Yiping Xun

Generation and evaluation of antibodies against human MGF E-peptide by reverse phase protein microarray and reverse competitive ELISA

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.

Prohibitin Is Involved in Patients with IgG4 Related Disease

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.

Identification of heat shock protein 27 as a novel autoantigen of Behçet’s disease

OBJECTIVE The aim of this study was to identify candidate pathogenic autoantigens of Behçets 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.

Electron Transfer Flavoprotein Subunit Beta Is a Candidate Endothelial Cell Autoantigen in Behçet’s Disease

Behçet’s disease (BD) is a chronic inflammatory disease with multisystem involvement, and it is listed as a rare disease in the United States but is common in the Middle East, China, and Japan. The aim of this study was to identify novel autoantigens in Chinese patients with BD. First, the candidate autoantigens were screened by Western blotting, and the sequences of putative antigens were identified by LC-MALDI-TOF/TOF mass spectrometry. Next, the screened protein was cloned, expressed and purified. Then, an optimized ELISA was developed, and the serological criteria were evaluated using a large number of confirmed patients. One antigen with a molecular weight of approximately 28 kDa was identified as electron transfer flavoprotein subunit beta (ETFB). Positive reactivity was detected in recombinant human ETFB sera from 38 of 92 BD patients (41 %) and 1 of 90 healthy controls (1 %).

Determination of Deoxynivalenol, Zearalenone, Aflatoxin B1, and Ochratoxin by an Enzyme-Linked Immunosorbent Assay

Despite efforts to control fungal contamination, some mycotoxins, including deoxynivalenol, zearalenone, aflatoxin, and ochratoxin, are ubiquitous in the rural China. The purpose of this study was to establish a simple method for noninstrumental detection of these residues by a multianalyte dot enzyme-linked immunosorbent assay. The multianalyte dot enzyme-linked immunosorbent assay was simple, inexpensive, and fast and is expected to have broad applications in the food processing industry in rural China.

Screening hybridomas for anabolic androgenic steroids by steroid analog antigen microarray

BACKGROUND Currently, dozens of anabolic androgenic steroids (AAS) are forbidden in the World Anti-Doping Agency Prohibited List, however, despite extensive investigation, there are still lots of AAS without corresponding monoclonal antibodies. RESULTS A steroid analog antigen microarray made up of ten AAS was fabricated to screen the hybridoma and it was found an original unsuccessful clone turned out to be a candidate anti-boldenone antibody, without any cross-reactions with endogenous AAS or 44 different AAS standard reference materials tested. CONCLUSION Our findings suggested that steroid analog antigen microarray could be a promising tool to screen and characterize new applications of antibodies for structure analogs, and this also exhibits the potential to fast identify effective epitopes of hybridomas in a single assay.

Identification of Moesin as a Novel Autoantigen in Patients with Sjögren’s Syndrome

BACKGROUND SjOgrens Syndrome (SS) is a systemic and chronic autoimmune disorder that affects the exocrine glands with massive autoantibody production. Although the pathogenesis of the disorder is incompletely understood, but some studies have reported that anti-moesin antibodies have been detected in autoimmune diseases with which SS is closely associated. Here, we have investigated moesins potential involvement in SS. OBJECTIVE This study aims to verify whether moesin is a specific autoantigen involved in Chinese Hans SS patients. METHODS First, recombinant human moesin was expressed and purified. Next, the protein was verified as antigen by Western blotting and immunoprecipitation. The positive protein band in the immunoprecipitation was identified by (MALDI-TOF/TOF). Finally, an optimized ELISA (Enzyme- Linked Immunosorbent Assay) kit was developed to measure the titer concentration of anti-moesin antibody-positive patients in a large cohort of clinical subjects. RESULTS Univariate analysis revealed that the proportion of individuals positive for serum IgG against recombinant human moesin was 42 % in a group of Chinese Hans SS patients (21 of 50), 22 % in systemic lupus erythematosus patients and (11 of 50), compared to only 4 % in healthy controls (2 of 50). CONCLUSION An association between anti-moesin antibodies and SS manifestation have been found which may be considered a suspected serum biomarker for the development of SS.

Circular IGFBP-3 level affected by the gene transfer of different growth hormone isoforms.

Abstract To evaluate whether through gene transfer, which growth hormone (GH) isoform can influence the circulation IGFBP-3 level, mice were injected three times with the human cytomegalovirus (CMV) mediated mammalian expression vector encoding different GH isoform gene. Then blood samples were drawn and the ELISA method was used to determine the circulation concentration of IGFBP-3. It was found that the 22 kDa GH gene could increase IGFBP-3 levels to 1.94 μg/mL whereas 20 kDa GH, 17 kDa GH and 5 kDa GH genes did not play a role in the circulation expression of IGFBP-3.