We have developed a cheaper an simple in house indirect ELISA that uses the live attenuated VZV vaccine as a coating antigen. The alternative ELISA had an agreement of 94% when compared with a commercial VZV ELISA kit. Moreover, our ELISA proved to be more reliable than the kit when assessing true negative samples. By adding a standard serum, we were able to produce results in international units per millilitre. Also, the addition of an extra step with 8M urea allowed the assessment of VZV IgG avidity without excessive costs. The cost per sample to test VZV IgG was 2.7 times cheaper with our ELISA, allowing the testing of many samples without the burden of production of VZV antigen in the laboratory.Desenvolvemos um ensaio imunoenzimático (ELISA) indireto simples e econômico para detecção de anticorpos contra o vírus da varicela zoster (VVZ) que utiliza a vacina contendo o vírus vivo atenuado como antígeno para recobrir a placa. Este ELISA mostrou uma concordância de 94% quando comparado com um kit de ELISA comercial para anticorpos contra varicela. Além disso, nosso ELISA mostrou ser mais confiável que o kit quando amostras comprovadamente negativas foram testadas. O uso de um soro padrão de referência, calibrado em unidades internacionais por mililitro, possibilitou também que os resultados pudessem ser comparados com outros estudos. O acréscimo de uma etapa extra com solução de uréia 8M permitiu avaliação de avidez de IgG para VVZ sem custos excessivos. O custo por amostra para testar IgG contra VVZ com nosso ELISA foi 2,7 vezes mais barato quando comparado com o kit comercial

Granato, Celso

Lafer, Manuel Mindlin

Moraes-Pinto, Maria Isabel de

Ono, Erika

Weckx, Lily Yin

English

Desenvolvemos um ensaio imunoenzimático (ELISA) indireto simples e econômico para detecção de anticorpos contra o vírus da varicela zoster (VVZ) que utiliza a vacina contendo o vírus vivo atenuado como antígeno para recobrir a placa. Este ELISA mostrou uma concordância de 94% quando comparado com um kit de ELISA comercial para anticorpos contra varicela. Além disso, nosso ELISA mostrou ser mais confiável que o kit quando amostras comprovadamente negativas foram testadas. O uso de um soro padrão de referência, calibrado em unidades internacionais por mililitro, possibilitou também que os resultados pudessem ser comparados com outros estudos. O acréscimo de uma etapa extra com solução de uréia 8M permitiu avaliação de avidez de IgG para VVZ sem custos excessivos. O custo por amostra para testar IgG contra VVZ com nosso ELISA foi 2,7 vezes mais barato quando comparado com o kit comercial.We have developed a cheaper an simple in house indirect ELISA that uses the live attenuated VZV vaccine as a coating antigen. The alternative ELISA had an agreement of 94% when compared with a commercial VZV ELISA kit. Moreover, our ELISA proved to be more reliable than the kit when assessing true negative samples. By adding a standard serum, we were able to produce results in international units per millilitre. Also, the addition of an extra step with 8M urea allowed the assessment of VZV IgG avidity without excessive costs. The cost per sample to test VZV IgG was 2.7 times cheaper with our ELISA, allowing the testing of many samples without the burden of production of VZV antigen in the laboratory

Cadernos Espinosanos (E-Journal)

