2. Immunity (resistance): It the sum of all naturally occurring
defense mechanisms that protect human from infectious disease Non
specific Specific Naturally acquired ( Innate ) ( Acquired )-
Mucous membranes- Phagocytic cells - Placental transfer of
antibodies( Passive )- Enzymes in secretion - Recovery from disease
( Active )-Interferons ( ,,) - - Administration of antitoxin (
Passive )- NKCs- - Vaccinations ( Active )-Skin-. Macrophages-
Artificially acquired
3. Naturally acquired Artificially acquiredactive passive
active passiveFirst: Non specific Immunity ( Innate):- This is a
physiologic mechanism which is inherent or innate with the -
following properties A single mechanisms Do not depends on specific
Protect It does not exhibit specificity recognition of a foreign
Against material many paths
4. Natural ( Innate ) Specific ( Adaptive )-Less specific . or-
Skin & mucous membrane . (Acquired)-NK cells .- Complement
cascade .- Phagocytosis .- C- reactive protein . Active
Passive-Induced by contact with foreign antigens .- - Induced by
antibody performed in -- Consist of clinical infection ,
immunization with live or - another host- killed infectious agents
or their toxins . - - Ab injected in the incubation period -- Long
term. - - Short term .
7. * B- Lymphocytes 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 ) .* Upon exposure to antigen , B-Lymphocytes are
stimulated to proliferate ,(large lymphocytes) differentiate and
mature into LARGE PLASMA CELLS * The large mature B-Lymphocytes
have short life span ( days to weeks ) . Secreted Immunoglobulins
or Humoral antibodies L-CHAIN Antibody H-CHAIN
8. * Some large mature B-Lymphocytes (B- cells ) can be
converted into small B- cells which have long life span This type
of cells isSecondary involved in the Immune Memory cells And serve
asResponseActivation & differentiation of B-Lymphocytes , in
certain instances , needs a Helper T- Lymphocytes activity to
enhance the above toprocesses in that B-Lymphocytes.
9. T-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 defense against malignant
cells graft rejection hyper sensitivity reactions bacteria &
protozoa Fungi viruses
10. T-CELLST-HELPER (TH) T-SUPRESSOR(TS) T-CYTOTOXIC (TCs)
T-DELAYEDT- Helper :Their Surface Antigen : is T4 (CD4) . Helper-
*They Promote Maturation Of Antigen . *Stimulated B and T cells.
Sensitivity And Cells ( TdH) Enhance their responseT suppressor
cells: * Their Surface antigen is T8(CD8). and * they suppress the
effect of T helper cells . T-CELL MEDIATED i.e. *Suppress T &B
response . IMMUNITYT cytotoxrc: * their Surface antigen T8(CD).
(Tcmi ) * they specifically destroy target cells.virus infected
cells unacceptable grafted cells tumor cellsT 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 substanceslymphokines that modulate the behavior of
other cells.
11. *- most antigens which have a small number of epitopes and
require carrier needT cell cooperation with B- cells for antibodies
production . * Deficiency of B cells (andor) T-helper cellsleads to
defective synthesis of antibodies.* its over activity lead to
Autoimmune disorders the majority of B-lymphocytes express both
surface IgM & IgD, very few expresssurface IgG & IgA or IgE
in the circulation.*the majority of B-cells also carry class 2
major histocompatibility complex (class MHC) products which are
functionally important in Regulation of immune response
12. 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
SPECIFIC IMMUNITY complement In direct lyses by C. + +BACTERIA
PHOGOCYTE PHOGOCYTE Opsonization B-Lymph INEFFECTIVE BACTERIA And
Phagocytosis Direct lyses + SERM Bacterial lyses + COMPLEMENT
BACTERIA B-Lymph a Ab b LYSISBACTERIA s Cell + MediatedBACTERIA
PHOGOCYTE bacteria T-Lymph response
13. classification of acquired Immunity:-- passive Acquired
Immunity :-Definition: acquired Immunity by given already form
antibodies or antitoxic serum or gammaglobulins from normal or
convalescent individuals or Trans placental or lactation . Trans
placental . NaturalTypes Lactation (Colostrum). Antitoxin serum
tetanus. (Anti_ cobra venom) Artificial Gamma globulins.-
characters :- * -Rapidly developed . * -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) .
