ABSTRACT
A range of fusion constructs (expressed in Escherichia coli) were produced that contained two or more HPV6b E proteins, producing a single continuous amino acid sequence corresponding to the sequences of the individual E proteins. The constructs also included a C-terminal hexahistidine tag fused in-frame to aid purification. The fusion proteins (polyproteins) were semipurified by Ni(++) metal affinity chromatography under denaturing conditions. Immunization of BALB/c mice with these polyproteins resulted in the production of specific E protein antibodies. The draining lymph nodes from these mice were used to produce monoclonal antibodies (MAbs). The specificity of the polyclonal and MAbs was confirmed by immunoblotting and by screening for reaction with a series of synthetic peptides of E proteins. HPV E polyproteins were found to be immunogenic and immunization with the polyproteins resulted in specific antibody responses to the component E proteins.
Subject(s)
Oncogene Proteins, Viral/chemistry , Oncogene Proteins, Viral/immunology , Amino Acids/chemistry , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Blotting, Western , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Hybridomas , Immunoblotting , Immunoenzyme Techniques , Lymph Nodes , Mice , Mice, Inbred BALB C , Nickel/pharmacology , Papillomaviridae/chemistry , Polyproteins/pharmacology , Protein Binding , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistryABSTRACT
The state of fimbriae type 2 (Fim 2) and fimbriae type 3 (Fim 3) preparations from Bordetella pertussis were examined by negative stain electron microscopy. Uranyl acetate induced clumping of Fim 3 regardless of pH and was unsuitable as a stain for establishing the state of fimbriae. Both ammonium molybdate and sodium phosphotungstate were able to show the differences in Fim 3 stored at pH 7.2 and pH 9.5.
Subject(s)
Bordetella pertussis/ultrastructure , Fimbriae, Bacterial/ultrastructure , Negative Staining/methods , Antibodies, Bacterial , Antibodies, Monoclonal , Antigens, Bacterial/ultrastructure , Hydrogen-Ion ConcentrationABSTRACT
Clostridium novyi type B alpha toxin was purified to homogeneity and shown to have a molecular weight of 200 kD by SDS polyacrylamide gel electrophoresis. The toxin was toxoided and used to produce a pair of non-interfering monoclonal antibodies. Their specificity was confirmed by immunoblotting and bioassay. The monoclonal antibodies were used to develop an enzyme immunoassay which was more sensitive than bioassay, and permitted less than 1 ng/ml toxin to be detected in a rapid 10 min assay format. Use of the assay can eliminate the requirement for in vivo testing of novyi toxin and toxoid, provided measurements of biological activity are not required. Because of its speed and sensitivity, the assay can be used to monitor toxin production during fermentation and as an alternative to bioassay to measure antigen content during toxoiding and vaccine formulation.
