Immunologists employ a number of techniques to assess the competence of immune system These techniques are common to other biological sciences eg purification of Abs apply the same methods for protein purification genetic basis of immunology have been elucidated by the standard techniques of ID: 774877
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Slide1
Laboratory Techniques in Immunology
Immunologists employ a number of techniques to assess the competence of immune system .
These techniques are common to other biological sciences e.g. purification of Abs apply the same methods for protein purification, genetic basis of immunology have been elucidated by the standard techniques of molecular biology. However, immunology has developed a number of its own techniques, particularly those based on the Ag-Ab interactions, identification and isolation of cell population as well as functional assays for lymphocyte function.
Slide2Indications for laboratory testing for immune competence
Clinical diagnosis, therapeutic monitoring, or prognosis of:
Congenital and acquired immunodeficiency diseases.
Immune reconstitution following bone marrow or other lymphoid tissue grafts.
Immunosuppression induced by drugs, radiation, or other means for transplant rejection, cancer treatment, or autoimmune diseases.
Autoimmune disorders, as possible adjunct to diagnosis or to monitor therapy.
Immunization, to monitor efficacy or immune status.
Clinical or basic research.
Slide3Ag-Ab Interactions
The reaction between Ag and serum Abs(serology) serves as the basis of many immune assays.
Because the immune responses are specific, the interaction between Ag and Ab
in vitro
is widely used for diagnostic purposes for the detection and identification of either Ag or Ab,e.g. serotyping of various microorganisms by the use of specific antisera.
Types of Reactions
Precipitation (if Ag is soluble)
Agglutination (if Ag is particulate)
Activation of Complement
Slide4Reactions depend on the interactions between multivalent Ags and Abs that have at least two combining sites per molecule.
The binding forces between Ag-Ab are relatively weak, they are:
Van der waals forces
Electrostatic forces
Hydrophobic forces
They require close fit between epitope and Ab( like lock and key).
Ag-Ab complexes can be easily dissociated by low or high pH, high salt concentration, or ions which interfere with H-bonding of water molecules e.g. cyanates.
Slide5Affinity
A measure
of the binding constant of a single Ag-combining site with a monovalent antigenic determinant(epitope or
hapten
). Affinity of Abs for Ag is increased with time following immunization.
Avidity
The summation of multiple affinities e.g. when a polyvalent
Ab
binds to a polyvalent Ag.
Titer
The
ability of an
Ab
to cause
Ags
to agglutinate requires an optimal proportion of
Ab
relative to Ag.
Slide6The level of serum
Ab
specific for a particulate Ag is determined sometimes by agglutination assay or by ELISA.
The highest dilution of serum that still causes agglutination or reaction but beyond which no agglutination occurs is termed the
titer
.
Prozone
no agglutination due to
excess Abs
Every epitope on a single particle of Ag may bind only to a single
Ab
molecule preventing cross-linking between different particles. To overcome
prozone
phenomenon, antiserum should be tested at several dilutions.
Slide7Passive agglutination
Agglutination takes place
between Abs and soluble Ag attached to an insoluble particle.
If soluble Ag is present in excess, Abs will not be able to bind with particulate (agglutination inhibition), to overcome this, the soluble Ag should be attached to an insoluble particles.
Slide8Slide9Precipitation Reactions
They take place between Abs and soluble Ag.
Prozone
-------------------No efficient cross linking
Equivalence Zone------Optimal Ag-
Ab
levels
Zone of Ag Excess------No efficient cross linking
Slide10Slide11Hemagglutination
and
Hemagglutination
inhibition
Some viruses have glycoprotein spikes(
hemagglutinins
) that agglutinate different species of RBCs, antibodies against these glycoproteins inhibit the agglutination of RBCs,
(
Hemagglutination
inhibition), presence of such Abs in patient serum indicates is diagnostic of infection.
Slide12Slide13Double Diffusion Method(
ouchterlony
Method)
It is used for establishing the antigenic relationship between various substances.
Three reaction patterns are seen in gel diffusion;
Pattern of identity form when the two
Ags
are identical, precipitin lines formed between
Ab
and two test
Ags
fuse to form arc.
Pattern of nonidentity, the precipitin lines cross each other.
Pattern of partial identity forms when the test antiserum reacts positively with
Ags
that contain epitopes that match and some that do not match, causing a precipitin spur to appear in the gel.
Slide14Slide15Antiglobulin
Tests( Coombs Test)
Ab
or complement are adsorbed onto RBCs are detected by using Abs to human serum globulins(AHG) which are produced either in animals or by
hybridoma
technique.
Direct
antiglobulin
test(DAT) detects
Ab
or complement coating the surface of RBCs.
Indirect
antiglobulin
test(IAT) identifies
Ab
in serum.
DAT
RBCs coated with auto-Abs + anti-
Ig
-----------agglutination of RBCs
DAT is used in investigation of autoimmune or drug-induced hemolytic anemia, hemolytic disease of newborn and suspected transfusion reactions.
Slide16IAT
RBCs uncoated with Abs + serum with Abs to RBCs------
Ab
-coated RBCs +anti-
Ig
---------
Agglutination of RBCs.
IAT is used to identify the presence and specificity of recipient serum
Ab
, select donor blood that is free of specific RBC
Ags
and confirm the absence of Ag-
Ab
reaction by testing recipient serum against donor blood cells(
crossmatch
).
