Production of monoclonal antibodies against conserved components of infectious bronchitis virus

Produção de anticorpos monoclonais contra componentes do vírus da bronquite infecciosa das aves

Production of monoclonal antibodies against conserved components of infectious bronchitis virus

[Produção de anticorpos monoclonais contra componentes do vírus da bronquite infecciosa das aves]

C.M. Souza, F.R.T. Rocha, N.R.S. Martins^*, J.S. Resende, M.A. Jorge, A.P. Rampinelli

Escola de Veterinária da UFMG

Caixa Postal 567

30123-970 - Belo Horizonte, MG

Recebido para publicação em 11 de agosto de 2000.

ABSTRACT

Murine hybridomas producing IgG1 monoclonal antibodies (Mabs) against N and S₂ proteins (53KDa and 82KDa, respectively) from avian infection bronchitis virus (IBV) strain M41 were generated by the fusion of a myeloma cell line (Sp2/0-Ag14) with spleen cells from Balb/c mice previously immunized with whole virus IBV M41. Post-fusion screening criterion was by ELISA and 36 positive hybrids were generated after fusions. Two hybrids specific to N (N3F10) and S₂ (S12B2) proteins from M41 (serotype Massachusetts) were selected by western blotting. These Mabs recognized the Ark-99 (serotype Arkansas) and A5968 (serotype Connecticut) IBV strains in addition to M41. By ELISA, the Mab against the S₂ (S12B2) recognized all reference and Brazilian strains (M41, SE-17, H52, 297, 283, PM-1, PM-2, PM-3, 351, 29-78 E 327) studied, while the Mab against N recognized only six (M41, SE-17, H52, 283, 327 e 297) strains. The Mab against S₂ may become a useful tool for IBV detection on the routine diagnosis of infectious bronchitis, especially for helping the differential diagnosis of clinically and pathologically confusing diseases, while the Mab against N (N3F10) recognized a probably less conserved region among the strains and may be interesting to comparing IBV isolates.

Keywords: Chicken, monoclonal antibody, infectious bronchitis, nucleoprotein, spike glicoprotein S₂.

RESUMO

Foram produzidos anticorpos monoclonais (AcM) da subclasse IgG1 contra as proteínas N (53KDa) e S₂ (82KDa) do vírus da bronquite infecciosa das galinhas (VBIG) amostra M41. Os híbridos secretores originaram-se da fusão entre células de mielomas da linhagem Sp2/0-Ag14 e linfócitos B de camundongos Balb/c previamente imunizados com o vírus completo. O primeiro critério de seleção foi por ELISA, no qual 36 híbridos originados de duas fusões reagiram positivamente; destes, foram selecionados dois AcM que reagiram contra as proteínas N (N3D4) e S₂ (S12B2) do VBIG da amostra M41 (sorotipo Massachusetts) no western blotting. Os mesmos AcM foram também capazes de reconhecer as estirpes Ark-99 (sorotipo Arkansas) e A5968 (sorotipo Connecticut) do VBIG no western blotting. No ELISA, o AcM contra S₂ (S12b2) reconheceu 11 estirpes das 11 estudadas (M41, SE-17, H52, 297, 283, PM-1, PM-2, PM-3, 351, 29-78 E 327), enquanto o AcM contra a proteína N reconheceu apenas seis estirpes (M41, SE-17, H52, 283, 327 e 297). O anticorpo monoclonal contra S₂ comportou-se como uma boa ferramenta de diagnóstico do VBIG, independentemente do sorotipo que pode ser aplicado em ensaios de rotina laboratorial, especialmente no diagnóstico das doenças que se confundem com a BIG nas galinhas, enquanto o AcM contra N (N3F10) demonstrou reconhecer uma região menos conservada entre as estirpes de VBIG e pode ser útil em estudos comparativos entre isolados de VBIG.

Palavras-chave: Galinha, anticorpo monoclonal, bronquite infecciosa, nucleoproteína, glicoproteína S₂.

