Antigen antibody reactions,

1. Dr Anuja Chandra By- Dr. Armaan SinghBy- Dr. Armaan Singh

2.  Introduction
 Immunity
 Antibody
 Antigen
 Antigen antibody reaction and their applications
 Diagnostic tests of periodontal disease activity
 Conclusion
 References

3. ▻ The term immunity is derived from immunitas (Latin for
exemption from civic duties or paying taxes)
▻ The term ‘immunity’ is defined as resistance exhibited
by the host against any foreign antigen including
microorganisms.
▻ The ability of an organism to resist a particular infection
or toxin by the action of specific antibodies or sensitized
white blood cells is called immunity.

5.  An antigen is a substance which when introduced into a
body evokes immune response to produce a specific
antibody with which it reacts in an observable manner.
 It can be classified as-
A. Complete Antigen
B. Incomplete Antigen (Haptens)
‣ Complete antigens are substances which can induce
antibody formation by themselves and can react
specifically with these antibodies.

6.  Haptens are substances unable to induce antibody
formation on its own but can become immunogenic
(capable of inducing antibodies) when covalently linked to
proteins, called carrier proteins. They can be simple or
complex.
 Proantigens are low molecular weight substances which do
not induce antibody formation but can cause delayed
hypersensitivity reaction.
 Epitope is the smallest unit of antigenicity.
 The combining site on the antibody molecule,
corresponding to the epitope is called Paratope.

8.  Foreignness
 Chemical nature
 Size
 Organ specificity
 Heterophile specificity
 Auto specificity
 Antigenic specificity
 Species specificity
 Susceptibility to tissue enzymes

9.  These are molecules that can interact with antigen
presenting cells and T lymphocytes in a non specific
manner.
 These antigens do not involve the endocytic processing
as required in typical antigen presentation.
 Viral proteins and staphylococcal enterotoxins are
examples of superantigens.

10.  These are substances which are formed in the serum and tissue
fluids in response to an antigen and react with that antigen
specifically and in some observable manner.
 Secreted by plasma cells, occur in two physical forms, a soluble
form that is secreted from the cell, and a membrane-bound form
that is attached to the surface of a B cell and is referred to as the
B cell receptor (BCR).
 The BCR is found only on the surface of B cells and facilitates
the activation of these cells and their subsequent differentiation
into either antibody factories called plasma cells or memory B
cells that will survive in the body and remember that same
antigen so the B cells can respond faster upon future exposure

13. Its uses are
1. In vivo
 Forms basis of immunity against infectious diseases
 May lead to tissue injury in hypersensitivity reactions and
autoimmune diseases
2. In vitro
 For diagnosis of infections
 Helpful in epidemiological studies
 For identification of enzymes
 Detection and quantitation of antigens or antibodies

14.  Reaction is specific, an antigen combines only with its
homologous antibody and vice versa. However cross
reactions may occur due to antigenic similarity.
 Entire molecules of antigen and antibody react and not
the fragments.
 Only the surface antigens participate in the antigen
antibody reaction.
 The reaction is firm but reversible. The firmness of
combination depends on the affinity and avidity.

15.  Precipitation reactions
 Agglutination
 Complement fixation test
 Neutralisation test
 Opsonisation
 Immunofluorescence
 Radioimmunoassay
 Enzyme linked immunosorbent assay
 Chemiluminescence assay
 Immunoelectronmicroscopic tests
 Immunoblotting

16.  When a soluble antigen reacts with its antibody in the
presence of electrolytes at an optimal temperature and
pH, antigen antibody complex forms an insoluble
precipitate that usually sediments at the bottom of the
tube and it is called precipitation.
 Precipitation may occur in liquid media or in gels such as
agar, agarose or polyacrylamide gels
 When instead of sedimenting, the precipitate is
suspended as floccules the reaction is called flocculation

18.  Identification of bacteria.eg Lancefield grouping of
streptococcus
 Detection of antibody for diagnostic purposes.eg VDRL
in syphilis
 Forensic application in identification of human blood and
seminal stains
 Testing for food adulterants
 To standardise toxins and antitoxins

20.  It is an antigen antibody reaction in which a particulate
antigen combines with its antibody in presence of
electrolytes at an optimal temperature and pH resulting in
visible clumping of particles.
 Lattice formation hypothesis holds good for agglutination
also.
 Occasionally incomplete antibodies (eg anti Rh and anti
Brucella) are formed that combine with the antigen but do
not cause agglutination. They act as “blocking antibodies”
inhibiting the agglutination by the complete antibody added
subsequently.

22.  Principle
The antigen antibody complexes have ability to ‘fix’
complement. This reaction has no visible effect.
To detect fixation of a complement, an indicator system
consisting of sheep erythrocytes coated with amboceptor
( rabbit antibody to sheep erythrocytes) is used.

