DIAGNOSTIC IMMUNOLOGY

1
DIAGNOSTIC IMMUNOLOGY

• Humoral immunity ? in vivo antigen-antibody
  reactions
• Diagnostic immunology ? in vitro
  antigen-antibody reactions
• Antigen-antibody reactions can be used to
  diagnose disease ? diagnostic immunology
• Assay for antigens or antibodies in serum ?
  serology

2
Antigen-Antibody Interactions

• - comformation of a haptan is more important than
  its
• chemical compond in immunization
• aminobanzen sulfunate gt aminobanzen arsenate gt
  aminobanzen carboccilate
• - a bimolecular association
• involving various noncovalent interactions
• Is similar to an enzyme-substrate interactions,
  
• but not lead to an irreversible chemical
  alteration

3
1/d2
Phe,Leu,Val
50
1/d7
4
Antigen-Antibody Interactions

• PH (2/2-8/6 7/2)
• Ionic strengh
• Temprature
• Incubation Time
• Mixing
• Ag-Ab concentration
• Affinity

5
(No Transcript)
6

• High affinity complexes have high Ka values
• Very stable complexes have very low values of Kd

7
(No Transcript)
8
(No Transcript)
9
(No Transcript)
10
(No Transcript)
11
Antigen-Antibody Interactions

• Three Type according to Ag (size, quantity,
  valency, MW) and nature of Ab
• Type 1not visible
• Type 2visible
• Type 3invivo biologic effect
• - Hemolytic Reaction
• - Autoimmune Diseases
• - Hypersensitivity Reactions
• - Cutaneous Tests
• - Glumerolonefritis caused by Immun complexes

12
IMMUNOASSAYS

• Immunoassays measure antigen-antibody reactions
  directly by employing tagged reactants. ?
  extremely sensitive
• Tagging methods
• Radioactive compound radioimmunoassay (RIA)
• Enzyme enzyme immunoassay (EIA)
• Fluorescent compound immunofluorescence assay
  (IF)

13
Radioimmuno Assay

• One of the most sensitive technique for
  measuring hormones, drugs, vitamins
• at conc. Oflt0.001 ?/?. first discovered by Dr.
  Berson Yalow in 1960
• (1977 Novel prize to Yalow)
• - The principle involves competitive binding of
  radiolabeled Ag and unlabeled Ag
• to the limited supply of a high affinity Ab.

14

• ELISA (enzyme-linked immunosorbent assay)
• sensitive, economical and safe
• Enzyme e.g. alkaline phosphatase (p-nitrophenyl
  phosphate ? p-nitrophenol (yellow color))

15
(No Transcript)
16
(No Transcript)
17
-gt precipitates forms a spot only on the areas
of the well where cytokine-secreting cells
had been deposited.
The ELISPOT assay, a modification of the ELISA
assay to determine qucontitatively the of
cells in a population that are producing specific
Ab or cytokine.
18
FLUORESCENT ANTIBODIES

• Fluorescent antibodies antibodies tagged with a
  fluorescent compound
• e.g. fluorescein isothiocyanate (FITC) visible
  green light emitted by irradiation with UV light.
• Direct or indirect fluorescent antibody test

• FITC-tagged antibodies green
• Rhodamine B-tagged antibodies orange

19
(No Transcript)
20
Immunofluorescence
mIgM-producing B cells indirectly stained with
rhodamine-conjurated secondary Ab under a
fluorescence microscope.
21
(No Transcript)
22
Floculation (micro Agg)

• Cloidal Ag
• anti-cardilypin, anti-animal serum
• VDRL and RPR

23
Agglutination Reactions

• Can detect antigens on the surface of cells or
  insoluble.
• Use antigen or antibody attached to a large
  particle, such as a cell or a latex bead.
• Widal test (typhoid fever) patient serum is
  mixed with a suspension of Salmonella typhi.
• Coagglutination antibodies attached to Protein
  A on surface of Staphylococcus aureus cells
• Latex agglutination antibodies attached to
  latex spheres

Indirect Coombs test
24

• Agglutination tests are extremely sensitive
• If agglutination occurs on a slide, the test is
  positive.

• Qualitative agglutination assays

• Hemagglutination reactions for blood typing

25
(No Transcript)
26

• Quantitative agglutination test progressive
  dilutions of a clinical sample until
  agglutination occurs no longer
• Titer the highest dilution of a test serum
  that causes agglutination

Titer is 1160
27
Hemagglutination

• immune (AbRBC)
• 1. active Abnatural RBC Ags
• 2. passive Ab soluble Ag coated to RBC
• very sensitive
• non-immune no Ab
• direct RBC Agg with lectins (PHA) or viruses
  (rubella)

28
Hemagglutination Inhibition

• viruseAb RBC no hemagg
• (Ab presence)

29

• visible clumping by interaction between Ab a
  particulate antigen such as RBC,
• latex beads.
• -depend on the crosslinking of polyvalent
  antigens, similar to precipitation rxns
• (lgM is a good agglutinin)
• -provide a way to type bacteria with a panel of
  typing antisera.
• -routinely performed to type RBCs for blood
  transfusion.

Demonstration of humaglutination using Ab against
sheep red blood cells (SRBCs) a constant of
SRBCs plus serial two-fold dilutions of anti-SRBC
serum
30
(No Transcript)
31
Precipitation Reactions

• Can detect soluble antigens or antibodies
• A visible insoluble precipitate is formed by
  reaction between soluble antigen and antibody
  molecules. ? precipitation reactions
• In optimal proportions of antigen and antibody,
  precipitate is formed. ? Equivalence zone.

