Characterization of Bangor virus proteins by using monoclonal antibodies

A new virus was isolated from a finch in quarantine in Northern Ireland in 1973. The virus had the morphological characteristics of a paramyxovirus, and was named Bangor virus (BaV). In order to identify the structural proteins of BaV and to investigate the biological characterization of the virus, 28 monoclonal antibodies (mAbs) directed against BaV were prepared. Eight of these mAbs reacted with the nucleocapsid protein (NP), 10 with hemagglutinin-neuraminidase (HN) protein, and 10 with fusion (F) protein. With the aid of these mAbs, the structural proteins of BaV were determined, namely, p52, gp74, gp63, and gp51 were identified as the NP, HN, F0, and F1 proteins, respectively. The biological activities of the mAbs directed against the envelope glycoproteins of BaV were examined. Intriguingly, it was found in the neutralization assay that four mAbs directed against the HN protein of BaV can enhance the fusion of HeLa cells infected with BaV, showing the presence of a potential third function of the HN protein that affects the fusion activity of the F protein. Furthermore, all of the anti-F protein mAbs showed neutralizing activity.     

The development of monoclonal antibodies for the therapy of cancer

Monoclonal antibodies (Mabs) were first described by K?hler and Milstein in 1975. Not only did this discovery lead to a Nobel prize, but it created an enormous scientific field that has now become a multimillion dollar industry. Mabs made the transition from laboratory reagents to clinical diagnostics very quickly. However, their development as therapeutic agents was, as predicted, more costly and time-consuming. Indeed, clinicians and scientists were required to learn a new set of rules for using these large, immunogenic, targeted agents in humans. Nevertheless, in 1997 the first Mab was licensed in the U.S. and several others will soon follow. In this review, we discuss Mab-based strategies for the treatment of cancer. We compare native, fragmented, recombinant and chimeric antibodies, bispecific antibodies, immunoconjugates, and immunoliposomes. The rationale for their development, their advantages, their in vitro and in vivo performance, and their clinical usefulness are discussed.     

Enzyme-linked immunosorbent assay, using staphylococcal protein A for detecting virus antibodies

A modification of the indirect enzyme-linked immunosorbent assay (ELISA) was developed which used staphylococcal protein A linked to horseradish peroxidase. Virus antibodies in equine, bovine, porcine, feline, canine, lagomorphic (rabbit), and human sera were detected, using the indirect ELISA in which the antiglobulin enzyme conjugate was replaced by protein A linked to horseradish peroxidase. Results of the ELISA were compared with the results of the serum-virus neutralization test. The application of the test in laboratories performing serologic assays with sera from diverse animal species is discussed.     

Detection by a solid phase independent enzyme immunoassay of monoclonal anti-idiotypic antibodies against monoclonal antibodies neutralizing Semliki Forest virus and encephalomyocarditis virus

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) is the rate limiting step in the mevalonate pathway that produces isoprenoids and cholesterol. Inhibitors of HMG-CoA reductase are teratogenic in vivo and induce neural tube defects in rat embryo culture, effects which appear unrelated to cholesterol deficiency. This study is the first to localize HMG-CoA reductase mRNA by in situ hybridization (ISH). Expression of reductase mRNA was examined in post-implantation rat embryos, and for control purposes in rat liver and UT-1 cells, using a digoxigenin-11 (dig-11) labelled cRNA probe. Eighteen-day fetal liver showed heavy but patchy hybridization, and adult rat liver showed strong hybridization only on some periportal hepatocytes, which was absent in livers of fasted animals. UT-1 cells stimulated to overexpress HMG-CoA reductase mRNA were strongly positive with the same probe. Control hybridizations with sense strand RNA probe, or with cRNA probe on pre-RNased tissue were negative. Strong hybridization signal for HMG-CoA reductase mRNA was observed in all tissues of the post-implantation rat embryo, from egg cylinder to 30 somite stages (7 to 12 days). Heavy signal was noted in primitive ectoderm and neural tube. The wide embryonic and extraembryonic distribution and abundance of HMG-CoA reductase mRNA may reflect developmental requirements for products of the mevalonate pathway, e.g., isoprenoids for post-translational farnesylation of p21ras.     

