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BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors nonprofit publishers academic institutions research libraries and research funders in the common goal of maximizing access to critical research Development of a Competitive Enzyme Linked Immunosorbent Assay for Detection of Turkey Coronavirus Antibodies Author s James S Guy Lynda G Smith Jamie J Breslin and Somsak Pakpinyo Source Avian Diseases 46 2 334 341 Published By American Association of Avian Pathologists DOI http dx doi org 10 1637 0005 2086 2002 046 0334 DOACEL 2 0 CO 2 URL http www bioone org doi full 10 1637 0005 2086 282002 29046 5B0334 3ADOACEL 5D2 0 CO 3B2 BioOne www bioone org is a nonprofit online aggregation of core research in the biological ecological and environmental sciences BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies associations museums institutions and presses Your use of this PDF the BioOne Web site and all posted and associated content indicates your acceptance of BioOne s Terms of Use available at www bioone org page terms of use Usage of BioOne content is strictly limited to personal educational and non commercial use Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder 334 AVIAN DISEASES 46 334 341 2002 Development of a Competitive Enzyme Linked Immunosorbent Assay for Detection of Turkey Coronavirus Antibodies James S Guy Lynda G Smith Jamie J Breslin and Somsak Pakpinyo Department of Microbiology Pathology and Parasitology College of Veterinary Medicine North Carolina State University Raleigh NC 27606 Received 18 July 2001 SUMMARY A competitive enzyme linked immunosorbent assay cELISA was developed for detection of turkey coronavirus TCV antibodies The cELISA utilized a recombinant baculovirus Autographa californica nuclear polyhedrosis virus expressed TCV nucleocapsid N protein and biotin labeled TCV N protein specific monoclonal antibody Sensitivity and specificity of the cELISA for detection of TCV antibodies were determined by comparison with the indirect fluorescent antibody test IFAT with 1269 reference experimentally de rived and field origin sera Sera with discordant cELISA and IFAT results were further evaluated by western immunoblot analyses The cELISA detected antibodies specific for TCV and infectious bronchitis virus a closely related coronavirus but did not detect antibodies specific for other avian viruses A high degree of concordance was observed between the cELISA and IFAT sensitivity and specificity of the cELISA relative to IFAT were 92 9 and 96 2 respectively Western immunoblot analyses provided additional evidence of cELISA specificity The findings indicate that the cELISA is a rapid sensitive and specific serologic test for detection of TCV antibodies in turkeys RESUMEN Desarrollo de un inmunoensayo competitivo ligado a enzimas para la de teccio n de anticuerpos contra coronavirus de los pavos Se desarrollo un inmunoensayo competitivo ligado a enzimas de las siglas en ingle s c ELISA para la deteccio n de anticuerpos contra coronavirus de los pavos La prueba de cELISA utilizo un baculovirus recombinante virus de la polihedrosis nuclear de Autographa californica que expresaba la prote na N de la nucleoca pside del coronavirus de los pavos adema s de un anticuerpo monoclonal contra la misma prote na marcado con biotina La sensibilidad y la especificidad de la prueba de cELISA para la deteccio n de anticuerpos fue comparada con la prueba indirecta de inmunofluorescencia en 1269 sueros de referencia provenientes de aves infectadas experimentalmente y de aves mantenidas bajo condiciones de campo Los sueros que mostraron resultados discordantes con cELISA y la te cnica de in munofluorescencia se evaluaron posteriormente por ana lisis de inmunoelectrotransferencia puntual western La prueba de cELISA detecto anticuerpos espec ficos para el coronavirus de pavo y el virus de la bronquitis infecciosa que es un coronavirus cercanamente