This document provides information on antigen-antibody reactions presented in an undergraduate lecture. It discusses the learning objectives which are to understand antigen-antibody principles and reaction types. It also describes various serological reactions used in immunological assays including precipitation, agglutination, complement fixation, and their applications in diagnosing infectious diseases.
Direct
Passive
Reverse Passive
Agglutination Inhibition
Coagglutination
Agglutination tests can be done :
On slides
In tubes
In microtritation plates
-Difference between precipitation and agglutination reaction.
Direct
Passive
Reverse Passive
Agglutination Inhibition
Coagglutination
Agglutination tests can be done :
On slides
In tubes
In microtritation plates
-Difference between precipitation and agglutination reaction.
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Antigen-antibody interaction, or antigen-antibody reaction, is a specific chemical interaction between antibodies produced by B cells of the white blood cells and antigens during immune reaction. It is the fundamental reaction in the body by which the body is protected from complex foreign molecules, such as pathogens and their chemical toxins. In the blood, the antigens are specifically and with high affinity bound by antibodies to form an antigen-antibody complex. The immune complex is then transported to cellular systems where it can be destroyed or deactivated.
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Antigen-antibody interaction, or antigen-antibody reaction, is a specific chemical interaction between antibodies produced by B cells of the white blood cells and antigens during immune reaction. It is the fundamental reaction in the body by which the body is protected from complex foreign molecules, such as pathogens and their chemical toxins. In the blood, the antigens are specifically and with high affinity bound by antibodies to form an antigen-antibody complex. The immune complex is then transported to cellular systems where it can be destroyed or deactivated.
This topic describes about antigen-antibody reaction in detail including their classification, mechanism of action, various examples of each reaction with labelled diagrams.
The lecture was presented to the students of Saudi board of Community Medicine to help them know about the various serological methods applicable in the diagnosis of infectious diseases in general with attention upon the specificity and sensitivity of various diagnostic modalities. The lecture covers the basic principles of each test and the clinical applications with the advantages and disadvantages of each.
This presentation explains about the Immunoassay ,radio immuno assay, definition, types, Principle , procedure, steps involved ,advantages ,disadvantages ,Application, RIA in insulin. RIA in Digitalis drug ligand etc....
Lab report that discusses the antigen-antibody precipitation reaction using the Ouchterlony Double Diffusion Technique.
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Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
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Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
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1. ANTIGEN & ANTIBODY
REACTIONS
UNDER GARADUATE STUDENT’S
LECTURE ON
BY
GUNJAL PN
ASSIST. PROF.
DEPT OF MICROBIOLOGY
DVVPF’S MEDICAL COLLEGE & HOSPITAL
AHMENDAGAR
6/7/2022 Department of Microbiology 1
2. Competency
Following are the competencies for this theory class :
MI 1.8 –Describe the mechanism of immunity and response of the host
to immune system to infections (K/KH/Y) (Lecture, small group
discussion) (Written/Viva).
MI 8. 13 – Choose the appropriate laboratory test in the diagnosis of
the infectious disease. (K/KH/Y) (Small group discussion, case
discussion) (Written/ Viva/OSPE).
6/7/2022 Department of Microbiology 2
3. Competency
MI 8.15 – (K/S / SH/Y) (Lecture/ small group discussion)
(Written/ Viva voce/ OSPE- Objective Structured Practical
Examination).
Domain - K- Knowledge / S- Skill.
Level of competency – K- Knows / KH-Knows How/ S-
Shows/ SH- Shows How/ P- Perform independently.
Core – Y – Yes (Necessary to complete requirement of
subject) / N- Non-core.
6/7/2022 Department of Microbiology 3
4. Learning Objectives
At the end of the session, the students will be able to understand:
Principles of Antigen and Antibody reactions.
Different types of Antigen and Antibody reactions.
Precipitation reactions – Principle, Types, Applications, Advantages,
Disadvantages, etc.
Agglutination reactions – Principle, Types, Applications, Advantages,
Disadvantages, etc.
