Technical Guide for ELISA

Technical Guide for ELISA - Protocols - Troubleshooting w w w . k p l . c o mWhere Better Science Begins KPL, Inc. • 800-638-3167 • 301-948-7755 • www.kpl.com Table of Contents for Chapters 1 - 4 Page 1. Introduction - What’s an ELISA? 3 2. Assay Formats - What are my choices? 4 3. How to Choose an Assay Format - I know what I want to measure. What do I do? 7 4. Factors to Consider When Developing an Assay - There are a lot but a few are critical. 9 5. Performing an Assay - What do I need to know? 26 6. Data Handling - What should I do with all this data? 28 7. Troubleshooting - I’m having trouble. Now what? 30 8. Resources - Where can I get more information? 34 9. Glossary 35 10. Related Products 38 Table of Contents T a b le o f C o n t e n t s 2 KPL, Inc. • 800-638-3167 • 301-948-7755 • www.kpl.com Who will this help? The KPL "ELISA Technical Guide" is a continuation of the series of guides and information from KPL to help researchers better understand the available tools for performing improved protein detection experiments. It is designed primarily for the beginner as it explains basic concepts, protocols and troubleshooting of ELISA assays. It will help you to determine which type of ELISA is most likely to give the needed information, how to set up and perform an ELISA and finally how to interpret the data. While the Guide is aimed at beginners, some of the hints, suggestions and troubleshooting should be useful reminders to experi- enced ELISA mavens. The Technical Guide is divided into ten sections starting with an overview of the different types of ELISA formats available. This is followed by more specific sections discussing the factors that need to be addressed to develop a successful ELISA: representative protocols, tips that will help improve the precision of an assay; and how to handle and interpret the data generated. Troubleshooting, and Resources sections provide information to help solve the problems encountered in an ELISA. Help for Beginners: √√ Which Type of ELISA √√ How to Set it Up √√ How to Interpret the Data Tips for the Experienced Who will this help? W h o w ill t h is h e lp ? 3 KPL, Inc. • 800-638-3167 • 301-948-7755 • www.kpl.com Introduction Introduction - What’s an ELISA? ELISA evolved in the late 1960s from RIA (radioim- munoassay) with the observation that either the antibody or the analyte (antigen) could be adsorbed to a solid surface and still participate in specific high affinity binding. The adsorp- tion process facilitated the separation of bound and free analyte, a situation that proved difficult to engineer for many analytes with RIA. Over the intervening years, the term ELISA has been applied to a wide variety of immunoassays, some of which do not employ enzymes and some of which do not require the separation of bound and free analyte. The distinguishing feature of all of these assays remains the use of antibodies to detect an analyte. In this guide, the discussion of assays is restricted to enzymatic systems that require the separation of bound and free analyte (heterogeneous assays). ELISA - Why use ‘em? ELISA has become extraordinarily useful because it allows rapid screening or quantitation of a large number of samples for the presence of an analyte or the antibody recognizing it. Variations on this theme are now used to screen protein- protein, protein-nucleic acid, nucleic acid-nucleic acid interac- tions in microarrays. Other solid supports have evolved such as nitrocellulose and PVDF for blotting (another variation albeit with less ability to quantitate). ELISA, however, remains popular because of its ease of performance and automation, accuracy, and the ready availability of inexpensive reagents. Some Limitations One limitation of the ELISA technique is that it provides information on the presence of an analyte but no information on its biochemical properties, such as molecular weight or its spatial distribution in a tissue. To obtain this information one needs to perform other types of assays. For example, blotting assays combine separations based on physical properties of the analyte with detection techniques. Immunohistochemical assays performed on tissue and cells provide information on the specific location of an analyte. (see Immunohistochemical Staining: Principles and Practices). Both of these techniques can also provide some quantitation of the analyte, but not as accurately as ELISA. Antibodies - The Key to an ELISA The antibody is the major factor determining the sensitivity and specificity of an assay. The structure of antibodies is discussed more thoroughly in "The Use of Antibodies in Immunoassays". Briefly, it is the three dimensional configura- tion of the antigen-binding site found in the F(ab) portion of the antibody that controls the strength and specificity of the interaction with antigen. The