14. Definition :- the body forms his OWN IMMUNITY when
stimulated (sensitized ) by introduction of immunogenic agent.
Natural InfectionTypes *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 Ab Cellular T_Cells
15. Embryo Liver stem cell In Bone marrow central or primary
lymphoid organs (tissues)SecondaryLymphoidOrgansSpleen or +Bone
marrow + A9 A9 T_Cells B_Cells Effector PLASMA Killer cells memory
cells CELLS B T HUMORAL ANTIBODIES
16. Specific memory and self-limitation of Immune response Ag A
Secondary anti A infection response Serum Primary Anti A AB
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
.
17. Specific immune response : It is developed as a result of
exposure to a variety of agents capable of inducing an immune
response ( i.e. immunogens ) vaccines microbes that colonize Macro
molecules in the diet in the bodyA special case Antigen in the form
of hapten 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 .
18. Specific immune response can be further Classified
according to its components intoprimary secondaryInitial exposure
to a particular on farther orInfectious agent or immunogen repeated
exposureInduction phase of lymphocytes to antigen ( same
)proliferation T-CELLS B-CELLS PLASMA CELLS increased resistance
develops Sensitized T-CELLS Antibodies Cellular Immune response
humoral through Humoral Cellular response response
22. VIRUS infection cell MHC Class I T-Cell receptor virus MHC
Killing Class II CD8 IgM CYTOTOXIC T-Cell RECEPTOR B-CELLS CD4 (
T_HELPER ) INTERLEUKIN-4 INTERLEUKIN-5VIRUS ANTIGEN Defense
mechanism against viral infectionVIRUS
23. *- Recognition of phases :- antigen recognition ( binding
of Ag to specificreceptor on mature lymphocyte ( exist prior to ag
exposure )*- activation proliferation & differentiation of
lymphocytes is the sequenceof events induced in lymphocytes as
result of Ag recognition .*- Effectors phases elimination of
antigen [ is the stage of the responseAt which the sensitized cells
perform the function that (eliminate of Ag)Some antigen stimulated
lymphocytes die by process called programmed celldeath ( apaptosis
). Elimination OF AgT OR + AgB Phagocytosis NATIVE complement
LYMPHOCYTES Recognition ACTIVATION Effector programmed cell phase
phase PHASE Death
24. Immunogenicety ability to induce immune
responseAntigenicity ability of the substance to react specifically
withimmune system must be Antigenic Immunogenic are not necessary
to beHappen is incomplete antigen ( di nitro phenol or
penicillin)It cannot stimulate humoral or cellular reactions but
can react with theseproducts specifically so it is Antigenic not
immunogenicIf they reacted with larger carrier protein (e.g.,
albumin , globulin orsynthetic poly peptide ) . It will be
ImmunogenicAnimals injected with this hapten proteinComplex will
make antibodies to this hapten ,Only if it is ( hapten ) covalently
linked tothe carrier (chemically bonded)
25. CARRIER HAPTEN ANTIBODYPRTOCAL PROTEIN i NO YES NO II YES
NO Anti carrier only III YES YES Anti carrier only ( not chemically
linked) IV YES YES Anti carrier (CHEMICALLY LINKED ) & Anti
hapten
26. Immune response :- its characterized by the produuction
ofproteins ( Igs) and specificially reactive lymphocytes (T-cells )
when ananimal encounters aforeign macromolecules or cells . The
inducing substances are called antigens 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 ANTIGENICITYHAPTEN PLUS PROTIEN CARRIER IS
IMMUNOGENAntigens are the aligands that react with the products of
an immuneresponse .
27. Epitope ( - antigenic determinants ) :- are the sites
either (on or) within the antigen with which antibodies or T-cells
receptor reactsparatope :- 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
vivovalency 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 .
28. The valence of A.g increases proportionally with molecular
size .Macro molecules are easily to induce phagocytic ( as example
) and easier to be phagocytosed Quaternary structure are the most
ImmunogenicThe more complexity , the more Immunogenicety
29. .factors Affecting ImmunogenicityForeigness chemical
complexity molecular sizeA foreigness :-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 incases of * peneumococcal polysaccharide . * Lip
polysaccharides in cell membrane of gram ( ve ) bacteria.
