ELISA assays are a heterogeneous enzyme immunoassay technique used in clinical analysis. Today, ELISA immunoassays are an integral component of clinical pharmacokinetics services. This assay format has one reaction component bound to a solid phase, such as plastic beads, magnetic particles, or microtiter well. This attachment is covalently bound or non-specifically adsorbed, which facilitates the detection of the target analyte.
With the growing demand for ELISA immunoassays, several laboratories, such as ELISA testing services and ELISA assay development service providers, are focused on providing robust ELISA development and validation solutions. Both ELISA assay development and ELISA assay validation are essential for instilling confidence in the generated data. The current article dives deep into the working of ELISA immunoassay and its necessity in biomedical research.
ELISA immunoassays for biomedical research
The four common ELISA assay formats include direct, indirect, sandwich, and competitive ELISA. Sandwich ELISA assays are one of the most commonly employed ELISA formats. In this approach, a sample containing the target analyte is added to a well and allowed to bind within an antibody attached to the solid surface. After a washing step, researchers add an enzyme-labeled antibody to form a sandwich complex. Any unbound antibody is washed away, and a substrate is added to generate a detectable signal. The amount of generated signal is directly proportional to the quantity of analyte in the sample.
The four primary steps in an ELISA immunoassay include coating with either antibody or antigen, blocking typically with bowing serum albumin, detection, and final read. ELISA detection is performed using a substrate that generates a color output. Multiple substrates are available for ELISA detection. However, alkaline phosphatase and horseradish peroxidase are the most commonly used substrates in ELISA immunoassays.
Must Read: Validation and Quality Control in ELISA Services: Ensuring Reliable Results
ELISA assays have multiple diagnostic applications, such as:
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Detecting and measuring antibodies in the blood, such as autoantibodies and antibodies against infectious diseases.
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Detecting and estimating tumor markers such as carcinoembryonic antigen and prostate-specific antigen.
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Detecting and estimating hormone levels of testosterone, prolactin, follicular stimulating hormone, luteinizing hormone, and human chorionic gonadotropin.
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Monitoring disease outbreaks of Cholera and influenza.
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Screening donated blood samples for possible contaminants and detecting past exposure.
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Detecting drug abuse such as cocaine and amphetamine.
Biological samples are inherently complex. Besides, they have endogenous components that may interfere with ELISA results. Multiple factors can interfere with ELISA assays at any phase of the experimental process. Factors that may interfere with ELISA immunoassay testing include the integrity and quality of the assay plate, capture antibody, blocking buffer, coating buffer, detection antibody, target antigen, enzyme conjugate substrate, washing step, and signal detection.
ELISA assays can be qualitative, semiquantitative, or quantitative. Qualitative ELISA confirms or denies the presence of an analyte in a biological sample. Semiquantitative ELISA compares signal intensity relative to an analyte level in the sample. On the other hand, quantitative ELISA plots sample concentrations and compares them to a standard curve.
Nevertheless, verifying the accuracy of quantitative and semiquantitative ELISA testing is critical. Each ELISA assay laboratory should have a quality control program with a professional and trained staff complying with standard operating procedures and regulatory requirements.