INTRODUCTION

Infectious bronchitis (IB) is a viral disease caused by infectious bronchitis virus (IBV) and the highly transmissible nature of the disease, the occurrence of multiple serotypes and the mutable character of genes of infection-related IBV antigens have complicated and increased the cost with disease outbreaks and have impaired its prevention through immunization (Cavanagh & Naqi, 1997). In order to obtain an adequate control of IB, fast, precise, sensitive and specific diagnostic methods are required, in order to reduce the economic losses, prompting sanitary measures to be taken. IBV typification may be performed by virus neutralization tests in embrionating chicken eggs (Cunningham, 1970) or in tracheal rings cultures (Darbyshire et al., 1979), hemagglutination inhibition (Alexander & Chettle, 1977; Lashgari & Newman, 1984), immunofluorescence in allantoic cells (Clarke et al., 1972) or in situ (Dhinakar et al., 1996), ELISA (Mockett et al., 1981) and more recently polimerase chain reaction (PCR) followed by restriction fragments length polymorphism analyses as tool to IBV detection and typing (Kwon et al., 1993). Monoclonal antibodies (Mab) have been used in both diagnosis and typing IBV (Karaca et al., 1992; Koch et al., 1990). Enzyme immunoassays using Mab enable fast and precise IBV detection (Koch et al., 1991b) and typing (Koch et al., 1991a), which are urgent for rapidly providing reliable answers to the field veterinarian. Considering the above, this study had the objective of producing Mab(s) specific to conserved structures (N e S₂) of the IBV M41 strain, to be used as tools to IBV diagnosis.

MATERIALS AND METHODS

IBV M41 strain (Massachusetts serotype), Ark-99 (Arkansas serotype) and A5968 (Connecticut serotype) were obtained from the American Tissue Type Collection (ATCC, USA) with permission from the Brazilian Ministry of Agriculture (MAA, Brazil). Brazilian isolates of IBV from natural IB field cases (Table 1) were evaluated using the Mab produced in this experiment.

Antigens were prepared as described previously (Cavanagh, 1984). Newcastle disease virus (NDV) strain La Sota was prepared similarly and used as negative antigen control. Viruses were grown in the allantoic cavity of SPF/ECE. IBV strains (10²DIE₅₀) and incubated for 72 hours/37°C. Allantoic fluids (AF) were collected in pools of each strain in ice bath and centrifuged at 5,000 x g/30 minutes for clarification. Supernatants were concentrated with buffered saturated ammonium sulfate solution to a final concentration of 36% and precipitated (10,000 x g/30 minutes) and the sediment reconstituted and dialysed in dialysis tubing (Thomas, USA) three times against PBS pH 7.2. PMSF (Phenylmethylsulfonyl fluoride, Sigma, USA) was added to 0.2 M and aliquots were conserved in liquid nitrogen. Virus purification was performed in sucrose density gradient (SDG) purification from 25 a 55% in NET (NaCl 100mM, EDTA 1mM, Tris-HCl 10 mM, pH 7.4), centrifuged at 40,000 x g/4ºC/16 hours (SORVALL Ultra Pro 80, rotor AH-629, DuPont, USA) with fractions evaluated at 280nm for protein detection. SDG fractions were assayed in ELISA for IBV specificity employing M41 specific Mab A38 (kindly donated by the Institute for Animal Health, UK) and those specifically detected were pooled and used as IBV concentrated purified antigen in ELISA and mouse inoculation. The protein content of the IBV antigen was determined (Lowry et al., 1951) in ELISA microplates (Corning, USA) and reader (Bio-Rad Model 550, USA).

