Establishment of a sandwich ELISA method for detection of reporter chloramphenicol acetyltransferase gene / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To establish a sandwich ELISA method for detection of reporter chloramphenicol acetyltransferase (CAT) gene.</p><p><b>METHODS</b>The full length sequence of CAT gene was amplified with PCR using plasmid pBLCAT6 as template, and inserted into the prokaryotic expression plasmid Pgex-2T. The purified fusion protein was emulsified with complete or incomplete Freund adjuvant and injected subcutaneously into rabbits. The antibody was labeled with biotin, and a sandwich ELISA technique with biotin streptavidin amplify system was established. Several CAT reporter plasmids containing different HPV 16 LCR sequences were generated and transfected transiently to monolayer cells in vitro. The cytoplasm proteins were extracted and the expressions of CAT were evaluated with the newly established ELISA assay.</p><p><b>RESULTS</b>SDS-PAGE displayed that the molecular weight of the expressed fusion protein was about 54,000. The prepared antiserum was able to recognize the CAT protein expressed by mammalian cells or prokaryote cells. Under the control of different promoters and their regulate sequences,two to eight folds CAT expression increased were evaluated in transiently transfected mammalian cells by the newly established sandwich ELISA method.</p><p><b>CONCLUSIONS</b>The established method could sensitively reflect the activities of the upstream promoters, as well as the influence of exchanges of nucleotides within the regulate region on the promoter activities. Therefore, it proposes a convenient assay for the studies using CAT as the reporter gene.</p>

Expression of human S100 protein and preparation of specific antiserum for S100 and establishment of a quantitative measurement for S100 protein in CSF specimens of patients with Creutzfeldt Jakob disease / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a method that can quantitatively detect S100 protein in CSF, and evaluate the possibility in diagnosis of Creutzfeldt-Jakob disease (CJD).</p><p><b>METHODS</b>S100 gene was amplified by PCR from a commercially supplied human brain cDNA library. After verified by sequence analysis, the full length of S100 DNA was subcloned into a (GST) expression vector Pgex-2T, and the expression of GST-S100 fusion protein was induced. Rabbits were immunized with the purified GST-S100 fusion protein, and the antiserum raised against S100 protein was collected and further evaluated. Using biotin-avidin system, a sandwich ELISA was established for quantitatively determining S100 protein, and further, used in screening for S100 protein in CSF and serum samples.</p><p><b>RESULTS</b>SDS-PAGE assays yielded a roughly 35,000 GST-S100 fusion protein. Using the established method, three CSF samples from probable CJD patients (14-3-3 protein positive in CSF) showed higher concentration of S100 protein (higher than 2.900 microg/L), whereas other CSF samples collected from patients with other CNS diseases showed lower concentration of S100 (less than 0.180 microg/L).Moreover, the sera S100 proteins from all the collected samples showed distinct individual difference.</p><p><b>CONCLUSIONS</b>The established method can be used in determining S100 protein in CSF quantitatively. The feasibility and significance of S100 protein in CSF for diagnosis of CJD should be further considered with more CSF samples.</p>

Preparation and primary application of 14-3-3 specific polyclonal antibody / 中华神经科杂志

Objective Preparation of specific antibody of neuron protein 14 3 3 and application for the diagnosis of prion diseases. Methods The rabbits were immunized with prokaryotic expressed GST 14 3 3 fusion protein and the collected antiserum was evaluated by ELISA and Western blot assays. Results ELISA revealed that the reactive titer of the prepared antibody against purified 14 3 3 protein was up to 1:4 096 000; Western blot assays confirmed that both prokaryotic expressed,and the natively presenting 14 3 3 proteins in the brain extracts were recognized by the prepared antibody. Two cases of clinically diagnosed Creutzfeldt Jakob disease (CJD)with typical EEG changes showed the positive 14 3 3 protein in cerebral spinal fluid (CSF). Four patients with dementia and narcoma but without typical EEG change were 14 3 3 negative in CSF,three of them being even further tested by brain biopsies without typical histological change of CJD and presentation of protease resistant PrP Sc protein. 35 samples CFS collected from patients without dementia were 14 3 3 negative in CSF. All the results were the same as that of standard 14 3 3 polyclonal antibody.Conclusion Using purified prokaryotic expressed GST 14 3 3 fusion protein as antigen,the specific antibody was elicited in the immunized animals. The prepared antibody can be used in identification of 14 3 3 protein in CSF for the diagnosis of CJD.