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﴾بسم ا الرحمن الرحيم﴿
سنريهم آياتنا فى اآلف اق وفى انفسهم حتى يتبين لهم الحق أو لم يكفى بربك أنه على كل شيء قدير أنه
صدق ا العظيم
Immunity (resistance): It the sum of all naturally occurring
defense mechanisms that protect human from infectious disease
Non – specific
( Innate )
- Mucous membranes
- Phagocytic cells
- Enzymes in secretion
--Interferons ( α,β,γ)
- - NKCs
--Skin
-. Macrophages
Specific
( Acquired )
Naturally acquired
- Placental transfer of antibodies( Passive )
- Recovery from disease ( Active )
- Administration of antitoxin ( Passive )
- Vaccinations ( Active )
Artificially acquired
-with the innate or inherentis which mechanism physiologicThis is a
following properties
It does not exhibit specificity Do not depends on specific
recognition of a foreign
material
A single
mechanisms
Protect
Against
many
paths
Artificially acquired Naturally acquired
active passive passive active
First: Non – specific Immunity ( Innate)
:-
Natural ( Innate ) Specific ( Adaptive )
or
(Acquired)
Less specific .-
- Skin & mucous membrane .
-NK cells .
- Complement cascade .
- Phagocytosis .
- C- reactive protein .
Active Passive
- -Induced by contact with foreign antigens .
-- Consist of clinical infection , immunization with live or
- killed infectious agents or their toxins .
-- Long term.
-- Induced by antibody performed in
-another host
-- Ab injected in the incubation period
- Short term .
- Cellular immunity .
- Cell mediated immunity .
- (T- lymphocytes-Mediated)
- Humoral immunity
Antibody mediated immunity.
( B- lymphocyte)
B- lymphocyte
(Protection is mediated by
the produced antibodies)
Helper T- LYMPH . Cytotoxic T-lymphocyte
TH2 TH1
help
CD4 CD8
help
Haematopoietic stem cell
Myeloerythroid progenitor Lymphoid stem cell
B-lymphocyte NK cell
T-lymphocyte
monocyte neutrophil eosinophil macrophage basophile RBC platelets
Antibody
L-CHAIN
H-CHAIN
Immunoglobulins or Humoral antibodies
* B-
Lymphocytes
to proliferate , stimulatedLymphocytes are -* Upon exposure to antigen , B
differentiate and mature into LARGE PLASMA CELLS (large lymphocytes)
* The large mature B-Lymphocytes have
short life span ( days to weeks ) .
This cell type consists ( 20 – 25%) of the total peripheral lymphocytes
n mammals , they mature in bone marrow , then, migrate to secondary
lymphoid organs ( e.g. spleen & Lymph nodes ) .
Secreted
This type of cells is
involved in the
* Some large mature B-Lymphocytes (B- cells ) can be converted
long life spancells which have -into small B
And serve as Memory cells
Secondary
Immune
Response
Activation & differentiation of B-Lymphocytes , in certain instances ,
needs a Helper T- Lymphocytes activity to enhance the above to
processes in that B-Lymphocytes.
*- THEY CONSTITUTE 65 -80 10 of total peripheral lymphocytes .
*- They have long life span ( months to years ).
*- They mature in thymus gland before migrating to lymphoid organs
*- Upon exposure to antigen , T -cell proliferate .
How ever , their specific effectors molecules are not secreted
and remains firmly Attached to their cellular membranes
Giving what is called
cell-mediated immune response
*- They are involved in a variety of cell-mediated immunological responses
T-Lymphocytes :-
defense against
malignant cells graft rejection
bacteria & protozoa
Fungi
hyper sensitivity
reactions
viruses
T-CELLS
T-HELPER (TH) T-SUPRESSOR(TS) T-CYTOTOXIC (TCs) T-DELAYED
T- Helper :Their Surface Antigen : is T4 (CD4) .
*They Promote Maturation Of Antigen .
*Stimulated B and T cells.
And
Enhance their response
T – suppressor cells: * Their Surface antigen is T8(CD8).
* they suppress the effect of T – helper cells .
i.e.
*Suppress T &B – response .
T –cytotoxrc: * their Surface antigen T8(CD).
* they specifically destroy target cells.
virus infected cells unacceptable grafted cells
cells tumor
T – delayed hypersensitivity & T cell mediated immunity.CD4 (T4)
*they are responsible for delayed hypersensitivity reactions to different
antigens , particularly those of intra cellular parasites & contact allergen .
In general : * some of the stimulated T-cells release soluble substances
lymphokines that modulate the behavior of other cells.
Helper-
Sensitivity
Cells ( TdH)
and
T-CELL MEDIATED
IMMUNITY
(Tcmi )
*- most antigens which have a small number of epitopes and require carrier need
T – cell cooperation with B- cells
for antibodies production .
* Deficiency of B – cells (and\or) T-helper cells
leads to defective synthesis of antibodies.
* its over activity lead to
the majority of B-lymphocytes express both surface IgM & IgD, very few express
surface IgG & IgA or IgE in the circulation.
*the majority of B-cells also carry class 2 major histocompatibility complex (class П
MHC) products which are functionally important in
Regulation of immune response
Autoimmune disorders
Co -operation of innate & specific Immunity
in
Host defense against infection
*Antibodies promote Phagocytosis or activate complement to kill microbes
*T-lymphocytes enhance phogocytic and microbial functions of macrophages
+
INNATE IMMUNITY
BACTERIA PHOGOCYTE PHOGOCYTE
INEFFECTIVE
BACTERIA
+ SERM
COMPLEMENT
BACTERIA
+
complement SPECIFIC IMMUNITY
Bacterial lyses
PHOGOCYTE
B-Lymph Opsonization
And Phagocytosis
T-Lymph
+ B-Lymph
Cell
Mediated
response
a
b
s
LYSIS A b BACTERIA
+
bacteria
Direct lyses
BACTERIA
In direct lyses by C.
classification of acquired Immunity:- - passive Acquired Immunity :- Definition: acquired Immunity by given already form antibodies or antitoxic serum or gamma
globulins from normal or convalescent individuals or Trans placental or lactation .
Trans placental .
Natural
Types Lactation (Colostrum).
Antitoxin serum tetanus. (Anti_ cobra venom)
Artificial
Gamma globulins.
Rapidly developed .-* -: characters -
* -Short duration .
[ Rapidly eliminated in 2-4 WKS due to the formation of anti – antibodies (a disadvantage )].
*-Heterogeneous antibodies .
* -Cellular mechanism not stimulated .
(No memory ).
-:Side effects -*
*- Hyper sensitivity reactions against the
foreign serum
*-Neurological affection in some cases
( Encephalitis ).
*-Superadded in infections
e.g. (AIDS & HEPAT) .
OWN IMMUNITY
2- Active Acquired Immunity
Definition :- the body forms his when
stimulated (sensitized ) by introduction of
immunogenic agent.
Natural Infection
Types
*living attenuated vaccine * killed vaccine .
Artificial bacterial products
*Endotoxins.
* Exotoxins.
Others .
Characters :- * slowly developed .
*longer duration
(and leave a potential immunity , so there is A rapid response
in the future to the Same antigen ) leads to ??
