M. J. Carter

New monoclonal antibodies to oestrogen and progesterone receptors effective for paraffin section immunohistochemistry.

Assessment of oestrogen and progesterone receptors (ER and PgR) in breast cancer is widely used for the prediction of response to endocrine therapy and as a prognostic marker. Cytosolic assays have been replaced in many centres by immunochemical techniques, which have many advantages including applicability to small samples, simplicity, and cost‐effectiveness. This study describes the generation and characterisation of two novel murine monoclonal antibodies recognizing ER and PgR, designated NCL‐ER‐6F11 and NCL‐PGR respectively, which are effective in heat‐treated formalin‐fixed, paraffin‐embedded tissue. The antibodies have been characterized by Western blotting and by immunohistochemistry on normal and pathological breast and other tissues. NCL‐ER‐6F11 has been shown to compare favourably with a currently available ER antibody. These antibodies may prove of value in the assessment of hormone receptor status in human breast cancer.

Defective translation of measles virus matrix protein in a subacute sclerosing panencephalitis cell line

Subacute sclerosing panencephalitis (SSPE) is a slowly progressing fatal human disease of the central nervous system (CNS) that is associated with measles virus persistence. Virus nucleocapsids are present in the brain1,2 and the patient is in a state of hyperimmunization towards this agent. However, although all other structural polypeptides are recognized by the immune system, there is a markedly decreased antibody response towards virus matrix or membrane protein3,4. Matrix protein has not been detected in brain cells5 and infectious virus is not present. The absence of this virus structural polypeptide is thought to account for the apparent restriction in virus maturation both in vivo and in vitro. SSPE viruses can only rarely be rescued from brain tissue by co-cultivation or cell fusion techniques using tissue culture cell lines susceptible to measles virus infection6. Often this procedure fails to yield a lytic budding virus but produces instead a carrier cell line in which the agent is cell associated. These lines (known as SSPE cell lines) also do not contain matrix protein7,8. However, the reason for this deficiency is unknown. We have therefore now examined an SSPE cell line which does not yield infectious virus in order to define this process further. We found that although messenger RNA for membrane protein was present, it was unable to form normal matrix protein in translation reactions.

Antigenic characterization of measles and SSPE virus haemagglutinin by monoclonal antibodies.

Hybrid cells secreting monoclonal antibodies directed against the haemagglutinin (H) protein of measles virus (Edmonston) were produced by fusion of mouse myeloma cells with spleen cells derived from immunized mice. Measles antibodies secreted by these cells were tested for their ability to react with measles virus in immunoprecipitation experiments and assays of binding, neutralization, haemagglutination inhibition and haemolysin inhibition. On this basis 21 out of 75 hybridomas could be defined and divided into five functional groups with different properties. However, when tested against other measles virus strains, including those isolated from subacute sclerosing panencephalitis (SSPE) patients, normalized radioimmunoassay (RIA) binding titres showed that the extent to which a given antibody bound could vary greatly with the virus strain examined. Moreover, the biological actions within a group were found to be very heterogeneous, even when high antibody binding titres were observed. These results suggest that different measles virus strains, which are not distinguishable by polyvalent sera, do in fact possess antigenic differences. Furthermore, the functional significance of a given virus epitope may vary from strain to strain. Hybridoma antibodies were also used to demonstrate the occurrence of antigenic changes within the H polypeptide of SSPE virus during the course of non-productive, persistent infection in vitro.

Identification and sequence determination of the capsid protein gene of human astrovirus serotype 1

Abstract We present the sequence of an open reading frame (ORF) at the 3′ end of human astrovirus serotype 1. Primer extension experiments showed that the RNA expressing this gene is shorter than the complete ORF, and could form a protein of M r 85 540. The protein was expressed by recombinant baculovirus and was recognized by anti-virion serum, indicating a structural role. Sequence comparison indicates that astrovirus serotypes 1 and 2 differ markedly in the C-terminal half of the protein but are well conserved towards the N-terminus.

Location of monoclonal antibody binding sites in the capsid protein of feline calicivirus.

We report the localization of three monoclonal antibody (MAb) binding sites in the capsid protein of feline calicivirus. Gene fragments were generated by restriction enzyme digestion or the polymerase chain reaction, and expressed as beta-galactosidase fusion proteins in Escherichia coli. These chimeric molecules were screened using three MAbs. A non-neutralizing MAb recognized a region within 36 amino acids of the C terminus. Two neutralizing MAbs bound to a different region of 37 amino acids in the centre of the protein. Comparative sequence analysis shows this area to be the major variable region of the capsid protein.

