1 / 21

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

lew
Download Presentation

Humoral immune status: Comparison of various serological assays

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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?

More Related