As defined previously, antibodies are proteins that the immune system produces in response to harmful substances called antigens. Antibodies are antigen-specific due to the matching binding sites on the antibody and antigen. Immunoassays were created to capitalize on the special properties of antibody-antigen binding and are one of the major methods of identifying and quantifying the proteins present in biological samples.
Main principle
There are multiple types of immunoassays that differ in their specific methods; however, most immunoassays have three important components: Antibody, analyte (antigen which we want to detect), and label. Here they are:
To analyze the sample using an immunoassay, researchers add an antibody specific to the target antigen (analyte) to the solution. The analyte and antibody bind to one another when combined, acting as a lock and key. This binding process takes some time, so the sample is incubated, and then the unbound antibodies are removed from the solution, or "washed off," as many biologists say. Then researchers can detect whether the sample contains analyte-antibody complexes based on the signal produced by a label that is attached to the antibody.
What does it mean for an antibody to be "labeled," and how do we detect the signal? Immunoassays are often classified by the label or labeling method, and some examples are:
- enzyme immunoassays (or ELISA, most popular type of immunoassay!) — label is an enzyme called horseradish peroxidase (or HRP). After incubating the HRP-antibody complex with the analyte and washing off unbound antibodies, the researcher adds a special chromogenic substrate to the antibody-analyte solution. The substrate undergoes an enzymatic reaction catalyzed by HRP molecules attached to the bound antibodies and changes color. The degree of color change can then be measured using a special device called a spectrophotometer, which lets the researcher calculate the analyte concentration;
- fluorescent immunoassays — label is a fluorophore which, when excited by light of a specific wavelength, emits light at a specific, higher wavelength. After incubation of the sample and the fluorophore-tagged antibody, researchers expose the mixture of antibodies and analyte to light at the wavelength that will excite the fluorophore, and the resulting light emission can be measured using a photodetector. The intensity of the emitted light is directly proportional to the amount of antigen present in the sample;
- chemiluminescent immunoassays — this one combines the principles of the previous two: the label is usually the same HRP enzyme BUT the substrate is chemiluminescent, meaning it can emit light under certain circumstances. When the researcher adds the substrate to the mixture of antibodies and analyte, the enzyme label reacts with the substrate components and light is emitted and detected by a photodetector;
- radioassays (the cheapest immunoassay) — label is a radioactive isotope. After incubation and thorough washing, the researcher detects the level of gamma radiation emitted by the radiolabeled antibodies using a special device called gamma counter.
The most widely used kind of immunoassay today is ELISA. Let's learn more about it!
ELISA
ELISA is not the name of scientist who discovered the method, it is an acronym that stands for Enzyme-linked immunosorbent assay. Due to its simplicity, high sensitivity, and ability to test various types of samples, including urine, saliva, or blood, ELISA remains the most popular immunoassay technology so far.
There are several sub-types of ELISAs: direct, indirect, competitive, and sandwich ELISA. What are the main differences, and how do you choose the most suitable one for your experiment?
- Direct ELISA: the simplest ELISA type. The sample is added to the well, and the antigen (and all of the other proteins in the sample) bind to the surface, then an enzyme-conjugated antibody is added, which will bind to only the antigen of interest. After incubation and several washes, the substrate is added and the colorimetric reaction happens. Simple as that! It is not, however, the most sensitive type of ELISA.
- Indirect ELISA: here, the sample is added to the well as in the direct ELISA, and the antigen and other proteins in the sample bind to the surface. The difference is that there is first a primary antibody added that is specific to the antigen. Then, there is a secondary or detection antibody that is added. The secondary antibody, which contains the label, binds only to the primary antibody. This way, the signal gets amplified, and the flexibility increases: secondary antibodies are reactive to many different primary antibodies and can be used with any of them, decreasing the number of antibodies you need to buy;
- Sandwich ELISA: the most common type of ELISA. Here, antibodies specific to the analyte are attached to the surface, not the analyte. The antibody attached to the surface is called the capture antibody. The sample is added, and any analyte molecules present are captured by the capture antibodies and bound to the surface. Then all of the unbound proteins are washed off, and a solution of secondary or detection antibodies is added. Why do we need a second antibody? Two types of antibodies target different regions of the antigen, therefore making the binding much more accurate. The secondary antibody can be labeled using any of the above methods, making the sandwich ELISA quite versatile along with its high sensitivity and specificity;
- Competitive (Inhibition) ELISA: the procedure is quite different here: first, capture antibodies are added to the sample itself, where they form antigen-antibody complexes. Note that unbound antibodies remain in this solution. Afterwards, the solution is added to the plate, where the reference antigen is attached to the surface. The remaining free primary antibodies bind to the reference antigen molecules. Then, the researcher can wash off the complexes which are not attached to the surface of the plate. After that, a solution of secondary antibodies with enzymes is added, then the second wash, then the substrate goes in and finally, the colorimetric reaction is produced. What does it mean if we get a large color change? It means there were many complexes with the reference antigen because there weren't a lot of analyte molecules present in the original sample -> more antibodies were free to connect with our reference antigen because they hadn't connected to the sample antigen. As a result, the stronger the signal, the less analyte was present in the sample.
Now that we know all the ELISA variations, we need to find out when immunoassay technology can be applied.
Immunoassay applications
Immunoassay technologies are used in a variety of applications, including:
- diagnostics, where immunoassays can be used to detect and quantify specific proteins or antibodies in patient samples. Ever wondered how pregnancy tests, rapid HIV tests, or coronavirus tests work? That's ELISA;
- drug development, where immunoassays can be used to screen for drugs that bind to specific targets, such as receptors or enzymes;
- research, where immunoassays can be used to measure the levels of specific proteins or antibodies in cell culture or animal models.
Here is what happens inside of a pregnancy test:
We have two lines on a test strip – one for the control, another for pregnancy detection. When a person is pregnant, their body releases a specific hormone called human chorionic gonadotropin (hCG), which is detectable in urine. When the test strip is placed in the urine sample, the flow brings targeting hCG secondary antibodies, located in the beginning of the stripe to the two lines. The test line area contains antibodies which will react ONLY in the presence of hCG, while the control line area contains antibodies that bind to the secondary antibodies from the test strip. This way, we will always see the control "negative" line and see two lines if there is hCG present in the sample.
Conclusion
There are many different types of immunoassay technologies available, each with its own advantages and disadvantages. ELISA is one of the most commonly used methods, due to its high sensitivity and specificity. However, it is important to select the right technology for the specific application, as each has its own strengths and weaknesses.