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Humoral immune status: Comparison of various serological assays

Humoral immune status: Comparison of various serological assays. Catherine Sadzot-Delvaux Laboratory of Fundamental Virology Pathology, B23 4000 Liège BELGIUM. Humoral immune status: Comparison of various serological assays. Immunity to VZV: Complex Not fully understood

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Humoral immune status: Comparison of various serological assays

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  1. Humoral immune status:Comparison of various serological assays Catherine Sadzot-Delvaux Laboratory of Fundamental Virology Pathology, B23 4000 Liège BELGIUM

  2. Humoral immune status:Comparison of various serological assays • Immunity to VZV: • Complex • Not fully understood • Natural immune response (NK cells,…) • Specific immune response • Humoral immunity: antibodies against glycoproteins, nucleocapsid and tegument proteins. • Cellular immunity: mainly Th1.

  3. Humoral immune status:Comparison of various serological assays • Serodiagnosis is very useful • for verifying the immune status prior to vaccination of healthy, at high-risk adults without history of chickenpox • for assessing the immunogenicity of varicella vaccine and assuring the follow-up of vaccination studies.

  4. Humoral immune status:Comparison of various serological assays • Specific antibodies can be detected by: • neutralization • complement fixation • enzyme-linked immunosorbent assays • ELISA • gpELISA • immunofluorescence • FAMA (Fluorescent antibody to membrane antigen) • IFAT (Indirect fluorescence antibody test) • latex agglutination (LA)

  5. Serological assays: ELISA(Enzyme-linked immunosorbent assay) • Solid-phase enzyme immunoassay • Antigen = extract of VZV-infected cells (ELISA) • Output: optical density • Simple and automated • Adaptable to detect IgA, IgM or IgG • Suitable for small- and large-scale testing The most frequently used assay in particular in the follow-up of vaccination studies

  6. Serological assays: ELISA (Enzyme-linked immunosorbent assay) BUT • Lacks sensitivity and /or specificity • Can be optimized if the preparation of antigens is optimized: the purer the antigens, the more sensitive and the more specific is the assay. • Determination of class-specific antibodies is sometimes difficult • Large amounts of IgG that interfere with less frequent IgM • False-positive frequently observed for IgM due to rheumatoid factor (RF) that can be produced in viral infections and interfere with IgM assays.

  7. Serological assays: VIDAS VZV(BioMerieux/Vitek) • Rapid: VIDAS VZG: 40min BUT • Requires an automate • Some equivocal results (23/625) even after retesting but no possibility to control since all steps performed in an automate

  8. Serological assays: gpELISA • Modified version of the « classical » ELISA • Antigen: purified VZV glycoproteins • 4x more sensitive than ELISA • Frequently used in post-vaccination studies. • Clinically relevant marker of functional immunity BUT • Not commercialized

  9. Serological assays: gpELISA • 6 weeks after immunization • 99% positivity (Mean titer:12.9 gpELISA Units) • Antibody titers correlate with levels of neutralizing antibody and the induction of cell-mediated immunity • Estimated vaccine efficacy: • 95.5% if 6-week post- vaccination titer > 5gpELISA • 83.5% if 6-week post- vaccination titer < 5gpELISA • 3.5 x more likely to develop breakthrough varicella • More lesions Li S, et al.Pediatr.Infect.Dis J 2002;21:337

  10. Serological assays: FAMA(Fluorescent Antibody Membrane Assay)Williams, et al. J Infect Dis,1974,130: 669-672. • Antigen: unfixed VZV-infected cells • Output: Fluorescence • Only serological test known to correlate protection from infection with a specific titer of antibodies • Highly sensitive, probably due to the use of unfixed cells in which conformational structures of VZV proteins are preserved • « Gold Standard » in sero- epidemiological surveys when low levels of antibodies By courtesy of Dr. A. Gershon

  11. Serological assays: FAMA(Fluorescent Antibody Membrane Assay) Williams, et al. J Infect Dis,1974,130: 669-672. BUT • Not widely available (one kit commercialized??) • Labor-intensive: cell-culture, live virus • Requires experience in reading and interpreting the results • Subjectivity of the output

  12. Serological assays: IFAT (Indirect Fluorescent Antibody Test)Sauerbrei A, et al. J. Virol. Methods, 2004;119:25-30 • Antigen: A549 (human lung carcinoma cells) + 0.0001 m.o.i, removed mechanically 7-10dpi (10% CPE) and fixed with acetone 1h -20°C (stable 6 months) • Output: fluorescence • Sera serially diluted (first dilution 1:5) (18h at RT + 3h 37°C)

  13. Serological assays: IFAT (Indirect Fluorescent Antibody Test) Comparison between FAMA and IFAT: Antibody Titer: 5x higher with IFAT, for low titer sera 8x higher with IFAT, for high titer sera Reproduced from Sauerbrei A et al. J Virol Methods 2004; 119: 25–30 with permission from Elsevier (http://www.sciencedirect.com/science/journal/01660934)

  14. Serological assays: IFAT (Indirect Fluorescent Antibody Test)Sauerbrei A, et al. J. Virol. Methods, 2004;119:25-30 • Validation of FAMA and IFAT using the British Standard for VZV antibodies (National Institute for Biological Standards and Control, Hertfordshire, UK): 4 IU anti-VZV IgG/ml • FAMA: 250mIU/ml (highest dilution showing a pos. Result: 1/16) • IFAT: 50mUI/ml (highest dilution showing a pos. Result: 1/80)  Both tests are sensitive BUT • Subjective output • Not commercialized

  15. Serological assays: LA(Latex Agglutination)Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527-9. • Antigen: VZV antigen coated on latex particles • Output: obvious clumping of latex particles Subjectivity • Easy and rapid to perform (15min) • Commercialized • Comparable to FAMA: Only 2% FAMA neg/LA pos (not true neg?)

  16. Serological assays: LA(Latex Agglutination)Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527-9.

  17. Serological assays: LA(Latex Agglutination)Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527-9.

  18. Serological assays: LA(Latex Agglutination)Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527-9. Comparison of VZV antibody determinations by LA and FAMA assay 48% 52% 44% 56% FAMA/LA: Sensitivity: 92% agreement Specificity: 93% agreement The rate of positivity is not significantly different between these assays (P>0.05)

  19. Serological assays: LA(Latex Agglutination)Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527-9. • Sensitive • Specific (no reactivity with other herpesviruses) • Reproducible Comparable to FAMA BUT • False negative (Prosone): need to retest the samples that appear neg at the 1:2 dilution

  20. Serological assays: conclusions • Need for harmonization of the tests used all over Europe to be able to compare results. • However, no serologic test is perfect. Which assay ??? • FAMA = « gold standard » but requires a lot of experience, and difficult to use for large-scale studies • IFAT? • LA? • ELISA? • First screening with one test and retesting with a second assay? • Comparison using the reference serum as control?

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