description of immune reactions and their applications in microbiology

1. Immunologic methods in the laboratory
diagnosis of infections

2. Definition of terms
Antigen = foreign substance that, when introduced into the
body, is capable of stimulating an immune reaction e.g.
foreign molecules in bacteria, viruses, protozoa, serum
components, etc.
Antibody aka immunoglobulin = large protein produced by
plasmocytes which identifies and neutralises antigens
Immune reaction = reversible binding of antigen to
homologous antibody (high specificity)

3. Immune reaction: Antigen-Antibody reaction
(Ag-Ab)
High specificity:
Antigen-binding site of the
antibody molecule perfectly
matches the antigen
(”key in hole”)

4. Ag-Ab Reactions: applications in laboratory
diagnosis of infections
• Same basic principle:
– specific detection and binding of antigen by
antibody → Ag-Ab complex
• Differences:
– the methods used to reveal the Ag-Ab
complex

5. Ag-Ab Reactions: applications in laboratory
diagnosis of infections (examples)
• Agglutination
• Immunofluorescence
• Enzyme-linked Immunosorbent Assay (ELISA)
• Immunoblotting, Western blot

6. Agglutination: on slide/in tubes
• clumping together by
antibodies of microscopic
foreign particles:
– red blood cells
– bacteria
– inert particles (latex)
• agglutinated particles are
visible with the naked eye
(pellet-like agglutination
product)

7. Agglutination: applications in microbiology
1. Serological diagnosis: detection (and quantification) of
unknown antibodies by use of known antigens
• Principle:
• serum from patient is put in contact with serial dilutions of Ag;
• if Ab present in serum →agglutination
• Titre of Ab = dilution of the tube with agglutination
2. Bacteriological diagnosis: identification of unknown
antigens (bacteria) by use of known antibodies
• Principle:
• Sera containing known Ab are put in contact with bacterial
suspension
• Identification of bacteria – indicated by the type of serum which
agglutinates the bacterial suspension

8. Antigen: isolated bacteria e.g. Salmonella
Antibody: kit with antibodies (antisera) against Salmonella
types

9. Passive agglutination (inert particles)
Ag on latex (latex-agglutination)/RBC (hemagglutination)
• If Ab are present in sample →agglutination of latex/RBC
Ab on inert particles: bacterial identification kits e.g.
Streptococcus pneumoniae, Neisseria gonorrhoeae,
Neisseria meningitidis, E.coli

10. Immunofluorescence
• Immunofluorescence:
– Bacterial culture (Ag) incubated with specific
antibody coupled with fluorescent dye
– if Ab matches Ag (bacterial culture) →Ag-Ab
complex + fluorescent dye
– under UV light bacteria covered with
antibodies coupled with fluorescent dye will
produce fluorescence

11. Treponema denticola – wet mount, dark field
microscopy + fluorescent dye staining

12. ELISA (Enzyme-linked Immunosorbent
Assay)
immune reaction (Ag-Ab)
linked to
enzymatic reaction (Enzyme-Substrate)
ELISA types:
- Direct
- Indirect
- ”Sandwich”

13. Solid support for ELISA:
96 microwell plastic plate

14. Direct ELISA
• Add serum on solid support (plastic
microwell plate); adherence to solid
support by charge interactions
• Add CONJUGATE: Ab conjugated
to enzyme
• Add SUBSTRATE of enzyme
• COLOUR = Ag present in serum

15. Indirect ELISA
• Solid support pre-coated with
Ag
• Add Patient serum
• if Ab in serum→formation of
Ag-Ab complex
• Add CONJUGATE: Ab anti-human
Ab+Enzyme
• Formation of Complex: Ag-Ab-
Ab anti-human Ab+Enzyme
• Add substrate of enzyme →
COLOUR develops as a
result of enzyme-substrate
reaction → presence of Ab in
patient serum (”primary
antibody”)

16. ”Sandwich” ELISA
• Solid support pre-coated with
”capture” Ab (speciffic for the Ag
tested for)
• Add patient serum; if Ag is
present, the Ab on plate
capture it
• Add ”detection” Ab which will
bind Ag (Ag bound between 2
Antibodies: sandwich)
• Add CONJUGATE: Ab anti-
”detection” Ab conjugated to
Enzyme
• Add SUBSTRATE of enzyme
• Colour develops

17. Western blot
• Confirmatory test to detect antibodies in ELISA-positive serum
samples e.g. HIV, Lyme disease
• Proteins from known infected cells are separated by electroforesis
and blotted on a nitrocellulose strip
• Serum applied to strip (primary antibody incubation step)
• if specific Ab are present in serum they will bind to the nitrocelulose
strip
• Add secondary anti-human antibody conjugated with enzyme signal
• stained bands will indicate the presence of Ab in patient serum

18. Western blot