32
(No Transcript)
33

• Liquid precipitation test a method for
  determining optimal concentration (the
  concentration of one reactant is fixed and that
  of the other is changed)

34
(No Transcript)
35

• Singel Radial diffusion test (Mancini)
• Can be used to estimate the relative
  concentration of antigen (or antibody).
• Antibody is incorporated into gel and antigens
  are added to wells cut into the gel.
• A ring of precipitate forms
• The diameter of the rings is proportional to the
  concentration of antigen.
• 1, 8 high concentration
• 2,3,9,10 low concentration
• 4,5,6,7 not detected

36
Simple Immunodiffusion Tests

• Gel precipitation test
• On gel antigen and antibody diffuse and react
  each other.
• At optimal concentration, the line of precipitate
  is formed.

37

• Double-diffusion method (Ouchterlony test)
• Can be used to determine if two
• antigens are identical, similar or different.
• Identical one continuous line
• Different two separate lines
• Partial identical a continuous line
• with a spur

38
(No Transcript)
39
Tests That First Separate Antigens
- Its sometimes necessary to separate the
individual antigens in a complex mixture.

• Immunoelectrophoresis
• Antigens are separated by electrophoresis
• Antibody mixture is added to a trough
• Antigens and antibodies diffuse toward one
  another, forming lines of precipitation.

40
Complement Fixation Reactions (vaserman test or
phipher phenomenon)

• Antibody and antigen complex activates complement
  and initiates the formation of MAC.
• Complement fixation assays are quantitative.
• A clinical specimen (antibody) is added to a test
  reagent (antigen)
• Complement is added. If antigen-antibody
  complexes formed, complement binds to them. ?
  Complement fixation
• Indicator system (erythrocytes coated with
  antibodies) is added. Lysis means that there
  wasnt any antibody in serum.

41
Complement Fixation

• Methodology

• Ag mixed with test serum to be assayed for Ab

• Standard amount of complement is added

• Erythrocytes coated with Abs is added

• Amount of erythrocyte lysis is determined

Ag
Ag

42
Indirect Coomb's Test If it is necessary to know
whether a serum sample has antibodies directed
against a particular red blood cell and you want
to be sure that you also detect potential non-
agglutinating antibodies in the sample, an
Indirect Coomb's test is performed. This test is
done by incubating the red blood cells with the
serum sample, washing out any unbound antibodies
and then adding a second anti-immunoglobulin
reagent to cross link the cells.
              Applications These include
detection of anti-rhesus factor (Rh) antibodies.
Antibodies to the Rh factor generally do not
agglutinate red blood cells. Thus, red cells from
Rh children born to Rh- mothers, who have
anti-Rh antibodies, may be coated with these
antibodies. To check for this, a direct Coombs
test is performed. To see if the mother has
anti-Rh antibodies in her serum an Indirect
Coombs test is performed.
43
Coomb's Test (Antiglobulin Test) Direct Coomb's
Test When antibodies bind to erythrocytes, they
do not always result in agglutination. This can
result from the antigen/antibody ratio being in
antigen excess or antibody excess or in some
cases electrical charges on the red blood cells
preventing the effective cross linking of the
cells. These antibodies that bind to but do not
cause agglutination of red blood cells are
sometimes referred to as incomplete antibodies.
In no way is this meant to indicate that the
antibodies are different in their structure,
although this was once thought to be the case.
Rather, it is a functional definition only. In
order to detect the presence of non-agglutinating
antibodies on red blood cells, one simply adds a
second antibody directed against the
immunoglobulin (antibody) coating the red cells.
This anti-immunoglobulin can now cross link the
red blood cells and result in agglutination.
44
(No Transcript)
45
(No Transcript)
46

• Western blots
• Useful for certain diagnoses (e.g. HIV infection)
• The mixture of antigen is separated by
  electrophoresis.
• The antigens are transferred onto a sheet of
  nitrocellulose.
• Labeled antibody is added to the sheet.
• Colored spots are detected.

47
Western blotting
separates the components according to their
molecular weight.
the proteins in the gel are transferred to the
sheet of nitrocellulose or nylon by the
passage of an electric current.
probed with Ab then radiolabeled or
enzyme-linked 2nd Ab.
a position is visualized by means of an ELISA
reaction.
48
(No Transcript)
49
(No Transcript)
50
Flow cytometry Fluorescence
Separation of fluorochrome-labeled cells with the
flow cytometer which uses a laser beam light
detector. different Ag in different cells /
different levels of Ag in the same type of cell
? fluorescence intensity / the size of cells.
51
(No Transcript)
52
Alternalives to Ag-Ab Reactions
53
Immuno EM.

• An immunoelectronmicrograph of the surface of a
  B-cell lymphoma was stained with two antibodies
  (Ab against class II MHC labeled sith 30nm gold
  particles, another Ab against class I MHC w/
  15nm gold particles.
• (The density of class I exceeds that of class II)
• Electron-dense label (ferritin or colloidal
  gold) is conjugated to the Fc
• portion.

54
CLINICAL FOCUS
Distribution of selected markers on some leukemic
cell types (leukemia can wise at any maturational
stage of any one of the hematopoietic lineages) ?
Immuno phenotyping the determination of the
profile of selected cell- surface markers
displayed by the leukemic cell, using flow
cytometry mAb
55
(No Transcript)