Archive for February, 2017

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).

ELISA Test: History, Types and Kits

What is ELISA Test

ELISA is the abbreviation of the enzyme-linked immunosorbent assay. Sometimes, you would see another terminology EIA (enzyme immunoassay) instead of  ELISA, they both represent the same meaning. It is firstly used in detecting autoimmune related antibodies in patients with autoimmune disease. With the help of linked enzyme, the reactions between antigens and antibodies could be showed in a particular color.  That is why it is named “enzyme-linked immunosorbent assay ”, because ELISA test is related to “immune” and “enzyme”.

But  as we can see, ELISA tests are not only used in antigen/antibody detection fields with its development, and some ELISA tests use other signal amplifying methods instead of enzyme” We still like to call those not related to “immune” or “enzyme tests as ELISA. Meanwhile, if  we prefer to keep using this terminology, we should expand the definition of ELISA..

We can define  the ELISA test by the following sentence, “In a ELISA test, ligand(s) conjugate with receptor(s) accomplished with a color alter which is detectable and measurable.” We use ligand and receptor to substitute antigen and antibody, as well as drop the enzyme related description, thus expand the range of the definition.

ELISA test is a typical “wet-laboratory” type test, though it uses a solid phase to detect the presence of the substance, the substance is usually in a liquid or wet sample. Generally the solid phase is a solid plate with 96 wells, while some ELISA tests are performed with 192 wells’ or 384 wells’ plate.

ELISA test has been wildly used in medical diagnostic. And, it has been used in detecting  plant diseases’ pathogen in plant research.  In addition, many industries, such as food industry, apply ELISA test in quality control and quality assurance process.

 

 

History of ELISA Test

From the late 1960s, ELISA tests play a role in diagnostic research over 50 years. The origin of ELISA was the idea of finding an alternative method to substitute radioimmunoassay (RIA) in immunoassay.  Before the invention of ELISA, RIA is the only method to conduct immunoassay. Actually, the invention of RIA was not much earlier than ELISA. The First paper introduced this technique was published in 1960 by Rosalyn Sussman Yalow and Solomon Berson.

However, the concerns of potential safety and health problems coming with the initiation of  RIA. Scientists proposed to find another labeling method to replace radioactive label.  Some of them came up with an idea to use enzyme labels in immunoassay. But, many thought it is impossible to link the enzyme, such as large molecule, to an antibody or antigen without affecting their bioactivity in conjugating reaction. Although met with skepticism and criticisms, Perlmann and Schuurs independently invented the method to prove the using of enzyme-linked immunoassay is feasible in 1966. And following in 1966-1970, other scientists continually polish and improve methods, including resolving two critical issues for ELISA, which are “reaction associated color changing” and “ removing non-specific conjugation”.

Finally in 1971, Peter Perlmann and Eva Engvall at Stockholm University in Sweden (Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G), and Anton Schuurs and Bauke van Weemen in the Netherlands (Immunoassay using antigen—enzyme conjugates)

independently published papers that systematically introducing EIA/ ELISA methods. ELISA tests developed rapidly in the 1970s and early 1980s, and revolute into commercial clinical used products what we use now.

In the above content, we have introduced the main history of ELISA. But the imaginations and insights from the other scientist also contributed to the ELISA test development. For example, the concept of immunoassay minaturisation is conceived in 1963 by J.G. Feinberg and A.W.Wheeler. They invented a “microspot” & cellulose acetate strips method to detect autoimmune antibody of thyroglobolin in patients. We can find the ideas and concept of today’s ELISA test plate from their devices.

 

Types of ELISA Test

 

1.Direct ELISA

Figure 1

We use Figure 1 as an example to show the principles of the direct ELISA.

(1). The plate surface is coated with the virus antigen.

(2). The enzyme-linked specific antibody interact with the antigen, and become a complex.

(3). Adding substrate, then it will react with the enzyme and induce a color change which is detectable.

Note: in this example, the antigen is the “ analyte”(what we want to detect), we use enzyme-linked antibody to detect the existence of the antigen. However, in some ELISA tests, the antibody is the analyte. In that situation, we use enzyme-linked antigen to detect antibody.

 

2. Indirect ELISA

 

Figure 2.

We use Figure 2 as an example to show the principles of the indirect ELISA.

(1). The antigen is coated on the plate surface.

(2). The capture antibody (first antibody) “capture” the antigen, and become a complex.

(3). The detector antibody, which is labeled, conjugate with the capture antibody, and become a larger complex. We can detect the label to indirectly detect the antigen.

Note:  the antigen is the analyte in this example, but in many situation the antibody is the analyte. So we should design a special antigen to “capture” the antibody and a specific detector antibody to interact with the antigen with another site rather than immunoactive site.(Figure 3)

Figure 3

 

 

3. Sandwish ELISA

 

Figure 4

We use Figure 4 as an example to show the principles of the Sandwish ELISA.

(1) The plate is coated with a capture antibody.

(2) Sample is added, the specific antigen (analyte) binds to the capture antibody.

(3) Then the detecting antibody is added, and it binds to the antigen.

(4) The enzyme-linked secondary antibody is added, and it binds to detecting antibody.

(5) Finally, we add substrate, and it react with the detecting antibody to induce the color changing.

Note: The antigen is the analyte in this example, in other situation antibody maybe the analyte.

 

4. Competitive ELISA

Figure 5

We use Figure 5 as an example to show the principles of the Competitive ELISA

(1) Incubate the Antibody with sample containing antigens we want to detect. And they become complex.

(2) The antibody-antigen complex is added to the micro-wells which are pre-coated with the antigen. So, the free antibody can binds to the antigen coated on the micro-wells. The antibody in the complex and the unbounded antibody can be removed with washing

(3) Then we add Enzyme linked secondary antibody which is specific to the primary antibody. Wash again.

(4) Finally we add substrate, which react with the enzyme to induce a color change. The higher the concentration of antigen in the sample, the weaker color will be displayed.

Note: Different to the other types of ELISA test, the less absorbance means higher analyte concentration in the sample.

 

ELISA Test Kits and Our products 

The most popular ELISA product is the ELISA kit. Usually, users only need to prepare the analyte sample for the experiments, all the other components, such as plates, antibodies or antigens, substrate solution, TMB solution, controls, calibrators and etc., are provided by the ELISA kit.

Our company, Diagnostic Automation/Cortez Inc., sells a very wide range of ELISA Kit. For example, our two major ELISA Kit products are Autoimmune Disease ELISA Kits and Infectious Disease ELISA Kits (Figure 6 and Figure 7). We supply hundreds kind of products for both United States and global customers, and we are in a good relationship with many customers for long years. Hope you can also find what you need from us!

      Figure 6 ENA Profile-6 ELISA Kit  

        Figure 7 Syphilis (TPA) IgG ELISA Kit

Reference:

1. J. G. Feinberg, Alan W. Wheeler. Detection of auto-immune antibody and tissue antigens by the `microspot’ technique. J Clin Pathol. 1963 May; 16(3): 282–284.

2. 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.

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