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Xun Y.,University of Science and Technology Beijing | Chen G.,ImmunoHunt Corporation | Du H.,University of Science and Technology Beijing
Current Pharmaceutical Analysis | Year: 2015

Currently more and more high-throughput biotechnologies have been armed by immunologists to explore the pathological mechanism of different autoimmune diseases. The new wave of approaches for the integrated understanding of human immune system based on high-throughput methods, corresponding mathematical and computational tools are increasing. This paper described the combination tendency of multiple methods to investigate the whole immune process at different levels including transcription, translation and post-translation modification, using microarray, Luminex and CyTOF as an example. When proper high-throughput technologies were used in combination, there will be more comprehensive recognition on immune system. The application of such techniques will promote deeper and more integrated study in the immunization field, and become a promising study strategy in this field. © 2015 Bentham Science Publishers.

Du H.,University of Science and Technology Beijing | Li C.,University of Science and Technology Beijing | Jin H.,University of Science and Technology Beijing | Chen G.,ImmunoHunt Corporation | Xun Y.,University of Science and Technology Beijing
Bioanalysis | Year: 2013

Background: Since 2005, as one of prohibited substances on the Prohibited List of the World Anti-Doping Agency (WADA), the occurence of mechano growth factor (MGF) abuse in sport has likely increased. However, there is still no WADA-validated and -approved method for its detection. Results: Four polyclonal antibodies (Ab-K01, Ab-B01, Ab-B02 and Ab-K02) against MGF C-terminal peptides were generated, purified and evaluated by western blot, ELISA and reverse-phase protein microarray, respectively. It was found that all the antibodies could identify their corresponding antigen in mouse serum by reverse-phase protein microarray, in particular, Ab-K01 showed the highest affinity among them and might be a potential tool for the detection of antibody affinity. Furthermore, Ab-B01 and Ab-K01 were successfully used for the determination of MGF-40 by reverse competitive ELISA. Conclusion: The quantitative measurement of MGF-40 has laid the foundation for doping detection of MGF and further biological research on MGF. © 2013 Future Science Ltd.

Chen P.,University of Science and Technology Beijing | Shi L.,University of Science and Technology Beijing | Jiang Y.,University of Science and Technology Beijing | Ji Y.,University of Science and Technology Beijing | And 7 more authors.
Biochemical and Biophysical Research Communications | Year: 2015

Objective: The aim of this study was to identify candidate pathogenic autoantigens of Behc¸et's disease (BD) in pathogen-stimulated target cells. Methods: First, three cell lines were used as target cells to screen autoantibody. Second, selected target cells were simulated with pathogens. Third, western blotting was used for detecting the auto-antigens in cell extracts. Next, immunoprecipitation was performed and the amino-acid sequences of target antigens were analyzed by LC-MALDI-TOF/TOF. Then, the potential target antigen was expressed, purified, and immunologically confirmed. And finally, an ELISA kit was developed and clinically validated through the assessments of 456 clinical samples with BD. Results: One antigen with a molecular weight of approximately 27-kDa was identified as heat shock protein 27 (HSP27). The reactivity of serum IgG against recombinant human HSP27 was detected in 52 of 91 BD patients (57%), 66 of 92 rheumatoid arthritis (RA) patients (72%), 32 of 90 Sjogren syndrome (SS) patients (36%), 22 of 92 systemic lupus erythematosus (SLE) patients (24%) and 0 of 91 healthy controls (HC). The reactivity of BD serum IgG antibodies against HSP27 was significantly higher than SLE (P < 0.0001) SS (P < 0.0001) and HC (P < 0.0001). Conclusions: This study identified HSP27 as a candidate endothelial cell autoantigen of BD, which is interesting and probably worth further exploration. © 2014 Elsevier Inc. All rights reserved.

Chen P.,University of Science and Technology Beijing | Yan H.,University of Science and Technology Beijing | Tian Y.,Chinese PLA General Hospital | Xun Y.,University of Science and Technology Beijing | And 14 more authors.
Scientific Reports | Year: 2015

Cell membrane proteins are believed to play a critical role in the pathogenesis of autoimmune diseases. However, few membrane autoantigens have been linked with Behçet's disease. Here, a cell-chip was performed to identify autoantibody target cells, and the suspected autoantigens were detected using immunoblotting. The amino acid sequences of the detected proteins were determined using LC-MALDI-TOF/TOF. Putative proteins were recombinantly expressed and purified, and a corresponding ELISA was developed and clinically validated using real clinical samples. It was found that a 36-kDa membrane protein - annexin A2 - was detected in approximately one-third of the patients' blood circulation. The immunohistochemistry results showed that annexin A2 was highly expressed in vascular endothelial cells. Moreover, vascular involvement was significantly higher in the anti-annexin A2 antibody-positive group versus the anti-annexin A2 antibody-negative group among all the clinical samples analyzed, indicating that annexin A2 is a novel endothelial cell membrane antigen involved in Behçet's disease.

Du H.,University of Science and Technology Beijing | Shi L.,University of Science and Technology Beijing | Chen P.,University of Science and Technology Beijing | Yang W.,University of Science and Technology Beijing | And 5 more authors.
PLoS ONE | Year: 2015

Objective: IgG4-related disease (IgG4-RD) is a chronic systemic disease involved in many organs and tissues. As only limited autoantigens have been found since the beginning of this century, the aim of this study was to reveal new candidate autoantigens of IgG4-RD. Methods: Multiple cell lines including HT-29, EA.hy926, HEK 293 and HepG2 were used to test the binding ability of circulating autoantibodies from IgG4-RD sera. The amino-acid sequence was then analyzed by matrix-assisted laser desorption/ionization time-of-flight tandem (MALDI-TOF/TOF) mass spectrometry. After the cloning and expression of recombinant putative autoantigen in a bacterial expression system, the corresponding immuno assay was set up and utilized to observe the prevalence of serum autoantibodies in a large set of confirmed clinical samples. Results: One positive autoantigen was identified as prohibitin. ELISA analysis showed that a majority of patients with IgG4-RD have antibodies against prohibitin. Anti-prohibitin antibodies were present in the sera of patients with definite autoimmune pancreatitis (25/34; 73.5%), Mikulicz's disease (8/15; 53.3%), retroperitoneal fibrosis (6/11; 54.5%), other probable IgG4-RD (26/29; 89.7%) and Sjögren's syndrome (4/30; 13.3%) but not in apparently healthy donors (1/70; 1.4%). Conclusions: An association between prohibitin and patients with some IgG4-RD was observed, although the results were quite heterogeneous among different individuals within autoimmune pancreatitis, Mikulicz's disease and retroperitoneal fibrosis. © 2015 Du et al.

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