ELISA test is an assay technique used for quantifying and detecting hormones, antibodies, proteins, and peptides. Its full name is Enzyme-linked Immunosorbent Assay. This assay requires a solid surface for immobilizing an antigen that is later complexed using an antibody.
This antibody has an enzyme link. Detections are executed by using substrate incubation to assess the activity of the conjugated enzyme. The vital element of this detection strategy involves an antibody-antigen interaction that is highly specific.
However, for these quantifications and detections of substances to be accurate, it’s important to perform controls designed for ELISA specifically.
3 Types of ELISAs
To help you understand these controls and why they are important, we first need to talk about different ELISA test types. All of them have different control needs because of different detection and sample preparation methods.
With Direct ELISAs, the sample is first added to the wells. The sample protein binding coats the wells to the plastic. In the next step, conjugated detection antibodies perform the binding of the immobilized analyte. In the end, an enzymatic color reaction occurs that is proportional to the antibody.
Before adding the sample into the wells, it’s essential to coat them using a capture antibody. After pre-coating, the sample is added, and the capture antibody binds the analyte. A conjugated detection antibody then bounds all the immobilized analytes. In the end, an enzymatic reaction and indirect detection occur proportionally to the analyte.
With Competitive ELISA, the wells also need to be pre-coated with a capture antibody. However, when adding a sample, another antibody is added at the same time, along with a conjugate. After that, the conjugate and analyte battle for binding and a different enzymatic color reaction occurs depending on the bond.
ELISA Controls and Their Importance
Even though there are different ELISA controls due to different nomenclature in terms of dependence and nature, there are three essential categories for confirming ELISA results: standard, positive, and negative ELISA controls.
Standard ELISA Controls
If you want to quantify experimental results, it’s necessary to use the standard ELISA control. This control has an accurate number of target molecules. This control gives vital information required for making a standard curve. This control type can be used for optimization as well since a poor standard curve can be an indicator of inadequate specificity of the protein or bad antibody binding.
Positive ELISA Controls
Positive controls help to confirm if the whole procedure is going as it should. There are three main functions of positive controls:
- To assist necessary optimizations and protocol adjustments.
- For checking whether negative results are valid.
- To check the validity of experimental results.
Negative ELISA Controls
Negative controls don’t have components required for detecting an analyte. They help to check whether a positive result is false and look for non-specific bindings. Every plate used should have a negative control sample to ensure that the results are valid.
To save time and resources, researchers can use ELISA test kits when preparing for the process.
In the interest of validity and accuracy, it’s important to perform these three controls with ELISA tests. There is no way to check whether the process went well and be 100% sure that the results are valid unless checked. Luckily, with the right ELISA kits and proper preparation, this can be done quickly.