ELISA stands for enzyme-linked immunosorbent assay. ELISA lab test is an antibody-based separation technique. ELISA lab test is usually performed in polystyrene plates via serial binding to the plate surface.

Bioanalytical services employ ELISA lab tests in numerous study samples as they are simple and quick to perform. Besides, ELISA assays can handle many  study samples in various diagnostic and research applications. 

Since its early inception in the 1970s, ELISA assays are now the primary detection technique in various biomedical applications. Moreover, newer formats have  modifications in the primary ELISAs to deliver high throughput and sensitive readouts. The current article explores the basics of the ELISA lab test. 

ELISA Lab Test

ELISA services begin the assay by coating the first layer on an ELISA plate. The initial coating can be an antibody or an antigen. Blocking and detection are the next steps in an ELISA assay protocol. As ELISA assays are surface binding techniques, each ELISA step has a washing step to remove unbound materials. 

In general, there are four different types of ELISA formats, including direct, indirect, sandwich, and competitive ELISA. In direct ELISA, the antigen is immobilized directly on the ELISA plate. On the other hand, indirect ELISA is a two-step process. First, specific antigens are attached with unlabeled primary antibodies, and then an enzyme-conjugated antibody is applied to the primary antibody. In sandwich ELISA, as the name suggests, the antigen is sandwiched between a pair of matched antibodies. Finally, competitive ELISA, also called inhibition ELISA, is a more advanced  ELISA technique. All the above assay formats can be modified into a competitive ELISA format. 

By definition, ELISA utilizes an enzymatic reaction to produce a detection assay signal. ELISA assay can employ direct or indirect detection. Indirect ELISA detection antibodies are labeled with HRP or alkaline phosphatase to produce a colorimetric output. On the other hand, in indirect ELISA, detection is biotin coupled with antibodies, followed by streptavidin-conjugated enzyme addition. 

ELISA assays can generate three types of data: quantitative, qualitative, and semi-quantitative. In quantitative detection, ELISA data is compared to a standard curve to calculate analyte concentrations. A qualitative ELISA test can help researchers generate a yes or no answer to determine the presence or absence of a particular analyte in the study sample. Finally, in semi-quantitative ELISA, researchers compare relative concentrations of analytes in study samples. 

ELISA components and other assay considerations are paramount for immunoassay development and validation. Generally, ELISA plates have 96 Wells. They are made from polyvinyl chloride or polystyrene material. However, it is vital to use assay plates optimized for specific experiments. ELISA plates are developed generally to deliver consistent research, minimize effects and provide optimal data. However, researchers should test multiple assay plates from different manufacturers to assess plate-to-plate and batch-to-batch variability.

Besides ELISA buffers, including standard, coating, blocking, and washing buffers and antibodies, such as monoclonal and polyclonal antibodies and matched antibody pairs, should be thoroughly considered during ELISA assay development and validation. Not to mention proper sample preparation and handling will remain crucial for generating reliable and reproducible ELISA results.