What is an ELISA kit?
ELISA stands for Enzyme Linked Immunosorbent Assay and is a biochemistry assay to quantitatively or qualitatively detect a specific analyte in a sample. The analyte is immobilized on a solid support (most commonly a 96-well microtiter plate). An enzyme (e.g a peroxidase) conjugated antibody recognizes and binds an epitope of the immobilized analyte, any unbound antibody is rinsed away. The enzyme helps to develop a colorimetric response - the degree of color change quantifies the analyte. ELISA is most frequently used when the researcher needs to rapidly detect or quantify the presence of a single target in a complex biological matrix. The targets are commonly but not exclusively proteins and sample types can range from raw biological fluids (e.g. plasma, serum, urine, perspirant) to refined cell culture media or purified recombinant protein in solution.
What are the advantages of using ELISA kits?
In order to perform an ELISA u need several materials. A pre-coated 96-well strip plate several diluents, a stop solution, a standard to test against a substrate in order to generate the color response detection reagents and last but not least a wash buffer. It is time consuming to gather all materials and mix the solutions prior to every experiment. ELISA kits are a convenient approach to perform your desired ELISA. The kit comes in a box containing all of the necessary reagents at once – no need to buy anything extra. Individual components are already tested, and have been proven effective and compatible with each-other. Together this safes a lot of time as no additional preparations have to be made. The enclosed step-by-step instructions allow to perform the assay without deep knowledge of the topic. Last but not least the cost-per-sample is generally lower than for de novo assay generation. Antibodies-online offers a huge variety of ELISA kits for different sample types and species.
The concept of Enzyme Linked Immunosorbent Assays has been constantly refined since the first description of the technique in 1971 by Peter Perlmann and Eva Engvall1 as well as Anton Schuurs and Bauke van Weemen2. They used antibodies and color change to identify a substance. The approach involves chromogenic reporters and substrates that produce some kind of observable color change to indicate the presence of antigen or analyte. Newer ELISA-like techniques use fluorogenic, electrochemiluminescent, and quantitative PCR reporters to create quantifiable signals. Today ultrasensitive, enzyme-based ELISA test using nanoparticles as a chromogenic reporter are able to give a naked-eye color signal from the detection (e.g. ABIN510317). Differences between main ELISA techniques are shortly described below, further information can be found on our resource page.
The immobilization of the antigen of interest can be accomplished by direct adsorption to the assay plate or indirectly via a capture antibody that has been attached to the plate. The antigen is then detected directly via a labeled primary antibody. Direct detection can be performed with antigen that is directly immobilized on the assay plate or with the capture assay format. Direct ELISAs are not widely used but are suitable for applications like immunohistochemical staining of tissues and cells or to test the efficiency of a new antibody against a known target. The assays tend to be quicker due to less steps and miss out cross-reactivity as no second antibody exists. On the other hand labeling primary antibodies for each specific ELISA system is time-consuming and expensive. Furthermore only minimal signal amplification can be achieved by this system.
Direct and indirect ELISA only differ in the detection step. In an indirect ELISA the primary antibody used to detect the analyte is not conjugated to an enzyme. Instead, an enzyme-conjugated secondary antibody which is reactive against the primary antibody is added to the mixture. Indirect ELISAs are prevalent in scientific use as they provide several benefits over a direct ELISA. The secondary antibody has specificity for the primary antibody and a wide variety of labeled secondary antibodies are available commercially. The maximum immunoreactivity of the primary antibody is retained as they are not labeled. The secondary antibody can bind to several epitopes of the primary, this leads to a much bigger signal amplification allowing a previously undetectable signal to be visualized and lowering the detection threshold of the assay in question. As mentioned above, when working with two antibodies, cross-reactivity might occur, resulting in nonspecific signal.
Sandwich ELISAs typically require the use of matched antibody pairs (capture and the detection antibody), where each antibody is specific for a different, epitope of the antigen molecule. The capture antibody often is a monoclonal antibody as they only bind a single epitope on an antigen. This increases the specificity and reduces background noises as well. Sandwich ELISA can be direct or indirect depending on whether the detection antibody is labeled or unlabeled. If an indirect detection method is chosen, special care must be taken to ensure that the secondary antibody used reacts only with the detection antibody and not with the capture antibody coated on the plate. The ELISA is suitable for complex samples, since the antigen does not require purification prior to measurement.
In a competitive format assay, as antigen concentration in a sample increases, signal intensity decreases. The central concept behind a competitive ELISA is that a larger quantity of analyte in a sample results in fewer free antibodies in solution and by extension a smaller number of labeled antibodies bound to the standard on the plate. The competitive assay is an appealing option if no suitable antibody pair can be identified for sandwich ELISA, or if the analyte in question is too small to permit binding of both a capture and detection antibody.
Validation of ELISA Kits
Over the last years reproducibility and antibody reliability has become more and more an urgent topic. Across biomedical research, the resulting waste in materials, time and money is vast — costing an estimated US$350 million annually in the United States alone3. This is why various projects have sprung up to try to make information about antibodies 4.
”The solution is validation”5
As the largest marketplace for research antibody reagents with more than 1.6 million products and customers in over 50 countries, we have introduced the Independent Validation Initiative to actively approach the problem. The IVI was launched in July 2013 to independently validate commercial antibodies and ELISA kits. Our aim is to increase the transparency of product quality in the antibodies and ELISA kits market by providing exhaustive background information on how the product was applied in the experiment, while also allowing our customers to review results.
Everybody can participate in this initiative. If you are interested in a non-validated product simply apply for validation. Upon completion you get a full refund of the product price and help the science community to improve the antibody situation on the same way!