Validation of a real-time RT-PCR assay for sensitive and specific detection of classical swine fever.

A fully validated, ready-to-use, real-time reverse transcription-polymerase chain reaction (RT-PCR) assay, multiplexed for simultaneous detection of an internal control, for the simple and rapid diagnosis of classical swine fever (CSF) was developed. Primers and FAM-labeled TaqMan-probes specific for classical swine fever virus (CSFV) were selected from the consensus sequence of the 5' non-translated region (5' NTR) of 78 different CSFV strains. For determining analytical sensitivity, an in vitro transcript (T7-PC3alf) of the 5' NTR was constructed and tested. In addition, the T7-PC3alf transcript was further used as a positive control and a standard for quantitation of CSFV genome copies. A second heterologous in vitro transcript based on a specific primer-probe HEX-system was designed as an internal positive control for the RNA isolation step and RT-PCR. By using limited primer concentrations for the internal control, no adverse effects on the sensitivity of the CSF-system could be observed, and the newly designed duplex real-time RT-PCR proved to have a sensitivity of approximately eight copies. The primer-probe combination selected was strictly CSFV-specific and no amplification was observed in all non-CSFV pestiviruses tested.

An actin-binding protein is involved in pestivirus entry into bovine cells.

Infection of bovine cells with bovine viral diarrhoea virus (BVDV) can be blocked by the monoclonal antibody (mab) BVD/CA 26, which is directed against a cellular membrane protein. To characterize this molecule, it was isolated and purified by column chromatography. It was found to be an acidic, glycosylated membrane protein consisting of two polypeptide chains of about 28 and 56 kDa. Under non-reducing conditions the chains formed multimers of about 200 kDa. In an actin binding assay the 56 kDa polypeptide chain bound to F-actin as judged by co-sedimentation with actin filaments. Since the target molecule of BVD/CA 26 is localized on the surface of living cells and additionally binds to F-actin, a possible biological function may be to connect the cortical actin filaments with the cellular plasma membrane. The blocking effect of BVD/CA 26 indicates that this cellular plasma membrane protein is involved in the endocytic pathway of BVDV particles.

Purification of a 28 kDa Babesia (Theileria) equi antigen and a 29 kDa spurious erythrocyte antigen from in vitro culture through ion exchange chromatography.

An extract of in vitro cultivated Babesia equi was fractionated using a MonoQ anion exchange column. Separation of a 28 kDa B. equi antigen from a 29 kDa spurious erythrocyte antigen, both of which were intensely immunoreactive, was achieved by chromatography of the infected erythrocyte proteins. Using tricine-SDS-PAGE, the 28 kDa antigen of B. equi showed multiple band resolution, while the 29 kDa antigen was consistently resolved as a single band. The 29 kDa antigen was identified in both infected and non-infected erythrocyte extracts. Moreover, B. equi antiserum recognised this antigen in the non-infected erythrocyte extract, and conversely serum from horses not infected with babesia detected the antigen in infected erythrocyte extract. This 29 kDa antigen could represent a horse erythrocyte isoantigen. The purified 28 kDa antigen is specifically recognised by B. equi antisera and therefore could be useful for the production of the recombinant replica and to employ these in further test systems.

Polysialylated neural cell adhesion molecule serum levels in normal children.

Serum concentrations of polysialylated neural cell adhesion molecule (PSA-NCAM), a developmentally regulated form of the NCAM, have been recently described to be elevated in children with rhabdomyosarcoma and neuroblastoma, proving PSA-NCAM to be a tumor marker of diagnostic relevance to these malignancies. The present investigation was undertaken to define age-dependent reference intervals in normal children. Serum concentrations of polysialylated NCAM were determined in 366 children aged newborn to 17 y and in 18 adult patients by an immunoluminescence assay using the polysialic acid-specific MAb 735. Serum levels in newborn children were 51.7 kU/L (mean +/- 12.0 kU/L SD), whereas in adult patients they were 9.9 kU/L (mean +/- 3.5 kU/l SD). Assigning the patients to 14 different age groups, a gradual decay of PSA-NCAM serum concentrations was observed, and therefore, mean levels and empirical interpolated percentiles were determined for every age group. Applying specially fitted logistic functions, two different sigmoid graphs were obtained describing the age-dependent decrease of serum PSA-NCAM during the neonatal period and during childhood. The age at which the levels reach half the initial value was located at 3.1 d (mean +/- 2 d SE) and 14 y (mean +/- 1 y SE), respectively. There was no difference between male and female individuals. Repeated measurements revealed variations below 10%. For the first time, our study describes serum levels of PSA-NCAM in children of different age and their gradual decay until adulthood.