*-Homogenous antibodies
*- Cellular defense mechanism play a role
Mechanism of Acquired immunity :-
Humeral A b
T_Cells Cellular
Derivation of T & B lymphocytes
Embryo Liver stem cell In Bone marrow
+
A 9 +
A 9
memory cells
HUMORAL ANTIBODIES
PLASMA
CELLS
central or
primary
lymphoid
organs
(tissues)
B_Cells T_Cells
Secondary
Lymphoid
Organs
Spleen or
Bone marrow
T B
Effector
Killer
cells
Specific memory and self-limitation of Immune response
Primary Anti A
RESPONSE
Ag A
infection
Secondary anti A
response
weeks 12 weeks
*- Antigen enhance THE production of specific Antibody A.
*- the secondary response to Ag A is more rapid and larger
then the primary response ( memory cells ) .
*- Antibodies Titer decline ( with time ) after each immunization .
Serum
AB
Specific immune response :
agents capable of It is developed as a result of exposure to a variety of
inducing an immune response
( i.e. immunogens )
Macro molecules microbes that colonize vaccines
in the body
Hapten is a micomolecule may be conjugate with a carrier protein in the
blood to be immunogen (antigen)
Specific immune response
Humoral cellular
B. Cells T-CELLS
*- They are two interrelated & interdependent mechanisms .
in the diet
in the form of hapten A special case Antigen
Specific immune response can be further
Classified according to its components into
primary secondary
Initial exposure to a particular on farther or
Infectious agent or immunogen repeated exposure
Induction phase of lymphocytes to antigen ( same )
proliferation T-CELLS
PLASMA CELLS increased B-CELLS
resistance
develops
through
Antibodies
humoral Sensitized T-CELLS
Cellular Immune response
Humoral
response
Cellular
response
Acquired immune response
Has both good ( desirable ) and
Bad ( undesirable ) consequence
undesirable Desirable
Immune
response
Protection
From infections
agents
Control of
Pre-cancerous
growths
Allergies (hypersensitivity)
Autoimmune diseases
Graft rejection
Interactions & functions of the major
components of the immune system
ANTIBODY – MEDIATED
IMMUNE RESPONSE
Main defense against
* exteracellular, encapsulated
pathogenic bacteria
e.g. streptococci & staphylococci
•*Neutralizations of toxins e.g.(
tetanus)
•* viruses ( Hepatitis C,A,B…….)
CELL MEDIATED REPONSE
Two major components
Cytotoxic
T-CELLS
Viruses
Acts by
Destroying
Virus- infected
cells
T-HLPER & MACROPHAGES
Intracellular bacteria
* ( mycobacterium &tuberculosis)
* Fungi
B-CELLS T-CELLS
HELPER
CD4
LYMPHOKINES CYTOTOXIC
CD8 PLASMA CELL
ANTIBODIES
+
COMPLEMENT
+
NEUTROPHILS
KILLING OF
BACTERIA
IL-2,IL-4.IL-5
IL_2
ACTIVATED HELPER
AND MACRO PHAGES
INHIBIT
INTRACELLULAR
Bacteria
&
fungi
Activated
Cytotoxic cells
Kill
Virus – infected
cells
IL_2
Neutralize
Toxins
Defense mechanism against viral infection VIRUS ANTIGEN
VIRUS
VIRUS infection
cell
MHC Class I
T-Cell
receptor
virus
IgM CD8
CYTOTOXIC
B-CELLS
INTERLEUKIN-4
INTERLEUKIN-5
MHC
Class II
T-Cell
RECEPTOR
CD4
( T_HELPER )
Killing
*- Recognition of phases :- antigen recognition ( binding of Ag to specific
receptor on mature lymphocyte ( exist prior to ag exposure )
*- activation proliferation & differentiation of lymphocytes is the sequence
of events induced in lymphocytes as result of Ag recognition .
*- Effectors phases elimination of antigen [ is the stage of the response
At which the sensitized cells perform the function that (eliminate of Ag)
Some antigen – stimulated lymphocytes die by process called programmed cell
death ( apaptosis ).
T
B OR + Ag
NATIVE
LYMPHOCYTES
Recognition
phase
ACTIVATION
PHASE programmed cell
Death
Effector
phase
Elimination
OF Ag
Phagocytosis
complement
Immunogenicety ability to induce immune response
Antigenicity ability of the substance to react specifically with
immune system
Happen is incomplete antigen ( di nitro phenol or penicillin)
It cannot stimulate humoral or cellular reactions but can react with these
products specifically so it is Antigenic not immunogenic
If they reacted with larger carrier protein (e.g., albumin , globulin or
synthetic poly peptide ) . It will be Immunogenic
Animals injected with this hapten – protein
Complex will make antibodies to this hapten ,
Only if it is ( hapten ) covalently linked to
the carrier (chemically bonded)
must be
Antigenic Immunogenic are not necessary to be
ANTIBODY
HAPTEN
CARRIER
PROTEIN PRTOCAL
NO
Anti carrier only
Anti carrier only
Anti carrier
&
Anti hapten
YES
NO
YES
YES
NO
YES
YES
( not chemically linked)
YES
(CHEMICALLY LINKED )
i
II
III
IV
Immune response :- its characterized by the produuction of
proteins ( Igs) and specificially reactive lymphocytes (T-cells ) when an
animal encounters
aforeign macromolecules or cells .
antigensThe inducing substances are called
i.e ( antibody generators ) or immunogens
*- Immunogenicity & antigenicity : Interchangeable terms used during
discussion of the immune reponse.
*- Immunogenicity : it the inherent ability of asubstance ( Immunogen
( complete antigen ) to induce a specific immune response .
*- Antigenicity : the ability to react with the products of that
response .
HAPTEN HAS AN ANTIGENICITY
HAPTEN PLUS PROTIEN CARRIER IS IMMUNOGEN
Antigens are the aligands that react with the products of an immune
response .
Epitope ( - antigenic determinants ) :-
are the sites either (on or) within the antigen with which antibodies or T-cells
receptor reacts
paratope :- the sites on antibodies which react with the antigen .
epitope size ( small )
conformational linear
conformational
site are on antigen surface
or internal that expressed only when the
antigen has been partially degraded in
vivo
valency of antigen :- e.g multivalent
i.e the antigen molecule carry a number of different epitopes
( some times 2 or>)
some of which specify antibody A others specify antibody B .
valency = total no . of epitopes the antigen pocesses .
with molecular size . proportionallyThe valence of A.g increases
Macro molecules are easily to induce phagocytic ( as example ) and easier to
be phagocytosed
Quaternary structure are the most Immunogenic
The more complexity , the more Immunogenicety
.factors Affecting Immunogenicity
Foreigness chemical complexity molecular
size
-: foreigness –A
the immunogenic substance must be forign to prduce immune response .
The greater the foreignness, the more will be the reponse
*- identical twins smaller or no response
*- brothers with the higher immune response
same tissues compatibility
the same blood groups .etc ………….
-B.CHEMICAL COMPLEXITY :
*- MOST of organic molecules are immunogenic expert lipids
*- proteins are the strongest immunogenic substance .
*-Polysaccharides most of them are haptens but they become complete Ag in
cases of
* peneumococcal polysaccharide .
