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Advanced Laboratory Operations and Automation > Immunoassay flashcards > Flashcards

Immunoassay flashcards

1
Q

Most common immunochemical techniques

A

‣ Add labeled antibody to
the patient’s sample
(analyte) contains
antigen
‣ Detecting patient’s
antibody

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2
Q

Examples of what immunochemical techniques are used for

A

Infectious disease, serology,
allergy testing, &
autoimmune testing

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3
Q

Antibodies in Immunoassays characteristics

A

1.)Specificity, 2) Affinity, 3) Cross-linking

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4
Q

A1 antitrypsin

A

Protects the bodys tissues from being damaged by infection fighting agents released by its immune system

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5
Q

Prozone

A

zone of antibody excess

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6
Q

Zone of equivalence

A

where you have the right amount of antigen and antibody

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7
Q

Post zone

A

Zone of excess antigen

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8
Q

Polymer effect

A

‣ Linear polymers enhance immune complex
precipitation

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9
Q

Polymer types

A

Dextran
* PVA
* PEG

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10
Q

methods for ag ab detection

A
  • Precipitation or Agglutination
  • Hemagglutination and Hemagglutination
    inhibition
  • Passive Gel Diffusion
  • Radio-immunoassays
  • ELISA
  • Immunofluorescence
  • Immmunoblotting
  • Immunochromatograph
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11
Q

Precipitation

A

‣ Antibodies react with soluble substances
ex) proteins, carbohydrates, etc
‣ Reaction visible with naked eye - particles

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12
Q

Agglutination

A

‣ Antibodies react with insoluble substances
ex) RBCs, bacterial cells, latex particles coated with antigen
‣ Reaction visible with naked eye – larger clumps
(aggregates)
‣ If agglutination target is RBCs, called hemagglutination
Precipitation or Agglutination?

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13
Q

Hemagglutination

A

If agglutination targets RBCs

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14
Q

Direct agglutination

Test patient serum against what

A

large, cellular antigens to
screen for the presence of antibodies in pt. serum.

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15
Q

Direct agglutination

Antigen is naturally what
and in this case what

A

Antigen is naturally present on the surface of the cells.
In this case, the Ag-Ab reaction forms an agglutination,
which is directly visible

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16
Q

Direct agglutination

The particle antigen may be

A

➡ The particle antigen may be a bacterium.
ex) Serotyping of E. coli, Salmonella
➡ The particle antigen may be a parasite.
ex) Serodiagnosis of Toxoplasmosis
➡ The particle antigen may be a red blood cell.
ex) Determination of blood groups
9
Direct Agglutination?

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17
Q

example of bacterium direct agglutination

A

ex) Serotyping of E. coli, Salmonell

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18
Q

Examples of parasite direct agglutination

A

x) Serodiagnosis of Toxoplasmosis

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19
Q

Example of RBC direct agglutination

A

ex) Determination of blood groups

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20
Q

Passive agglutination

A
  • An agglutination reaction that employs particles that
    are coated with antigens not normally found in the
    cell surfaces
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21
Q

Passive agglutination particle carriers include

A

– Red blood cells
– Polystyrene latex
– Bentonite
– charcoal

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22
Q

Reverse passive agglutination principle

A

‣ Antigen (in serum) binds
to antibody (from kit)
coated on carrier particles
and results in
agglutination
ex) detecting cholera toxin

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23
Q

Reverse passive agglutination example

A

detecting cholera toxin

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24
Q

Passive gel diffusion

A

Performed on semisolid (agarose)
✦ Precipitin band dependent on:
* Solubility of antigen/antibody
* Relative concentration of each
* Temperature
* Time
* Gel viscosity
Passive Gel Diffusion

25
Q

Passive gel diffusion

Precipitation band dependent on

A
  • Solubility of antigen/antibody
  • Relative concentration of each
  • Temperature
  • Time
  • Gel viscosity
    Passive Gel Diffusion
26
Q

Simple gel diffusion

Simple diffusion ( radial Immunodiffusion

A

❖ Antibody: suspended uniformly in gel
❖ Antigen: applied to well cut in gel
‣ Diffusion away from well dilutes antigen
‣ Zone of equivalence results in precipitin ring
‣ Area of zone can be compared to known antigen concentrations
(standards)
Passive Gel Diffusion: Simple Diffusion (Radial Immunodiffusion)

27
Q

Simple diffusion ( radial immunodiffusion)

Antibody is what in gel

Antigen is applied

A

Antibody: suspended uniformly in gel
❖ Antigen: applied to well cut in gel

28
Q

Diffusion away from what dilutes what

Simple Diffusion (Radial Immunodiffusion)

A

Diffusion away from well dilutes antigen

29
Q

zone of equivalence results in

A

Zone of equivalence results in precipitin ring

30
Q

Passive Gel Diffusion: Simple Diffusion (Radial Immunodiffusion)

Area of zone can be what

A

compared to known antigen concentrations
(standards)