Polysialylated neural cell adhesion molecule as a marker for differential diagnosis in pediatric tumors.

BACKGROUND/PURPOSE: Some malignant pediatric tumors express the polysialylated form of the neural cell adhesion molecule (PSA-NCAM), which normally becomes restricted to a few regions of neural plasticity and regenerating nerve tissue after embryogenesis. Recently, serum concentrations of polysialylated NCAM have been shown to be tumor associated in children with rhabdomyosarcoma and neuroblastoma. This study was undertaken to evaluate polysialylated NCAM as a marker helpful in distinguishing between various embryonal tumors and other lesions suspicious of a malignancy. METHODS: Fresh frozen specimens from exemplary tumors of the thorax, adrenal glands, and kidneys of 17 children were investigated immunohistochemically by the Alkaline Phosphatase anti-Alkaline Phosphatase (APAAP) technique. Simultaneously, the patients' serum was investigated by a chemiluminescent immunoassay, which likewise used the polysialic acid-specific monoclonal antibody 735. RESULTS: Serum concentrations correlated with the expression of PSA-NCAM in immunohistochemistry. They were elevated in children with PSA-NCAM-positive tumors as alveolar rhabdomyosarcoma, undifferentiated neuroblastoma, and anaplastic Wilms tumor, but negative in all other patients. CONCLUSION: PSA-NCAM serves as a useful marker for differential diagnosis during workup of tumors suspicious of a malignant neoplasm.

Serum polysialylated neural cell adhesion molecule in childhood neuroblastoma.

Neuroblastoma cells express the polysialylated form of the neural cell adhesion molecule (NCAM), which normally becomes restricted to a few neural tissues after embryogenesis. In this study, we investigated serum levels of polysialylated NCAM in 14 children with different grades and stages of neuroblastoma using an immunoluminescence assay, and compared the results to 269 healthy control subjects. Simultaneously, the polysialylated NCAM content of the tumours was determined by immunohistochemistry. Serum levels were dramatically elevated (more than sixfold) in children with advanced stages and fatal courses of disease, whereas children with differentiated tumour types and limited disease had low or normal levels. Serum concentrations correlated with the polysialylated NCAM content of the tumours, and they decreased during successful therapy. We therefore suggest polysialylated NCAM to be a useful marker monitoring childhood neuroblastoma.

Polysialylated neural cell adhesion molecule in childhood rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) cells express the polysialylated (PSA) form of the neural cell adhesion molecule (NCAM). During embryogenesis, PSA-NCAM is widespread and dynamically regulates embryonal developing processes, whereas postnatally, PSA-NCAM becomes restricted to a few regions of neural plasticity and regenerating neural tissues. Recently, PSA-NCAM has been shown to be a diagnostic and prognostic marker in adult patients with small cell lung cancer and multiple myeloma, both PSA-NCAM-expressing tumors. In this study, we determined the amount of PSA-NCAM in tumor specimens of nine children with different histologic types and clinical stages of RMS immunohistochemically, using the polysialic acid-specific MAb 735. In seven children, serum levels were investigated by an immunoluminescence assay using the same MAb. Patients with extensive disease showed strong staining of the tumor specimens, whereas patients with limited stages or after chemotherapy had distinctly a lesser amount of PSA-NCAM or almost no staining. Simultaneously, the serum levels were very high (up to 9-fold) in patients with extensive disease, whereas patients with limited disease or after successful therapy had normal serum levels. We conclude that PSA-NCAM expression is high in tumor specimens and serum of patients with advanced stages of RMS and decreases during successful therapy. PSA-NCAM might therefore serve as a marker for diagnosis and monitoring childhood RMS.

Measures to overcome HAMA interferences in immunoassays.