ELISAs were developed as proposed previously by Mockett & Cook (1986), Harlow & Lane (1988), Hudson & Hay (1989) and Martins et al. (1990) and performed with modifications as described in brief as follows. The IBV M41 S₁ glycoprotein specific A38 Mab (Mockett et al., 1984) was included in ELISA for IBV detection in purified fractions or as positive Mab reference in ELISA for antibody detection in hybridomas supernatants. NDV La Sota as negative and M41 as positive control antigens reacting against plasmacitoma supernatants (Mab negative) and A38 (Mab positive), were always included in all microplates (Hemobag, Brazil or Corning, USA) coated with 3.43 mg/ml (IBV M41) or 4.1 mg/ml (NDV La Sota). Conjugate alkaline phosphatase goat IgG anti-mouse IgG whole molecule (Sigma, USA) and substrate Pnpp (p nitrophenyl phosphate, Sigma, USA) at 1mg/ml in magnesium chloride diethanolamine pH 9.8 were used to detect bound Mab with reactions read at 405nm wavelength (Bio-Rad Model 550, USA).

Mab were prepared according to Köhler & Milstein (1975), Campbell (1991), Harlow & Lane (1988) and Hudson & Hay (1989). The Sp2/0-Ag14 plasmacitoma cell line (kindly donated by the The University of Florida) and grown in RPMI 1640⁷ added with 10% fetal calf serum-FCS (Gibco, USA) in 5% CO₂ in a CO₂ incubator (Model 5100, Napco, USA). Balb/c female mice were inoculated intraperitoneally with 250ml containing 85.75mg of IBV M41 purified antigen protein mixed with 250ml of Freund’s complete adjuvant⁷ to complete 500ml per inoculum. Inoculations were repeated at 14, 21 and 42 days post primary inoculation, via the same route, protein concentration and volume in Freund’s incomplete adjuvant (Sigma, USA). Serum samples were obtained at day 52 for ELISA antibody evaluation. Best responder mice were chosen as spleen donours for immediate fusion or had spleen cells frozen in liquid nitrogen as described previously (Marusich, 1988). Logarythmically growing Sp2/0-Ag14 cells and spleen cells at a 10:1 ratio, respectively, were fused using 0.8ml PEG 1450 at 50% (v/v in serum free RPMI 1640) (Hybrimax, Sigma, USA), washed in 10ml serum free RPMI 1640 (Hybrimax, Sigma, USA) (250 x g/10 min) and re-suspended in 20% FCS HAT medium (Hybrimax, Sigma, USA). The cellular suspension was distributed onto seven 96-well culture microplates and incubated at 37ºC/5% CO₂ (Model 5100, Napco, USA). Unfused Sp2/0-Ag14 cells were maintained in A1, A2 and A3 wells to monitor the efficiency of HAT medium in eliminating unfused plasmacitomas. Two weeks after fusion, healthy large hybrid cell colonies observed at microscopy (100X, Olympus IX, Japan) were assayed by ELISA for IBV specific antibody in supernatants. ELISA IBV-positive antibody synthesizing cells were cloned twice by limiting dilution onto 24-48 hour macrophage cultured 96-well microplates (Corning, USA) to contain 0.5 cell/100ml. The antibody synthesis and monoclonal purity of clones was assayed and confirmed by ELISA and western blotting. Growing positive clones (hybridomas) were expanded in 25cm² flasks and frozen in liquid nitrogen.

Mab in supernatants were typed for immunoglobulin classes and subclasses in an ELISA using a sub-isotyping kit (American Qualex/Sigma, USA), processed acording to the manufacter’s instructions.

Mab specificity characterization was determined by western blotting onto nitrocellulose sheats containing IBV proteins after purified IBV electrophoresis (Laemnli, 1970; Promega, 1996). Electrophoresis was performed with 600mg of protein of IBV strains M41, A5968 or Arkansas-99 and electrotransference (BioRad, USA) was performed onto 0.2mm nitrocellulose membrane according to Towbin et al. (1979), Bjerrum & Schafer-Nielsen (1986) and Trans-Blot...(1995). Immunoblotting of Mab onto nitrocellulose sheats containing IBV proteins was performed for three hours in a immunoblotting system (BioRad Mini–Proteanâ II Multi Screen (BioRad, USA)). Bound Mab was detected by goat anti-mouse whole molecule IgG alkaline phosphatase conjugate⁷ and revealed by Western Blueâ (Promega, USA) substrate.