30. *- Glycoproteins :-Are immunogenic ex blood group Ags (
A,B,AB,O,RH )*- POLYPEPTIDES & nucleic acids :-Are weak
immunogens*- lipids :- are not antigenic or immunogenicC.molecular
size :- usually the larger the molecule the stronger the
Immunogenicety .M.Wt below 5000 DA ARE NOT IMMUNOGENICMACRO
MOLECULES are the most potent immunogens .( e.g. albumin m.wt
40.000 DaGlobulin m.wt 160 kDaMacrocyanin m.wt 1000 kDa
31. EPITOPE (ANTIGENIC DETEREMNANT):- The portion of Ag that
binds specifically withthe binding site of Ab (paratope) or a
receptor(s) on T_lymphocyteSIZE CONFORMATIONAL STRUCTURE The size
and the structure of epitope are complementary to that of
paratope.i.e. they must have approximately the same dimensionsWITH
RESPECT TO THEIR STRUCTURE ,A g MAY HAVE THE FOLLOWINGCHRACTES :-
Ag may have only a single epitope of a given specificity on its
surface which iscapable 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 calledin the case MULTIVALENT (which
determine the number).If the epitopes are of the same type called
also UNIDETERMINANT and if they are ofdifferent types called
MULTIDETERMINANT (specificity ). UNIVALENT MULTIVALENT MULTIVALENT
UNIDETREMNANT UNIDETREMINANT MULTIDETRMENANT
32. Hapten-carrier conjugates have nativeantigenic determinants
of the carrier aswell as new determinants of the hapten
33. Antigenic determinants are usually limited to those
portions of the antigenthat are accessible to antibodies shownin
black for this iron-containing protein
34. In an antigen, the same antigenic determinant repeated many
times
35. T-dependent antigens are characterized by a few copies of
many different antigenic determinants
36. MULTIVALENT since it has only one kind of determinant but
many of such determinant on each molecule Ex. Many poly saccharides
& homo polymer (e-g peptide chain of the some .A. Acids .)*-
some antigens are multi determinant & valent such molecules
have manyepitopes of different kinds (multi specificity ) but only
one of each kind ( monovalent ) Ex. Most proteins .*- High M.WT ,
chemically complexed compounds or polymerized proteins(quaternary
structure or heteropolymerized proteins are usually .*-Multi
determinant Ag ( multi specific) , multivalent Ag (more than
oneepitope of each kind) )What kind &How many of such kind
(
37. Antibody binding site ( Paratope ). Binding of Ag &
AbAffinity :- the strength of attraction and binding between an
epitope( monovalent ) of an Ag and the antigen combining site of Ab
molecule ( Paratope).Avidity :- The strength with which (
multivalcnt ) Ag bind to itsantibodies ( Abs). ( chemical
complexity ) This depends on the affinities of the individual
combining sites of the determinants on the antigen
38. ANTIBODIES and their STRUCTURESElectrophoretic separation
of serum proteins
39. ANTIBODY STRUCTUR *Classes of antibodies . IgM , IgG , IgE
, IgA & IgD . CH1A = COMPLEMENT BINDING SITE Constant A
Constant A CH2 Heavy chainB = NEUTROPHILS & MACRO- PHAGE
BINDING SITE Hinge bonds Constant B Constant BVARIBLE = ANTIGEN
BINDING SITE . CH3
40. ANTI BODIESPOLYCLONAL ANTI BODIES MONOCLONAL ANTIBODIES-
INDUCED AGAINST WHOLE ANTIGEN . INDUCED AGAINST ONE EPITOPE .- LESS
SPECIFIC (I.E . SMALL PART OF ANTIGEN )- PRESENT IN SERUM - MORE
SPECIFIC . - PRODUCCED BY HYBRIDOMA EPITOPE EPITOPE TECHNOLOGY .
INFECTION POLYCLONAL Ab. MONOCLONAL Ab MAb MAb MAb
42. Immunoglobulins "Humoral antibodiesThey are formed of two
identical units each of them is formed of :- A) heavy chain B)
light chain C) hing regionA) Light chain 2( lambda) but never 1 and
1K K ( Kappa ) 2B) 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.
