Background to ELISA- Becky Flashcards
(18 cards)
What is ELISA?
The ELISA assay is a widely used immunological assay to detect in a sample the presence of and quantity of proteins, such as hormones and antibodies and bacteria or viruses.
How does the ELISA assay work?
The ELISA assay uses the coupling of antigens and antibodies and relies on the specificity and affinity of antibodies for antigens.
What are antigens?
Antigens include “non-self” molecules and cells, such as:
*viruses, bacteria and parasites (foreign proteins/ carbohydrate)
*food
*environmental pollutants and other foreign substances like asbestos, tattoo ink, and cigarette smoke
*foreign transplanted tissue
*cancerous cells
What is an antibody?
*An antibody, also known as an immunoglobulin, is a large, Y-shaped protein
*Part of the adaptive immune response
*Secreted by a plasma cell
*Recognises antigens
Humoral immune response:
Plasma B-lymphocytes produce antibodies in response to antigens (any foreign substance)
*Each B-cell makes its own distinct antibody in response to a specific antigen which comes in contact with it.
*Each antibody is specific for a surface binding site or epitope on the antigen.
Polyclonal vs Monoclonal antibodies:
Most antigens have several epitopes.
What are polyclonal antibodies?
mixtures of antibodies, each specific for one of the various epitopes on an antigen.
What are monoclonal antibodies?
are all identical, produced by clones of a single antibody-producing cell. They recognize one specific epitope.
Monoclonal antibodies:
Hybridoma Technology to Manufacture
Hybridoma technology:
1 Immunisation of animals
2. Isolated B cells collect
3. Fusion from partner myeloma
5. Hybridoma forms
6. Selection in HAT medium
6.Screening
7. Expansion
8. Monoclonal antibodies
They recognise one specific epitope.
Basic principle of ELISA:
Antibody interacts with antigen
We have a method to detect this interaction
*Usually by conjugating the antibody to an enzyme
*Amount of signal is correlated with the amount of antigen bound by the antibody
Key facts on ELISA:
One of the most sensitive immunoassays available.
Used to detect analytes
*e.g to determine cytokine levels in fluids, used as diagnostic tests (e.g HIV)
Typical detection range is 0.01 ng to 0.1 ng
Sensitivity dependent upon the particular characteristics of the antibody-antigen interaction
There are a number of ELISA formats…
What happens in direct ELISA?
Antigen is bound to a plate
*Detection antibody recognises the antigen
*Detection antibody is conjugated to an enzyme – the enables the amount of antigen to be determined
What happens in Indirect ELISA?
*Antigen is bound to a plate
*Primary antibody recognises the antigen
*Detection antibody recognises the primary antibody
*Detection antibody is conjugated to an enzyme – the enables the amount of antigen to be determined
What happens in Sandwich ELISA?
At least two antibodies required:
1.Capture Antibody:
*the first antibody
*often polyclonal to capture as much antigen as possible.
2.Detection Antibody:
*second antibody
*often conjugated to an enzyme
*Antibodies must recognize different epitopes on the same antigen
*The assay can be very sensitive (up to 2 to 5 times more sensitive than direct or indirect ELISA)
Optimisation of antibody concentration:
*The optimum concentration of both capture and detection antibody is required
*Too little antibody and the signal will be too low
*Too much antibody and may get non specific signals
*All ELISAs will have undergone optimisation prior to be used.
Sandwich ELISA to detect Staphylococcus Infection:
As the substrate is added, the enzyme converts it into a colored product. The colour formation is proportional to the amount of antigen present.
The colour is measured on a plate reader
Optical Density is Measured on a Plate Reader:
OD measures absorbance.
The greater the OD, the darker the colour, this is proportional to the analyte concentration.
