antigen-antibody reactions im m unoaglutina tion im m unoprecipita tion

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