Immunodiagnosis
Antigen antibody reactions
-Antigen antibody reactions in vitro are known as serological
reactions.
-They can be used for detection and quantification of either antigens
or antibodies(Diagnosis of infectious diseases, autoimmune
diseases, determination of blood groups).
- Antigens & antibodies combine with each other specifically and in
observable manner.
- Measurement of antigen and antibody is in terms of
*mass (example mg)
*Units or titre.
-The antibody titre is the highest dilution of the serum which shows
an observable reaction with the antigen in a particular test.
- Antigens also can be titrated against sera.
Antigen antibody reactions
(serological tests)
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Agglutination reactions
Precipitation reactions
Toxin-antitoxin neutralization test
Virus neutralization test
Complement fixation test
Immuno fluorescence test
Radioimmuno assay
Enzyme- linked immunosorbent assay(ELISA
Immunoelectroblot eg., westren blot
Immunochromatographic test.
Agglutination reactions
The antigen is in form of particles(Suspension
suspended antigen)e.g. RBCs, bacteria, latex
particles coated with antigens.
• When a particulate antigen is mixed with its
specific antibody the particles are clumped or
agglutinated.
• If one of the reactants(Ag or Ab) is known, the
reaction can be used for identification of the other.
Types of agglutination reactions
I- Direct agglutination:
A-Slide agglutination test:
On a clean slide, place a suspension of unknown
culture of an organism, then add to it a drop of
known antiserum(AB), and mix well→
clumping(Agglutination) →Positive
→No clumping → Negative
This test is commonly used in blood grouping.
negative
positive
B-Tube agglutination test
- Is a quantitative test, used to determine the amount of antibodies
in the patient serum (titer).
- Done by mixing serial dilutions of the patient serum (1/20, 1/40,
1/80, etc.) with fixed amount of a known bacterial suspension,
suspected to be the cause of the disease. The tubes are incubated over
night, then examined for presence of clumping at the bottom of the
tubes with clear supernatant fluid
- The last tube showing visible agglutination will reflect the serum
antibody titer.
-The antibody titre is the highest dilution of the serum which shows an
observable agglutination reaction with the antigen.
NB: Paired serum samples separated by 1-2 weeks are recommended
in serodiagnosis. A rising titre of 2 folds or more is diagnostic.
e.g. Widal test in diagnosis of enteric fever, in diagnosis of Malta fever, ………
2- Antiglobulin agglutination test
(Coomb's test)
- Used to detect Rh incompatibility which causes erythroblastosis
fetalis.
- Also used to detect incomplete Abs in autoimmune hemolytic
anemia.
- Most anti-RH antibodies are IgG incomplete antibodies which
can only coat the Rh positive RBCs, but can not cause agglutination.
1- Indirect Comb's test: detects anti-Rh Abs or autoantibodies in the
serum. Mother’s serum is mixed with washed Rh+ve RBCs blood
group (O)→incubate for 1-2 hr→ Centrifuge→ wash the deposit
→add antihumanglobulon →again incubate →Agglutination(+Ve).
2- Direct Comb's test: detects incomplete Abs coating the RBCs of
The newborn. The antihuman globulon is added directly to a washed
suspension of newborn RBCS.
3- Passive agglutination test
Agglutination reaction in which inert particles
as latex or RBCs are coated with various Ags
or Abs. These particles are aggregated in the
presence of specific Abs or Ags respectively.
e.g.
1- Pregnancy test.
2- Rheumatoid factor (RF).
3- C-reactive protein.
4- ASOT
4- Coagglutination(CoA)
This test is based on that, Staph.aureus is rich in
protein A. Staph.aureus is coated with Abs and
mixed with unknown Ag →Agglutination(+Ve).
5- Virus haemagglutination inhibition(HAI)
Serum(?Abs) + known Ag(Virus)→incubate for
1 hr, then add washed RBCs→ Incubate
for ¼ hr +ve =No HA(No agglutination for RBCs)
-ve =HA(agglutination for RBCs)
5- Virus haemagglutination and HA
inhibition(HAI)
Prozone & zone of equivalence & zone of
antigen excess (postzone)
• Precipitation will occur more rapidly and
abundantly when antigens & antibodies are
present in optimal or equivalent proportions.
• When antibody excess or antigen excess the
precipitation is absent or weak.
• Prozone – antibody excess, many antibodies coat
all antigen sites- results in false negative
• Postzone – antigen excess, antibody coats
antigen but cannot get lattice formation, results
in false negative
Precipitation
- The antigen is soluble in solution, Ag-Ab reaction results in
formation of a precipitate.
- Methods of PPT reactions:
A-Tube precipitation test(Ring test):
this simplest type of precipitation test consists of layering of
antiserum in narrow tube (preferably capillary tube). A
precipitate forms at junction of the two liquids.
