Antigen – Antibody reactionsPart II

Dr.M.MalathiFinal year PG

Time to recollect !!!

• Antigen?

• Antibody?

• Antigen antibody reactions?

Complement Fixation Test (CFT)

• What is a complement ?

• Antigen antibody complex – bound by complement – mediates bacterial clearance, viral neutralisation, lysis of RBC

Im the source of complement

Procedure

• Heat inactivation of the antiserum – to inactivate the complementary activity of serum

• The source of the complement is guinea pig serum.

• Complement – heat labile

• Freshly draw the serum

Ingredients

• CFT has two steps and five reagents• Antigen• Antibody• Complement• Sheep erythrocytes• Amboceptor ( Rabbit antibody to sheep red

cells)

Amboceptor ( Rabbit antibody to sheep red cells)

Wassermann reaction

• Formerly used for the serodiagnosis of syphilis• Procedure

1. Lysis of erythrocytes – Negative CFT2. No lysis of erythrocytes – Positive CFT• For every test, appropriate controls should be

added.

Other complement dependent tests:

• Immune adherence-T.pallidum, V.cholerae

• Immobilisation test – Treponema pallidum

• Cytolytic or cytocidal test- V.cholerae

Conglutination

• Some donot fix the guinea pig serum complement

• Use indirect CFT or conglutination test• Use sheep RBC with bovine serum• Bovine serum – conglutinin – beta globulin –

antibody to complement

NEUTRALISATION TESTS

• Virus neutralisation tests:1. Neutralisation of viruses by their antibodies

and are demonstrated in cell cultures, eggs and animals

2. Neutralisation of bacteriophages by the plaque inhibition test

Toxin neutralisation tests

• Bacterial exotoxins - induce neutralising antibodies(antitoxins) protection against diseases such as diphtheria and tetanus.

• In vivo tests:1. Toxigenicity test-detection of toxin of C.diphtheriae2. Schick test

• In vitro tests:1. Anti streptolysin O (ASO) test2. Virus neutralisation test3. Nagler reaction-rapid detection of C.welchii

OPSONISATION TEST

• Opsonisation - process by which a particulate antigen becomes more susceptible to phagocytosis.

• Opsonin – is then identified as a complement.• Bacteriotrophin – heat stable serum factor

with similar activity Specific antibody

• Opsonic index ratio of the phagocytic activity of the patients blood for a given bacterium, to the normal individual.

• Measured by incubating fresh citrated blood with the bacterial suspension at 37deg C for 15 minutes

• Phagocytic Index: Estimating the average number of phagocytosed bacteria per PMN from stained blood films

Immunofluorescence

• Fluorescence ?• Immuno fluorescence ?• Types – Direct and indirect

• The commonly used fluorescent dyes are:i) Fluorescin isothiocyanate (Blue green

fluorescence)ii) Lissamine rhodamine (orange red fluorescence)

Direct immunofluorescence test

Uses

1. It is commonly employed for detection of bacteria, viruses or other antigens in blood, CSF, urine, faeces, tissues and other specimens

2. It is a sensitive method to diagnose rabies by detection of the rabies virus antigens in brain smears

Disadvantage

• Separate specific fluorescent labelled antibody has to prepared against each antigen to be tested

Indirect immunofluorescence test

RADIOIMMUNOASSAY (RIA)

• Berson and Yallow (1959) first described RIA• Radioimmunoassay is widely-used because of

its great sensitivity• RIA detect antigens upto picogram (10−12 g)

quantities

RADIOIMMUNOASSAY(RIA)

Uses of RIA

• RIA have applications in biology and medicine-

1. Quantitation of hormones, drugs, tumour markers,

2. IgE and viral antigens

Disadvantages

• Radiation hazards: Uses radiolabelled reagent

• Radioactive waste disposal

ELISA

Micro-plate reader

96-well micro-plate

Positive result

ENZYME LINKED IMMUNOSORBENT ASSAY

Types of ELISA

– Direct ELISA

– Indirect ELISA

– Competitive ELISA

– Sandwich ELISA

– Capture ELISA

– Cylinder or cassette ELISA

Types of ELISA

Immunochromatographic Tests

• A colored band appears at the second window

• Control also can be recorded

Chemiluminescence immunoassay (CLIA)

• Chemiluminescence - Chemical reaction emitting energy in the form of light

• Chemilumiscence compounds - Luminol or acridinium esters .

• Signal can be amplified, measured, and the concentration of the analyte calculated

• The method is fully automated• Uses - where the volume of work is large - drug sensitivity testing of M.tuberculosis

CLIA

Immunoelectroblot/Westernblot

• Immunoblot or westernblot techniques combine the sensitivity of enzyme immunoassay with much greater specificity.

• Western blotting – Proteins• Northern blotting – RNA• Southern blotting - DNA

Western blotting (Immunoblotting)

Western Blot for detection of HIV antibody

HIV-1 Western Blot

• Lane1: Positive Control• Lane 2: Negative Control• Sample A: Negative• Sample B: Indeterminate• Sample C: Positive

Immunoelectronmicroscopic tests

Immunoelectronmicroscopy :viral particles are mixed with specific antisera and observed under the electron microscope which are seen as clumps.

Use: study of some viruses such as hepatitis A and viruses causing diarrhoea

Flow cytometry

• An instrument capable of analyzing single cells as they pass through an orifice at high velocity.

• Measures the properties of light scattering by the cells.

• Detection by the emission of light from flourescently labelled mAb bound to the surface of the cell

• ELISA ?• CLIA ?• EIA ?• RIA ?• CFT ?

What are the types of ELISA?

Enumerate the antigen antibody reactions?

Enumerate the antigen antibody reactions?

• Precipitation• Agglutination• Complement fixation test• Neutralisation test• Opsonisation • Radioimmunoassay (RIA)• Enzyme immunoassay (EIA)

• Chemiluminescence immunoassay (CLIA)• Immunoelectroblot / western blot• Immuno chromatographic test• Immuno electron microscopic tests• Immunoflourescence

THANK YOU