2. â«Definition
ï The interactions b / w antigens and antibodies.
ï Highly specific .
ï Antigens react only with antibody produced by itself or with
closely related antigens .
3. â«Purposes
â«The basis of antibody mediated immunity in infectious
diseases .
â«Help in diagnosis of infections in lab.
â«In epidemiological surveys .
â«Detection & quantitation of either Ags or Abs
â«Invitro â serological reactions
4. â« Ag â Ab reactions
Occur in 3 stages
1 . Primary stage
2 . Secondary stage
3 . Tertiary stage
5. Primary Stage
âą Initial interaction
âą Without visible effect
âą Low temperature
âą Reaction reversible
âą Vander Waalâs forces , ionic bond and hydrogen bonding
âą Detection - markers â
ïŒ radio isotopes
ïŒ fluorescent dyes
ïŒ ferritin
6. Secondary Stage
âą Demonstrable events
âą Precipitation , Agglutination
âą Lysis of cells , killing of live antigens
âą Neutralisation of toxin
âą Complement fixation
âą Immobilisation of motile organisms
âą Enhancement of phagocytosis
âą Agglitinin , Precipitin ,
âą Agglutinogen , precipitoinogen
7. Tertiary Reaction
ï” Ag-Ab reaction in vivo - chain reaction
ï” â neutralisation , destruction of injurious Ag, tissue damage
9. Precipitation
When a soluble antigen combines with its antibody in the
presence of electrolytes at a suitable
âą Temp
âą PH
The Ag â 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 .
12. Applications
Qualitative or quantitative test sensitive in detection of
antigen , 1 ”g of protein - can be detected
1. Forensic application - Detection of blood/serum
stains
2. Testing - Food adulterants
3. Grouping of Streptococci â Lancefield technique
4. VDRL test for syphilis
5. To standardize toxins and toxoids
6. To test toxigenicity in diphtheria bacillus
14. Precipitation in solution :
1 . Ring test
2 . Flocculation tests
Ring test :
Ex
1 . C â 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 â 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
30. 4 . Laurellâs two dimensional immunoelectrophoresis
It is a 2 step procedure
31. 2 ) AGGLUTINATION :
â«An Ag â 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
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:
Hâ - flagellar antigen - large, loose, fluffy
clumps
âOâ - somatic antigen - tight compact
deposit
36. 3 . Heterophile agglutination test :
Ex : 1 . Weil â Felix test
2 . Paul â Bunnel test
3 . Streptococcus MG
agglutination test
4. Cold agglutination test
37. 4 . Antiglobulin ( Coombs ) test
â« 2 types
1 . Direct coombs test
2 . Indirect coombs test
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
44. USES :
â«For detection of Cryptococcal Ags
â«For diagnosis of Amoebic & Hydatid
Ags
â«For grouping of Streptococci ,
and Mycobacteria
â«For typing of N . Gonorrhoea ,
Legionella
45. 3 ) COMPLEMENT â DEPENDENT
SEROLOGICAL TESTS
1 . Complement fixation test
2 . Immune adherence test
3 . Immobilization test
4 . Cytolytic or cytocidal reaction
46. âą Complement - Ag â Ab
âą Lyses erythrocytes Kills / lyse
bacteria, immobilises motile
organism
âą Promotes phagocytosis
âą Immune adherence
âą 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
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:
â«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.
54. Virus Neutralisation Tests
Neutralisation of viruses
Neutralisation of bacteriophages
ïŻ
Plaque inhibition
Toxin Neutralisation
Bacterial exotoxins - Tetanus,
diphtheria
Diphtheria toxin - Schick test
55. Anti streptolysin O (ASO) test â Streptococcal O
hemolysin
Naglerâs reaction â Clostridium
perfringens - alpha toxin
56. OPSONISATION
âOpsoninâ â Wright - 1903
â«Heat-labile substance - Facilitates
phagocytosis (complement)
â«Heat-stable serum factor â
âbacteriotrophinâ
â«âOpsonic indexâ- Progress of resistance
- ratio of phagocytic activity of
patientâs blood for given bacterium to
phagocytic activity of blood from
normal individual
57. Radioimmunoassay (RIA)
â«Radioisotopes conjugated to antigen or
antibody
â«Binder - Ligand assay
â«Analyte or ligand (Ag) â Substance
whose concentration is to be
determined
â«Binder (Ab) â Binder protein which
binds to the ligand
â«RIA â Berson and Yallow, 1959
59. Homogenous EIA :
âą Does not require bound and free
fractions to be separated
âą EMIT
âą Assay of haptens
âą Drugs â opiates , cocaine
barbturates ,amphetamine -
serum
60. Heterogenous EIA
âąRequires the separation of free and
bound fractions either by
centrifugation or absorption on solid
surfaces and washing
ELISA
61. ELISA
â«Involves the use of immunosorbent
â« for one of the components of the
reaction: antigen or antibody
â«96 â well microtitre plate
â«Principle illustrated by outlining its
application for detection of Rotavirus
antigen in feces
68. â«Competitive ELISA â Similar to RIA,
unknown antigen (sample) and known
antigen (standard) compete with each
other for fixed antibody
â«Hapten detection
â«Cylinder or cassette ELISA â Each
sample tested in a separate disposable
cassette, in-built controls, result read
visually
â«Ex : Tri - dot test
71. Applications
â«Detection of infectious diseases â HIV,
Hepatitis, EBV, CMV , Dengue, TORCH ,
Influenza
â«Rota virus , ET of E .coli in feces
â«Syphilis IgG /IgM , H pylori IgG , Ag
â«Food toxins â aflatoxins
â«Food adulterants â E.coli, Campylobacter,
Salomonella Ag
â«Mycobacterial antibody detection
â«Human allergic specific IgE & IgA ELISA
72. â«Chemiluminescent compounds are used
in CLIA as a label to provide signal
during antigen - antibody reaction
â«The signal (light) can be amplified,
measured and concentration of analyte
calculated
Chemiluminescence immunoassay (CLIA)
73. Immunoelectroblot / western blot
techniques
â«The technique is a combination of
three procedures
â«Separation of ligand and antigen by gel
electrophoresis
â«Blotting of electrophoresed ligand
fraction
â«Enzyme immunoassay to detect
antibody â varius ligand fraction bands
â«Confirmatory test - HIv
75. IMMUNOFLUORESCENCE
â«Fluorescence - Property of absorbing light rays of one
wavelength and emitting rays of a different wavelength
â«Direct immunofluorescence test - Specific antiserum
labelled with a fluorescent dye, used for identification of
antigens , bacteria , viruses
78. â«Immunohistochemical technique â Helps to visualise
antigen - antibody reactions in situ
â«Flow cytometry âFluorescence technique used to
identify and enumerate cells bearing a particular
antigen or surface marker
â«Cells are made to flow in a single stream through an
electronic detection apparatus
80. Applications
Size ,granularity , DNA or RNA content,
Cellular Ags , receptor levels
â«Differential leucocyte count
â«T cell subsets - CD4 and CD8 counts in
HIV patients
â«Diagnosis, prognosis and treatment of
cancer
â«To study the cell cycle , apoptosis