Enzyme-linked Immunosorbent Assays (ELISAs)


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a) Diagram of a sandwich ELISA showing what happens in both a positive and a negative sample. First, the primary antibody binds to the well. This is shown as Ys bound to a surface. Next, blocking agent is added. This is shown as a black covering on the surface between the antibodies. Next, the sample is added; if the correct antigen is present it binds to the antibody. In the positive well a circle binds to the antibodies; in the negative well nothing binds to the antibodies. Next, any unbound sample is washed away. Next, antibody-enzyme conjugate is added. This is shown in the positive well as another Y shape that binds to the circles. This new Y shape has a purple circle at the end. These antibodies are also in the negative sample but they are not attached to anything. Next, unbound antibody-enzyme conjugate is washed away; these remain in the positive sample (because they are attached to the antigen) but are washed away in the negative sample. Finally, a substrate is added to both the positive and negative samples. The enzyme in the positive sample turns this substrate blue. b) A plastic plate with many wells. Some are clear, some are blue.
(a) In a sandwich ELISA, a primary antibody is used to first capture an antigen with the primary antibody. A secondary antibody conjugated to an enzyme that also recognizes epitopes on the antigen is added. After the addition of the chromogen, a spectrophotometer measures the absorbance of end product, which is directly proportional to the amount of captured antigen. (b) An ELISA plate shows dilutions of antibodies (left) and antigens (bottom). Higher concentrations result in a darker final color. (credit b: modification of work by U.S. Fish and Wildlife Service Pacific Region)

OpenStax Microbiology

The enzyme-linked immunosorbent assays (ELISAs) are widely used EIAs. In the direct ELISA, antigens are immobilized in the well of a microtiter plate. An antibody that is specific for a particular antigen and is conjugated to an enzyme is added to each well. If the antigen is present, then the antibody will bind. After washing to remove any unbound antibodies, a colorless substrate (chromogen) is added. The presence of the enzyme converts the substrate into a colored end product. While this technique is faster because it only requires the use of one antibody, it has the disadvantage that the signal from a direct ELISA is lower (lower sensitivity).

In a sandwich ELISA, the goal is to use antibodies to precisely quantify specific antigen present in a solution, such as antigen from a pathogen, a serum protein, or a hormone from the blood or urine to list just a few examples. The first step of a sandwich ELISA is to add the primary antibody to all the wells of a microtiter plate. The antibody sticks to the plastic by hydrophobic interactions. After an appropriate incubation time, any unbound antibody is washed away. Comparable washes are used between each of the subsequent steps to ensure that only specifically bound molecules remain attached to the plate. A blocking protein is then added (e.g., albumin or the milk protein casein) to bind the remaining nonspecific protein-binding sites in the well. Some of the wells will receive known amounts of antigen to allow the construction of a standard curve, and unknown antigen solutions are added to the other wells. The primary antibody captures the antigen and, following a wash, the secondary antibody is added, which is a polyclonal antibody that is conjugated to an enzyme. After a final wash, a colorless substrate (chromogen) is added, and the enzyme converts it into a colored end product. The color intensity of the sample caused by the end product is measured with a spectrophotometer. The amount of color produced (measured as absorbance) is directly proportional to the amount of enzyme, which in turn is directly proportional to the captured antigen. ELISAs are extremely sensitive, allowing antigen to be quantified in the nanogram (10–9 g) per mL range.

In an indirect ELISA, we quantify antigen-specific antibody rather than antigen. We can use indirect ELISA to detect antibodies against many types of pathogens, including Borrelia burgdorferi (Lyme disease) and HIV. There are three important differences between indirect and direct ELISAs as shown in the image below. Rather than using antibody to capture antigen, the indirect ELISA starts with attaching known antigen (e.g., peptides from HIV) to the bottom of the microtiter plate wells. After blocking the unbound sites on the plate, patient serum is added; if antibodies are present (primary antibody), they will bind the antigen. After washing away any unbound proteins, the secondary antibody with its conjugated enzyme is directed against the primary antibody (e.g., antihuman immunoglobulin). The secondary antibody allows us to quantify how much antigen-specific antibody is present in the patient’s serum by the intensity of the color produced from the conjugated enzyme-chromogen reaction.

As with several other tests for antibodies, there is always concern about cross-reactivity with antibodies directed against some other antigen, which can lead to false-positive results. Thus, we cannot definitively diagnose an HIV infection (or any other type of infection) based on a single indirect ELISA assay. We must confirm any suspected positive test, which is most often done using either an immunoblot that actually identifies the presence of specific peptides from the pathogen or a test to identify the nucleic acids associated with the pathogen, such as reverse transcriptase PCR (RT-PCR) or a nucleic acid antigen test.

Diagram of indirect ELISA showing what happens in both a positive and a negative sample. First the antigens are bound to the well. This is shown as diamonds on a surface of both the positive and negative wells. Next, blocking agent is added. This is shown as a black covering on the surface between the antigens. Next, the sample is added. If the correct antibody is present it binds to the antigen. This is shown as Ys in both the positive and negative wells. Otherwise, nothing binds to the antigen. Next, any unbound sample is washed away. In the positive well, there is an antibody bound to the antigen, in the negative well, there is nothing bound to the antigen. Next, anti-human enzyme-linked antibody is added. This is shown as a Ys with a purple circle in both wells. Next, unbound antigen in washed away. In the positive sample this Y remains bound to the old antibody. In the negative well it is no longer present. Finally, substrate is added to both wells. In the positive well, the enzyme changes the substrate to a blue color.
The indirect ELISA is used to quantify antigen-specific antibodies in patient serum for disease diagnosis. Antigen from the suspected disease agent is attached to microtiter plates. The primary antibody comes from the patient’s serum, which is subsequently bound by the enzyme-conjugated secondary antibody. Measuring the production of end product allows us to detect or quantify the amount of antigen-specific antibody present in the patient’s serum.

Source: OpenStax Microbiology

Source:

Parker, N., Schneegurt, M., Thi Tu, A.-H., Forster, B. M., & Lister, P. (n.d.). Microbiology. Houston, Texas: OpenStax. Access for free at: https://openstax.org/details/books/microbiology