Transcript IMMUNOLOGIC METHODS шкільна

IMMUNOLOGICAL
METHODS IN DERMATOLOGY
Shkilna M.I.
1.QUALITATIVE AND QUANTITATIVE TESTS FOR
ANTIGENS AND ANTIBODIES
2. Tests for cellular immune function
3. Immunofluorescence-used to detect
antibodies in tissue or fluids
QUALITATIVE AND QUANTITATIVE TESTS
FOR ANTIGENS AND ANTIBODIES
1. Immunodiffusion-detection of antigen-antibody
complex, visible as a precipitate in a medium at
optimal pH and temperature:
a. Single immunodiffusion-either antibody or antigen remains
fixed and the other diffuses through semisolid medium,
either linearly or radially, and is allowed to complex with
the prescribed reactant
b. Double immunodiffusion - both antibody and antigen
diffuse toward each other, either linearly or radially, and
are allowed to complex and precipitate; commonly used
as the Ouchterlony radial immunodiffusion technique (or
test)
Single immunodiffusion
a. Single
immunodiffusioneither antibody or
antigen remains fixed
and the other diffuses
through semisolid
medium, either
linearly or radially, and
is allowed to complex
with the prescribed
reactant
Double immunodiffusion
b. Double
immunodiffusion - both
antibody and antigen
diffuse toward each other,
either linearly or radially,
and are allowed to
complex and precipitate;
commonly used as the
Ouchterlony radial
immunodiffusion
technique (or test)
2. Electrophoresis-protein separation in an
electric field:
a. Zone-proteins separated by surface charge; used to
quantitate serum protein and immunoglobulins.
b. Immunoelectrophoresis-proteins are separated by
diffusion and precipitation of proteins
c. Radioimmunoelectrophoresis-immunoelectrophoresis
combined with radiolabeled antigens
Immunoelectrophoresis
b. Immunoelectrophoresisproteins are separated by
diffusion and precipitation
of proteins
3.
a.
b.
Electroimmunodiffusion-use of an electric
field to rapidly drive reactants toward each
other:
Counterimmunoelectrophoresis (one dimensional double
electroimmunodiffusion) -antigen and antibody rapidly diffuse
in gel medium toward each other, leading to semiquantitative
measurement of antigen in biological fluid; particularly useful
in CSF and serum diagnosis of several diseases.
Rocket electrophoresis (one-dimensional single
electroimmunodiffusionl-antigens are electrophoresed from a
well through gel medium with fixed antibody.
4. Radioimmunoassay-very sensitive and specific
technique to measure small quantities of
hormones, immunoglobulins, etc
5. Agglutination-A very sensitive test of antigen
reaction with insoluble antigen or antigencoated particle which is performed in tubes or
microtiter plates:
a. Direct agglutination-antibody titer determined by
agglutination of serially-diluted unknown quantity of
antibody with fixed quantity of insoluble antigen
(RBC, bacteria, fungi,)
Direct agglutination
a. Direct agglutinationantibody titer
determined by
agglutination of seriallydiluted unknown
quantity of antibody
with fixed quantity of
insoluble antigen (RBC,
bacteria, fungi,)
b. Indirect (passive) agglutination-soluble antigen coupled to RBCs
or inert particles
C. Hemagglutination inhibition-prevent agglutination of antigencoated RBCs by homologous antigen
d. Coombs' test (antiglobulin test)-measures nonagglutinating
antibodies or antibodies too small to effectively agglutinate. Test
is used in blood typing, evaluation of hemolytic diseases of
newborns, and autoimmune hemolytic anemias.
i. Direct Coombs' test detects serum proteins adherent to RBCs
taken from a sensitized
person
ii. Indirect Coombs' test detects incomplete antibodies in serum.
Serum is incubated with
RBCs. The antibody-coated cells are then agglutinated by a
Coombs' antiglobulin serum
Indirect (passive)
agglutination
b. Indirect (passive)
agglutinationsoluble antigen
coupled to RBCs or
inert particles
Hemagglutination inhibition
C. Hemagglutination
inhibition-prevent
agglutination of
antigen-coated RBCs
by homologous
antigen
Indirect Coombs'
ii. Indirect Coombs' test
detects incomplete
antibodies in serum.
Serum is incubated with
RBCs. The antibodycoated cells are then
agglutinated by a
Coombs' antiglobulin
serum
6. Complement assays:
a. Hemolytic assay (CH50)-antibody-mediated hemolysis
of RBCs by complement
b. Functional assays-determines the activity of
individual complement components
c. Immunoassays-antibodies to complement
components and then immunochemical determination
is made
Hemolytic assay (CH50)
a. Hemolytic assay
(CH50)-antibodymediated hemolysis
of RBCs by
complement
Functional assays
b. Functional assaysdetermines the
activity of individual
complement
components
Immunoassays
c. Immunoassaysantibodies to
complement
components and
then
immunochemical
determination is
made
7. Complement fixation-consumption of
complement used to measure
antibodies and antigen.
8.
Monoclonal antibodies-refers to
production of identical antibodies.
9. Latex fixation-latex particles are used as
passive carriers for adsorbed antigen. Test
detects rheumatoid factor (RF), which is a
pentameric IgM directed against IgG. IgG is
passively absorbed to latex particles, and
determinants on IgG then react with IgM
rheumatoid factor.
10.Enzyme-linked immunosorbent assay (ELISA)
Enzyme-linked immunosorbent
assay (ELISA)