Enzyme-linked immunosorbent assay (ELISA), is a popular format of a “wet-lab” type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay (EIA) to detect the presence of a substance in a liquid sample.
“Wet lab” analytic biochemistry assays involves detection of an “analyte” (i.e. the specific substance whose presence is being quantitatively or qualitatively analyzed) in a liquid sample by a method that continues to use liquid reagents during the “analysis” (i.e. controlled sequence of biochemical reactions that will generate a signal which can be easily measured quantified and interpreted as a measure of the amount of analyte in the sample) that stays liquid and remains inside a reaction chamber or well that is needed to keep the reactants contained; as opposed to “dry lab” that can use dry strips – and even if the sample is liquid (eg a measured small drop), the final detection step in “dry” analysis involves reading of a dried strip by methods such as reflectometry and does not need a reaction containment chamber to prevent spillover or mixing between samples.
ELISA is similar in principle to RIA but uses an enzyme rather than a radioactive label. In its simplest format, called direct ELISA, a solid-phase binding support such as a polystyrene plate is used to capture the tested antigen, which is then detected with the aid of a specific enzyme-lined antibody (E-Ab). The detection involves conversion of a colorless substrate solution into an intense-colored product that can be measured directly in the reaction plate. The increase in optical density during the reaction is recorded at different time points for several sample dilutions, and the collected data is then used in conjunction with a standard curve to calculate the concentration of the antigen in the original sample.
Although E-Abs can be obtained by covalently binding enzymes to antibodies through a coupling agent such as glutaraldehyde, this method is not commonly used, because it often yields products with impaired immunological and/or enzymatic activity. A superior method to prepare fully active E-Abs is by exploting the high binding affinity of avidin for biotin. The biotinylated antibody used in this method contains on average 6-8 biotin moieties, which due to their small size do not interfer with the interaction between the antibody and the captured antigen. Once unboudn antiboyd has been rinsed form the plate, the biotin moieties are used in-situ to form stable complexes with avidin-enzymes conjugates.
A major limitation of the direct ELISA is that certain antigens like low molecular weight cytokines bind poorly to polystyrene plates. To by-pass this limitation, the plate can be pre-coated with a specific antibody that recognizes the tested antigen through epitopes that are not used by the E-Ab. Using a matched pair of antigen-specific antibodies, one for capturing and one for detection, significantly improve both the sensitivity and specificity of the assay. This format is called a Sandwich ELISA and it enables detection of picogram quantities of antigens. It is the preferred method for measuring cytokines and hormones in biological samples.
Applications
Identification of Epitopes:
Wilhelmus (US 13/982970) discloses identification of VHH fragments which bind to the CH1 domain of IgG antibodies. First, llamas were immunized with human IgG antibodies and/or Fab fragments thereof. Screening of individual VHH fragments from constructed expression libraries were performed by ELISA using different human antibody isotype and subclasses and fragments thereof, which resulted in identification of a panel of VHH fragments binding to the CH1 domain of IgG from different mammalian species and human IgG1 to 4 in particular. For the screening, Maxisorp binding plates were coated with human antibody antigens and subsequently blocked. Bound VHH fragments were detected by either a mouse anti-Myc mAb in combintation with a polyclonal goat anti mouse HRP conjugate or a polyclonal rabbit anti-llama-VHH serum in combination with a polyclonal swine anti rabbit IgG HPO conjugate. From this screening a set of VHH fragments were identified that showed binding to polyclonal human IgG and Fab fragments thereof and to human IgG antibodies; IgG1-4. No binding wa observed for polyclonal human IgG Fc fragments and human IgM. These results indicated that this panel of VHH fragments recognize an epitope present on the Fab portion of human IgG antibodies and that binding to this epitope enables binding of all 4 human IgG subclasses independent of its type of light chain (kappa or lamda).