Major uses of serologic (antibody-based tests)

1. Diagnosis of infectious diseases

- when organisms cannot be cultured, e.g., syphilis and hepatitis A,
B, and C

IMMUNOLOGIC METHODS - when the organism is too dangerous to culture, e.g., rickettsial
diseases
-When culture techniques are not readily available, E.G., HIV, EBV
-When organism takes too long to grow, e.g., Mycoplasma

* Problem with this approach: it takes time for antibodies to form,

* 2 serum samples (acute and convalescent sera):
4-fold or greater rise in Ab titer
Example: 1st sample 1:80 2nd sample 1:320
By this time the patient has recovered and the diagnosis becomes a
retrospective one.

* Detection of IgM: current infection.

Major uses of serologic (antibody-based tests) The Precipitation Curve

2. Diagnosis of autoimmune diseases

Use Ab against various normal body components are used

e.g., Ab vs DNA in SLE
Ab vs human IgG (rheumatoid factor) in RA
rheumatoid arthritis.

3. Determination of blood type and HLA type

1
The Precipitation Curve Antibodies
 Polyclonal antibody – formed in response to multi-
determinant Ag (several epitopes)
-e.g., bacterial cell has flagella, O Ag, capsule
 Monoclonal antibody – specific for one epitope
- Capsular Ag only

Double Immunodiffusion Ouchterlony Method

 Determines relationship between

2 Ags
1

 Identity
precipitin lines fuse
 Non-Identity
precipitin lines cross
 Partial identity
a ‘spur’ is formed
spur

Both the Ag and Ab diffuse.

2
 Single Radial Immunodiffusion Rocket Immunoelectrophoresis

 Ab is mixed with the gel  Single immunodiffusion

 Ag in the well diffuses in facilitated by an electric
radial pattern field
 Interpolate concentration  Ab in gel
from a standard curve  Ag moves in one direction
 Used to determine  Ag Concentration: height
concentration of Ag in a of rocket
sample  Interpolated from a
 Ig concentration standard curve
 apoproteins

Classical Immunoelectrophoresis Countercurrent Immunoelectrophoresis (CIE)

 double
1. Separation of Ag by immunodiffusion
electrophoresis technique facilitated
by an electric field
2. Antiserum in a trough
 Ag and Ab are placed
3. Ag and Ab diffuse in the gel
in parallel wells
4. Precipitin arc/s formed
 pH is adjusted so that
Ag is (-) and Ab is (+)
 Ag & Ab move toward
one another
 Precipitin band or arc Application: detection of capsular
Ag of H. influenzae, N.
meningitidis, C. neoformans,
etc in CSF or other fluids

3
 AGGLUTINATION

• Intrinsic Ags on microbes,

cell or other particulate
Ags
• Detect Ag or Ab
• Application:
•Widal Test
•Weil-Felix Test

Direct (Active) •serotyping

Agglutination

Agglutination

Known soluble Ag or
Ab is adsorbed to a
particle
• Particle: latex, rbc,
Staphylococus
Protein A
• Application:
• Soluble Ag or
Ab in body
fluids, e.g., CSF
Indirect (Passive) Agglutination

4
 Hemagglutination COMPLEMENT
FIXATION
1st Step
Unk Ag + Specific Ab → Ag-Ab complex

2nd Step
+ C’ → Ag-Ab-C’

3rd Step

RBC-Ab complex → no hemolysis (+)

Direct Indirect
IMMUNOFLUORESCENCE ELISA

Sandwich Method

•2nd Ab is F*-labeled,
•Anti-human IG (Specific
for Fc region)

5
 WESTERN BLOTTING
Different Indicator Labels (IMMUNOBLOTTING)
 Fluorochromes label Protein molecules
 e..g. Fluorescein isothiocyanate
 Fluorescent microscope
 Enzymes
SDS denaturation
 e.g., Alkaline phosphatase
 Chromogenic substrate
 Spectrophotometer

Western blot

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