Establishment of a Sandwich ELISA Method for Detection of Vascular Endothelial Growth Factor in Serum Samples of Hepatocellular Carcinoma Patients

Objective To establish a sandwich ELISA method for detecting vascular endothelial growth factor (VEGF) in sera of population and the patients with hepatocellular carcinoma (HCC). Methods Full length and two truncated human VEGF cDNA sequences were amplified from a commercial plasmid pBLAST49-hVEGF by PCR and inserted into the prokaryotic-expression plasmid pET-32a or pGEX-2T. Various VEGF proteins were expressed and purified from E. coli in His-Trx or GST fusion forms. The specific VEGF antibodies were elicited in experimental rabbits and mice by immunization of the full length VEGF fusion protein His-Trx-VEGF1-165. After purification of antibodies with chromatograph of Protein G, a sandwich ELISA technique was established. Serum VEGF levels were evaluated in 229 adults and 291 HCC patients. Results SDS-PAGE displayed that the molecular weights of the expressed full length (His-Trx-VEGF1-165), N-terminal (His-Trx-VEGF1-100) and C-terminal (GST-VEGF100-165) human VEGF fusion proteins were about 38KD, 31KD, and 33KD, respectively. Western blots confirmed that the prepared antisera were able to recognize both prokaryoticly and eukaryoticly expressed recombinant VEGF proteins. Assays of serially diluted His-Trx-VEGF1-100 by the established sandwich ELISA method showed that the linear range of the standard curve was 0.625-320 ng/mL, with the squared correlation coefficient R2=0.991. Screening of a serum panel containing 291 serum samples of HCC patients and 229 health adults revealed that the average VEGF level in HCC patients was higher than that in healthy controls, with a statically significant difference. Conclusion The established sandwich ELISA reflects the level of serum VEGF and provide scientific basis for screening metastasis and recurrence of HCC using serum VEGF as an index.

Treatment of Scrapie Pathogen 263K With Tetracycline Partially Abolishes Protease-resistant Activity in vitro and Reduces Infectivity in vivo

Objective To study the possible effect of tetracycline on protease-resistant activity in vitro and infectivity in vivo of a scrapie strain 263K. Methods Scrapie pathogens were incubated with tetracycline at different concentrations for various periods of time and protease-resistant PrP signals were evaluated with proteinase K-treatment and Western blots. The preparations treated with tetracycline were intracerebrally inoculated into golden hamsters and typical TSE manifestations were noted. PrPSc in brain tissues of the infected animals was detected by PrP specific Western blot assays. Results Protease-resistant PrP was significantly reduced in or removed from the preparations treated with tetracycline in a dose-dependant manner. Compared with the control group after incubated for 53.75±0.50 days, the preparations treated with 5 mmol/L and 20 mmol/L tetracycline prolonged the incubation time of 61.5±1.73 and 59.5±0.58 days (P<0.05). Conclusion Treatment of scrapie pathogen 263K with tetracycline reduces or removes its protease-resistant activity in vitro.

Establishment of Cell Free Conversion System With Biotin-labelled Recombinant PrP^(sen) Expressed in E.coli

Objective To report a protocol using biotin-labelled PrP protein in cell free conversion assay instead of isotope. Methods A hamster PrP protein (HaPrP) was expressed in E. coli and purified with HIS-tag affinity chromatograph. After being labelled with biotin, HaPrP was mixed with PrPSc preparation from scrapie strain 263K. Results Protease-resistant bands were detected after four-day incubation. Conclusion The new conversion model provides a reliable, easily handling, and environment-friendly method for studies of prion and transmissible spongiform encephalopathies.

Preparation of Monoclonal Antibodies Against Prion Proteins With Full-length Hamster PrP

Objective To prepare the PrP specific monoclonal antibodies (mAbs) that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases. Methods Several BALB/c mice were immunized with recombinant hamster prion protein (HaPrP). Three hybridoma cell lines designated as B7, B9, and B10, secreting monoclonal antibodies against HaPrP, were established by hybridoma technique. The mAbs reactivities were evaluated with ELISA, Western blot, and immunohistochemistry. Results The mAbs produced by these cell lines reacted well with different recombinant hamster PrP proteins. Western blot analyses showed that mAbs B7 and B9 reacted with PrPSc from the scrapie-infected animals after proteinase K digestion with three glycosylated forms. The mAbs exhibited cross-reactivity with various PrPC from several other mammalian species, including humans and cattles. Immunohistochemistry assays confirmed that mAbs B7 and B9 could recognize not only extracellular but also intracellular PrPSc. Conclusion The mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.

T188K-Familial Creutzfeldt–Jacob Disease, Predominant Among Chinese, has a Reactive Pattern in CSF RT-QuIC Different from D178N-Fatal Familial Insomnia and E200K-Familial CJD

Dear Editor,Human prion diseases consist of sporadic, genetic/familial, and acquired forms. The familial form accounts for 5%-15% of all human prion diseases, including familial Creutzfeldt- Jacob disease (fCJD), Gerstmann-Straussler-Scheinker syndrome, and fatal familial insomnia (FFI)[1-3]. All genetic prion diseases are directly associated with mutations (point-mutation or insertion) in the PRNP gene located on human chromosome 20 and encodes prion protein (PrP). So far,>55 mutations in the PRNP gene have been described [4]. Some PRNP mutations and their related genetic prion diseases have been reported worldwide, while others show clear region- or ethnicity-associated features.