SN2 SHORT NOTE 5M ★ High-yield

Describe the antigen-antibody reactions used in the laboratory and the interpretation of serological results.

Microbiology · 2nd Professional — Para-Clinical

Principles

Antigen-antibody reactions are the specific, reversible, non-covalent combinations of antigen with antibody, used to detect either partner and forming the basis of serology.

Bonds are non-covalent — hydrogen bonds, electrostatic, van der Waals and hydrophobic forces — so the reaction is reversible
Affinity is the strength of binding of a single site to a single epitope; avidity is the overall strength of the whole molecule, which is why pentameric IgM has high avidity despite low affinity
The zone phenomenon — visible reaction occurs only in the zone OF equivalence. In antibody excess (prozone) or antigen excess (postzone) the lattice does not form and the test is falsely negative

CLINICAL PEARL

The prozone phenomenon is a false negative caused by too much antibody, which is exactly the opposite of what intuition suggests. Each antigen becomes coated with separate antibody molecules and no cross-linking lattice forms, so nothing is seen. It is a recognised cause of a falsely negative VDRL in secondary syphilis, when antibody levels are at their highest — and the remedy is simply to dilute the serum and repeat.

Types of Reaction

Reaction	Principle	Examples
Precipitation	Soluble antigen + antibody form a visible lattice	Ring test (Ascoli, Lancefield grouping); immunodiffusion (ouchterlony double diffusion, showing patterns of identity, non-identity and partial identity); radial immunodiffusion (Mancini) for quantification; immunoelectrophoresis; ELEK test for diphtheria toxigenicity
Agglutination	Particulate antigen clumps; more sensitive than precipitation	ABO blood grouping; WIDAL test; slide and tube agglutination; COOMBS (antiglobulin) test; latex agglutination; passive haemagglutination; Weil-Felix
Complement fixation	Antigen-antibody complexes consume complement, so added sensitised sheep red cells are not lysed	Historically important — the Wassermann test for syphilis; largely superseded
Neutralisation	Antibody blocks the biological activity of a toxin or virus	Antistreptolysin O (ASO) titre; virus neutralisation tests
Immunofluorescence	Antibody labelled with a fluorochrome and viewed by UV microscopy	Direct for antigen in tissue (rabies in brain, respiratory viruses); indirect for antibody in serum, and more sensitive
ELISA	Enzyme-labelled antibody produces a colour change	HIV, hepatitis, dengue; the workhorse of modern serology
Radioimmunoassay	Radioisotope label	Very sensitive; superseded by non-radioactive methods
Immunochromatography (lateral flow)	Antigen or antibody captured on a strip, read visually	Rapid point-of-care tests — malaria, dengue NS1, HIV, pregnancy; no equipment required, which makes them invaluable in field conditions

Interpretation of Serological Results

A fourfold rise in titre between acute and convalescent samples taken 10 to 14 days apart is the standard evidence of recent infection
A single titre is difficult to interpret in an endemic area, where much of the population is already seropositive from past exposure — a persistent problem with the Widal test in India
IgM indicates recent infection; IgG indicates past infection or immunity
Antibody appears only after a window period, so early serology is negative; antigen detection or nucleic acid testing is required in that interval, which is critical in HIV and hepatitis
False positives arise from cross-reacting antigens, rheumatoid factor, pregnancy, autoimmune disease and other infections; false negatives from the window period, the prozone effect, and immunosuppression
Sensitivity and specificity govern use — a highly sensitive test is used to screen and a highly specific test to confirm, which is why a reactive HIV ELISA is confirmed by a second assay before any result is given

Applied Aspects

Never report a positive HIV screening test as a diagnosis; India’s national algorithm requires reactivity on three different assays, and pre- and post-test counselling are part of the procedure
Dilute and repeat when a strongly suspected serological test is negative, to exclude the prozone effect
Rapid tests have transformed field diagnosis in India — malaria, dengue, HIV, hepatitis B, syphilis and visceral leishmaniasis can all be diagnosed without a laboratory, which changes what is possible at primary care level
The Widal test is over-used and over-interpreted; blood culture remains the definitive test for typhoid, and a single raised Widal titre in an endemic population is weak evidence
Take paired sera whenever serology is used for diagnosis, and label the dates clearly; a single sample often cannot answer the question
Store and transport sera correctly; haemolysed, lipaemic or contaminated samples give unreliable results, and repeated freeze-thaw cycles destroy antibody

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