* Lip polysaccharides in cell membrane of gram (– ve ) bacteria.
-Glycoprotein's : -*
Are immunogenic ex blood group Ags ( A,B,AB,O,RH )
-POLYPEPTIDES & nucleic acids : -*
Are weak immunogens
are not antigenic or immunogenic -: lipids -*
-:C.molecular size
usually the larger the molecule the stronger the Immunogenicety .
M.Wt below 5000 DA ARE NOT IMMUNOGENIC
MACRO MOLECULES are the most potent immunogens .
( e.g. albumin m.wt 40.000 Da
Globulin m.wt 160 kDa
Macrocyanin m.wt 1000 kDa
EPITOPE (ANTIGENIC DETEREMNANT):-
The portion of Ag that binds specifically with
the binding site of Ab (paratope) or a receptor(s) on T_lymphocyte
SIZE CONFORMATIONAL STRUCTURE The size and the structure of epitope are complementary to that of paratope
.i.e. they must have approximately the same dimensions
WITH RESPECT TO THEIR STRUCTURE ,A g MAY HAVE THE FOLLOWING
CHRACTES :-
Ag may have only a single epitope of a given specificity on its surface which is
capable to bind with antibodies , such Ag is called UNIVALENT AND UNIDETRMINANT
(one kind of specificity ) for example hapten
Ag may have two or more epitopes (which determine the specificity ) the A g called
in the case MULTIVALENT (which determine the number).
If the epitopes are of the same type called also UNIDETERMINANT and if they are of
different types called MULTIDETERMINANT (specificity ).
UNIVALENT
UNIDETREMNANT
MULTIVALENT
UNIDETREMINANT
MULTIVALENT
MULTIDETRMENANT
Hapten-carrier conjugates have native
antigenic determinants of the carrier as
well as new determinants of the hapten
Antigenic determinants are usually
limited to those portions of the antigen
that are accessible to antibodies shown
in black for this iron-containing protein
In an antigen, the same antigenic determinant
repeated many times
T-dependent antigens are characterized by a few copies
of many different antigenic determinants
of determinant but many of such one kindMULTIVALENT since it has only
determinant on each molecule
Ex. Many poly saccharides & homo polymer (e-g peptide chain of the
some .A. Acids .)
are multi determinant & valent such molecules have many some antigens -*
epitopes of different kinds (multi specificity ) but only one of each kind ( mono
valent )
Ex. Most proteins .
, chemically complexed compounds or polymerized proteins High M.WT -*
(quaternary structure or heteropolymerized proteins are usually .
Ag ( multi specific) , multivalent Ag (more than one Multi determinant-*
epitope of each kind)
(What kind &How many of such kind )
Antibody binding site ( Paratope ).
Binding of Ag & Ab
Affinity :- the strength of attraction and binding between an epitope( mono
valent ) of an Ag and the antigen combining site of Ab molecule ( Paratope
) .
Avidity :- The strength with which ( multivalcnt ) Ag bind to its
antibodies ( Abs).
This depends on the affinities of the individual
combining sites of the determinants
on the antigen
( chemical complexity )
Electrophoretic separation of serum proteins
ANTIBODIES and their STRUCTURES
*Classes of antibodies .
IgM , IgG , IgE , IgA & IgD .
A = COMPLEMENT BINDING
SITE
B = NEUTROPHILS & MACRO- PHAGE
BINDING SITE
VARIBLE = ANTIGEN BINDING
SITE .
Con
sta
nt
B
Consta
nt A
Consta
nt A
C
on
sta
nt
B
Heavy
chain
ANTIBODY STRUCTUR
CH3
CH2
CH1
Hinge bonds
ANTI BODIES
POLYCLONAL ANTI BODIES
- INDUCED AGAINST WHOLE ANTIGEN .
- LESS SPECIFIC
- PRESENT IN SERUM
MONOCLONAL ANTIBODIES
INDUCED AGAINST ONE EPITOPE .
(I.E . SMALL PART OF ANTIGEN )
- MORE SPECIFIC .
- PRODUCCED BY HYBRIDOMA
TECHNOLOGY . EPITOPE
EPITOPE
POLYCLONAL Ab.
INFECTION
MONOCLONAL Ab MAb MAb MAb
i-Immunization
antigen
HybrIdoma technique
+
fusion B-CELLS
MYELOMA
CELLS
HYPRID CELLS Tissue culture supernatant
Fluid
Ascetic fluid selection of
Desired
Clone
+ MICE
II_ FUSION
TUMOR MICE MICE
Immunoglobulins
"Humoral antibodies”
They are formed of two identical units each of them is formed of :-
A) heavy chain B) light chain C) hing region
A) Light chain 2( lambda)
but never 1 and 1K
K ( Kappa )
2
B) Heavy chains :
* M-Wt 53.000 - 75.000 Da
*- heavy chains are hold together with (disulphide bonds) .
*- Fixed region contain 2k or 2 .
*- The variable region contain a mixture of K,.
*- both L& H chains contains the following region :
Light chain contain variable (VL) and hyper variable (VH) regions
Heavy chain contain variable and hyper variable regions.
VARIABLE REGION CONSTANT REGION
* Amino terminal * carboxyl terminal
* The amino acids differ * A. As are similar in different
on to another specificity. * it contain the effectors domain which
is responsible for the
* The VL & VH are adjacent to initiation of the process
each other forming paratope . by which the body gets-rid of Ag. .
* They have sub-regions of the
variable region (hypervariable)
-
-
-.
-
)hyper variable(
It is responsible for
Designation of Ab class & its
distribution. These regions have extreme
variability in their A .As
sequence in different antibodies
and they are responsible for
binding with Ag(s)
{CDRs}
comptementary
detemining
regions
C) Hinge region :-
* CH 1, CH 2 , CH 3 : occupies ¾ that of Heavy chains the other ¼ is
VH .
* The Hinge region lies between CH 1 & CH 2 .
* It is flexible & allows movement between the two antibody binding
sites .
* The hinge region is digested by protease (e.g. pa pain ) which splits it
into :-
( i ) antigen binding fragments (fab) = They are 2 identical fragments containing the
antigen binding site .
)ii ) crystallization fragment (FC ):-It contains the effectors (
Structures and function
Of
Specific Immunoglabulins
*- Ig(s) are glycoprotein's in the gamma globulin fraction of serum proteins (albumin ,
fibrinogen , globulins ( , and ) .
*- they are produced by B- lymphocytes or plasma cells in response on
immunogen (or Ag ).
General Ig structure :-
*- 4 poly peptide chains.
*- they are linked covalently by disulphide bonds
*- the 4 chains , monomeric Ig structure ,are
composed of 2 identical heavy poly peptide chains (H)
2 identical light poly peptide chains (L)
*- Heavy and light chains : *- H- chain :
*- Have a M.wt of 50-75 KD (Twice that of L chain )
*- H chains contain 400 A.As (Twice that of L chain )
*-A. As differences in the .COOH terminal portion of the heavy chain (CH) identify 5
distinct H-chains isotypes .