24.  Bacterial exotoxins are capable of producing
neutralising antibodies (antitoxins) which play a
role in protection against diseases such as
diphtheria and tetanus.
 The toxicity of bacterial endotoxins is not
neautralised by antisera.

27.  It is a process by which a particulate antigen becomes more
susceptible to phagocytosis.
 Opsonic index is defined as ratio of phagocytic activity of the
patient’s blood for a given bacterium to that of a normal
individual.
 Phagocytic index is the average number of phagocytosed
bacteria per polymorphonuclear leukocytes from stained
blood films.
 Phagocytic index denotes the phagocytic activity of blood
and thus helps in measuring opsonic index.

29.  Fluorescence is the property of certain dyes which
absorb rays of one particular wavelength(ultraviolet light)
and emit rays with a different wavelength(visible light).
 Most commonly used dyes are
 1.fluorescin isothiocyanate
 2.lissamine rhodamine
 Two types are Direct and Indirect.

30. Specimen (Positive for antigen)
+
labelled antibodies
▼
Flurescence observed
▼
antigen is present in the specimen

31.  It is a sensitive method to diagnose rabies by detection
of rabies virus antigen in brain smears.
 It is commonly employed for detection of bacteria,viruses
or other antigens in blood, CSF,urine,faeces,tissues and
other specimens.
 Note – A separate specific fluorescent labelled antibody
has to be prepared against each antigen to be tested.

32.  Unlike direct method, antigen is known in this
method.
 Lets have a look at the flowchart for clear
understanding.

34.  Berson and Yallow(1959) first described
radioimmunoassay and since then it has been utilised for
quantitation of hormones, drugs, hepatitis B surface
antigen, IgE and viral antigens.
 This test can detect antigens upto picogram quantities.
 It is based on competition for a fixed amounts of specific
antibody between a known radiolabelled antigen and an
unknown unlabelled test antigen.

36. The RAST test (radioallergosorbent
test) is an example of
radioimmunoassay. It is used to
detect the causative allergen for an
allergy.

37. Advantages are-
 Highly sensitive
 Can be used for detection of small quantities
 Quantification possible
Disadvantages are –
 Expensive
 Requires isotopes

38.  Enzyme Linked Immunosorbent Assay is as sensitive as
radioimmunoassay.
 Requires only microlitre quantities of test reagents
 The enzyme acts on substrate to produce a color in a
positive test.
 It can be used for detection of antigen or antibody
 Types are Sandwich, Indirect, Competitive or Cassette
Elisa

44.  Detection of HIV Antibodies in serum
 Detection of mycobacterial antibodies in tuberculosis
 Detection of Rotavirus in faeces
 Detection of Hepatitis B markers in serum
 Detection of enterotoxin of E.coli in faeces

46.  Immunoferritin tests
 Immunoelectron microscopy
 Immunoenzyme tests
 Immunoblotting

47.  Ferritin (electron dense substance) conjugated antibody
is used to react with an antigen.This antigen antibody
reaction can be visualised under microscope
 Used in identification of Legionella pnemophila

48.  Viral particles are mixed with specific antisera and are
observed under electron microscope. These are seen as
clumps.
 Used in detection of Hepatitis A virus

49.  Tissue sections are treated with peroxidase labelled
antisera to detect corresponding antigen. The
peroxidase bound to antigen is visualised under electron
microscope.

50.  In immunoblots, antibodies can detect proteins in
mixtures.The mixture of proteins is electrophoretically
separated in a gel.
 The separated proteins are transferred from gel to
nitrocellulose paper.
 These nitrocellulose paper strips are reacted with test
sera and subsequently with enzyme conjugated anti
human immunoglobulin.

52.  This test has been widely used to confirm the ELISA
positive HIV antibody cases.
 The above procedure may also be applied to analyse
DNA or RNA. When DNA is transferred on nitrocellulose
strips from gel, this test is referred to as Southern Blot
test. Similarily, if RNA is transferred it is named as
Northern Blot test.

53.  Potential biomarkers of the disease activity would need
to be involved in the disease process in some way and
therefore need to undergo extensive and careful basic
research investigation before undergoing clinical
evaluation.
 Only when the source,precise nature and the role of
potential marker are known and understood can clinical
evaluation be meaningful.

