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SERO > Primary Reactions 2.2 EIA > Flashcards

Primary Reactions 2.2 EIA Flashcards

1
Q

an antibody labeled with an enzyme, is used as a reagent to detect a specific antigen.

A

Immunoenzyme

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2
Q

______ can be applied to all Ag-Ab systems, including those involving serum protein, hormones, drugs, and other antigens and the antibodies directed against pathogens.

A

Enzymeimmunoassays

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3
Q

____ can be developed by the amplification effect of enzymes.

A

Sensitive assays

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4
Q

Enzymes used as labels for immunoassay are chosen according to:

A

a. the number of substrate molecules converted per molecule of enzyme
b. ease and speed of detection
c. stability
d. availability and cost of enzyme and substrate

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5
Q

Frequently used enzyme labels:

A
  1. Horseradish peroxidase
  2. Alkaline phosphatase
  3. Lysozyme
  4. G-6-PD
  5. D-galactosidase
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6
Q

_____ using enzymes as labels were developed as alternatives to radioisotopes

A

Quantitative immunoassays

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7
Q

The most widely performed tests are:

A

a. Enzyme-linked immunosorbent assay (ELISA)

b. Enzyme-multiplied immunoassay technique (EMIT)

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8
Q

2 Main Methods of Enzymeimmunoassay (EIA):

A
  1. Qualitative Slide Test -

2. Quantitative Tube Test

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9
Q

Main Methods of Enzymeimmunoassay (EIA):

It is a test for Antinuclear Antibody

A

Qualitative Slide Test

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10
Q

Quantitative Tube Test – 2 Major Types:

A

Heterogeneous EIA

Homogeneous EIA

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11
Q

Quantitative Tube Test – Major Types:

● Antigen-antibody reaction does not affect the activity of the enzyme label.
● Requires physical separation of bound and unbound antigen.

A

Heterogeneous EIA

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12
Q

Quantitative Tube Test – Major Types:

○ Antigen-antibody reaction modulates the activity of the enzyme label.
○ No separation of bound and free antigen is required.

A

Homogeneous EIA

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13
Q

Methods of Heterogeneous ELISA:

A

Direct ELISA
Indirect ELISA
Sandwich ELISA
Competitive ELISA

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14
Q

In _________, only an enzyme-labeled primary antibody is used. The enzyme labeled antibody “directly” binds to the (target) antigen that is immobilized to the plate (coated surface).
Next, substrate is added. The enzyme linked to the primary antibody then reacts with the substrate to produce a visible signal that can be measured. In this way, the antigen of interest is detected.

Advantages:

  1. ______.
  2. _____ possible non-specific binding of secondary antibody. Only one antibody is involved.
A

Direct ELISA,

Fast
Eliminates

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15
Q

In this technique, antigen is coated on the microtiter well. Serum or other sample containing primary antibody is added to the microtiter well and allowed to react with the coated antigen.
Any free primary antibody is washed away and the bound antibody to the antigen is detected by adding an __________that binds to the primary antibody. Unbound secondary antibody is then washed away and a specific substrate for the enzyme is added.

_____ hydrolyzes the substrate to form colored products. The amount of colored end product is measured by _________ that can measure the absorbance of all the wells.

Advantages:

  1. ________, since more than one labeled antibody is bound per primary antibody.
  2. _______ of the primary antibody is retained because it is not labeled.
  3. _____, since different primary detection antibodies can be used with a single labeled secondary antibody.
  4. ____, since fewer labeled antibodies are required.
  5. _________ markers can be used with the same primary antibody.
A

Indirect ELISA
enzyme-conjugated secondary antibody

Enzyme; spectrophotometric plate readers

Increased sensitivity
Maximum immunoreactivity
Flexibility
Cost savings
Different visualization
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16
Q

Antigen can be detected by this. In this technique, antibody is coated on the microtiter well. A sample containing antigen is added to the well and allowed to react with the antibody attached to the well, forming _________.
After the well is washed, a ________ specific for a different epitope on the antigen is added and allowed to react with the bound antigen. Then, unbound secondary antibody is removed by _____.
Finally substrate is added to the plate which is hydrolyzed by enzyme to form colored products.