31
Q

Ouchterlony double diffusion

Both what

Position of precipitin band indicates what

A

✦ Both Antibody and antigens added to separate wells
of gel
✦ Position of precipitin bands indicates identity
1. Identity (common epitopes)
2. Non-identity (different epitopes)
3. Partial identity (some epitopes in common

32
Q

Identity

A

(common epitopes

33
Q

Non identity

A

(different epitopes)

34
Q

Partial identity

A

(some epitopes in common)

35
Q

Immunoelectrophoresis

A

✦ Immunoelectrophoresis (IEP)
‣ Serum proteins are
electrophoretically separated
‣ Reagent antibody is placed in
a trough running parallel
‣ Diffusion to precipitin arc

36
Q

Immunoelectrophoresis

2 dimensional crossed IE

A

✦ Two dimensional crossed IE (aka 2D IE, CRIE)
* Serum separation by charge/size first
* Turn 90°, electrophorese into gel containing ab
Immunoelectrophoresis

37
Q

Electroimmunoassay

Rocket electrophoresis

A
  • Antigen applied to wells in lower gel
  • Upper gel contains antibody
  • Electrophorese – quantitative when compared
    to calibrators
  • Rapid detection & quantification
38
Q

Immunofixation electrophoresis (IFE)

A

✦ Electrophorese proteins
✦ Antibody placed on gel
– precipitation
✦ All other proteins washed
out
✦ Stain gel with dye
(Coomassie blue, etc)
Immunofixation electrophoresis (IFE)

39
Q

Indicator labeled Immunoassays

Labels

A

‣ Radioactive isotopes (rarely)
‣ Enzymes (common)
* HRP, ALP, glucose oxidase
* Substrate ! Product (colored)
* If cleavage of substrate produces photon of light
➡ “Chemiluminescent”
‣ Fluorescent labels (common)

40
Q

Competitive immunoassays

Typical use what

Carried out in

A

Typically use labeled antigen (reagent)
Carried out in presence of “excess antigen

41
Q

LABELED IMMUNOASSAYS

Patients unknown analyte and what compete

A

Patient’s unknown analyte & the labeled analyte
compete for binding sites on a known/limited amount of
antibody

42
Q

Labeled Immunoassay

A

Value of patient analyte is inversely proportional to the
signal generated by the bound labeled antigen

43
Q

Noncompetitive immunoassay

Labeled entity is

Typically use what

Carried out in what

A

-Labeled entity is antibody
-Typically use an unlabeled capture antibody and a labeled
indicator antibody (reagent)
-Carried out in presence of “excess antibody

44
Q

LABELED IMMUNOASSAYS

Patient unknown analyte is what

Value of patient analyte is

A

-Patient unknown analyte antigen is held between the two
antibodies (‘sandwich’)
-Value of patient analyte is directly proportional to the signal
generated by the bound labeled antibody

45
Q

Heterogeneous immunoassays

A

‣ Usually require washing step to separate antibody-
bound antigen from remaining free antigen.

46
Q

Homogenous Immunoassays

A

‣ No separation between antibody-bound antigen with
free antigen
L ABELED IMMUNOASSAYS

47
Q

Free

A

Free: floating in mixture, sometimes washed out,
sometimes precipitated or adsorbe

48
Q

Bound

A

Bound: stuck to surface in some way

49
Q

Know what

A

✦ Know relationship of free to bound for each assay
✦ Know what you are measuring – free vs. bound
✦ Know relationship of signal to unknown concentration
(directly or indirectly proportional)

50
Q

Seperation techniques

Solid phase

A

✦ Solid phase*
✦ Adsorption with dextran-treated charcoal or dextran
gel (Sephadex)
✦ Precipitated with ammonium sulfate
✦ Precipitated via double antibody technique (bound
fraction)
SEPARATION TECHNIQUES

51
Q

Standards

A

✦ Regardless of assay type, standards MUST BE USED to
convert signal to amount
✦ In case of saturation – dilute sample

52
Q

Radioisotopes

Requirments

A

✦ Requirements:
‣ Licensing
‣ Specialized equipment
‣ Speci alized handling and disposa

53
Q

Radioisotopes are

A

Inexpensive, sensitive

54
Q

Common radiolables

A

‣ Iodine (gamma)
‣ Tritium (beta)

55
Q

RADIOIMMUNOASSAY
uses

A

‣ Hormones in plasma
‣ Drug levels (digoxin, drugs of abuse)
‣ Hepatitis B Surface Antigen (HBsAg)
‣ anti-DNA Antibodies (SLE)
‣ Allergy Testing (RAST)
‣ Essentially any antibody test

56
Q

RADIOLABELED ANTIBODIES

Additional uses

A

PET Scanning, infectious disease, cancer therapy

57
Q

Luminescent Immunoassays

Luminescent compounds emit what

are highly what

A

✦ Luminescent compounds emit a photon of light as
the result of an electrical biochemical or chemical
reaction
✦ “Highly sensitive

58
Q

LUMINESCENT IMMUNOASSAYS

ofter used as what

A

Often used as substrate, and coupled with enzymes
as immunoassay label

Advanced Laboratory Operations and Automation (17 decks)

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