54. Potential sources from which markers can be obtained
are-
 1.blood or serum
 2.saliva
 3.subgingival plaque sample
 4.gingival crevicular fluid

55. 1) Bacteria and their product
2) Inflammatory and immune products
3) Hydrolytic enzymes of tissue origin and those released
from dead cells
4) Connective tissue degradation products
5) Products of bone resorption

56.  Despite the complex interaction that exists between
bacteria and host, a number of possible pathogens have
been selected on the basis of their association with
disease progression, animal pathogenicity and their
possesion of virulence factors which could damage the
tissues(Genco et al 1988, Listgarten 1992, Socransky
and Haffajee 1992).

57.  Porphyromonas gingivalis
 Prevotella intermedia
 Bacteroides forsythus
 Actinobacillus actinomycetamcomitans
 Capnocytophaga ochracea
 Eikenella corrodens
 Campylobacter recta
 Fusobacterium nucleatum
 Treponema denticola

58.  There is no evidence for any one specific pathogen in
chronic periodontitis and therefore it may be considered
as a non specific bacterial disease (Theilade 1986).
 The bacteria listed here tend to be present in higher
numbers at active disease sites (Socransky and Haffajee
1992).
 Bacterial species numbers may be determined in variety
of ways (Listgarten 1992) and these include the following
–

59.  1.Dark ground or Phase contrast microscopy
 2.Culture techniques
 3.Immunological assays
 4. DNA probes
 5. BANA assays

60. They use either paper points or curette bacterial
sampling from pocket and include the following-
Evalusite (Kodak)
 This utilizes ELISA’s using antibody against P.gingivalis,
P intermedia, A. actinomycetemcomitans.
 This reaction is carried out in simple chairside kit.
 Subgingival plaque samples are reacted with the
antibodies and detection substrate in a multiwell reaction
dishes.

61. Omnigene and BTD(Bio technical diagnostics)
 These are DNA probe systems for a number of
subgingival bacteria.
 A paper point sample of subgingival plaque is placed in
a container and sent to company for assay.
 Probes are available for A. actinomycetemcomitans,
P.gingivalis, P intermedia, E.corrodens, F.nucleatum,C.
Recta and T.denticola

62. Perioscan (Oral B Laboratories)
 This is a chairside test kit system
which utilises BANA Test for bacterial
trypsin like proteases.
 They are mainly produced by
P.gingivalis and in lesser amounts by
B.forsythus and T.denticola.
 A subgingival plaque sample is reacted
in the kit with the substrate linked to a
color detection system.

63. Diamond Probe/Perio 2000 system
 Combines feature of periodontal probe with detection of
volatile sulphur compounds in periodontal pocket.

64. Prototek
Given by Cox et al in1990, this can be used to detect
bacterial proteases arg-gingipain/gingivain and DPP in
GCF.
This is not commercially available yet.

65.  Predictive of disease activity
 Simple
 Results available in short time
 Visual result

66.  Polymicrobial nature of the disease
 Most are not predictive of the disease activity
 Site
 Can only detect bacteria we look for
 Special laboratory required
 Cost

67. Those of possible relevance to periodontal pathology are-
 Immune response
 Antibody
 Total IgG and IgG subgroups
 Complement
 Inflammatory mediators
 Arachidonic acid derivatives
 Cytokines

68.  Porphyromonas gingivalis has been implicated as major
periodontal pathogen and it has been reported that a positive co
relation exists between IgG levels to P.gingivalis and severity of
periodontal disease(Gmur et al 1986;Lamster et al
1998;Blackburn 1992)
 Furthermore elevation in P.gingivalis specific IgG2, IgG1 and
IgG4 in rapidly progressing periodontitis and adult periodontitis
have been reported.(Kinane et al 1999)
 Complement proteins are present in GCF from sites with
inflammation and the split fragments C3 and Factor B have been
detected during gingivitis(Patters et al 1989). However, none of
these factors have been associated with disease activity.

69. Periotemp
 It has been developed to measure small changes in
sublingual and subgingival temperatures and positive
cross sectional comparision with clinical parameters have
been found.
 Increased subgingival temperature has been positively
corelated with increased pocket depths, decreased
attachment levels, clinical parameters of gingival
inflammation, higher proportions of putative periodontal
pathogens and gingival crevicular fluid enzymes( Dinsdale
et al 1997, Haffajee et al 1992, Wolff et al 1997)

70.  Although GCF PGE2 has considerable potential as a
screening test for periodontal activity, no commercial
efforts are currently underway to develop one.
 Cytokines are also assayed using ELISA techniques
which could be developed into chairside kits. However at
present the predictive ability of these markers is still in
doubt.

71.  Only GCF PGE2 has been shown to be predictive of
disease activity in longitudinal studies.
 ELISA techniques can be used to detect cytokines and
PGE2 which could be developed into chairside kits which
are simple to use.
 ELISA can be read after short time.
 They can be shown to the patient and related to the
tooth site.