Rev. Inst. Med. trop. S. Paulo46(3):165-168, May-June, 2004Research supported by FAPESP (Grants: 01/05481-0 and 97/06118-9)(1) Division of Pediatric Infectious Diseases, Federal University of São Paulo.(2) Division of Adult Infectious Diseases, Federal University of São Paulo(3) Immunology Department, Fleury Laboratory, São PauloCorrespondence to: Maria Isabel de Moraes-Pinto, Division of Pediatric Infectious Diseases, Federal University of São Paulo (UNIFESP-EPM). Rua Pedro de Toledo 781, 9. andar, VilaClementino, 04039-032 São Paulo, SP, Brasil. Tel/Fax: +55-11-5574-6471. E-mail:m.isabelmp@uol.com.brA SIMPLE AND CHEAPER IN HOUSE VARICELLA ZOSTER VIRUS ANTIBODY INDIRECT ELISAErika ONO(1), Manuel Mindlin LAFER(1), Lily Yin WECKX(1), Celso GRANATO(2,3) & Maria Isabel de MORAES-PINTO(1)SUMMARYWe have developed a cheaper an simple in house indirect ELISA that uses the live attenuated VZV vaccine as a coating antigen.The alternative ELISA had an agreement of 94% when compared with a commercial VZV ELISA kit. Moreover, our ELISAproved to be more reliable than the kit when assessing true negative samples.By adding a standard serum, we were able to produce results in international units per millilitre. Also, the addition of an extra stepwith 8M urea allowed the assessment of VZV IgG avidity without excessive costs.The cost per sample to test VZV IgG was 2.7 times cheaper with our ELISA, allowing the testing of many samples without theburden of production of VZV antigen in the laboratory.KEYWORDS: Varicella; ELISA; Antibody avidity.INTRODUCTIONAssessment of varicella zoster virus (VZV) antibodies is performedroutinely by enzyme immunoassays (ELISA) with commerciallyavailable kits. A cheaper and as accurate alternative is the use of “inhouse” ELISAs. However, that requires the production of VZV antigenin tissue culture and its subsequent extraction from VZV-infected cells.Moreover, most in house ELISAs use a control for nonspecific antibodybinding, a similarly prepared extract of uninfected cells14.Production of VZV antigen is usually performed in cell lines derivedfrom human fibroblasts (e.g., MRC5 cell line), known to be timeconsuming and very often not so easy to handle21.The increasing use of a VZV vaccine in different populations hasadded to the necessity of measuring anti-VZV antibodies as an indicatorof prior or recurrent infection, to predict susceptibility to disease, and toevaluate immune response to vaccination3,4,5,10,12,16,17,18,22,23.We have developed a cheaper and simple alternative indirect ELISAthat uses the live attenuated VZV vaccine as a coating antigen. The useof a standard reference serum allowed us to produce results ininternational units per millilitre (IU/mL). Finally, with the addition ofan extra step with 8M urea, we could also assess VZV IgG avidity.MATERIAL AND METHODSSerum samples: Two hundred and thirty-nine serum samples weretested: 221 were from healthy adults, 122 (55%) of them who referredhaving had varicella in the past and 99 (45%) who denied previous clinicalVZV infection; the remaining 18 sera were drawn from 12-month-oldinfants without clinical or serologic evidence of previous VZV infection.Serum samples were collected to investigate VZV seroprevalence indifferent populations. All studies were approved by the Ethics Committeeof the Federal University of São Paulo, in São Paulo, Brazil.VZV ELISA kit: A Hemagen indirect ELISA kit to detect IgG VZVantibodies (Columbia, USA) was used according to the manufacturer’sinstructions.High, medium and low callibrators were added together with serumsamples diluted 1 in 41, all in duplicates. A standard curve with acorresponding linear regression curve fit equation was obtained andresults were calculated by interpolation of mean optical density (OD)values onto the curve fit.Results were considered positive if they were higher than 20 arbitraryunits per millilitre (AU/mL), as suggested by the kit’s manufacturer.166ONO, E.; LAFER, M.M.; WECKX, L.Y.; GRANATO, C. & MORAES-PINTO, M.I. - A simple and cheaper in house varicella zoster virus antibody indirect ELISA. Rev. Inst. Med. trop. S.Paulo, 46(3):165-168, 2004.