ELISA Testing Service, ELISA Assay Validation and Development, by PhDs at 20+ Year, FDA-Inspected Lab

ELISA is a plate-based immunoassay that uses an antibody to capture the analyte and an enzyme-linked detection step to quantify it, which makes it a workhorse for measuring proteins, peptides, antibodies, cytokines and other large molecules in biological samples. In drug development it carries PK quantitation, immunogenicity tiers and biomarker panels. Its precision, speed and adaptability across formats are why ELISA method development remains the standard route for large molecule bioanalysis.

While there are various formats of ELISA method, the most common is a sandwich ELISA method. In this format, the analyte whose concentration is to be measured is sandwiched in-between two antibodies that bind to a different region (epitopes) on the antigen. These antibodies are referred to as detection antibody and capture antibody. In this assay, the capture antibody is coated to a microtiter plate in a 96-well format. The antigen binds to the capture antibody, and a detection antibody is used to measure the analyte. The antibody is conjugated to an enzyme, typically horseradish peroxidase (HRP), and detected via catalysis of a substrate which yields a colored product. When the antigen is small, or two antibodies that can simultaneously bind to the antigen are unavailable, then another variation of ELISA, termed competitive ELISA method, is utilized using a single epitope.

Enzyme Linked Immunosorbent Assay or ELISA Method development is a complex task involving multiple sequential steps for the measurement of analyte concentration in a sample.

Plate Coating

Whenever an ELISA method is developed for any analyte, a surface attachment strategy is established first. Once the analyte is affixed to the surface of the plate, it is immobilized and helps with the sequential addition of other reagents and washing cycles in the next steps, without mixing. The process of immobilizing an antibody or antigen to the plate is essential, as the method of immobilization utilized is what determines the efficacy of the ELISA method. Many times, the immobilization may cause a conformational change in the analyte or antibody, preventing the detection antibody from binding.

Antibody and Antigen Pairs

The specificity and sensitivity of the assay are determined using a range of concentrations of antibody and/or antigen. The affinity of the detection antibody and sensitivity of the detection reagents corresponding to a specific antigen are principally responsible for defining the performance of an optimized ELISA method. Selection of the best capture and detection antibody is critical for effective assay development.

Detection

The antibodies which are labeled with an enzyme (detection antibodies) contribute to the signal output of this assay. Different types of enzymes can be utilized, such as alkaline phosphatase, horseradish peroxidase, etc., each of which produces a color upon reacting with their substrates. These colored products are detected with spectrophotometry, where the signal intensity is proportional to the amount of antigen in the microwell. Afterward, a standard curve along with positive and negative controls is used to quantify the amount of analyte within a given sample.

NorthEast BioLab offers various client-centric ELISA assay development services.

Bioanalytical Assay ELISA Development and Validation

Proper ELISA method development and validation are critical for the accurate quantification of an analyte. NorthEast BioLab helps you select the right ELISA method format and develop a robust protocol for analysis.

Pharmacokinetics (PK) Studies

Our pharmacokinetic studies for quantitation of drug-responsive levels of a specific serum protein using ELISA protein assay development allow accurate and reliable pharmacokinetic data to help regulate dosage regimens.

Toxicokinetic (TK) Studies

As in pharmacokinetic studies, an ELISA method can be used in toxicokinetic studies to quantitate a drug or compound when administered at high dosage.

Biomarker Testing

ELISA method is hugely popular as a fundamental, cost-effective technique that offers high precision and broad dynamic range in addition to specificity, selectivity, and reliability for biomarker testing and quantitation.

Immunogenicity Testing Assay

An ELISA method can be used for immunogenicity testing. These studies are carried out to assess how drug exposure induces an immune response, and how anti-drug antibodies (ADA) can affect immunogenicity.

Cell-Based Assay

Our scientists help clients with the successful execution of cell-based assays to assess the toxicity of the compounds and ensure reliable drug manufacturing.

ELISA immunoassays include a direct, indirect, sandwich, and competitive variants. In the direct ELISA method, an antigen is immobilized to the surface of a multi-well plate and detected with an antigen-specific antibody directly conjugated to HRP or another detection tag.

For an indirect ELISA assay, as with the direct ELISA method, an antigen is immobilized to the surface of the multi-well plate, but we need a two-step process for detection. The primary antibody binds to the antigen of interest, and then a secondary antibody labeled with a detection reagent is used for quantification. The sandwich ELISA method, the most widely used format, requires an antibody pair where each antibody is specific to a different epitope of the antigen. Competitive ELISA methods, often referred to as an inhibition ELISA assay, measure the concentration of an antigen by detecting signal interference, where the antigen in the sample competes with a reference antigen and lower concentrations of analyte produce a higher level of the signal.

In the Sandwich ELISA method, the specificity and selectivity offered by matched antibody pairs provide improved sensitivity and broad dynamic range. These ELISA assays require two antibodies, attaching with different epitopes on the same antigen, making them suitable for accurately isolating and quantitating antigens of interest within complex biological matrices such as plasma, serum, CSF, tissue lysates, etc. Furthermore, sandwich ELISA development and validation are highly adaptable. We can optimize various assay conditions for the antibody pairs to meet or exceed our immunoassay validation and sample analysis acceptance criteria. One of the most significant challenges faced by a sponsor or ELISA lab working on a sandwich ELISA method is narrowing down a proprietary or commercially available antibody pair that works well together for the desired assay parameters.

Direct ELISA method offers the advantages of easy experimental setup and execution, saving on time and reagent costs, and minimizes concerns around cross-reactivity since there is no secondary antibody. Disadvantages of the direct ELISA method are the potential for high background due to all proteins in the sample binding the plate, the need to conjugate the primary antibody, and no signal amplification.

