Immunologic methods in the laboratory

diagnosis of infections

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

(”key in hole”)

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

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

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

ELISA (Enzyme-linked Immunosorbent

Assay)

immune reaction (Ag-Ab)

linked to

enzymatic reaction (Enzyme-Substrate)

ELISA types:

- Direct

- Indirect

- ”Sandwich”

Solid support for ELISA:

96 microwell plastic plate

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

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”)

”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

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

Western blot