Discover a Clear and Easy Guide to ELISAs

ELISA GUIDE A CLEAR AND EASY GUIDE TO ELISAS 2 Table of Contents What is an ELISA? ............................................................................3 The Highest Quality ELISAs Available.......................................3 What's at the core of your immunoassay?.............................3 ELISA Types Direct ELISA.........................................................................................4 Indirect ELISA ......................................................................................4 Sandwich ELISA...................................................................................4 Competitive ELISA...............................................................................5 Why use an ELISA over other techniques?.......................5 ELISA Formats ....................................................................5 Which Immunoassy is Right for You?.................................................5 The Citation Pack Leader: Quantikine® ELISAs.................................6 Quantikine® Colorimetric Sandwich ELISA Kits ...........................6 Quantikine® QuicKit Colorimetric Sandwich ELISA Kits ..............6 Quantikine High Sensitivity Colorimetric Sandwich ELISA Kit ....6 Quantikine IVD Colorimetric ELISA Kits........................................6 QuantiGlo Chemiluminescent Sandwich ELISA Kits....................6 Parameter Colorimetric Competitive ELISAs................................6 The DIY Solution: DuoSet® ELISAs .....................................................7 DuoSet® ELISA Development Systems.........................................7 DuoSet IC (Intracellular) Assay ELISA Development Systems ....7 DuoSet IC (Intracellular) Phospho-specific ELISA Development Systems ..........................................................................................7 Your Next Generation ELISA: Simple PlexTM Assay Simple Plex Assays ........................................................................8 New Quantikine QuicKit Colorimetric Sandwich ELISA Kits .............9 Ensuring ELISA Performance and Consistency..................10 Accurate Detection of Natural Proteins ...........................................10 Confirmed Lot-to-Lot Consistency ....................................................10 Precision & Reproducibility: Providing Confidence in Your Results ..............................................11 Sensitivity: Measuring Proteins at the pg/ml Range ......................11 Linearity Experiments Idenfity False Positive Signals.....................12 What is the Importance of ELISA Controls?.........................13 Positive ELISA Controls .....................................................................13 ELISA Spike Controls.........................................................................13 Negative ELISA Controls ...................................................................13 Sample Preparation.......................................................................14 Sample Collection & Storage............................................................14 ELISA Assay Timelines..................................................................15 Data Analysis: Calculation of Results.....................................16 Calculating concentration of target protein in the sample.............16 Calculating the coefficient of variation............................................16 Best Practices and Techniques ................................................17 Quantikine® ELISA FAQs...............................................................18 Troubleshooting your Quantikine® ELISA..............................20 Duoset® ELISA Assay Optimization..........................................21 Troubleshooting your DuoSet® ELISA .....................................23 ELISA Kits Offerings.......................................................................25 Supplemental ELISA Development Products......................25 3 ELISAs (Enzyme Linked Immunosorbent Assays) are a type of immunoassay that are commonly used to quantify levels of a specific target within a sample. Samples routinely used in ELISAs include serum, plasma, cell culture supernates, cell lysates, saliva, tissue lysates, and urine. ELISAs are usually run in 96-well microplates 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 binds to a different epitope on the target analyte. A substrate solution is subsequently added to produce a signal that is proportional to the amount of analyte bound. ELISAs can have different formats. Descriptions and diagrams of these can be found in the next section. R&D Systems®, a Bio-Techne brand, has over 30 years of experience designing, testing, and optimizing immunoassay kits to ensurethehighestlevel ofperformanceinanalytequantification. We currently offer more than 600 complete, ready-to-use Quantikine ELISA Kits, 1,000 DuoSet ELISA Development Systems for numerous different analytes and species, including human, mouse, rat, canine, primate, and porcine, and an automated Simple Plex platform with over 200 assays available. Choosing quality reagents that will lead to results you can trust is one of the most critical aspects of scientific research. Your results matter, so what’s inside your immunoassay should too. R&D Systems® antibodies and proteins are the core of every Bio-Techne immunoassay platform. Our antibodies and proteins are highly specific, manufactured in-house to ensure reproducibility and tested for suitability on every application we develop. In addition, the proteins used for the immunoassay standard and as antibody immunogens are typically fulllength, recombinant proteins that are confirmed to be biologically active. The ensures that our standard closely mimics the natural protein and that the antibodies will recognize the native for of the analyte. What is an ELISA? The Highest-Quality ELISAs Available What’s at the core of your immunoassay? • Most Referenced ELISA Manufacturer • Flexible Formats available • Extensive Analyte Selection • Rigorous Validation Testing • Extensive Quality Control Testing • Long-term Consistency • Bulk Packaging Available 4 ELISA Types The four main types of ELISAs are indirect, direct, sandwich, and competitive. Each type of ELISA has its own advantages and disadvantages. Indirect ELISA An indirect ELISA is similar to a direct ELISA in that an antigen is immobilized on a plate, but it includes an additional amplification detection step. First, an unconjugated primary detection antibody is added and binds to the specific antigen. A conjugated secondary antibody directed against the host species of the primary antibody is then added. Substrate then produces a signal proportional to the amount of antigen bound in the well. Sandwich ELISA Sandwich ELISAs are the most common type of ELISA. Two specific antibodies are used to sandwich the antigen, commonly referred to as matched antibody pairs. Capture antibody is coated on a microplate, sample is added, and the protein of interest binds and is immobilized on the plate. A conjugated-detection antibody is then added and binds to an additional epitope on the target protein. Substrate is added and produces a signal that is proportional to the amount of analyte present in the sample. Sandwich ELISAs are highly specific, since two antibodies are required to bind to the protein of interest. Direct ELISA In a direct ELISA, an antigen or sample is immobilized directly on the plate and a conjugated detection antibody binds to the target protein. Substrate is then added, producing a signal that is proportional to the amount of analyte in the sample. Since only one antibody is used in a direct ELISA, they are less specific than a sandwich ELISA. When