Introduction: ELISA Kit

Background

Not long after the invention of the ELISA, scientists admired the efficiency it brought to the experiments. They could perform hundreds of tests one day individually, which was unable to image before. However, the difficulties in the preparation and procedures make it not available to most laboratories. Thus, it was come up with an idea to package the components of ELISA Test into a product (ELISA Kit) that can perform ELISA by individuals without complicate preparation and procedure steps.

 

Invention and Development

As we mentioned in the last article, RIA test systems were widely performed before the invention of ELISA test system. However, during the late 1960s and early 1970s, there were some difficulties when performing RIA test. In that period, most individual researchers use home-brew methods to perform RIA test. That is not because home-brew methods were more efficient (actually they were not). The reason is that they could not keep pace with the possibilities and facilities ( for some financial and technical reasons) of commercial manufactures such as Boehringer-Manheim, Abbott, and Organon Teknika. These companies also noticed this phenomenon, then they found that ELISA test systems.

 

As we mentioned, ELISA test systems were invented in the same period. Compared to RIA, ELISA is much cheaper, less technical recommend, and much safer than RIA, because of non-radiation. But the complex preparation and procedure steps blocked the spread of this new test system. If someone could make a ELISA kit, it provide the important materials used in the test and they are ready to use. The difficulties would be resolved. These manufacture came up with the same idea and they started to commercialize the ELISA Kit which contains the critical components for the ELISA test, which could substantially reduce the difficulties in the preparation and procedures.

 

One critical step of the ELISA Kit development is the use of solid-phase techniques. By the help of solid-pace techniques, scientists could noncovalently bind an antigen or an antibody in the wells of microtiter. In addition, the invention of automated machines, ,such as microtiter plate reader and washing instruments, promoted the population of ELISA Kit.

 

The Dutch company Organon Tenika is the first manufacture made progress in this field. They invented different ELISA Kits for reproductive endocrinology, etc, human chorionic gonadotropin, total estrogens and human placental lactogen from plasma sample. However, the new ELISA tests out of the fields we mentioned above, did not be commercialized until the late 1970s and early 1980s, when they could compare the exquisite sensitivity of the RIS test system for the same sample.

 

In 1976, Organon developed and marketed a very successful ELISA Kit for the hepatitis B surface antigen (HbsAg), with a 960well micotitier plate format (Figure 1). It became the first commercial ELISA Kit. And it rapidly replaced the use of RIA or nonradioactive but rather cumbersome hemagglutination test in the blood-bank screening for the B surface antigen. Other same kind of ELISA Kit followed soon, such as hepatitis B “e” (Hbe) antigens, rubella antibodies, toxoplasma antibodies, and HIV antibodies ELISA Kit.

Figure 1. HbaAg ELISA kit by Organon Teknika

 

The use of ELISA kit significantly increased from 1976 to 1990s, and there is not sign of decrease till now (Figure 2).

Figure 2. Black line—ELISA Kit, grey line—RIA system.

 

In addition to the successfully commercialization of ELISA Kit,  there is another thing for ELISA worth to be noted in 1976. Perlmann, Schuurs, Engvall, and van Weemen were honored for their inventions when they received the German scientific award of the “Biochemische Analytik” in 1976. It was 5 years after they had published their first papers which systematically introduced ELISA Systems.

 

 

Components in the ELISA Kit

The most common components in the ELISA kit include Microplate, sample Diluent, Control(s), Standard(s) or Calibrator(s), Conjugate, Substrate, Stop Solution, Wash Buffer(s).

Note: most commercial ELISA kits apply indirect or sandwich ELISA test system.

 

Now let us take a close look at the function of these components :

 

Microplate: A solid phase most commonly has 96-well, and each well is noncovalently coated with antigen or antibody.

Sample Diluent: A solution used to dilute the sample to the concentration proper for the ELISA test.

Control(s); Usually there is a positive control and a negative control. The OD value of the negative control should not be higher than a standard value, and the OD value of the positive control should not be lower than a standard value, or the test is invalid.

Standard(s) or Calibrator(s): Manufacture would provide different known-concentrate standards or same concentrate Calibrator(s). Using standards, researchers could get a curve, and read the sample concentrate value from the curve. Using calibrator, researcher could get a cut-off value, and use it to get the sample/calibrator ratio, fatherly the concentrate value.

Conjuagte: It contains the secondary antibody, which will bind to the antibody-antigen complex. And it is linked with an enzyme to react with substrate.

Substrate: Substrate react with enzyme to produce a color change, which is mean to measure the amount of the antibody or antigen. TMB (3,3’,5,5’-Tetramethylbenzidine) is a often used substrate in the ELISA Kit.

Stop Solution: Stop the reaction, it usually is the H2SO4 and HCI mixed solution.

Wash Buffer(s): When users remove the non-bound antibodies or antigens, they choose the proper concentrate wash buffer.

 

The following picture (Figure 3) is a components list from our ANA Screen ELISA Kit.

Figure 3. Components list from ANA Screen ELISA Kit

Reference:

1. White AM, Collett JR, Seurynck-Servoss SL, Daly DS, Zangar RC. ELISA-BASE: an integrated bioinformatics tool for analyzing and tracking ELISA microarray data. Bioinformatics. 2009 Jun 15;25(12):1566-7. doi: 10.1093/bioinformatics/btp182. Epub 2009 Apr 3.

2. Rudolf M. Lequin. Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA). Clinical Chemistry 51:12 2415–2418 (2005).

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