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Abstract: 2A Peptide sequences are now being widely used to construct multicistronic expression vectors. It is suggested that when only the first 2A-linked protein bears a signal sequence, the signal-less protein(s) downstream of 2A can also be translocated into the mammalian endoplasmic reticulum system through a “slipstreaming” mechanism. By using flow cytometry and cell surface CD90 as a localization indicator, we show here that slipstreaming translocation does not occur in mammalian cells; that is, the second protein downstream of 2A still requires signal sequence for secretary or membrane-anchored expression. [Copyright &y& Elsevier]

Invasive Aspergillosis is a high-risk illness with a high death rate in immunocompromised people due to a lack of early detection and timely treatment. Based on immunology study, we achieved an efficient production of anti-galactomannan antibody by Chinese hamster ovary (CHO) cells and applied it to time-resolved fluoroimmunoassay for Aspergillus galactomannan detection. We first introduced dual promoter expression vector into CHO host cells, and then applied a two-step screening strategy to screen the stable cell line by methionine sulfoximine pressurization. After amplification and fermentation, antibody yield reached 4500 mg/L. Then we conjugated the antibodies with fluorescent microspheres to establish a double antibody sandwich time-resolved fluoroimmunoassay, which was compared with the commercial Platelia™ Aspergillus Ag by clinical serum samples. The preformed assay could obtain the results in less than 25 min, with a limit of detection for galactomannan of approximately 1 ng/mL. Clinical results of the two methods showed that the overall percent agreement was 97.7% (95% CI: 96.6%–98.4%) and Cohen's kappa coefficient was 0.94. Overall, the assay is highly consistent with commercial detection, providing a more sensitive and effective method for the rapid diagnosis of invasive aspergillosis. [Display omitted] • An efficient and stable cell line expression system was constructed to produce of anti-galactomannan monoclonal antibodies. • The antibody yield reached 4500 mg/L by scaling up fermentation, a 1.3-fold increase. • A time-resolved fluorescence immunoassay was established by conjugating the antibodies to detect aspergillosis. • The assay is highly consistent with commercial Platelia™ Aspergillus Ag according to clinical serum results. [ABSTRACT FROM AUTHOR]

Objective To establish a high throughput, low cost, and simple nanotechnology-based method for the detection of single nucleotide polymorphism (SNP) loci in type 2 diabetes mellitus (T2DM). Methods Multiplex ligase detection reaction (LDR) amplification was performed using fluorescently labeled magnetic nanosphere-bound upstream LDR probes and downstream probes labeled with a unique fluorescent group for each SNP locus. The amplified LDR products were separated by magnetic nanospheres and then scanned by fluorescence spectroscopy. Four SNP loci associated with T2DM were detected, including the rs13866634 locus in SLC30A8, rs10811661in CDKN2A/2B, rs1111875 in the HHEX gene, and rs7903146 in the TCF7L2 gene. The SNP genotype was also determined by DNA sequencing as a control. Results The SNP genotypes of the four gene loci determined by the nanosphere-based multiplex LDR method were consistent with the DNA sequencing results. The accuracy rate was 100%. Conclusion A method based on multiplex PCR and LDR was established for simultaneous detection of four SNP loci of T2DM susceptibility genes. [ABSTRACT FROM AUTHOR]

Adenosine deaminase (ADA) is a key enzyme of adenosine metabolism. There are currently various kits and systems available for ADA measurement, and all yield variable results. This study optimized a reference measurement procedure (RMP) for serum ADA for the standardization of routine methods. ADA coupled with purine-nucleoside-phosphorylase, xanthine-oxidase and peroxidase was selected as the basic method and was optimized using Response Surface Methodology. Then the performance was validated and the results were compared after replication by 3 other reference laboratories. A reference interval was also developed. In addition, this optimized method was applied to calibrate a routine system. The intra-assay precision was 0.44% at both concentrations of 29.8 and 100.4 U/L, and inter-assay precision was 1.01% and 0.95% at 30.1 and 100.3 U/L, respectively. The linearity was up to 351.9 U/L (R2 = 0.9998), with no significant interference or carryover (<5%). A Comparison among 4 reference laboratories showed good reproducibility (R2 ≥ 0.9975). The procedure proved valid for a reference interval of 11.7–38.5 U/L. The mean relative deviation for a routine system was −55.9% and −3.7% before and after calibration. This candidate RMP for serum ADA can potentially be used for standardization of clinical systems. Image 1 • This candidate reference method for serum ADA will facilitate the implementation and standardization of clinical systems. • The enzyme coupling method used in this work is simple, easy, fast, non-toxic and has the potential for automation. • Measurement conditions were optimal, determined by response surface methodology. [ABSTRACT FROM AUTHOR]