43. * 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 processeach other
forming paratope . by which the body gets-rid of Ag. .* They have
sub-regions of the variable region (hypervariable) It is
responsible for Designation of Ab class & its These regions
have extreme - distribution. variability in their A .As sequence in
different antibodies - and they are responsible for binding with
Ag(s) -. - (hyper variable ) CDRs}{ comptementary detemining
regions
44. C) Hinge region :- * CH 1, CH 2 , CH 3 : occupies that of
Heavy chains the other 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
theantigen binding site .(ii ) crystallization fragment (FC ):-It
contains the effectors )
45. Structures and function Of Specific Immunoglabulins*- Ig(s)
are glycoproteins 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 ,arecomposed 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 .
46. * Each H chain has 4 or 5 domains :1 domain in the variable
and 3 or 4 in the constant3 IgG ( ), IgA( )&IgD( ) Or 4 Igm (
)&IgE( )Total = 1 Variable + 3 constant or = 4constant +1
Variable 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 ) 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 .itssequence is unique
(sole) for each Ig type and subclass
47. 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 butnot 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
48. The subdivision is based on the greater similarity of A.As
sequence shown bysubclasses of the same classi.e. 1 , 2 , 3 etc,.
Than is shown by different classes (i.e. , , or )*- The heavy chain
subclasses determine immunoglobulin subclassese.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-:
49. i.Interchain disulphide bonds : occurs between 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 JonesThey can be 1:15 depending
disulphide bond protein ) seen inOn the class & subclass types
urine of some patients with multiple myeloma
50. 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 ofdomains 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 its 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
inheritedchanges
51. *- 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 oneH Chain class ( i.e
IgG, IgM , IgD ,IgA and IgE ) .*- Thus during class switching in an
immune response e.g when B cells changetheir production from IgM to
IgG heavy chainonly the constant regions of the H (CH) changes and
the antibody specificityremains the same .HYPER variable regions*-
they are particular areas within the variable regionsThat 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 invariantmolecules
.
52. CDRs :- they are short polypeptide segments lining near A.
As positions 30,50 CDR1 CDR2 CDR3 AND 90 in the variable regions of
both L and H chains . FR1 FR2 FR3 FR4 variabilityFRs CDRS
89-97variable region 24-34 50-56 NO OF AgS 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 ) .
54. Immunoglobulin are glycoproteins :- (3-13 % of their M.WT )
OLIGOSACCHARIGES + PROTEIN*- THESE oligosaccharides are present in
CH2 or CH3 .*- N -glycosidic bonds usually link N-
acetylglucosamine in the carbohydrate moietyto asparagine residue
in the peptide c-chain of Ab[ linkage with the enzyme N
-acetylglucosamine .- Asparagine transglycosylase ] transferase*- t
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 acidis bind .*- when Abs have the sialic
acid removed by the enzyme neuraminidase , theybecome susceptible
to degradation in the liver .*- in this case the terminal galactase
bind to a receptor on hepatocytes and theentire molecule is , then
, interenalized to the cell for degradation viaProteolytic enzymes
in lysosomes of the cells .
55. Restriction enzymes digestion of Abs :1) Papain : digest
above hinge region so it leaves 2 Fab fragments each is monovalent
S-SAnd crystalline fragment (FC) papain Fab FC Monovalent 2)Pepsin:
digest away most of FCFragments below the Interchain disulphide
bond(below the hinge region) it give one large fragmentsF(AB)2
which is consist of two Fabfragments joined by the disulphide
bondThus , it is bivalent ,possessing the ability to bind and form
agglutination S-S F(ab)2 FC Ab
57. Classes of antibodiesThey are 5 isotypesThe class of Ab
depends on the A.A: sequenceof the constant regions of the heavy
chain . IgM*- 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.
58. *- 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 tohypersensitivity .* 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 ,andmaking them more phagocytosed .
60. Laboratory Methods SerologyIn vitro Ag & Ab reactions
called serologyIt provide methods for i) Identification
(Diagnosing) ( ii ) quantization of titre of Ab (and or) AgTitre :
or the level of Ab (s) in the serum can be measured by using known
AgThe titre may have diagnostic or prognosticEx. A rise in Ab titre
between acute &convalescent serum can be used as adiagnostic
tool for a specific diseaseThe titre is defined as the greatest
dilution of serum (which contain the Ab underconsideration ) that
reacts which the antigen ( i.e. gives +ve result ) .
