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ANTIGEN-ANTIBODY
REACTIONS
Immunoaglutination
Immunoprecipitation
Ag-Ab reactions (general features)
Reaction between antigen and specific antibodies detectable in
vitro
The presence of antigen (using known antibody) or antibodies
(using known antigen) in the sample (e.g. serum, cerebrospinal fluid
cell culture…) is tested
Ag - infectious agent or its part (e.g. HbsAg of hepatitis B virus)
- other (e.g. cytokines, hormons, tumor-markers, Ig...)
Ab - total Ab in the sample (e.g. total IgG in serum)
- specific Ab for certain Ag (e.g. IgM for hepatitis A virus)
Traditionally called serologic reactions
Ag-Ab reactions (general features)
Can be:
Result:
1. Qualitative
POSITIVE
NEGATIVE
2. Quantitative
Ag - CONCENTRATION (e.g. 2 mg/ml ili IU)
Total Ab - CONCENTRATION (e.g. IgG 13,5 mg/ml)
Specifc Ab - TITAR (npr. 1/64 ili 64)
TITER is a measure of quantity of specific antibodies. It represents
the highest dilution which gives the positive result.
Controls
- positive
- negative
Test is invalid without controls
Primary and secondary humoral immune response
Serum Ab concentration
First pathogen
encounter
IgM,I IgG
Following
pathogen
encounter
IgG
I
IgM
I
Time (weeks)
more IgG
I
little IgM
IgA i IgE
IMMUNOAGLUTINATION
IMMUNOAGLUTINATION is a reaction between particulate
(insoluble) antigen and specific antibodies
Antigen is tipically on the surface of the cell (e.g. bacterial) or it
is a solubile molecul bound for inert insoluble particle (carrier)
Result of reaction is aglutinate, visible structure created by
aggregation, i.e. aglutination of particles
It is tipically done on a slide (e.g. microscopic)
Maximal amount of aglutinate is observed when the concentrations of
antigen and antibody are approximately equal
EXCESS OF
ANTIBODIES
Ag-AB COMPLEXES:
AGLUTINATION:
RESULT:
ZONE OF
EQUIVALENCE
EXCESS OF
ANTIGEN
SMALL
LARGE
SMALL
ABSENT
PRESENT
ABSENT
UNRELIABLE
(FALSE NEGATIVE)
PROZONE
RELIABLE
(POSITIVE)
UNRELIABLE
(FALSE NEGATIVE)
Application (Example)
Detection of Ag
DIRECT
Detection of Ab
IMMUNOAGLUTINATION
INDIRECT
(passive)
Identification
of bacteria
(enterobacteria)
BAB test
(Brucella sp.)
Direct aglutination
(identification of bacteria)
Application (Example)
Detection of Ag
DIRECT
Identification
of bacteria
(enterobacteria)
Detection of Ab
IMMUNOAGLUTINATION
Detection of Ab
(Ag bound to a carrier)
INDIRECT
(passive)
Detection of Ag
(Ab bound to a carrier)
BAB test
(Brucella sp.)
Indirect aglutination
Ab detection
(Ag bound to a carrier particle)
Ag detection
(Ab bound to a carrier particle)
Application (Example)
Detection of Ag
DIRECT
Detection of Ab
Identification
of bacteria
(enterobacteria)
BAB test
(Brucella sp.)
IMMUNOAGLUTINATION
INDIRECT
(passive)
Latex RF test
Detection of Ab
(Ag bound to a carrier) (reumatoid
factor)
Detection of Ag
HBsAg test
(Ab bound to a carrier) (hepatitis B virus)
IMMUNOPRECIPITATION
IMMUNOPRECIPITATION is a reaction between soluble
antigen and specific antibody
Antigen is some product of a microbe (e.g. toxin) or plasma protein
(immunoglobulins, complement components etc.)
As a result of Ag-Ab reaction soluble Ag is precipitated by
an antibody
Maximal amount of preciptate is observed when the concentrations of
antigen and antibody are approximately equal
Immunoprecipitation can be performed in:
in fluid
fast diffusion (precipitate forms after minutes)
in semisolid medium (gel)
slow diffusion (precipitate forms after days)
electrophoresis (precipitate forms after hours)
Example
RING test
(qualitative)
IN FLUID
Nephelometry
(quantitative)
IMMUNOPRECIPITATION
IN SEMISOLID MEDIUM
(GEL)
Antrax detection
(history)
Interfacial (interphase) RING test
(principle)
Example
RING test
(qualitative)
Antrax detection
(history)
IN FLUID
Nephelometry
(quantitative)
IMMUNOPRECIPITATION
IN SEMISOLID MEDIUM
(GEL)
determination of
plasma protein
concentration
(e.g. Ig)
Nephelomety
(principle)
Example
RING test
(qualitative)
Antrax detection
(history)
IN FLUID
Nephelometry
(quantitative)
IMMUNOPRECIPITATION
Double
immunodiffusion
(qualitative)
IN SEMISOLID MEDIUM
(GEL)
RID test
(quantitative)
determination of
plasma protein
concentration
(e.g. Ig)
Diphteric toxin
detection
(history)
Double immunodiffusion – Diphteric toxin detection
(principle)
Example
RING test
(qualitative)
Antrax detection
(history)
IN FLUID
Nephelometry
(quantitative)
IMMUNOPRECIPITATION
Double
immunodiffusion
(qualitative)
IN SEMISOLID MEDIUM
(GEL)
RID test
(quantitative)
determination of
plasma protein
concentration
(e.g. Ig)
Diphteric toxin
detection
(history)
determination of
plasma protein
concentration
(e.g. Ig)
Radial immunodifusion – RID test
(principle)
Ab in gel
2
3
4
Ag in wells:
1
1
standard (low concentration)
RID
2
standard (medium concentration)
IgG
3
standard (high concentration)
4
sample
5
sample
5
Quantification
(principle)
Standard (calibration) curve
D2 (mm2)
3
2
1
4
5
Ag – concentration (g/l)
Example
RING test
(qualitative)
Antrax detection
(history)
IN FLUID
Nephelometry
(quantitative)
IMMUNOPRECIPITATION
Double
immunodiffusion
(qualitative)
IN SEMISOLID MEDIUM
(GEL)
RID test
(quantitative)
determination of
plasma protein
concentration
(e.g. Ig)
Diphteric toxin
detection
(history)
determination of
plasma protein
concentration
(e.g. Ig)
1. Aglutination is
2. Prozone is
a. nephelometry and radial
immunodiffusion
b. direct immunoaglutionation
3. For final identification of Entrobacteriaceae
family is used
c. false negative result in presense of
excessive Ab concentration
4. Latex agglutination is
d. liquid or gel
5. Solubilan antigen cannot be measured by
e. qualitative technique
6. Immunoprecipitation can take place in
f. in liquid
7. Ring test is an example of
immunoprecipitation in
g. direct immunoaglutination
8. Nephelometry is
h. quantitave technique
9. Double immunodifusion is an example of
i. indirect immunoaglutination
10. C3 complement component in blood can be
measured by
j. imunoprecipitation in gel
1._____
3._____
4._____
5._____
6.____
7.____
e 2._____
c
b
i
g
d
f
8.____
h
9.____
j
10.____
a