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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
Passive gel diffusion Precipitation band dependent on
* Solubility of antigen/antibody * Relative concentration of each * Temperature * Time * Gel viscosity Passive Gel Diffusion
26
Simple gel diffusion Simple diffusion ( radial Immunodiffusion
❖ 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
Simple diffusion ( radial immunodiffusion) Antibody is what in gel Antigen is applied
Antibody: suspended uniformly in gel ❖ Antigen: applied to well cut in gel
28
Diffusion away from what dilutes what Simple Diffusion (Radial Immunodiffusion)
Diffusion away from well dilutes antigen
29
zone of equivalence results in
Zone of equivalence results in precipitin ring
30
Passive Gel Diffusion: Simple Diffusion (Radial Immunodiffusion) Area of zone can be what
compared to known antigen concentrations (standards)
31
Ouchterlony double diffusion Both what Position of precipitin band indicates what
✦ 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
Identity
(common epitopes
33
Non identity
(different epitopes)
34
Partial identity
(some epitopes in common)
35
Immunoelectrophoresis
✦ Immunoelectrophoresis (IEP) ‣ Serum proteins are electrophoretically separated ‣ Reagent antibody is placed in a trough running parallel ‣ Diffusion to precipitin arc
36
Immunoelectrophoresis 2 dimensional crossed IE
✦ Two dimensional crossed IE (aka 2D IE, CRIE) * Serum separation by charge/size first * Turn 90°, electrophorese into gel containing ab Immunoelectrophoresis
37
Electroimmunoassay Rocket electrophoresis
* Antigen applied to wells in lower gel * Upper gel contains antibody * Electrophorese – quantitative when compared to calibrators * Rapid detection & quantification
38
Immunofixation electrophoresis (IFE)
✦ Electrophorese proteins ✦ Antibody placed on gel – precipitation ✦ All other proteins washed out ✦ Stain gel with dye (Coomassie blue, etc) Immunofixation electrophoresis (IFE)
39
Indicator labeled Immunoassays Labels
‣ 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
Competitive immunoassays Typical use what Carried out in
Typically use labeled antigen (reagent) Carried out in presence of “excess antigen
41
LABELED IMMUNOASSAYS Patients unknown analyte and what compete
Patient’s unknown analyte & the labeled analyte compete for binding sites on a known/limited amount of antibody
42
Labeled Immunoassay
Value of patient analyte is inversely proportional to the signal generated by the bound labeled antigen
43
Noncompetitive immunoassay Labeled entity is Typically use what Carried out in what
-Labeled entity is antibody -Typically use an unlabeled capture antibody and a labeled indicator antibody (reagent) -Carried out in presence of “excess antibody
44
LABELED IMMUNOASSAYS Patient unknown analyte is what Value of patient analyte is
-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
Heterogeneous immunoassays
‣ Usually require washing step to separate antibody- bound antigen from remaining free antigen.
46
Homogenous Immunoassays
‣ No separation between antibody-bound antigen with free antigen L ABELED IMMUNOASSAYS
47
Free
Free: floating in mixture, sometimes washed out, sometimes precipitated or adsorbe
48
Bound
Bound: stuck to surface in some way
49
Know what
✦ 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
Seperation techniques Solid phase
✦ 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
Standards
✦ Regardless of assay type, standards MUST BE USED to convert signal to amount ✦ In case of saturation – dilute sample
52
Radioisotopes Requirments
✦ Requirements: ‣ Licensing ‣ Specialized equipment ‣ Speci alized handling and disposa
53
Radioisotopes are
Inexpensive, sensitive
54
Common radiolables
‣ Iodine (gamma) ‣ Tritium (beta)
55
RADIOIMMUNOASSAY uses
‣ 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
RADIOLABELED ANTIBODIES Additional uses
PET Scanning, infectious disease, cancer therapy
57
Luminescent Immunoassays Luminescent compounds emit what are highly what
✦ Luminescent compounds emit a photon of light as the result of an electrical biochemical or chemical reaction ✦ “Highly sensitive
58
LUMINESCENT IMMUNOASSAYS ofter used as what
Often used as substrate, and coupled with enzymes as immunoassay label

Advanced Laboratory Operations and Automation (17 decks)

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