* Each H chain has 4 or 5 domains :
1 domain in the variable and 3 or 4 in the constant
3 IgG ( ), IgA( )&IgD( ) Or 4 Igm ( )&IgE( )
Total = 1 Variable + 3 constant or = 4constant 1 + Variable
The Hinge region *- It is the portion of the H-chain between CH 1& CH 2.
*- there is no homology between it and the other H- chain domains, thus .its
sequence is unique (sole) for each Ig type and subclass
Notes (1) -Each L- chain has 2 domains 1 VL
1 CL
(2)- Folding of the polypeptides chains brings the hyper
variable regions of the VH and VL domains into close
proximity .
(3)- this folding creates a 3-dimensional structure that is
complementary to the epitope (last figure )
IgM & IgE do not possess a hinge region but have one more CH domain.
These structure explain why both IgM & IgE have 4 domains on the CH chains but
not like the other types (which have only 3 domains on CH)
*-In this region (hinge), inter chain disulphide bonds forms between the arms of the
Fab fragments preventing them from folding and therefore
, rendering this portion of the molecule highly susceptible to fragmentation by
enzymatic attach .
* - The hinge region is highly flexible and allows for movement of the Fab arms in
relation to each other .This motility explain why native antibody
molecule do not activate complement , whereas those in an immune
complex do .This is because , the native Ab is not in the appropriate
configuration t1/2 or half life of( Abs) .
*- These heavy chain isotypes form the basis of the 5 Class of Immunoglabulins
molecule IgG () ,IgA ( ) ,IgM ( ),IgD ( )and IgE ( ).
*- H chains Classes and are subdivided into subclasses of molecules
1 , 2 , 3 , and 4
And 1 and 2
The subdivision is based on the greater similarity of A.As sequence shown by
subclasses of the same class
i.e. 1 , 2 , 3 etc,. Than is shown by different classes
(i.e. , , or )
*- The heavy chain subclasses determine immunoglobulin subclasses
e.g. 1 = IgG1
2 = IgG2 , 3 = IgG3 etc,.
*-L-chains :-
* Are composed of 200 A. As .
* They are of 2 types ( K= Kappa or = lambda ) .
{ based on their structural (antigenic) differences }
* All Igs classes have 2K or 2 chains but not k or k .
ex. * The proportion of K/ = 3/2 (human Ig) .
- chain Isotypes :- *- There is no isotypic variations in K chains
*- There are 4 distinct chains 4 different isotypes .
*- All the 4 subclasses are present in each of the Ig classes
i.e. in IgM , IgE , IgD etc.
*- Disulphide bonds Hold together the 4 polypeptide chains
in Ig molecules .
*- There are 2 types of disulphide bonds :-
i.Interchain disulphide bonds :
H – H chains H – L chains L – L chains
Single L-L only in
Hinge But also in Ig A2M (1)
region COOH-terminal such bond can of the H chain occurs in all Ig(s)except occur under path-
Ig A2M (1) which ogenic conditions.
Lacks an inter chain (e.g. Bence Jones
They can be 1:15 depending disulphide bond protein ) seen in
On the class & subclass types urine of some
patients with
multiple myeloma
occurs between
INTRA CHAIN DISULPHIDE BONDS :
*- occurs within an individual chain .
*- they are stronger than inter chain bonds .
*- they no. of intra-chain disulphide bonds varies depending only on the number of
domains in the molecular .
Light chain have 2 intra-chain bonds .
*- human IgG, IgA, IgD heavy chains have 4 intra_chain bonds
*- human IgM , IgE heavy chains have 5 intra-chain bonds .
*- Each H& L-Chain has a variable (v) and constant (c) region
*- V region lies in the – NH2 terminal portion of the molecule .
*- The V region has a wide variation in it’s A.A composition .
*- The C region lies in the - COOH terminal end of the molecule .
*- The C region has a much more constant A.A Sequence except for minor inherited
changes
*- The variable regions associate with appropiate constant regions .
so that a variable H – Chain regions (VH) does not occur in an variable L – Chain
(VL) and Vise versa .
*- However , a particular VH chain sequence may occur in more than one
H – Chain class ( i.e IgG, IgM , IgD ,IgA and IgE ) .
*- Thus during class switching in an immune response e.g when B – cells change
their production from IgM to IgG heavy chain
only the constant regions of the H (CH) changes and the antibody specificity
remains the same .
HYPER variable regions
*- they are particular areas within the variable regions
That are highly variable in A. As sequence .
*- THESE hyper variable regions often called complentary determining regions
*- THESE regions occurs at simillar A.A positions in an relatively invariant
molecules .
CDRs :- they are short polypeptide segments lining near A. As positions 30,50
AND 90 in the variable regions of both L and H chains .
Note :- the variability range ( index ) used is an arbitrary scales of the no. of
different A.AS found in each position if 100 different Light chain were analyzed .
*- the hyper variable regions are important in the structure of the Ag binding site
( paratope ) .
*- L – chain have 3 hyper variable regions ( the last figure )
*- H – chain have 4 hyper variable regions although,
ONLY 3 OF THE 4 have been associated with epitope recognition
*- each Ig chain consists of a series of globular regions or domains enclosed by
disulphide bonds ( intra or inter ) ?? Chain disulphide bond .
*- The A.AS sequence of the domains show a high degree of homology
( i.e the sequences are very similar ) .
24-34 50-56 89-97
variability FR1 FR2 FR3
FR4
CDR1 CDR2 CDR3
NO OF AgS
CDRS
FRs
variable region
-Properties of Ig :
IgE IgD IgM IgA IgG
-
190
11
0.05
2
-
-
?
+
-
180
13
3
3
-
-
-
-
-
900
12
120
10
++++
-
+
-
1 2
160-400
7
200
6
-
+
+++
-
H – CHAIN
H – CHAIN SUBCLASS 1, 2
M.Wt 150
Carbohydrate (%) 3
Serum conc(mg %) 1200
Seru t ½ ( days) 21
-: Functions
Complement activation ++
Opsonization ++++
Antiviral activation ++
Mast cell sensitization -
% of their M.WT )13 -3( -: Immunoglobulin are glycoproteins
OLIGOSACCHARIGES + PROTEIN
*- THESE oligosaccharides are present in CH2 or CH3 .
*- N -glycosidic bonds usually link N- acetylglucosamine in the carbohydrate moiety
to asparagine residue in the peptide c-chain of Ab
[ linkage with the enzyme N -acetylglucosamine .
- Asparagine transglycosylase ] transferase
*- t ½ of Abs in the circulation depends on the status of oligosaccharide side chain
*- the oligosaccharide side chain of Ab terminate with galactose to which sialic acid
is bind .
*- when Abs have the sialic acid removed by the enzyme neuraminidase , they
become susceptible to degradation in the liver .
*- in this case the terminal galactase bind to a receptor on hepatocytes and the
entire molecule is , then , interenalized to the cell for degradation via
Proteolytic enzymes in lysosomes of the cells .
Restriction enzymes digestion of Abs :
1) Papain : digest above hinge region so it leaves 2 Fab fragments each is monovalent
And crystalline fragment (FC)
2)Pepsin: digest away most of FC
Fragments below the Interchain disulphide bond
(below the hinge region) it give one large fragmentsF(AB)2 which is consist of two Fab
fragments joined by the disulphide bond
Thus , it is bivalent ,possessing the ability to bind and form agglutination
papain Fab FC
Monovalent
S-S
Ab
F(ab)2 FC
S-S
Figure 4 Immunoglobulin fragments:
Structure/function relationships
Classes of antibodies
They are 5 isotypes
The class of Ab depends on the A.A: sequence
of the constant regions of the heavy chain .