Complement Fixation Test- Principle, Application, Advantages,
Disadvantages,
etc.
6/7/2022 Department of Microbiology 4
5. Antigen – Antibody Reactions
Antigen(Ag) – Antibody(Ab) reactions in vitro are
known as Serological / Immunological reactions or
Immunoassays.
Ag-Ab reactions is a biomolecular association where
the Ag & Ab combines with each other specifically &
in an observable manner.
Help in - the diagnosis of infections.
6/7/2022 Department of Microbiology 5
6. Stages of Ag-Ab reactions
3 stages - Primary
- Secondary
- Tertiary
Primary Stage
Initial interaction between Ag & Ab
No visible effect
Rapid & reversible
Held by weak forces
6/7/2022 Department of Microbiology 6
7. Secondary stage
Follows 10 stage.
Visible effects - Precipitation
- Agglutination
- Cell lysis
- Killing of live Ags
- Neutralisation of toxins
- Complement fixation
- Phagocytosis
6/7/2022 Department of Microbiology 7
8. Tertiary Stage Reactions
Seen in vivo
Ag- Ab reactions initiate chain reactions that
lead to
Neutralization or destruction of injurious Ags.
Tissue damage.
Include humoral immunity against infectious
diseases as well as clinical allergy & other
immunological diseases.
6/7/2022 Department of Microbiology 8
9. General features of Ag-Ab
reactions
Specific reaction, but cross reactions may occur due to
antigenic relatedness.
Entire molecule of Ag & Ab reacts and participates in the
formation of agglutinates or precipitates.
No denaturation of Ag or Ab occurs during the reaction.
Combination occurs at the surface.
Combination is firm but reversible.
6/7/2022 Department of Microbiology 9
10. Diagnostic Use
As these reactions are specific and observable hence
extensively used for diagnosis of infectious diseases in
laboratories.
Ag and Ab reactions are also called as Immunoassays.
Most immunoassays uses serum samples, however other
samples can be used such as Urine, CSF, etc.
6/7/2022 Department of Microbiology 10
11. Diagnostic Use
Immunoassays can be broadly categorized into
Antigen detection assays – Detecting Ags in patients
sample using specific Abs.
Antibody detection assays – Detecting Abs in patients
sample employing specific Ags.
6/7/2022 Department of Microbiology 11
12. Qualitative and Quantitative
Assays
Immunoassays can be performed by both qualitative and
quantitative assays.
QUALITIATIVE ASSAYS –In these undiluted specimen
containing Abs is directly mixed with suspension of an Ag or vice
versa.
Results are read as “ POSITIVE” or “NEGATIVE” based on
presence of or absence of an Ag or Ab in specimen.
Exact amount of Ag or Ab present in specimen can not be
estimated.
6/7/2022 Department of Microbiology 12
13. Quantitative Assays
When qualitative test is positive.
Exact amount of Ab in specimen can be detected by serial
dilution of specimen and adding known amount of Ag to each
dilution of Ab suspension.
Ab titre – Highest dilution of serum showing observable
reaction with Ag. Ag titre can be measured similarly.
Quantitative tests are more reliable as they can differentiate
between true negative and false negative results.
6/7/2022 Department of Microbiology 13
14. Evaluation of
Immunoassays
6/7/2022 Department of Microbiology 14
Sensitivity and specificity are the two most important statistical
parameters.
Sensitivity is defined as ability of a test to identify correctly all
those who have the disease i.e. true positives.
Sensitivity is calculated as =
True positives
True positives + False negatives
15. Evaluation of
Immunoassays
6/7/2022 Department of Microbiology 15
Specificity is defined as ability of a test to identify correctly all
those who do not have disease i.e. true negatives.