relacionado sin embargo la prueba no detecto anticuerpos espec ficos contra otros virus aviares Se ob servo un alto grado de concordancia entre cELISA y la prueba de inmunofluorescencia la sensibilidad y especificidad de cELISA con relacio n a inmunofluorescencia fueron de 92 9 y 96 2 respectivamente El ana lisis de inmunotransferencia aporto evidencia adicional acer ca de la especificidad de cELISA Los resultados indican que la te cnica de cELISA es una prueba serolo gica ra pida sensible y espec fica para la deteccio n de anticuerpos contra coro navirus de los pavos Key words turkey coronavirus baculovirus enzyme linked immunosorbent assay Abbreviations cELISA H11005 competitive enzyme linked immunosorbent assay ELISA H11005 enzyme linked immunosorbent assay IBV H11005 infectious bronchitis virus IFAT H11005 indirect fluorescent antibody test IgG H11005 immunoglobulin G M H11005 membrane MAb H11005 monoclonal Resources used to support this research were provided by United States Poultry and Egg Association 335cELISA for turkey coronavirus antibody N H11005 nucleocapsid PBS H11005 phosphate buffered saline PBST H11005 phosphate buffered saline containing 0 05 Tween 20 PE H11005 postexposure rBTCV N H11005 recombinant baculo virus containing TCV N gene SDS PAGE H11005 sodium dodecyl sulfate polyacrylamide gel electrophoresis SF 9 H11005 Spodoptera frugiperda SPF H11005 specific pathogen free TCV H11005 turkey coronavirus Turkey coronavirus TCV is the cause of an acute highly contagious enteric disease of tur keys that initially was referred to as bluecomb disease 13 Bluecomb disease was first iden tified in turkeys in 1951 and a coronavirus was determined to be the cause of the disease in 1973 13 In recent years TCV has been in creasingly recognized as an important cause of enteric disease in turkeys resulting in economic loss due to impaired growth and poor feed con version TCV is a member of the family Coronaviri dae The Coronaviridae comprise a large group of RNA viruses that infect a wide variety of avian and mammalian species 16 19 Coro naviruses have a distinctive morphology they are pleomorphic enveloped particles 80 220 nm in diameter with long club shaped surface projections approximately 20 nm in length 16 19 The coronavirus genome consists of a positive sense single stranded RNA molecule 27 30 kb in size Coronavirus virions are com posed of three major structural proteins surface glycoprotein 90 180 kD integral membrane M protein 20 35 kD and nucleocapsid N protein 50 60 kD Additionally some coro naviruses also contain a fourth major structural protein the hemagglutinin esterase protein 120 140 kD 16 The coronavirus N protein binds to virion RNA and provides the structural basis for the helical nucleocapsid 12 It is the most abun dant viral polypeptide in coronavirus infected cells and it is immunodominant 11 12 Re cently the amino acid sequences of TCV M and N proteins were determined to be very sim ilar H1102290 identity to infectious bronchitis vi rus IBV M and N proteins 1 2 Addition ally the TCV N protein was determined to have a molecular weight of approximately 52 kD 3 Sequence analysis of other TCV pro teins has not been reported Serologic diagnosis of TCV infection cur rently is accomplished by indirect fluorescent antibody IFAT procedures 8 14 These IFAT procedures are labor intensive and time consuming In addition they require expensive equipment highly trained personnel and an antigen obtained from frozen sections of TCV infected turkey embryo intestines or epithelial cells exfoliated from bursae of Fabricius of TCV infected turkeys 8 14 A TCV specific enzyme linked immunosorbent assay ELISA would be an improved method for serologic di agnosis however the production of large quan tities of high quality antigen for this procedure has been hampered by the inability to propa gate TCV in cell culture Recently the TCV N protein was cloned and expressed in a baculovirus expression system 