The enzyme-linked immunosorbent assays (ELISAs) from EUROIMMUN use antigens or antibodies coated on polystyrene plates. With 96 wells as a solid phase, they bind specific antibodies or antigens in patient samples resulting in an enzymatic color reaction following the addition of a conjugate. The processing can be manual, semi-automated, or fully automated.
Monospecific ELISAs are helpful for the semi-quantitative or quantitative determination of antibodies or antigens. Semi-quantitative detection of various antibodies on a single microplate strip is possible through profile ELISAs. Here, the solid phase is coated with an antigen mixture. The antibodies can be detected semi-quantitatively. Afterward, a differentiated detection must be performed with the respective monospecific assay.
Antibody detection by ELISA
The process begins with the placement of microplates coated with antigens along with diluted patient sample in the incubation. If the sample contains specific antibodies directed against the antigen, these bind to the antigen-coated plates. As a next step, peroxidase-labelled antibodies are added to the sample and these bind to the specific antibodies one aims to detect.
The addition of a peroxidase substrate tetramethylbenzidine (TMB) causes the peroxidase to catalyze a color reaction. The intensity of the resulting color solution is positively correlates with the antibody concentration in the patient samples within the measurement range. This can be used to formulate a concentration curve in quantitative and a ratio value in semi-quantitative tests.
Antibody detection by ELISA (competitive ELISA)
Competitive ELISA differs from a normal ELISA in that a competitive molecule like biotin is added to the sample to see if the antibodies compete with it in binding the antigens on the microplate.
It has the following steps:
- The antigen-coated reagent wells of the microplate undergo incubation with diluted patient samples.
- If a sample contains specific antibodies directed against the antigen, these bind to the antigen-coated reagent wells. They also inhibit the binding of a biotin-labeled artificial molecule to the reagent well in a subsequent step.
- Afterward, peroxidase-labeled avidin binds to the biotin-labeled molecules.
- In the third incubation step, the peroxidase and the peroxidase substrate tetramethylbenzidine (TMB) catalyze a color reaction.
- The intensity of the resulting color solution positively correlates with the antibody concentration in the patient samples within the measurement range. This can used to formulate a concentration curve in quantitative tests and a ratio value in semi-quantitative tests.
Antigen detection by ELISAs (sandwich ELISA)
Unlike during the ELISA for detection of antibodies, when detecting for antigens, the reagent microplates are coated with mono-specific antibodies and incubated with diluted patient samples. If the sample contains the respective antigens, these bind to the antibody-coated reagent well. In a further step, a peroxidase-labeled antibody (conjugate) is added, which binds to another epitope of the antigen.
When the peroxidase substrates tetramethylbenzidine (TMB) are added, the peroxidase catalyzes a color reaction. The intensity of the results color solution is proportional to the antigen concentration in the patient sample. It is within the measurement range and converted into a concentration using a calibration curve in the quantitative tests.
Antigen detection by ELISA (competitive ELISA)
In competitive ELISA, the antigens in the patient’s sample are made to compete with biotin-labelled antigens in binding the antibodies on the reagent microplate. The steps include the following:
- Reagent wells of the microplate coated with monospecific antibodies are incubated with diluted patient samples and a defined amount of biotin-labeled antigen.
- If the sample contains the corresponding antigens, these compete with the biotin-labeled antigen for the binding sites in the antibody-coated reagent well.
- Afterward, the peroxidase-labeled streptavidin (conjugate) binds to the biotin-labeled antigens.
- With the addition of peroxidase substrate tetramethylbenzidine (TMB), the peroxidase catalyzes a color reaction. The intensity of the results color solution is inversely proportional to the antigen concentration in the patient sample. It lies within the measurement range and is converted into a concentration utilizing a calibration curve in quantitative tests.