Rome, Italy - March 18-19, 2005
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Humoral immune status:Comparison of various
serological assays
Catherine Sadzot-DelvauxLaboratory of Fundamental VirologyPathology, B234000 Liège BELGIUM
Rome, Italy - March 18-19, 2005
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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.
Rome, Italy - March 18-19, 2005
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• 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.
Humoral immune status:Comparison of various
serological assays
Rome, Italy - March 18-19, 2005
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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)
Rome, Italy - March 18-19, 2005
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. . . .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
testingThe most frequently used assay in The most frequently used assay in
particular in the follow-up of vaccination particular in the follow-up of vaccination studiesstudies
Rome, Italy - March 18-19, 2005
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. . . .Serological assays: ELISA
(Enzyme-linked immunosorbent assay)
BUTBUT• 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.
Rome, Italy - March 18-19, 2005
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. . . .Serological assays:
VIDAS VZV (BioMerieux/Vitek)
• Rapid: VIDAS VZG: 40min
BUTBUT• Requires an automate• Some equivocal results (23/625) even
after retesting but no possibility to control since all steps performed in an automate
Rome, Italy - March 18-19, 2005
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. . . .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 Clinically relevant marker of functional immunityimmunity
BUTBUT• Not commercialized
Rome, Italy - March 18-19, 2005
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. . . .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
Rome, Italy - March 18-19, 2005
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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- » in sero- epidemiological surveys whenepidemiological surveys when low levels of antibodieslow levels of antibodies
By courtesy of Dr. A. Gershon
Rome, Italy - March 18-19, 2005
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BUTBUT• Not widely available (one kit
commercialized??)• Labor-intensive: cell-culture, live virus• Requires experience in reading and
interpreting the results• Subjectivity of the output
Serological assays: FAMA(Fluorescent Antibody Membrane Assay)
Williams, et al. J Infect Dis,1974,130: 669-672.
Rome, Italy - March 18-19, 2005
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. . . .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)
Rome, Italy - March 18-19, 2005
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Comparison between FAMA and IFAT:
Antibody Titer: 5x higher with IFAT, for low titer sera 8x higher with IFAT, for high titer sera
Serological assays: IFAT (Indirect Fluorescent Antibody Test)
Reproduced from Sauerbrei A et al. J Virol Methods 2004; 119: 25–30 with permission from Elsevier (http://www.sciencedirect.com/science/journal/01660934)
Rome, Italy - March 18-19, 2005
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• 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 sensitiveBoth tests are sensitive
BUTBUT• Subjective output• Not commercialized
Serological assays: IFAT (Indirect Fluorescent Antibody Test)
Sauerbrei A, et al. J. Virol. Methods, 2004;119:25-30
Rome, Italy - March 18-19, 2005
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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?)
Rome, Italy - March 18-19, 2005
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FAMA LA
Healthy children 0% 0%
Sera prior to or on the day of onset of varicella 0% 0%
Convalescent-phase (2 sera from the same individual)
100% 96%
Post-vaccine 77% 61%
Serological assays: LA (Latex Agglutination)
Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527-9.
Rome, Italy - March 18-19, 2005
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Serological assays: LA (Latex Agglutination)
Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527-9.
Percentage of positive samples
FAMA assay LA ELISA
Healthy adults 69 52 36
Healthy children 93 76 61
Leukemic children 72 57 48
Total 77 61 47
P vs FAMA – 0.001 0.0001
P vs LA – – 0.01
Rome, Italy - March 18-19, 2005
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FAMA/LA:
Serological assays: LA (Latex Agglutination)
Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527-9.
Number of FAMA results
LA result
Positive Negative Total
Positive 379 46 425
Negative 11 442 453
Total 390 488 878
48%
44%
Sensitivity: 92% agreement
Specificity: 93% agreement
52%
56%
Comparison of VZV antibody determinations by LA and FAMA assay
The rate of positivity is not significantly different between these assays (P>0.05)
Rome, Italy - March 18-19, 2005
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• Sensitive• Specific (no reactivity with other herpesviruses)• Reproducible
Comparable to FAMAComparable to FAMA
BUTBUT• False negative (Prosone): need to
retest the samples that appear neg
at the 1:2 dilution
Serological assays: LA (Latex Agglutination)
Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527-9.
Rome, Italy - March 18-19, 2005
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. . . .ANTIGENS
Commer-
cializedDISADVANTAGES ADVANTAGES
ELISA
(Enzyme linked immunosorbent assay)
Extract from VZV-infected cells
+
- Lack of sensitivity- Lack of specificity
-Automated- Objectivity of the output- Useful for numerous samples analysis
gpELISA VZV purified Glycoproteins
- oversensitive? See ELISA
LA(Latex Agglutination)
VZV proteins coated on latex particles +
- Subjectivity of the output - false- negative results in Prosone
- Rapid- Useful for numerous samples
FAMA(Fluorescent Antibodies to Membrane Antigens)
Live, unfixed VZV-infected cells
+?
- Subjectivity of the output: need of experience for reading and interpretation- cannot be automated
-Sensitive-Specific-Good correlation with protection
IFAT(Immuno-Fluorescent Antibody Test)
Acetone-fixed VZV-infected A549 cells
-
- Subjectivity of the output: need of experience for reading and interpretation- cannot be automated
-Sensitive-Specific-Good correlation with protection
Rome, Italy - March 18-19, 2005
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. . . .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?