Avidity determination of IgG in
diagnosis of tick-born encephalitis
Hana Zelená
Jiří Januška
Jan Raszka
Virology department, National Reference Laboratory for Arboviruses,
Institute of Public Health, Ostrava, Czech Republic
Diagnosis of tick-born encephalitis
• 1st phase (fever): direct detection
- PCR
- virus isolation from blood
• 2nd phase (neurological): indirect detection
- IgG, IgM ELISA, IFA
- Complement fixation test (CFT)
- Virus neutralizing antibodies (VNA)
Dynamics of diagnostic markers in tickborn encephalitis
Dynamics of diagnostic markers in tickborn encephalitis
Avidity of IgG antibodies
• Avidity is a measure of the strenght of the
antibody-antigen interactions, it increases with
their binding affinity and with their valence
• Avidity reflects the maturity of the antibodies.
Low avidity antibodies are synthetized during the
primary infection, in time avidity gradually
increases.
• High avidity antibodies are produced by memory
B-cells during the secondary infection or
reactivation or infection in people that were
vaccinated.
Low avidity occures also in immunosupression.
Usefullness of IgG avidity
measurement
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Rubella
CMV
VZV
Toxoplasmosis
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VCA-EBV
HIV
Viral hepatitis
WNV
Dynamics of diagnostic markers in tickborn encephalitis
Principle of IgG avidity measurement
• Test of the strenght of antigen-antibody
interaction by incubation with denaturing
agent (urea)
• Low avidity antibodies dissociate from
complexes and are washed away.
• High avidity antibodies withstand urea
treatment and remain bound to the antigen.
Protocol for the determination of antiTBEV IgG avidity
• Anti-TBEV IgG test kit is based on indirect ELISA.
• Patient serum is tested in two parallel wells.
• During the 1st incubation step antibodies found in
the serum sample bind to the antigen. One of the
two wells is incubated with urea solution (8
mol/L) for 5 minutes, while the other well
remains empty.
• Absorbance ratio between the two parallel wells
is calculated at the end of the test (the well with
urea/the well without urea)
IgG avidity measurement
results and interpretation
• Avidity (%) = absorbance of the well with
urea/absorbance of the well without urea
Avidity
Result
Interpretation
<40%
low avidity
Recent infection
(< 3 weeks)
40-60%
„gray zone“
>60%
high avidity
Infection in the
past, infection in
people that were
vaccinated
IgG avidity in time
(patients)
Dynamics of anti-TBEV antibody levels
(patient with typical TBE)
Anti-TBEV positive samples (april to september 2010)
29 samples IgG+ IgM+ 59 samples IgG+ IgM-
Pitfalls in TBE serology
– relevance of avidity testing
• Infection in vaccinated people
- min. 4-fold increase in CFT and VNA – must be paired samples
- IgM positive or negative
- high IgG avidity
• Elevated IgM pesistence after primary infection (up to 1 year)
- IgG and IgM positive
- high IgG avidity
• Atypical or subclinical course of the disease
- IgM and IgG positive
- low IgG avidity
• Early disappearance of IgM in primary infection
– low IgG avidity
Conclusion
• Avidity determination of IgG is a complementary tool
that makes serological diagnosis of tick-born
encephalitis more accurate
• It increases the testing reliability in acute infections,
posses high importance in cases of:
- atypical course of the disease
- atypical antibody response
- infection in vaccinated people
• Interpretation of serological result that is
complemented with IgG avidity measurement has
clearly higher validity.