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ANACHEM LEC > 3rd Exam ELISA and Western Blotting > Flashcards

3rd Exam ELISA and Western Blotting Flashcards

1
Q

ELISA meaning

A

ENZYME-LINKED IMMUNOSORBENT ASSAY

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2
Q

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.

A

ELISA

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3
Q

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

A

specificity and affinity

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4
Q

is the ability to discriminate among diverse proteins

A

Specificity

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5
Q

ability to tightly bind to molecules

A

Affinity

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6
Q

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.

A

True

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7
Q

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

A

Antibodies

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8
Q

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

A

Lymphocytes

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9
Q

Lympocytes include (2)

A

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

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10
Q

Antibodies bind to antigens following the __________.

A

lock-and-key model

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11
Q

Where does antigens bind?

A

receptor sites

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12
Q

T or F

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

A

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.

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13
Q

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

A
  • sorbent
  • immuno
  • enzyme-linked
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14
Q

sorbent property of ELISA

A

Antigen/antibody of interest is adsorbed on a plastic surface

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15
Q

immuno property of ELISA

A

Antigen is recognized and binds to a specific antibody

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16
Q

enzyme-linked property of ELISA

A

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

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17
Q

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

A

colored

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18
Q

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.
A

ELISA method

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19
Q

INDIRECT ELISA steps (9)

A
  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
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20
Q
  • The indirect ELISA method begins with a sample of known antigen being bound to the wells of a microtiter plate
A

a) Binding Known Antigen

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21
Q
  • 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.
A

b) Blocking

casein or bovine serum albumin,

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22
Q

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

A

c) Washing

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23
Q
  • 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.
A

d) Adding Test Sample Primary Antibody

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24
Q

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

A

e) Washing

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25
Q
  • An enzyme-linked secondary antibody is added next to bind to the test sample antibodies.
A

Adding Enzyme-linked Secondary Antibody

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26
Q

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

A

proteins

horse radish peroxidase or alkaline phosphatase.

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27
Q

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

A

g) Washing

28
Q
  • A _____ is then applied which is converted by the enzyme to give a color or fluorescence or electrochemical signal.
A

h) Adding Substrate

29
Q

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

A

TMB

30
Q
  • i) Reading Results
  • By using a _____________(3), the results can be read and recorded.
A

spectrophotometer, spectrofluorometer, or electrochemical device

31
Q

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.

A

True

32
Q

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

A

TMB (3,3’,5,5’-Tetramethylbenzidine)

33
Q

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

A

colorless; blue-green

34
Q

are commonly used to read ELISA results.

A

Spectrophotometers (microplate reader)

35
Q

T or F

Direct ELISA has the advantage of signal amplification than indirect ELISA.

A

False

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

36
Q
  • is the most commonly used format that detect antigens in the sample.
  • this format requires two antibodies specific for different epitopes of the antigen.
A

SANDWICH ELISA

37
Q

The two antibodies in sandwich ELISA are normally referred to as

A

matched antibody pairs

38
Q

T or F

SANDWICH ELISA is highly specific which minimizes false positives

A

True

39
Q

SANDWICH ELISA steps (5)

A
  1. Capture Antibody, Blocking Buffer
  2. Antigen
  3. Biotin Labeled Detection Antibody
  4. Streptavidin-HRPO
  5. TMB Substrate to produce colored product
40
Q

ELISA ADVANTAGES (5)

A
  • High sensitivity and specificity
  • High throughput
  • Easy to perform
  • Quantitative
  • Possibility to test various sample types
41
Q

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.

A

T

42
Q

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

A

High throughput

43
Q

T or F

ELISA protocols are hard to follow and is time consuming

A

F

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

44
Q

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

A

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

45
Q

Disadvantages of ELISA

A
  • Temporary readouts
  • Limited antigen information
46
Q

Why is ELISA has only temporary readouts?

A

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

47
Q

ELISA provides limited antigen information (2)

A

amount or presence of the antigen in the sample

48
Q

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

A

Human Chorionic Gonadotropin (hCG)

49
Q

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.

A

Blots / BLOTTING

50
Q

Southern blot

A

DNA

51
Q

Northern blot

A

RNA

52
Q

Western blot

A

proteins (immunoblot)

53
Q

Eastern blot

A

post-translational proteins

54
Q

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

A

Protein blotting

55
Q

is a prerequisite for Western blotting

A

SDS PAGE technique

56
Q

is an electrophoresis method that allows protein separation by mass.

A

SDS-PAGE (Polyacrylamide Gel Electrophoresis)

57
Q

is a negatively charged detergent used to denature and linearize proteins

A

Sodium dodecylsulfate (SDS)

58
Q

SDS-PAGE process

A
  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
59
Q

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

A

electric field

60
Q

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

A

Polyacrylamide

61
Q

most common stain used to visualize protein bands

A

Coomassie Blue

62
Q

most sensitive test used to visualize protein bands

A

Silver stain

63
Q

WESTERN BLOT - TRANSFERRING

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

A

nitrocellulose or polyvinylidene difluoride (PVDF)

64
Q

two types of transfer for antibody detection during western blot

A
  • Diffusion transfer
  • Electro transfer
65
Q

Western Blot steps (4)

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

ANACHEM LEC (10 decks)

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