to Use Assessing antibody affinity and specificity. Investigating blocking/ inhibitory interactions Advantages • Fast and simple protocol Disadvantages • Less specific since you are only using 1 antibody. • Potential for high background if all proteins from a sample are immobilized in well. When to Use Measuring endogenous antibodies Advantages • Amplification using a secondary antibody Disadvantages • Potential for cross-reactivity caused by secondary antibody When to Use Determining analyte concentration in a biological sample Advantages • Highest specificity and sensitivity • Compatible with complex sample matrices Disadvantages • Longer protocol • Challenging to develop 5 ELISA Formats Why use an ELISA over other techniques? Which Immunoassay is Right for You? Competitive ELISA Competitive ELISAs are commonly used for small molecules, when the protein of interest is too small to efficiently sandwich with two antibodies. Similar to a sandwich ELISA, a capture antibody is coated on a microplate. Instead of using a conjugated detection antibody, a conjugated antigen is used to complete for binding with the antigen present in the sample. The more antigen present in the sample, the less conjugated antigen will bind to the capture antibody. Substrate is added and the signal produced is inversely proportional to the amount of protein present in the sample. KIT Quantikine ELISA Quantikine QuicKit Quantikine HS ELISA DuoSet ELISAs Simple Plex Assays Format 96-well plate 96-well plate 96-well plate Flexible Cartridge Benefit • Most Published • Low CVs • Fastest Plate-based ELISA • Highest Sensitivity • Economical • Largest Menu • Flexible • Hands Free • Sensitive Sample Volume 10-200 mL 50 mL 10-200 mL 100 mL 2.5-25 mL Number of Analytes 1 1 1 1 Up to 8 Assay Time 3-5 Hours 90 minutes 4-4.5 hours 20 hours 90 minutes Pre-coated Yes Yes Yes No Yes When to Use Determining concentrations of small molecules and hormones Advantages • Ability to quantitate small molecules Disadvantages • Less specific since you are only using 1 antibody • Requires a conjugated antigen There are many different immunoassay platforms available to measure protein levels in biological fluids. ELISAs are preferred in many cases due to their sensitivity, specificity, accuracy, and ability to tolerate harsh buffers or pretreatments. Comparing an ELISA to a Western blot, sandwich ELISAs use 2 specific antibodies rather than one and allow for completely quantitative results, while a Western blot can see non-specific bands and are semi-quantitative at best. An advantage of ELISAs over different multiplexing platforms is the ability to customize the assay for the target analyte and not having to worry about interference caused by many other antibodies and proteins working together. The diluents used in our Quantikine ELISA kits are fully optimized to achieve the best performance for that analyte in complex sample matrices. The potential of observing cross-reactivity or interference is minimized and you can push the sensitivity limits with this technique. Analyte from sample HRP - conjugated recombinant analyte Analyte from sample HRP - conjugated recombinant analyte 6 Quantikine® HS (High Sensitivity) Colorimetric Sandwich ELISA Kit Quantikine HS ELISA Kits are complete assays generally used when very low levels of the target protein are expected. These kits utilize distinct protocols to achieve their impressive levels of analyte detection. Two different amplification systems are available. NEW Quantikine® QuicKit Colorimetric Sandwich ELISA Kits QuicKit ELISAs allow for rapid quantitation of target protein in only 90 minutes. With a shortened protocol and only one wash step, these ready-to-use kits allow you to accomplish more in your day, while getting the same quality of a Quantikine ELISA. Quantikine® IVD Colorimetric ELISA Kits Quantikine IVD (in vitro diagnostic) ELISA Kits are complete ready-to-run ELISA kits that have been 510(k) cleared for in vitro diagnostic use. QuantiGlo® Chemiluminescent Sandwich ELISA Kits QuantiGlo ELISA Kits use a chemiluminescent substrate for analyte detection and require a luminometer for output reading. These kits have a broad dynamic range in comparison to standard colorimetric immunoassays. Parameter Colorimetric Competitive ELISAs Parameter ELISA kits are complete microplate-based assays designed to accurately measure the levels of small molecules using the competitive ELISA format. The Citation Pack Leader: Quantikine® ELISAs Kit Components • Pre-coated 96-well Microplate • Conjugated Detection Antibody • Calibrated Immunoassay Standard • Assay Diluent • Calibrator Diluent(s) • Wash Buffer • Color Reagent A and B • Stop Solution • Plate Sealers Quantikine Colorimetric Sandwich ELISA Kits Quantikine ELISA Kits are complete, ready-to-use kits that represent the gold standard in single analyte detection. Kits are available for measuring a wide range of molecules including cytokines, chemokines, growth factors, proteases, and more. 7 Learn more | rndsystems.com/elisas The DIY Solution: DuoSet® ELISAs DuoSet Development Systems DuoSet ELISA Development Systems contain the basic components required to develop a sandwich immunoassay for accurately measuring analytes in biological fluids. When complete kits are not an option, DuoSet ELISA Development Systems are an economical alternative to buying separate antibodies and protein standards. DuoSet IC (Intracellular) Assay Development Systems DuoSet IC ELISA Reagents are sensitive and convenient assays used to measure intracellular protein levels in cell lysates.R&D Systems DuoSet IC assays measure kinases, apoptosis-related and genotoxic stress proteins, heat shock proteins, and more. These signal transduction assays make an excellent alternative to Western blot, especially when used in combination with R&D Systems DuoSet IC Phospho-specific ELISAs. DuoSet IC (Intracellular) Phospho-specific ELISA Development Systems The DuoSet IC Phospho-specific ELISA format is a sensitive and convenient phosphorylation assay for use with cell lysates. Extensive validation work is done in-house to ensure specificity. These signal transduction assays are an excellent alternative to Western blot, especially when used in combination with DuoSet IC ELISAs designed to measure the levels of total protein. Kit Components • capture antibody • biotinylated detection antibody • mass-calibrated standard • streptavidin-HRP • detailed protocol B B P Accelerate your Biomarker discovery and qualification with the full support of the R&D Systems ELISA development team. •Novel Target ELISA Development' •New Sample Type Qualification •New Species Qualification •DuoSet Serum or Plasma Optimization •Modification of Existing Kit Contents and Sizes Learn more | www.rndsystems.com/services/assay-production-custom-development-services 8 Your Next Generation ELISA: Simple PlexTM Assays Simple Plex Assays Simple Plex Assays on Ella bring your immunoassays to the next level. In just 90 minutes you get highly reproducible validated assay data with no manual steps. The assay performance behind that data includes sub-picogram level sensitivity, 4+ logs of dynamic range and reproducibility that rivals the best laboratory automation. The entire assay happens within the microfluidic cartridge which allow Simple Plex assays to achieve a high level of sensitivity, precision and throughput. Testing across 3 sites, 11 users and 9 Ella instruments. Four different assays were processed for CCL2, IL-6, TNF-a and VEGF A. Eight unique serum samples with 2 controls for a total of 704 answers. How Simple Plex Assays Work Highly Standardized and Reproducible Simple Plex assays give you single digit reproducibility — even across multiple users and labs. Simple Plex assays remove the manual steps from executing an immunoassay and enable a high level of precision and standardization even at very low analyte levels. This ensures that no matter where you measure a sample you get the same result. And because Simple Plex assays are correlated against our gold standard Quantikine ELISAs, it’s easy to transfer to another platform whenever you need to. Sandwich immunoassays happen in the Glass Nano Reactor (GNR) Sample Fluorescent Label dAb Target/Sample Capture Ab GNRs GNRs Waste dAb-4 dAb-3 dAb-2 dAb-1 Fluor Running Buffer 1.5 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 Normalized Concentration Multi-Site Test (All Results Histogram) CV= 7.5% 1. Sample is routed through microfluidic channels. 