Therapeutic use of monoclonal antibodies: a new era?

The serum pyruvate and lactate levels were studied after exercise on a bicycle ergometer in a family of diabetes mellitus (DM) associated with a mutation at nucleotide 3243 in the mitochondrial gene. A 56-year-old Japanese woman with the mutation at a percentage of 5% in the blood had insulin-dependent DM and sensory hearing loss without muscle symptoms. Her serum lactate and pyruvate levels increased markedly during and after exercise on a bicycle ergometer. Two of her sons were found to have the same mutation at a percentage of 17% and 18%, respectively. Her 26-year-old son was found to have borderline DM after oral glucose loading, although he showed no abnormalities of the metabolism of pyruvate and lactate. Her 31-year-old son showed no abnormalities after oral glucose loading and after exercise on a bicycle ergometer. Although the same mutation causes more severe MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), little is known about whether these diabetic patients are subclinically involved with myopathy. The noninvasive ergometer exercise with determination of serum pyruvate and lactate may be useful in evaluating the severity of myopathy in these patients.     

An immunohistochemical method of detecting Mycoplasma species antigens by use of monoclonal antibodies on paraffin sections of pneumonic bovine and caprine lungs

Lung samples from pneumonic lesions in cattle and goats, naturally or experimentally infected with strains of the Mycoplasma mycoides cluster, were fixed in formalin and embedded in paraffin. An immunohistochemical technique using monoclonal or polyclonal antibodies was performed on tissue sections in order to detect Mycoplasma antigens. Four monoclonal antibodies (MAbs), one (2A3) raised against M. mycoides ssp. mycoides small colony (SC) and large colony (LC), two (1D3 and 5E5) against M. mycoides ssp. capri, and one (5A10) against M. bovis, were used. A range of polyclonal antibodies, raised to the individual subspecies of the M. mycoides cluster, and one to Pasteurella haemolytica, was also used. The MAb 2A3 showed positive immunostaining in lung sections from cattle and goats naturally and experimentally infected with M. mycoides ssp. mycoides SC and LC, but not with pneumonic lesions of cattle and goats due to other members of the M. mycoides cluster, M. bovis or Pasteurella spp. The MAb 1D3 showed immunostaining in lung sections from goats naturally and experimentally infected with M. mycoides ssp. capri, but again not with pneumonic lesions caused by other members of the M. mycoides cluster, M. bovis or Pasteurella spp. The MAb 5E5 immunoreacted in sections from pneumonic lesions from all animals infected with one of the three M. mycoides cluster subspecies used in the study, but not with M. bovis or Pasteurella infected tissue. Immunoreaction was mainly found in the cell debris around necrotic areas, as well as in macrophages, neutrophils and epithelial cells. The localization of antigens of the M. mycoides cluster using polyclonal antisera followed basically the same pattern as that obtained with the monoclonals. However, a wide cross reactivity was found between different antisera and relatively high background immunostaining was also seen, especially in necrotic areas. The results suggest that immunohistochemical methods using monoclonal antibodies are useful tools for the diagnosis and study of the pathogenesis of pneumonia caused by the Mycoplasmas of the M. mycoides cluster.     

Evaluation of a competitive ELISA for detection of antibodies against avian influenza virus nucleoprotein