-Immunoglobulin M (IgM) : -*
* it is a pentamer ( 5 molecules ) .
* they are linked together by disulphide bridges at the COOH terminal end
of the heavy chains as well as an additional poly peptide chain ( joining chain)
* this type of Ab account for 8-10% of the total PLASMA ANTIBODIES .
* it is the most abundant Ab produced by the faetus .
* it binds with viruses and bacteria
-Immunoglobulin g ( IgG ) : -*
* it is a monomer
* it accounts of ~ 75 % of the total antibodies .
* it is important for elicit ting the immune response to Ags
* it is only antibody which pass through the placenta to protect the faetus.
IgM
*- immunoglobulin D ( IgD):-
* It is a monomer ACCOUNTING FOR < 1% & TOTAL ANTIBODIS .
* Its function is controversial .
*- immunoglobulin E ( IgE):-
* It is a monomer ( below 0.004 % & the total Abs)
* It is present in spleen , tonsils , mucus membrane of lungs GI
* On binding with ag it releases histamine from mast cells leading to
hypersensitivity .
* It provides immunity to intestinal parasites .
*- immunoglobulin A ( IgA):-
* MONOMER , DIMER or TRIMER( mostly dimer )
* Like IgM the units are linked by disulphide and j chain
* it is found in tears , saliva , intestinal treat secretions
* it binds with Ag preventing them from tissue adherence , colonization ,and
making them more phagocytosed .
-Properties of Ig :
IgE IgD IgM IgA IgG
-
190
11
0.05
2
-
-
?
+
-
180
13
3
3
-
-
-
-
-
900
12
120
10
++++
-
+
-
1 2
160-400
7
200
6
-
+
+++
-
H – CHAIN
H – CHAIN SUBCLASS 1, 2
M.Wt 150
Carbohydrate (%) 3
Serum conc(mg %) 1200
Seru t ½ ( days) 21
-: Functions
Complement activation ++
Opsonization ++++
Antiviral activation ++
Mast cell sensitization -
Laboratory Methods
Serology
In vitro Ag & Ab reactions called serology
It provide methods for
i) Identification (Diagnosing) ( ii ) quantization of titre of Ab (and \ or) Ag
Titre : or the level of Ab (s) in the serum can be measured by using known Ag
The titre may have diagnostic
prognostic
Ex. A rise in Ab titre between acute &convalescent serum can be used as a
diagnostic tool for a specific disease
The titre is defined as the greatest dilution of serum (which contain the Ab under
consideration ) that reacts which the antigen ( i.e. gives +ve result ) .
or
- the forces involved in Ag-Ab reactions are greatly affected by various environmental factors :-
*- The Ag- Ab complex is not bound firmly together .
*_This complex may even dissociate spontaneously .
* physiologic ph & salt concentration promote optimal union of them .
*- the force of attraction tend to be weaker in
a) very acidic .e.g. 0.01M
b) very alkaline medium
i.e pH 4 and alkaline ( i.e. above pH 10 )
- temperature :- it plays an important role :
* the higher the temp ( up to 50 – 55 0 c ) , the more rapid is the rate of reaction
between Ag & Ab .
* the reason is the increase in kinetic motions of the reactants ( Ag & Ab )
various forces act to hold the Ag-Ab complex together :- * The maximum attractive forces stabilizing Ag-Ab complexes
Are van der weal forces
Ionic bonds
1- van der weal forces :-
* occurs because of spatial fit ( the below fig )
* these forces of attraction hold Ag to Ab only
When the two molecules have complementary shapes (a)
2Epitope puratope 2
(a) (i) significant changes
In the shape of epitope 2
Into 2a
puratope 2
a2Epitope
( b )
these change precludes its ( 2a ) interactions with the matching binding site of the
original Ab .
* When the molecules have less similar shapes ( b) , these forces are less effective
(b)
2-Ionic bonds :-
* They are patterns of complementary electric charges on the molecule .
* The electrostatic interactions tend to hold the molecules together .
COO
COO
COO
Affinity :- the strength of attraction between a single epitope and its matching
paratope is the referred to as the affinity of the reaction between the two reactants .
Ag-Ab complex of low affinity dissociate readily
Avidity :-
* It is a related term to affinity
* It refers to the strength of the interactions between multivalent antigens and the
population of Abs that they have included .
NH3+
NH3
+NH3
*- Avidity is influenced by the affinity of individual Abs for their
(A) epitope
(B) the valency of Ag and
(C) the valency of Ab
tertiary structure of protein :
*- the ability of Ab to bind with Ag can be affected by altering the tertiary
structure of any of them
ex.
insulin which is composed of A&B chains Ab to either one of these chains can
be produced by
(a) splitting the chains
(b) purifying tem
.e.g. a pig) ((b) injecting
them into foreign host
the pig will produces Ab to the particular chain that was injected
*- if the host (pig) Abs are injected back into the animal species that supplied the
original insulin (man) , the abs will not react with intact insulin molecules .
*- This is because the tertiary structure of native insulin is such that the
epitopes on the A & B chains are not accessible ..
Now , it is generally accepted that in a given poly peptide
the A .As that are spatially accessible because of
Tertiary structure of this protein are only immune
reactive
Large enough to be visible
with naked eye
*- The physical state of the antigen is responsible for the identification of Ag –Ab
reactions and the naming of Abs .
*- The same Ab molecule could , in fact , be described by each of the following
terms : (1) Agglutinins are Abs that aggregate cellular Ags.
(2) Lysins are abs that cause dissolution of cell membrane .
(3) Precipitins are abs that form precipitate with soluble Ags .
(4) Antitoxins are abs that neutralize toxins .
procedures must be involving direct demonstration and observation of
reaction ..
The relative sensitivities of the tests for Ags and Abs are
Presented in table 8.1 page 156 [ immunology , 3rd edn ].
A- Agglutination Reactions :- cellular Agsand identify AgglutininsServe to detect and quantities
Bacterial cell white blood cells red blood cells .
**-- when the cells intact with the appropriate Ab , they clump together and eventually
b a
form masses
*- When Ab agglutinates bacteria in the body opsonization occur .
*- Agglutination occurs because Abs and at least bivalent .
*- Two sites on the Ab and multiple sites on the Ag
Ag – Ab lattice formation
that can
build up into increasingly larges coupled
lattice structure
Example widal test :- (diagnostic test of typhoid )
*-Ab of patient serum is measured by adding a constant mount Ag
(e.g. salmonella typhi ) to serially diluted serum .
*- After incubation , the test tubes are examined for visible agglutination .
*- the last tube (i.e the highest dilation of serum ) showing agglutination
is referred as the titre .
-lyses Reactions : -B
In the presence of a complement an Ag – Ab reaction , on a cell
membrane , may result in membrane damage that leads to cell lyses
This phenomenon is important in the host's defense against condition
such as microbial infection or cancer ( graft cell , virus infected cells
, etc…………….)