2. Capture antibody captures target analyte 3. Stringent wash removes unbound analyte 4. Detection antibody migrates through microfluidic channel 5. Stringent wash removes unbound detection antibody 6. Scan GNRs 9 Accomplish more in your day without compromising quality. The Quantikine QuicKit ELISAs provide quick, accurate quantitation of proteins in serum, plasma, and cell culture supernates. Unlike traditional ELISAs, QuicKit ELISAs have a fast, simplified protocol that only takes 90 minutes to results with just one wash step. You can expect the same high quality results, sensitivity, and lot-to-lot consistenct to our existing goldstandard Quantikine ELISA kits. New Quantikine QuicKit Colorimetric Sandwich ELISA Kits How Quantikine QuicKit ELISA Assays Work Anti-tag Antibody Anti-tag antibody coated plates B Add 50 µL sample Add Antibody Cocktail HRP-conjugated detection antibody Protein of interest Non-specific proteins Tag-conjugated capture antibody Incubate for 1 hour C Wash Plate Add Substrate D Incubate for 20 minutes Stop and Read plate Superior Linearity and Recovery Linearity and recovery are key performance criteria to ensure your assay is accurately detecting samples, identifying false positives, and demonstrating your assay is ideal for your sample matrix. R&D Systems Quantikine ELISA kits excel in this area and the Quantikine QuicKit ELISAs are no different. Add data and legends from linearity and recovery document attached to email. Get Quantikine quality in a fraction of the time! Figure 1. The IL-18 Quantikine® QuicKitTM ELISA has superior linearity to Brand A. At dilute concentrations, Brand A is susceptible to matrix effects. Figure 2. The R&D Systems® IP-10 Quantikine® QuicKitTM has superior linearity to the Brand A counterpart. Figure 3. The R&D Systems® G-CSF Quantikine® QuicKitTM has superior linearity to the Brand A counterpart. IL-18 Spike Linearity Dilution Serum - R&D Systems 250% Percent Recovery 200% 150% 100% 50% 0% 1:2 1:4 1:8 1:16 Serum - Brand A Plasma - R&D Systems Plasma - Brand A IP-10 Spike Linearity Dilution IP-10 Spike Linearity Plasma - R&D Systems IP-10 Spike Linearity Plasma - Brand A Percent Recovery 200% 150% 100% 50% 0% 1:2 1:4 1:8 G-CSF Spike Linearity Dilution 250% Percent Recovery 200% 150% 100% 50% 0% 1:2 1:4 1:8 1:16 Plasma - R&D Systems Plasma - Brand A 10 Confirmed Lot-to-Lot Consistency All lots are tested to ensure low background, a linear standard curve, consistent assay sensitivity, and a broad dynamic standard curve range. Consistent standard curve O.D.s, control values, and natural sample values ensure that your samples run consistently over time. Corrected O.D. Human β-NGF (pg/mL) 0.01 0.1 1 10 10 100 1000 10000 2013 Data 2003 Data Corrected O.D. 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 1 10 100 10000 1000 Low Control High Control Quantitation of Human β-NGF in High and Low Controls. Controls are assayed with every manufactured lot of β-NGF DuoSet ELISA. Controls must read within a set range of ± two standard deviations from the mean. Controls for the b-NGF DuoSet ELISA Development System have remained consistent across 13 years. Comparison of Human β-NGF Standard Curve O.D.s from 2003 and 2013. Using the Human β-NGF DuoSet ELISA Development System (Catalog # DY256), standard curve values generated in 2003 and 2013 were compared for lot-to-lot consistency. Standard curve O.D.s remained consistent over ten years. Ensuring ELISA Performance and Consistency Accurate Detection of Natural Proteins Antibody pairs recognize the supplied recombinant standard and the natural proteins in biological samples in a parallel manner, confirming that this kit can be used to measure the relative mass values of the natural analyte. R&D Systems has determined the ideal standard curve range for each assay, ensuring peak sensitivity and reproducibility of results. Recognition of Recombinant and Natural Human IL-6. Serial dilutions of rhIL-6 standard (dark green line) or natural IL-6 produced by unstimulated monocytes (light green line) were quantitated using the Human IL-6 DuoSet ELISA Development System (Catalog # DY206). DuoSet ELISAs detect both recombinant and natural proteins in a parallel manner across a range of concentrations. Corrected O.D. IL-6 (pg/mL) 0.01 0.1 1 1 10 10 rhIL-6 Standard Natural IL-6 100 1000 11 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) determine 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, intraassay 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. 50 150 Concentration (pg/mL) 0 0 100 200 6 9 12 15 Time (months) 50 150 Lot 1 Lot 2 Lot 3 Lot 4 Concentration (pg/mL) 0 0 100 200 A B 4 6 9 12 Time (months) Precision & Reproducibility: Providing Confidence in Your Results 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 # D6050) over a 15 month period. B. Three samples with differing IL-6 concentrations (colored lines) were assayed using four different lots of the Human IL-6 Quantikine ELISA Kit (Catalog # D6050) over a 12 month period. 4 Number of Observations 0 8 12 16 30–39 40–49 60–69 80–89 100–109 120–129 140–149 160–169 50–59 70–79 90–99 110–119 130–139 150–159 170–179 IL-12 p40 Concentration (pg/mL) A 2 Number of Observations 0 4 6 8 10 <0.199 0.400–0.599 0.600–0.799 1.200–1.399 1.800–1.999 2.400–2.599 4.600–4.799 0.800–0.999 1.000–1.999 1.400–1.599 1.600–1.799 2.200–2.399 2.000–2.199 2.800–2.999 4.800–4.999 10.000–10.199 IL-6 Concentration (pg/mL) B 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. Sensitivity: Measuring Proteins at the pg/mL Range 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). 12 Quantikine ELISA Kits Are Developed to Detect Natural and Recombinant Proteins. A serum sample containing activated human TGF-β1 was serially diluted (blue line) and compared to the TGF-β1 standard curve (red line). Results show that the Human TGF-β1 Quantikine ELISA Kit (Catalog # DB100B) measures recombinant and natural TGF-β1 with equal effectiveness. Interference Testing of the Human TNF-α Quantikine ELISA. TNF-α, 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-α 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-α concentration determined using the Quantikine ELISA Kit. 40 100 TNFα Measured (% of expected) 0 0 20 60 80 120 1 10 100 1000 sTNF R present (ng/mL) sTNF RI sTNF RII Optical Density 100 0.01 TGF-β1 Standard Natural TGF-β1 0.1 1.0 10 102 103 104 TGF-β1 Concentration (pg/mL) Sample Dilution Quantikine Kit Competitor Kit Analyte Concentration Detected (ng/mL)* 4.16 20.87 1:2 105% 73% 1:4 108% ND 1:8 106% ND Linearity claim 85–115% 89–118% * Samples were diluted prior to the assay as directed in the product data sheet. All samples and dilutions were within the standard curve range. Linearity Experiments Identify False Positive Signals Learn more about identifying and eliminating false positive results. Review our application note. 