ELISA can be defined as a qualitative or quantitative solid-phase immunological method to measure an analyte following its binding to an immunosorbent surface and its subsequent detection by the use of enzymatic hydrolysis of a reporter substrate, either directly (as with an analyte that has enzymatic properties or is directly labeled with an enzyme) or indirectly (by means of an enzyme-linked antibody that binds to the immunosorbed analyte). Qualitative results provide a simple positive or negative result for a sample. Converting quantitative to qualitative results based on a cutoff value that separates positive and negative results is common practice. Because the performance properties of the assay depend heavily on the cutoff value, the process used to determine the cutoff should be evidence-based and well documented. Quantitative assays determine the quantity of the analyte based on the interpolation of a standard calibration curve with known analyte concentration, run simultaneously in the same assay. This standard should be an appropriate, preferably homologous, reference or calibration material that is representative of the analyte(s) of interest. The power of immunoassays has been demonstrated by the variety of procedures that have evolved, including alternative solid surfaces such as beads of different sorts, various plastics in plates of different configurations, and alternative detection methods, e.g., chemiluminescence and fluorescence. ELISA assays are widely used in the biopharmaceutical industry for various applications such as identity, purity, potency, detection or quantitation of antibody or antigen, and other purposes.

The essential steps of an ELISA can be broken down as follows (see Figure 1):

In this assay an antigen is coated onto a solid surface and then, after blocking, a solution containing a specific antibody is added (Figure 2B). After incubation, the unbound antibody is washed away, followed by the addition of an anti-immunoglobulin (anti-Ig) detector antibody. Anti-Ig detectors are available commercially for specific Ig classes and subclasses from a variety of species, which makes this assay format useful for isotyping of antibodies. In addition, the use of a labeled anti-Ig detector amplifies the signal compared to a Direct ELISA, thereby increasing assay sensitivity.

Solid phases are available in a variety of forms (e.g, membrane, plate, or bead) and chemistries (e.g, nylon, nitrocellulose, polyvinylidine fluoride (PVDF), polyvinyl, polystyrene, or a chemically derivatized surface). The selection of the solid phase determines the most likely binding mechanism, i.e., hydrophobic, hydrophilic, or covalent interactions. In general, compared to plates, beads offer higher capacity and are more commonly used in clinical assays whereas plates are more commonly used to test biotechnology products. Additional information on plates is provided below.

Samples and reagents generally are pipetted into the ELISA plate wells. Care should be taken to avoid cross-contamination, frothing, or bubbles. Labor-saving equipment such as electronic pipets, automated liquid handlers, plate washers, and robotic pipets also can be used to improve precision, reduce analyst-to-analyst variability, and increase throughput.

Wash steps are included throughout the ELISA procedure to remove the unbound coating antigen, sample, and detection reagents. Washing is critical for assay performance, can be a source of assay failure, and is important to evaluate during method development. Multiple approaches can be used for washing. Manual procedures include using a squeeze bottle, dipping the microtiter plate in wash buffer, and adding wash buffer with a multichannel pipet or hand-held multi-channel (8- or 12-pin) manifolds. Analysts should wash carefully to avoid cross-well contamination. Automatic microplate washers generally provide more washing consistency. Strip-well and multiwell washers are available. Most automatic washers can be programmed for different dispensing volumes and speeds, number of washes, speed of buffer aspiration, and amount of residual buffer left in the well. Incorrectly programmed or maintained as well as incompletely cleaned automatic washers can cause assay variation and elevated assay background.

ELISAs are incubated following the addition of samples and reagents. The optimal time, conditions, and temperature of each incubation step should be determined during method development. Incubation times vary from minutes to overnight. Commonly used incubation temperatures are ambient temperature, 4, and 37. ELISA plates commonly are sealed or placed in a secondary container to avoid evaporation or contamination during incubation. Atmospheric conditions such as dry or humidified incubation should be evaluated during method development. Rocking, shaking, or rotating the microtiter plates may be necessary or desirable depending on the kinetics of binding.

After immobilization and removal of the unbound antigen or antibody, unoccupied binding sites are blocked to ensure that the measured analyte in the test article or subsequent (detection) reagents does not bind nonspecifically to the solid surface or to the coated antigen or antibody. If nonspecific binding occurs, any reported signal could bias the measurement and may reduce the sensitivity and dynamic range of the assay. Blocking is critical to ensure the sensitivity and/or specificity of the assay. Sources of nonspecific binding fall into two general categories:

The choice of blocking agent (examples are found in the Blocking Agents and Buffers section above) is determined empirically, and the balance between the reduction in nonspecific binding and the impact on assay sensitivity should be assessed during method development. Cross-reactivity with other assay reagents should be considered; for example, endogenous biotin is found in milk and serum, and serum may contain antibody to viral or bacterial proteins. Therefore, screening of serum lots may be necessary. The volume of blocking solution added to the well should be greater than the maximum reaction volume used for later steps so that all of the potential surface area that may interfere with the binding reaction is blocked.

In addition, Ig in the test materials can be removed by using buffers that inhibit antibody conformation or aggregate the heterophilic antibodies, by blocking with nonimmune serum, or by removing Fc regions in critical antibody reagents, thereby reducing or eliminating undesired immunological interactions that cannot be addressed by the blocking reagents described above. Negative control wells can be included to monitor nonspecific reactions. The nature of the negative control wells depends on the assay but can include blocked wells without coating antigen, eliminating the primary or secondary antibody, or using buffer in place of sample. Control wells also can be useful as part of system suitability testing.