B- PPT in agar gel (Immunodiffusion):
Diffusion of Ag and Ab is allowed to occur in agar gel.
1- Double diffusion (Ouchterlony):
Wells are punched in agar.
- The Ag is placed in one well and the Ab in another. Both
will diffuse towards each other and where they meet at
optimal proportions, PPT bands (Lines)will form.
- 3 types of lines:a) line of identity,b) line of non-identity, c)
line of partial identity
2-Radial (Single)immunodiffusion
• The antibody is incorporated in the agar gel and the
antigen is placed in a well cut in the agar.
• The antigen reacts with the antibody to give
precipitate ring around the well.
• The diameter of the ring is proportional to the
concentration of each antigen
• Estimations can be done by comparing with known
concentration of antigen.
Uses of radial immunodiffusion
• Estimation of plasma proteins
• Immunoglobulins (IgG, IgM,)
• Complement proteins.etc.,
3- Precipitation in agar with an electric field:
A- Immunoelectrophoresis:
- A serum sample is placed in a well in agar on a slide.
- An electric current is passed through the agar, the serum
proteins will move in the electric field according to their
molecular weight and charge.
- A trough is cut into the agar and filled with antiserum
antibody.
- As the Ags and Abs diffuse towards each other, PPT are
will be formed.
- This permits serum proteins to be characterized in terms
of presence or absence or unusual patterns as in human
myloma proteins.
B- Western Blot:
- Antigen, or mixture of antigens is first separated
electrophoretically in gel.
- The separated materials are then blotted (transferred)
onto nitrocellulose sheet, to which antigen bind
strongly.
- Antibody is then applied and will bind to its specific
antigen.
- To visualize the result of test, the antibody is labeled
with enzyme or radioactive substance (direct test), or a
labeled anti-immunoglobulin is added.
e.g. WB test used to confirm diagnosis of HIV infection.
Complement fixation test(CFT)
Serum(? Abs)+ known Ag+C →Incubate for 1 hour,
then add indicator system(Sheep RBCs+Ani-sheep
RBCs).→Incubate for 1 hour
+ve = No lysis
-ve = lysis
Uses:
Diagnosis of:
Syphilis, Viral
Infections,
Gonorrhoea,….
Immunofluoresence
1-Direct: For detection of Ag
Specimen(? Ag) fixed on a slide + Fluorescein labeled
Abs.→ Incubate for 1 hour then wash and examine
under flu.microscope
+ve = green fluoresence
-ve = absence of fluoresence
2-Indirect: For detection of Abs
Serum(? Abs)+Known Ag,→ incubate for 1 hr, and then
wash. Add specific labeled anti-human γ
globulin,→ incubate for 1 hr, and then wash. Examine
under flu.microscope.
Uses: Syphilis, viral infection,………..
Virus neutralization test
Serum(? Abs) + Ag(virus)→incubate for 1 hr. →
Inoculate this mixture to tissue culture→
Incubate for several days.
+ve = No CPE
-ve = Presence of CPE
Enzyme linked immunosorbent assay(ELISA)
1- Double Ab technique (sandwich technique) :For detection of Ag
Known Ab is fixed to the solid phase +
Specimen(? Ag),→incubate for 1 hr and then
wash. Add specific labelled Abs,→incubate for
1 hr and then wash. Add substrate , →measure the
colour +ve = Colour
-ve = No colour
2- Indirect: For detection of Abs:
Serum(?Abs)+known Ag fixed to the solid
phase, → incubate for 1 hr and then wash. Add labelled
anti-human γ- globulin,→incubate for 1 hr and then wash.
Add substrate →measure the colour
+ve = Colour
-ve = No colour
ELISA
PRINCIPLE OF ELISA
Radioimmunoassay
• In radioimmunoassay instead of fluorescent
dyes radioactive material is used
• Radioactivity is measured after the reaction
in gamma counter.
• Used for detection of thyroid hormones T3 &
T4 & HBs Ag.
Toxin-antitoxin neutralization test
Toxin + Serum(Antitoxin)→ ½ hr →RBCs
suspension(Blood group O) → ½ hr
Result: No hemolysis →+ve
Hemolysis → -Ve
e.g.Antistreptolysin O titre
(ASOT) used for diagnosis of
rheumatic fever
Immunochromatographic test(IC)
• It is produced in card with immunological
reagents(either Ag or Ab) fixed on the card
membrane.The specimen to be tested is applied
to the well(S)
• Result:
• 2 pink bands (C & T) →+Ve
• 1 pink band(Only in c) →-Ve
• Uses: For HIV, HBV, HCV,
Pregnancy,H.pylori, AFP…..