3.2 Enzyme-linked immunosorbent assay
This method can be classified into several distinct ELISA types
Direct ELISA: Involves the primary antibody-enzyme conjugate interacting directly with an antigen-coated plate.
Indirect ELISA: Utilizes enzyme-linked secondary antibodies specifically designed to interact with the primary antibodies that have already bound to the antigen-coated plates.
Competitive ELISA: Engages in a competition of sample antigen with plate-coated antigen for binding to the primary antibody, followed by the introduction of a secondary antibody-enzyme conjugate.
Sandwich ELISA: ELISA involves adding the antigen to an antibody-coated plate, followed by attaching a detection antibody and a secondary antibody-enzyme conjugate to the antigen recognition site. ELISA offers versatile and precise antigen detection methods [26], used in clinical practice for various analytes in diverse sample types. Since its invention, ELISA has found applications in fields like food quality, environmental monitoring, biotechnology, microbiology, and chemistry. Engvall and Perlmann pioneered ELISA research in 1971 by using enzyme marker, alkaline phosphate, to quantify IgG levels in rabbit serum [27]. ELISA assays detect allergic proteins in wheat that cause hypersensitive reactions in sensitive individuals [28], identify Bacillus thuringiensis (Cry1Ac) protein in recombinant BT cotton [29], and detect Botrytis cinerea in fruit tissues [30].
Digital ELISA detects mono molecules in the blood, like prostate-specific antigen (PSA) at low concentrations (14 fg/ml). It’s also used to identify p, p′-DDE, an insecticide metabolite, aiding in persistent organic pollution detection [31]. However, ELISA has limitations, including time-consuming testing and limited sensitivity for challenging biomolecules.