3 The present paper describes the develop ment of a competitive ELISA cELISA with recombinant baculovirus expressed TCV N protein MATERIALS AND METHODS Virus TCV NC95 was isolated from enteritis affected turkeys and propagated by amniotic inocu lation of embryonated turkey eggs 9 Recombinant antigen Recombinant baculovi rus Autographa californica nuclear polyhedrosis virus expressing the TCV N protein rBTCV N 3 was propagated in suspension cultures of serum free adapted Spodoptera frugiperda SF 9 insect cells Gibco BRL Grand Island NY SF 9 cells were grown in serum free medium SF 900 SFM medium Gibco BRL supplemented with penicillin 100 units ml streptomycin sulfate 100 H9262g ml and am photericin B 0 25 H9262g ml Cells were grown in or bital shaker flasks with stirring at approximately 150 revolutions per minute 28 C in a non CO 2 ambi ent air incubator SF 9 cells were grown to a density of approximately 2 H11003 10 6 cells ml and infected with rBTCV N at a multiplicity of infection of approxi mately 1 After incubation for 52 54 hr cells were pelleted by centrifugation 800 H11003 g for 10 min at 4 C and resuspended in lysis buffer consisting of 0 1 Triton X 100 in phosphate buffered saline PBS with protease inhibitors phenylmethyl sulfonylfluo ride 100 H9262g ml leupeptin 0 5 H9262g ml pepstatin A 1 H9262g ml Cell debris was removed by centrifuga tion 800 H11003 g for 10 min at 4 C and supernatant was stored at H1100275 C Western immunoblotting was done to confirm expression of TCV N protein 336 J S Guy et al Monoclonal antibody MAb MAbs specific for TCV N protein were prepared by the procedure de scribed by Carter et al 5 Briefly recombinant TCV N protein for immunization of mice was harvested from rBTCV N infected SF9 cells as described above and concentrated by ultrafiltration with a filter with a molecular weight cutoff of 10 000 Splenocytes were collected from immunized BALB c mice and fused with murine myeloma cells Hybridoma colo nies secreting antibodies specific for TCV were de tected by assay of culture supernatant fluids by IFAT 3 Each positive hybridoma colony was cloned twice by limiting dilution and ascites fluid was produced by intraperitoneal injection of approximately 10 7 hy bridoma cells into pristane primed mice Specificity of MAbs for TCV proteins was deter mined by western immunoblot analysis The immu noglobulin subclass of TCV specific MAbs was de termined by a commercial ELISA test system MonoAb ID EIA kit Zymed Laboratories San Francisco CA used according to the manufacturer s instructions Biotin labeling of TCV specific MAb MAb 4 23 was purified from mouse ascites fluid and con jugated to biotin by Kirkegaard and Perry Labora tories Gaithersburg MD 10 Reference experimentally derived and field origin sera Reference antisera against TCV strains NC95 Minnesota were prepared in 4 wk old spe cific pathogen free SPF chickens SPAFAS Inc Norwich CT as previously described 9 Antisera prepared in SPF chickens against IBV Massachu setts Arkansas Connecticut JMK avian reovirus avian influenza avian adenovirus 1 avian paramyxo virus 3 Newcastle disease virus and avian encepha lomyelitis virus were obtained from SPAFAS Inc Negative control serum from unimmunized SPF chickens was obtained from SPAFAS Inc Sera n H11005 67 were collected from turkeys exper imentally infected with TCV NC95 at designated times postexposure PE Briefly 10 2 wk old turkeys were orally inoculated with approximately 2 8 H11003 10 4 embryo infectious doses of TCV NC95 Serum samples were collected from each turkey on days 0 4 7 10 14 21 and 28 PE A total of 1189 field origin sera were obtained from turkeys in North Carolina South Carolina and Virginia Field origin sera originally were received for serologic evaluation by the IFAT All sera were stored at H1100220 C until tested cELISA Optimal concentrations of antigen bio tin labeled MAb 4 23 and streptavidin horseradish peroxidase were determined by checkerboard