3rd Exam ELISA and Western Blotting

Question 1

ELISA meaning

Answer 1

ENZYME-LINKED IMMUNOSORBENT ASSAY

Question 2

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.

Answer 2

ELISA

Question 3

ELISA uses the coupling of antigens and antibodies and relies on the ________ and ________ of antibodies for antigens.

Answer 3

specificity and affinity

Question 4

is the ability to discriminate among diverse proteins

Answer 4

Specificity

Question 5

ability to tightly bind to molecules

Answer 5

Affinity

Question 6

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.

Answer 6

True

Question 7

are large glycoprotein molecules produced by B- lymphocytes during the humoral immune response to antigens introduced into the body.

Answer 7

Antibodies

Question 8

are white blood cells form from the hematopoietic (blood) stem cells in the bone marrow.

Answer 8

Lymphocytes

Question 9

Lympocytes include (2)

Answer 9

B-lymphocytes (B-cells) and Tlymphocytes (T-cells)

Question 10

Antibodies bind to antigens following the __________.

Answer 10

lock-and-key model

Question 11

Where does antigens bind?

Answer 11

receptor sites

Question 12

T or F

Secondary antibodies does not follow the lock-and-key model and bind to the receptor sites.

Answer 12

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.

Question 13

Three properties of ELISE (it is where the name ELISA is derived)

Answer 13

• sorbent
• immuno
• enzyme-linked

Question 14

sorbent property of ELISA

Answer 14

Antigen/antibody of interest is adsorbed on a plastic surface

Question 15

immuno property of ELISA

Answer 15

Antigen is recognized and binds to a specific antibody

Question 16

enzyme-linked property of ELISA

Answer 16

The antibody is recognized by the second antibody which has an enzyme attached

Question 17

Substrate react with the enzyme to produce a product, usually ______

Answer 17

colored

Question 18

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.

Answer 18

ELISA method

Question 19

INDIRECT ELISA steps (9)

Answer 19

1. binding known antigen
2. blocking
3. washing
4. adding test sample primary antibody
5. washing
6. adding enzyme-linked secondary antibody
7. washing
8. adding substrate
9. reading results

Question 20

• The indirect ELISA method begins with a sample of known antigen being bound to the wells of a microtiter plate

Answer 20

a) Binding Known Antigen

Question 21

• 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.

Answer 21

b) Blocking

casein or bovine serum albumin,

Question 22

– Rinse to remove any unbound antigen and non-interacting protein

Answer 22

c) Washing

Question 23

• 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.

Answer 23

d) Adding Test Sample Primary Antibody

Question 24

– Rinse to remove any antibodies that did not bind to the known antigen.

Answer 24

e) Washing

Question 25

- An enzyme-linked secondary antibody is added next to bind to the test sample antibodies.

Answer 25

Adding Enzyme-linked Secondary Antibody

Question 26

The enzyme on the secondary antibodies are _______, such as (2)

Answer 26

proteins horse radish peroxidase or alkaline phosphatase.

Question 27

– Rinse to remove any secondary antibodies that did not bind to the primary antibody

Answer 27

g) Washing

Question 28

- A _____ is then applied which is converted by the enzyme to give a color or fluorescence or electrochemical signal.

Answer 28

h) Adding Substrate

Question 29

a substrate that turns blue in the presence of horse radish peroxidase

Answer 29

TMB

Question 30

* i) Reading Results - By using a _____________(3), the results can be read and recorded.

Answer 30

spectrophotometer, spectrofluorometer, or electrochemical device

Question 31

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.

Answer 31

True

Question 32

plays the role of a chromogenic substrate, and is also one of the most sensitive substrates for HRP

Answer 32

TMB (3,3',5,5'-Tetramethylbenzidine)

Question 33

When a TMB solution is added to HRP, HRP will reduce hydrogen peroxide and oxidize TMB, turning it from_______ to ________

Answer 33

colorless; blue-green

Question 34

are commonly used to read ELISA results.

