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Markossian S, Grossman A, Arkin M, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-.
Karen L. Cox , BS, Viswanath Devanarayan , PhD, Aidas Kriauciunas , Joseph Manetta , BS, Chahrzad Montrose , PhD, and Sitta Sittampalam , PhD.
Karen L. Cox , BS, 1 Viswanath Devanarayan , PhD, 2 Aidas Kriauciunas , 1 Joseph Manetta , BS, 1 Chahrzad Montrose , PhD, 1 and Sitta Sittampalam , PhD 3 .
Published May 1, 2012 ; Last Update: July 8, 2019 .
Immunoassays are used to quantify molecules of biological interest based on the specificity and selectivity of antibody reagents generated. In HTS and lead optimization projects, assays are designed to detect molecules that are produced intracellularly or secreted in response to compounds screened. This chapter describes the basics of designing and implementing robust, automation friendly immunoassays for HTS, modes of immunoassay formats (competitive and sandwich), instrumentation, reagent selection, experimental design and detailed data analysis concepts. The importance of an appropriate curve-fitting model for calibration curves used for quantification is also addressed in detail. This is an excellent primer for beginners as well as for experienced investigators.
The intent of this document is to provide general guidelines to aid in the development, optimization and validation of an immunoassay. Following these guidelines will increase the likelihood of success in developing a robust immunoassay that will measure consistent values for unknown samples.
Immunoassays are used when an unknown concentration of an analyte within a sample needs to be quantified. To obtain the most accurate determination of the unknown concentration, an immunoassay must be developed based not only on the usual assay development criteria (standard deviation or optimal signal window) but also on how well the immunoassay can predict the value of an unknown sample. First, one needs to establish the assay critical success factors. Then the immunoassay needs to be developed, which establishes proof of concept. During the optimization phase, the quantifiable range of the immunoassay method is determined by calculating a precision profile in the matrix in which the experimental samples will be measured. A spiked recovery is then performed by spiking the analyte into the matrix and determining the percent recovery of the analyte in the matrix. If the precision profile is within the desired working range, then assaying spiked recovery samples over several days completes the validation of the immunoassay. If the precision profile limits are not within the desired working range, further optimization of the immunoassay is required prior to validation.
Establish assay critical success factors (i.e. sensitivity required).
Ensure appropriate antibody and antigen reagents are available.
Adsorb antigen or capture antibody to a solid surface.
Wash off unbound reagents.
Block nonspecific binding sites to reduce background.
Incubate the secondary antibody with the sample.
Wash off unbound reagents.
Incubate secondary antibody-conjugate with sample.
Wash off unbound reagents.
Incubate substrate to generate signal.
Calibration curve fitting, data analysis and quantitation by non-linear regression.
Immunoassays are used in screening to quantify the production or inhibition of antigens/haptens related to a disease target. These antigens or haptens are characteristic of the disease process and mediated by the target, such as cytokines or growth factors. Hence the screening procedure will involve incubating compounds with the specified target, usually expressed in cells, and collecting the cell medium or lysates to quantify the activity of the compounds. Several examples of this approach for using immunoassay procedures have been described in the literature (1-5). The critical steps in setting up a screen are as follows:
Develop a validated immunoassay as described above.
Acquire antibody, antigen/calibrator, label and buffer reagents in quantities needed for HTS.
Establish liquid handling and automation procedures for screening and immunoassay methods.
Establish stability of the capture antibody or antigen bound to a plate. Determine compound collections to be tested.
Develop and validate a method for incubation of compounds with a relevant target in the screening mode.
Develop a sample collection procedure from screening experiments.
Develop data analysis procedures to use immunoassay data to derive compound potency such as IC50 or EC50.
It is important to define the relevant immunoassay parameters before one begins the development, optimization and validation of an immunoassay:
Analyte (hapten or antigen) to be measured.
Sample matrices in which measurements will be made (serum, plasma, cell lysates, culture media, etc.).
Source of antibody, analyte standards and detection reagents (labeled antibody, enzyme substrates, etc.). Availability of these reagents is a critical requirement.
Detection mode (colorimetric, fluorescence or chemiluminescence) and appropriate plate readers.
Type of immunoassay to develop: Sandwich, competitive or antigen-down formats.
