Immunoglobulins and antibodies

Immunoglobulins

Immunoglobulins or antibodies are

glycoproteins present in the serum and
bodyfluids, that are induced when
immunogenic molecules are introduced into
the hosts lymphoid system.
They specifically bind to the antigen that
induced their formation.

 WHO Definition: proteins of animal

origin endowed with known antibody
activity and certain proteins related to
them by chemical structure
Antibody globulins, abnormal proteins,
sub units of Igs.
Antibody-1/5th carbohydrate and
polypeptide
Antibody binds to antigen specifically
and initiate secondary biological
phenomena-Bi-functional molecule

 Von Berhing and Kitasato-(1890 )Proved

their physical presence
Tiselius andKabat (1939 ) identified
antibody molecule
Porter(19590 ) cleaved the antibody
molecule by papain
WHO (1969)coined the term Ig
The immunoglobulins include antibodies
and abnormal proteins

Functions of immunoglobulins
1. Prevent attachment of microbes to
mucosall surfaces of the host
2. Reduce the virulence of
microorganisms by neutralizing toxins
and viruses
3. Facilitate phagocytosisby
opsonizationof microbes
4. Activate compliment

 Porter, Elderman and Nisanoff

Immunoglobulin antigenic
determinants
Isotypes: the particular constant region
of light or heavy region of Ig
Immunoglobulins are classified on basis of
heavy chains isotypes
Light chains have isotype markers kappa
and lamda
Isotypes are present in all members of a
species

IgM:Stucture : pentamer

Serum conc.: 10-15%

Location:
Sedimentation coefficient :19
MW:900,000
Carbohydrate content:12
Serum conc;(mg/ml) :1.2
Half life:5days
Complement binding:classical pathway
Placental transfer:Present in milk:Seromucous secretions:Heat stability(560c): stable. Heavychain:mu
Susceptibility to mercatoethanol:susceptible

IgG:Stucture :Monomer
Percentage in total serum:80%
Location: Intra and extra vascular
Sedimentation coeffient:7
MW :150,000
Carbohydrate %:3
Serum conc.(mg/ml): 12
Half life:23days
Heavy chain: gamma 1,2,3,4
Complement binding : classical pathway
Placental transfer: +
Present in milk: +
Heat stability:+
Susceptibility to mercaptoethanol: not susceptible

IgA:
Structure:Dimer in mucosal surfaces and monomer in
serum
% of total serum: 5-10%
Sedimentation coefficient: 7
MW:
160,000
Carbohydrate content%:8
Serum conc.(mg/ml): 2
Heavy chain: alpha
Complement binding : Alternate pathway
Placental transfer: Present in milk: +
Seromucous secretion : +
Heat stability(560c)

IgE: Reaginic antibody ,heat labile, affinity

for mast cells of the same species
(homocytotrophism), antibody in P-K reactionare the unique features of IgE
Sedimentation coefficient:8S
M.W:190,000
Serum con.: Very less. Mainly present in extra
vascular compartments. Increase in con. In
atopic conditions
Carbohydrate content is more
H chain :epsilon, valency two

 Placental transfer :no

Susceptibility to 0.2 mercaptoethanol:
susceptible
Increased levels are seen heavy
parasitic infestations
Half life: 1-5 days
Action: by release of inflammatory
mediators by mast cell degranulation
IgD: structurally resembles IgG mostly
intravascular with half life 3 days.
Present on the surface of B-cells as
antigen receptor

IgG: protects body fluids

IgA:body surfaces
IgM: blood stream
IgE: antibody in type 1 hypersensitivity
reactions
IgD: recognition molecule on B- cells.

 Generation of ab. diversity: Antibodies

produced in an individual are more than total
no. of proteins in the body
C,JandV regions code for L chain and C,D,J
and V code for H chain
Few genes code for C-region,many genes code
for V-region
Immunoglobulin specifities: The specific ag.
Determinants on paratope are called
idiotopes. The sum total of idiotopes on
antibody molecule is idiotype
Three categories of antigenic determinants
are present on Igs.

 The antigenic specificities which

distinguish different classes and sub
classes of Igs. in normal individualISOTYPE specificities
The antigenic specificities which
distinguish of the same class between
different groups of individuals of the
same species ALLOTYPIC specificities
Gm system(gamma,alfa,epsilon&K chains)
and InV system (kappa light chains)

Abnormal immunoglobulins:
Multiple myeloma: A plasma cell tumour
producing excess amounts of single class of
Igs. with IgG,IgM
IgE myelomas are extremely rare
Bence-Jones proteins:light chains of
immunoglobulins either K or L. coagulate at
50 degrees and re dissolve at 70degrees
Heavy chain disease: abnormal proliferation of
Fc portion of heavy chains
Cryoglobunaemia: presence of cryoglobulin