stronger the interaction, the lower the concentration of antigen that can be detected (other factors being equal). A competing factor is the specificity of binding or the cross-reactivity of the antibody to serum proteins other than the the target antigen. Depending on whether the antibodies being used are polyclonal or monoclonal, cross-reactivity will be caused by different forces. In either case driving the assay to the limit of sensitivity may result in cross-reactivity, and one is faced with the conflicting needs of sensitivity versus specificity. We will discuss this further in Section IV. 1 . W h a t ’s a n E L I S A ? ELISAs can be qualitative or quantitative but they all need highly specific and sensitive ANTIBODIES. At first glance, the choices in ELISA formats may be overwhelming, but don’t dispair. This chapter will help you make sense of the options. There are a wide variety of ELISA formats available that vary depending on the sensitivity required and whether one is trying to detect an analyte or the antibody response to it. In the following section we will discuss these various configurations and when to use them. Homogeneous vs. Heterogeneous Homogeneous ELISA formats do not require separation of reacted from unreacted material in order to detect measure target antigen, usually a hapten. Bound analyte can modify the activity of a labeled detection reagent (e.g. up regulating or down regulating enzyme activity upon binding). In a hetero- geneous assay format the bound analyte does not modify the activity of the detection reagent, thus the bound and free must be separated by a washing step after binding in order to distinguish them. A Basic ELISA 1. Coat solid phase with either antibody or analyte. 2. Block remaining binding sites on the solid phase. 3. Add either analyte or anti-analyte antibody to be detected. 4. Wash out excess reagent. This separates bound from free analyte. 5. If reagent in step 4 is an analyte, add a second anti-analyte antibody with detection molecule attached. If reagent is an anti-analyte antibody, add an anti-Ig antibody with detection molecule attached. 6. Wash out excess reagent. 7. Add substrate. The color change or amount of light emitted is proportional to the level of target analyte. Capture vs. Direct Within the heterogeneous type of assay, several different formats can be distinguished based on which component is immobilized. As illustrated in Figure 1, either antibody or antigen (the analyte to be detected) can be immobilized. Antibody immobilized formats are generally referred to as capture or sandwich assays. Either a primary antibody recog- nizing an epitope of the molecule to be detected, or an anti-Ig (or protein A/G) can be immobilized. This is the preferred format in situations where the antigen is being detected. In contrast, an antigen or epitope can be immobilized and is 4 KPL, Inc. • 800-638-3167 • 301-948-7755 • www.kpl.com Assay Formats referred to as a direct assay format. This format is commonly used when the immune response to an antigen is being analyzed. Competitive vs. Noncompetitive Each of the above assay types can be adapted to a competitive or noncompetitive format. The distinguishing feature of a competitive assay format is that the combination of an unknown amount of analyte introduced from the sample and the reference analyte compete for binding to a limited number of antibody binding sites. This assay can be performed with either the analyte or the antibody adsorbed to the solid phase. As shown in Figure 2A, added sample analyte is competing Primary Ab Bound Anti Ig Bound Analyte Bound 2 A Labelled Antibody Labelled Analyte Figure 1. Figure 2. Assay Formats Capture/Sandwich Direct Competitive Assay Format 2A 2B 2 . W h a t a r e m y c h o ic e s ? Antibodies with Various Specificities Protein to Block Nonspecific Binding Sites Streptavidin EnzymeAntigens with Various Epitopes Epitope Figure Legend Labeled Antibody Labeled Analyte with solid phase adsorbed reference analyte for binding to a limited amount of labeled antibody. In Figure 2B labeled reference analyte in solution combined with sample analyte competes for binding to a limited amount of solid phase -- adsorbed antibody. If a saturating amount of antibody were present, adding a small amount of the sample competitor may not effect a detectable change in activity in the assay. Thus the sensitivity of a competitive assay depends on having slightly fewer antibody binding sites than the number of reference analyte sites. It provides the most accurate quantitation of the different formats available. Because only a limiting amount of antibody can be used, the sensitivity of this format is strictly limited by the affinity of the interaction between the antibody and the analyte. In practice it is not possible to accurately quantitate analyte at a concentration