Expected analyte concentration ranges to be measured: pg/ml, ng/ml or µg/ml in the sample matrix of choice. This would determine the detection limits and the measurable range that should be achieved in a validated assay.
Data analysis models and format for reporting results.
Validation and optimization criteria using statistical experimental design tools.
Recovery, accuracy and precision expected at the limits of quantification and the measurable range.
Sample throughput, frequency of use, automation and the number of laboratories that would run the assay.
Control samples that would be used for optimization, validation and quality control runs.
Reagents are a critical piece of any assay development process. This refers to all of the reagents that will be used in the assay. There are certain items that need to be considered when obtaining reagents:
Quality of standards and antibodies.
Quantity of standards and antibodies.
Purity of standards and antibodies (when possible antibodies are affinity purified).
Selectivity and specificity of antibodies.
Greiner high binding plates, Costar EIA/RIA high-low binding plates, Immunotech, Falcon, Nunc
Note: Other plate types can also be used based on the experience of the investigator and appropriate quality control to demonstrate acceptability.
50 mM sodium bicarbonate, pH 9.6
0.2 M sodium bicarbonate, pH 9.4
PBS - 50 mM Phosphate, pH 8.0, 0.15 M NaCl
TBS - 50 mM TRIS, pH 8.0, 0.15 M NaCl
1% BSA or 10% host serum in TBS, or TBS with 0.05% Tween-20
Phosphate Buffer: 73 mM Sucrose, 1.7 mM NaH2PO4, 98 mM Na2HPO4·7H2O, 0.1% NaN3, pH 8.5
Casein Buffer: Pierce Blocker cat# 37528
Protein Free Block: Pierce cat# 37573
Pierce has many blocking buffers that are available in their catalog.
Heterophilic Blocking Reagent (HBR): Scantibodies Laboratory, Inc., cat# 3KC533
Scantibodies has many other blocking reagents that are available in their catalog.
PBST, 0.05% Tween-20
TBST, 0.05% Tween-20
1% BSA or 10% host serum in TBS, or TBS with 0.05% Tween-20
1% BSA or 10% host serum in PBS, or PBS with 0.05% Tween-20
50 mM HEPES, 0.1 M NaCl, 1% BSA, pH 7.4
Serum or plasma from the sample species (this might contain the analyte to be measured which will interfere with the assay)
Serum or plasma from a species different from the sample (the analyte, if present, might not cross react with the antibody)
0.1 M HEPES, 0.1 M NaCl, 1% BSA, 0.1% Tween-20
Tissue culture medium for samples
Cell lysates (these might contain SDS or other denaturing reagents that might interfere with the assay)
Horseradish peroxidase (HRP) substrates:
TMB: 3, 3’, 5,5'-tetramethyl benzidine (colorimetric)
OPD: o-phenylene diamine (colorimetric)
ABTS: 2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (colorimetric)
Pierce Supersignal (chemiluminescent)
Pierce QuantaBlu (chemifluorescent)
Pierce QuantaRed (chemifluorescent)
Pierce has other substrates that provide strong signal and sensitivity with HRP enzyme conjugates that are available in their catalog.
Alkaline phosphatase substrate:
pNpp (p-Nitrophenyl Phosphate)
HRP/TMB: 2M H2SO4 solution (at a 1:1 volume with the HRP/TMB substrate/enzyme solution)
OPD: 3M H2SO4 solution, (at a 1:1 volume with the OPD substrate/enzyme solution)
An immunoassay technique where the antibody or the antigen is labeled with a molecule capable of emitting light during a chemical reaction. For detection, a luminescent plate reader is required (available from PerkinElmer).
Sandwich Immunoassay: matched pair of antibodies, one for analyte capture on a solid surface and one for detection that binds to the antigen/hapten/analyte. Antibodies need to be affinity purified for optimal results.
Competitive Immunoassay: a single antibody specific for the hapten/analyte. For optimal results affinity purified reagents are preferred.
The analyte to be measured is typically a recombinant form of the natural analyte or peptide.
Enough standard should be obtained for use in the development phase, validation phase and the continued support of the method to avoid changing lots and/or running out of standard.
Standard quality: Can vary from vendor to vendor and from lot to lot from a vendor.
Standard stability: Information on the stability of a standard can be obtained from the vendor and their recommendations should be followed in storing the standards.