“In House” VZV indirect ELISA: Varicella IgG antibodies wereassessed by an “in house” indirect ELISA. Immulon 2 96-well microtiterplates (Dynex, USA) were coated with varicella vaccine batchVA254A41A, 103,3 PFU/mL (Varilrix, SmithKline Beecham, Belgium)diluted 1:100 in 0.1M carbonate-bicarbonate buffer, pH 9.6, andincubated overnight at 4 °C. Two-fold serial dilutions of serum samplesand of varicella reference serum [in house standard calibrated against“Varicella zoster virus antibody human immunoglobulin- NIBSC reagent(90/690)”] in 0.01M phosphate buffered saline (PBS), pH 7.2 and 0.05%Tween 20 with 1% bovine serum albumin (BSA) were added to the plateand incubated for 1 h at 37 °C. Reference serum was added to 10 wellsand serum samples were added to 3 wells, in all twofold dilutions startingat 1:100.In the next step, alkaline phosphatase conjugated rabbit anti-humanIgG, specific for γ-chains (Dako, Denmark) diluted 1:500 in 0.01M PBS,pH 7.2 and 0.05% Tween 20, was incubated for 1 h at 37 °C. p-nitrophenyl-phosphate disodium (Sigma, USA) in 0.1M diethanolamine,0.005M magnesium chloride buffer, pH 9.8, was used as substrate in aconcentration of 1 mg/mL. OD was read at 405 nm in an immunoreaderELX-800, using 630 nm as a reference filter (Bio-Tek Instruments, USA).Between steps, the plate was washed five times in 0.01M PBS, pH7.2 and 0.05% Tween 20. All solutions were added in a 100 µL volumeto microplate wells. Varicella zoster antibodies were expressed in IU/mL using the curve comparison method to transform optical density inconcentration units. In all plates two blank wells we always present, andmean values were subtracted from all other wells.IgG antibody avidity: To assess varicella IgG avidity, an extra stepwas added to the test. An 8M urea solution in PBS was dispensed to halfof all wells after serum samples had been washed out. IgG avidity indexwas calculated by dividing results obtained with urea addition by resultswithout urea and multiplying them by 100. Serum samples with avidityindices below 30% were considered with low avidity.Protective antibody levels: Because an internationally acceptedantibody level that predicts previous contact with wild varicella zostervirus or vaccine is not available, we have established a cutoff based onthe following. Nineteen children who had been regularly followed upfrom birth and did not have evidence of previous varicella infectionassessed both by clinical exam and repeated serologic testing during thefirst year of life were tested for varicella at 12 months of age. Cutoff of0.2 IU/mL was calculated by adding mean antibody levels (0.04 IU/mL)to 3 standard deviations (3 x 0.04 IU/mL).Assessment of VZV ELISA specificity: Because cross-reactivityamong Herpesvirus antibodies has been described1,2, we have tested 8serum samples for cytomegalovirus (CMV) IgG (CMV-IgG, Asxym,Immunofluorimetry with microparticles) and Epstein Barr viral capsidantigen (VCA) IgG (Indirect Immunofluorescence, “home made” withH3PR1 cells). Five sera were from adults without history of VZVinfection who proved to be seronegative for IgG VZV by our “in houseELISA”; the other 3 sera were from 15 month old infants who had beenvaccinated at 12 month of age with VZV vaccine (Varilrix, SmithKlineBeecham, Belgium) and were seropositive for IgG by our “in house”VZV ELISA.RESULTSComparison of “in house” ELISA with commercial ELISA kit: Inhouse ELISA and commercial ELISA kit showed an agreement of 94%.When compared with commercial kit, our in house ELISA showed asensitivity of 95% and specifity of 87%. Only 13 out of 239 samples testedproduced discordant results when assessed by both ELISAs (Table 1).True positive and true negative samples assessed by “in house”ELISA and commercial ELISA kit: As described in the Materials andMethods session, we sellected among the samples tested by both methodsthose that were supposedly positive (adults who referred previous clinicalVZV infection) and those that were supposedly negative (12-monthinfants without clinical and laboratory evidence of VZV infection).While the commercial ELISA kit detected 98% (120 in 122)supposedly positive samples and 89% (16 in 18) supposedly negativesamples, our in house ELISA detected 98% (120 in 122) and 100% (18in 18) of the same serum samples (Table 2).Assessment of VZV ELISA specifity: Among the 5 samples fromVZV IgG seronegative adults, 4 were positive from CMV IgG and onewas negative. All 5 sera were also positive for VCA IgG antibodies.Among the 3 samples from VZV IgG seropositive children, 2 wereseronegative for CMV IgG and one was positive; 2 were seronegativefor VCA IgG and one was weakly positive (Table 3).Cost assessment of “in house” ELISA and commercial ELISAkit: Our in house ELISA proved to be cheaper than the commercial kit,with a cost per sample of US$0.99 and US$2.70, respectively(Table 4).Table 1Relative performance of in house indirect ELISA in detecting varicella zosterIgG antibodies on the basis of a commercial indirect ELISACommercial Kit of indirect ELISAPositive Negative TotalIn house Positive 172 08 180indirect ELISA Negative 005 54 059Total 