ELISA Kit Development

More Information

What Materials Are Needed for ELISA Kits?

If an ELISA kit for the protein of interest is not commercially available, it can be created in the laboratory. Below are the materials needed to create custom-made sandwich ELISA, the most commonly used ELISA.

1. Capture antibody
2. Detection antibody
3. Enzyme-linked antibody (if unavailable, antibody labeling kit)
4. Standard
5. Blocking reagent
6. Washing solution
7. Detection reagent
8. Stop solution
9. ELISA plate

Overview of Each ELISA Kit Component and Key Points for Selection

1. Capture antibody

Capture antibodies are immobilized on the ELISA plate to capture the antigen to be measured. The antibody is immobilized through the attachment of the Fc region to the ELISA plate. Monoclonal antibodies are suitable, as they specifically capture the target antigen.

　

2. Detection Antibody

Detection antibodies are used to detect antigens captured by the capture antibodies. Polyclonal antibodies are preferred, because of their high antigen-binding ability. It is critical that the detection antibodies bind to epitopes that are different from those recognized by the capture antibody.

When an enzyme-linked antibody is directed toward the detection antibody, the enzyme-linked antibody must not bind to the capture antibody. In principle, antibodies derived from different animal species are used for the capture and detection antibodies.

In some cases, the enzyme is directly linked to the detection antibody. However, the molecular weight of the enzyme is large, which may affect the binding ability of the antibody.

　

3. Enzyme-linked Antibody

By using an enzyme-linked antibody that specifically binds to the detection antibody, the enzyme necessary for color development can be introduced without compromising the binding ability of the detection antibody. In addition to antibodies, the biotin-avidin system is also available. In this system, a biotin-conjugated detection antibody is used and then reacted with an enzyme-linked avidin (or streptavidin). Many of the ELISA kits from Fujifilm Wako and Fujifilm Wako Shibayagi use a biotin-avidin system.

Biotin-conjugated detection antibodies can be easily prepared; biotin can be conjugated to the amino group (-NH₂) or sulfhydryl group (-SH) of the antibody by using a commercially available kit.

In ELISA, peroxidase (especially horseradish peroxidase; HRP) and alkaline phosphatase (AP) are the most common linked enzymes. HRP is more often selected than AP, because of its stability and greater number of substrates. However, HRP is inhibited by fluoride in anticoagulants and azide ion in sodium azide, a preservative, and the use of these agents should be avoided.

　

4. Standard

When quantifying a protein of interest in ELISA, a standard of known concentration (a standard protein solution) is required. The standard is diluted to a series of concentrations and each concentration is measured in the same manner as the sample. The target protein in the sample is quantified by comparing the absorbance of the sample with the absorbance values of the standard dilution series.

　

5. Blocking Reagent

Blocking reagents prevent detection antibodies from binding to the plate and to nonspecific proteins. Various blocking reagents are available; some are based on bovine serum albumin (BSA) or casein, and others are made from polymers. When a biotin-avidin system is used for the ELISA, skim milk, which may contain biotin, should be avoided.

　

6. Washing Solution

Washing solutions are used to remove samples and unreacted antibodies. PBS or PBS-T is used as a washing solution. PBS-T, which contains a detergent, is effective in removing nonspecific adsorption. The washing solution must be removed thoroughly at each step of the process, because washing solution left in the wells could cause variation in the measurement.

　

7. Detection Reagent

In ELISA, the amount of antigen can be indirectly determined by adding a substrate of the enzyme linked to the antibody and measuring the absorbance of the dye produced. When HRP-linked antibodies are used, tetramethylbenzidine (TMB) or o-phenylenediamine (OPD) is used as a substrate. For AP-linked antibodies, p-nitrophenyl phosphate (pNPP) is used.

Detection by chemiluminescence, instead of absorbance, provides higher detection sensitivity. Fujifilm Wako offers ELISA-Star™ Peroxidase Chemiluminescent Substrate, which is a chemiluminescent peroxidase substrate with sustained luminescence and excellent quantitative performance.

　

8. Stop Solution

HRP decomposes hydrogen peroxide, and the resulting reactive oxygen species oxidize the substrate, causing the color change. Sulfuric acid (1 M) is added to stop the reaction.

　

9. ELISA Plates

Polystyrene 96-well plates are often used for ELISA. Polystyrene tends to adsorb proteins well, and immobilization will take place by simply adding an antibody solution to the plate. Wells/plates used for ELISA must be as uniform as possible, and plates specifically for ELISA are commercially available from various manufacturers.

References

1. “Detection and quantification tips” ed. by Moriyama, T., Yodosha, Japan, (2005). (Japanese)
2. Wakabayashi, K.: “ELISA A to Z 5th edition”, FUJIFILM Wako Shibayagi Corporation, Japan, (2017). (Japanese)
3. 「はじめての抗体標識プロトコル」ed. by Dojindo Laboratories. (Japanese)