﴾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