177 62 239Sensitivity: 97%; Specificity: 87%; Agreement: 94%Table 2Relative performance of in house indirect ELISA and a commercial kit ofindirect ELISA in detecting varicella zoster antibodies in samples fromindividuals with previous varicella infection and in children without previousvaricella infection at 12 months of ageSamples In house ELISA Commercial KitSupposedly positive  120/122 (98%)  120/122 (98%)(previous varicella infection)Supposedly negative  18/18 (100%)  16/18 (89%)(12 months of age withouthistory of varicella infection)ONO, E.; LAFER, M.M.; WECKX, L.Y.; GRANATO, C. & MORAES-PINTO, M.I. - A simple and cheaper in house varicella zoster virus antibody indirect ELISA. Rev. Inst. Med. trop. S.Paulo, 46(3):165-168, 2004.167IgG VZV antibody avidity: By the addition of an extra 8M ureastep, we were able to assess IgG VZV antibody avidity using the inhouse ELISA. As shown on Table 5, individuals with past primary VZVinfection had high (above 60%) or intermediate IgG avidity (between30% and 60%). Those with recent or concurrent infection showed lowIgG avidity (below 30%).DISCUSSIONWe have developed a modified indirect ELISA that proved to besimple, cheap and easy to perform. Most “in house” ELISAs that detectantibodies against viruses require the production and extraction of virusantigen6,20. Many laboratories do not have tissue culture facilities,precluding the use of such techniques.However, previous immunoassays well accepted in the literature haveused alternatives for less accessible reagents. In special, toxoid vaccineshave been routinely used in ELISAs to detect tetanus and diphtheriaantibodies6-9,15.Live attenuated vaccines against measles and mumps have also beenused as antigens for “in vitro” T cell proliferation assays, with goodresults11.Our modified ELISA had a good agreement when compared with acommercial ELISA kit and did not show cross-reactivity with antibodiesfrom other Herpesviruses. Moreover, it proved to be superior than thekit when true negative samples were tested. As suggested by others19,we used sera from 12 month-old infants without evidence of previousVZV infection.Another advantage of the test we have developed was the use of areference serum with a known concentration antibodies measured in IU/mL. As it is well known, that allows the comparison of results obtainedin different countries13.Finally, introducing an extra step allowed the assessment of VZVIgG avidity without excessive costs.In sum, we have developed a cheap and accurate alternative ELISAto measure VZV antibodies that proved suitable for most laboratorieswithout the cumbersome needs of tissue culture, that produced results inIU/mL and permitted the assess of IgG avidity.RESUMODesenvolvimento de ELISA indireto simples e de baixo custo paradetecção de anticorpos anti-varicela zosterDesenvolvemos um ensaio imunoenzimático (ELISA) indiretosimples e econômico para detecção de anticorpos contra o vírus davaricela zoster (VVZ) que utiliza a vacina contendo o vírus vivo atenuadocomo antígeno para recobrir a placa.Este ELISA mostrou uma concordância de 94% quando comparadocom um kit de ELISA comercial para anticorpos contra varicela. Alémdisso, nosso ELISA mostrou ser mais confiável que o kit quando amostrascomprovadamente negativas foram testadas.O uso de um soro padrão de referência, calibrado em unidadesinternacionais por mililitro, possibilitou também que os resultadospudessem ser comparados com outros estudos. O acréscimo de uma etapaextra com solução de uréia 8M permitiu avaliação de avidez de IgG paraVVZ sem custos excessivos.O custo por amostra para testar IgG contra VVZ com nosso ELISAfoi 2,7 vezes mais barato quando comparado com o kit comercial.Table 3 Assessment of cross-reactivity between VZV IgG antibodies detected by ourELISA and cytomegalovirus (CMV) IgG and Epstein Barr viral capsidantigen (VCA) IgGVZV IgG antibodies CMV IgG VCA IgGNegative (n = 5) 4 positive 5 positive1 negative 0 negativePositive (n = 3)* 2 negative 2 negative1 positive 1 weakly positive* Serum sample tested positive for CMV IgG was not the same tested weaklypositive for Epstein Barr VCA IgG.Table 4Compared costs of varicella zoster in house indirect ELISA andcommercial KitCosts and sample In house Commercialtested per microplate ELISA kitCost per microplate US$ 27.67 US$ 243.33Number of sample per microplate 28 90Cost per sample US$ 0.99 US$ 2.70Table 5IgG VZV antibody avidity assessed in different patientsPatient VZV antibodies VZV IgG(IU/mL) avidity (%)Adult A with VZV 1.4 62infection in childhoodAdult B with VZV 1.1 48infection in childhoodAdult C with concurrent 1.1 62herpes zoster infectionChild A with concurrent 11.1 1.3VZV infectionChild B 2 months 3.2 6.1after infection VZV168ONO, E.; LAFER, M.M.; WECKX, L.Y.; GRANATO, C. & MORAES-PINTO, M.I. - A simple and cheaper in house varicella zoster virus antibody indirect ELISA. Rev. Inst. Med. trop. S.Paulo, 46(3):165-168, 2004.REFERENCES1. AMERICAN ACADEMY OF PEDIATRICS - Human Herpesvirus 6 (including Roseola)and 7. In: PICKERING, L.K., ed. Red Book: 2003 Report of the Committee onInfectious Diseases. 