There are two types of ELISA: two-site sandwich and competitive ELISA
Sandwich ELISA principle for analyte determination
High specificity and sensitivity of the test system
Capable to withstand the temperature of +37o С for 14 days and the temperature of +25o С for 50 days when transported
It may be used in the field
A broad measurement range for analyte concentrations ranging from several tens of picograms to several micrograms, which allows for, on the one hand, high sensitivity of the method and, on the other hand, detection of relatively high concentrations of a target analyte with a minimum quantity of dilutions, thus reducing the probability of a sample preparation error
The method ensures high accuracy in determining the analyte: the deviation of a measurement result does not exceed 10% in one determination and 15% between determinations
Owing to the use of highly specific monoclonal and polyclonal antibodies, a low level of false-positive reactions is achieved. This means that ELISA-based test systems, apart from high sensitivity, have the quality of outstanding reactivity with a particular analyte or group of analytes only
High precision of the measurement result: when studying the samples in several repetitions, the variations of a measurement result do not exceed 8 %.
An opportunity to perform both quantitative and qualitative assessment of an analyte. The second situation does not require any additional equipment, i.e. the assessment of a result is made visually
The analysis has a high tolerance to different matrices: the analyte may be extracted from a sample by way of preliminary extraction, after which the isolation obtained is studied in the test system. Such an approach provides for the possibility to detect the analyte both in solid and in liquid samples, regardless of their initial content
The possibility to simultaneously test up to 40 samples under quantitative assessment and 46 samples under qualitative assessment of the analyte content
There are two types of ELISA: two-site sandwich and competitive ELISA
Sandwich ELISA principle for analyte determination
The sandwich format of ELISA is mostly used to determine protein antigens and high-molecular compounds.
The studied sample is placed in the microplate wells, the surface of which is coated with the antibodies against a target antigen (analyte). The sample antigen binds to the antibodies on the surface of a well, while any unbound antigens and parts of matrix are removed by washing. After that, second antibodies against the same antigen are introduced in the wells, having a label (conjugate), which is most commonly horseradish peroxidase. The wells are washed one more time to remove the excess of conjugate. Then, a special chromogen-substrate mixture is added, which is capable of forming a colored product upon interaction with the enzyme. The quantity of such product and, therefore, the intensity of coloring are directly proportional to the quantity of the analyte in the sample. The reaction is then stopped and optical density is measured in the wells by photometry.
The two-phase format of implementation of ELISA reactions has an undisputed advantage: applying such approach in sandwich test systems ensures that no so-called hook effect takes place–an inhibition of sandwich formation when there are high concentrations of the analyte. In its turn, under the one-phase format of implementation of sandwich systems, despite a greater convenience in terms of procedure, the hook effect is possible and, therefore, a false negative result may be obtained in such above-mentioned case.
Competitive ELISA Principle for Analyte Determination
The sandwich competitive ELISA is mostly used to determine low-molecular analytes: hormones, peptides, mycotoxins, etc.
When assessing the content of analytes by competitive ELISA method, the final purpose as well is to obtain an antigen-antibody complex immobilized in a well of the microplate. However, there are some principal distinctions in this case.
Both the studied sample, presumably containing the antigen of interest, and the horseradish-peroxidase labelled antibodies to the same antigen are simultaneously placed in the microplate wells, where the antigen is sorbed onto their surfaces (the same as the sought-for analyte). As a result of the interactions taking place, the sample antigen binds to the labelled antibodies, thus resulting in the inhibition of formation of the antigen-antibody complex with the antigen on the surface of the well. Once the incubation is complete, the wells are washed to remove the reaction mixture, after which, as is in the first case, a special chromogen-substrate mixture is added, which is capable of forming a colored product upon interaction with an enzyme. Here, the quantity of such product and the intensity of coloring are inversely proportional to the quantity of the analyte in the sample. The reaction is then stopped and optical density is measured in the wells by photometry.