much less than 10 fold below the K (equilibrium constant) of the antibody. Quantitation can be obtained by generating a "standard curve" of concentration of added competitor analyte vs activity. To do this in the format illustrated in Figure 2A, one would add aliquots of known increasing concentrations of analyte to wells containing the solid phase adsorbed analyte. To each of these wells one would add an aliquot of labeled antibody and generate the curve illustrated in Figure 3. For the format illus- trated in 2B, one would mix aliquots of known increasing concentrations of analyte with aliquots of labeled reference analyte and add each mix to a well containing the solid phase adsorbed antibody and generate the curve illus- trated in Figure 3. The standard curve should be run each time an unknown is analyzed. Increasing Concentration of Competitor Analyte A ct iv it y A ct iv it y Concentration of Competitior Analyte n ea t 1/ 2 1/ 4 Various Dilutions of Sample with Unknown Concentration of Analyte Activiity Obtained after Unknown Sample AddedStandard Curve A ct iv it y Figure 3. Figure 4. Standard Curve in a Competitive ELISA Antibodies with Various Specificities Protein to Block Nonspecific Binding Sites Streptavidin EnzymeAntigens with Various Epitopes Epitope Figure Legend Quantitation in a Competitive ELISA Figure 5. Noncompetitive Format Highest Sensitivity Assay Formats 2 . W h a t a r e m y c h o ic e s ?The sensitivity of a competitive assay depends on having slightly less antibody binding sites than the number of reference analyte sites. 5 KPL, Inc. • 800-638-3167 • 301-948-7755 • www.kpl.com To quantitate an unknown, compare the activity obtained with the aliquot of unknown to the standard curve as in Figure 4. If the unknown has enough activity, it is advisable to run several dilutions of the unknown. In an ideal situation the activity generated by these dilutions should parallel the dilutions that were made (e.g. a 1/2 dilution should yield 1/2 the activity). If it does not, there are likely to be interfering components in the sample matrix. In addition, by a careful choice of analyte or epitope, a competitive assay can be made highly specific even in the presence of cross-reactive antibodies. Noncompetitive ELISA formats are illustrated by the capture and direct assays shown in Figure 5. The distinguishing feature of this format is that antibody-binding sites are present in excess over the analyte being detected. As a result, this format is the most sensitive and can be performed with either the antibody adsorbed to the solid phase or the analyte or epitope adsorbed. Detection limits up to 104 less than the K of the antibody are possible. The first format has been successfully used to quantitate multi epitope molecules (e.g. cytokines) and depends on preparing antibodies to at least two different and non overlapping epitopes, usually monoclonal antibodies. Assays with detection limits of 10-15M have been reported. The amount of signal generated by the binding of the second antibody is proportional to the amount of antigen present and is often referred to as a propor- tional assay. Quantitation can be obtained by generating a standard curve as illustrated in Figure 6 and comparing the activity obtained with a sample to the activity on the standard curve. Indirect The sensitivity of an ELISA can be increased by amplifying the label bound to the detection partner. This is referred to as an indirect ELISA. In the simplest format, an antibody labeled with one or more detection molecules is bound to the immobilized antigen. In order to increase the sensitivity, the bound antibody is biotinylated in several locations on its surface. It can thus bind multiple streptavidin molecules, each labeled with one or more detection molecules, as illustrated in Figure 7. Alternatively, labeled protein A/G or anti-Ig labeled with detection molecules can be bound to the primary antibody. 6 KPL, Inc. • 800-638-3167 • 301-948-7755 • www.kpl.com Assay Formats Concentration of Analyte A ct iv it y b b b b b b Figure 6. Figure 7. Quantitation in a Proportional ELISA Sensitivity Enhancement 2 . W h a t a r e m y c h o ic e s ? Antibodies with Various Specificities Protein to Block Nonspecific Binding Sites Streptavidin EnzymeAntigens with Various Epitopes Epitope Figure Legend 7 KPL, Inc. • 800-638-3167 • 301-948-7755 • www.kpl.com What is being detected? A protein or other large molecule Now that you know the various formats, let’s apply them to what you want to measure. If a protein with multiple epitopes is being detected, a sandwich assay as illustrated in Figure 8 is a good choice. This assay format has been used to both detect and quantitate cytokine molecules. It usually requires two antibodies that react with different epitopes. However, if the molecule has multiple repeating epitopes, it is