72.  The choice of most appropriate biomarker may still be
difficult at present state of knowledge.
 There is difficulty in determining the sites to sample and
when to sample them.
 Cost

73. 1. Proteolytic enzymes
Collagenases
Elastase
Cathepsin G, B and D
Dipeptidylpeptidases
Tryptase
2. Hydrolytic enzymes
Aryl sulphatase
Beta glucuronidase

74.  Alkaline phosphatase
 Acid phosphatase
 Myeloperoxidase
 Lysozyme
 Lactoferrin

75.  Collagenase 2(MMP 8), collagenase 1(MMP 1) and
collagenase 3(MMP 13) activity are present in gingival
tissue,saliva and gcf and can be assayed biochemically
with collagen substrates(Sorsa et al 1990) or with ELISA
technique (Ingman et al 1996, Matsuki et al 1996).
 Elastase in gingival tissues is produced by PMN’s and
is held in an inactive form probably bound with an
inhibitor.It is inhibited by secretory leukocyte protease
inhibitor (SLPI) and skin antileukoproteinase (SKALP)
(Cox et al 2001)

76.  In humans, GCF tryptase activity corelates with clinical
parameters of disease severity including probing
attachment and bone lossand significantly reduces
following periodontal treatment (Eley and Cox 1992).
 Both tissue DPP 2 AND 4 corelate with clinical
parameters of disease severity and significantly reduce
following periodontal treatment (Cox and Eley 1992)

77. Periocheck
 These system detects presence of neutral proteinases
such as collagenases in GCF.
 A paper strip used to obtain GCF sample
is placed in contact with collagen gel to
which a blue dye has been covalently bonded.
It is then incubated at 43 degrees.
 Intensity α amount of enzyme present in the sample

78. Prognostik (Dentsply)
 This system detects presence of serine proteases,
elastases in GCF samples
 Intensity of fluorescence α amount of enzyme in the
sample

79.  β glucuronidase
It is a diagnostic kit that uses a histochemical substrate
for the enzymes coupled to a color detection system
which is releasedif the enzyme attacks the
substrate( Lamster et al 1988, 1991, 1994)
 Cysteine and serine proteinases
Developed in conjugation in researchers from Prototek
has following advantages over it-

80.  Can be modified to detect number of different
proteinases including DPP 2 and 4, tryptase etc.
 GCF sample is collected on a normal paper strip and
therefore leaving group are not introduced into the
gingival crevice unlike Dentsply
 Color detection system for this method is more
convenient foe the use in dental practice as it requires
no special apparatus.

81.  Simple
 Can be read after short time
 Can be shown to patient and related to tooth site

82.  Cost
 Selection of site
 When to sample
 Choice of most appropriate biomarker may still be
difficult at present state of knowledge

83. Since cell death is an essential component of
periodontal tissue destruction they should be released
during this process and should pass with the
inflammatory exudate into GCF.
 Aspartate amino transferase (AST)
 Lactate dehydrogenase (LDH)

84.  Periogard AST in GCF test kit

85.  Both of these potential markers associate with disease
activity but do not predict it
 Simple
 Can be shown to patient
 Can be read after short period
 Can be related to tooth site affected

86.  Cost
 Selection of site
 When to sample
 Choice of most appropriate biomarker may still be
difficult at present state of knowledge

87.  The detection of breakdown products of normal
components of extracellular matrix could be indicative of
tissue breakdown.

88.  Most complex and expensive
 Long collection times
 Not suitable for chairside

89. Possible markers of bone resorption and hence
periodontal disease activity are-
 Osteonectin
 Bone phosphoprotein (N-propeptide)
 Osteocalcin
 Telopeptides of type 1 collagen
 Collagen 1
 Proteoglycans

90.  Most of the potential markers in this group could be
easily adapted into test kits as their detection involves
use of specific monoclonal or polyclonal antibodies.
 Osteocalcin- ELISA(Kunimatsu et al 1993) or
RIA(Giannobile 1995)
 CTP- RIA(Giannobile 1995)
 Osteonectin, N Propeptide- ELISA(Bowers et al 1989)

91.  Some of these potential biomarkers associate with
the disease but do not predict it
 Can be read after relatively short periods
 Can be shown to the patient

92.  Choice of most appropriate biomarker is difficult at
the present state of knowledge
 Difficulty in determining sites to sample and when
to sample them
 Cost

93.  Therefore we see the application of antigen
antibody reactions in development of variety of
diagnostic tests.
 Only markers with credentials should be used in
clinical practice for the reasons listed below-
1. To prevent destructive disease
2. To prevent progression of the disease
3. To identify high risk patients
4. To target treatment to specific sites
5. To monitor the effects of periodontal treatment

94. Thank
you !!
 Thank you 