Specificity is calculated as = True negatives
True negatives + False positives
16. Mechanism of precipitation
Lattice formation - lattice hypothesis by
Marrack
6/7/2022 Department of Microbiology 16
Prozone Postzone
Zone of
Equivalence
17. Antibody excess
Optimal
proportions Antigen excess
Serial Dilution of Serum containing Abs-
decreasing concentration of Ab
6/7/2022 Department of Microbiology 17
Prozone Postzone
18. Serological Reactions /
Immunoassays
Precipitation
Agglutination
Complement fixation test (CFT)
Neutralisation
Immunofluorescence (IF)
Enzyme immunoassay (EIA/ ELISA)
6/7/2022 Department of Microbiology 18
19. PRECIPITATION
When a soluble Ag combines with its Ab in the presence of
optimum concentration of electrolytes (NaCl) at a suitable
temperature & optimum pH, the Ag-Ab complex forms an
insoluble precipitate.
When instead of sedimenting, the precipitate remains
suspended as floccules, the reaction is called Flocculation.
It can take place in liquid media or in gels such as agar,
agarose or polyacrylamide.
6/7/2022 Department of Microbiology 19
21. Clinical Application of Precipitation
Reactions
Earlier precipitation reactions were one of the widely used
serological tests.
However with the advent of simple and rapid newer techniques
their application is greatly reduced.
There are only limited situations where precipitation reactions is
still in use
VDRL Test
Elek’s Gel Precipitation test
6/7/2022 Department of Microbiology 21
22. Applications
Very sensitive in Ag detection (1µg of protein).
Forensic – identification of blood & seminal stains.
Food adulteration.
Types of Precipitation Test
Ring test.
Slide / Tube flocculation test.
Immunodiffusion (precipitation in gel).
Electro immunodiffusion.
6/7/2022 Department of Microbiology 22
23. Types of Precipitation & Flocculation
1. RING TEST
- Simplest.
- Layering Ag solution over a column of antiserum
in a narrow tube.
- Ppt forms at the junction.
e.g. Ascoli’s thermopreciptin test.
Lancefield grouping of streptococci.
2. SLIDE TEST – A drop each of Ag & antiserum are
placed on a slide & mixed – floccules appear.
e.g. VDRL test for syphilis.
6/7/2022 Department of Microbiology 23
24. Types
3. TUBE TEST – Tube flocculation.
e.g. Kahn test for syphilis.
Standardisation of toxins & toxoids.
4. IMMUNODIFFUSION (Precipitation in gel)
Advantage
– Distinct band of ppt form which is stable & can
be stained for preservation.
- Number of different Ags in the reacting mixture
can be identified.
6/7/2022 Department of Microbiology 24
25. Immunodiffusion
Single diffusion in one dimension (Oudin
procedure)
Antigen
Precipitation band
Ab in agar gel
6/7/2022 Department of Microbiology 25
26. Elek’s Gel Precipitation
Test
The Corynebacterium diphtheriae strain is isolated and streaked
on to a medium containing a filter paper soaked with diphtheria
antitoxin.
If the strain is toxigenic, it produces the toxin, which diffuses in
the agar, meets with the antitoxin and produces arrow-shaped
precipitation band.
This test can also be used to know the relatedness between the
strains isolated during an outbreaks.
6/7/2022 Department of Microbiology 26
27. e.g. Elek’s test for C. diphtheriae
- most widely used.
- helps to compare different Ags & antisera directly.
- reaction of identity.
6/7/2022 Department of Microbiology 27
Double diffusion in
two dimensions
(Ouchterlony
procedure)
29. AGGLUTINATION
When a particulate Ag is combines with its
specific Ab in the presence of electrolytes at a
suitable temperature & pH, the particles are
clumped or agglutinated.
More sensitive than precipitation for the
detection of Abs.
Incomplete or monovalent Abs do not cause
agglutination– “Blocking Abs”.
6/7/2022 Department of Microbiology 29
30. Types of Agglutination- Direct
SLIDE AGGLUTINATION
A drop of antiserum + a drop of uniform suspension of
Ag Mixed (direct Ag + Ab) clump formation.
Control is must.
