Dr. Ajit Surya Singh

1. ANTIGEN – ANTIBODY
REACTIONS
Dr. Ajit Kumar Singh
MD Laboratory Medicine
Chittaranjan national cancer institute
Kolkata 700160

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 .Western blotting
10.Chemiluminescence assay
11.Immunoelectron microscopic tests

9. 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 between the Ags and Abs .
Flocculation
When instead of sedimenting .
The precipitate remains suspended as floccules .

10. Zone phenomenon : lattice hypothesis

11. Mechanism of precipitation
Lattice hypothesis

12. Applications
Qualitative or quantitative test sensitive in detection of
antigen , 1 µg of protein - can be detected
1. Forensic application - Detection of blood/serum
stains
2. Testing - Food adulterants
3. Grouping of Streptococci – Lancefield technique
4. VDRL test for syphilis
5. To standardize toxins and toxoids
6. To test toxigenicity in diphtheria bacillus

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

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

15. 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

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

17. 1 . Single diffusion in one dimension Oudin procedure

18. 2 . Single diffusion in two dimensions Radial immunodiffusion

19. 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

20. 3 . Double diffusion in one dimension Oakley – Fulthrope procedure

21. 4 . Double diffusion in two dimensions Ouchterlony procedure

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

23. 3 . Precipitation in agar with electric field
1 . Immnunoelectrophoresis
2 . Counter current immunoelectrophoresis
3 . Rocket electrophoresis
4 . Two dimensional immunoelectrophoresis

24. 1. Immunoelectrophoresis

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

26. 2 . Counter current immuno eletrophoresis

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

28. 3 . Rocket electrophoresis :
One dimensional single elctroimmunodiffusion

29. Uses :
⚫Mainly for quantitative estimation of Ag in
serum

30. 4 . Laurell’s two dimensional immunoelectrophoresis
It is a 2 step procedure

31. 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

32. 1 . Direct agglutination :
1 . Slide agglutination
2 . Tube agglutination
3 . Heterophile agglutination
4 . Antiglobulin ( Coombs ) test

33. 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

34. 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

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

36. 3 . Heterophile agglutination test :
Ex : 1 . Weil – Felix test
2 . Paul – Bunnel test
3 . Streptococcus MG
agglutination test
4. Cold agglutination test

37. 4 . Antiglobulin ( Coombs ) test
⚫ 2 types
1 . Direct coombs test
2 . Indirect coombs test

38. 1 . Direct Coombs test :

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

40. 2 . Passive agglutination :
Types :
1 . Latex agglutination tests
2 . Haemagglutination tests
3 . Coagglutination tests

41. 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

42. 2 . Haemagglutination tests :
Rose – waaler test :
Rheumatoid arthritis

43. 3 ) Coagglutination test :
Reverse passive agglutination

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

45. 3 ) COMPLEMENT – DEPENDENT
SEROLOGICAL TESTS
1 . Complement fixation test
2 . Immune adherence test
3 . Immobilization test
4 . Cytolytic or cytocidal reaction

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

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

49. Complement fixation test—Wassermann reaction

50. 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

51. 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 RNPAg 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.

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

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

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

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

56. 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

57. 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

58. ⚫Enzyme-labelled conjugates
⚫Homogenous EIA
⚫Heterogenous EIA
ENZYME IMMUNOASSAY

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

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

61. 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

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

63. Sandwich 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

64. Sandwich ELISA

65. Indirect ELISA
Anti HIV Ab
• 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 )adde
⚫Positive – colour change
⚫Negative – no colour change

66. Indirect ELISA

67. Dengue ELISA

68. ⚫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

69. TRI –DOT TEST

70. Enzyme linked immunosorbent assay (EL ISA)

71. Applications
⚫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

72. ⚫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)

73. Immunoelectroblot / western blot
techniques
⚫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

74. Western blotting

75. 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

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

77. Nuclear
Fine Speckled

78. ⚫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

79. Schematic diagram of a flow cytometer

80. Applications
Size ,granularity , DNA or RNA content,
Cellular Ags , receptor levels
⚫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

81. THANK YOU