Slide17Slide18Complement fixation Test
This test is used to identify both Ag and
Ab
if one of them is not known.
Procedure includes adding standard amount of complement to a tube containing both
Ag and suspected
Ab
(e.g. serum ), and then the mixture is incubated as instructed.
If Ag and
Ab
are specific to each other, complement will disappear(fixed), on the other hand, if Ag and
Ab
are not specific, complement will remain in the reaction mixture.
RBCs + anti-RBCs Abs are added to the reaction mixture:
.
Slide19If hemolysis takes place, complement fixation test is negative
i.e
Ag and
Ab
are not specific to each other as the complement remains and causes hemolysis.
No hemolysis indicates that both Ag and
Ab
are specific to each other as the complement has been consumed in the first step.
N.B RBCs will not
hemolyze
except in the presence of both anti RBCs Abs and complement.
Slide20Slide21Slide22Slide23Immunofluorescence
Slide24Cellular Assays
Slide25Slide26Flow Cytometry and cell Sorting
Slide27*FITC: Fluorescein
isothiocyanate
*PE:
Phcoerythrin
Fluorescence
Assessment of Lymphocyte Function
B and T cells stimulation in response to mitogen.
Production of
Ab
/cytokines after stimulation.
Functional integrity of a particular subset especially in immunodeficiency diseases.
Mitogens that selectively activate B cells
LPS induce polyclonal activation which is measured using H3 as radiolabel of DNA
Mitogens that selectively activate T cells
Lectins
:
concanavalin
A”con
A”
Phytohemagglutinin
“PHA”
POKEWEED MITOGEN “PWM” activates both B and T cells.
Slide28ELISPOT Assay
Individual B cells producing specific
Ab
or individual T cells secreting particular cytokines may be detected by enzyme linked
immunospot
(ELISPOT) assay
.
T helper function focuses on B cells and macrophage activation.
Cytotoxicity assay of T cells measures the ability of T and NK cells to kill
radiolabled
target cells expressing an Ag to which the cytotoxic cells were synthesized.
ADCC of NK cells could be measured as NK cells express membrane Fc receptors that
Bind to Fc of certain
Ig
isotypes
.
Slide29Slide30B-Cell
Hybridomas
and Monoclonal Antibodies
See Lecture of
Ab
structure and function(
Ig
new
)
Experimental Animal Models
Inbred
Mice,Rats,Guinea
pigs
Selective inbreeding of littermates for more than 20 generations usually leads to the production of an inbred strain.
All members are genetically identical.
They are syngeneic.
Experiments using inbred strains led to the identification of class I and
clas
II MHC genes.
Slide31SCID Mice
Severe combined Immunodeficiency disease(SCID)
B and T
cells fail to develop.
Individuals become compromised with respect to lymphoid defense mechanisms.
In 1980s an inbred strain of mice spontaneously developed an autosomal recessive mutation resulted in SCID in homozygous
scid
/
scid
mice.
Because of the absence of B and T cells, SCID mice are able to accept cells and tissue grafts from other strains of mice or other species.
SCId
mice can be engrafted with human hematopoietic stem cells to create SCID-human
Chimeras.
Slide32Such chimeric mice develop mature functional T and B cells derived from infused human stem cell precursors.
This animal model has become a valuable research tool, since it allows immunologist to manipulate the human immune system
in vivo
and investigate the development of various lymphoid cells.
Slide33Thymectomized
and Congenitally
Athymic
(Nude) Mice
Mice that have been
neonatally
thymectomized
irradiated and then reconstituted with syngeneic bone marrow , such mice fail to develop T cells.
Mice homozygous for a mutation in a gene called nu also fail to develop mature T cells because the mutation results in an
athymic
(and hairless, hence, the term nude) phenotype.
Slide34T cell development can be restored by grafting these mice with
thymic
epithelial tissue.
Like SCID mice, these animal models have been useful in the study of T-cell development.
They have also been useful for the
in vivo
propagation of tumor cell lines due to absence of T cells required for the rejection of foreign cells.
Slide35Transgenic Mice
They are made by injecting a cloned gene(transgene) into fertilized mouse eggs.
The eggs are then microinjected into mice rendered
pseudopregnant
using hormone therapy. However, the success rate is 10-30%.
Transgene is integrated into both somatic and germ cells, it is transmitted to offspring as a
mandelian
trait.
Transgenic mice have been used to study genes that are not usually expressed
in
vivo
e.g. oncogenes as well as effects of trans genes encoding particular
Ig
molecule, MHC class I and MHC class II molecules and
varaiety
of cytokines.
Slide36In some
transegenic
mice, the entire mouse
Ig
locus has been replaced by human
Ig
genes to generate human Abs in mice.
Two disadvantages of transgenic method
Transgene integrates randomly within the genome.
It is
unphysiological
to express high quantities of transgenes in wrong tissues.
Slide37Slide38Knockout Mice
The aim is to study how the removal of a particular gene product affects the immune system using a gene-targeting method.
It is possible to replace a normal gene with one that has been mutated or disrupted to generate the so-called Knockout mice.
Slide39Unlike transgenic mice, Knockout mice express transgenes that integrate at specific endogenous genes through a process known as homologous recombination.
Silence the expression of a variety of important genes, including those encoding particular cytokines and MHC molecules.
Knockout mice have also been used to identify the parts of genes essential for normal gene function.