Ag ab reactions part i

ANTIGEN & ANTIBODY
REACTIONS
UNDER GARADUATE STUDENT’S
LECTURE ON
BY
GUNJAL PN
ASSIST. PROF.
DEPT OF MICROBIOLOGY
DVVPF’S MEDICAL COLLEGE & HOSPITAL
AHMENDAGAR
7/8/2021 Department of Microbiology 1

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).

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.

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.

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.

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

Secondary stage
 Follows 10 stage.
 Visible effects - Precipitation
- Agglutination
- Cell lysis
- Killing of live Ags
- Neutralisation of toxins
- Complement fixation
- Phagocytosis

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.

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.

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.

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.

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.

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.

Evaluation of
Immunoassays
 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

Evaluation of
Immunoassays
 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

Mechanism of precipitation
 Lattice formation - lattice hypothesis by
Marrack
Prozone Postzone
Zone of
Equivalence

Antibody excess
Optimal
proportions Antigen excess
Serial Dilution of Serum containing Abs-
decreasing concentration of Ab
Prozone Postzone

Serological Reactions /
Immunoassays
 Precipitation
 Agglutination
 Complement fixation test (CFT)
 Neutralisation
 Immunofluorescence (IF)
 Enzyme immunoassay (EIA/ ELISA)

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.

PRECIPITATION

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

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.

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.

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.

Modifications of Immunodiffusion
 Single diffusion in one dimension (Oudin
procedure)
Antigen
Precipitation band
Ab in agar gel

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.

 e.g. Elek’s test for C. diphtheriae
- most widely used.
- helps to compare different Ags & antisera directly.
- reaction of identity.
Double diffusion in
two dimensions
(Ouchterlony
procedure)

Agglutination Reactions

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”.

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.

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.

Tube Agglutination Test

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

Microscopic Agglutination
 Agglutination test performed on
microtitre plates.
 Results are read under microscope.
 Example – Microscopic Agglutination
Test (MAT) for – Leptospirosis.

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.

INDIRECT OR
PASSIVE AGGLUTINATION

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
7/8/2021 Department of Microbiology

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

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.

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

Quantitative Micro
Hemagglutination Test
(HA)
Haemagglutination Test (HA)

Haemagglutination
 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

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

Direct Haemagglutination Tests
 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

 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.

 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,

 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.

 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.
Viral Hemagglutination Test

Viral Haemagglutination
test

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.
51

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.
52

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.
53

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

http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter31/animation_quiz_4.html

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)

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.

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.

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.

Thank You

Ag ab reactions part i

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