titration as described 4 The rBTCV N antigen was diluted 1 1280 in 0 2 M carbonate 0 2 M bicarbonate buff er pH 9 6 75 H9262l was added to each well in 96 well ELISA plates Pro BindH23042 Assay Plate FalconH23041 Bec ton Dickinson and Co Lincoln Park NJ and in cubated overnight at 4 C Antigen coated plates were washed three times with 0 01 M PBS pH 7 2 con taining 0 05 Tween 20 PBST then 200 H9262lof block buffer PBST containing 1 nonfat dry milk was added to each well and incubated for 1 hr at 25 C Plates were washed three times with PBST Posi tive and negative control sera and test sera were di luted 1 10 in block buffer and 50 H9262l samples of each were placed in duplicate wells a diluent control con sisting of block buffer 50 H9262l also was placed in du plicate wells Plates were incubated for 60 min at 25 C with gentle shaking then washed three times with PBST Biotin labeled MAb 4 23 50 H9262l diluted 1 160 in block buffer was added to each well except diluent wells and incubated for 60 min at 25 C with gentle shaking Plates were washed three times with PBST and 75 H9262l streptavidin horseradish peroxidase Kirkegaard and Perry Laboratories Inc diluted 1 200 in block buffer was added to each well Plates were incubated for 30 min at 37 C then washed three times with PBST ABTS 2 2H11032 azino bis 3 ethylbenzthiazoline 6 sulfonic acid substrate Kir kegaard and Perry Laboratories Inc 100 H9262l was added to each well color development was stopped after 20 min with 1 sodium dodecyl sulfate w v in water Optical densities of wells were read on an ELISA reader BT 2000 MicroKinetics Reader Fisher Scientific Norcross GA at 405 nm Optical densi ties of duplicate wells including positive and negative control sera wells and diluent wells were averaged Percentage of inhibition of optical densities of test serum wells relative to negative control serum was calculated after subtracting the diluent control which was subtracted from all test and control well averages to yield a corrected value Percentage of inhibition was calculated as 100 H11002 100 H11003 test serum H11002 dil uent negative control H11002 diluent Sera were consid ered to be positive if inhibition H1135045 was observed and negative if inhibition H1102145 was observed IFAT The IFAT was performed as previously de scribed with epithelial cells exfoliated from bursae of Fabricius of TCV infected turkeys 3 Sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS PAGE and western im munoblot assay TCV was partially purified from infected turkey embryo intestines as described 1 Proteins were analyzed on a 4 stacking 10 re solving gel by a discontinuous SDS PAGE system 15 Electrophoresis was performed with the Mini ProteanH23041 II Dual Slab Cell apparatus BioRad Lab oratories Richmond CA Electrophoretic separation of proteins was performed for 1 hr at 200 volts After SDS PAGE proteins were transferred onto a 0 45 H9262m Trans BlotH23041 nitrocellulose membrane BioRad Laboratories with a Mini Trans BlotH23041 Electropho retic Transfer Cell BioRad Laboratories Transfer was accomplished in 1 hr at 100 volts in transfer buffer 25 mM Tris pH 7 5 192 mM glycine 20 337cELISA for turkey coronavirus Fig 1 Western immunoblot analyses showing specificity of MAbs 1 01 and 4 23 for TCV proteins TCV proteins were separated by SDS PAGE and an alyzed by immunoblotting Lane 1 chicken anti TCV hyperimmune serum lane 2 MAb 1 01 lane 3 MAb 4 23 lane 4 infectious laryngotracheitis vi rus specific MAb negative control Molecular weight scale kilodaltons shown at left TCV N pro tein is indicated by arrowhead methanol Membranes were air dried and incubated for 3 hr at room temperature in block solution con sisting of 1 nonfat dried milk and 2 fetal bovine serum in PBS Block solution was decanted and membranes were incubated for 2 hr at room tem perature with MAb chicken serum or turkey serum diluted 1 25 in block solution Membranes were washed for 15 min with four changes of TNT