This document discusses antigen-antibody reactions and various serological techniques used to detect them. It defines antigen-antibody reactions and notes their high specificity. Various purposes and stages of antigen-antibody reactions are described. Several techniques are summarized, including precipitation reactions, agglutination tests, complement-dependent tests, neutralization tests, immunoassays, and immunofluorescence. Specific applications and methods are provided for many common serological techniques.

1. ANTIGEN – ANTIBODY
REACTIONS
DR.KRISHNA AGARWAL
ASST PROFESSOR
MICROBIOLOGY

2.  Definition : The interactions b / w
antigens and antibodies .
 Highly specific .
 Antigens react only with antibody
produced by itself or with closely
related antigens .

3.  Purposes :
 The basis of antibody mediated
immunity in infectious diseases .
 Help in diagnosis of infections in lab.
 In epidemiological surveys .
 Detection & quantitation of either
Ags or Abs
 Invitro – serological reactions

4.  Ag – Ab reactions
Occur in 3 stages
1 . Primary stage
2 . Secondary stage
3 . Tertiary stage

5. Primary Stage
• Initial interaction
• Without visible effect
• Low temperature
• Reaction reversible
• Vander Waal’s forces , ionic bond and
hydrogen bonding
• Detection - markers - radio isotopes ,
fluorescent dyes , ferritin

6. Secondary Stage
• Demonstrable events
• Precipitation , Agglutination
• Lysis of cells , killing of live antigens
• Neutralisation of toxin
• Complement fixation
• Immobilisation of motile organisms
• Enhancement of phagocytosis
• Agglitinin , Precipitin ,
• Agglutinogen , precipitoinogen

7. Tertiary Reaction
Ag-Ab reaction in vivo - chain reaction
– neutralisation , destruction of
injurious Ag, tissue damage

8. Types :
1 . Precipitation
2 . Agglutination
3 . Complement dependent serological
tests
4 . Neutralization tests
5 . Opsonization
6 . Immunofluorescence
7 . Radioimmunoassay
8 . Enzyme immunoassay

9. 9 . Western blotting
10 . Chemiluminescence assay
11 . Immuno electron microscopic tests

10. PRECIPITATION :
When a soluble antigen combines with
its antibody in the presence of
electrolytes at a suitable temp , PH , the
Ag – Ab complex forms an insoluble
precipitate .
A lattice is formed b / w the Ags and Abs .
FLOCCULATION : When instead of
sedimenting , the precipitate remains
suspended as floccules .

11. ZONE PHENOMENON :
LATTICE HYPOTHESIS :

12. Mechanism of precipitation
Lattice hypothesis

13. Qualitative or quantitative test
sensitive in detection of antigen, 1 µg
of protein - can be detected
Forensic application - Detection of
blood/serum stains
Testing - Food adulterants
Applications

14. Grouping of Streptococci – Lancefield
technique
VDRL test for syphilis
To standardise toxins and toxoids
To test toxigenicity in diphtheria
bacillus

15. Types :
1 . Precipitation in solution
2 . Precipitation in agar gel
3 . In agar with electric field

16. Precipitation in solution :
1 . Ring test
2 . Flocculation tests
RING TEST :
Ex 1 . C – reactive protein
2 . Streptococcal grouping by
Lancefield

17. Flocculation tests
1. Slide flocculation test :
Ex : VDRL test
2 . Tube flocculation test
Ex : 1 . Kahn test for Syphilis
2 . For standardization of toxins
and toxoids

18. Precipitation in ager gel :
Is termed as Immunodiffusion
Types : Based on the
1 . Number of reactants diffusing
2 . Direction of diffusion

19. 1 . Single diffusion in one dimension
Oudin procedure

20. 2 . Single diffusion in two dimensions
Radial immunodiffusion

21. Uses :
1 . For quantitative estimation of Abs &
Ags in the serum
2 . To measure IgG , IgM , IgA , and
Complement
3 . To measure Abs to Influenza virus in
sera
4 . To estimate serum transferrin & Alfa
fetoprtein