Characterized Mab were used in an ELISA against Minas Gerais State field isolates 297, 283, PM1, PM2, PM3, 351, 29-78, 327 or M41 (Massachusetts serotype), SE-17 (Georgia), H52 (Holland, Massachusetts serotype) reference IBV strains (American Type Culture Collection (ATCC), USA), as described by Souza (1999).

RESULTS AND DISCUSSION

Fresh spleen cells and frozen spleen cells fusions were performed with similar results. 14 days post-fusion 36 hibridomas (2.6% of the total) from fresh spleen cells and 17 (2.5% of the total) hibridomas from frozen spleen cells were detected by ELISA. Similar results were observed by Marusich (1988) and point to the possibility of using a stock of designed specificity frozen spleen cells for future fusions. The advantages include also the preservation of spleen cells at the plateau of the immune response and no necessity to maintain mice for very long in the laboratory.

As determined by ELISA and immunoblotting, two hydridomas, one of which producing Mab capable of recognizing N (N3F10) and glicoprotein S₂ (S12B2) of M41 were described. Mab N3F10 and S12B2, as evaluated against the Ark-99 and A5968 (Arkansas and Connecticut serotypes, respectively) in immunoblotting gave similar results as found for the M41 strain (Fig. 1). The Sp2/0-Ag14 supernatant did not react in the immunoblotting test. Based on the immunoglobulin class and subclass typification both Mab were typed as IgG1.

Ignjatovic & Galli (1995) have described the kinetics of the immune response to IBV structural proteins after infection, reporting that the N and S2 specific responses were dominant and appeared the earliest. We have obtained most hybridomas producing Mab specific to these proteins (data not shown) and speculate if the immunodominant behaviour had prevailed in our mice inoculation experiments, even though IBV was given inactivated and into an oil emulsion to mice.

Three (297, 283 e 327) out of eight field IBV isolates and all reference strains employed were detected using the Mab N3F10 specific to the N protein (Table 2 and Fig. 2). The N3F10 Mab did not detect all field isolates and this may be due to its specificity being directed to a less conserved region of N. Ignjatovic & McWaters (1991) have also detected differences in N protein sequence of several isolates using Mab. However, Koch et al. (1986) have described Mab capable of recognising all strains studied.

The homology of the N protein was described to be from 94.4 to 98.3% among IBV strains Massachusetts 41, Arkansas 99, H52, Beaudette, Gray e KB8523 (Collisson, 1992), being more conserved in the middle than on the extremities and for all strains being strongly conserved at residues 165 to 192. Thus, selecting S2 and N proteins Mab was based on the conserved character of these antigens among strains and serotypes (Cavanagh, 1984; Sneed, 1989), in contrast to the S1 and M proteins, in order to obtain reagents which would react to any antigenic type of IBV, enabling the development of assays for the detection of any IBV isolate in diagnosis and research. The assays with the N3F10 Mab did, however, display surprising results, by evidentiating diferences among strains in the N protein. It is assumed that N3F10 might be specific to a less conserved amino acid sequence of N, considering that variations in N sequence have been previously reported in the composition of both the N precursor gene and in the protein (Koch et al., 1986; Cavanagh, 1991; Ignjatovic & McWaters, 1991; Koch et al., 1991).

The N3F10 Mab may be important in studies of the immune response during the infection, when the immune system is demanded for eliminating infected cells. It was recently realized that cytotoxic T cells are involved in destroying IBV infected cells through a specific response against N protein (Seo et al., 1998), interrupting the infection and collaborating to cure. Vaccinal virus replication should elicit, as well as surface projections (S1 and S2) specific responses, a response to N protein in order to prepare the immunity to eliminating infected cells. The evaluation of the immune response to N protein as an IBV replication product may be an important information on the capacity of the chickens to rapidly cure an infection.

