ELISA Testing Service, ELISA Assay Validation & Development by PhD Scientists at 20+ Year, FDA-Inspected Lab

ELISA Method: Accurate and Flexible Analysis in 96 well plates

Enzyme Linked Immunosorbent Assay (ELISA Assay) method is a widely utilized immunoassay for quantitating and detecting proteins, hormones, peptides(through ELISA peptide), cytokines, antibodies, and other drugs along with their metabolites. ELISA Assay methods are effective in quantitatively detecting any molecule, or antigen, that can be ascertained by an antibody. For example, ELISA methods are used in pregnancy testing, infectious disease identification, and detection of cytokines, and soluble receptor proteins, etc. Due to the precision, sensitivity, assay speed, and ease of quantitation, ELISA method development is a common choice for several diagnostic and research applications.

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. Spectrophotometry is used to monitor the colored product, and a standard curve is utilized for calculating the antigen concentration in the sample. 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.

It is notable that the ELISA method typically uses 96 microwell plates for parallel analysis of many samples, standards, and controls in a single experiment. The surface of these plates is treated with special absorbents such that the antigen or antibody can adhere properly. ELISA method offers increased sensitivity and specificity compared to other standard antibody-based assays. In ELISA method, the interactions with analyte or antibody occur through serial binding that is done to some solid surface such as polystyrene in the 96 microwell plates discussed above.

Step-by-step ELISA Method Development

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. ELISA method is thus a solid-phase assay.

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. Thus, it is critical to immobilize the antibody or antigen while preserving its conformational structure.

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.

Extensive ELISA Method Development by NorthEast BioLab

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. Our specialists optimize the assay by titrating various components to ensure accurate results.

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. TK studies allow estimation of the level of toxicity that can be produced by these drug compounds.

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 in the body of humans or other animals. It further evaluates 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. For example, we perform cytotoxicity testing and mechanism of action (MOA) assay to understand the biochemical reactions triggered by drug compounds.

Frictionless ELISA Method from scratch or validated Research Use Only (RUO) kits

At NorthEast BioLab, we ensure robust ELISA methods to quantify large molecule analytes such as biologics, proteins, and antibodies in your samples. As often as it takes, our veteran scientists with 30+ years experience discuss and study your ELISA method development and validation in depth. Our team continues without yielding until we have customized these assays per your requirement and satisfaction.

We encourage intensive collaboration between the client and our scientists for complete transparency throughout your study with us. NorthEast BioLab offers quick turnaround times, scalability, and flexibility, given our extensive experience with ELISA method development and a thorough understanding of the science behind it. We enable our clients to expedite drug approval through efficient assay development, validation, and optimization while maintaining 100% regulatory compliance.

ELISA Method, based on colorimetric or chemiluminescent reaction, is the best-in-class ligand binding assay (LBA) predominantly used for large molecule bioanalytical method development and sample analysis. Scientists and regulatory agencies widely trust the direct, indirect, sandwich, and competitive variants of ELISA Development and Method Validation for diverse PK, BA/BE, TK, Immunogenicity (ADA), and Biomarker testing. Our veteran team undertakes Enzyme Linked Immunosorbent Assay development from scratch using available antibodies, diluents, buffers, substrates, reagents, control matrices, as well as research use only (RUO) kits. NorthEast BioLab delivers research mode, and fully GLP validated ELISA Method and Assay Development at an unmatched turnaround and value to sponsors.

ELISA Method is an abbreviation for enzyme-linked immunosorbent assay, also called enzyme immunoassays (EIA). The fundamental principle used in ELISA method development is the use of antibodies to detect and quantify proteins, peptides (through ELISA peptide), hormones, or other small molecules in preclinical or clinical samples. In an ELISA method development, antigens are immobilized on solid support either directly or via the use of a capture antibody immobilized on the surface. Subsequently, we add a primary detection antibody. If the detection antibody binds to the analyte of interest, the complex can be directly detected and quantified by the chromogenic tag or a secondary enzyme-conjugated antibody. In our GLP ELISA lab, we typically perform method development, validation, and sample analysis in multi-well plates (96- or 384-wells) for capture antigen binding. This characteristic makes the ELISA Assay Method easy to perform on multiple samples simultaneously with excellent selectivity.

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. The first antibody, often called the capture antibody, is coated on the 96 well plate and binds to the analyte of interest. Once the unbound materials in the sample matrix get washed away, the second antibody conjugated with a detection tag quantitates the analyte. Competitive ELISA methods, often referred to as an inhibition ELISA assay or competitive immunoassays, measure the concentration of an antigen by detecting signal interference. We can adapt direct, indirect, or sandwich ELISA assays to the competitive format. Here, the antigen in the sample competes with a reference antigen pre-coated on a multi-well plate or added in the same plate wells. Consequently, lower concentrations of analyte produce a higher level of the signal. Some competitive ELISA assay, however, utilize a slightly different approach. These kits substitute a labeled antigen for the labeled antibody, but the principle of the ELISA protein assay remains the same.