Although ELISA methods are designed to measure an analyte in complex mixtures, the presence of other materials can prove problematic if they interfere with analyte detection. In order to ensure assay specificity, the specific procedure to treat samples to remove nonspecific interfering substances (e.g., reducing agents or precipitates) can be determined empirically during method development and then can be incorporated into the validated assay. Any sample-processing step should be evaluated against the potential that the treatment will alter the test article’s properties and/or introduce further variability that results in biased measurements. Samples, standards, and controls should be prepared and handled in processes as similar to each other as possible. Analysts should verify that sample pretreatments have not damaged the sample so much that it can no longer be measured (e.g., by spiking experiments).

Depending on ELISA format, detector antibodies labeled with enzyme or other labels can be used as primary or secondary reagents to enable detection of the immobilized analyte. In a direct or competitive ELISA (Figure 2A and Figure 2C), after the analyte is bound to the immunosorbent surface, excess analyte is washed away and the immobilized analyte is detected using a detector antibody that is considered to be the primary antibody. In other ELISA formats (Figure 2B, 2D, and 2E), the analyte-specific Ig (nonconjugated primary antibody) is allowed to bind to the immobilized analyte, and any excess antibody is washed away before the addition of a detector antibody, which is termed the secondary antibody.

To facilitate detection, in all ELISA formats that use enzyme-conjugated antibodies, a substrate specific for the conjugated enzyme is introduced into the assay system. An enzymatic reaction ensues, converting a substrate into a soluble product that can be measured using appropriate wavelengths and a suitable reader.

ELISA sensitivity depends on the quality of the reagents and the detection system, including the label and substrate. If multiple differently conjugated antibodies are available, analysts should select one appropriate for the assay. During this evaluation, the dilution of each conjugate that yields desirable sensitivity and specificity should be determined using appropriate controls.

The most commonly used labeling enzymes for conjugating to antibodies include alkaline phosphatase (AP), horseradish peroxidase (HRP), and galactosidase. These enzymes are highly specific, sensitive, and stable in catalyzing chromogenic, luminescent, or fluorescent reactions. para-Nitrophenyl phosphate (pNPP) is a commonly used substrate for AP. Commonly used substrates for HRP include TMB, OPD (o-phenylenediamine dihydrochloride), and ABTS [2,2¢-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt] (see Table 2). The substrates for AP and HRP are chromogenic and result in the formation of a colorimetric product that can be measured using a spectrophotometer. Chemiluminescent and fluorescent substrates for AP and HRP also are available, and in many cases they are available as commercial kits. Disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2¢-(5¢-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate (CSPD) is a known chemiluminescent substrate for AP (see Table 2). Well-known fluorescent substrates for galactosidase include MG (4-methylumbelliferyl galactoside) and NG (nitrophenyl galactoside). If a chemiluminescent substrate is used, then a luminometer is required to quantitate the formed product. A fluorometer is needed if a fluorescent substrate is used in the ELISA.

Table 2 also provides a summary of the advantages and disadvantages of different types of ELISA substrates. Colorimetric substrates have been prevalent since the origin of ELISAs and may yield robust assays that generally are more cost efficient than assays that use chemiluminescent and fluorescent substrates. Nevertheless, chemiluminescent and fluorescent ELISA methods may yield more rapid and sensitive assays with a wider dynamic range than assays that use a colorimetric readout. The final choice of readout should be governed by the assay’s purpose and the requirements of the assay.

Key considerations for critical reagents are source, purity, specificity, and stability. For quality measurements, ITMs use reference standards along with critical reagents for analyte capture and detection. Any changes of critical biological reagents should be evaluated (see, for example, guidance contained in Design and Development of Biological Assays 1032.)

The steps of the process by which an ELISA method is developed, validated, and used in routine sample analysis are described below:

During assay development, the critical parameters and reagents that are required for the assay should be assessed and set at levels that yield desired assay performance. In many instances several parameters may be evaluated, and well-designed experiments can accelerate assay development, particularly for assessing the potential interaction of several inputs.

Many ELISA procedures are product specific, and external reference/calibration standards may not be available. The preparation and stability of reference/calibration standards should be considered early in assay development.

Assay validation is executed according to guidances from appropriate regulatory bodies (e.g., ICH Q2) to demonstrate that the particular test used for an analyte is appropriate for its intended use. More information about assay validation can be found in the general information chapter Validation of Compendial Procedures 1225 or in general information chapter Biological Assay Validation 1033 if the ELISA is used as a surrogate potency assay. See Appendix 2 for additional information.

Basic statistical methods are not detailed here. General information chapter Analytical Data—Interpretation and Treatment 1010 addresses important fundamentals, including data handling; computation of means, standard deviations, and standard errors; detection of and methods to address nonconstant or nonnormal variation; detection of and management of outliers; and procedures for and interpretation of statistical tests and confidence intervals. The concepts behind validation, goals, designs, analysis, and practical methods for validation are described in general information chapters Analytical Data—Interpretation and Treatment 1010, Validation of Compendial Procedures 1225, and Biological Assay Validation 1033. General test chapter Design and Analysis of Biological Assays 111 contains guidance on combining results from independent assays.

Nonlinear calibration for immunoassays draws on many sources for statistical design and analysis. These include methods for assessing and addressing nonconstant variance, designs and analysis methods for experiments with complex structures, and validation. The concepts behind linear calibration design, analysis, and inverse regression apply in nonlinear calibration, and professional statisticians can help apply these appropriately.

Reported estimates of concentration should be understood as having an associated confidence interval based on the results of the validation. The reported value or estimate used to describe a sample can be based on a combined result from multiple assays.