The incidence of human anti-mouse antibodies (HAMA) in different patient sera panels may be debatable, the interference of HAMA, if present, in immunochemically based assays is, however, a fact. This interference can lead to falsely elevated or depressed results depending on the nature of the HAMAs involved and the particular assay format chosen. For several reasons, assays for serum tumor markers may be especially prone to HAMA interference. Consequently, in the development of such assays, special attention should be given to the HAMA issue. In our experience, the degree of HAMA interference varies greatly from one assay to another. Nevertheless, the HAMA issue has to be addressed. Several methods have been described to remove HAMA (and other interfering substances) via sample pretreatment. Alternatively, there are also some options to counterbalance the potential effect of HAMA by using assay reagents optimized for minimal HAMA susceptibility, e.g. inclusion of an excess of non-relevant mouse antibodies. However, there is no guarantee that any given assay will not be affected by HAMA. This is especially true if a portion of the HAMA in a patient's sample is comprised of anti-idiotypic "internal image" antibodies.

A new tumour marker assay for ovarian cancer on the OPUS immunoassay system.

The human tumour associated cancer antigen CA 125 is a glycoprotein with high molecular weight. The determination of this antigen has been proven to be useful in the monitoring of patients with ovarian cancer. OPUS OV, the tumour marker assay for the ovarian cancer antigen CA 125 is an ELISA that was developed for the family of fully automated random-access analyzers, OPUS, OPUS Plus and OPUS I Magnum. The assay uses a double monoclonal sandwich format (antibodies B27.1 and B43.13, Biomira/Canada). The first antibody is immobilized on the solid phase of the OPUS modules. Sample is automatically added and incubated for 5 minutes. The addition of a solution of the second antibody conjugated to the enzyme alkaline phosphatase leads to the formation of a sandwich complex within 5 minutes. The last step, the addition of the fluorogenic substrate 4-methylumbelliferyl phosphate, serves both as washing procedure and for the development of the fluorescence signal (kinetic measurement). OPUS OV has an assay range from 0-1000 kU/L with a detection limit of 5 kU/L. Within run cv's are 6-8%. A good correlation was found to commercially available kits for the determination of CA 125. We conclude that this new OPUS OV assay is a valid alternative for use in the routine determination of the cancer associated antigen CA 125 and allows more reliable determinations in terms of random access, speed, and ease of operation.

Cloning and expression of two genes from Babesia equi merozoites and evaluation of their diagnostic potential.

High-titre equine immune sera were used to screen a lambda gt 11 expression library of Babesia equi cDNA fragments. Two cDNA clones which did not cross-hybridize to each other were studied. Both clones hybridized specifically to DNA from B. equi but not to DNA from B. caballi, B. divergens or B. ovis. Recombinant proteins were expressed as glutathione S-transferase (GST) fusion proteins with apparent molecular weights of 40 kDa and 75 kDa. Polyclonal antibodies directed against the 40 kDa and 75 kDa recombinant proteins detected native antigens of 55 kDa and 50 kDa respectively in crude lysates of B. equi merozoites indicating that neither cDNA clone was full length (GST = 26 kDa). In Western blotting experiments the 75 kDa protein showed cross-reactivity with sera from horses infected with B. caballi and was not further investigated. The 40 kDa protein was additionally tested in an enzyme-linked immunosorbent assay (ELISA). A test was developed which had a calculated specificity of 99% and a sensitivity of 88% with sera from horses infected with the homologous strain of B. equi. The ELISA did not recognize sera from horses infected with B. equi strains from Brazil and Morocco.

Identification of cell membrane proteins linked to susceptibility to bovine viral diarrhoea virus infection.

Three monoclonal antibodies directed against cell surface molecules of bovine cells inhibited subsequent infections with bovine viral diarrhoea virus (BVDV). They specifically blocked the infectivity of three non-cytopathogenic and three cytopathogenic BVDV strains. These results showed that an important mechanism for virus uptake was inhibited. The ligand of the monoclonal antibody BVD/CA 17, which blocked infectivity most efficiently, was found on leukocytes from a wide range of domestic and wild even-toed ungulates using flow cytometric analysis. In contrast, the monoclonal antibodies BVD/CA 26 and BVD/CA 27 appeared to be specific for bovine cells. Immunoprecipitation of labelled bovine cell surface proteins showed that the three monoclonal antibodies bound to proteins with identical relative molecular masses (M(r)). Proteins of an apparent M(r) of 93 K and 60 K were precipitated from lysates of fetal bovine kidney cells irrespectively of the MAbs used.

