051520 ITC Blog – Immunoassays

Talk about powerful. Immunoassay tests are used beyond the hospital. There they help medical professionals detect and assess the progression of disease. In the lab they sniff out contaminants in our food and water.

These tests also are crucial in the progress of life sciences, where immunoassays help to track how proteins, hormones, and antibodies act or react in our bodies. Immunoassays are amazing detectives – at the molecular level.

 

Also known as ELISA

Immunoassays have been a diagnostic tool for more than 40 years now. These tests can detect specific molecules and are used both in laboratory environments and on site at medical establishments for point of care testing. The test relies on the inherent ability of an antibody to bind to the specific structure of a molecule.

Animals and humans generate antibodies when their bodies detect antigens – or foreign molecules. Protein-based antibodies bind to antigens, making them a perfect detection device because antibodies will only bind to the specific antigen.

 

Rapid Assay

Some immunoassay tests can be done quickly, such as a home pregnancy test. Known as rapid assays, these tests utilize the antibody/antigen relationship and porous membranes. These membranes react with positive samples by channeling excess fluid to non-reactive areas of the membrane.

Benefits of this type of immunoassay include minimal or no processing steps, along with the ability to quickly determine a positive or negative result without the need of instrumentation. These assays are also often less expensive, and the results can be read by nonprofessionals.

 

Antibody Sandwich Immunoassay

There are generally four steps to these types of immunoassays:

1. Prior to a sample being added, capture antibodies are adsorbed onto the well of a plastic microtiter plate with a coating buffer. A blocking buffer is subsequently used to block remaining protein-binding sites on the microtiter plate and stabilize coated antibodies.

2. The sample to be tested is added, and the target antigen binds to the antibody absorbed on the plate. The antigen is retained in the well.

3. An enzyme-conjugated detection antibody is added, and the conjugate binds to the target antigen, which is itself bound to the capture antibody on the plate. This creates an “antigen sandwich” between the two types of antibodies.

4. A colorimetric substrate is added to detect the enzyme-conjugated antibodies. It generates a specific color signal which is proportional to the amount of antigen present in the sample.

Antigen-Down Immunoassay

In this ELISA format, an analyte is coated onto the microtiter plate instead of an antibody. When a sample is added, the antigen on the plate is bound by antibodies from the sample and retained in the well. A species-specific antibody (anti-human IgE for example) labeled with HRP is added next. It also binds to the antibody bound to the antigen on the plate. A higher presence of antibodies creates a stronger signal. These tests can be done quickly and are commonly used to diagnose allergies or various infectious diseases.

 

Competitive Inhibition Immunoassay

We’ve already walked through the steps of the Antibody Sandwich Assay, which is the foundation of a standard ELISA format. There are times when it is necessary, however, to measure the binding of only one antibody. This test makes it possible. Competitive Inhibition assays measure the concentration of an antigen by detection of signal interference. In one version, labeled antigen competes with sample antigen (unlabeled) for binding to a primary antibody. The colored signal is inversely proportional to the concentration of unlabeled target analyte in the sample – the lower the amount of antigen in the sample, the stronger the signal due to the presence of more labeled antigen in the well.

 

We offer a comprehensive line of coating buffers, blockers, sample and assay diluents, conjugate stabilizers, and wash buffers. Our products are designed to work together to minimize the build up of unwanted proteins to generate a very clean signal. These products work together to address common issues during ELISA development, such as specificity, sensitivity, reproducibility, and shelf-life. Learn more.