3rd Exam ELISA and Western Blotting Flashcards
ELISA meaning
ENZYME-LINKED IMMUNOSORBENT ASSAY
a widely used biochemical assay to detect in a sample the presence of and quantity of proteins, such as hormones and antibodies and bacteria or viruses.
ELISA
ELISA uses the coupling of antigens and antibodies and relies on the ________ and ________ of antibodies for antigens.
specificity and affinity
is the ability to discriminate among diverse proteins
Specificity
ability to tightly bind to molecules
Affinity
T or F
One can determine how much antibody is present by starting with an antigen, or one can determine how much antigen or hormone is present by starting with an antibody.
True
are large glycoprotein molecules produced by B- lymphocytes during the humoral immune response to antigens introduced into the body.
Antibodies
are white blood cells form from the hematopoietic (blood) stem cells in the bone marrow.
Lymphocytes
Lympocytes include (2)
B-lymphocytes (B-cells) and Tlymphocytes (T-cells)
Antibodies bind to antigens following the __________.
lock-and-key model
Where does antigens bind?
receptor sites
T or F
Secondary antibodies does not follow the lock-and-key model and bind to the receptor sites.
False
Secondary antibodies can also bind to antibodies following the lock-and-key model. These can either bind to the receptor site or the tail part.
Three properties of ELISE (it is where the name ELISA is derived)
- sorbent
- immuno
- enzyme-linked
sorbent property of ELISA
Antigen/antibody of interest is adsorbed on a plastic surface
immuno property of ELISA
Antigen is recognized and binds to a specific antibody
enzyme-linked property of ELISA
The antibody is recognized by the second antibody which has an enzyme attached
Substrate react with the enzyme to produce a product, usually ______
colored
has been used
- to detect hepatitis B, rabies, and HIV through antibodies in the blood serum, just to name a few diseases, or
- to measure the amount of various other proteins in the blood serum, such as hormones, toxins, and allergens.
ELISA method
INDIRECT ELISA steps (9)
- binding known antigen
- blocking
- washing
- adding test sample primary antibody
- washing
- adding enzyme-linked secondary antibody
- washing
- adding substrate
- reading results
- The indirect ELISA method begins with a sample of known antigen being bound to the wells of a microtiter plate
a) Binding Known Antigen
- The other unoccupied sites in each well are then bound by a concentrated solution of non-interacting protein, like ______ or ______ to block or prevent other proteins in the test sample from adhering.
b) Blocking
casein or bovine serum albumin,
– Rinse to remove any unbound antigen and non-interacting protein
c) Washing
- The test sample of serum containing the primary antibodies is added to each well. Antibodies could be HIV, rabies, or hepatitis B antibodies, for example.
d) Adding Test Sample Primary Antibody
– Rinse to remove any antibodies that did not bind to the known antigen.
e) Washing
- An enzyme-linked secondary antibody is added next to bind to the test sample antibodies.
Adding Enzyme-linked Secondary Antibody
The enzyme on the secondary antibodies are _______, such as (2)
proteins
horse radish peroxidase or alkaline phosphatase.
– Rinse to remove any secondary antibodies that did not bind to the primary antibody
g) Washing
- A _____ is then applied which is converted by the enzyme to give a color or fluorescence or electrochemical signal.
h) Adding Substrate
a substrate that turns blue in the presence of horse radish peroxidase
TMB
- i) Reading Results
- By using a _____________(3), the results can be read and recorded.
spectrophotometer, spectrofluorometer, or electrochemical device
T or F
In indirect ELISA, the amount of color produced is proportional to the amount of primary antibody bound to the antigen proteins on the bottom of the wells.
True
plays the role of a chromogenic substrate, and is also one of the most sensitive substrates for HRP
TMB (3,3’,5,5’-Tetramethylbenzidine)
When a TMB solution is added to HRP, HRP will reduce hydrogen peroxide and oxidize TMB, turning it from_______ to ________
colorless; blue-green
are commonly used to read ELISA results.
Spectrophotometers (microplate reader)
T or F
Direct ELISA has the advantage of signal amplification than indirect ELISA.
False
Indirect ELISA has the advantage of signal
amplification because multiple secondary antibodies can bind the analyte-specific antibody.
- is the most commonly used format that detect antigens in the sample.
- this format requires two antibodies specific for different epitopes of the antigen.
SANDWICH ELISA
The two antibodies in sandwich ELISA are normally referred to as
matched antibody pairs
T or F
SANDWICH ELISA is highly specific which minimizes false positives
True
SANDWICH ELISA steps (5)
- Capture Antibody, Blocking Buffer
- Antigen
- Biotin Labeled Detection Antibody
- Streptavidin-HRPO
- TMB Substrate to produce colored product
ELISA ADVANTAGES (5)
- High sensitivity and specificity
- High throughput
- Easy to perform
- Quantitative
- Possibility to test various sample types
T or F
it is common for ELISAs to detect antigens at the picogram level in a very specific manner due to the use of antibodies.
T
commercial ELISA kits are normally
available in a 96-well plate format. But the assay can be easily adapted to 384-well plates
this allows automated testing of large numbers of chemical and/or biological compounds for a specific biological target
High throughput
T or F
ELISA protocols are hard to follow and is time consuming
F
protocols are easy to follow and involve little
hands-on time
sample types where you can use ELISA to test the presence or possibility (5)
serum,
plasma,
cellular and tissue extracts,
urine, and
saliva among others
Disadvantages of ELISA
- Temporary readouts
- Limited antigen information
Why is ELISA has only temporary readouts?
detection is based on enzyme/substrate reactions and therefore readout must be obtained in a short time span
ELISA provides limited antigen information (2)
amount or presence of the antigen in the sample
ELISA ARE WIDELY AVAILABLE AS TEST KITS such as pregnancy test. What is the antigen used for pregnancy test?
Human Chorionic Gonadotropin (hCG)
are techniques for transferring DNA, RNA and proteins onto a carrier so they can be separated, and often follows the use of a GEL ELECTROPHORESIS.
Blots / BLOTTING
Southern blot
DNA
Northern blot
RNA
Western blot
proteins (immunoblot)
Eastern blot
post-translational proteins
is an analytical method that involves the immobilization of proteins on membranes before detection using antibodies.
Protein blotting
is a prerequisite for Western blotting
SDS PAGE technique
is an electrophoresis method that allows protein separation by mass.
SDS-PAGE (Polyacrylamide Gel Electrophoresis)
is a negatively charged detergent used to denature and linearize proteins
Sodium dodecylsulfate (SDS)
SDS-PAGE process
- the disulfide bonds in proteins are reduced by 2-mercaptoethanol
- SDS with negative charge is added which linearize proteins
- Molecular weight markers
In SDS-PAGE, Application of _______ causes the migration and separation of proteins
electric field
is used to form a gel that provides a matrix of pores through which molecules migrate at different rates in SDS-PAGE
Polyacrylamide
most common stain used to visualize protein bands
Coomassie Blue
most sensitive test used to visualize protein bands
Silver stain
WESTERN BLOT - TRANSFERRING
In order to make proteins accessible to antibody detection, they are transferred
onto a membrane made of ________________.
nitrocellulose or polyvinylidene difluoride (PVDF)
two types of transfer for antibody detection during western blot
- Diffusion transfer
- Electro transfer
Western Blot steps (4)
- SDS PAGE (stain)
- Transfer to a membrane
- Blocking and incubation with antibodies
- Detection of bands
