Simple is univalent complex is polyvalent. pptn occurs when antigen as two or more antibody combining sites.
Flocculation VDRL Syphilis
Antibody dependent cellular cytotoxicity
Nobel prize 1977 yallow :10 to power minus 12
DIPEPTIDYLPEPTIDASE – ALL DPP ACTIVE AT ACID ph except DPP 4 ALKALINE ph present in gingival tissue and GCF
7 amino trifluromethylcoumarin
METHOD OF ISOLATION IS HPLC high pressure liquid chromatography
Antigen antibody reactions
Dr Anuja Chandra By- Dr. Armaan SinghBy- Dr. Armaan Singh
Antigen antibody reaction and their applications
Diagnostic tests of periodontal disease activity
▻ 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
▻ 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.
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.
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
Proantigens are low molecular weight substances which do
not induce antibody formation but can cause delayed
Epitope is the smallest unit of antigenicity.
The combining site on the antibody molecule,
corresponding to the epitope is called Paratope.
Susceptibility to tissue enzymes
These are molecules that can interact with antigen
presenting cells and T lymphocytes in a non specific
These antigens do not involve the endocytic processing
as required in typical antigen presentation.
Viral proteins and staphylococcal enterotoxins are
examples of superantigens.
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
Its uses are
1. In vivo
Forms basis of immunity against infectious diseases
May lead to tissue injury in hypersensitivity reactions and
2. In vitro
For diagnosis of infections
Helpful in epidemiological studies
For identification of enzymes
Detection and quantitation of antigens or antibodies
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
Only the surface antigens participate in the antigen
The reaction is firm but reversible. The firmness of
combination depends on the affinity and avidity.
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
Identification of bacteria.eg Lancefield grouping of
Detection of antibody for diagnostic purposes.eg VDRL
Forensic application in identification of human blood and
Testing for food adulterants
To standardise toxins and antitoxins
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
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
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.
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.
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
Phagocytic index is the average number of phagocytosed
bacteria per polymorphonuclear leukocytes from stained
Phagocytic index denotes the phagocytic activity of blood
and thus helps in measuring opsonic index.
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
Two types are Direct and Indirect.
Specimen (Positive for antigen)
antigen is present in the specimen
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
Note – A separate specific fluorescent labelled antibody
has to be prepared against each antigen to be tested.
Unlike direct method, antigen is known in this
Lets have a look at the flowchart for clear
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.
The RAST test (radioallergosorbent
test) is an example of
radioimmunoassay. It is used to
detect the causative allergen for an
Can be used for detection of small quantities
Disadvantages are –
Enzyme Linked Immunosorbent Assay is as sensitive as
Requires only microlitre quantities of test reagents
The enzyme acts on substrate to produce a color in a
It can be used for detection of antigen or antibody
Types are Sandwich, Indirect, Competitive or Cassette
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
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
Viral particles are mixed with specific antisera and are
observed under electron microscope. These are seen as
Used in detection of Hepatitis A virus
Tissue sections are treated with peroxidase labelled
antisera to detect corresponding antigen. The
peroxidase bound to antigen is visualised under electron
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
These nitrocellulose paper strips are reacted with test
sera and subsequently with enzyme conjugated anti
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.
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
Only when the source,precise nature and the role of
potential marker are known and understood can clinical
evaluation be meaningful.
Potential sources from which markers can be obtained
1.blood or serum
3.subgingival plaque sample
4.gingival crevicular fluid
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
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).
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
Bacterial species numbers may be determined in variety
of ways (Listgarten 1992) and these include the following
1.Dark ground or Phase contrast microscopy
4. DNA probes
5. BANA assays
They use either paper points or curette bacterial
sampling from pocket and include the following-
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
Omnigene and BTD(Bio technical diagnostics)
These are DNA probe systems for a number of
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
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.
Diamond Probe/Perio 2000 system
Combines feature of periodontal probe with detection of
volatile sulphur compounds in periodontal pocket.
Given by Cox et al in1990, this can be used to detect
bacterial proteases arg-gingipain/gingivain and DPP in
This is not commercially available yet.
Predictive of disease activity
Results available in short time
Polymicrobial nature of the disease
Most are not predictive of the disease activity
Can only detect bacteria we look for
Special laboratory required
Those of possible relevance to periodontal pathology are-
Total IgG and IgG subgroups
Arachidonic acid derivatives
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
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.
It has been developed to measure small changes in
sublingual and subgingival temperatures and positive
cross sectional comparision with clinical parameters have
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)
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
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
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.
1. Proteolytic enzymes
Cathepsin G, B and D
2. Hydrolytic enzymes
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)
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)
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
This system detects presence of serine proteases,
elastases in GCF samples
Intensity of fluorescence α amount of enzyme in the
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-
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.
Can be read after short time
Can be shown to patient and related to tooth site
Selection of site
When to sample
Choice of most appropriate biomarker may still be
difficult at present state of knowledge
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)
Both of these potential markers associate with disease
activity but do not predict it
Can be shown to patient
Can be read after short period
Can be related to tooth site affected
Selection of site
When to sample
Choice of most appropriate biomarker may still be
difficult at present state of knowledge
The detection of breakdown products of normal
components of extracellular matrix could be indicative of
Most complex and expensive
Long collection times
Not suitable for chairside
Possible markers of bone resorption and hence
periodontal disease activity are-
Bone phosphoprotein (N-propeptide)
Telopeptides of type 1 collagen
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
CTP- RIA(Giannobile 1995)
Osteonectin, N Propeptide- ELISA(Bowers et al 1989)
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
Choice of most appropriate biomarker is difficult at
the present state of knowledge
Difficulty in determining sites to sample and when
to sample them
Therefore we see the application of antigen
antibody reactions in development of variety of
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