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Avidity determination of IgG in diagnosis of tick-born encephalitis

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.

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Avidity determination of IgG in diagnosis of tick-born encephalitis

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  1. Avidity determination of IgG in diagnosis of tick-born encephalitis Hana ZelenáJiří Januška Jan Raszka Virology department, National Reference Laboratory for Arboviruses, Institute of Public Health, Ostrava, Czech Republic

  2. 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)

  3. Dynamics of diagnostic markers in tick-born encephalitis

  4. Dynamics of diagnostic markers in tick-born encephalitis

  5. 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 synthetizedduring the primary infection, in time avidity gradually increases. • High avidity antibodies are produced by memory B-cells duringthe secondary infection or reactivation or infection in people that were vaccinated. Low avidity occures also in immunosupression.

  6. Usefullness of IgG avidity measurement • Rubella • CMV • VZV • Toxoplasmosis • VCA-EBV • HIV • Viral hepatitis • WNV

  7. Dynamics of diagnostic markers in tick-born encephalitis

  8. 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.

  9. Protocol for the determination of anti-TBEV 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)

  10. IgG avidity measurementresults and interpretation • Avidity (%) = absorbance of the well with urea/absorbance of the well without urea

  11. IgG avidity in time(patients)

  12. Dynamics of anti-TBEV antibody levels(patient with typical TBE)

  13. Anti-TBEV positive samples (april to september 2010)29 samples IgG+ IgM+ 59 samples IgG+ IgM-

  14. 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

  15. Conclusion • Avidity determination of IgG is a complementary tool that makes serological diagnosis of tick-born encephalitis more accurate • It increases the testing reliability inacute 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 measurementhas clearly higher validity.

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