61. - 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
ina) very acidic .e.g. 0.01Mb) 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 )
62. various forces act to hold the Ag-Ab complex together :- *
The maximum attractive forces stabilizing Ag-Ab complexesAre 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 onlyWhen the two molecules have
complementary shapes (a) puratope 2 puratope 2 Epitope 2 (a) (i)
significant changes Epitope 2a In the shape of epitope 2 (b) Into
2a
63. these change precludes its ( 2a ) interactions with the
matching binding site of theoriginal 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 NH3 COO NH3+ COO
+NH3Affinity :- the strength of attraction between a single epitope
and its matchingparatope is the referred to as the affinity of the
reaction between the two reactants . Ag-Ab complex of low affinity
dissociate readilyAvidity :-* It is a related term to affinity* It
refers to the strength of the interactions between multivalent
antigens and thepopulation of Abs that they have included .
64. *- 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 themex. 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(b) injecting ( .e.g. a pig)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 on the A & B
chains are not accessible .. epitopes 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
65. *- The physical state of the antigen is responsible for the
identification of Ag Abreactions 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 :- a b Serve to detect and quantities Agglutinins and
identify cellular Ags Bacterial cell white blood cells red blood
cells .**-- when the cells intact with the appropriate Ab , they
clump together and eventually Large enough to be visible form
masses with naked eye
66. *- 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.
67. B- lyses Reactions :- 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 hosts
defense against condition such as microbial infection or cancer (
graft cell , virus infected cells , etc.)*i)- Haemolysis :- In
which the Hemoglobin is released from R.B.C, is a requisite
phenomenon for the complement fixation test .*ii)-- bacteriolysis
:- cells of gram ( ve) bacteria are undergoes immune lyses under
certaincondition .*iii)-- cytolysis :- involves the destruction of
other cells types (e.g. lymphocytes ). C- precipitation:- * occurs
when the Ag is soluble instead of cellular *therefore a large
number of molecules are required for lattice formation and alarge
no .of lattice must be formed for an aggregate to be formed and
visibly seen.
68. *when soluble Ag (s) come intact with specific Ab. They
aggregate (i.e precipitate ) Three conditions are presentA- where
the (Ag) is very low with excess Ab (zone of Ab excess ), Formation
of complex occurs But Residual Ab remains in the supernatantB- As
more Ag is added , large aggregate is formed In the (zone of
equivalence) ,maximal Ag-Ab complex are formed and precipitatedC-
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
+2AgNote:- the soluble Immune complex are not processed efficiently
by the reticuloendothelial system ,and ,this cause damage
(how??)
69. Amount of precipitate Zone Of equivalence Effects of
increasing amounts of Ag on the total immune precipitate obtained
from a mixture of soluble Ag and its homologous AbINDIRECT 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 pregnancyA state of Ag excess a state of equivalence
will be reachedTherefore, no precipitation therefore, precipitation
occurDirect 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 )
70. Hyaluronic acid (HA) assay using excessHA 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 separatedand quantities by radio- immune assay technique ,in
case of no precipitation, thesample is negativeImmune 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 ofprecipitate will be formed .* The
reason of precipitation , is the diffusion of the components (Ag
& Ab ) towardseach other .* A useful example is a double immune
diffusion technique :-
71. Procedure :-* Ag& Ab preparations are placed in
separate wells that are cut into a thin layer ofagar in a Petri
dish .* The reactants diffuse towards each other through the agar
until they meet anoptimal proportions [ zone of equivalence ] and
forms ( ppt ) bands Solid Chevron Fig (a) Ag PPT Ag Zone of Ab
equivalenceThe advantages of the procedure is that antigenic
relationshipcan be detected by the precipitation pattern (s)
72. 3 basic patterns are given :(a)- in reaction of identity ,
the 2 Ags are similar , they will diffuse at the same rateand the
two precipitations bonds merges into a solid chevron ( fig b) Aga
Aga Fig b Ab2- in reactions of non-identity , the two Ags are
completely different and the linesof the precipitate cross (fig c)
Ayab Agac Aga Agb Fig c Aba Abb Aba Abb3- reaction of partial
identity :-* It is indicated by spur formation indicating that one
of theAg(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-reactingAg (Ag ac) but migrate past that Ag (Ag ac )
until it reaches an Ag (Ag ab ) that
73. 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 aconcentric ring or halo precipitate .* The diameter of
the halo of precipitate directly correlate with the [ IgG] in
thesample .Thus , the levels of IgG in the sample can be determined
by referring a standardcurve 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 .