-: Haemolysis -*i)
In which the Hemoglobin is released from R.B.C, is a requisite
phenomenon for the complement fixation test .
-bacteriolysis : --*ii)
cells of gram (– ve) bacteria are undergoes immune lyses under certain
condition .
-: cytolysis --*iii)
involves the destruction of other cells types (e.g. lymphocytes ).
-:precipitation -C * occurs when the Ag is soluble instead of cellular
*therefore a large number of molecules are required for lattice formation and a
large no .of lattice must be formed for an aggregate to be formed and visibly seen .
*when soluble Ag (s) come intact with specific Ab. They aggregate
(i.e precipitate )
Three conditions are present
A- where the (Ag) is very low with excess Ab (zone of Ab excess ),
Formation of complex occurs
But
Residual Ab remains in the supernatant
B- As more Ag is added , large aggregate is formed
In the (zone of equivalence) ,
maximal Ag-Ab complex are formed and precipitated
C- Instead of reaching a plateau , this curve comes back down to zero
with increasing the mount of Ag (zone of Ag excess )
* this is because the lattice size becomes too small to precipitate .
* In extreme Ag excess . the complex will be trimmer
i.e one Ab +2Ag
Note:- the soluble Immune complex are not processed efficiently by
the reticuloendothelial system ,and ,this cause damage (how??)
INDIRECT HCG :
Examples 1 :- determination( and\ or) detection of HCG by using indirect methods .
(i) an Ag will be added ( HCG ) .(from the kit)
(ii) Urine will be added ( excess Ag ) from a female may be pregnant .
(iii) Ab to HCG will be added
In case of positive In case of negative pregnancy
A state of Ag excess a state of equivalence will be reached
Therefore, no precipitation therefore, precipitation occur
Direct HCG assay :
* (i) Ab to HCG will be added (from the kit)
* (ii) Ag ( HCG of the test sample will be added) .
If precipitation occur ( positive) if, no precipitation occur ( negative )
Am
ou
nt
of
pre
cip
ita
te
Zone
Of
equivalence
Effects of increasing amounts of Ag on the total immune precipitate obtained from a mixture of soluble Ag and its homologous Ab
Hyaluronic acid (HA) assay using excess
HA binding protein (HABP) :-
* HABP will be added in excess ( known excess ) (ACT AS Ab)
* Sample will be added ( containing HA) (ACT AS Ag)
* [ A state of Ab excess no ppt ]
* An radiolabelled HA will be added ( Ag )
Thus, precipitation occur ( IF +ve sample) and immune complex will be separated
and quantities by radio- immune assay technique ,in case of no precipitation, the
sample is negative
Immune diffusion
* It used for quantization of Ag (s)
* Thus, precipitate will also be demonstrated .
* If an Ag – Ab reaction takes place in semisolid medium (e.g. agar ) , band of
precipitate will be formed .
* The reason of precipitation , is the diffusion of the components (Ag & Ab ) towards
each other .
* A useful example is a double immune diffusion technique :-
Procedure :-
* Ag & Ab preparations are placed in separate wells that are cut into a thin layer of
agar in a Petri dish .
* The reactants diffuse towards each other through the agar until they meet an
optimal proportions [ zone of equivalence ] and forms
( ppt ) bands
The advantages of the procedure is that antigenic relationship
can be detected by the precipitation pattern (s)
Ab
Ag Ag
Fig (a)
Solid
Chevron
PPT
Zone of
equivalence
3 basic patterns are given :
(a)- in reaction of identity , the 2 Ags are similar , they will diffuse at the same rate
and the two precipitations bonds merges into a solid chevron ( fig b)
2- in reactions of non-identity , the two Ags are completely different and the lines
of the precipitate cross (fig c)
3- reaction of partial identity :-
* It is indicated by spur formation indicating that one of the
Ag(s) is cross-reactive ( but not identical ) to the other one .
* The spur occurs because one the Abs (b) does not react with the cross-reacting
Ag (Ag ac) but migrate past that Ag (Ag ac ) until it reaches an Ag (Ag ab ) that
Aga Aga
Ab
Fig b
Fig c
Aba Abb
Aga Agb Ayab Agac
Abb Aba
Has an epitope for which it has specificity .
B- quantitative radial immune diffusion
* It is used routinely to quantities Ab in serum .
* For this purpose , an agar coated slide is used .
* The agar being impregnated by anti sera ( antibody to human IgG )
* SERUM samples are placed in wells in the sugar .
* As it diffuse through the agar and encounters the Ab, the IgG in the sample form a
concentric ring or halo precipitate .
* The diameter of the halo of precipitate directly correlate with the [ IgG] in the
sample .
Thus , the levels of IgG in the sample can be determined by referring a standard
curve based on halo diameter (s) of known concentration (s) of IgG
C-immune electrophoresis :-
* It was developed because the double immune diffusion technique.
(i) Could not resolve high complex mixtures of Ags .
(ii) and, a more sophisticated technique was needed .
In this procedure :
(a) Ag is placed in wells in agar on a glass slide and then , subjected to
electrophoresis through application of an electric current .
(b) Under these conditions , the individual Ags or antigenic components ( in
the same sample ) migrate through the agar at variable rates .
(c) If Ab is placed in a well that runs the length of the slide parallel to the path
of migration , the reactants will diffuse towards one another and form separate arcs
of precipitate for each antigenic determinant
D- counter-immune ectrophoresis (CIE)
This technique Is
the double diffusion method + an electric current
Which plays as the migratory force which:
(i) – amplify the speed of reaction ( 24 hrs to 30 min )
(ii) Intensifies the precipitation bonds .
(iii) Increasing the sensitivity of the assay about 10 fold .
Procedure :-
(i) Ag & Ab are placed in wells and the current is applied .
(ii) in suitable buffer ( eg ph 8.6 ) the negativity charged Ags migrate
towards the anode , whereas the Ab [ which has no sufficient net charge ] migrate in
the opposite or counter-direction , as a result of endosmosis .
•Precipitation occurs where the reactants melt .
D- Antitoxin :-
* If a serum contain an antitoxin ( i.e. antibody to a toxin ) , the Ab . Will neutralize
the toxin examples :-
- Suppose serum containing antitoxin is mixed with toxin ( in vitro ).
- Then , after a few minutes , a small amount of the mixture is injected into an
experimental animal ( in vivo ) .
- The animal will be protected against the introduced toxin , and thus , its deleterious
affects disappear because of antitoxin is present .
( the virus haemagglutinate R.BCs) -: Clinical example
* To examine the serum of a patient suspected of having influenza ,
*1) The patient serum is mixed with known influenza
2)add red blood cells
i- if Ab is present haemagglutination will be prevented
.i.e the sample is positive
this is due to the ability of Ab to bind with the virus and block its ability to
haemagglutinate the R.B.Cs
ii- if no Ab is present , haemagglutinate will occur .
virus + R.B.Cs haemagglutination occur
.i.e the sample is negative
E-Flacculation :-
it is another form of Ag –Ab reaction that
occurs if the Ag is neither cellular
nor soluble
but it is an insoluble particulate
The venereal disease research laboratory (VDRL) test is a slide flocculation test
used for the diagnosis of syphilis .