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. Figure 1. The importance of blocking reagents when detecting human GDNF. The absence of our proprietary blocking reagents resulted in false positives in 9 of 11 samples using the Quantikine ELISA (1A). Conversely, there were no false positive results in Competitor A’s ELISA when our blocking reagents were added to the diluent (1B). Note that Competitor A lacked the sensitivity needed to detect GDNF in positive control supernates from U-118MG and U-87MG cell cultures. TABLE 1 – FALSE POSITIVE HUMAN GDNF READINGS IN 2 COMPETITOR ELISA KITS We next determined whether removing blockers from the diluents in the Human GDNF Quantikine ELISA Kit would lead to false positive detection. The Competitor B ELISA kit was no longer commercially available, so they were not included in the additional testing. Eleven serum samples from apparently healthy individuals were tested on the Human GDNF Quantikine ELISA Kit according to protocol. Samples diluted in the Quantikine diluent were compared to identical samples in diluents that lacked blocking components. Identical samples were also tested using Competitor A’s Human GDNF ELISA with our blocking reagents added to the diluent. U-118MG and U-87MG glioblastoma conditioned media supernate samples were also tested on both kits with and without our blocking reagents. These were used as a positive control7 to ensure appropriate binding with and without blocking reagents When using the Quantikine diluents as provided in the GDNF kit, no serum samples are detectable. When the blocking reagents are removed from the Quantikine diluents, 9 out of 11 serum samples were detectable, giving false positive sample values (Figure 1A). The U-118MG and U-87MG conditioned supernate samples are detectable whether blocking reagents are used or not, indicating that the blocking reagents are not suppressing the real GDNF detection in these positive controls. When testing the ELISA kit from Competitor A, following the kit protocol and using the diluents provided, 9 out of 11 serum samples were detectable (Figure 1B). When blocking reagents were added to Competitor A’s diluents, the serum samples were now undetectable. The U-118MG and U-87MG conditioned supernate samples are not detectable whether blockers are added to the diluents or left as provided in the kit. This indicates lower assay sensitivity with Competitor A when compared to the Quantikine ELISA, although the standard curve range is actually lower on Competitor A (Quantikine standard curve range: 9.38 – 300 pg/mL, Competitor A standard curve range: 2.74 – 2000 pg/mL). QUANTIKINE COMPETITOR A COMPETITOR A + BLOCKERS COMPETITOR B COMPETITOR B + BLOCKERS Standard Curve Range: 9.38 - 300 pg/mL 2.74 - 2000 pg/mL 15.6 - 1000 pg/mL Sample Dilution Neat 1:2 (AT) 1:10 Serum 1 ND 692 ND 44.4 ND Serum 2 ND 614 ND 53.9 ND Serum 3 ND ND ND ND ND Serum 4 ND ND ND 18.2 ND Serum 5 ND ND ND ND ND Serum 6 ND ND ND 15.6 ND ND = non-detectable, sample values << the lowest standard curve point Quantikine Serum Values Quantikine Diluent 0 50 100 150 200 250 300 350 400 450 500 Serum 7 Serum 8 Serum 9 Serum 10 Serum 11 Serum 12 Serum 13 Serum 14 Serum 15 Serum 16 Serum 17 U-118 MG U-87 MG Human GDNF Concentration (pg/mL) Quantikine Diluent w/o Blockers 1A 0 100 200 300 400 500 600 700 800 Serum 7 Serum 8 Serum 9 Serum 10 Serum 11 Serum 12 Serum 13 Serum 14 Serum 15 Serum 16 Serum 17 U-118 MG U-87 MG Human GDNF Concentration (pg/mL) Competitor A Serum Values Competitor A Diluent Competitor A Diluent + Blockers 1B HUMAN GDNF SERUM VALUES ON R&D SYSTEMS ELISA VS COMPETITOR A ELISA. AVOID FALSE POSITIVE ELISA DATA: IMPROVE PERFORMANCE WITH THE R&D SYSTEMS® QUANTIKINE® ASSAY APPLICATION NOTE INTRODUCTION Enzyme Linked Immunosorbent Assays (ELISAs) are commonly used to quantify biomarkers in serum, plasma, and cell culture supernates. These samples contain a variety of proteins, blood components, and other factors that can interfere with the ELISA results, which is commonly referred to as a matrix effect. Eliminating these factors is essential to obtaining accurate sample values. Blocking reagents are commonly used in ELISA kits to reduce interference from proteins in samples which may produce false positive results. False positives can also occur from non-specific binding on the microplate. Solid phase blocking agents are specifically designed to saturate unoccupied binding sites on the ELISA plate surface and prevent this nonspecific binding. R&D Systems uses specialized diluents that are specifically designed to alleviate false positives to give the most accurate results. Here are some kit-specific examples on how blocking reagents were used improve performance. THE IMPORTANCE OF BLOCKING REAGENTS WHEN DETECTING HUMAN GDNF Glial Cell Line-derived Neurotrophic Factor (GDNF) is a neurotrophic factor that has been shown to promote the survival of various neuronal subpopulations in both the central and peripheral nervous systems at different stages of their development. Neuronal subpopulations shown to be affected by GDNF include motor neurons, midbrain dopaminergic neurons, Purkinje cells and sympathetic neurons. GDNF is produced by Sertoli cells, type 1 astrocytes, Schwann cells, neurons, pinealocytes, and skeletal muscle cells. GDNF binding to GFR alpha 1 induces the recruitment of Ret, NCAM-1/CD56, various integrins, Syndecan-3, or N-Cadherin1-3. When developing the Human GDNF Quantikine ELISA (Catalog # DGD00), it was discovered that the serum sample values were not matching reported literature values using commercially available ELISAs4-6. These competitor assays were achieving values that ranged from non-detectable to > 600 pg/mL for serum samples from apparently healthy individuals, while the same samples were undetectable with the Quantikine ELISA. FALSE POSITIVE DETECTION IN TWO COMMERCIALLY AVAILABLE ELISA KITS Knowing that blocking agents alleviate non-specific binding, a further investigation into our competitors’ assays was conducted by adding blocking agents to their diluents and testing them side by side with the diluent provided in their kit. As instructed in their respective inserts, serum samples were diluted 1:2 in Competitor A and 1:10 with acid treatment in Competitor B. Samples were tested neat on the Quantikine® ELISA. As shown in Table 1, none of the samples were detectable in using the Quantikine kit. However, competitor kits detected false positive GDNF levels ranging from 15.6 pg/ml to 692 pg/ml. Two of the samples were detectable with Competitor A using their diluent, but when blockers were added, the samples were no longer detectable. Four of the samples were detectable with Competitor B, but when blockers were added, the samples were no longer detectable. 13 Quantitation of Human IL-6 in High, Medium, and Low Controls. High (blue line), medium (red line), and low (green line) controls are assayed with every manufactured lot of the Human IL-6 Quantikine® ELISA Kit (Catalog # D6050). Controls for the Human IL-6 Quantikine® ELISA Kit fall within acceptable ranges (gray bars) and remain consistent from lot to lot. What is the importance of ELISA controls? The importance of including ELISA controls, both positive and negative, in your immunoassay helps to verify that the assay was run properly and everything is performing accurately. Positive ELISA Controls A positive ELISA control can be a recombinant or natural sample that you know will be detectable in the assay. Positive controls help to show that a negative sample is truly negative. The standard curve is one form of positive control and you can compare your results to the standard curve data that is provided in your product insert. R&D Systems also sells ELISA controls for the Quantikine ELISAs . Most Human Quantikine kits have a lyophilized tri-level control with expected ranges that are validated by our Quality Control and our Mouse/Rat Quantikine ELISAs include one control in the kit. These are great when running multiple plates or when you have multiple users running the assay, to verify that values are all within the expected ranges. ELISA Spike Controls When using complex sample matrices, it is also important to make sure that there is nothing present in the matrix that interferes in the assay. It is recommended to spike in recombinant or natural protein into your matrix and verify that the amount you spike in is what you read out. For more information, refer to Section 4f. Our Quantikine ELISAs are already validated for many complex sample types, so refer to the kit insert for details. Negative ELISA Controls Negative controls help to verify that you are not obtaining any false positive results or non-specific binding. Use a sample that you know will does express the protein you are measuring. If you are quantitating a cell culture supernate, a good negative control would be to test your cell culture media. 