Indirect ELISA methods allow for signal amplification since multiple secondary antibodies can bind to the primary antibody, and are adaptable because the same secondary antibody serves several primary antibodies. Disadvantages are potential cross-reactivity to the secondary antibody and higher time and cost.

Sandwich ELISA methods offer excellent specificity as they employ two distinct antibodies for capture and detection, though they can be difficult to develop since two antibodies that recognize different epitopes on the same target must work well together. We can modify any ELISA method to a competitive format with a reference antigen and appropriate detection, which our team often employs for low molecular weight antigens such as small molecule drug compounds.

ELISA labs perform sensitive and selective quantitation of various antigens during drug discovery and development. We rely on ELISA method development to evaluate pharmacokinetics (PK) or anti-drug antibody (ADA) for novel biological treatments and determine biomarkers predictive of preclinical or clinical disease, therapeutic treatment, or toxicity adverse events. For many of your drug R&D assays, we can optimize commercial ELISA kits for performance, selectivity, sensitivity, and adaptability to multiple biological sample matrices.

Enzyme-linked immunosorbent assay (ELISA) method validation should be fit for purpose and follow the FDA Bioanalytical Method Validation Guidance for Industry, specifically related to ligand binding assays (LBAs). Generally, ELISA method validation includes –

• All applicable experiments for Stock Solutions, Reference Standards, Quality Controls, Calibration Curve, Minimum Required Dilution, Spike Recovery, etc
• Six independent validation runs over multiple days for estimating intraday and interday Accuracy and Precision
• Applicable experiments for Method Selectivity, Specificity, Matrix Effect, Matrix Suppression, etc.
• Process Stability experiments for Short-term Bench-Top, Freeze-Thaw, and Long-Term Cycles

ELISA labs perform method development and validation for your analyte of interest as per assay requirements such as calibration range and limitations such as antibody pair affinity, minimum required dilution, observed background, etc. In sandwich ELISA methods, the antibodies selected for capture and detection must bind to two distinct epitopes of the antigen and work well to achieve the quantitative assay goals. Also, we must choose appropriate plate binding surfaces, assay blockers, and buffers for diluents and washing to suit the sample matrix and yield selective and sensitive quantitation. Among other factors, our scientists pay close attention to signal-to-noise ratio, false positives due to endogenous compounds, interference from endogenous or exogenous compounds, and substrate selection, for enhancing your ELISA method’s sensitivity, selectivity, specificity, and dynamic range.

ELISA quantitation, like any other highly sensitive bioanalytical quantitation, should be handled with care and precision. To begin with, we must review proper calibration and verification of instrumentation, pipettes, and other equipment before use. Next, we must intensively train all ELISA lab personnel in protocol reproducibility, pipetting techniques, and pipette tip handling. Generally, ELISA analysis plate readouts and incubation steps are highly time-dependent, so plates must be analyzed quickly following the addition of substrate for any pre-reads or final reads after stopping the reaction. We can automate sample analysis with liquid handlers, plate washers, and other robotics adaptable to 96- or 384-well plates.

ELISA methods and enzyme immunoassays are valuable tools for analyzing large molecule antigens, proteins, and antibodies but are not suitable for all small molecules and alternate biomolecules. For proper execution, an ELISA protein assay must include reasonable incubation times, appropriate binding of antibodies to the intended epitopes for capture and detection, compatible buffers, and a suitable level of plate washing. Furthermore, ELISA methods do not generally include an internal standard as in chromatographic assays. That said, we may use a visible dye in the plate reader’s detection range to track dilutions throughout the assay as a surrogate to verify that the assay steps and dilutions are properly executed.

Once development and validation are complete, our lab runs two to four 96-well plates of study samples in a typical workday, with incubation and readout windows controlled for consistency. For the phase before that: single-analyte large molecule validation typically takes four to six weeks, plus about a week per added analyte, and a written scope with timeline comes back within two business days of your inquiry.

Usually, yes, and finding out is cheap relative to abandoning the assay. Kit failures in a real matrix are most often blocking, minimum required dilution or interference problems rather than bad antibodies, and those are solvable. We run a short diagnostic, tell you whether the kit can be optimized, converted to another format, or genuinely needs a custom pair, and quote only the path we would actually take.

When the study needs sensitivity or dynamic range the plate cannot give, or when sample volume has to feed many endpoints. A working antibody pair usually converts: capture and detection are adapted to electrode plates and the assay is requalified fit for purpose. The escalation is linked from this page, and because both platforms run in this building the conversion does not restart your program.

Our GLP ELISA lab regularly performs immunogenicity studies that look for the potential immune response to therapeutic agents as needed for clinical drug studies. A bridging enzyme linked immunoassay is one of the most common methods to test for the presence and quantification of these anti-drug antibodies (ADA). In this sandwich ELISA assay development, a biotinylated drug compound gets attached to the streptavidin coated ELISA plate, and another labeled drug compound gets utilized as a detection agent. Ultimately, the anti-drug antibody of interest bridges these drug compounds together. Among other antibodies, this ELISA method gets frequently used for IgG and IgM ADA detection.

Research use only (RUO) label indicates in vitro diagnostic products that can be used in the laboratory research phase of development. RUO ELISA kits are helpful for early drug discovery or preliminary analyses but are not yet fully validated for compliance under GLP or clinical regulations. Commercially available RUO kits are typically qualified to some degree to ensure specificity, accuracy, and precision. Still, we need to fully validate these kits for the stability of test articles and critical reagents, cross-reactivity, or matrix interference. RUO ELISA quantification procedures can be validated if the need arises, typically for novel biologic therapeutics reaching the preclinical toxicology phase or for novel clinical biomarkers.