22. 3 . Double diffusion in one dimension
Oakley – Fulthrope procedure

23. 4 . Double diffusion in two dimensions
Ouchterlony procedure

24. Uses :
1 . Small pox serodiagnosis
2 . Identification of fungal antigens
3 . Antibodies to extractable nuclear
antigens
4 . Eleks gel precipitation test

25. 3 . Precipitation in agar with electric
field
1 . Immnunoelectrophoresis
2 . Counter current
immunoelectrophoresis
3 . Rocket electrophoresis
4 . Two dimensional
immunoelectrophoresis

26. 1. Immunoelectrophoresis

27. Main advantage :
1 . The number of antigens can be
identified
2 . To detect normal as well as abnormal
proteins - serum & urine

28. 2 . Counter current immuno
eletrophoresis

29. Uses :
 Commonly for Hepatitis B surface
antigens
 Alfa feto protein
 Hydatid and amoebic Ags
 Cryptococcal & meningococcal Ags in
CSF

30. 3 . Rocket electrophoresis :
One dimensional single elctroimmunodiffusion

31. Uses :
 Mainly for quantitative estimation of Ag
in serum

32. 4 . Laurell s Two dimensional
immunoelectrophoresis
It is a 2 step procedure

33. 2 ) AGGLUTINATION :
 An Ag – Ab reaction in which a
particulate Ag combines with its
antibody resulting in formation of
visible clumping of particles .
TYPES :
1 . Direct agglutination
2 . Passive agglutination

34. 1 . Direct agglutination :
1 . Slide agglutination
2 . Tube agglutination
3 . Heterophile agglutination
4 . Antiglobulin ( Coombs ) test

35. 1 . Slide agglutination :
Uses :
1 . As a routine procedure to identify
bacterial strains such as Salmonella ,
Shigella , Vibrio etc
2 . For blood grouping & cross matching

36. 2 . Tube agglutination test :
Uses :
1 . It is a standard method for quantitative
estimation of antibodies in the serum
2 . Routinely used for serodiagnosis of
Enteric fever , Brucellosis , typhus fever
Widal Test:
H’ - flagellar antigen - large, loose, fluffy
clumps
‘O’ - somatic antigen - tight compact
deposit

37. Complications :
• Prozone phenomenon
• Incomplete or blocking Abs

38. 3 . Heterophile agglutination test :
Ex : 1 . Weil – Felix test
2 . Paul – Bunnel test
3 . Streptococcus MG agglutination
test
4. Cold agglutination test

39. 4 . Antiglobulin ( Coombs ) test
 2 types
1 . Direct coombs test
2 . Indirect coombs test

40. 1 . Direct Coombs test :

41. Uses :
1 . Detection of anti Rh antibodies
2 . For detection of incomplete
antibodies in Brucellosis and other
diseases

42. 2 . Passive agglutination :
Types :
1 . Latex agglutination tests
2 . Haemagglutination tests
3 . Coagglutination tests

43. 1 . Latex agglutination tests :
Uses :
1 . For rapid identification of antigens of
group B Streptococcus , Staphylococcus
Neisseria meningitidis and Cryptococcus
neoformans
2 . For detection of soluble microbial
antigens in urine , spinal fluid , serum
3 . To detect RA factor , ASO , CRP ,
HCG

44. 2 . Haemagglutination tests :
Rose – waaler test :
Rheumatoid arthritis

45. 3 ) Coagglutination test :
Reverse passive agglutination

46. USES :
 For detection of Cryptococcal Ags
 For diagnosis of Amoebic & Hydatid
Ags
 For grouping of Streptococci , and
Mycobacteria
 For typing of N . Gonorrhoea ,
Legionella

47. 3 ) COMPLEMENT – DEPENDENT
SEROLOGICAL TESTS
1 . Complement fixation test
2 . Immune adherence test
3 . Immobilization test
4 . Cytolytic or cytocidal reaction