Control samples are real samples where the antigen analyte level has been determined by another validated method. Samples are aliquoted, frozen and used as control samples in each experiment to track assay performance.
Spiked controls are created by adding a known concentration of the standard analyte into the matrix (for example: tissue culture, serum, plasma, or cell lysates). Spiked controls can be used to determine assay performance based on calculating the percent recovery.
The instrument used to read the output of the immunoassay should be tested initially for both linearity and performance. Instrument performance should be regularly calibrated according to manufacturer’s specifications. The majority of plate readers employ UV-Vis Absorbance, fluorescence or chemiluminescence signals as the measured response, because the products of enzyme labels are chromophores, fluorophores or emit luminescent signals. Linearity in response to the specific enzyme product of an enzyme-linked immunoassay (ELISA) should be checked at the appropriate wavelengths and instrument settings.
Lamp sources and Photomultiplier Tubes (PMT) vary in quality and performance in many plate readers. The linear range of many plate readers is generally between 0-3 Absorbance Units (AU), but other instruments have a linear range up to 4.0 AU. A malfunctioning lamp source or photomultiplier tube can significantly affect the linear response range.
These readers employ excitation and emission filter sets in addition to excitation lamp sources and PMTs. In addition to the lamps and PMTs, the filter sets also vary in quality, light throughput and bandwidth. Fluorescence signals are generally in Relative Fluorescence Units (RFU) and linearity should be verified with appropriate filter sets for the fluorophore employed according to instrument specifications.
These instruments have sensitive photomultipliers to detect light emitted from a chemical reaction. No Lamp sources are necessary. These readers usually have a much larger dynamic range, thus allowing for the increase in sensitivity. Signals or responses are measured in Relative Light Units (RLU) and can be significantly different depending on the instrument design.
An ELISA is one of several methods used in the laboratory to detect and quantify specific molecules. ELISAs rely on the inherent ability of an antibody to bind to the specific structure of a molecule. In order to optimize an ELISA and obtain the sensitivity and dynamic range required for the particular assay being developed, all the various components of the assay must be evaluated. The components will vary depending on the immunoassay format selected. The following is a description of the various types of ELISA formats as well as reagents that need to be optimized in order to obtain a robust assay.
Three frequently used types of ELISA are: sandwich assays, competitive assays and antigen down assays. The format selected depends on the reagents that are available and the dynamic range required for the particular assay. Sandwich assays tend to be more sensitive and robust and therefore tend to be the most commonly used.
A sandwich immunoassay is a method using two antibodies, which bind to different sites on the antigen or ligand (Figure 1). The capture antibody, which is highly specific for the antigen, is attached to a solid surface. The antigen is then added, followed by addition of a second antibody referred to as the detection antibody. The detection antibody binds the antigen at a different epitope than the capture antibody. As a result, the antigen is ‘sandwiched’ between the two antibodies. The antibody binding affinity for the antigen is usually the main determinant of immunoassay sensitivity. As the antigen concentration increases, the amount of detection antibody increases, leading to a higher measured response. The standard curve of a sandwich-binding assay has a positive slope. To quantify the extent of binding, different reporters can be used. These reporters (i.e. enzyme, fluorophore, or biotin) can be directly attached to the detection antibody or to a secondary antibody which binds the detection antibody (i.e. goat, anti-mouse IgG – HRP). In this latter case, the capture antibody and the detection antibody must be from different species (i.e. if the capture antibody is a rabbit antibody, the detection antibody would be from goat, chicken, etc., but not rabbit). If the detection antibody is directly labeled, then the capture and detection antibodies can be from the same species. Polyclonal antibodies often contain multiple epitopes and the same affinity purified polyclonal can be used as the capture and labeled detection antibody. The substrate for the enzyme is added to the reaction that forms a colorimetric readout as the detection signal. The signal generated is proportional to the amount of target antigen present in the sample.
Diagram of a sandwich ELISA. The addition of the enzyme’s substrate leads to color development. The amount of color (absorbance) is directly proportional to the analyte concentration.
The antibody linked reporter used to measure the binding event determines the detection mode. For an ELISA, where the detection is colorimetric, a spectrophotometric plate reader is used. Several types of reporters have been developed in order to increase sensitivity in an immunoassay. For example, chemiluminescent substrates have been developed which further amplify the signal and can be read on a luminescent plate reader. Also, a fluorescent readout where the enzyme step of the assay is replaced with a fluorophore tagged antibody is becoming quite popular. This readout is then measured using a fluorescent plate reader. When the detection antibody is labeled with biotin, you have the flexibility to use a number of different types of streptavidin conjugated reporters.