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 (e.g., type of assay plates, coating/washing/dilution buffer, coating-detection concentration, calibration standard/quality control/sample preparation, incubation times, etc.) to meet/exceed our immunoassay validation and sample analysis acceptance criteria. One of the most significant challenges faced by sponsor or ELISA Lab working on sandwich ELISA method is narrowing down a proprietary/commercially available antibody pair that works well together for desired assay parameters.

In our GLP ELISA Lab, we perform both direct and indirect ELISA method development that differ in their means of detection. In the direct ELISA assay, an antigen is immobilized to the surface of a multi-well plate and detected with an antigen-specific antibody directly conjugated to HRP or other detection molecules. For an indirect ELISA assay, as with the direct ELISA method, the 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 analyte of interest and the secondary antibody labeled with the detection agent is used for quantitation. Direct ELISA methods are simple to set up and have no cross-reactivity from a secondary antibody but may have poor sensitivity due to high background as all proteins in the sample bind the well surface and there is no signal amplification to read the antigen. Indirect ELISA methods offer signal amplification to improve sensitivity but are more challenging to set up and perform and may have cross-reactivity from the secondary antibody.

The principle of ELISA involves antigen detection via binding to specific antibodies that we can apply in various formats. Direct ELISA method offers the advantages of easy experimental setup and execution, saving on time and reagent costs, and minimize 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. Also, the primary antibody must be conjugated, and there is no signal amplification for analyte detection.

Indirect ELISA methods allow for signal amplification since multiple secondary antibodies can bind to the primary antibody. Indirect methods are also adaptable, allowing us to use the same secondary antibody for several primary antibodies. Disadvantages of an indirect ELISA method are potential cross-reactivity to the secondary antibody and higher time and cost needs relative to the direct ELISA method.

Sandwich ELISA methods offer excellent specificity as they employ two distinct antibodies for capture and detection. These ELISA, however, can be difficult to develop since discovering two antibodies that recognize two different epitopes on the same target and work well together can at times be difficult.

We can modify any ELISA method to a competitive format with a reference antigen and appropriate detection. Our team often employs Competitive ELISA to detect and quantitate low molecular weight antigens, such as small molecule drug compounds (e.g., Gentamicin).

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. In summary, our ELISA labs and assay kit vendor ecosystem can be immensely advantageous to your bioanalytical method ELISA development and validation. For many of your drug R&D assays, we can optimize commercial ELISA kits for performance, selectivity, sensitivity, and are 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 (May, 2018), 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 Intraday 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. Furthermore, we need to carefully optimize antibody and sample concentration and incubation times for ELISA development and validation. For instance, in ELISA Peptide is important for mapping of epitopes or definition of protein interaction sites. Among other factors, our scientists pay close attention to signal-to-noise (SNR) ratio, false-positive due to endogenous compounds, interference from endogenous or exogenous compounds, substrate selection, etc., 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 to perform ELISA Development and Validation. Generally, ELISA analysis plate readouts and incubation steps are highly time-dependent. Thus, we must emphasize that 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 for ELISA assay 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, 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 (IS) 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 ELISA IS to verify that your ELISA Lab properly executes the assay steps and dilutions.

Generally, our ELISA lab can perform sample analysis from 2-to-4 96-well plates within a typical workday if we have ELISA method development and immunoassay validation completed already. That said, sample preparation can vary per the complexity of the biological matrix and ELISA analysis itself (e.g., tissues or whole cells vs. serum). Similarly, sample incubation times can vary, typically overnight or less. Our scientists closely monitor and control the incubation and readout times during ELISA Method Development and sample analysis for consistency in your ELISA immunoassay results. Since the ELISA assay readouts are enzyme-dependent, lab personnel should read plates in the optimal reaction time window as the signal can decay markedly over time.

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.

ELISA methods and other types of enzyme immunoassays are typically used to quantitate large molecules in biological matrices with excellent sensitivity and specificity. It can be quite challenging to develop an ELISA method for a novel biologic molecule in-house. Thus, you would be in the superb company of pioneering biotech to rely on our ELISA lab to characterize your drug pharmacokinetics or its correlation to pharmacodynamic responses related to efficacy or toxicity. Generally, proper ELISA lab method development offers versatility and utility for biomarker determinations in relation to disease states and disease treatment and modulation. As with any other quantitative bioanalytical method, ELISA assay should be fully validated or at least partially qualified before use as appropriate to your study objectives.

Research use only (RUO) label indicates in vitro diagnostic (IVD) products that can be used in the laboratory research phase of development. RUO ELISA kits are helpful for early drug discovery or preliminary diagnostic testing analyses but are not yet fully validated for compliance under GLP or clinical regulations. Commercially available RUO kits for ELISA lab protocols 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 of disease or therapy.

Enzyme linked fluorescent assay (ELFA) is a similar enzyme immunoassay that uses the same principles as the ELISA assay but differs in the sample detection method. We use a fluorogenic substrate for detection in the ELFA assay instead of the chromogenic substrates used in ELISA assays. In general, ELFA is more sensitive than ELISA, making it helpful in detecting antigen biomarkers in earlier time windows relative to exposure for diagnostic disease testing.