Shikari (Q-AFA) Filgrastim Quantitativer ELISA-Kit

Enzyme Linked Immunosorbent Assay (ELISA) has been around for 50 years and ELISAs with different technical solutions are still being developed, improving and expanding the scope.

The test was first described by Engvall and Perlmann in 1971 and was based on the work of Avrameas who used enzyme-linked antibodies in histochemistry. The method was quickly developed for the serodiagnosis of trichinosis and antibodies against   Plasmodium vivax  and   P. falciparum to be used in epidemiological studies of malaria.

• Since its discovery, there have been numerous applications of ELISA, used to detect both antigens and antibodies. In addition to the detection of protein antigens, ELISAs have been optimized that enable the determination of antibodies against native and denatured DNA, polysaccharide antigens and phospholipids.
• In fact, the name  ELISA is sometimes used for tests that do not have antibodies present, but instead use specific protein-protein interactions. From the point of view of optimization, validation and standardization, such tests can be treated the same. In the case of protein antigens, the sensitivity of the ELISA is usually in the pg/ml range.
• When developing a diagnostic test, precise and optimal performance conditions must be found for all steps within the test protocol. This ensures that the entire process is optimal. Before it can be used routinely, for example in diagnostics, the newly developed or newly modified method must prove to be accurate, precise and reproducible.
• In addition, in order to measure the values ​​​​obtained with the test, it is necessary to standardize the test. Therefore, the Optimization, Validation and Standardization (OVS) of ELISA are extremely important and necessary, especially if they are to be used in clinical or veterinary medicine. In this chapter, the procedures that characterize ELISA are presented in a clear and concise manner.
• When reviewing the literature, we noticed that the described boundaries between optimization, standardization and validation are not clear enough.
• The reason for this is  that in certain situations performing a single ELISA can lead to a completion of both validation and optimization features,  which is fully valid. Before going into more detail, and to avoid confusion, it is appropriate to clearly define these three terms.

ACCORDING TO THE MERRIAM-WEBSTER DICTIONARY,

Optimization  is:   „  An action, process, or method to make something such as a design, system, or decision as completely perfect, functional, or effective as possible.“

Validation  is:   „  an action, process or instance of validation,   in particular  : determining the degree of validity of a measuring device.“

Standardization  is: „to bring into line with a standard, particularly to ensure consistency and regularity… to compare to a standard: to determine the strength, value, or quality of (something) by comparison to a standard.“

ELISA is mostly used to measure the presence or amount of antibodies or antigens or, in general, biomolecules that can be recognized by antibodies. In biological matrices (such as serum, plasma, blood, urine and saliva) ELISA is an important diagnostic tool for the detection of various antigens and antibodies. Indirect or direct ELISAs are used in medical device development, especially for testing vaccines and new drugs. ELISA with specific antibodies can be developed to measure impurities in the medical devices resulting from the manufacturing process.

Antibody assays against these contaminants should also be developed and validated to test contaminant levels, which should be kept to a minimum to avoid adverse immune responses. The sandwich ELISA is used for immunogenic substances with expected low concentrations such as cytokines, hormones, toxins, etc.

Regardless of the ELISA design (indirect, direct, or sandwich), the OVS principles are the same. Of paramount importance for any bioanalytical method is that it is well characterized, fully validated and documented to a satisfactory standard in order to produce reliable results.

The first step in ELISA development is optimization, followed by standardization and finally validation.