•The VDRL make use of heterogenetic ( heterophillic ) antigen shared between the
Spirochete of syphilis & normal beef heart .
* The Ag used is a water insoluble cardiolipin that had coated the surface of
cholesterol particles that were added to the system .
* These form visible aggregate indicate to the presence of Ab ( reagin ) in the serum
of patient for syphilis
[ reagin is Ab type which flocculate (or ppt) an Ag that is neither cellular nor soluble
but it is insoluble ]
* The test can be performed on a glass slide .
VDRL TEST FOR Syphilis
Technique :-
cholesterol particles + normal beef heart extract
( inert support ) ( antigen like substance )
Insoluble antigen
serum
( A.Bsource )
visible aggregate
Which can either seen by
The naked eye Using a microscope
and green filter
Ag-Ab reactions in which the visible manifestation requires Participation of:
a) Accessory factors
b) Indicators system
c) Specialized equipment
COMPLEX SEROLOGICAL PROCEDURES
-fluorescent dyes : -A
e.g fluorescein isothiocyanate ( FITC)
* FITC can be conjugated to Ab. Molecules to visualize of the molecule under (uv) or
a fluorescence microscope .
* such labeled Ab. May then be used to identify Ag(s)
(i) Direct immunofluorescence assay :-
* The method uses Ab. That is specific for a particular Ag
parasite
* This Ab is labeled with a fluorescent dye (FITC)
* This conjugate is allowed to react with unknown tissue or organism .
* IF the Ab reacts ( i.e +VE the result ) , it will visualized as green stain on the
fluorescence microscopespecimen when it examined under the
using uv light
(b)
or
by
Examples :-
Identification of Trepenoma palladium ( syphilis ) in an extracted from a patient
suspected of having syphilis .
- Procedure :-
*1) The slide is coated with the Ag .
*2) Ab tagged with FITC is added .
* 3)Excess Ab is then washed .
* 4)Then , the slide is examined with uv fluorescent microscope .
* Trepenoma palladium is fluoresce against the black back ground
this methods can be extended for other pathogens .
(ii)-immune peroxides technique : If viral antigen in tissues will be detected, horse radish peroxides
is conjugated with the Ab .
*-(1) After the enzyme – Ab complex has reacted with the tissue (Ag) .
*- (2) Excess Ab is washed .
*-(3) And , an appropriate enzyme substrate is added to the tissue
section .
*- the bound Ab. Is detected by the presence of a dark precipitate
*- Advantage of immune peroxides technique over the immune fluorescent
technique:-
*- The specimen can be stained with conventional Histochemical dyes
So structural details can be seen ( noted )
. light microscopethe tissue con be examined by standard -*
(iii)- Indirect immune fluoresce technique:-
*- The procedure use Ab ( secondary ) ( against ) another Ab
( primary ) of patient .
*- the primary Ab is the patient' serum detection of Abs.
*- the secondary Ab is covalently conjugated whit fluorescent
compound (FITC)
antihuman( INJECTED Ab in host is FROM rabbitex. Of secondary Ab is -*-*
gamma globulin anti sera . HUMAN )
.i.e Produced against Ab used to immunize rabbit and that Ab
which will be examined latter on (unknown conc.)
Technique :- (this procedure allows for detection of Abs,)
Example serodiagnosis of syphilis by the fluorescent trepenomal antibody
absorption (FTA-Abs) test
*-(i) -T. pallidum is fixed to a slide
*-(ii)- the slide is flooded with the patient serum (staining Ab )
*-(iii)-If Ab to spirochete are present, the Ab will reacts (bind) with the
organism on the slide
* (iv)-Excess Ab (serum) must be removed with washing , to detect the bound Ab
only .
*-(v) the Ag-Ab complex formed is them treated with the fluorescein – tagged Ab to
human gamma globulin, the excess Ab is washed carefully.
*-If the patient's serum contains Ab (+ v e) against the T. pallidum, fluorescein
organism will be seen when the slide is examined with fluorescence microscope
**-- Ab fluorescence
conjugate is
binded .
(uv ) is used Microscope
Indirect Immune fluorescence assay is also used for -
-Detection of Antinuclear Antibodies (Ana) :
( e.g. DNA , RNA & His tone)
ANA are present in systemic lupus erythroMatosis ( SLE ) , some
Times in rheumatoid arthritis and other autoimmune collagen –
Vascular diseases .
-Example (SLE ) : * The procedure is similes to that of T . palladium .
* The Ag is ( DNA ) histone in form such as
Animal Buffy coat calls
Human Buffy coat calls
Rat kidney section
beef thymus
Lymphoid Thymus
Lymphoid organs
FITC
Patient’s
Serum is
added
Slide coated
with ag in
(excess)
-:Haem agglutination Inhibition teat
It involves The agglutination of R.B.cs by
(haemagglutinin(s))
Ab(s) Certain virus
particles (influenza)
Or
other
substances
It demonstrates the presence of serum Ab to haemagglutinating viral substance .
-Technique : R.B.C s
serum sample Ab from the
Which contain Ab kit that make
Prevent haemagglutination haemagglutination
Agglutination occur no agglutination
The sample is negative the sample is positive
Similar test can be used to detect soluble Ag(s) which able to
react with and neutralize a haemagglutinating Ab .
R.B.Cs from kit
Abs from kit which
Capable of haemagglutination
serum Ag which
Prevent haemagglutination
haemagglutination
Inhibition
Sample is positive
haemagglutination
takesplace
Sample is negative
- passive agglutination:-
in the conversion of a reaction system from one that
precipitate one that agglutinate
Thus yields a more sensitive indication of
the presence of antibodies .
The detectable antibody is called rheumatoid factor
-RHEUMATOID ARTHRITISExample :
The use of latex particles in the diagnosis of rheumatoid arthritis (soluble Antibodies
) is an example of passive agglutination .
Principle :-
In this disease ,the patient produces an Ab (Mainly IgM) to his own IgG
Technique :-
*-(i) latex particles were coated with IgG.
*-(ii) patient 's serum is added (which contains antibodies IgM)
*-(iii) Agglutination indicates the presence of Antibodies (Ig?)
(i.e The test is positive )
Bis-diazotized diphenyl :-
it is a coupling reagent that can be used to
proteins
conjugate : or R.B.Cs
Haptens
and thus Passive haemagglutination
Occur.
to
Thus :
*- Addition of serum containing Antibodies to these substances (proteins or
haptens ) allow the detection of these specific antibodies to these substance by a
technique called Passive Haemagglutination
rose –waaler Test :
which detect rheumatoid factor in serum of the patients suspected to
have an anti-IgG auto-antibody .i.e. .(IgM to an accumulated IgG
- Tannic acid –treated a sheep R.B.Cs (S R.B.Cs) are coated with rabbit IgG
Antibodies specific for these S. R.B.Cs
haemagglutination
the sample is positive IgM
from
the kit
serum from a patient suspected
to have autoantibodies R.B.Cs
Tannic acid
coated by IgG
Coomb's (antiglobulin) Test :-
* In certain people .Abs directed against antigenic determinants (e.g. R.B.Cs
antigens )are able to form visible aggregates when subjected to :
1- precipitation 2- Agglutination
*-To demonstrate the presence of Abs in such cases the coomb's (antiglobulin)test
may be used .
