This document discusses various direct and indirect methods used for the diagnosis of infectious diseases. Direct methods involve detecting the pathogen itself through microscopy, culture techniques, or molecular methods on a specimen. Indirect methods involve detecting the immune response to the pathogen through serology techniques like detecting IgM or rising titers of IgG antibodies. It then discusses various antigen-antibody reactions that can be used for serological diagnosis, including agglutination, precipitation, complement fixation, viral neutralization, immunofluorescence, ELISA, and radioimmunoassay.

1. Diagnosis of infectious diseases
INDIRECT METHOD
DIRECT METHOD
(Serology)
Specimen
Detection of
-IgM
-rising titre of IgG
Microscopy
Culture technique
Identification e.g.
-Microscopy
-Bioch. reactions
- Serology
- DNA probes.
Antibiotic sensitivity
Non-culture technique
- Molecular methods
- Serology.

2. ANTIGENANTIBODY
INTERACTIONS

3. Antigen-Antibody reactions in vitro = Serology
Diagnosis of diseases
Identification
of Ag /Ab
Quantitation of Ag / Ab

4. METHODS OF DETECTION OF
ANTIGEN-ANTIBODY
REACTIONS
Reactions
Accompanied by
Visible Phenomena
Reactions
Detected by
Labeled Reagents

5. Reactions Accompanied by
Visible Phenomena
Resulting Ag-Ab complexes seen directly
• By naked eye
• By microscope

6. A- AGGLUTINATION
Ag: cellular or particulate
antigen = “agglutinogen”
antibody = “agglutinin”

7. AGGLUTINATION

8. Agglutination
Slide Agglutination. 1
Tube Agglutination. 2

9. 1. Slide agglutination
(Qualitative Test)
Unknown Ag
 Add known
antiserum (Ab)


13. 2. Tube agglutination
(Quantitative Test)
Detection of Ab against a certain pathogen
= indirect evidence for diagnosis of disease
= serological diagnosis


14. Tube Agglutination
1/10
1/20
1/40
1/80
1/160
1/320
1/640

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)

17. Soluble known antigen coated on latex particle
+
Unknown antibody (IgM)

18. (Soluble) known antibody coated on latex particle
+
Unknown soluble antigen

19. Examples:

Rheumatoid arthritis:
(Patient produces Abs against IgG)
Latex-IgG (known Ag) + serum from patient (Ab?)

C-Reactive protein: (inflammatory conditions)
Latex-anti-CRP (known Ab) + serum (CRP?)

Pregnancy Test (Test for HCG)
Latex-anti-HCG (known Ab) + urine (HCG?)

20. C- COOMBS (ANTIGLOBULIN) TEST
Non-agglutinating
antibodies
Examples:


Anti-Rh antibodies
Abs in autoimmune
haemolytic anaemia

22. Direct
Coombs Test
Done for cases of:
- Newborn with erythroblastosis foetalis
- Patients with haemolytic anaemia
(RBCs have attached Abs already)

24. Indirect Coombs Test
Done to detect non-agglutinating antibodies in
serum of Rh-negative mother sensitized
with Rh antigen

26. D- HAEMAGGLUTINATION INHIBITION
Some viruses agglutinate RBCs in vitro.
Antibodies (if present) prevent haemagglutination
= Haemagglutination inhibition

27. E- PRECIPITATION
• Ag: soluble
Applications:
Agar gel diffusion
a. Double diffusion
b. Single radial immunodiffusion

28. Agar gel diffusion
a. Double Immunodiffusion
Semi-solid
medium
Precipitation Line
Ag
well
Ab
well

29. Elek’s Test
(double diffusion)

30. Elek’s Test

31. b. Single radial immunodiffusion
• Method:
- Ab in media
- Ag in well
Antibody
Ag
Precipitation Ring

32. b-Single radial immunodiffusion

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

41. ANTIGEN-ANTIBODY
REACTIONS
Reactions
Accompanied by
Visible Phenomena
Reactions
Detected by
Labeled Reagents

42. A. Immunofluorescence
B. ELISA
C. RIA

43. A. Immunofluorescent Techniques:
Principle:
Fluorescent substances (e.g. Fluorescein isothiocyanate)
attached to known Ab
→ seen by fluorescence microscope
using UV light
→ fluorescence = positive reaction

44. i) Direct Immunofluorescence
• Detects unknown Ag
e.g.: rabies virus in
brain of dead animal
• Tissue (Ag?)
+ labeled known Ab
→ binding
→ fluorescence

45. Positive test

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.

49. Direct Method
(Double Antibody Technique)
For detection and quantitation of Ag

50. Indirect Method
For detection and quantitation of Ab in serum.
Unknown Ab in
test serum
Known Ag
Enzyme labeled
anti-human Ig
Substrate is
added

51. ELISA

52. ELISA
plates

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

54. MCQs

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