Answer 34

Spectrophotometers (microplate reader)

Question 35

T or F Direct ELISA has the advantage of signal amplification than indirect ELISA.

Answer 35

False Indirect ELISA has the advantage of signal amplification because multiple secondary antibodies can bind the analyte-specific antibody.

Question 36

- is the most commonly used format that detect antigens in the sample. - this format requires two antibodies specific for different epitopes of the antigen.

Answer 36

SANDWICH ELISA

Question 37

The two antibodies in sandwich ELISA are normally referred to as

Answer 37

matched antibody pairs

Question 38

T or F SANDWICH ELISA is highly specific which minimizes false positives

Answer 38

True

Question 39

SANDWICH ELISA steps (5)

Answer 39

1. Capture Antibody, Blocking Buffer 2. Antigen 3. Biotin Labeled Detection Antibody 4. Streptavidin-HRPO 5. TMB Substrate to produce colored product

Question 40

ELISA ADVANTAGES (5)

Answer 40

* High sensitivity and specificity * High throughput * Easy to perform * Quantitative * Possibility to test various sample types

Question 41

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.

Answer 41

T

Question 42

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

Answer 42

High throughput

Question 43

T or F ELISA protocols are hard to follow and is time consuming

Answer 43

F protocols are easy to follow and involve little hands-on time

Question 44

sample types where you can use ELISA to test the presence or possibility (5)

Answer 44

serum, plasma, cellular and tissue extracts, urine, and saliva among others

Question 45

Disadvantages of ELISA

Answer 45

* Temporary readouts * Limited antigen information

Question 46

Why is ELISA has only temporary readouts?

Answer 46

detection is based on enzyme/substrate reactions and therefore readout must be obtained in a short time span

Question 47

ELISA provides limited antigen information (2)

Answer 47

amount or presence of the antigen in the sample

Question 48

ELISA ARE WIDELY AVAILABLE AS TEST KITS such as pregnancy test. What is the antigen used for pregnancy test?

Answer 48

Human Chorionic Gonadotropin (hCG)

Question 49

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.

Answer 49

Blots / BLOTTING

Question 50

Southern blot

Answer 50

DNA

Question 51

Northern blot

Answer 51

RNA

Question 52

Western blot

Answer 52

proteins (immunoblot)

Question 53

Eastern blot

Answer 53

post-translational proteins

Question 54

is an analytical method that involves the immobilization of proteins on membranes before detection using antibodies.

Answer 54

Protein blotting

Question 55

is a prerequisite for Western blotting

Answer 55

SDS PAGE technique

Question 56

is an electrophoresis method that allows protein separation by mass.

Answer 56

SDS-PAGE (Polyacrylamide Gel Electrophoresis)

Question 57

is a negatively charged detergent used to denature and linearize proteins

Answer 57

Sodium dodecylsulfate (SDS)

Question 58

SDS-PAGE process

Answer 58

1. the disulfide bonds in proteins are reduced by 2-mercaptoethanol 2. SDS with negative charge is added which linearize proteins 3. Molecular weight markers

Question 59

In SDS-PAGE, Application of _______ causes the migration and separation of proteins

Answer 59

electric field

Question 60

is used to form a gel that provides a matrix of pores through which molecules migrate at different rates in SDS-PAGE

Answer 60

Polyacrylamide

Question 61

most common stain used to visualize protein bands

Answer 61

Coomassie Blue

Question 62

most sensitive test used to visualize protein bands

Answer 62

Silver stain

Question 63

WESTERN BLOT - TRANSFERRING In order to make proteins accessible to antibody detection, they are transferred onto a membrane made of ________________.

Answer 63

nitrocellulose or polyvinylidene difluoride (PVDF)

Question 64

two types of transfer for antibody detection during western blot

Answer 64

* Diffusion transfer * Electro transfer

Question 65

Western Blot steps (4)

Answer 65

1. SDS PAGE (stain) 2. Transfer to a membrane 3. Blocking and incubation with antibodies 4. Detection of bands