Researchers in research widely use the enzyme-linked immunosorbent assay (ELISA) kits to detect different proteins, including growth factors and cytokines. They are also useful for detecting a wide range of diseases such as infectious diseases, tumors, diabetes, small molecules, drugs, and other related diseases, including detecting antibodies. ELISA kits make an investigation into specific findings quick. These kits provide convenience and accuracy of tools for detecting and quantitation of interest targets in culture and samples.
What Is An ELISA Kit?
These kits are the ready-to-use immunoassay kits that contain all reagents for carrying out the ELISA (Enzyme-Linked Immunosorbent Assay). These kits are built to detect and quantify molecules such as antibodies, peptides, hormones, and molecules or vitamins. For quantifying the result or outcome of specific research, antibodies linked with enzymes are required.
How Does The ELISA Kit Work?
ELISA kit detects various molecules and antibodies. However, the first step in how the ELISA kit works is immobilizing the antigen of interest. Accomplishing this can by directly or indirectly absorbing the assay plate through a capture antibody fixed to the plate. The researcher or investigator then detects the antigen. However, this can either be detected directly (enzyme-labeled primary antibody) or indirectly (enzyme-labeled secondary antibody). Usually, when antibodies are detected, they are labeled with alkaline phosphatase (AP) or horseradish peroxidase (HRP). A large selection of substrates is available to perform the ELISA with an HRP or AP conjugate.
However, substrate choice depends on the sensitivity of assay needed, including the available equipment or instrumentation to detect signal (spectrophotometer, fluorometer, or luminometer). The measurement of enzyme activity helps to achieve detection. One of these methods is incubation with TMB substrate, which can be detected through the converted substrate’s light absorbance at 450nm. On the other hand, enzymes can be responsible for catalyzing chemiluminescent substrate, which triggers a strong reaction of luminescent, which can be quantified.
Many elisa testing services offer researchers or investigators multiple assay formats, including the conventional sandwich ELISA kits. Also, there are different competitive Elisa kits, including cell-based ELISA Kits for many targets. This ELISA comes with pre-coated plates with capture/detection antibodies, standards, buffers, and all necessary accessory reagents. Each kit passes through fit-for-purpose validation and stability testing, which helps to ensure precise outcomes of research, accuracy, sensitivity, specificity, and reproducibility.
The Advantages and disadvantages of ELISA
Elisa provides investigators with a wide range of advantages, making it one of the reasons researchers use it during research. Here are some of the advantages researchers can derive from Elisa
- It provides users with a simple procedure to achieve the best research result
- Most commercial Elisa provides users with high specificity and sensitivity. This is a result of an antigen-antibody reaction
- Elisa provides users with high efficiency as users can perform analysis without complicated pre-treatment sample
- Users are generally safe when they use Elisa
- These testing tools are eco-friendly as designed by ELISA service because there is no need for radioactive substances and large sums of organic solvents
- Users do not need to spend much as these tools are cost-effective. They are designed with low-cost reagents.
While most commercial Elisa comes with huge advantages, they also have some disadvantages to users while carrying out their findings. Some of the disadvantages ELISA exhibits include
- Preparation of antibody is very expensive and labor-intensive as a result of the need for a sophisticated technique and expensive culture cell media needed for obtaining certain antibody
- The chances of having false positive or negative results are high. This is a result of the insufficient blocking surface of microtiter plate immobilized with antigen
- An antibody is not stable due to the antibody being a protein that needs refrigerated transport and storage
Types of Elisa
The direct Elisa was the base for all kinds of ELISA as it was first developed in 1971. It is a type of Elisa where antigen or an antibody is immobilized on the microtiter plate surface. After proteins such as albumin or casein and other related proteins block the microtiter plate surface to avoid other kinds of protein absorption, the corresponding enzyme-labeled antibody or antigen is allowed for a reaction with the immobilized targets followed by color development with appropriate substrates.
Developed in 1973, this type of Elisa helps in detecting rat α-fetoprotein. However, it involves the development of two other types of Elisa- Sandwich and indirect. There is a major event in competitive Elisa: the competitive reaction between targets in the sample and enzyme-labeled targets against a corresponding immobilized antigen.
The development of this type of Elisa is based on direct Elisa to evaluate the presence of antibodies in antisera. The major step of the system is a two-binding process of the primary antibody and enzyme-labeled secondary antibody.
Indirect competitive Elisa
This ELISA type combines both the indirect and competitive Elisa. The immobilization of the target antigen is on a microtiter’s solid plate and is blocked. In this Elisa type, free target antigen and antibody are not restricted from incubation, with immobilized antigen and free antigen competing against antibodies.
This ELISA has been the most sought-after for an exhibition of higher specificity, including a wider working range than other types of Elisa, even for many Elisa test services. Comparing these two antibodies with varying epitopes can be very difficult, especially for haptens, because steric hindrance disturbs the reaction of antigen-antibody due to haptens’ small size. As a result, developing true sandwich assays for hapten can be rare.
Elisa kits help in detecting various diseases. They come with different advantages and disadvantages at the same time. While different Elisa kits are available for executing research works, it is important to find out if these kits will fit your needs.