Immunoassay flashcards
Most common immunochemical techniques
‣ Add labeled antibody to
the patient’s sample
(analyte) contains
antigen
‣ Detecting patient’s
antibody
Examples of what immunochemical techniques are used for
Infectious disease, serology,
allergy testing, &
autoimmune testing
Antibodies in Immunoassays characteristics
1.)Specificity, 2) Affinity, 3) Cross-linking
A1 antitrypsin
Protects the bodys tissues from being damaged by infection fighting agents released by its immune system
Prozone
zone of antibody excess
Zone of equivalence
where you have the right amount of antigen and antibody
Post zone
Zone of excess antigen
Polymer effect
‣ Linear polymers enhance immune complex
precipitation
Polymer types
Dextran
* PVA
* PEG
methods for ag ab detection
- Precipitation or Agglutination
- Hemagglutination and Hemagglutination
inhibition - Passive Gel Diffusion
- Radio-immunoassays
- ELISA
- Immunofluorescence
- Immmunoblotting
- Immunochromatograph
Precipitation
‣ Antibodies react with soluble substances
ex) proteins, carbohydrates, etc
‣ Reaction visible with naked eye - particles
Agglutination
‣ 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?
Hemagglutination
If agglutination targets RBCs
Direct agglutination
Test patient serum against what
large, cellular antigens to
screen for the presence of antibodies in pt. serum.
Direct agglutination
Antigen is naturally what
and in this case what
Antigen is naturally present on the surface of the cells.
In this case, the Ag-Ab reaction forms an agglutination,
which is directly visible
Direct agglutination
The particle antigen may be
➡ 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
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Direct Agglutination?
example of bacterium direct agglutination
ex) Serotyping of E. coli, Salmonell
Examples of parasite direct agglutination
x) Serodiagnosis of Toxoplasmosis
Example of RBC direct agglutination
ex) Determination of blood groups
Passive agglutination
- An agglutination reaction that employs particles that
are coated with antigens not normally found in the
cell surfaces
Passive agglutination particle carriers include
– Red blood cells
– Polystyrene latex
– Bentonite
– charcoal
Reverse passive agglutination principle
‣ Antigen (in serum) binds
to antibody (from kit)
coated on carrier particles
and results in
agglutination
ex) detecting cholera toxin
Reverse passive agglutination example
detecting cholera toxin
Passive gel diffusion
Performed on semisolid (agarose)
✦ Precipitin band dependent on:
* Solubility of antigen/antibody
* Relative concentration of each
* Temperature
* Time
* Gel viscosity
Passive Gel Diffusion
Passive gel diffusion
Precipitation band dependent on
- Solubility of antigen/antibody
- Relative concentration of each
- Temperature
- Time
- Gel viscosity
Passive Gel Diffusion
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)
Simple diffusion ( radial immunodiffusion)
Antibody is what in gel
Antigen is applied
Antibody: suspended uniformly in gel
❖ Antigen: applied to well cut in gel
Diffusion away from what dilutes what
Simple Diffusion (Radial Immunodiffusion)
Diffusion away from well dilutes antigen
zone of equivalence results in
Zone of equivalence results in precipitin ring
Passive Gel Diffusion: Simple Diffusion (Radial Immunodiffusion)
Area of zone can be what
compared to known antigen concentrations
(standards)
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
Identity
(common epitopes
Non identity
(different epitopes)
Partial identity
(some epitopes in common)
Immunoelectrophoresis
✦ Immunoelectrophoresis (IEP)
‣ Serum proteins are
electrophoretically separated
‣ Reagent antibody is placed in
a trough running parallel
‣ Diffusion to precipitin arc
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
Electroimmunoassay
Rocket electrophoresis
- Antigen applied to wells in lower gel
- Upper gel contains antibody
- Electrophorese – quantitative when compared
to calibrators - Rapid detection & quantification
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)
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)
Competitive immunoassays
Typical use what
Carried out in
Typically use labeled antigen (reagent)
Carried out in presence of “excess antigen
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
Labeled Immunoassay
Value of patient analyte is inversely proportional to the
signal generated by the bound labeled antigen
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
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
Heterogeneous immunoassays
‣ Usually require washing step to separate antibody-
bound antigen from remaining free antigen.
Homogenous Immunoassays
‣ No separation between antibody-bound antigen with
free antigen
L ABELED IMMUNOASSAYS
Free
Free: floating in mixture, sometimes washed out,
sometimes precipitated or adsorbe
Bound
Bound: stuck to surface in some way
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)
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
Standards
✦ Regardless of assay type, standards MUST BE USED to
convert signal to amount
✦ In case of saturation – dilute sample
Radioisotopes
Requirments
✦ Requirements:
‣ Licensing
‣ Specialized equipment
‣ Speci alized handling and disposa
Radioisotopes are
Inexpensive, sensitive
Common radiolables
‣ Iodine (gamma)
‣ Tritium (beta)
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
RADIOLABELED ANTIBODIES
Additional uses
PET Scanning, infectious disease, cancer therapy
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
LUMINESCENT IMMUNOASSAYS
ofter used as what
Often used as substrate, and coupled with enzymes
as immunoassay label