14 Plasma Collect plasma using EDTA, heparin, or citrate as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Cell Culture Supernates Remove particulates by centrifugation and assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Cell Lysates Solubilize cell in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20°C. Platelet-poor Plasma Collect plasma using EDTA, heparin, or citrate as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. An additional centrifugation step of the plasma at 10,000 x g for 10 minutes at 2-8°C is recommended for complete platelet removal. Assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes at room temperature before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Saliva Collect saliva in a tube and centrifuge for 5 minutes at 10,000 x g. Collect the aqueous layer, assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Urine Aseptically collect the first urine of the day (mid-stream), voided directly into a sterile container. Centrifuge to remove particulate matter. Assay immediately or aliquot and store at ≤ -20°C. Avoid repeated freezethaw cycles. Human Milk Centrifuge for 15 minutes at 1000 x g at 2-8°C. Collect the aqueous fraction and repeat this process a total of 3 times. Assay immediately. Tissue Homogenates The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood, homogenized in 20 mL of 1X PBS and stored overnight at ≤ -20°C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 x g. The supernate was removed immediately and assayed. Alternatively, aliquot and store samples at ≤ -20°C. Avoid repeated Tissue Lysates Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20°C. Sample Preparations Sample Collection & Storage The sample collection and storage conditions listed below are intended as general guidelines. Sample stability has not been evaluated. (Some proteins require the presence of fetal calf serum for stability) 15 ELISA Assay Timelines SimplePlex QuicKit Quantikine DuoSet Sample/reagent Prep KEY Analyte Capture Detection Antibody Incubation Substrate Incubation Plate Prep Wash Step 90 minutes 4.5 hours 5.5 hours Plate Prep O/N 90 minutes 16 The values of the unknown samples are assigned in relation to the standard curve. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Always run ELISA samples in duplicate or triplicate This will provide enough data for statistical validation ofthe results. Average the duplicate or triplicate readings for each standard, control, and sample and subtract the average zero standard optical density (O.D.). The coefficient of variation (CV) of duplicates should be ≤ 20%. Create a standard curve by reducing the data using computer software capable of plotting the mean absorbance (y axis) against the protein concentration (x axis). When possible, utilize the recommended data reduction method specified in the assay protocol. If the recommended data reduction method is unavailable, it is recommended that various methods (e.g. linear, semi-log, log/log, 4 or 5 parameter logistic) be tried to see which curve best fits the data. One way to determine if the curve fit is correct is to backfit the standard curve O.D. values. To do this, first plot the standard curve.Next, treat standards as unknowns and interpolate the O.D. values from your standard curve. They should read close to the expected values (+/- 10%). Use the data reduction method that gives the best correlation value and backfit. If software is unavailable, the data may be linearized by plotting the log of the concentrations versus the log of the O.D. on a linear scale. The best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. A representative standard curve is shown in the figure below from Human IL-6 HS Quantikine ELISA (Cat# HS600C). (pg/mL) O.D. Average Corrected 0 0.051 0.059 - 0.067 0:156 0.101 0.103 0.044 0.105 0.313 0.148 0.149 0.090 0.149 0.625 0.246 0.251 0.192 0.255 1.25 0.431 0.432 0.373 0.433 2.5 0.798 0.804 0.745 0.809 5 1.407 1.418 1.359 1.429 10 2.485 2.498 2.439 2.510 10 1 0.1 0.1 1 Human IL-6 Concentration (pg/mL) Optical Density 10 0.01 Calculating the coefficient of variation The coefficient of variation (CV) is the ratio of the standard deviation to the mean, which is usually expressed as a percentage. CV = standard deviation mean Calculating CV is important as it can indicate any inconsistencies or inaccuracies in your ELISA results. The CV of duplicates should be ≤ 20%. A larger CV indicates greater inconsistency and possible error. 10 1 0.1 0.1 1 Human IL-6 Concentration (pg/mL) Optical Density 10 0.01 To determine the concentration of each sample, first find the absorbance value on the y-axis and extend a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the x-axis and read the corresponding concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Calculating concentration of target protein in the sample Data Analysis: Calculation of Results 17 Best Practices and Techniques While R&D Systems builds Quantikine ELISA kits to be robust in the hands of even inexperienced users, there are several tips and tricks that can help even the experienced user get the most from their assay. Make sure all reagents are brought to room temperature before using (unless instructed to keep them cold). If you are not going to run the entire plate, ensure that the remaining strips are sealed in the plate bag with the desiccant to prevent moisture from degrading the plate. For standards that are not single use, it is best to aliquot the remaining standard into smaller volumes and freeze. This allows you to avoid repeated freeze-thaws. 1 2 3 4 5 6 7 8 9 10 11 12 ✔ Multichannel pipettes speed the ability to plate your standard and samples and lead to more consistent results. ✔ When pipetting, dispense liquid with the pipette tips held at an angle and not touching the bottom of the well. ✔ While it is not necessary to change your pipette tips between each replicate, it is recommended that you change them between different samples or standards to prevent contamination. ✔ 1 2 3 4 5 6 7 8 9 10 11 12 Plate washer It is highly recommended that a plate washer is used as manual plate washing can lead to higher backgrounds. 30 sec When washing plates, either manually or with a plate washer, be sure to give the wash buffer time to work by adding a 30 second soak time in between washes. 5min Pay close attention to the incubation times. As a general guide the incubation time should not vary by more than +/- 5 minutes per hour of incubation time. ✔ If the assay calls for incubation in a cold environment, at 2–8 °C, and you are running multiple assays, do not stack the plates on top of each other instead placing them individually on the shelf. 