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What do I need to know about ELISA - Enzyme linked immunosorbent assay
Terms in this set (64)
enzyme-linked immunosorbent assay
In the ELISA, the purpose of the solid phase is to
determine the presence and concentration of the target protein
An antigen-binding immunoglobulin, produced by B cells, that functions as the effector in an immune response.
A protein that, when introduced in the blood, triggers the production of an antibody
When an antibody molecule binds to its proper antigen.
Two parallel pairs of polypeptide chains:
-pair of heavy chains
-pair of light chains
Each chain contains:
-Variable segments - antigen binding sites
Why is ELISA called a solid phase immunoassay?
antigen is bound to a solid surface such as a microplate wall
microplate or microwell plate
a flat plate with many wells; used in ELISA
Why called the primary antibody?
It is the first AB to bind to the target antigen in an ELISA assay
added to protein sample before putting the samples into the microplate wells
the target protein bound to the plate well
block open surface area from being bound with reactive proteins by adding inert proteins such as casein, BSA or gelatin
why add sample diluent buffer to the protein extract?
to dilute the sample to be within the detection range of ELISA kits
why use 96-well (or 384-well) polystyrene plates?
The plates passively bind antibodies and proteins - result in immobilization of targeted protein
Why wash the plates with PBS buffer?
Washing removed the non-bound material so only the bound proteins remain
What is the KEY step of ELISA?
immobilization of the antigen of interest so its presence and concentration can be measured
why called direct ELISA?
Detects antigen directly with a single enzyme-labeled (conjugated) primary antibody
alkaline phosphatase (AP)
enzyme used to produce the yellow signal color in an ELISA - binds with PNPP substrate
horseradish peroxidase (HRP)
enzyme used to produce the signal color in an ELISA - binds with OPD, TMB and AMTS substrates
substrate used in ELISA, binds with AP enzyme
Why Colorimetric ELISA enzyme-substrate reactions in ELISA?
generate soluble products with an absorbance which can be measured in a spectrophotometer
the absorbance of a specific wavelength of light by a solution
Relation between color of the substrate and concentration of the bound analyte?
proportional, positive relationship
the target protein - the protein being quanitified
purpose of Lyophilized Recombinant Standard?
provides the standard absorbance/concentration for finding the equation of the line
stable biological substance prepared by rapid freezing and dehydration under high vacuum
uses of PBS buffer
Keep balanced pH and ion concentration; wash buffer; rinsing agent, diluting reagent
Phosphate Buffered Saline reagent
PBS (a nontoxic buffer)
facts about PBS as a buffer in assays
"The 1X final solution is isotonic and pH balanced with a buffering capacity aiding in the preservation of tissue integrity and proteins functionality" (bosterbio.com)
A solution with the same concentration of water and solutes as inside a cell, resulting in the cell retaining its normal shape because there is no net movement of water.
Antibody diluent buffer
solutions for diluting of antibodies that will maintain their solubility, activity, and stability
optical density of each well is read by a spectrophotometer
Direct Enzyme-Linked Immunosorbent Assay (ELISA)
bound antigen is detected by an antibody that has been directly conjugated to an enzyme
in ELISA, what do we want to be bound to the well surface?
the target protein - protein we want to detect/quantify
bovine serum albumin
benefits of indirect ELISA vs. direct ELISA?
indirect is cheaper, more versatile b/c getting an enzyme-conjugated primary antibody made/produced is expensive
sandwich ELISA steps
1. coat wells of plate w/ diluted capture antibodies (3x):
- rows with standards
- rows with samples
3. wash the plate 2 x w/ PBS
4. block remaining protein sites in the coated wells w/ unreactive protein
5. wash plate
6. Add the diluted protein samples (antigens) to the wells, including the standards and unknown samples
9. add diluted detection antibodies to wells
12. add diluted conjugate
15. add an HRP enzyme substrate
16. in dark room for up to 30 min
17. add acid to wells to stop the oxidative reaction
18. put plate in microreader set at 405 nm to quantify the results
binds to the immobilized antigen in direct/indirect ELISA
The detection antibody can be enzyme conjugated, in which case this is referred to as a direct sandwich ELISA. If the detection antibody used is unlabeled, a secondary enzyme-conjugated detection antibody is required. This is known as an indirect sandwich ELISA.
What's an ELISA sandwich?
The target antigen is sandwiched between a capture antibody and a detection antibody (may/maynot be conjugated w/ enzyme)
matched antibody pairs
pair of capture and detection antibodies used in sandwich ELISA - each binds with a specific epitope (binding site) of the antigen
key benefits of sandwich ELISA
"The key advantage of a sandwich ELISA is its high sensitivity; it is 2-5 times more sensitive than direct or indirect ELISAs. Sandwich ELISA also delivers high specificity as two antibodies are used to detect the antigen."
a collection of identical antibodies that interact with a single antigen site
purpose of the linked enzyme?
the detection method and possible signal amplifier (if target antigen is present)
Why a substrate for the enzyme is added?
Substrate is added so that an enzymatic reaction btw the substrate and the antibody-linked enzyme takes place, producing a change in color, fluorescence, or luminescence that is "read" to determine the quantity of the target analyte in each sample.
Direct ELISA -
ELISA in which one enzyme-conjugated antibody binds to the immobolized antigen.
Indirect ELISA -
ELISA in which an unlabeled primary antibody binds to the antigen. A secondary conjugated antibody is then used to detect the bound antibody.
Sandwich ELISA -
ELISA in which the antigen is trapped between two antibodies. The detection can be direct or indirect.
Competition/inhibition ELISA -
A more complex ELISA in which a control antigen "competes" for antibody-binding sites with the target antigen. The color signal is inversely proportional to the concentration of the target antigen.
X and Y axes of an ELISA standar curve
protein concentration (x) and absorbance (y)
synonym for enzyme-conjugated
synonym for primary antibody
In sandwich ELISA, what is the first substance added to the plate wells?
diluted capture antibodies
In sandwich ELISA, what substance is added to the plate wells following the addition of the capture antibodies?
unreactive proteins for surface blocking
In sandwich ELISA, what substance is added to the plate wells when the surface blocking procedures are completed?
the protein standards and unknown sample(s)
What do you have to do to the microplate wells BEFORE adding the standards and unknown samples?
wash the wells multiple times with PBS buffer to remove any unbound proteins and other contaminants
The standards/unknown samples have been incubated and the wells have been washed; what substance do you add to the wells next?
diluted detection antibodies
In sandwich ELISA, what substance is added to the wells AFTER adding the detection antibodies (following incubation and more washing of course!)?
diluted enzyme-conjugate (AP or HRP)
What substance must be added at the end of any ELISA assay in order to generate a detection signal?
a substrate for the assay's conjugate-enzyme that was previously added
To prevent "color saturation", stop the enzyme-substrate chemical reaction by rinsing the wells with what substance?
The ELISA reader (spectrophotometer) may be set to read the optical density at what frequency?
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