BACKGROUND: Growth hormone (GH) has been shown to promote wound healing and to improve protein metabolism in burned patients. Through immunomodulation, GH has also protected rats infected with Salmonella typhimurium and mice infected with Escherichia coli. In spite of advances in the management of patient care for those with thermal injuries, high mortality rates of burned patients as a result of infections are of special concern. An improvement in the resistance of burned patients to certain infections will make the beneficial role of GH very clear. In this study, therefore, the immunomodulating effects of recombinant human GH (rhGH) in thermally injured mice exposed to opportunistic herpesvirus infections were investigated. METHODS: (1) Burned mice, exposed to herpes simplex virus type 1 (HSV-1), were treated subcutaneously with rhGH (4 mg/kg) and observed for 21 days to determine the protective antiviral effect of rhGH. (2) Because of reports describing a lack of interferon-gamma (IFN-gamma) responsiveness in burned mice, the IFN-gamma-producing ability of the splenic mononuclear cells (SMNC) from burned mice treated with rhGH was examined. (3) Because the generation of burn-associated suppressor macrophages that can inhibit the IFN-gamma production by SMNC has been previously described, the suppressor cell activities of macrophages from burned mice treated with rhGH were examined. RESULTS: After exposure to lethal amounts of HSV-1, mice treated with rhGH displayed a reduced mortality rate compared with control mice treated with saline. SMNC from burned mice treated with rhGH produced IFN-gamma, whereas this cytokine was not produced by SMNC from burned mice treated with saline. Also, an inhibition of the generation of burn-associated suppressor macrophages was displayed in burned mice treated with rhGH. CONCLUSION: Exogenous administration of rhGH caused an improvement in the resistance of burned mice to HSV-1 infection. In burned mice treated with rhGH, the impaired IFN-gamma responsiveness was restored and the generation of burn-associated suppressor macrophages was inhibited. IFN-gamma, a typical antiviral cytokine induced by rhGH through the regulation of the suppressor macrophage generation, may therefore play a role in the protection of burned mice infected with a lethal amount of HSV-1.     

Evaluation of multiple antibodies to Epstein-Barr virus as markers for detecting patients with nasopharyngeal carcinoma

Five serological tests were assessed for their sensitivity for screening and early detection of nasopharyngeal carcinoma (NPC). The tests included the detection of antibodies to various gene products of EBV: viral capsid antigen (VCA) using an indirect immunofluorescence assay (FA), DNase using an activity neutralisation test (NT), Dnase using an enzyme-linked immunosorbent assay (ELISA), DNA polymerase (DP) using NT, and major DNA binding protein (MDBP) by ELISA. Sera from 100 NPC outpatients and 20 NPC patients, who were detected in a prospective study, were examined. The results showed that levels of antibody to DNase detected by ELISA and to DP detected by NT and the positivity rate for VCA by FA increased with NPC stage. More species of EBV antibody became detectable as NPC progressed. The detection of anti-MDBP antibody by ELISA was suitable for screening for NPC. Anti-DP antibody detected by NT was a valuable marker both for early detection and prognosis of NPC. Detection of anti-DNase antibody by ELISA was the most sensitive method for detection of NPC. No single test was sufficient to detect all the NPC patients and a combination of anti-DNase by ELISA with other tests are recommended to identify NPC patients.     

Mechanism of protective immunity against influenza virus infection in mice without antibodies

We describe a hypothalamus-specific mRNA that encodes preprohypocretin, the putative precursor of a pair of peptides that share substantial amino acid identities with the gut hormone secretin. The hypocretin (Hcrt) protein products are restricted to neuronal cell bodies of the dorsal and lateral hypothalamic areas. The fibers of these neurons are widespread throughout the posterior hypothalamus and project to multiple targets in other areas, including brainstem and thalamus. Hcrt immunoreactivity is associated with large granular vesicles at synapses. One of the Hcrt peptides was excitatory when applied to cultured, synaptically coupled hypothalamic neurons, but not hippocampal neurons. These observations suggest that the hypocretins function within the CNS as neurotransmitters.     

Characterization of three novel monoclonal antibodies against hepatitis C virus core protein

Three novel monoclonal antibodies (MAbs) were established against a recombinant hepatitis C virus (HCV) core protein derived from cloned genotype 1b HCV cDNA. MAbs C7-50 and C8-59 recognize a conserved linear epitope represented by amino acid residues 21 to 40 of the nucleocapsid protein. MAb C8-48 is directed against a strain-specific conformational epitope located within the first 82 amino acids. A sensitive two-site MAb-based immunoradiometric assay was established using antibodies directed against distinct epitopes on the nucleocapsid protein. Processed 21 kDa core protein was detected by immunoblotting in human hepatocellular carcinoma cell lines and primary adult rat hepatocytes transfected with a cytomegalovirus promoter-driven expression construct. Immunofluorescence microscopy studies revealed a granular and vesicular cytoplasmic staining pattern. MAb C7-50 was used successfully to detect HCV core antigen in chronically infected chimpanzee liver tissue. These MAbs represent important reagents for the study of HCV biology and for the development of immunodiagnostic assays.     

