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Humoral immune status:
Comparison of various
serological assays
Catherine Sadzot-Delvaux
Laboratory of Fundamental Virology
Pathology, B23
4000 Liège BELGIUM
Rome, Italy - March 18-19, 2005
Humoral immune status:
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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
Humoral immune status:
. .. .
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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.
Rome, Italy - March 18-19, 2005
Humoral immune status:
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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)
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• 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
Rome, Italy - March 18-19, 2005
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Serological assays: ELISA
(Enzyme-linked immunosorbent assay)
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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.
Rome, Italy - March 18-19, 2005
Serological assays:
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VIDAS VZV (BioMerieux/Vitek)
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• 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
Rome, Italy - March 18-19, 2005
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•
•
•
•
Serological assays:
gpELISA
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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
Rome, Italy - March 18-19, 2005
Serological assays:
gpELISA
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• 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 postvaccination titer > 5gpELISA
• 83.5% if 6-week postvaccination 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
Serological assays: FAMA
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(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 seroepidemiological surveys when
low levels of antibodies
By courtesy of Dr. A. Gershon
Rome, Italy - March 18-19, 2005
Serological assays: FAMA
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(Fluorescent Antibody Membrane Assay)
Williams, et al. J Infect Dis,1974,130: 669-672.
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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
Rome, Italy - March 18-19, 2005
Serological assays: IFAT
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(Indirect Fluorescent Antibody Test)
Sauerbrei A, et al. J. Virol. Methods, 2004;119:25-30
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• Antigen: A549 (human lung
carcinoma cells) + 0.0001
m.o.i, removed mechanically 710dpi (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
Serological assays: IFAT
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(Indirect Fluorescent Antibody Test)
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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
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
Serological assays: IFAT
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(Indirect Fluorescent Antibody Test)
Sauerbrei A, et al. J. Virol. Methods, 2004;119:25-30
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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 antiVZV 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
Rome, Italy - March 18-19, 2005
Serological assays: LA
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(Latex Agglutination)
Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527-9.
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• 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
Serological assays: LA
(Latex Agglutination)
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Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527-9.
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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%
Rome, Italy - March 18-19, 2005
Serological assays: LA
(Latex Agglutination)
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Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:15279.
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
Serological assays: LA
(Latex Agglutination)
. .. .
Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527-9.
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Comparison of VZV antibody determinations by LA and FAMA assay
Number of
FAMA results
Positive
Positive
379
LA result
Negative
46
11
390
44%
442
488
56%
Negative
Total
FAMA/LA:
Total
425 48%
453 52%
878
Sensitivity: 92% agreement
Specificity: 93% agreement
The rate of positivity is not significantly different between these assays (P>0.05)
Rome, Italy - March 18-19, 2005
Serological assays: LA
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(Latex Agglutination)
Steinberg SP, Gershon AA. J. Clin. Microb 1991;29:1527-9.
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• 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
Rome, Italy - March 18-19, 2005
ANTIGENS
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ELISA
(Enzyme linked
immunosorbent
assay)
Extract from VZVinfected cells
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Commercialized
+
DISADVANTAGES
ADVANTAGES
- Lack of sensitivity
- Lack of specificity
-Automated
- Objectivity of the
output
- Useful for numerous
samples analysis
gpELISA
VZV purified
Glycoproteins
- oversensitive?
See ELISA
LA
VZV proteins coated
on latex particles
+
- Subjectivity of the
output
- false- negative results
in Prosone
- Rapid
- Useful for numerous
samples
+?
- Subjectivity of the
output: need of
experience for reading
and interpretation
- cannot be automated
-Sensitive
-Specific
-Good correlation with
protection
(Latex
Agglutination)
FAMA
(Fluorescent
Antibodies to
Membrane
Antigens)
IFAT
(ImmunoFluorescent
Antibody Test)
Live, unfixed VZVinfected cells
Acetone-fixed VZVinfected A549 cells
-
- Subjectivity of the
-Sensitive
output: need of
-Specific
experience for reading
-Good correlation with
and interpretation
protection
- cannot be automated
Rome, Italy - March 18-19, 2005
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Serological assays:
conclusions
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• 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?
Rome, Italy - March 18-19, 2005