Effect of Measles Virus Antibodies on a Measles SSPE Virus Persistently Infected C6 Rat Glioma Cell Line

Maintenance of measles (SSPE-Lec) virus persistently infected C6 rat glioma cells in medium containing polyclonal measles antiserum resulted in the loss of detectable expression of all measles virus proteins. Removal of these cells from antiserum, however, led to a re-expression of virus proteins and the production of infectious virus. Cloning of antibody-modulated non-expressing cells in the presence of antiserum showed that re-expression of virus proteins was not due to an incomplete curing process following the addition of antiserum, as a large number of non-expressing cell clones developed the capacity to express measles virus antigen at different periods after removal of antiserum. Irradiation of persistently infected cells to give a non-growing culture showed that modulation was not mediated by a selection and outgrowth of a small percentage of non-expressing cells originally present in the culture. Antibody directed against C6 membrane proteins did not lead to modulation and it was also shown that only monoclonal antibodies with neutralizing activity could affect intracellular antigen expression. Immunoglobulin Fab fragments with neutralizing activity also had modulating activity. Although all modulated cell clones were more susceptible to homologous virus infection than control C6 cells, it was not possible to rescue any defective measles virus which may have been maintained in the culture.

Relationships between Monoclonal Antibody-binding Sites on the Measles Virus Haemagglutinin

Twenty-one monoclonal antibodies directed against the measles virus haemagglutinin have recently been obtained. These were known to fall into five groups, each defined by its effects on the biological functions of the H protein. A representative of each group was selected and examined by competitive radioimmunoassay in an attempt to deduce the relationships between antibody-binding sites on the antigen. It was found that these five antibodies formed three binding groups which recognized different but overlapping areas of the molecule. These three areas formed a series of sites which traversed the active region of the H polypeptide. A haemagglutinin-directed monoclonal antibody which displayed a haemolysin-inhibiting activity was also examined. This antibody fitted into the binding group scheme determined here and there was no additional binding site for this molecule on the F protein itself.

A dot-blot hybridization procedure for the detection of astrovirus in stool samples.

We have developed a nucleic acid dot-blot hybridization test for the detection of astroviruses in stool samples. The test was not as sensitive as electron microscopy for the detection of low numbers of well preserved astrovirus particles, but was able to identify astroviruses in stools containing particles of indistinct morphology. In total, this procedure identified astroviruses in more samples than did electron microscopy, and the data indicate that the incidence of astroviruses may be substantially underestimated.

Restriction enzyme analysis of faecal adenoviruses in Newcastle upon Tyne

Adenovirus DNA was isolated directly from virus-containing stools and digested with restriction endonucleases. The resulting fragments were separated by polyacrylamide gel electrophoresis (PAGE) and visualized by silver staining. This enabled us to assign most of the viruses detected to subgenus, serotype and, sometimes, unique strains. Although less sensitive than electron microscopy, the method allowed more information about the infecting virus to be obtained and no cultivation was necessary. Comparison with culture also allowed dual infections to be recognized. A 2-year survey of faecal adenoviruses in Newcastle upon Tyne showed that type 41 (strain 41a) was the predominant type and strain 41p was not recorded. Heterogeneity in strain 41a was also noted as found elsewhere. Adenovirus type 40 was common prior to 1985 but was absent during the last 2 years.

Adaptation of Coronavirus JHM to Persistent Infection of Murine Sac(-) Cells

Coronaviruses can establish persistent infections in the central nervous system of rodents, and these are associated with demyelinating encephalomyelitis. The effects of persistence on the virus are difficult to study in vivo but may have a crucial influence on the course of infection. We therefore produced a persistent infection in vitro using the neurotropic coronavirus JHM, in order to investigate the events underlying the establishment of such an infection and the adaptation of the virus to persistence. The persistent infection was maintained for over 115 passages and continued to release high levels of infectious virus. During the 18 months of culture the number of cells expressing virus antigen detected by indirect immune fluorescence decreased to 40%. Analysis showed that the carried virus contained a significant proportion of heterogeneous temperature-sensitive mutants. All virus clones isolated possessed the capacity to induce a more productive growth cycle, a less pronounced cytopathic effect and showed a much reduced neurovirulence when inoculated into newborn and weanling rats. Evidence for structural changes involving the surface peplomer protein (E2) was obtained using hybridoma antibodies, which neutralized the parental JHM virus but not the JHM-Pi virus. Defective interfering particles and interferon activities have been excluded as possible agents instrumental in the establishment and maintenance of the chronic infection, and we suggest that the emergence of virus variants of lowered virulence is central to these processes.