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Analytical Chemistry Lecture > ELISA and Western Blotting > Flashcards

ELISA and Western Blotting Flashcards

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1
Q

The acronym ELISA stands for:

A

Enzyme-Linked Immunosorbent Assay

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2
Q

ELISA is a widely used biochemical assay to detect in a sample the presence of and quantity of ____, such as hormones and antibodies and bacteria or viruses.

A

proteins

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3
Q

ELISA uses the ____ and relies on the _____.

A

coupling of antigens and antibodies; specificity and affinity of antibodies for antigens

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4
Q

____ is the ability to discriminate among diverse proteins.

A

Specificity

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5
Q

____ is the ability to tightly bind to molecules.

A

Affinity

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6
Q

One can determine how much antibody is present by starting with an ____, or one can determine how much antigen or hormone is present by starting with an ____.

A

antigen; antibody

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7
Q

Antibodies are large glycoprotein molecules produced by ____ during the humoral immune response to antigens introduced into the body.

A

B-lymphocytes

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8
Q

The name ELISA: (4)

A
  1. Antigen/antibody of interest is adsorbed on a plastic surface (sorbent).
  2. Antigen is recognized and binds to a specific antibody (immuno).
  3. The antibody is recognized by the second antibody which has an enzyme attached (enzyme-linked).
  4. Substrate react with the enzyme to produce a product, usually colored.
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9
Q

Indirect Elisa
Steps to detect antibodies in a sample: (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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10
Q

TMB or _____

A

3, 3’, 5, 5’-Tetramethylbenzidine

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11
Q

When a TMB solution is added to HRP, HRP will reduce hydrogen peroxide and oxidize TMB, turning it from ____.

A

colorless to blue-green

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12
Q

____ are commonly used to read ELISA results.

A

Spectrophotometers (Microplate Reader)

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13
Q

___ is the most commonly used format to detect antigens in a sample.

A

Sandwich ELISA

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14
Q

Sandwich ELISA requires two antibodies specific to different epitopes of the antigen. These two antibodies are normally referred to as _____.

A

matched antibody pairs

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15
Q

Advantages of ELISA (5)

A
  1. High sensitivity and specificity: it is common for ELISAs to detect antigens at the picogram level in a very specific manner due to the use of antibodies.
  2. High throughput: commercial ELISA kits are normally available in a 96-well plate format. But the assay can be easily adapted to 384-well plates.
  3. Easy to perform: protocols are easy to follow and involve little hands-on time.
  4. Quantitative: it can determine the concentration of antigen in a sample.
  5. Possibility to test various sample types: serum, plasma, cellular and tissue extracts, urine, and saliva among others.
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16
Q

Disadvantages of ELISA (2)

A
  1. Temporary readouts: detection is based on enzyme/substrate reactions and therefore readouts must be obtained in a short time span.
  2. Limited antigen information: information is limited to the amount or presence of the antigen in the sample.
17
Q

____ are techniques for transferring DNA, RNA, and proteins onto a carrier so they can be separated, and often follows the use of gel electrophoresis.

A

Blots

18
Q

Types of Blots (4)

A
  1. Southern Blot - DNA
  2. Northern Blot - RNA
  3. Western Blot - Proteins
  4. Eastern Blot - Post-translational Proteins
19
Q

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

A

Protein Blotting

20
Q

The _____ is a prerequisite for Western blotting.

A

SDS Page Technique

21
Q

SDS-PAGE (or ______)

A

Polyacrylamide Gel Electrophoresis

22
Q

_____ is an electrophoresis method that allows _____.

A

SDS; protein separation by mass

23
Q

SDS or _____

A

Sodium Dodecylsulfate

24
Q

_____ is a negatively-charged detergent used to denature and linearize proteins.

A

SDS

25
Q

Application of electric field causes the _____ of proteins.

A

migration and separation

26
Q

_____ is used to form a gel that provides a matrix of pores through which molecules migrate at different rates.

A

Polyacrylamide

Analytical Chemistry Lecture (3 decks)

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