The development and certification of the Hepabest anti-HBc IgG test system for detecting antibodies to the core antigen of the hepatitis B virus]

Postembryonic development of the nestlings in microgravity has revealed their capacity for orientation and stabilization of their position in space which called for developing the special devices. Under terrestrial laboratory conditions the facility for maintaining the nestlings aged up to 10 days has been developed and tested. The results of tests have indicated that despite the restricted volume of individual cages the nestlings can grow without significant variations from the norm as evidenced by the dynamics of nestlings body mass during 10 days and the amount of consumed food.     

Antigenic differentiation of turkey rhinotracheitis virus strains using monoclonal antibodies and polyclonal antisera

Monoclonal antibodies (mAbs) were prepared against the 8597/CV94 strain of turkey rhinotracheitis virus (TRTV). These mAbs were used to investigate antigenic relationships among three strains (8597/CV94, 1162/92 and CVL14/1 strain) of TRTV, together with polyclonal chicken and rabbit antisera to 8597/CV94 strain, and guinea pig antisera to each of the three strains. Thirty mAbs to the glycoprotein (G:3 clones), fusion (F1:6 clones), phosphorylated (P:6 clones), nucleocapsid (N:12 clones), and matrix (M:3 clones) proteins of viral antigen were obtained by cell fusion. Among these, two mAbs to F1 protein showed virus neutralizing activity. The results of ELISA test indicated that some mAbs only reacted to the 8597/CV94 strain, some reacted to 8597/CV94 and 1162/92 strains, and others reacted to all three viral strains. In neutralization tests with the three virus strains, polyclonal chicken and rabbit antisera against the 8597/ CV94 strain showed the same antibody titers. Results with four neutralizing mAbs including two previously reported mAbs [Ref. 21] indicated the titers of two mAbs (Pn2-2E and Pn3-2F) to 8597/CV94 were much higher than those to the other two viral strains. No differences were observed in the titers of the other two mAbs (Pn01-8E and Pn06-4D) against any viral strains. In cross-neutralization tests with polyclonal guinea pig antisera, there was some variations among viral strains. This work demonstrated that the Japanese isolate 8597/CV94 of TRTV is somewhat different in antigenicity from two British isolates from chickens and turkeys.     

Analysis of the bovine respiratory syncytial virus fusion protein (F) using monoclonal antibodies

Seven monoclonal antibodies (MAbs) directed against bovine respiratory syncytial virus (BRSV) fusion (F) protein were produced and characterized by radioimmunoprecipitation and immunofluorescence assays. These seven MAbs together with the previously described MAbs (Beeler and Van Wyke Coelingh, 1989) to the F protein of human respiratory syncytial virus (HRSV) were used to study the antigenic variation of 12 strains of ungulate RSV. All except one MAbs specific for the HRSV-F protein reacted with ungulate RSV strains less efficiently, indicating that some epitopes are conserved, and others are not conserved on the F proteins of HRSV and BRSV strains. Three MAbs specific to the BRSV-F protein neutralized virus infectivity and reacted with all the ungulate RSV strains, suggesting that these epitopes are well conserved. Based on the reactivity of three other MAbs specific to the BRSV-F protein, ungulate RSVs could be grouped into two subgroups. The results indicated that there are antigenic variations in the F protein among ungulate RSV strains.     