The diagnosis of IBV infection could be implemented using the N3F10 Mab. A rapid detection of IBV was proposed by Koch et al. (1991b) using a N specific Mab in na antigen capture assay.

The S2 glycoprotein presents also a conserved amino acid sequence among IBV strains and cross reactions have been demonstrated among isolates (Ignjatovic & Ashton, 1996; Collisson et al., 1992; Koch et al., 1990; Kusters et al., 1989; Lenstra et al., 1989). The Mab S12B2 was capable of detecting all IBV strains employed in the ELISA and western blotting, including all field (297, 283, PM1, PM2, PM3, 351, 29-78, 327 and reference strains (M41 Massachusetts, SE-17 Georgia and H52 Holland Massachusetts serotype reference strains) of IBV, indicating its specificity to a very conserved region of S2 (Table 2 and Fig. 2). The reactivity of S12B2 to the various IBV employed in this study is supporting the conclusion that it may be very useful to detecting IBV isolates of whichever serotype, best than N3F10, and is recommended for the routine diagnosis of field IBV isolates being replicated in the laboratory.

Mab S12B2 will also be useful for establishing infected tissue staining techniques, in which IBV would be detected in situ, enabling infection and pathogenicity studies irrespective of serotype. Capture or indirect ELISA for rapidly detecting IBV in culture material, such as embryo allantoic fluid (AF) or infected tissue extracts may be developed for providing rapid diagnosis of IBV infection.

CONCLUSIONS

It is concluded that the Mab S12B2, being specific to the IBV S2 glycoprotein, is a valuable tool for detecting IBV irrespective of serotype and that the Mab N3F10, which has detected variation in N protein, would enable grouping of the IBV isolates of different serotypes.

ACKNOWLEDGEMENTS

The authors are grateful to FAPEMIG, CNPq and FEPMVZ-Coordenação Preventiva for the financial support. We are indebted to Dr. Maurício Resende, Dr. Cristiano Gontijo, Dr. Andrey Pereira Lage and Dr. Zélia Inês Portela Lobato for the valuable advice. The technical assistance of Mrs. Tânia Mara Gomes de Pinho and Dr. Elizabeth Dias Bontempo (ICB-UFMG) is acknowledged.

Trans-Blotâ Semi-Dry Electrophoretic Transfer Cell Instruction Manual. Hercules (CA): (BIO-RAD cat#170-3940), 1995.