18 What is included in a Quantikine Kit? Quantikine 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. How many samples can be assayed in a Quantikine kit? Most Quantikine Kits will run the standard curve and 40 samples in duplicate. Please refer to the datasheet for details on each kit. What samples can be tested in the kit? Typically the R&D Systems Quantikine 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 product-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. Has this kit ever been tested with my sample type? Unfortunately, R&D Systems has not routinely tested many sample types such as tissue homogenates or bronchoalveolar lavage for ELISA kits. This does not mean that the ELISA kit is not suitable for other sample types. 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 may 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. Please see the Citations tab for peer-reviewed papers utilizing a wide range of sample types. Why can I not detect any of my samples? 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. 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 review the literature to find out if there is an established normal range for your target. It is important to recognize that assay platforms and manufacturers differ in their calibrations for their unique assay products and reported measurements may not directly correlate. 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. Quantikine® ELISA FAQs 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. Values which are greater than the sensitivity can be distinguished as separate from the background or the noise of the assay, however the confidence level for reporting these values is lower than if the sample values fall within the standard curve range. Why is a sample dilution necessary in some kits? 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. Won’t addition of Assay Diluent cause further dilution of the 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. Is there enough Calibrator Diluent for all of my sample preparations? 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. My diluents appear to contain 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 will be noted in the specific protocol booklet. If it is not noted in the protocol booklet, please contact Technical Service. The assay protocol specifies to use the shaker at 500 rpm. This is too fast for my shaker. Is this 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. R&D Systems recommends the ThermoFisher Model # 4625 microtiter plater shaker. Assays requiring shaker incubations have been optimized for performance with these shaker specifications only. 19 Are controls available for 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). Please inquire for specific ordering information. What is the stability of supplemental ELISA controls? Controls are assigned an expiration date of 6 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. 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. Why must I use polypropylene tubes for standard curve dilutions in certain assays? Certain proteins or analytes will bind to glass and polystyrene, but do not readily bind to the polypropylene tubes. 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 is there 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 orangebrown 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. 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. 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 an 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 reagents from different 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. 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 am I seeing high variability between sample duplicates? The two main reasons for high variability in an assay is related to pipetting & washing technique. 20 Problem Possible Cause Solution No signal or low signal Reagents added in incorrect order, or incorrectly prepared • Repeat assay • Check calculations, standard reconstitution, etc. Standard has been damaged (if there is a signal in the sample wells) • Check that standard was handled according to directions. Avoid vortexing. • Use new vial Incorrect incubation conditions • Check incubation conditions were for the specified length, at the appropriate temperature, and shaker specifications were met if required. Incorrect filters • Check specified signal and correction wavelengths in the protocol Incorrect Storage/Handling • Check that kit was stored properly according to conditions indicated on the box label Too much signal – whole plate turned uniformly blue Insufficient washing/washing step skipped – unbound peroxidase remaining • See washing procedure Substrate Solution mixed too early and turned blue • Substrate Solution should be mixed and used immediately Plate sealers or reagent reservoirs reused, resulting in presence of residual HRP. This will turn the TMB blue non-specifically • Use fresh plate sealer and reagent reservoir for each step Work surface cleaned with bleach • Residual bleach fumes can oxidize TMB and cause non-specific high signal Standard curve achieved but poor discrimination between points (low or flat curve) Plate not developed long enough • Increase Substrate Solution incubation time • Use recommended time Incorrect procedure • Eliminate modifications, if any Improper calculation of standard curve dilutions • Check calculations, make new standard curve Insufficient washing • See washing procedure • If using an automatic plate washer, check that all ports are clean and free of obstructions, add a 30 second soak step and rotate plate halfway through the wash Plate sealer reused • Use a fresh plate sealer for each step Poor Duplicates No plate sealers used • Use plate sealers Insufficient washing • See washing procedures • If using an automatic plate washer, check that all ports are clean and free of obstructions Variations in incubation temperature • Avoid incubating plates in areas where environmental conditions vary Variations in protocol • Adhere to the same validated assay protocol Variation in pipetting • Ensure all pipette tips are securely fastened and dispensing consistent volumes • Establish use of either forward or reverse pipetting for entirety of the assay Improper shaker • Check that shaker orbit and speed meet specifications indicated in the kit insert. Any splashing on the plate sealer or foaming of liquid in the sample can also result in poor precision. Saliva contamination • Wear a mask to avoid contamination Poor assay to assay reproducibility Plate sealers reused • Use fresh plate sealer for each step Improper calculation of standard curve dilutions • Check calculations, make new standard curve • Use internal controls No signal when a signal is expected, but standard curve looks fine No cytokine in sample or levels below assay range • Repeat experiment • Reconsider experimental parameters • Obtain fresh samples, minimize freeze-thaw cycles • Use enzyme inhibitors Sample matrix is masking detection • Dilute samples at least 1:2 in appropriate diluent, or preferably do a series of dilutions to look at recovery • If specified in the kit protocol, the assay may only recognize the sample after specific treatment. Follow any sample treatments specified in assay insert. Samples are reading too high, but standard curve looks find Samples contain cytokine levels above assay range • Dilute samples further and run again Very low readings across the plate Incorrect wavelengths • Check filters/reader Insufficient development time • Increase development time Green color develops upon addition of stop solution when using streptavidin-HRP Reagents not mixed well enough in wells • Tap plate Edge Effects Uneven temperatures around work surfaces • Avoid incubating plates in areas where environmental conditions vary • Use plate sealers Drift Interrupted assay set-up • Assay set-up