Anti-idiotype monoclonal antibodies against anti-microcystin antibody and their use in enzyme immunoassay

Two different makes of bioimpedance spectrometer (UniQuest-SEAC SFB-3 and Xitron 4000B) were used for a series of measurements on volunteers and patients in intensive care. Although each machine was accurate over the frequency range 5 to 500 kHz when bench tested on model resistor-capacitor circuits, significant differences in their recorded impedance parameters appeared when used in vivo, especially on intensive care patients. A series of laboratory tests was performed on each machine simulating the situation in vivo to identify possible reasons for these differences. Whilst stray capacitance in the environment was identified as the major contributor to variability in high-frequency performance, interaction between electrode impedance and lead positioning was also a factor. The observed phase shift with frequency or time delay (Td) used in the Xitron modeling software appears to be the result of a time constant caused by stray capacitance and so is unlikely to have any biological meaning. Significant differences in the in vivo numerical values produced by bioimpedance spectrometers may be attributed to instrument design, data processing and, in particular, the clinical environment.     

Antigenic variation of the Epstein-Barr virus nuclear antigen EBNA1 as revealed by monoclonal antibodies

To obtain specific immunological probes for studying molecular mechanisms involved in cell renewal, cell differentiation, and pattern formation in intact and regenerating planarians, we have produced a hybridoma library specific for the asexual race of the fresh-water planarian Dugesia (Girardia) tigrina. Among the 276 monoclonal antibodies showing tissue-, cell-, cell subtype-, subcellular- and position-specific staining, we have found monoclonal antibodies against all tissues and cell types with the exception of neoblasts, the undifferentiated totipotent stem-cells in planarians. We have also detected position-specific antigens that label anterior, central, and posterior regions. Patterns of expression uncovered an unexpected heterogeneity among previously thought single cell types, as well as interesting cross-reactivities that deserve further study. Characterization of some of these monoclonal antibodies suggests they may be extremely useful as molecular markers for studying cell renewal and cell differentiation in the intact and regenerating organism, tracing the origin, lineage, and differentiation of blastema cells, and characterizing the stages and mechanisms of early pattern formation. Moreover, two position-specific monoclonals, the first ones isolated in planarians, will be instrumental in describing in molecular terms how the new pattern unfolds during regeneration and in devising the pattern formation model that best fits classical data on regeneration in planarians.     

Primary influenza A virus infection induces cross-reactive antibodies that enhance uptake of virus into Fc receptor-bearing cells

Sera of young children who had had a primary infection with influenza A virus or were immunized with a live attenuated influenza A virus vaccine were examined for antibody responses that neutralized virus or enhanced uptake of virus into Fc receptor-bearing cells, because antibodies that enhance uptake of influenza virus into Fc receptor-bearing cells have been reported using mouse immune serum and monoclonal antibodies. The neutralizing antibody titers to the homologous infecting virus and to another H1N1 virus isolated several years later were higher after natural infection than after infection with the live attenuated virus. Natural infection and the attenuated vaccine induced antibodies that enhanced uptake of homologous virus and H1N1 virus isolated several years later. These results demonstrate that primary influenza A virus infection results in the induction of infection-enhancing antibodies.     

The reactivity of monoclonal antibodies against orf virus with other parapoxviruses and the identification of a 39 kDa immunodominant protein

A panel of 27 mouse monoclonal antibodies (Mabs) was raised against orf virus. Sixteen of these Mabs reacted with a protein with a molecular mass of 65 kDa, 8 reacted with a protein with a molecular mass of 39 kDa and three remain uncharacterised. Reactivity of the Mabs with a library of recombinant vaccinia viruses expressing various regions of the NZ-2 orf virus genome identified the approximate positions of the genes encoding these 2 immunodominant orf virus proteins. The gene encoding the 39 kDa protein was identified and sequenced. The protein was detected in an envelope fraction of orf virus and was shown to be homologous to the envelope protein encoded by the H3L gene of vaccinia virus. The 65 kDa protein has not been fully chracterised, but the gene encoding it has been localised to a 10 kbp region of the orf virus genome. The Mabs were used to discriminate 4 parapoxviruses derived from sheep, 2 from cattle and 1 each from a seal and squirrel. Eighteen Mabs reacted with all 4 sheep viruses, 19 Mabs reacted with both cattle viruses, 6 recognised seal parapoxvirus and 2 recognised the squirrel parapoxvirus. Only one of the 27 Mabs reacted with all 8 parapoxviruses suggesting it recognises a conserved epitope within the genus.