possible in a sandwich assay to use the same antibody for both capture and detection. Alternatively, if there is a supply of the analyte to be detected in pure form that can adsorb effectively to a microwell, then one can set up a competitive assay in which the purified analyte is immobilized and analyte in the sample competes with the immobilized analyte for binding to labeled antibody. solid phase, or may be masked by the blocking protein added. However, small molecules can often be attached to larger proteins which provide a means to attach the desired epitope to a solid phase in a configuration that allows the epitope to be bound by an antibody. In order to analyze the immune response to a single epitope, the format illustrated in Figure 9A can be employed. If detecting or quantitating the epitope is desired, typically a competitive format is required, as illustrated in Figure 9B. Another variation, not illustrated, is to add the small molecule as a competitor in format 9A. Is the measurement qualitative or quantitative? Screening assays where the results are "eyeballed" can easily be performed in noncompetitive formats where a positive result must be discerned over background, especially when the background has been controlled to a non-observable level. On the other hand, in competitive assays a difference in the amount of color is much more difficult to discern by eye. Unless one is looking only for gross differences, it is best to rely on plate readers for quantitation. Detecting a Protein or an Organisim A B In this case it is essential to titrate the antibody so that it is limiting, or else the assay sensitivity will be lowered. An organism Bacterial or viral assays that detect whole organisms can also use sandwich assays with the same antibody for both capture and detection as illustrated in Figure 8. A small molecule If the target molecule is small or consists of a single epitope, a modification of the formats described above is needed. Small molecules by themselves either do not adsorb well to a Figure 8. Figure 9. Detecting a Small Molecule Detecting a Protein or an Organism 3 . I k n o w w h a t I w a n t t o m e a s u r e . W h a t d o I d o ? Antibodies with Various Specificities Protein to Block Nonspecific Binding Sites Streptavidin EnzymeAntigens with Various Epitopes Epitope Figure Legend How to Choose an Assay Format Is the measurement of the antibody response to a molecule? Antibody responses to an epitope, especially if the epitope is on a large molecule, are typically easy to follow in a direct assay format. Responses to haptens are easily analyzed by attaching the epitope to a large protein that can be adsorbed to the solid phase as illustrated in Figure 9A. What level of sensitivity is required? The factors that determine the ultimate sensitivity of a competitive assay are the antibody affinity constant and the experimental errors. The detection limit of the substrate is not typically limiting. It has been calculated theoretically that with a K = 10 12 M -1 (an extraordinarily high equilibrium constant for an antigen-antibody interaction) and a 1% coeffi- cient of variation (CV) for the response at zero dose, the lowest detection limit possible would be 10 -14 M. A more easily achievable limit would be 10-9 - 10-10 M. The factors limiting the sensitivity of a sandwich assay are the affinity of the antibody, the experimental errors and the nonspecific binding of the labeled antibody, expressed as a percentage of the total antibody. It has been estimated that with a K = 10 12 M -1 , 1% CV of the response at zero dose, and a 1% nonspecific binding of the labeled antibody, the detection limit can be as low as 10 -16 M . In addition, this can be enhanced further by using more sensitive detection substrates. 8 KPL, Inc. • 800-638-3167 • 301-948-7755 • www.kpl.com Need more Sensitivity? To get the most sensitivity from an assay, the following factors must be addressed: • Background noise, can usually be minimized by optimizing the blocking and washing steps. The lower the signal, the lower the background must be in order to detect a positive result. • Low signal due to low level attachment of the bound molecule can often be overcome by testing different plates or by switching to covalent linkage to the plate. • Low signal can be amplified by incorporating indirect labeling techniques or by switching from chromogenic to chemiluminescent substrates. • Low signal can sometimes be amplified by increasing the incubation times, allowing the binding steps to come to equilibrium. 3 . I k n o w w h a t I w a n t t o m e a s u r e . W h a t d o I d o ? Measure Analyte Immune Response to an Analyte Use Capture or Competitive Direct or Indirect Which format for me? How to Choose an Assay Format 9 KPL, Inc. • 800-638-3167 • 301-948-7755 • www.kpl.com Factors to Consider When Developing