Rome, Italy - March 18-19, 2005 masque.... Humoral immune status: Comparison of various serological...

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


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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.


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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.


serological assays


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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)


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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


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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.


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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


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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


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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


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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


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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.


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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)


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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)


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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


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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?)


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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%




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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


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FAMA/LA:



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)


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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




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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


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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?

Rome, Italy - March 18-19, 2005 masque.... Humoral immune status: Comparison of various serological...

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