ABBEXA ELISA KITS INDIA SUPPLIER

Enzyme-linked Immunosorbent Assay (ELISA) Kits are used to detect and quantify analytes. A reactant is immobilised to the assay plate either by direct dsorption or adsorption of a capture antibody. Once the desired analyte has been captured by the immobilised reactant, a detection antibody linked to an enzyme or other tag is added. This antibody is specific and will only bind to the analyte. The substrate is then introduced and the enzyme catalyses a reaction to produce a measurable result, e.g. a colour change.

The four main types of ELISA Kits are: Direct, Indirect, Sandwich and Competitive

Direct ELISAs

Direct ELISAs are considered the simplest form of ELISA.

The sample is coated onto the plate either directly or via a capture antibody. Detection is via a labelled primary antibody which produces a colour change upon the addition of the substrate. Direct ELISAs are beneficial as no secondary antibody is required thus preventing cross reactivity between antibodies. This ELISA can also use a labelled antigen to detect an antibody coated onto the plate. Compared to Indirect ELISAs, sensitivity is recognised to be lower due to the signal being less amplified. However, they can be performed much faster as only one step is required for detection.

Indirect ELISAs

Indirect ELISAs use a secondary antibody conjugated to an enzyme to detect the primary antibody. The analyte is first coated onto the plate before primary antibody is added which will specifically bind to the analyte. Labelled secondary antibody is then introduced which will bind the primary antibody at a different epitope to the analyte. The addition of
substrate results in a colour change, the intensity of which correlates with the concentration of analyte.

This type of ELISA has increased sensitivity due to the presence of multiple epitopes on the primary antibody that can be bound by the labelled secondary antibody. Flexibility may also be increased as more than one secondary detection antibody can be used with a single primary detection antibody. There may also be a reduction in cost to perform this assay as only one type of antibody will need to
be labelled.

Competitive ELISAs

Competitive ELISA’s are commonly used to determine small molecules such as lipids, hormones and small peptides. A purified antigen is labelled and competes with the unlabelled antigen in the sample to bind the capture antibody. Signal output is measured and compared with an expected signal output to determine the concentration of analyte present in the sample.

Sandwich ELISAs

Sandwich ELISAs tend to be the most readily recognised type of ELISA. The analyte to be measured is sandwiched, as the name suggests, between two antibodies, one for capture and one for detection. Detection can either be directly or indirectly. The analyte for Sandwich ELISA’s should be fairly large in size to ensure the antibodies used are able to bind at different epitopes. When carrying out a Sandwich ELISA it is important that the
antibodies used are matched pairs. Matched pairs refers to antibodies being specifically tested together to ensure that they bind to different epitopes of an antigen. This prevents the chance of the antibodies binding to the same site or recognising each other.

This technique is beneficial when the target analyte concentration is low. Following binding to the capture antibody, any other proteins within the sample that have not bound will be washed away.