ANTIGEN-ANTIBODYREACTIONS
ImmunoaglutinationImmunoprecipitation
Reaction between antigen and specific antibodies detectable in vitro
Traditionally called serologic reactions
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)
Ag-Ab reactions (general features)
The presence of antigen (using known antibody) or antibodies (using known antigen) in the sample (e.g. serum, cerebrospinal fluid cell culture…) is tested
1. Qualitative
2. Quantitative
TITER is a measure of quantity of specific antibodies. It represents the highest dilution which gives the positive result.
POSITIVE
NEGATIVE
Ag - CONCENTRATION (e.g. 2 mg/ml ili IU)
Specifc Ab - TITAR (npr. 1/64 ili 64)
Can be: Result:
Ag-Ab reactions (general features)
Total Ab - CONCENTRATION (e.g. IgG 13,5 mg/ml)
Controls - positive
- negativeTest is invalid without controls
Time (weeks)
Seru
m A
b c
once
ntr
ati
on
First pathogen encounter
Following pathogen encounter
Primary and secondary humoral immune response
IgA i IgE
IIgM
IIgM, IgGIIgG
Imore IgGlittle IgM
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 ANTIGEN
EXCESS OF ANTIBODIES
ZONE OF EQUIVALENCE
Ag-AB COMPLEXES:
AGLUTINATION:
RESULT:
SMALL
ABSENT
UNRELIABLE(FALSE NEGATIVE)
PROZONE
LARGE
PRESENT
RELIABLE(POSITIVE)
SMALL
ABSENT
UNRELIABLE(FALSE NEGATIVE)
IMMUNOAGLUTINATION
INDIRECT(passive)
DIRECT
Detection of Ag
Detection of Ab
Identification of bacteria
(enterobacteria)
BAB test(Brucella sp.)
Application (Example)
Direct aglutination(identification of bacteria)
IMMUNOAGLUTINATION
INDIRECT(passive)
DIRECT
Detection of Ag
Detection of Ab
Detection of Ab (Ag bound to a carrier)
Detection of Ag (Ab bound to a carrier)
Identification of bacteria
(enterobacteria)
BAB test(Brucella sp.)
Application (Example)
Indirect aglutination
Ab detection (Ag bound to a carrier particle)
Ag detection (Ab bound to a carrier
particle)
IMMUNOAGLUTINATION
INDIRECT(passive)
DIRECT
Detection of Ag
Detection of Ab
Detection of Ab (Ag bound to a carrier)
Detection of Ag (Ab bound to a carrier)
Identification of bacteria
(enterobacteria)
BAB test(Brucella sp.)
Latex RF test(reumatoid
factor)
HBsAg test(hepatitis B virus)
Application (Example)
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
in semisolid medium (gel)
fast diffusion (precipitate forms after minutes)
slow diffusion (precipitate forms after days)
electrophoresis (precipitate forms after hours)
IMMUNOPRECIPITATION
IN SEMISOLID MEDIUM(GEL)
IN FLUID
RING test(qualitative)
Nephelometry(quantitative)
Antrax detection(history)
Example
Interfacial (interphase) RING test(principle)
IMMUNOPRECIPITATION
IN SEMISOLID MEDIUM(GEL)
IN FLUID
RING test(qualitative)
Nephelometry(quantitative)
Antrax detection(history)
determination of plasma protein concentration
(e.g. Ig)
Example
Nephelomety(principle)
IMMUNOPRECIPITATION
IN SEMISOLID MEDIUM(GEL)
IN FLUID
RING test(qualitative)
Nephelometry(quantitative)
Doubleimmunodiffusio
n(qualitative)
RID test(quantitative)
Antrax detection(history)
determination of plasma protein concentration
(e.g. Ig)
Diphteric toxin detection(history)
Example
Double immunodiffusion – Diphteric toxin detection (principle)
IMMUNOPRECIPITATION
IN SEMISOLID MEDIUM(GEL)
IN FLUID
RING test(qualitative)
Nephelometry(quantitative)
Doubleimmunodiffusio
n(qualitative)
RID test(quantitative)
Antrax detection(history)
determination of plasma protein concentration
(e.g. Ig)
Diphteric toxin detection(history)
Example
determination of plasma protein concentration
(e.g. Ig)
RID
IgG
1
2
3
4 5
Radial immunodifusion – RID test(principle)
1 standard (low concentration)
2 standard (medium concentration)
3 standard (high concentration)
4 sample
5 sample
Ab in gel
Ag in wells:
Ag – concentration (g/l)
D2 (mm2)
1
2
3
4 5
Quantification(principle)
Standard (calibration) curve
IMMUNOPRECIPITATION
IN SEMISOLID MEDIUM(GEL)
IN FLUID
RING test(qualitative)
Nephelometry(quantitative)
Doubleimmunodiffusio
n(qualitative)
RID test(quantitative)
Antrax detection(history)
determination of plasma protein concentration
(e.g. Ig)
Diphteric toxin detection(history)
Example
determination of plasma protein concentration
(e.g. Ig)
10.____9.____8.____7.____5._____4._____3._____2._____
imunoprecipitation in gelj.C3 complement component in blood can be measured by
10.
indirect immunoaglutination i.Double immunodifusion is an example of9.
quantitave techniqueh.Nephelometry is 8.
direct immunoaglutinationg.Ring test is an example of immunoprecipitation in
7.
in liquid f.Immunoprecipitation can take place in6.
qualitative technique e.Solubilan antigen cannot be measured by 5.
liquid or geld.Latex agglutination is4.
false negative result in presense of excessive Ab concentration
c.For final identification of Entrobacteriaceae family is used
3.
direct immunoaglutionation b.Prozone is2.
nephelometry and radial immunodiffusion
a.Aglutination is1.
1._____ 6.____c b i g d f h je a