*- The test involves the addition of Ab direction against gamma globulin : which
provides a bridge between two antibodies coated call or particle
Thus ,The major use of the coomb's test is to detect the Non agglutinating
(haemagglutinating )anti-red blood Cs Abs.
(1) – Direct coomb's test * It is used to detect call bound antibodies
Technique :-
You must use EDTA BLOOD then centrifugation
*-(1) The red blood calls (bound antibodies )are washed free from serum and the
unbound antibodies (to be leaving the bound ones ).
*- (2) Antiglobulin serum is added directly to this call suspension
*- The direct coomb's test is of value in the detection of antibodies to
R.B.Cs associated with hemolytic disease of new born (e.g.
erythroblastosis fetalis ) and auto immune anemia or disease .
*- The Abs associated with these diseases have the ability to attach to but
not agglutinate the target R.B.Cs .
*- These absorbed Abs can be detected by the use of Ab (i.e. coomb's Kit
serum ) to This human gamma globulin .
(2) indirect coomb's test :- *- It is used to detect the presence of Circulating Antibodies.
*- It is of value in detecting IgG - associated antibodies in the serum of
woman who is though to be (a) sensitized to Rh antigen And
(b) at risk for carrying an erythoblastbotic febus .
Technique :-
*(1)- Serum sample (containing Ab ) is incubated with donor R.B.Cs( contain
Rh antigen).
*- (2) Then ,the cells R.B.Cs are washed off ( to remove excess Ab ) .
)(3) The anti globulin (coomb reagent) reagent is added ( kit -*
Faetus like blood
Serum
serum
Anti globulin commb reagent
Ab is absorbed haemagglutination ( + ve ).
No Ab .(No absorption ) haemagglutination ( - ve ).
Viral Neutralization :-
It is very similar to haemagglutination Inhibition on ( i.e. it is a
neutralization event ) .
*- Principle :-
The assays is based on the ability of specific Abs to interfere with
Some biological function of the virus under consideration ( usually
The infective property is blacked ) .
( 1 )- Cyotpathic effect ( CPE ) :-- certain virus + cells ( in tissue culture ) cell destruction .
2 )- the CPE is useful in the search for virus neutralizing Abs in serum sample.
-Technique :-*
**_ serum suspected of containing Ab is added to a virus suspension .
**_ Susceptible cell culture is inoculated with the mixture .
If the culture fail to develop
CPE (no killing ) ( + ve )
CPE developed (killing )
no Neutralizations
( culture Cell death ) ( - ve )
– Radio immune assay (RIA ):- * - It is and extremely sensitive method for quantization
of any substance that is Immunogenic or heptenic and can be
labeled with radioactive isotope e .g. I (25I) .
Liquid phase RIA :- It depends on the competition between labeled (Known ) and
Unlabeled ( unknown )antigen for the same antibody .
Abs are interfere with the
Ability of the virus to kill
Tissue culture cells
Unlabled Ag[unknown amount] Labeled Ag (known amount)
Ab specific known amount
The radioactivity of
either
Is then determined
( i ) Separation of this complex by immunological method by
secondary Ab or by Precipitating agent . [ (NH4)(SO4)]
ii) Separation of this complex by physiological methods
(a) centrifugation
(b) Decantation .
PPT (commonly used)
The supernatant .
A Calibration curve based on using serial dilution of known
unlabeled standard ( instead of the serum ) is used for
calculating of un known samples
CP
M
conc.
Immune reaction product
(Immune complex)
*- Solid phase RIA :- Liquid phase is modified by :-
( i ) adsorption or covalently linkage of Ab to solid matrix ( solid phase
RIA) .
( ii ) The unlabeled Ag (sample ) is added followed by the labeled one
( Antigen or Antibody ) .
Then
The bound versus free Ag can be determined by
Using reference calibration curve ( as before) [ washing steps]
Enzyme linked Immune Sorbet Assays { ELISA } :-
*- ELISA is both highly sensitive ( > 99% ) and specific ( > 99% in
high – risk populations ) .
*- It can be for the assay of either Ag(s) or Ab(s) .
*- Ag or Ab can be attached to solid phase support ( plastic
surfaces , paper disks ) and still retains its immunologic activity. *- Either Ag or Ab can linked with an enzyme e.g. ( horse
reddish peroxidease alkaline phosphates ) .
*- substrate is added and the color absorbed by the enzymatic
procluct is then quantization and compared with a calibration
curve .
Example :- detection of Ab(s) to the human HIV :- *- The virus is grown in vitro in a human T-cell culture .
*- purified whole virus is disrupted 8 viral proteins are immobilized
onto plastic beads or multi well trays .
*- Abs to any of these antigens will bind with them & immobilized .
*- Excess proteins are removed by washing the beads ( or wells ) and
an enzyme linked anti human gamma globulin antibody is added .
*- The presence of this second Ab can be detected calorimetrically
by adding a substrate for the enzyme that will yield a colored end
product .
*- The rate of substrate degradation is determined by the amount of
enzyme – labeled Ab that is bound which is proportional with the
amount of Ag in the solution being tested.
*- the color change can be measured quantitatively in a
spectrophotometer .
* Double Antibody sandwich ELISA :- It is used for the assay of Ag ( e.g. HBSAg ) uses tow Abs as
below :-
( i ) first Ab ( specific e.g. HBs Ag ) is coated on a plastic surface
( poly styrene), the solution being tested for HBs Ag is then
applied to the surface .
( ii ) Washing of any un reacted material .
( iii ) The second Ab ( ie enzyme linked anti HBs Ag specific Ab is
then applied .
( iv ) Any excess conjugate is rewove by washing .
(v) finally substrate is added to the detest the present of En2
ABO group & Transfusion Reactions
*- ALL human erythrocytes contain all antigens ( i.e. Antigens that vary
among individual members of a species ) of the ABO group .
*- This is important system , which . is the basis for blood typing &
transfusions .
*- The A & B antigens are carbohydrates that differ by a single sugar .
*- Despite this small difference , A & B antigens do not cross – react
*- R.B.Cs have 3 terminal sugars ( in common ) on there are surface..
**-- Type A cells have an additional N –acetylgalatose .
**-- Type B cells have an additional galactose.
N.B . Type A & B genes code for transferaes that add
the respective Sugar .
*-* Type O have only the H antigen :
To avoid Ag – Ab reactions that would result in transfusion, all blood
for transfusion must be carefully cross matched .
*-* So , Ag the corresponding Ab do not coexist in the some person's
blood .
*-* Transfusion reactions result when incompatible donor's R.B.Cs
are transfused e . g . group A group B .
N – acetyl glucose amine
Galactose Fucose H antigen
In to
Ag – Ab – Reaction Involving R . B . Cs antigens :-
ABO blood group . Structure of the terminal sugars that
Determine ABO blood groups .
4 POSSIBILITES
OF
CONBINATION
Antibody in plasma Antigen on R.B.Cs Group
Anti B A A
Anti A B B
No Anti A NOR Anti B A & B AB
Anti A & Anti B No A nor B O
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