Enzyme-Linked Immunosorbent assay 1- Becky Flashcards
(17 cards)
What does ELISA stand for?
Enzyme-linked
Immunosorbent Assay
What does ELISA involve?
Involves antigen/antibody interactions
Uses reaction of an enzyme with a substrate to generate a coloured reaction product
How many antibodies are required?
2
What is the first antibody?
- Capture antibody:
- often polyclonal to capture as much antigen as possible.
What is the second antibody?
- Detection antibody:
- often conjugated to an enzyme
Why are antibodies important for the assay?
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
Outline a generalised scheme of a typical sandwich ELISA protocol.
- Incubate standard and sample. - aspirate and wash x4
- Incubate detection antibody. -aspirate and wash x4
- Incubate HRP conjugate.- aspirate and wash 4x
- Incubate stabilised chromogen.
- Add stop solution and read at 450nm.
What is sandwich ELISA?
sandwich ELISA requires a mutually compatible pair of specific antibodies, called antibody-matched pairs.
Important facts on sandwich ELISA?
*If a researcher is developing an ELISA from scratch, it is often because the target of interest is “new” to research, in which case a matched pair of antibodies may not be available.
*Custom antibody production may be required.
*Generally, unless new ELISA development is necessary for specialized research needs, one should check the availability of commercial ELISA kits for the target of interest.
ELISA optimisation:
*ELISA development involves choosing a format
*Direct vs indirect vs sandwich
*Gathering the needed components
*Antibodies, buffers, substrate
*ELISA optimization involves systematically adjusting and testing the many components and variables to help ensure results are robust and accurate.
*The most important part of optimization is testing different concentrations (i.e., dilutions) of antibodies, samples, and buffers.`
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.
How is elisa measured on a plate reader:
OD measures absorbance.
The greater the OD, the darker the colour, this is proportional to the analyte concentration.
What is the aim of the ELISA practical?
Use a sandwich ELISA to determine the concentration of your antigen (rabbit IgG) a sandwich ELISA
-We have already optimised the concentration of the capture antibody and other reagents
-ELISA 1 – determine optimum concentration of the detection antibody
What is ELISA practical 1:
ELISA 1, OPTIMISATION
*A direct ELISA format will be used to titrate the detection antibody
*You will use this data to determine the most suitable dilution to use in your sandwich ELISA
Week1 :ELISA 1, Optimisation of Detection Antibody
Titration of detection antibody to establish the best dilution to use (direct ELISA)
Basic Principle of Antibody Concentration Optimisation
*Coat plate with rabbit IgG
*Prepare different concentrations of the detection antibody in standard diluent
*Apply an equal volume of each concentration to the plate and proceed with the ELISA protocol.
*Check for strong signal vs. low background.
Week1 :ELISA 1, Optimisation of Detection Antibody Steps:
1.Coat wells with doubling dilutions of rabbit IgG and incubate overnight
at room temperature
2. Then add varying concentrations of the detection antibody
3. Then add the substrate.
4. Then produce graph of results