48. COMPLEMENT FIXATION TEST
• Complement - Ag – Ab
• Lyses erythrocytes
• Kills / lyse bacteria, immobilises
motile organism
• Promotes phagocytosis
• Immune adherence
• Tissue damage
Complement fixation test

49. CFT
Ag-Ab complex - fix complement
Versatile, sensitive
CFT - Two steps
Five reagents - Ag
Ab
Complement
Sheep RBC
Amboceptor
Principle

51. CFT
Complement fixation test—Wassermann reaction

52. Uses : 1 . Wassermann test for syphilis
2 . Test for antibodies to
Mycoplasma pneumoniae
Bordetella pertusis
Many viruses
Fungi such as Cryptococcus spp ,
Histoplasma ,
Coccidiodes immitis

53. Applications of CFT:
 Virology:
Herpes simplex virus
Picorna virus
CFT is useful to identify exposure to
Poliovirus
but not for type-specific diagnosis.
Influenza virus-CFT with the RNP Ag of influenza
virus
type A,B & C are very useful as the antibodies
are
formed during infection only.CFT can also be
done
using V antigens for demonstration of strain
specific Abs.

54. Contd….
Mycology:
 Histoplasmosis
 Coccidioidomycosis
 Sporotrichosis
 Pneumocystis jirovecii
Parasitology:
 Protozoans- Trypanosoma cruzi(Machado-
Guerreiro )
Trypanosoma brucei
Leishmania donovani(WKK
antigen )
Toxoplasma gondii(Sabin

55. Contd….
 Platyhelminthes- Schistosoma haematobium
Fasciola hepatica
Clonorchis sinensis
Paragonimus westermanii
 Nemathelminthes-Trichinella spiralis
Strongyloides stercoralis
Loa loa
Onchocerca volvulus

56. THANK YOU

57. NEUTRALISATION TESTS
Virus Neutralisation Tests
Neutralisation of viruses
Neutralisation of bacteriophages

Plaque inhibition
Toxin Neutralisation
Bacterial exotoxins - Tetanus,
diphtheria
Diphtheria toxin - Schick test

58. NEUTRALISATION
Anti streptolysin O (ASO) test –
Streptococcal O hemolysin
Nagler’s reaction – Clostridium
perfringens - alpha toxin

59. OPSONISATION
‘Opsonin’ – Wright - 1903
 Heat-labile substance - Facilitates
phagocytosis (complement)
 Heat-stable serum factor –
‘bacteriotrophin’
 ‘Opsonic index’- Progress of resistance
- ratio of phagocytic activity of
patient’s blood for given bacterium to
phagocytic activity of blood from
normal individual
OPSONISATION

60. RADIOIMMUNOASSAY(RIA)
 Radioisotopes conjugated to antigen or
antibody
 Binder - Ligand assay
 Analyte or ligand (Ag) – Substance
whose concentration is to be
determined
 Binder (Ab) – Binder protein which
binds to the ligand
 RIA – Berson and Yallow, 1959
Radioimmunoassay (RIA)

61. ENZYME IMMUNOASSAY
 Enzyme-labelled conjugates
 Homogenous EIA
 Heterogenous EIA
ENZYME IMMUNOASSAY

62. Homogenous EIA :
• Does not require bound and free
fractions to be separated
• EMIT
• Assay of haptens
• Drugs – opiates , cocaine
barbturates ,amphetamine - serum

63. Heterogenous EIA
• Requires the separation of free and
bound fractions either by
centrifugation or absorption on solid
surfaces and washing
ELISA

64. ENZYME LINKED IMMUNOSORBENT
ASSAY (ELISA)
 Involves the use of immunosorbent
 for one of the components of the
reaction: antigen or antibody
 96 – well microtitre plate
 Principle illustrated by outlining its
application for detection of Rotavirus
antigen in feces
ELISA