Visible to the naked eye.
Uses :
Blood grouping.
Identification of bacterial isolates.
6/7/2022 Department of Microbiology 30
31. TUBE AGGLUTINATION
Standard quantitative method for the
measurement of Abs.
Uses : WIDAL test for Typhoid fever.
Heterophile agglutination test : Weil-Felix
Haemagglutination – RBCs are used as
antigens.
e.g. Paul Bunnell test, Cold agglutination test.
6/7/2022 Department of Microbiology 31
33. No agglutination
Agglutination
1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl
In this case, the titre is 1/40
Tube Agglutination Test
6/7/2022 Department of Microbiology 33
34. Microscopic Agglutination
Agglutination test performed on
microtitre plates.
Results are read under microscope.
Example – Microscopic Agglutination
Test (MAT) for – Leptospirosis.
6/7/2022 Department of Microbiology 34
35. INDIRECT OR
PASSIVE AGGLUTINATION
Soluble Ags are attached to the surface of carrier
particles to convert precipitation tests into agglutination
tests.
More convenient & sensitive.
Carrier particles : RBC’s, Latex, Bentonite.
e.g. ASO test.
6/7/2022 Department of Microbiology 35
37. Passive Agglutination Test
Converting a precipitation test to an agglutinating test.
Chemically link soluble antigen to inert particles such as
LATEX or RBC.
Addition of specific antibody will cause the particles to
agglutinate.
Reverse PAT: antibody linked to LATEX.
e.g. Lancefield grouping in Streptococci.
37
6/7/2022 Department of Microbiology
38. Passive Agglutination
Passive agglutination has been used in the
detection of :
Rheumatoid factor.
Antinuclear antibody in Lupus Erythematosus.
Ab to Group A streptococcus antigens.
Ab to Trichinella spiralis.
38
6/7/2022 Department of Microbiology
39. Reversed Passive Agglutination
Instead of Ag, the Ab is adsorbed on the surface of carrier
particles.
These Abs must be still reactive and is join in such way that the
active sites are facing outwards.
This type of testing of used to detect microbial antigens.
Latex particle coated with Ab (Known) + serum looking for
particular Ag.
If present then visible agglutination observed.
Very sensitive.
May give false positive results.
6/7/2022 Department of Microbiology 39
40. Reverse passive agglutination
Principle
Antigen binds to soluble
antibody coated on carrier
particles and results in
agglutination.
Detects antigens.
Example
Detecting cholera toxin.
Detection of HBsAg.
40
6/7/2022 Department of Microbiology
42. Haemagglutination
6/7/2022 Department of Microbiology 42
Refers to the agglutination tests that use red blood cells
(RBCs) as source of antigen.
Various types of hemagglutination tests include:
Direct hemagglutination test
Indirect hemagglutination test
43. 6/7/2022 Department of Microbiology 43
Direct Hemagglutination Test
Serum antibodies directly agglutinate with surface
antigens of RBCs to produce a matt.
Examples include-
Paul Bunnell test (for Epstein-Barr Virus)
Cold agglutination test (for Mycoplasma pneumoniae)
Blood grouping
Coombs test or Antiglobulin test
44. Direct Haemagglutination Tests
6/7/2022 Department of Microbiology 44
Paul Bunnell Test- Heterophile agglutination
test Employs ship RBC’s as Ag to detect the Epstein-Barr
Virus Abs in the serum. The test is performed in tubes.
Cold agglutination Test: Uses human RBC’s as Ag to
detect Mycoplasma pneumoniae Abs in pt. serum. Tube
test.
Blood grouping – ABO or RH grouping
45. Direct Haemagglutination Tests
6/7/2022 Department of Microbiology 45
Coombs test or Antiglobulin test:
It is performed to detect Rh incompatibility by detecting Rh Abs
from mother’s and baby’s serum.
Rh incompatibility is a condition when a Rh negative mother
delivers a Rh positive baby (Rh Ag +ve).