should be continuous – have all standards and samples prepared appropriately before commencement of the assay Reagents not at room temperature • Ensure that all reagents are at room temperature before pipetting into the wells unless otherwise instructed in the antibody inserts Troubleshooting your Quantikine® ELISA 21 There are many parameters which influence the results obtained in an ELISA. These include: antibody quality and concentrations, incubation times, incubation temperatures, detection reagent quality and concentration, and substrate type and quality. For this section, it is assumed that all recommended reagents are being used. Antibody concentration—the best way to determine the optimal capture and detection antibody concentrations is to perform a grid experiment. A grid experiment provides a method to test many antibody pair concentrations using only one plate. Antibody starting concentrations will vary depending on antibody type (monoclonal versus polyclonal) used for capture and detection, see Table 1. Refer to the product inserts for capture and detection antibody types as well as recommended starting concentrations. Table 1. Recommended antibody starting concentrations Table 2. Grid experiment for monoclonal capture-polyclonal detection assay Monoclonal Capture/ Polyclonal Detection Monoclonal Capture/ Monoclonal Detection Polyclonal Capture/ Polyclonal Detection Capture Concentration 1, 2, 4 and 8 µg/mL 0.5, 1, 2 and 4 µg/mL 0.2, 0.4 and 0.8 µg/mL Detection Concentration 50, 100, 200 and 400 ng/mL 0.25, 0.5, 1 and 2 µg/mL 50, 100, 200 and 400 ng/mL To form the grid, divide a 96-well plate into 4 quadrants. See Figure 2 for an example of a monoclonal capture-polyclonal detection grid experiment. The 6 columns in each quadrant represent capture antibody concentrations, the 4 rows in each quadrant represent standard curve points, and each of the 4 quadrants represents a different detection antibody concentration. Each quadrant is a “minigrid”, identifying different capture antibody and standard concentrations at one particular detection antibody concentration. In the grid experiment in Figure 2, each quadrant contains all the possible combinations of capture antibody at 1, 2 and 4 µg/mL and standard curve points of ø (Diluent stated on the product insert), 1000, 2000, and 4000 pg/mL, at one detection antibody concentration. From the multiple combinations of antibody pair concentrations illustrated on the grid, select the concentrations that give the best signal to noise ratio. The ø standard points give the “noise” or the background value that can be expected at each of the antibody pair concentrations. The 1000, 2000 and 4000 pg/mL standard curve points give the “signal” resulting from each of the many antibody pair concentrations. Select the highest signal to noise ratio that still gives an acceptable background. A signal to noise ratio of at least 10 is excellent, but the ratio should be at least five. 50 ng/mL detection 100 ng/mL detection 1 2 3 4 5 6 7 8 9 10 11 12 1 µg/mL capture 1 µg/mL capture 2 µg/mL capture 2 µg/mL capture 4 µg/mL capture 4 µg/mL capture 1 µg/mL capture 1 µg/mL capture 2 µg/mL capture 2 µg/mL capture 4 µg/mL capture 4 µg/mL capture A Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø B 1000 pg/mL standard 1000 1000 1000 1000 1000 1000 pg/mL standard 1000 1000 1000 1000 1000 C 2000 pg/mL standard 2000 2000 2000 2000 2000 2000 pg/mL standard 2000 2000 2000 2000 2000 D 4000 pg/mL standard 4000 4000 4000 4000 4000 4000 pg/mL standard 4000 4000 4000 4000 4000 E Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø F 1000 pg/mL standard 1000 1000 1000 1000 1000 1000 pg/mL standard 1000 1000 1000 1000 1000 G 2000 pg/mL standard 2000 2000 2000 2000 2000 2000 pg/mL standard 2000 2000 2000 2000 2000 H 4000 pg/mL standard 4000 4000 4000 4000 4000 4000 pg/mL standard 4000 4000 4000 4000 4000 200 ng/mL detection 400 ng/mL detection DuoSet® ELISA Assay Optimization 22 Background <0.2 O.D. units. Factors that influence background include: blocking reagent, capture and detection antibody concentrations, detection system, incubation times, diluents and washing technique. Curve height preferably above 1.0, usually between 1.0 and 3.0 O.D. units. Factors that influence curve height include: capture and detection antibody concentrations (see grid experiment in Figure 2), incubation times and temperatures, detection system concentration, avidity of antibodies for antigens, pH, diluents and quality of reader. Detection system assay sensitivity may increase with increasing detection reagent concentration or alternate detection system. However, this may result in higher background readings. Dilution of serum and plasma samples serum and plasma samples may require a dilution of at least 2-fold in an appropriate buffer to overcome matrix effects. Empirically determine the dilution of the samples required to result in linearity of dilution. When diluting samples, remember that the diluent used for the standard curve should be the same as that used for samples. If samples are diluted, include the appropriate dilution factor when calculating results. BSA bovine serum albumin, used as a blocking and carrier protein. Since different grades of BSA exist and may contribute to background, an ELISA grade BSA should be chosen and validated. Incubation temperatures the sample and detection antibody incubations should be performed at room temperature. Sample incubation overnight at 4°C or 1 hour at 37°C may increase assay sensitivity, but may also increase the background. Interfering substances it is important to be aware of the possible presence of interfering substances such as heterophilic antibodies or rheumatoid factors. Please refer to The Immunoassay Handbook, edited by David Wild, Nature Publishing Group, copyright 2001, for suggestions on how to control for these substances. Reagent reconstitution and storage conditions reconstitution and storage instructions provided with each reagent must be followed to ensure proper reagent perfomance. Sample preparation and storage while not every analyte has the same stability within a given matrix, there are general precautions which should be followed. Samples that are not used immediately after preparation should be stored in single use aliquots at -70°C. A -20°C freezer may be acceptable, depending on analyte, if it is a manual defrost freezer. It is best if the samples contain carrier protein. Multiple freeze-thaw cycles should be avoided. Samples/standard volume use of a larger sample/standard size (200 µL per well vs. 100 µL per well) may increase sensitivity. Substrate substrates can vary. However, choosing an alternate substrate will require additional assay condition optimization. Some substrates require a longer incubation time to get the curve to a reasonable height. If the substrate is functioning as expected, sensitivity may be enhanced by increasing incubation time. Monitor the plate as it is developing to avoid excessively high backgrounds. Typically, the incubation time ranges from 10 to 30 minutes. Use the correct filters required to read the appropriate wavelength for the substrate chosen. This information is available from the substrate vendor.Incubation times sensitivity may be increased with a longer incubation time at room temperature. Be aware that the top of the curve may flatten out and become unusable, limiting the assay range. Additionally, background may increase. Use of a shaker at room temperature may increase sensitivity. Shakers may be used for some or all of the incubation steps. Incubation times should be determined empirically. Washing, follow instructions given in your ELISA Protocol. Insufficient washing can result in high coefficients of variation (CVs), high background