How to Choose Your ELISA Kit

ELISA kits can be extremely useful for your research, but only when they perform well. It is important to select the right type of ELISA kit for your research needs, and then once you have it, perform your experiment carefully to ensure your results are accurate and reliable.

ELISA Overview

What is an ELISA kit? ELISA is an acronym for enzyme-linked immunosorbent assay. This is a laboratory technique to detect, and often quantify, the presence of a target antigen in a sample. ELISA assays use interactions between antigens and antibodies to create a multi-part complex that allows for detection. For example, in a sandwich ELISA, the antigen of interest binds to a capture antibody, which is then bound by a detection antibody. The detection antibody is often conjugated with biotin, which can then bind to Streptavidin-HRP, to create a reaction that results in colour change.

Types of ELISA

There are four main types of ELISA kits: Direct, Indirect, Sandwich, and Competitive Inhibition. In a Direct ELISA, the antigen of interest in detected directly by an antibody which is conjugated to the detection molecule. In an indirect ELISA, a primary antibody is used to bind to the antigen of interest, but then a secondary antibody specific for the primary antibody is conjugated to the detection molecule. Both of these types of ELISA require the antigen of interest to bind to the well of the plate used for the experiment. The Sandwich ELISA uses two different antibodies to create a ‘sandwich’ with the antigen of interest in the middle. The antibodies bind to two different epitopes of the target antigen, with the secondary (or detection) antibody conjugated to the detection method. The sandwich ELISA is currently the most commonly used format. The Competitive Inhibition ELISA is different than the other formats as the results are reverse proportional to the concentration of the antigen of interest. The sample antigen competes with a reference antigen for the binding site on the capture antibody. The detection antibody is specific for the reference antigen and is conjugated to the detection molecule.

Pros and Cons

Like most research techniques, ELISAs have both advantages and disadvantages that you should be aware of before performing your experiment. ELISAs are often highly sensitive and specific, and can be used to quantify the concentration of target antigen in the sample. They are relatively easy to perform and can often be modified to fit a high throughput format. However, ELISA experiments can be lengthy due to the extended incubation times, and can be challenging to troubleshoot as results are only gathered and analyzed at the end and samples cannot be purified or reused. Many ELISAs can be purchased in a ready to use kit format, or offered in parts that allow you to set up your ELISA experiment independently. Making your own ELISA kit allows you more flexibility and is often more economical, but ready to use ELISA kits are more convenient and require less optimization.

Specifications

When looking for an ELISA, you want to consider the specifications to ensure the product is right for your experiment. Many ELISA kits can detect in the nanogram or picogram range, but this sensitivity is not necessary if your samples are much more concentrated, and the results could be effected by extreme dilutions. Alternatively, if your samples are expected to have a very low antigen concentration, you want to ensure the kit will be sensitive enough to detect them. You also want to consider the sample type you will be using. Many ELISA kits can, in theory, work with a variety of different samples, but are not always validated for less common options. If you have limited samples, or a unique sample type, it’s best to consider what sample types are recommended or validated for an ELISA kit. Lastly, you should also consider any additional supplies or instruments necessary for use with an ELISA. Most colorimetric ELISAs will require a spectrophotometer to read the OD values from the kit. You want to ensure you have all necessary reagents and supplies before beginning your experiment.

Tips and Tricks

There are a variety of problems that could arise from any ELISA experiment, and it is often best to deal with those directly with the manufacturer on a case-by-case basis. However, there are some general tips to keep in mind to make the most of any ELISA kit.

Make sure to read the manual before beginning the experiment. This will ensure you are familiar with how to prepare the necessary reagents and execute the procedure. You will also be aware of any specific needs or differences for that particular ELISA kit. Always run a standard curve with each independent ELISA experiment. This is how you will measure your sample readings to ensure the data is accurate, and a good standard curve can indicate proper functioning of the ELISA principle in the kit. Pipetting and washing technique can make or break your ELISA experiment. You want to make sure your pipettes are properly calibrated, and all pipetting steps are performed in a routine, timed manner for uniformly accurate results. It is equally important that the washing technique used is uniform and thorough to get an accurate reading of your samples.

Reddot Biotech currently carries both Sandwich and Competitive Inhibition ELISAs for a variety of targets. Browse our full range of ELISA kits here.