buffer 10 mM Tris HCl pH 7 5 0 5 M NaCl and 0 05 Tween 20 then incubated for 1 5 hr at room tem perature with horseradish peroxidase labeled goat anti mouse immunoglobulin G IgG or horseradish peroxidase labeled goat anti chicken IgG Kirkegaard and Perry Laboratories diluted 1 2000 in block so lution Membranes were washed as above and reacted with a solution of 3 3H11032 diaminobenzidine Vector Laboratories Burlingame CA for 2 5 min The re action was stopped by washing membranes in dH 2 O for 5 min Statistics Sensitivity and specificity were calcu lated with standard formulae 6 RESULTS MAb production and characterization Two hybridoma cell lines were identified that secreted antibodies MAb 1 01 MAb 4 23 spe cific for TCV These cell lines were selected on the basis of a strong reaction of antibody to TCV antigens as determined by IFAT and ab sence of specific reaction when IFAT was per formed with uninfected cells Western immunoblot analysis demonstrated specificity of MAbs 1 01 and 4 23 for TCV N protein Fig 1 MAbs 1 01 and 4 23 specifi cally reacted with TCV proteins approximately 52 and 46 kD in size Both MAbs 1 01 and 4 23 were determined to be IgG1 isotypes MAb 4 23 was arbitrarily chosen for conjuga tion to biotin and use in the cELISA Comparison of cELISA and IFAT test re sults All cELISA and IFAT test results for ref erence antisera were in agreement Table 1 The cELISA and IFAT detected antibodies in all antisera prepared against TCV strains NC95 Minnesota and IBV strains Connect icut Massachusetts Arkansas JMK No anti bodies inhibition H1102145 were detected in an tisera prepared against avian reovirus avian in fluenza virus avian adenovirus 1 avian para myxovirus 3 Newcastle disease virus avian encephalomyelitis virus and negative control serum The cELISA IFAT and western immunoblot test results for sera collected from turkeys ex perimentally infected with TCV are shown in Table 2 Sera were collected from experimen tally infected turkeys on days 0 4 7 10 14 21 and 28 PE No antibodies to TCV were detected in turkeys on days 0 4 and 7 PE by cELISA and IFAT Seroconversion to TCV was detectable by both cELISA and IFAT beginning on day 10 PE however cELISA detected more antibody positive birds at this time 10 10 100 than did IFAT 5 10 50 All birds tested by cELISA and IFAT at subsequent time intervals days 14 21 and 28 PE were positive by both tests Western immunoblot analysis detected sero conversion to TCV in 3 of 10 30 experi mentally infected turkeys beginning on day 7 PE Table 2 Fig 2 All birds tested at subse quent time intervals were positive by western immunoblot analysis Table 2 338 J S Guy et al Table 1 cELISA and IFAT results for reference antisera prepared in SPF chickens against TCV IBV and other avian viruses Antiserum cELISA inhibition A IFAT TCV NC95 TCV Minnesota IBV Massachusetts IBV Connecticut IBV Arkansas IBV JMK Reovirus Adenovirus 1 Paramyxovirus 3 Avian influenza virus Newcastle disease virus Avian encephalomyelitis Negative serum H11001 84 H11001 98 H11001 99 H11001 86 H11001 94 H11001 96 H11002 H110023 H11002 19 H11002 2 H11002 H110022 H11002 8 H11002 H110027 H11002 H110024 H11001 H11001 H11001 H11001 H11001 H11001 H11002 H11002 H11002 H11002 H11002 H11002 H11002 A H11002H11005negative H1102145 inhibition H11001H11005positive H1135045 inhibition Table 2 Detection of TCV specific antibodies in sera of experimentally infected turkeys by cELISA IFAT and western immunoblotting A Days post exposure No positive no tested positive cELISA IFAT Immunoblotting 0 4 7 10 14 21 28 0 10 0 0 10 0 0 10 0 10 10 100 10 10 100 10 10 100 7 7 100 0 10 0 0 10 0 0 10 0 5 10 50 10 10 100 10 10 100 7 7 100 0 10 0 0 10 0 3 10 30 10 10 100 10 10 100 10 10 100 7 7 100 A Two week old turkeys were orally inoculated with TCV NC95 Serum was collected at intervals from day 0 to day 28 postexposure On the basis of cELISA IFAT and western immunoblot results for sera collected from ex perimentally infected turk