13. 2. Tube agglutination
(Quantitative Test)
Detection of Ab against a certain pathogen
= indirect evidence for diagnosis of disease
= serological diagnosis
15. B- PASSIVE AGGLUTINATION
Soluble antigen + antibody → precipitation
Not very sensitive!!
How can we convert:
precipitation
agglutination ??
more sensitive !!!
16. Both Ag & Ab are soluble
One known!
The other not known!
+
↔
Soluble known reactant
is coated onto inert particles e.g. Latex particles
Known reactant becomes particulate (instead of soluble)
Reaction becomes Agglutination (instead of
precipitation)
33. b. Single radial immunodiffusion
Interpretation:
Diameter of ring is proportional
to concentration of Ag in
sample.
1
2
3
Diameter2
Note: Standards (1-4)of known
concentrations are included
in the test to create a curve.
Ab in gel
Ag Concentration
4
34. • Used to quantitate
various immunoglobulin
classes
• Immunogloblin acts as an
Ag.
Standard 3
Standard 2
Pt. 2
Pt. 3
• In well: patient’s serum
(Ag?)
• In gel: anti-IgG, anti-IgM,
……etc
Pt. 1
Standard 1
35. F- FLOCCULATION
Antigen: Small insoluble particulate
Examples:
• Venereal Disease Research Laboratory (VDRL)
test
• Rapid plasma regain (RPR) test
36. VDRL: slide flocculation test for diagnosis of syphilis
Cardiolipin used as antigen instead of Treponema
antigen
Antibody detected is a heterophil Ab called Reagin
Negative
Positive
Microscopical aggregates
37. RPR rapid plasma reagin
Small carbon particles added → easier visibility
(by naked eye)
-ve control
+ve control
+ve Test
-ve control
+ve control
-ve Test
38. H- Complement fixation:
• Test depends on the fact that:
Ag - Ab → complement fixation
• Sensitive
• Used to:
Detect and quantitate antibody
Detect and quantitate antigen (less commonly)
• Many applications in diagnosis of diseases caused by
bacteria, viruses, fungi, etc.
• Famous Test: Wassermann Test for serologic diagnosis of
syphilis
39. I- Viral Neutralization:
Certain viruses cause cytopathogenic (CPE) effect on
certain cell cultures
Uninfected cells
Viral-infected cells, showing
cytopathic effect (CPE).
Fusion of infected cells→
multinucleated giant cells
CPE are inhibited by virus-neutralizing Abs.
40. I. Viral Neutralization (cont.):
First step:
Serum (virus-neutralizing Ab?) + known Virus
Second step:
Cell culture inoculated with mixture:
No Ab (in serum) + Virus → no neutralization → CPE
Ab ( in serum) + Virus
→ neutralization
→ No CPE
44. i) Direct Immunofluorescence
• Detects unknown Ag
e.g.: rabies virus in
brain of dead animal
• Tissue (Ag?)
+ labeled known Ab
→ binding
→ fluorescence
46. ii) Indirect Immunofluorescence
• Detects Ab in serum
→ indirect diagnosis of
disease
e.g. Syphilis
• Known antigen
(Treponema pallidum)
bound to slide
+ serum (Ab against
Treponema?)
• Labeled antihuman
globulin added
→ fluorescence
47. B.Enzyme-Linked Immunosorbent Assay
(ELISA)
•
•
•
•
•
Very sensitive
Very specific
Used to detect Ag or Ab
Label used: Enzyme
Example: Horseraddish peroxidase or alkaline
phosphatase
• Enzyme can be conjugated to Ag or Ab
• Ag-enzyme or Ab-enzyme is called conjugate
• Enzyme acts on colorless substrate
→ colored product
48. • Colour change = +ve test.
• Colour intensity (by spectrophotometer)
is proportional to the amount of Ag or Ab
(quantitative).
• Many variations in test procedure.
53. C. Radioimmunoassay (RIA)
• Same idea as ELlSA but different label:
Radioactive isotope (instead of enzyme)
• Measurement of Degree of radioactivity
(instead of degree of colour change)
• As sensitive as ELISA
• Disadvantage: Hazards of radioactivity
• Applications:
– Measurement of biological substances (Ags)
(e.g. drugs, hormones, tumour markers)
– Measurement of antibodies
55. 1. If you mix bacteria with specific Abs, it
would result in:
a) Cell lysis
b) Agglutination
c) Haemagglutination
d) Precipitation
e) Fluorescence
56. 2. In passive agglutination reaction, soluble
antigens or antibodies coat:
a) Latex particles
b) White blood cells
c) Platelets
d) Carbon particles
e) None of the above
57. 3. Regarding direct Coomb's test:
a) Maternal RBCs are used
b) Group O Rh +ve RBCs are used
c) Anti-human globulins are added directly to
foetal RBCs
d) Mother's serum is used
e) Foetal serum is used
58. 4. In Elek’s test:
a) The antigen is mixed with the agar gel.
b)Lines of precipitate will form in positive cases.
c) The antibody is incorporated in the agar.
d) A strip of filter paper is soaked with the antigen.
e) Agglutination can be observed.
59. 5. Single radial immunodiffusion test:
a) Is an example of an agglutination reaction
b) Utilizes agar gel mixed with the antigen
c) Depends on complement fixation during Ag-Ab
reaction
d) Is used to detect non-agglutinating antibodies
e) Is a precipitation reaction
60. 6. Direct ELISA requires:
a. Known Ab
b. Known Ag
c. Complement
d. Patient’s Ab
e. Sheep RBCs