complement fixation, immunoflourescence, ELISA

1. Antigen – Antibody reactions
Part II
Dr.M.Malathi
Final year PG

2. Time to recollect !!!
• Antigen?
• Antibody?
• Antigen antibody reactions?

3. Complement Fixation Test (CFT)
• What is a complement ?
• Antigen antibody complex – bound by
complement – mediates bacterial clearance,
viral neutralisation, lysis of RBC

4. Im the source of complement

5. 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

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

7. Amboceptor ( Rabbit antibody to
sheep red cells)

9. Wassermann reaction
• Formerly used for the serodiagnosis of syphilis
• Procedure
1. Lysis of erythrocytes – Negative CFT
2. No lysis of erythrocytes – Positive CFT
• For every test, appropriate controls should be
added.

10. Other complement dependent tests:
• Immune adherence-T.pallidum, V.cholerae
• Immobilisation test – Treponema pallidum
• Cytolytic or cytocidal test- V.cholerae

11. 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

12. 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

13. 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.diphtheriae
2. Schick test
• In vitro tests:
1. Anti streptolysin O (ASO) test
2. Virus neutralisation test
3. Nagler reaction-rapid detection of C.welchii

14. 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

15. • 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

16. 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)

17. Direct immunofluorescence test

18. 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

19. Indirect immunofluorescence test

20. 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

21. RADIOIMMUNOASSAY(RIA)

22. Uses of RIA
• RIA have applications in biology and medicine-
1. Quantitation of hormones, drugs, tumour
markers,
2. IgE and viral antigens

23. Disadvantages
• Radiation hazards: Uses radiolabelled reagent
• Radioactive waste disposal

24. ELISA
Micro-plate reader
96-well micro-plate
Positive result
ENZYME LINKED
IMMUNOSORBENT ASSAY

26. Types of ELISA
– Direct ELISA
– Indirect ELISA
– Competitive ELISA
– Sandwich ELISA
– Capture ELISA
– Cylinder or cassette ELISA

27. Types of ELISA

28. Immunochromatographic Tests
• A colored band
appears at the
second window
• Control also can be
recorded

29. 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

30. CLIA

31. 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

32. Western blotting (Immunoblotting)

33. 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

34. 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

35. 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

37. • ELISA ?
• CLIA ?
• EIA ?
• RIA ?
• CFT ?

38. What are the types of ELISA?

39. Enumerate the antigen antibody
reactions?

40. Enumerate the antigen antibody
reactions?
• Precipitation
• Agglutination
• Complement fixation test
• Neutralisation test
• Opsonisation
• Radioimmunoassay (RIA)
• Enzyme immunoassay (EIA)

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

42. THANK YOU