[In vitro study of the properties of bioresorbable lactic acid polymer materials]. / Etude du comportement in vitro des matériaux biorésorbables en polymère d'acide lactique.

PURPOSE OF THE STUDY: The potential applications of biodegradable osteosynthesis implants present many advantages over conventional metallic devices. Polyesters of the poly and hydroxy-acid type were recognized early as serious candidates. These polymers have demonstrated a very good biocompatibility and are biodegradable in vivo. After biological and chemical testing poly L. lactic acid 98 (PLA 98) was selected as a candidate. We used a static and dynamic investigation in vitro to assess firstly the material properties of PLA 98 and secondly how its characteristics could be modified within a physiological environment. MATERIAL: Michel Vert and colleagues have shown that polymers of lactic acid have a similar time to resorption providing they contain 98 per cent of the "L" form of the polymer. In vitro studies were assessed on bars made in PLA 98. METHODS: In a first time in vitro studies in traction and flexion on bars allowed an assessment of mechanical properties of PLA 98. In a second time stresses were applied on bars using a physiological environment (Haemacel - 37 degrees C). In a third time we assessed the mechanical properties at the temperature of 37 degrees C with dynamic tests on bars in traction and flexion. RESULTS: The stress-strain curves on bars showed that the material is fragile. Sterilisation with ethylene-oxide did not affect the mechanical properties. When bars were placed in a thermostatically controlled (37 degrees C) physiological environment, the stress-strain curve showed that the material became ductile. With a temperature of 37 degrees C and with a frequency better than one hertz, the dynamic tests on bars showed that the material endurance is good up to 20,000 cycles. At 37 degrees C and at the end of one month, the Young modulus and the maximal strain before breaking lose 50 per cent of their initial value. DISCUSSION: All things considered and as the digital value showed, the PLA 98 appear to be ten times less strong than steel. In a physiological environment the mechanical properties improved due to hydratation of the polymer. The material become quickly ductile or malleable. This allowed transient loading without causing breakage. CONCLUSION: The mechanical properties of bioresorbable materials are very different from those of stainless steel and there is a learning curve in their utilisation. The PLA 98 polymer has demonstrated a very good biocompatibility and is totally biodegradable in vivo. With these results we think that PLA 98 can be used in clinical practice. Indications and clinical use should remain limited to bones regions with low applied stresses.

The monomer covalently closed linear replicative form DNA of Aleutian disease parvovirus is infectious after transfection into permissive cells.

The recently described monomer covalently closed linear replicative form DNA (Mccl RF DNA) of Aleutian disease parvovirus (ADV) is an infectious intermediate of the viral DNA replication cycle. Transfection of highly purified Mccl RF DNA into susceptible feline kidney cells (CCC clone 81 cells) resulted in viral DNA replication, expression of viral proteins and synthesis of infectious progeny virus. Mccl RF DNAs generated under permissive (32 degrees C) or non-permissive (37 degrees C) conditions were shown to be biologically indistinguishable. The accumulation of the Mccl RF DNA form at the non-permissive temperature in vitro strongly resembles that in bone marrow cells of naturally infected mink and may reflect one mechanism contributing to virus persistence of ADV in vivo.

Demonstration of extrachromosomal DNA from Babesia equi merozoites.

An extrachromosomal nucleic acid element was detected in high-molecular-weight DNA preparations form Babesia equi merozoites. This extrachromosomal element was shown to be DNA rather than RNA and had an apparent fragment size of about 9 kilobase-pairs (kb). Hybridization experiments using purified 9-kb DNA as a probe revealed sequence homologies with extrachromosomal DNA from two other Babesia species.

Differentiation of pestiviruses by a hog cholera virus-specific genetic probe.

A hog cholera virus (HCV)-specific genetic probe has been generated after cloning of the genomic viral RNA. This probe distinguished between HCV and the closely related bovine viral diarrhoea virus (BVDV). Furthermore, it detected a broad spectrum of HCV strains and isolates which differ in their phenotype such as virulence.

Enhanced full-length transcription of Sindbis virus RNA by effective denaturation with methylmercury hydroxide.

49-S Sindbis virus RNA was reverse transcribed into a complementary DNA. The RNA templates were denatured by three different methods prior to DNA synthesis. Efficient full-length transcription was only achieved after treatment with methylmercury hydroxide.