• ALEXANDER, D.J., CHETTLE, N.J. Procedures for the haemagglutination and haemagglutination-inhibition tests for avian infectious bronchitis virus. Avian Pathol, v.5, p.9-17, 1977.
• BJERRUM, O.J., SCHAFER-NIELSEN, C. Analytical Electrophoresis In: DUNN, M.J. (Ed.). Berlim: Verlag Chemie, Weinheim, 1986. 315p.
• BRAUNE, M.O., GENTRY, R.E. Standardization of the fluorscent antibody technique for the detection of avian respiratory viruses. Avian Dis., v.9, p.535-545, 1965.
• CAMPBELL, A.M. Laboratory techniques in biochemistry and molecular biology: Monoclonal antibody and immunosensor technology Elsevier Science Publishers B.V, NY-USA, 1991. 427p.
• CAMPBELL, A.M. Laboratory techniques in biochemistry and molecular biology: Monoclonal antibody technology the production and characterization of rodent and human hybridomas Elsevier Science Publishers B.V, NY-USA, vol. 13, 1986. 265p.
• CAVANAGH, D. Coronavirus IBV: Further evidence that the surface projections are associated with two glycopolypeptides. J. Gen. Virol., v.64, p.1787-1791, 1983.
• CAVANAGH, D. Sequencing aproach to IBV antigenic variation and epizootiology. II International Symposium on Infectious Bronchitis. Rauischholzhausen, Germany, p. 147-153, 1991.
• CAVANAGH, D., DARBYSHIRE, J.H., DAVIS, P.J. et al. Induction of humoral neutralising and haemagglutination-inhibiting antibody by the spike protein of avian infectious bronchitis virus. Avian Pathol., v.13, p.573-583, 1984.
• CAVANAGH, D., DAVIS, P.J. Coronavirus IBV: removal of spike glycopeptide S1 by urea abolishes infectivity but not attachment to cells. J. Gen. Virol., v.67, p.1443-1448, 1986.
• CAVANAGH, D., DAVIS, P.J., DARBYSHIRE, J.H. et al. Coronavirus IBV: virus retaining spike glycopeptide S2 but not S1 is unable to induce virus neutralizing or hemagglutination inhibiting antibody or induce chicken tracheal protection. J. Gen. Virol., v.67, p.1435-1442, 1986.
• CAVANAGH, D., NAQI, S.A Infectious bronchitis. In: CALNEK, B.W., BARNES, H.J., BEARD, C.W. et al. Diseases of poultry 10.ed., Ames: Iowa State University Press, 1997, Chap. 17, p.511-526.
• CLARKE, J.K., McFERRAN, J.B., GAY, F.W. Use of allantoic cells for the detection of avian infectious bronchitis virus. Arch. Ges. Virusfor., v.36, p.62-70, 1972.
• COLLISSON, E.W., PARR, R.L., LI, W. et al. An overview of the molecular characteristics of avian infectious bronchitis virus. Poult. Sci. Rev., v.4, p.41-55, 1992.
• COOK, J.K.A. The classification of new serotypes of infectious bronchitis virus isolated from poultry flocks in Britain between 1981 and 1983. Avian Pathol., v.13, p.733-741, 1984.
• CUNNINGHAM, C.H. Avian infectious bronchitis. Adv. Vet. Sci. Comp. Med., v.14, p.105, 1970.
• DARBYSHIRE, J.H., ROWELL, J.G., COOK, J.K.A. et al. Taxonomic studies on strains of infectious bronchitis virus using neutralization tests in tracheal organ cultures. Arch. Virol., v.61, p.227-238, 1979.
• DHINAKAR, G., JONES, R.C., Infectious bronchitis virus: immunopathogenesis of infection in the chicken. Avian Pathol, v.26, p.677-706, 1997.
• HARLOW, E.D., LANE, D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, p.139-242, 1988.
• HUDSON, L., HAY, F. C. Practical Immunology. 3.ed. Blackwell Scientific Publications, p.367-378, 1989.
• IGNJATOVIC, J., ASHTON, F. Detection and differentiation of avian infectious bronchitis viruses using monoclonal antibody-based ELISA. Avian Pathol., v.25, p.721-736, 1996.
• IGNJATOVIC, J., GALLI, L. Immune responses to structural proteins of avian infectious bronchitis virus. Avian Pathol., v.24, p.313-332, 1995.
• IGNJATOVIC, J., McWATERS, P.G. Monoclonal antibodies to three structural proteins of avian infectious bronchitis virus: characterization of epitopes and antigenic differentiation of Australian strains. J. Gen. Virol., v.72, p.2915-2922, 1991.
• JACKWOOD, M.W., KWON, H.M., HILT, D.A. et al Infectious bronchitis virus detection in allantoic fluid using the polymerase chain reaction and a DNA probe. Avian Dis., p.403-409, 1992