A competitive binding assay is based upon the competition of labeled and unlabeled ligand for a limited number of antibody binding sites (Figure 2). Only one antibody is used in a competitive binding ELISA. Competitive binding assays are often used to measure small analytes. These assays are also used when a matched pair of antibodies to the analyte does not exist. A fixed amount of labeled ligand (tracer) and a variable amount of unlabeled ligand are incubated with the antibody. According to the law of mass action, the amount of bound labeled ligand is a function of the total concentration of labeled and unlabeled ligand. As the concentration of unlabeled ligand is increased, less labeled ligand can bind to the antibody and the measured response decreases. Thus the lower the signal, the more unlabeled analyte there is in the sample. The standard curve of a competitive binding assay has a negative slope. Alternatively, the antigen can be coated on the plate with the antibody and the sample in solution. Fewer antibodies will be available to bind the coated antigen as the amount of antigen in the sample increases. The antibody and labeled antigen concentrations are the important parameters that need to be optimized.
Diagram of a competitive binding assay. After addition of both the analyte and the enzyme-conjugated analyte, competition occurs between the two for binding to the antibody. The addition of the enzyme’s substrate leads to color development. The (more. )
An antigen-down immunoassay or immunometric assay involves binding the antigen to a solid surface instead of an antibody (Figure 3). This is done by coating the solid surface with the antigen, allowing for passive absorbance to the solid surface. Antigen-down immunoassays are used to bind antibodies found in a sample or in a competitive ELISA format (discussed above). When the sample is added (such as human serum), the antibodies (IgE for example) from the sample bind to the antigen coated on the plate. A species-specific antibody (anti-human IgE for example) labeled with HRP is added next. The signal is directly proportional to the amount of antibody present in the sample; the more antibodies there are in the sample, the higher the signal.
Diagram of an antigen-down ELISA. The addition of the enzyme’s substrate leads to color development. The amount of color (absorbance) is directly proportional to the antigen specific antibody concentration.
A single antibody ELISA format is considered when there is only one antibody available that recognizes the analyte (Figure 4). The assay is configured using the same antibody as both capture and detection. The antibody is biotinylated for use as the detection antibody. The method also utilizes two plates. The basic concept is to capture the antibody to an ELISA plate and allow the analyte of interest to bind to the capture antibody. Unbound material is removed by washing the plate and then adding an acid solution to elute the analyte from the capture antibody. The eluted analyte is then transferred to another ELISA plate containing the neutralization solution. The eluted analyte is then allowed to bind to the second ELISA plate. The unbound material is removed and the plate is blocked followed by a wash step. The detection antibody, which is biotinylated, is then added to the plate, followed by an incubation period. Another wash step is performed to remove excess detection antibody, followed by addition of a streptavidin reporter. The last wash step is performed to remove the excess reporter, followed by addition of the substrate. Below is a generic protocol that can be used to set up a Single Antibody Two Plate ELISA Method.
Diagram of a Single Antibody Two Plate ELISA. The addition of the enzyme’s substrate leads to color development. The amount of color (absorbance) is directly proportional to the analyte concentration.
Included in the list below are the plate type and buffers that are a good starting point for single antibody ELISA assays.
One antibody that recognizes the analyte.
The optimal capture and detection antibody concentrations need to be determined experimentally.
Nunc immunoassay plate
Coating buffer: TBS
Blocking buffer: 1% BSA, TBS, 0.1% Tween-20
Antibody diluent buffer: 1% BSA, PBS or TBS, or 0.1% Tween-20
Acid elution buffer: 200 mM Acetic Acid
Neutralizing buffer: 1M Tris pH 9.5
Wash buffer: TBS 0.1% Tween-20
TMB and HRP are used for enzyme/substrate readout.
Acid stop buffer
The conditions for the following need to be tested and optimized for optimal assay performance: pH, buffers, incubation times, concentrations of antibodies, volumes used, etc.