and poor results. Sensitivity varies for each antibody pair. Sensitivity is defined by reliable discrimination from the zero standard. Factors which influence sensitivity include: capture and detection antibody concentrations (refer to the grid experiment shown in Figure 2), incubation times and temperatures, avidity of antibodies for antigens, sample/standard volumes, pH, diluents and wash buffer formulation. However, there is a limit to the sensitivity that can be achieved with each antibody pair. 23 Problem Possible Cause Solution High Background Insufficient washing • See washing procedure • Increase number of washes • Add a 30 second soak step in between washes Too much streptavidin-HRP or equivalent • Check dilution, titrate if necessary Insufficient blocking • Check blocking solution calculations • Increase blocking time BSA impurities • Use high-quality BSA and consider evaluating a different preparation of BSA Incubation times too long • Reduce incubation times Interfering substances in samples or standards • Run appropriate controls Buffers contaminated • Make fresh buffers No signal Reagents added in incorrect order, or incorrectly prepared • Repeat assay • Check calculations and make new buffers, standards, etc. Contamination of HRP with azide • Use fresh reagents Not enough antibody used • Increase concentration Standard has gone bad (if there is a signal in the sample wells) • Check that standard was handled according to directions • Use new vial Buffer containing FCS used to reconstitute antibodies • Requalify your reagents of choice BSA impurities • Use high-quality BSA and consider evaluating a different preparation of BSA Capture antibody did not bind to plate • Use an ELISA plate (not a tissue culture plate) • Dilute in PBS without additional protein Buffers contaminated • Make fresh buffers Too much signal—whole plate turned uniformly blue Insufficient washing/washing step skipped – unbound peroxidase remaining • See washing procedure Substrate Solution mixed too early and turned blue • Substrate Solution should be mixed and used immediately Too much streptavidin-HRP • Check dilution, titrate if necessary Plate sealers or reagent reservoirs reused, resulting in presence of residual HRP. This will turn the TMB blue non-specifically • Use fresh plate sealer and reagent reservoir for each step Buffers contaminated with metals or HRP • Make fresh buffers Standard curve achieved but poor discrimination between points (low or flat curve) Not enough streptavidin-HRP • Check dilution, titrate if necessary Capture antibody did not bind well to plate • Use an ELISA plate (not a tissue culture plate) • Dilute in PBS without additional protein Not enough detection antibody • Check dilution, titrate if necessary Plate not developed long enough • Increase Substrate Solution incubation time • Use recommended time Incorrect procedure • Go back to General ELISA Protocol; eliminate modifications, if any Improper calculation of standard curve dilutions • Check calculations, make new standard curve Poor Duplicates Insufficient washing • See washing procedure • If using an automatic plate washer, check that all ports are clean and free of obstructions, add a 30 second soak step and rotate plate halfway through the wash Uneven plate coating due to procedural error or poor plate quality (can bind unevenly) • Dilute in PBS without additional protein • Check coating and blocking volumes, time and method of reagent addition. Check plate used • Use an ELISA plate (not a tissue culture plate) Plate sealer reused • Use a fresh plate sealer for each step No plate sealers used • Use plate sealers Buffers contaminated • Make fresh buffers Troubleshooting your DuoSet® ELISA continued on next page 24 Problem Possible Cause Solution Poor assay to assay reproducibility Insufficient washing • See washing procedures • If using an automatic plate washer, check that all ports are clean and free of obstructions Variations in incubation temperature • Adhere to recommended incubation temperature • Avoid incubating plates in areas where environmental conditions vary Variations in protocol • Adhere to the same protocol from run to run Plate sealers reused, resulting in presence of residual HRP which will turn TMB blue • Use fresh plate sealer for each step Improper calculation of standard curve dilutions • Check calculations, make new standard curve • Use internal controls Buffers contaminated • Make fresh buffers No signal when a signal is expected, but standard curve looks fine No cytokine in sample or levels below assay range • Use internal controls • Repeat experiment, reconsider experimental parameters Sample matrix is masking detection • Dilute samples at least 1:2 in appropriate diluent, or preferably do a series of dilutions to look at recovery Samples are reading too high, but standard curve looks fine Samples contain cytokine levels above assay range • Dilute samples and run again Very low readings across the plate Incorrect wavelengths • Check filters/reader Insufficient development time • Increase development time Coated plates are old and have gone bad • Coat new plates Capture antibody did not bind to the plate • Use an ELISA plate (not a tissue culture plate) • Dilute in PBS without additional protein Buffer containing FCS used to reconstitute antibodies • Requalify your reagents of choice Green color develops upon addition of stop solution when using streptavidin-HRP Reagents not mixed well enough in wells • Tap plate Edge Effects Uneven temperatures around work surfaces • Avoid incubating plates in areas where environmental conditions vary • Use plate sealers Drift Interrupted assay set-up • Assay set-up should be continuous – have all standards and samples prepared appropriately before commencement of the assay Reagents not at room temperature • Ensure that all reagents are at room temperature before pipetting into the wells unless otherwise instructed in the antibody inserts Troubleshooting your DuoSet® ELISA continued 25 ELISA Kit Offerings Quantikine Kits Species # of Kits Human 458 Mouse 140 Rat 53 Porcine 15 Canine 15 Multi-Species 7 Cynomologus Monkey 1 Rhesus Macaque 1 Viral 1 DuoSet Kits Species # of Kits Human 732 Mouse 297 Rat 61 Canine 17 Porcine 13 Feline 10 Equine 8 Bovine 5 Rabbit 5 Multi-Species 5 Cotton Rat 4 Guinea Pig 3 Primate 2 Viral 1 C. botulinum 1 Viral 1 Supplemental ELISA Development Products Product Catalog # DuoSet ELISA Ancillary Reagent Kit 1 DY007 DuoSet ELISA Ancillary Reagent Kit 2 DY008 DuoSet ELISA Ancillary Reagent Kit 3 DY009 Clear Microplates DY990 Black Microplates DY991 ELISA Plate Sealers DY992 ELISA Plate-Coating Buffer DY006 Glo Substrate Reagent Pack DY993 Stop Solution DY994 Reagent Diluent Concentrate 3 DY004 Reagent Diluent Concentrate 2 DY995 Reagent Diluent Concentrate 1 DY997 Reagent Additive 1 DY005 Streptavidin-HRP DY998 Substrate Reagent Pack DY999 Wash Buffer Concentrate WA126 Sample Diluent Concentrate 1 (5X) DYC001 Sample Diluent Concentrate 1 (2X) DYC002 EvenCoat™ Streptavidin Microplates CP003, CP004 EvenCoat™ Goat Anti-mouse IgG Microplates CP001, CP002 Sample Activation Kit 1 DY010 Cell Lysis Buffer 1 890713 Cell Lysis Buffer 2 895347 Cell Lysis Buffer 3 895366 Cell Lysis Buffer 5 895890 Lysis Buffer 6 895561 Lysis Buffer 16 895935 Lysis Buffer 17 895943 26 Notes _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ 27 Notes _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ bio-techne.com Global info@bio-techne.com bio-techne.com/find-us/distributors TEL +1 612 379 2956 North America TEL 800 343 7475 Europe | Middle East | Africa TEL +44 (0)1235 529449 China info.cn@bio-techne.com TEL +86 (21) 52380373 For research use or manufacturing purposes only. 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