74. In this procedure : (a) Ag is placed in wells in agar on a
glass slide and then , subjected toelectrophoresis through
application of an electric current . (b) Under these conditions ,
the individual Ags or antigenic components ( inthe 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 pathof
migration , the reactants will diffuse towards one another and form
separate arcsof precipitate for each antigenic determinantD-
counter-immune ectrophoresis (CIE)This technique Is the double
diffusion method + an electric currentWhich 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 .
75. 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 migratetowards the anode , whereas the Ab [
which has no sufficient net charge ] migrate inthe 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
neutralizethe 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 anexperimental animal
( in vivo ) .- The animal will be protected against the introduced
toxin , and thus , its deleteriousaffects disappear because of
antitoxin is present .
76. Clinical example :- ( the virus haemagglutinate R.BCs)* To
examine the serum of a patient suspected of having influenza ,*1)
The patient serum is mixed with known influenza2)add red blood
cellsi- 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 tohaemagglutinate the
R.B.Csii- if no Ab is present , haemagglutinate will occur . virus
+ R.B.Cs haemagglutination occur .i.e the sample is
negativeE-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
77. VDRL TEST FOR SyphilisThe venereal disease research
laboratory (VDRL) test is a slide flocculation testused 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 ofcholesterol particles
that were added to the system .* These form visible aggregate
indicate to the presence of Ab ( reagin ) in the serumof patient
for syphilis[ reagin is Ab type which flocculate (or ppt) an Ag
that is neither cellular nor solublebut it is insoluble ]* The test
can be performed on a glass slide .
78. 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 COMPLEX
SEROLOGICAL PROCEDURESAg-Ab reactions in which the visible
manifestation requires Participation of: a) Accessory factors b)
Indicators system c) Specialized equipment
79. A- fluorescent dyes :-e.g fluorescein isothiocyanate (
FITC)* FITC can be conjugated to Ab. Molecules to visualize of the
molecule under (uv) or (b)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
or 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 thespecimen when it examined under the fluorescence
microscope by using uv light
80. Examples :- Identification of Trepenoma palladium (
syphilis ) in an extracted from a patientsuspected 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 groundthis methods
can be extended for other pathogens .
81. (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 fluorescenttechnique:-*-
The specimen can be stained with conventional Histochemical dyesSo
structural details can be seen ( noted )*- the tissue con be
examined by standard light microscope .(iii)- Indirect immune
fluoresce technique:-*- The procedure use Ab ( secondary ) (
against ) another Ab ( primary ) of patient .
82. *- the primary Ab is the patient serum detection of Abs.*-
the secondary Ab is covalently conjugated whit fluorescent compound
(FITC)*-*- ex. Of secondary Ab is rabbit antihuman( INJECTED Ab in
host is FROMHUMAN ) gamma globulin anti sera . .i.e Produced
against Ab used to immunize rabbit and that Abwhich 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 Abonly .*-(v) the Ag-Ab complex formed is them treated with
the fluorescein tagged Ab tohuman gamma globulin, the excess Ab is
washed carefully.*-If the patients serum contains Ab (+ v e)