Prepare the capture antibody in coating buffer
Add 100 µl of the capture antibody in coating buffer to Plate 1- Nunc 96-well microtiter plate
Incubate plate with gentle shaking for 1 hour at room temperature
Remove the unbound capture antibody from the microtiter plate by dumping the plate
Wash the plate 3 times with wash buffer
Prepare serial dilutions of the analyte in dilution buffer
Add 100 µl of the analyte to the plate
Incubate plate with gentle shaking for 1 hour at room temperature
Wash the plate 3 times with wash buffer
Add 65 µl of 200 mM Acetic Acid for 5 minutes at room temperature with gentle shaking
Prepare Plate 2 by adding 100 µl of 1 M Tris pH 9.5 to the microtiter plate
After 5 minutes of incubation transfer the 65 µl of acidified analyte from Plate 1 to Plate 2
Incubate overnight at 4°C with gentle shaking
Wash Plate 2, 3 times with wash buffer
Add 200 µl of blocking buffer to the plate
Incubate for one hour at room temperature with gentle shaking
Wash the plate 3 times with wash buffer
Add 100 µl of the biotinylated detection antibody (this is the same antibody that was used as the capture antibody)
Incubate for one hour at room temperature with gentle shaking
Wash the plate 3 times with wash buffer
Add Streptavidin HRP
Incubate for 20-30 minutes at room temperature with gentle shaking
Wash the plate 3 times with wash buffer
Add TMB substrate to the plate
Incubate according to manufacturer’s suggestions with gentle shaking
Add Stop Solution, mix thoroughly
Read optical density at 450 nm
Biomarker research has expanded over the years, producing a need to quantitatively measure multiple analytes simultaneously from one sample. In the pre-clinical research area there is a need to measure endpoints from rodents and non-human primates to determine safety and efficacy of drug candidates. Typically the samples from these animal models are limited in volume and expensive to obtain, which produces a challenge in obtaining data if more than one analyte needs to be quantified. The same issues apply to clinical samples being assayed for Biomarker, Pharmacokinetic, and Pharmacodynamic studies. A single endpoint ELISA tends to use a larger volume of sample than a multiplexed assay.
Two of the widely used multiplex technologies that have been developed are the Luminex xMAP technology (LMX) and the Meso Scale Discovery (MSD). Both technologies have well validated immunoassays that cover a wide range of secreted and intracellular proteins. In most cases, numerous analytes can be measured with sample volumes of less than 50 µl.
Meso Scale Discovery is a multiplexed technology based on MULTI-ARRAY ® technology. The technology utilizes a proprietary electrochemiluminescence detection system and an array of spots in a standard 96-well format. The electrochemiluminescence technology allows for an increase in dynamic range of the standard curve as well as an increase in sensitivity over normal ELISA readouts, such as HRP/TMB. The MSD technology utilizes a 96- or 384-well microtiter plate, allowing an immunoassay to be developed and optimized using the same variables of antibody concentrations, buffers, and incubation times that are used in a standard ELISA. The plates are read using a Sector 6000 instrument yielding Relative Light Units that can be back calibrated off the standard curve to the analyte concentration that is being analyzed. Additional information on the MSD platform can be found at: http://www.mesoscale.com/.
Luminex xMAP is a bead based technology that allows capture antibodies to be coupled to color coded beads or microspheres that contain different emission spectra. A sandwich assay format is performed with the analyte added to the capture antibody bound beads, followed by the addition of a biotinylated antibody. The detection occurs by adding a streptavidin-conjugated flourochrome to the complex containing the sandwiched immunoassay. The fluorescent readout is detected using a Luminex xMAP which is a flow cytometry based instrument. The microspheres are classified based on their emission spectra and the amount of analyte detected is directly proportional to the fluorescent signal. Additional details on the LMP technology can be found at: http://www.luminexcorp.com/TechnologiesScience/xMAPTechnology/.
Studies have been performed by numerous laboratories directly comparing the MSD technology to the Luminex xMAP as well as to commercially available enzyme-linked immunosorbent assays (6-12). Validation data has been generated for spiked recovery in various matrixes, including other validation parameters for both the MSD and Luminex technologies. Overall the results from numerous studies show that while there usually is a quantitative difference between the technologies (most likely due to the use of different antibodies), the relative differences are comparable. Data from these numerous studies demonstrate that multiplexed technologies are suitable for screening for trends in cytokine profiles and other secreted proteins to support pre-clinical and clinical studies.
