Immunoassays and the different options

Be it for determining a Vitamin-D deficiency or the presence of COVID-19 or HIV; there are many types of immunoassays for measuring specific biomarkers.

When discussing immunoassays and the various options in the field, it is important to be aware of the way an immunoassay functions. The immunoassays developed verify the presence of specific markers in a specimen by using antibodies and antigens. By setting up the right composition a reaction is triggered. In short, any reaction indicates the presence of a specific substance. If there is no reaction, the marker for which the test is created is not identifiable in the specimen.

A biomarker can be measured in different kinds of immunoassays. Although the outcome is similar, the method and technology behind the test may differ entirely. To illustrate this, we will highlight some assay models. Colorimetric immunoassay, Fluorescence and Chemiluminescence Immunoassays, IHC Immunohistochemistry, Turbidimetric Immunoassay, Radioimmunoassay

Colorimetric immunoassay

The enzyme-linked immunosorbent assay determines various entities such as the presence of antibodies to an infectious disease. EIA (Enzyme Immunoassay) also known as ELISA (Enzyme Linked Immune Sorbent Assay), detects and measures antibodies in samples as a reaction to specific antigens, or vice-versa. To name only a few as an example, ELISA-tests are available to test for the presence of Vitamin D, insulin, COVID-19, or hepatitis antigens in samples.

A basic ELISA-test can be executed by coating antigens or antibodies to a surface. The decision to coat an antigen or antibody on the surface will be made depending on whether target detection will be of an antibody or antigen. Next, in the instance of an antigen coated on the surface, a sample potentially containing matching antibodies is applied to the surface. Only antibodies present to a specific marker bind to the antigens. Actual binding depends on the presence or absence of these antibodies, and all antibodies that do not bind are discarded. What is left is the pure antibodies binding of interest.

In an ELISA-test a detecting or secondary antibody or antigen is also utilized. These are typically linked to enzymes, which interact in some fashion with the antibody/antigen binding discussed above. The final step in ELISAs consists of adding a substrate that reacts with this enzyme. This reaction produces a color formation that is used to determine the binding interactions that have occurred.

Enzyme-linked Immunosorbent Assay (ELISA) was developed in 1970 and rapidly became accepted as a standard routine assay technology. The assay format is sometimes referred to as EIA (Enzyme Immuno Assay).

The assay format combines the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily assayed enzyme. ELISAs can provide a useful measurement of antigen or antibody concentration. There are two main variations on this method: The ELISA can be used to detect the presence of antigens that are recognized by an antibody, or it can be used to test for antibodies that recognize an antigen.

A simplified ELISA procedure:

1. Coat the microtiter plate wells with antigen

2. Block all unbound sites to prevent false positive results

3. Add antibody to the wells

4. Add anti- IgG conjugated to an enzyme

5. Reaction of a substrate with the enzyme to produce a colored product, thus indicating a positive reaction. The color development is related to bound ligand and is measured spectrophotometrically.

There are many different types of ELISAs. Currently the most important ones are:

Competitive methods

Sandwich methods

Antibody capture methods

Fluorescence and Chemiluminescence Immunoassays

Besides the use of immunoassays based on color intensity to detect antibodies in specimens, detection can also be done by fluorescent tracers and light-generating molecules. These are called Fluorescent Immunoassay (FIA) and Chemiluminescence Immunoassay (CLIA). The basis of these immunoassays is a variant of ELISA. The substrate used does not generate color but emits light.

IHC Immunohistochemistry

Visualization of the antigen present in tissue sections is accomplished in a multi-step immunohistochemical staining process, in conjunction with a horseradish peroxidase (HRP) or alkaline phosphatase (AP) linked detection system. The process involves the addition of the stated antibody (primary antibody) to a tissue slide, followed by a secondary antibody (link antibody) which specifically binds to the primary antibody. A chromogenic substrate is then added which reacts with the enzyme complex, resulting in a colorimetric reaction at the site of the antigen. Results are interpreted using a light microscope.

Turbidimetric Immunoassay

Turbidimetry is a method of determining the number of cells in a solution by measuring the turbidity. This turbidimetric approach is a quick one-step method to follow the complex formed by antigen-antibody reactions. Moreover, Turbidimetric-based immunoassays are less sensitive compared to enzyme-based methods.

Turbidimetry-based immunoassays are applied to detect the presence of, for example, calprotectin or COVID-19 in blood.

Radioimmunoassay

Radioimmunoassay (RIA) is a very sensitive assay technique used to measure the concentrations of antigens (for example, hormone levels in the blood) using antibodies to those antigens.

In an RIA, a known quantity of an antigen is made radioactive and mixed with a known quantity of antibody to that antigen. When a sample containing the antigen of interest is added, the non-radioactive antigen displaces the bound radioactive antigen. By washing away unbound antigen and measuring the ratio of the radioactivity in the supernatant versus known standards, a standard curve can be developed and the amount of antigen in the sample determined.

*****Radioactive materials may only be purchased by persons / Facilities who have a specific license from the NRC (Nuclear Regulatory Commission). IBL-America requires that a copy of the NRC license or State Radioactive Materials License is uploaded before the order is processed.

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If the stock is sold out, we will unfortunately have to postpone your shipment on the next iodination date. 

IBL-America has a product catalog that incorporates many of the aforementioned methodologies. Please contact us to discuss how we can collaborate with you to find a solution to your testing needs.