against the T. pallidum, fluoresceinorganism will be seen when the
slide is examined with fluorescence microscope
83. FITC **-- Ab fluorescence conjugate is Patients binded .
Slide coated with ag in Serum is Microscope (uv ) is used (excess)
added- 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 Rat kidney section Human Buffy coat calls
beef thymus Lymphoid Thymus Lymphoid organs
84. Haem agglutination Inhibition teat :- It involves The
agglutination of R.B.cs by Or (haemagglutinin(s)) Certain virus
other Ab(s) particles (influenza) substancesIt 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
85. 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 serum Ag which Abs from kit whichPrevent haemagglutination
Capable of haemagglutination haemagglutination haemagglutination
takesplace Sample is negative Inhibition Sample is positive
86. - passive agglutination:- in the conversion of a reaction
system from one thatprecipitate one that agglutinate Thus yields a
more sensitive indication of the presence of antibodies .Example
:RHEUMATOID ARTHRITIS- 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 IgGTechnique :-*-(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 )The
detectable antibody is called rheumatoid factor
87. Bis-diazotized diphenyl :- it is a coupling reagent that
can be used to proteinsconjugate : or to R.B.Cs Haptens and
thusPassive haemagglutination Occur.Thus :*- Addition of serum
containing Antibodies to these substances (proteins orhaptens )
allow the detection of these specific antibodies to these substance
by atechnique called Passive Haemagglutinationrose waaler Test :
which detect rheumatoid factor in serum of the patients suspected
tohave an anti-IgG auto-antibody .i.e. .(IgM to an accumulated
IgGTannic acid treated a sheep R.B.Cs (S R.B.Cs) are coated with
rabbit IgG -Antibodies specific for these S. R.B.Cs
88. fromTannic acid the kit coated by IgG serum from a patient
suspected R.B.Cs to have autoantibodies haemagglutination the
sample is positive IgM
89. Coombs (antiglobulin) Test :-* In certain people .Abs
directed against antigenic determinants (e.g. R.B.Csantigens )are
able to form visible aggregates when subjected to : 1-
precipitation 2- Agglutination*-To demonstrate the presence of Abs
in such cases the coombs (antiglobulin)testmay be used .*- The test
involves the addition of Ab direction against gamma globulin :
whichprovides a bridge between two antibodies coated call or
particleThus ,The major use of the coombs test is to detect the Non
agglutinating(haemagglutinating )anti-red blood Cs Abs.(1) Direct
coombs test * It is used to detect call bound antibodiesTechnique
:- You must use EDTA BLOOD then centrifugation*-(1) The red blood
calls (bound antibodies )are washed free from serum and theunbound
antibodies (to be leaving the bound ones ). (2) Antiglobulin serum
is added directly to this call suspension -*
90. *- The direct coombs test is of value in the detection of
antibodies toR.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 butnot agglutinate the target R.B.Cs .*- These absorbed
Abs can be detected by the use of Ab (i.e. coombs Kitserum ) to
This human gamma globulin .
91. (2) indirect coombs test :- *- It is used to detect the
presence of Circulating Antibodies.*- It is of value in detecting
IgG - associated antibodies in the serum ofwoman 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( containRh antigen). Faetus
like blood*- (2) Then ,the cells R.B.Cs are washed off ( to remove
excess Ab ) .*- (3) The anti globulin (coomb reagent) reagent is
added ( kit)
92. Serum Ab is absorbed haemagglutination ( + ve ). serum No
Ab .(No absorption ) haemagglutination ( - ve ). Anti globulin
commb reagent 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.
93. *-Technique:- **_ serum suspected of containing Ab is added
to a virus suspension . **_ Susceptible cell culture is inoculated
with the mixture . CPE developed (killing ) If the culture fail to
develop no Neutralizations CPE (no killing ) ( + ve ) ( culture
Cell death ) ( - ve ) Abs are interfere with the Ability of the
virus to kill Tissue culture cells 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 .
94. Labeled Ag (known amount) Unlabled Ag[unknown amount] Ab
specific known amount Immune reaction product (Immune complex) ( 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 radioactivity of either . The
supernatantIs then determined CPMA Calibration curve based on using
serial dilution of known unlabeled standard ( instead of the serum
) is used for calculating of un known samples conc.
95. *- 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]
96. 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 .
97. *- 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 .
98. *- Despite this small difference , A & B antigens do
not cross react*- R.B.Cs have 3 terminal sugars ( in common ) on
there are surface.. N acetyl glucose amine Galactose Fucose H
antigen**-- 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 persons blood .*-* Transfusion reactions result when
incompatible donors R.B.Cs are transfused e . g . group A In to
group B .
99. Ag Ab Reaction Involving R . B . Cs antigens :-ABO blood
group . Structure of the terminal sugars that Determine ABO blood
groups .
100. Group Antigen on R.B.Cs Antibody in plasma A A Anti B B B
Anti A AB A&B No Anti A NOR Anti B O No A nor B Anti A &
Anti B 4 POSSIBILITES OF CONBINATION
