Precipitation and Immunodiffusion Tests
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Direct answer
Precipitation occurs when a soluble antigen and its antibody combine in optimal proportions to form a visible lattice, and immunodiffusion tests make that lattice visible in agar gel. Ouchterlony double diffusion compares antigens qualitatively, while Mancini radial immunodiffusion quantifies a protein such as IgG. Counter-immunoelectrophoresis and VDRL flocculation are older formats that remain useful in examinations.
What you must remember
- Zone of equivalence: a visible precipitate forms only where antigen and antibody are in optimal proportion, so excess of either one prevents it.
- Prozone phenomenon: antibody excess blocks lattice formation, seen in high titre syphilis serology, and is resolved by diluting the serum.
- Ouchterlony test: double diffusion in agar, where lines of identity, partial identity or non-identity compare antigens.
- Mancini test: single radial immunodiffusion in which the square of the ring diameter is proportional to antigen concentration.
- Counter-immunoelectrophoresis: an electric field drives antigen and antibody towards each other, a method once used for bacterial antigens in CSF.
- Immunoelectrophoresis: serum proteins are separated by electrophoresis and then tested against antibody, used to identify paraproteins in myeloma.
- VDRL: a non-treponemal flocculation test using a cardiolipin antigen, used to screen for syphilis and monitor treatment response.
- Ascoli thermoprecipitation: heat-extracted anthrax antigen from tissue forms a precipitate at the interface with specific antiserum.
Running a Mancini radial immunodiffusion assay
Radial immunodiffusion is the quantitative member of this family, and it is the format most often described in examinations. The steps below follow the usual laboratory sequence from gel preparation to reading the result.
- Gel preparation: antiserum is mixed into molten agar and poured as a uniform gel, so antibody is evenly distributed.
- Well loading: standard antigen concentrations and the unknown samples are placed into wells cut into the gel.
- Diffusion: antigen moves outward from each well, and a ring of precipitate forms where antigen and antibody reach the zone of equivalence.
- Ring measurement: after an adequate incubation period, the diameter of each ring is measured, because a larger ring means more antigen.
- Calibration: the square of the diameter is plotted against the concentration of the standards, producing a straight line.
- Quantification: the unknown concentration is read from the standard line, which is how serum immunoglobulin levels are reported.
Where the exam tries to catch you
Precipitation questions often test whether the candidate understands the zone of equivalence, so the proportion of antigen and antibody matters more than the names of the tests.
- Trap: reading a negative precipitation test as proof that antigen is absent Correct: a prozone effect from excess antibody can prevent precipitation, so the serum must be diluted before a negative result is trusted.
- Trap: choosing VDRL as the confirmatory test for syphilis Correct: VDRL is a non-treponemal screening test, and a treponemal test such as TPHA is used to confirm the diagnosis.
- Trap: using counter-immunoelectrophoresis for quantitative antibody measurement Correct: it is a qualitative antigen detection method, whereas Mancini radial immunodiffusion gives quantitative results.
Frequently asked questions
Why does a prozone effect cause a false negative precipitation result?
A prozone effect causes a false negative because excess antibody coats antigen molecules and prevents the lattice from forming a visible precipitate. Diluting the serum shifts the proportions back into the zone of equivalence, so the precipitate becomes visible.
What is the purpose of the VDRL test in syphilis?
The VDRL test is a non-treponemal flocculation screen used to detect syphilis and to monitor treatment response, because titres usually fall after successful therapy. A positive result is confirmed with a treponemal test, since biological false positives occur in conditions such as SLE and pregnancy.
Which test quantifies immunoglobulin levels in serum?
Mancini single radial immunodiffusion quantifies immunoglobulin levels in serum, by measuring the diameter of the precipitin ring formed around each sample. The square of the diameter is plotted against the concentration of known standards to read the unknown level.
What was counter-immunoelectrophoresis used to detect in cerebrospinal fluid?
Counter-immunoelectrophoresis was used to detect bacterial antigens in cerebrospinal fluid, such as those of Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae type b. Latex agglutination and PCR have largely replaced it in routine laboratories.