• KARACA, K., NAQI, S., GELB Jr., J. Production and characterization of monoclonal antibodies to three infectious bronchitis virus serotypes. Avian Dis., v.36, p.903-915, 1992.
• KOCH, G., HARTOG, L., KANT, A. et al. Antigenic differentiation of avian bronchitis virus variant strains employing monoclonal antibodies. Isr. J. Vet. Med., v.42, 89-97, 1986.
• KOCH, G., HARTOG, L., KANT, A. et al. Antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions. J. Gen. Virol., v.71, p.1929-1935, 1990.
• KOCH, G., KANT, A., COOK, J.K.A. et al. Epitopes of neutralizing antibodies are located within three regions of the S1 spike protein of infectious bronchitis virus. II International Symposium on Infectious Bronchitis Rauischholzhausen, Germany, p.154-160, 1991a.
• KOCH, G., KANT, A., JAPINE, G. vos et al. Rapid detection of infectious bronchitis virus by nucleoprotein specific antigen capture assay. II International Symposium on Infectious Bronchitis. Rauischholzhausen, Germany, p.168-172, 1991b.
• KOHLER, G., MILSTEIN, C. Continuous culture of fused cells secreting antibody of predefined specificity. Nature, v.256, p.495-497, 1975.
• KUSTERS, J.G., JAGER, E.J., LENSTRA, J.A. et al. Analysis of an immunodominant region of infectious bronchitis virus. J. Immunol., v.143, 2692-2698, 1989.
• KWON, H.M., JACKWOOD, M.W., GELB Jr., J. et al. Differentiation of infectious bronchitis virus serotypes using polymerase chain reaction and restriction fragment length polymorphism analysis. Avian Dis., v.37, 194-202, 1993.
• LAEMMLI, U.K Cleavage of strutural proteins during the assembly of the head of bacteriophage T4. Nature, v.227, p.680-686, 1970.
• LASHGARI, M.S., NEWMAN, J.A. Determination of the antigenic relationships within the Massachusetts (M41) type of infectious bronchitis virus using the hemagglutination-inhibition test. Avian Dis., v.28, p.444-452, 1983.
• LENSTRA, J.A., KUSTERS, J.G. Antigenicity of the peplomer protein of infectious bronchitis virus using the haemagglutination-inhibition test. Avian Dis., v.28, p.435-443, 1984.
• MARUSICH, M.F. Efficient hybridoma production using previously frozen splenocytes, J. Immunol. Meth., v.144, p.155-159, 1988.
• MOCKETT, A.P.A. Envelope proteins of avian infectious bronchitis virus: purification and biological properties. J. Virol. Meth., v.12, p.271-278, 1985.
• MOCKETT, A.P.A., CAVANAGH, D., BROWN, T.D.K. Monoclonal antibodies to the S1 spike and membrane proteins of avian infectious bronchitis coronavirus strain Massachusetts M41. J. Gen. Virol., v.65, p.2281-2286.
• MOCKETT, A.P.A., DARBYSHIRE, J.H. Comparative studies with an enzyme-linked immunosorbent assay ELISA for antibodies to avian infectious bronchitis virus. Avian Pathol., v.10, p.1-10, 1981.
• PROMEGA Protocols and applications guide Ed. Promega Corporation. 3.ed., (cat#p1610), p.279-304, 1996.
• SEO, S.H., COLLISSON, E.W. Cytotoxic T lymphocyte responses to infectious bronchitis virus infection Coronaviruses and arteriviruses. New York: Plenum Press, 1998. p.455-460.
• SHULMAN, M., WILDE, C.D., KÖHLER, G. A better cell line for making hybridoma secreting antibodies. Nature, v.276, p.269-270, 1978.
• SNEED, L.W., BUTCHER, G.D., PARR, R. et al. Comparations of the structural proteins of avian infectious bronchitis virus as determined by western blot analysis. Viral Immunol., v.2, p.221-227, 1989.
• SOUZA, M.B. Afinidades antigęnicas de amostras de campo do vírus da bronquite infecciosa das galinhas com a amostra Massachusetts M4.1. Belo Horizonte: Escola de Veterinária da UFMG, 1999. 75p. (Dissertaçăo, Mestrado).
• TOWBIN, H., STAEHLIN, T., GORDON, G. Eletrophoretic transfer of proteins from polycrylamide gels to nitrocellulose sheet: procedure and some applications. Proc. Natl. Acad. Sci., v.76, p.4330-4335, 1979.
• VILLEGAS, P. Laboratory manual of avian diseases. University of Georgia, 1985. 56p.

Publication Dates

• Publication in this collection
  18 June 2002
• Date of issue
  Oct 2001

History

• Received
  11 Aug 2000