Advantages:

  1. _____, since two antibodies are used, the antigen is specifically captured and detected.
  2. ____ for complex samples, since the antigen does not require purification prior to measurement.
  3. Flexibility and _____
A

Sandwich ELISA
antigen-antibody complex
second enzyme-linked antibody
washing

High specificity
Suitable
sensitivity

17
Q

Test is used to measure the concentration of an antigen in a sample.

Antibody is first incubated in solution with a sample containing antigen. The antigen antibody mixture is then added to the micro-titer well coated with antigen.

The more the antigen present in the sample, the ____ free antibody will be available to bind to the antigen-coated well. After the well is washed,_________ specific for isotype of the primary antibody is added to determine the amount of primary antibody bound to the well.

The higher the concentration of antigen in the sample, the _____ the absorbance.

A

Competitive ELISA

less
enzyme conjugated secondary antibody

lower

18
Q

Competitive ELISA

Advantages:

  1. ______, since two antibodies are used.
  2. _______, since both direct and indirect detection methods can be used.
  3. _____ samples, since the antigen does not require purification prior to measurement.
A

High specificity
High sensitivity
Suitable for complex

19
Q

Application of ELISA:

  1. Presence of _____ or the presence of _____ in a sample can be evaluated.
  2. Determination of ____________in a virus test.
  3. Used in food industry when detecting _______
  4. Applied in _____- tracking the spread of disease e.g. HIV, bird flu, common, colds, cholera, STD etc.
A

antigen; antibody
serum antibody concentrations
potential food allergens.
disease outbreaks

20
Q

Advantages of EIA:

  1. Sensitive assays can be developed by the ______ of enzymes.
  2. Reagents are relatively cheap and can have a _____.
  3. _____ simultaneous assays can be developed.
  4. A ______ of assay configurations can be developed.
  5. ______ is inexpensive and widely available.
  6. _______ occur during labelling or disposal of wastes.

➢Disadvantages of EIA:

  1. Measurement of enzyme activity can be more _____ than measurement of the activity of some radioisotopes.
  2. ________ as radioimmunoassays.
  3. Enzyme activity may be affected by ____ constituents.
  4. Homogeneous EIAs for large protein molecules have been developed but require complex immunochemical reagents
A 
amplification effect
long shelf- life
Multiple
wide variety
Equipment
No radiation hazards

complex
Not as sensitive
plasma

21
Q

________ are less sensitive than heterogeneous assays, but are rapid, simple to perform, and easily adapt to automation.

A

homogeneous assays

22
Q

No separation step, no washing step are necessary.

Its chief use has been in the determination of low-molecular-weight analytes, such as, hormones, therapeutic drugs, and drugs of abuse in both serum and urine.

A

HOMOGENEOUS ENZYME IMMUNOASSAY

23
Q

________is an example of a homogeneous immunoassay

A

Enzyme multiplied immunoassay technique (EMIT)

24
Q

Are based on the principle of change in enzyme activity as specific antigen antibody combination occurs.

A

HOMOGENEOUS ENZYME IMMUNOASSAY

25
Q

❑ Applied to any antigen-antibody system which measure the degree of immune reaction that is carried out without a separation of the free and the antibody-bound components.

❑ Chief application is the determination of low molecular weight analytes, such as hormones and drugs; for the detection of small molecules, i.e., haptens (Morphine, Digoxin and many other drugs)

A

Enzyme Multiple Immunoassay Technique (EMIT)

26
Q

Enzyme Multiple Immunoassay Technique (EMIT):

● This is a homogeneous competitive binding assay.
● When antibody binds to the labeled antigen, it blocks enzymatic activity, reducing the amount of color development
● The more patient antigen that binds to the antibody the more enzyme tagged antigen remains free to react with the chromogen.
● Color development is _____ proportional to the concentration of antigen
● The drug in the sample and the drug labeled with _____ complete for antibody binding sites
● Binding inhibits enzyme activity, while free enzyme remains active to interact with the substrate.
● Enzyme activity/absorbance is _____proportional to drug concentration.

A

directly
G6PD
directly

SERO (6 decks)

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