 Immunoglobulin class switching: Initial

antibody is IgM fallowing primary or
initial antigenic challenge and B- cell
proliferation
Class switch occurs later with gene
rearrangement producing either IgG or
IgA with the same specificity
Same VH genes associated with
different CH genes

Monoclonal antibodies
Antibodies directed against single
epitope single clone of cells get
activated producing monoclonal
antibodies with single antigenic
specificity
Microbial infection and immunization
leads to polyclonal antibody production
Naturally occurring monoclonal
antibodies are myeloma proteins

Milstein and Kohler devised a technique

of hybridoma production for large
scale production of monoclonal
antibodies artificially
Hybridoma : somatic hybrids are
developed by fusing antibody forming
spleen cells with myeloma cells
1.
Spleen cells are immunized with
desired antigen
2.
Myeloma cells do not produce and
deficient in HPRT enzyme

Enzyme HPRThypoxanthine
phosphoribsoyl transferase
3. Exposure to polyethylene glycol
Applications:wider diagnostic
therapeutic
research
Commonly available monoclonal antibodies
Mouse monoclonal abs. can be humanized by
genetic manipulation- chimeric antibodiesmurine variable region and human constant

 Cleaved Fab fragment coupled to various toxic

substances and enzymes
By fusing genes coding for abs.fragments to
bacteriophage genes
Advantages: limitless
growthand10,000moleculesof Ig /min.

Applications: Diagnostic
leucocyte identification
HLA typing
viral detection sub typing
parasitic ag. Identification
detection of tumor related ags.

Therapeutic: Radiotope tagging

antitumor therapy
immunosuppresion
treatment of auto immune
and Hyper sensitivity diseases
treatment of GVH
Identification and purification of
microbial products

MONOCLONAL ANTIBODIES:
HPRT negative,
HPRT positive,
Ig positive
spleen
Ig negative myeloma
cells
cells
Fused by rapid exposure
To polyethelyne glycol

Myeloma cells

Hybrid cells

Spleen cells

Multiply indefinitely
Die because they
lack HPRT enzyme

HAT medium

Die because of
senescence

ANTIGEN- ANTIBODY
REACTIONS
Ags.and Abs. combine with eac other
specifically and in observable manner
Inivivo AMI is effective in extracellular
pathogens mainly resulting in immune
elimination or in tissue injury either
hypersensitivity or autoimmunity
Invitro ag-ab reactions serological reactions
useful inIdentification of I.agents
purification of ags and
abs.
Diagnosis of diseases
epidemiological surveys

 Ag-Ab reactions occur in three stages:

Primary stage reactions are rapid and occur at
low temp. detected byusing markers
Secondary reactions : some primary reactions
lead to secondary reactions demonstrable by
precipitation
agglutination
lysis of cells
killing of live antigens
neutralization
fixation of complement
immobilization of motile cells
enhancement of
phagocytosis

Tertiary stage: some ag-ab reactions invivo

leadto chain of reactions
neutralization or destruction of injurious ags.
or to tissue injury resulting in AMI or
clinical allergy
or other immunological diseases.
General features of ag-ab reactions:
1. Specific
2. Entire molecule takes part in the reaction
3. No denaturation of ag. or ab. during the
reaction
4. Reaction occurs at the surface .surface ags
are immunologically relevant
5. Reaction is firm but reversible. Firm ness
depends on affinity and avidity

6. Both ag and ab participate in formation

of ag-ab complexes
7. Valency for ag or ab are not fixed
Measurement of ag-ab:in terms of
mass of nitrogen or in units
Ab titre of the particular test :titre of
serum is the highest dilution of serum
which gives observable reaction.
Nature and quantity of the Ag
influence the titre
Two parameters of serological test are
sensitivity and specificity
Another parameter is reproducibility

Sensitivity:
ability of the test to detect even very
minute amounts of Ag or Ab
Specificity:
ability of the test to detect reactions
between homologous Ag and Ab only
Ina serological test the sensitivity,
specificity and reproducibility depend
on the standard of Ag and Ab and
facilities in the laboratory

SEROLOGICAL reactions detected by

1. Precipitation
2.Agglutination
3. Complement fixation
4.Neutralization
5. Opsonization
6.IF
7.RIA
8. EIA
9.Electro immunoblot
10.Immunochromatographic
tests
11.IEM

PRECIPITATION: when soluble Ag

combines with its Ab in the presence of
electrolytes at a suitable temperature
and PH Ag-Ab complexes form an
insoluble precipitate
FLOCCULATION: remains as a floccule.
Example : VDRL
Precipitation can takes place in liquid
media or in gels such as agar, agarose
and polyacrylamide

Amount of precipitation is influenced by

relative proportions of Ag and Ab.
LATTICE HYPOTHESIS: