Recombinant baculovirus-expressed rotavirus protein (VP4) in an ELISPOT assay of antibody secretion.

Studies on the protein specificity of the intestinal antibody response to rotavirus infection have been hampered by lack of antigenically conserved isolated proteins to serve as antigens in immunochemical assays. In this report, the use of an antigenically conserved baculovirus-expressed rotavirus protein (VP4) as a capture antigen in the ELISPOT assay is described. Anti-VP4 antibody-secreting hybridoma cells are used as a test population to show that expressed VP4 as the capture antigen detects numbers of antibody secreting cells comparable to intact rotavirus particles. Hybridoma cells specific for other rotavirus proteins are used to ensure the specificity of the expressed VP4 in the assay. The flexibility and ease of use of a recombinant expressed protein product as a capture antigen in this assay dramatically enhances the ability to quantitate intestinal antibody responses to specific viral proteins.

[1]  N. Legendre.  Immobilon-P transfer membrane: applications and utility in protein biochemical analysis. , 1990, BioTechniques.

[2]  C. Stokes,et al.  Whole Cholera Toxin and B Subunit Act Synergistically as an Adjuvant for the Mucosal Immune Response of Mice to Keyhole Limpet Haemocyanin , 1990, Scandinavian journal of immunology.

[3]  H. Greenberg,et al.  Immunization with baculovirus-expressed VP4 protein passively protects against simian and murine rotavirus challenge , 1990, Journal of virology.

[4]  G. Losonsky,et al.  The immune response in primary asymptomatic and symptomatic rotavirus infection in newborn infants. , 1990, The Journal of infectious diseases.

[5]  W. Strober,et al.  Cholera holotoxin and its B subunit enhance Peyer's patch B cell responses induced by orally administered influenza virus: Disproportionate cholera toxin enhancement of the IgA B cell response , 1990, European journal of immunology.

[6]  J. Vaerman,et al.  Use of anti-cholera toxin IgA-secreting hybridoma cells for the elaboration of an antigen-specific IgA-ELISPOT-assay. , 1990, Immunological investigations.

[7]  M. Estes,et al.  Rotavirus gene structure and function. , 1989, Microbiological reviews.

[8]  H. Ushijima,et al.  Detection of anti rotavirus coproantibodies by immunoblotting technique , 1989, Journal of medical virology.

[9]  D. Haines,et al.  Development of a murine model to study the pathogenesis of rotavirus infection , 1989, Experimental and Molecular Pathology.

[10]  M. Estes,et al.  Nucleotide sequence of bovine rotavirus gene 1 and expression of the gene product in Baculovirus☆ , 1989, Virology.

[11]  H. Greenberg,et al.  The rhesus rotavirus outer capsid protein VP4 functions as a hemagglutinin and is antigenically conserved when expressed by a baculovirus recombinant , 1989, Journal of virology.

[12]  A. Ulmer,et al.  Dipeptidyl Peptidase IV in Human T Lymphocytes , 1989, Scandinavian journal of immunology.

[13]  A. Osterhaus,et al.  Detection of IgA Antibodies and Quantification of IgA Antibody‐Producing Cells Specific for Ovalbumin or Trichinella spiralis in the Rat , 1988, Scandinavian journal of immunology.

[14]  R. Shaw,et al.  Characterization of homotypic and heterotypic VP7 neutralization sites of rhesus rotavirus. , 1988, Virology.

[15]  H. Greenberg,et al.  The rhesus rotavirus gene encoding protein VP3: location of amino acids involved in homologous and heterologous rotavirus neutralization and identification of a putative fusion region. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[16]  W. Stok,et al.  Improved procedure for the isolation of functionally active lymphoid cells from the murine intestine. , 1987, Journal of immunological methods.

[17]  R. F. Ramig,et al.  Protection between different serotypes of bovine rotavirus in gnotobiotic calves: specificity of serum antibody and coproantibody responses , 1987, Journal of clinical microbiology.

[18]  Y. Matsuura,et al.  Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins. , 1987, The Journal of general virology.

[19]  M. Summers,et al.  A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures. , 1987 .

[20]  R. Shaw,et al.  Antigenic mapping of the surface proteins of rhesus rotavirus. , 1986, Virology.

[21]  N. Lycke.  A Sensitive Method for the Detection of Specific Antibody Production in Different Isotypes from Single Lamina Propria Plasma Cells , 1986, Scandinavian journal of immunology.

[22]  J. Butler,et al.  The immunochemistry of sandwich ELISAs--I. The binding characteristics of immunoglobulins to monoclonal and polyclonal capture antibodies adsorbed on plastic and their detection by symmetrical and asymmetrical antibody-enzyme conjugates. , 1986, Molecular immunology.

[23]  P. Holt,et al.  ELISA plaque assay for the detection of antibody secreting cells: observations on the nature of the solid phase and on variations in plaque diameter. , 1984, Journal of immunological methods.

[24]  P. Offit,et al.  A murine model for oral infection with a primate rotavirus (simian SA11) , 1984, Journal of virology.

[25]  R. Yolken,et al.  Kinetics of viral replication and local and systemic immune responses in experimental rotavirus infection , 1984, Journal of virology.

[26]  O Ouchterlony,et al.  A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. , 1983, Journal of immunological methods.

[27]  P. Holt,et al.  A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells. , 1983, Journal of immunological methods.

[28]  D. Graham,et al.  Rotavirus stability and inactivation. , 1979, The Journal of general virology.