streptavidin-HRP. These kits are usually validated for cell culture supernatant samples only and require further development and validation for accurate measurement in more complex samples such as serum and plasma.The DuoSet Kits offer an economical, flexible alternative for the experienced ELISA user.
  • What is the recommended dilution for cell culture supernate samples in the High Sensitivity Quantikine ELISA Kits?
  • The High Sensitivity Quantikine  ELISA kits are not validated for use on cell culture supernate samples because cell culture supernate sample levels are expected to be high and easily detectable by the corresponding standard Quantikine ELISA. The expected values may be so high in cell culture supernate samples that the dilution you would perform to get levels within the standard curve range would change the matrix of the assay, and the sample values would not be accurate. Additionally, it may be difficult to choose the correct dilution factor to get a high value sample to fall within the narrow standard curve range of a high sensitivity kit. The high sensitivity kits may be useful for cell culture supernate samples with very low expected levels of the target, but may require sample validation when used for this purpose.
  • What is the stability of supplemental ELISA kit controls (Quantikine, Quantikine HS, or QuantiGlo ELISA kits)?
  • Controls are assigned an expiration date of 3 months from date of receipt. They are to be used once and discarded. If the lyophilized controls are stored properly, it is possible that they will remain stable for an extended period of time, although we have not conducted extended stability testing. The controls have not been tested for stability after reconstitution.
  • Why is my wash buffer yellow?
  • R&D Systems wash buffers contain ProClin® 300. ProClin 300 is a preservative used in diagnostic and research reagents that is clear to pale yellow in color. The yellow color may become more pronounced over time and occurs more rapidly at room temperature than at 2-8 °C. This color change has no effect on efficacy of the preservative or the performance of the assay. 
  • Will a Logit-Log curve fit work for ELISA data analysis?
  • R&D Systems develops and QCs most of our ELISA Kits using a 4-parameter logistic (4-PL) curve-fit, which is an extension of the Logit-Log curve fit equation.
  • Won't addition of assay diluent cause a further dilution of a sample?
  • Since the assay diluent is added to all wells, standards and specimens are treated equally. Therefore, sample concentration can be read from the standard curve without adjusting for this dilution.
  • Will your Quantikine ELISA kit detect normal sample values of my target?
  • You will be able to quantify samples down to the lowest point on the standard curve. In some cases, the standard curve does go down low enough to detect normal samples. In other cases, normal samples have levels of the target that are just too low to detect. You can check the Sample Values section in your kit booklet to find out what kind of sample values we obtained from apparently healthy individuals. You may also want to check the literature to find out if there is an established normal range for your target. We also have Quantikine HS kits for some of the cytokines that are known to be very low in normal samples. Even in the high sensitivity (HS) kits, it is important to check the standard curve range and the sample values section to determine if the kit is likely to detect the target in your samples

Quantikine IVD In Vitro Diagnostic Colorimetric ELISAs

Supplemental ELISA Products

  • What grade BSA is recommend for use in ELISA and ELISpot Development Systems?
  • The grade of the BSA used has been found to be a critical component for running a successful ELISA. BSA with a minimum purity of 98% is recommended. R&D Systems recommends using the same BSAs used in development of the DuoSet; Catalog # DY995 Reagent Diluent Concentrate 2, Catalog # DY997 Reagent Diluent Concentrate 1. Millipore Probumin® Diagnostic Grade BSA (Millipore, Catalog # 820451) is another example.
  • Which ELISA plate is recommended for DuoSet® ELISA Development Kits?
  • R&D Systems offers clear (Catalog # DY990) and black (Catalog # DY991) microtiter plates, and plate sealers (Catalog # DY992). For added convenience, EvenCoat™ goat anti-mouse IgG microplates are available (Catalog # CP001; Catalog # CP002). EvenCoat microplates are clear, pre-blocked, 96-well polystyrene microplates coated with goat-derived antibody specific for the Fc region of mouse IgG. These plates may be used as a solid support for most sandwich ELISAs utilizing a mouse IgG capture antibody and a non-mouse IgG detection antibody. Other applications include competitive ELISA, IgG isotyping, and hybridoma screening/selection. EvenCoat Microplates may also be used with most DuoSet® ELISA Development Kits. Check your DuoSet ELISA datasheet to see if it can be used with EvenCoat Microplates. If there is no indication that it has been tested, please contact our Technical Service Department for more information.  
  • Why is my wash buffer yellow?
  • R&D Systems wash buffers contain ProClin® 300. ProClin 300 is a preservative used in diagnostic and research reagents that is clear to pale yellow in color. The yellow color may become more pronounced over time and occurs more rapidly at room temperature than at 2-8 °C. This color change has no effect on efficacy of the preservative or the performance of the assay. 

Surveyor IC (Intracellular) ELISAs