Preparation Of Monospecific Antisera Research Articles

Preparation of monospecific antisera against fowl cholera, fowl coryza and Newcastle disease reference strains in laboratory animals

Preparation of monospecific antisera against fowl cholera, fowl coryza and Newcastle disease reference strains in laboratory animals

An antigenically related polypeptide family is a major structural constituent of a stable acrosomal matrix assembly in bovine spermatozoa.

The apical and principal segments of the bovine acrosome contain a stable matrix complex that is bound to the outer acrosomal membrane and exhibits hydrolase-binding activity. The present study was undertaken to determine whether the outer acrosomal membrane-associated matrix complex (OMC) is composed of a unique set of acrosomal proteins and to define its fate during both capacitation and the acrosome reaction. A purified OMC fraction was isolated from ejaculated spermatozoa, and one polypeptide of 32 kDa (OMC32) was purified to homogeneity and used for N-terminal sequence analysis and preparation of monospecific antisera. Immunofluorescence staining of sperm with anti-OMC32 demonstrated that the polypeptide localized specifically to the apical and principal segments of the acrosome. Immunoelectron microscopy further revealed that OMC32 was restricted to the stable matrix assembly and was not associated with the inner acrosomal membrane or the equatorial segment. Immunoblot analyses of sperm lysates and of the purified OMC fraction revealed that anti-OMC32 recognized an antigenically related family of polypeptides between 38 and 19 kDa. These polypeptides exhibited no size processing during capacitation or the acrosome reaction, and they were not released during the acrosome reaction but remained in the particulate cell subfraction, associated with the hybrid membrane complex. N-terminal sequence analysis of OMC32 indicated a structural relationship to the SP-10 polypeptide family of human and baboon spermatozoa. The potential function of the OMC complex and differences in the intraacrosomal distribution of bovine OMC32-related polypeptides from that reported for acrosomal SP-10 polypeptides in other species are discussed.

A polycistronic mRNA specified by the coronavirus infectious bronchitis virus

The third largest of the nested set of subgenomic mRNAs (mRNA3) from the coronavirus infectious bronchitis virus (IBV) contains three separate open reading frames (3a, 3b, and 3c) which are not present on the next smallest of the mRNAs, suggesting that this mRNA may be functionally polycistronic. However, although a protein product has been identified from the 3c open reading frame, to date the coding function of 3a and 3b has not been established. We present nucleotide sequence data suggesting that each of the three open reading frames is conserved in a variety of different IBV strains and further show, through the preparation of monospecific antisera against bacterial fusion proteins, that IBV-infected cells contain small amounts of the products of these ORFs. In vitro translation studies using synthetic mRNAs containing the 3a, 3b, and 3c open reading frames suggest strongly that all three proteins can be translated from a single molecular species, and expression studies carried out in intact cells support this conclusion. Thus mRNA3 of IBV appears to be functionally tricistronic.

Separation of Sendai virus glycoproteins by using glutaraldehyde-treated erythrocytes and preparation of monospecific antisera against the glycoproteins.

Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins, F and HN, were separated from Triton X-100- or Nonidet P-40-solubilized envelopes as unadsorbed and eluted fractions, respectively, by using glutaralde-hyde-treated chicken erythrocytes. These separated glycoproteins were biologically active. Monospecific antisera (in terms of monoreactivity to virus glycoproteins in gel diffusion precipitation patterns) were prepared by using these fractions as immunogens. Anti-HN rabbit serum inhibited all of the viral activities tested (infectivity, neuraminidase, hemagglutinating, and viral hemolysis), whereas anti-F serum definitely inhibited viral hemolysis only, although the two antisera enhanced neutralization in the presence of complement. The advantages and disadvantages of this separation method were discussed.

B-6. 正常ないし軽度高脂血症の血清を用いたアポリポ蛋白-Cの分離精製及び抗アポ-C血清の作成

B-6. 正常ないし軽度高脂血症の血清を用いたアポリポ蛋白-Cの分離精製及び抗アポ-C血清の作成

Identification of an antigen associated with malignant melanoma.

Xeno-antisera, prepared by immunization with soluble membrane material obtained from melanoma tumours and partially purified by column chromatography, were found to detect an antigenic specificity common to some tumour extracts and sera of melanoma patients. The presence of this antigen in patients' sera allows speculation for its possible use for early diagnosis of metastases and its role in the blocking of the immunological defences of the melanoma patients. Preparation of monospecific antisera by immunization with insoluble serum-antigen/antibody complexes has been successfully undertaken.

Further Studies on Porcine Enteroviruses Isolated at Cambridge: I.—Infections in SPF Pigs and the Preparation of Monospecific Antisera

SUMMARY To enable more critical serological studies to be undertaken with various porcine enteroviruses, attempts were made to produce monospecific antisera to 13 selected strains. Each selected strain was cloned 3 times by the plaque technique and then used to infect 2 first-generation specif c pathogen free (SPF) pigs. Before inoculation, faecal samples from these pigs were examined for the presence of cytopathogenic agents and blood samples were tested for antibodies against porcine enteroviruses. Elaborate precautions were taken to prevent accidental contamination. Most of the pigs were killed 2 or 3 weeks after infection. All the selected strains before and after triple-cloning were tested by cross-neutralization tests in tissue culture against sera already available and against the new sera. Each new serum was checked also against an early passage of the selected strain as near as possible to the original isolation. Virus recovered from the faeces and tissues of each serum-producing pig was tested against sera already available. The results of all these tests indicated that, in relation to the porcine enteroviruses, the new sera were monospecific. It appeared probable that the pre-cloning sample of one selected strain, F59, had comprised a number of viruses, at least one of which was eliminated by the cloning procedure. All the selected strains multiplied in the lower portions of the intestinal tract and were excreted in the faeces. In addition to the Talfan and T80 viruses, which are known to produce polioencephalomyelitis, the enterovirus, F59, produced mild lesions of polio-encephalomyeiitis.