The Enzyme-Linked Immunosorbent Assay (ELISA) is a fundamental biochemical technique used in medical diagnostics and biological research. This assay uses the specific binding properties of an antibody to its target antigen to detect and quantify substances like proteins, hormones, or antibodies within a liquid sample. ELISA provides a sensitive and specific way to measure the concentration of a particular molecule within a complex biological mixture. The reaction is visualized through an enzyme linked to an assay component, which produces a measurable signal, typically a change in color, proportional to the amount of target substance present.
Understanding the Four Main Types of ELISA
The structure of the assay determines its utility, utilizing four standard formats: direct, indirect, sandwich, and competitive. The direct ELISA is the simplest, immobilizing the antigen onto the microplate surface, followed by the addition of an enzyme-labeled primary antibody. This format is fast but has low sensitivity because it lacks a signal amplification step.
The indirect ELISA also coats the antigen onto the plate, but uses an unlabeled primary antibody first, followed by an enzyme-labeled secondary antibody that binds to the primary antibody. This secondary binding step allows for signal amplification, making the indirect format more sensitive than the direct method.
The sandwich ELISA is the most common format, known for its high sensitivity and specificity. Here, a capture antibody is coated onto the plate, which binds the antigen between itself and a second, enzyme-labeled detection antibody.
Competitive ELISA is chosen for detecting small antigens that cannot be bound by two antibodies simultaneously. In this assay, the antigen in the sample competes with a known amount of labeled antigen for limited binding sites on a capture antibody. A higher concentration of the target antigen results in a lower final signal, establishing an inverse relationship between signal and concentration.
Essential Materials and Pre-Assay Preparation
Successfully running an ELISA requires careful preparation of several specialized components. The assay is performed in a polystyrene microplate, typically with 96 wells, where the plastic surface passively adsorbs proteins. Central to the assay are the antibodies: a primary antibody that recognizes the target molecule and a secondary antibody, often called the detection antibody, linked to an enzyme like horseradish peroxidase (HRP) or alkaline phosphatase (AP).
The enzyme conjugate provides the measurable signal when it reacts with a specific substrate, such as TMB. Preparation also involves various buffers. A washing buffer, typically Phosphate-Buffered Saline with detergent (PBS-T), is used repeatedly to remove unbound materials.
A separate blocking buffer, often containing a protein like Bovine Serum Albumin (BSA), is prepared to coat any remaining exposed plastic surface in the well. This prevents non-specific binding of assay components or sample proteins to the plate, which would generate background noise.
Sample preparation is also important, as biological fluids like serum or plasma often need dilution to ensure the target concentration falls within the assay’s measurable range. Samples should be stored at cold temperatures to preserve the integrity of the target molecule. Diluting samples into a specific assay buffer minimizes interference from other components found within the biological matrix.
Step-by-Step Procedure for Running an ELISA
The first step is Plate Coating, where the capture antibody or target antigen is immobilized onto the microplate wells. The coating reagent is added and incubated, often overnight, to facilitate passive adsorption of the protein to the plastic surface. After incubation, the coating solution is removed, and the plate is thoroughly washed with wash buffer to eliminate unbound material.
Next is the Blocking step, where blocking buffer is added to fill any surface areas not occupied by the coated protein. This prevents subsequent assay components from sticking non-specifically to the well walls. The plate is incubated, typically for 30 minutes to one hour, before the wells are washed again.
The Sample and Antibody Incubation steps follow. First, the diluted sample is added and incubated, allowing the target antigen to bind to the immobilized capture antibody. After washing, the detection antibody is introduced; this antibody binds to the captured antigen, completing the “sandwich.” A final incubation with the enzyme-conjugated secondary antibody then binds to the detection antibody, unless a direct detection scheme is used.
Washing Steps are performed between almost every stage to ensure only specifically bound molecules remain attached. This repeated washing removes all unbound reagents, which is necessary for achieving a low background signal and accurate results.
The final step is Substrate Addition and Color Development. The enzyme’s specific substrate is added, catalyzing a reaction that changes the substrate into a colored or luminescent product. The intensity of this signal is directly proportional to the amount of the target substance captured in the well.
Analyzing and Interpreting the Results
The final step involves adding a stop solution, such as a dilute acid, which halts the enzymatic reaction and stabilizes the color. The microplate is then placed into a plate reader, a specialized spectrophotometer that measures the intensity of the color, or optical density (OD), in each well at a specific wavelength. This OD value is the raw data representing the amount of target molecule detected.
To translate OD values into a quantifiable concentration, a Standard Curve must be run on every plate. This curve uses a series of wells containing known, decreasing concentrations of the purified target substance. Plotting the OD reading (Y-axis) against the known concentration (X-axis) generates a reference curve.
The OD value of each unknown sample is located on the standard curve, allowing the corresponding concentration to be interpolated. If the original sample was diluted, the interpolated concentration must be multiplied by the dilution factor to determine the true concentration in the starting material. Running positive and negative controls validates the assay performance and ensures reliable results. For competitive ELISA, the analysis is different, as the signal is inversely related to the concentration.