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ANTIGEN-ANTIBODY REACTIONS Im m unoaglutina tion Im m unoprecipita tion. Ag-A b rea ctions ( general features ). Reaction between antigen and specific antibodies detectable in vitro.

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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 fluidcell culture…) is tested

Ag

- infectious agentor 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 Abfor 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)

TITERis a measure of quantity of specific antibodies. It represents the highest dilution which gives the positive result.

- positive

Controls

Test is invalid without controls

- negative


Primary and secondary humoral immune response

Following pathogenencounter

First pathogen encounter

I

more IgG

little IgM

I

I

IgG

IgM

IgA i IgE

I

IgM, IgG

Serum Ab concentration

Time (weeks)



IMMUNOAGLUTINATIONis a reaction betweenparticulate (insoluble) antigen and specific antibodies

Antigen is tipicallyon the surface of the cell (e.g. bacterial) or it is a solubile moleculboundfor inert insolubleparticle (carrier)

Result of reaction isaglutinate, visible structurecreated by

aggregation, i.e. aglutination of particles

It is tipically done on a slide (e.g. microscopic)

Maximalamount of aglutinateis observed when the concentrations of

antigen and antibody are approximately equal


EXCESS OF

ANTIBODIES

EXCESS OF

ANTIGEN

ZONE OF

EQUIVALENCE

Ag-AB COMPLEXES:

AGLUTINATION:

RESULT:

SMALL

ABSENT

UNRELIABLE

(FALSE NEGATIVE)

PROZONE

LARGE

PRESENT

RELIABLE

(POSITIVE)

SMALL

ABSENT

UNRELIABLE

(FALSE NEGATIVE)


Detection of Ab

Application (Example)

Identification

of bacteria

(enterobacteria)

Detection of Ag

DIRECT

BAB test

(Brucella sp.)

IMMUNOAGLUTINATION

INDIRECT

(passive)


Direct aglutination

(identification of bacteria)


Detection of Ab

Application (Example)

Identification

of bacteria

(enterobacteria)

Detection of Ag

DIRECT

BAB test

(Brucella sp.)

IMMUNOAGLUTINATION

Detection of Ab

(Ag bound to a carrier)

INDIRECT

(passive)

Detection of Ag

(Ab bound to a carrier)


Indirect aglutination

Ab detection

(Ag bound to a carrier particle)

Ag detection

(Ab bound to a carrier particle)


Detection of Ab

Application (Example)

Identification

of bacteria

(enterobacteria)

Detection of Ag

DIRECT

BAB test

(Brucella sp.)

IMMUNOAGLUTINATION

Latex RF test

(reumatoid

factor)

Detection of Ab

(Ag bound to a carrier)

INDIRECT

(passive)

Detection of Ag

(Ab bound to a carrier)

HBsAg test

(hepatitis B virus)



IMMUNOPRECIPITATIONis a reaction betweensoluble antigen and specific antibody

Antigen is some product of a microbe (e.g. toxin) or plasma protein (immunoglobulins, complement componentsetc.)

As a result of Ag-Ab reaction soluble Ag is precipitated by

an antibody

Maximalamount ofpreciptateis 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)

Antrax detection

(history)

IN FLUID

Nephelometry

(quantitative)

IMMUNOPRECIPITATION

IN SEMISOLID MEDIUM

(GEL)



Example

RING test

(qualitative)

Antrax detection

(history)

IN FLUID

determination of

plasma protein

concentration

(e.g. Ig)

Nephelometry

(quantitative)

IMMUNOPRECIPITATION

IN SEMISOLID MEDIUM

(GEL)


Nephelomety

(principle)


Example

RING test

(qualitative)

Antrax detection

(history)

IN FLUID

determination of

plasma protein

concentration

(e.g. Ig)

Nephelometry

(quantitative)

IMMUNOPRECIPITATION

Double

immunodiffusion

(qualitative)

Diphteric toxin

detection

(history)

IN SEMISOLID MEDIUM

(GEL)

RID test

(quantitative)



Example

RING test

(qualitative)

Antrax detection

(history)

IN FLUID

determination of

plasma protein

concentration

(e.g. Ig)

Nephelometry

(quantitative)

IMMUNOPRECIPITATION

Double

immunodiffusion

(qualitative)

Diphteric toxin

detection

(history)

IN SEMISOLID MEDIUM

(GEL)

determination of

plasma protein

concentration

(e.g. Ig)

RID test

(quantitative)


Radial immunodifusion – RID test

(principle)

Abin gel

2

Ag in wells:

3

1

RID

IgG

4

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

determination of

plasma protein

concentration

(e.g. Ig)

Nephelometry

(quantitative)

IMMUNOPRECIPITATION

Double

immunodiffusion

(qualitative)

Diphteric toxin

detection

(history)

IN SEMISOLID MEDIUM

(GEL)

determination of

plasma protein

concentration

(e.g. Ig)

RID test

(quantitative)


1.

Aglutination is

a.

nephelometry and radial immunodiffusion

2.

Prozone is

b.

direct immunoaglutionation

3.

For final identification ofEntrobacteriaceaefamilyis 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

Nephelometry is

h.

quantitave technique

8.

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._____

e

2._____

c

3._____

b

4._____

i

5._____

g

6.____

d

7.____

f

8.____

h

9.____

j

10.____

a


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