26. ed. Elk Grove Village, American Academy of Pediatrics,2003. p. 357-358.2. AMERICAN ACADEMY OF PEDIATRICS - Varicella-zoster infections. In:PICKERING, L.K., ed. Red Book: 2003 Report of the Committee on InfectiousDiseases. 26. ed. Elk Grove Village, American Academy of Pediatrics, 2003. p. 672-686.3. AMPOFO, K.; SAIMAN, L.; LARUSSA, P. et al. - Persistence of immunity to liveattenuated varicella vaccine in healthy adults. Clin. infect. Dis., 34: 774-779, 2002.4. ASANO, Y.; SUGA, S.; YOSHIKAWA, T. et al. - Experience and reason: twenty-yearfollow-up of protective immunity of the Oka strain live varicella vaccine. Pediatrics,94: 524-526, 1994.5. BOGGER-GOREN, S.; BABA, K.; HURLEY, P. et al. - Antibody response to varicella-zoster virus after natural or vaccine-induced infection. J. infect. Dis., 146: 260-265,1982.6. DE MORAES-PINTO, M.I.; ORUAMABO, R.S.; IGBAGIRI, F.P. et al. - Neonatal tetanusdespite immunization and protective antitoxin antibody. J. infect. Dis., 171: 1076-1077, 1995.7. DE MORAES-PINTO, M.I.; ALMEIDA, A.C.M.; KENJ, G. et al. - Placental transferand maternally acquired neonatal IgG immunity in human immunodeficiency virusinfection. J. infect. Dis., 173: 1077-1084; 1996.8. GALAZKA, A.M. - The immunological basis for immunization series. Module 2:Diphtheria. Geneva, World Health Organization, 1993.9. GALAZKA, A.M. - The immunological basis for immunization series. Module 3: Tetanus.Geneva, World Health Organization, 1993.10. GALIL, K.; LEE, B.; STRINE, T. et al. – Outbreak of varicella at a day-care centerdespite vaccination. New Engl. J. Med., 347: 1909-1915, 2002.11. GANS, H.; YASUKAWA, L.; RINKI, M. et al. - Immune responses to measles andmumps vaccination of infants at 6, 9 and 12 months. J. infect. Dis., 184: 817-826,2001.12. GERSHON, A.A.; STEINBERG, S.P. & GELB, L. - Live attenuated varicella vaccineuse in immunocompromised children and adults. Pediatrics, 78: 757-762, 1986.13. KEMENY, D.M. - Quantitation. In:___________ A pratical guide to ELISA. Oxford,Pergamon, 1991. p. 57-67.14. KRAH, D.L. - Assays for antibodies to varicella-zoster virus. Infect. Dis. Clin. N. Amer.,10: 507-527, 1996.15. KRISTIANSEN, M.; AGGERBECK, H. & HERON, I. - Improved ELISA fordetermination of anti-diphtheria and/or anti-tetanus antitoxin antibodies in sera. Actapath. microbiol. immunol. scand., 105: 843-853, 1997.16. KUTER, B.; MATTHEWS, H.; SHINEFIELD, H. et al. - Ten year follow-up of healthychildren who received one or two injections of varicella vaccine. Pediat. infect. Dis.J., 23: 132-137, 2004.17. LEVIN, M.J.; GERSHON, A.A.; WEINBERG, A. et al. - Immunization of HIV-infectedchildren with varicella vaccine. J. Pediat., 139: 305-310, 2001.18. REIS, A.D.; PANNUTI, C.S. & SOUZA, V.A.U.F. - Prevalência de anticorpos para ovírus da varicela-zoster em adultos jovens de diferentes regiões climáticas brasileiras.Rev. Soc. bras. Med. trop., 36: 317-320, 2003.19. ROBERTSON, P.W.; BELL, S.M. & FERSON, M.J. - A method for determining the cut-off value of a varicella-zoster virus IgG enzyme immunoassay for immune status. J.virol. Meth., 26: 115-118, 1989.20. SHANLEY, J.; MYERS, M.; EDMOND, B. & STEELE, R. - Enzyme-linkedimmunosorbent assay for detection of antibody to varicella-zoster virus. J. clin.Microbiol., 15: 208-211, 1982.21. SHEHAB, Z. & BRUNELL, P.A. - Enzyme-linked immunosorbent assay for susceptibilityto varicella. J. infect. Dis., 148: 472-476, 1983.22. WATSON, B.; KELLER, P.M.; ELLIS, R.W. & STARR, S.E. - Cell-mediated immuneresponses after immunization of healthy seronegative children with varicella vaccine:kinetics and specificity. J. infect. Dis., 162: 794-799, 1990.23. ZERBONI, L.; NADER, S.; AOKI, K. & ARVIN, A.M. - Analysis of the persistence ofhumoral and cellular immunity in children and adults immunized with varicellavaccine. J. infect. Dis., 177: 1701-1704, 1998.Received: 5 January 2004Accepted: 14 May 2004

A simple and cheaper in house varicella zoster virus antibody indirect ELISA

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A simple and cheaper in house varicella zoster virus antibody indirect ELISA

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