Immune tests in the laboratory diagnosis of infections

- brief overview for dentistry students -

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)

Immune reaction: Antigen-Antibody reaction (Ag-Ab)

High specificity:

Antigen-binding site of the

antibody molecule perfectly

matches the antigen

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

Ag-Ab Reactions: applications in laboratory diagnosis of infections (examples)

• Agglutination

• Immunofluorescence

• Enzyme-linked Immunosorbent Assay (ELISA)

• Immunoblotting, Western blot

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)

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

1. 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

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

types

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

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

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

ELISAimmune reaction (Ag-Ab)

linked to

enzymatic reaction (Enzyme-Substrate)

• Solid support coated with Ag - Add Patient serum • if specific Ab present→ Ag-Ab complex• Add Ab anti-human Ab conjugated to Enzyme

• Complex: Ag-Ab-(Ab anti-human Ab)-Enzyme

• Add substrate of enzyme → COLOUR develops:– action of enzyme on substrate AND – presence of specific Ab in patient serum

Western blot