65. ELISA – Types
Sandwich ELISA
Indirect ELISA
Competetive ELISA
Capture ELISA
Cylinder or casstte ELISA

66. ELISA
 Microtitre wells coated with goat
antirotavirus antibody
 Fecal samples added after washing
 Incubated, wells washed
 Guinea pig anti rotavirus antiserum
labelled with alkaline phosphatase added
 Incubated, wells washed
 Substrate (Para – nitrophenyl phosphate)
added
 Positive – colour change
 Negative – no colour change
Sandwich ELISA

67. Sandwich ELISA

68. S Indirect ELISA
Anti HIV Ab
A: TYPES
• Antigen adsorbed onto microassay
plate wells
 Test serum added, incubated, washed
 Goat anti-human immunoglobulin
antibody conjugated with horse radish
peroxidase enzyme is added , incubate
 Wash, substrate (O- phynelene diamine
dihydrochloride )added
 Positive – colour change
 Negative – no colour change

69. Indirect ELISA

70. Dengue ELISA

71.  Competitive ELISA – Similar to RIA,
unknown antigen (sample) and known
antigen (standard) compete with each
other for fixed antibody
 Hapten detection
 Cylinder or cassette ELISA – Each
sample tested in a separate disposable
cassette, in-built controls, result read
visually
 Ex : Tri - dot test
ELISA: TYPES

72. TRI –DOT TEST

73. Enzyme linked immunosorbent assay (EL ISA)
ELISA: TYPES

74. U OF ELISA
 Detection of infectious diseases – HIV,
Hepatitis, EBV, CMV , Dengue, TORCH ,
Influenza
 Rota virus , ET of E .coli in feces
 Syphilis IgG /IgM , H pylori IgG , Ag
 Food toxins – aflatoxins
 Food adulterants – E.coli, Campylobacter,
Salomonella Ag
 Mycobacterial antibody detection
 Human allergic specific IgE & IgA ELISA
Applications

75. CHEMILUMINESCENCE IMMUNOASSAY
(CLIA)
 Chemiluminescent compounds are used
in CLIA as a label to provide signal
during antigen - antibody reaction
 The signal (light) can be amplified,
measured and concentration of analyte
calculated
Chemiluminescence
immunoassay (CLIA)

76. IMMUNOELECTROBLOT (WESTERN
BLOT)
 The technique is a combination of
three procedures
 Separation of ligand and antigen by gel
electrophoresis
 Blotting of electrophoresed ligand
fraction
 Enzyme immunoassay to detect
antibody – varius ligand fraction bands
 Confirmatory test - HIv
Immunoelectroblot / western blot
techniques

77. Western blotting

78. IMMUNOFLUORESCENCE
 Fluorescence - Property of absorbing
light rays of one wavelength and
emitting rays of a different wavelength
 Direct immunofluorescence test -
Specific antiserum labelled with a
fluorescent dye, used for identification
of antigens , bacteria , viruses
IMMUNOFLUORESCENCE

79. Direct immunofluorescence tests
Diagnosis of rabies Ag – Brain smears
Disadvantages – separate F conjugate >
each Ag
IMMUNOFLUORESENCE

80. Nuclear
Fine Speckled

81. IMMUNOFLUORESCENCE
 Immunohistochemical technique –
Helps to visualise antigen - antibody
reactions in situ
 Flow cytometry –Fluorescence
technique used to identify and
enumerate cells bearing a particular
antigen or surface marker
 Cells are made to flow in a single
stream through an electronic detection
apparatus

82. FLOW CYTOMETER
Schematic diagram of a flow cytometer

83. APaPLIATIONS
 Differential leucocyte count
 T cell subsets - CD4 and CD8 counts in
HIV patients
 Diagnosis, prognosis and treatment of
cancer
 To study the cell cycle , apoptosis
Applications
Size ,granularity , DNA or RNA content,
Cellular Ags , receptor levels

84. THANK YOU