During birth, some Rh Ag +ve RBC’s may pass from fetus to the
maternal circulation and may induce Rh Ab formation in the
mother.
Which may affect future Rh positive pregnancies.
46. Direct Haemagglutination Tests
6/7/2022 Department of Microbiology 46
Coombs test or Antiglobulin test:
These Rh Abs are incomplete or blocking Abs of IgG type.
These can pass placenta and bind to Rh Ag on fetal RBC’s.
But does not results in agglutination; instead they blocks
the sites on fetal RBC’s.
The reaction can be visualized by Coombs test,
47. Direct Haemagglutination Tests
6/7/2022 Department of Microbiology 47
Coombs test or Antiglobulin test:
Which is carried out by adding Coombs Reagent.
It contains Abs to human IgG, which bind to Fc portion of
Rh Abs bound on RBC’s, resulting in visible agglutination.
49. In strict sense, it is not an antigen antibody reaction.
The hemagglutinin antigens (HA) present on surface of some
viruses.
Hemagglutinating viruses (e.g. influenza virus) can agglutinate
with the receptors present on the surface of RBCs.
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Viral Hemagglutination Test
51. Co-agglutination
Co agglutination is similar to the latex agglutination
technique for detecting antigen (described above).
Protein A, a uniformly distributed cell wall component of
Staphylococcus aureus, is able to bind to the Fc region
of most IgG antibodies leaving the Fab region free to
interact with antigens present in the applied specimens.
The visible agglutination of the S. aureus particles
indicates the antigen-antibody reactions.
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52. Co agglutination Test
Agglutination test in which inert
particles (latex beads or heat-
killed S. aureus Cowan 1 strain
with protein A) are coated with
antibody to any of a variety of
antigens.
Then used to detect the antigen
in specimens or in isolated
bacteria.
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53. Weil-Felix reaction – A Heterophile
agglutination Test
A Weil-Felix reaction is a type of heterophile
agglutination test.
In which patients serum is tested for agglutinins to
O antigen of certain non-motile Proteus
strains(OX19, OX2, OXk).
OX19, OX2 are strains of Proteus vulgaris.
OXk is the strain of Proteus mirabilis.
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54. COMPLEMENT FIXATION TEST
Ability of Ag-Ab complexes to fix complement.
Consists of two steps & five reagents.
e.g. Wasserman reaction for Syphilis
1. ANTIGEN + TEST SERUM
(contains Ab) COMPLEMENT FIXED
+ COMPLEMENT
+ HEMOLYTIC SYSTEM Result – No Hemolysis
(Sheep RBC + Amboceptor) POSITIVE CFT
2.ANTIGEN + TEST SERUM COMPLEMENT NOT FIXED
(contains no Ab)
+ COMPLEMENT
+ HEMOLYTIC SYSTEM Result – Hemolysis
NEGATIVE CFT
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56. NEUTRALISATION TESTS
Ability of the Ab to neutralize various effects of
micro organisms mediated through toxins,
enzymes or microbial Ags.
e.g. Nagler’s test for
Clostridium perfingens
(test to detect alpha toxin)
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57. Expected Questions
Enumerate the properties and types of Ag-Ab reactions.
Describe in detail about the principle, types, and application
of Precipitation reactions.
Enumerate the properties and types of Ag-Ab reactions.
Describe in detail about the principle, types, and application
of Agglutination reactions.
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58. Expected Questions
Write Short Notes on :
Precipitation Reactions, types and applications.
Agglutination Reactions, types and applications.
Complement Fixation Test.
Neutralization Test.
Haemagglutination Test.
Co-agglutination Test.
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59. MCQ’s
Prozone phenomenon is due
to:
A. Excess Antigens.
B. Excess Antibodies.
C. Hyper immune reaction.
D. Both Ag and Ab excess.
Ans: B.
All are agglutination Reaction
except:
A. VDRL
B. Standard Agglutination Test.
C. Widal Test
D. Paul Bunnell Test
Ans: A.
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