Yuying Wei

Highly Sensitive Microplate Chemiluminescence Enzyme Immunoassay for the Determination of Staphylococcal Enterotoxin B Based on a Pair of Specific Monoclonal Antibodies and Its Application to Various Matrices

A highly specific and sensitive microplate chemiluminescent enzyme immunoassay (CLEIA) was established and validated for the detection of staphylococcal enterotoxin B (SEB). A pair of monoclonal antibodies (mAbs) that recognizes different epitopes of SEB was selected from 20 SEB-specific mAbs, and the experimental conditions were examined and optimized for the development of the CLEIA. This method exhibited high performance with a dynamic range of 0.01-5 ng/mL, and the measured limit of detection (LOD) was 0.01 ng/mL. Intra- and interassay coefficient variations were all lower than 13% at three concentrations (0.2, 0.4, and 2 ng/mL). For specificity studies, when this method was applied to test staphylococcal enterotoxins A, C1, and D, no cross-reactivity was observed. It has been successfully applied to the analysis of SEB in a variety of environmental, biological and humoral matrices such as sewage, tap water, river water, roast beef, peanut butter, cured ham, 10% nonfat dry milk, milk, orange juice, and human urine and serum. The aim of this article is to show that the highly sensitive, specific, and simple microplate CLEIA, based on a pair of highly specific monoclonal antibodies, has potential applications for quantifying SEB in public health and military reconnaissance.

miR-30c-1* promotes natural killer cell cytotoxicity against human hepatoma cells by targeting the transcription factor HMBOX1

Natural killer (NK) cells play a critical role in antitumor immunity, and the activation of NK cells is regulated by a series of NK cell receptors. Here, we show that crosslinking CD226, an important NK cell receptor, with the anti‐CD226 mAb LeoA1 on NKL cells, regulated the expression of several microRNA and transmembrane tumor necrosis factor‐α. Among them, miR‐30c‐1* was noticed because overexpression of miR‐30c‐1* triggered upregulation of transmembrane tumor necrosis factor‐α expression and enhanced NK cell cytotoxicity against hepatoma cell lines SMMC‐7721 and HepG2. Furthermore, we proved that the inhibitory transcription factor HMBOX1, which depressed the activation of NK cells, was the direct target gene of miR‐30c‐1*. In conclusion, our results revealed a novel regulatory mechanism: miR‐30c‐1* promoted NK cell cytotoxicity against hepatoma cells by targeting HMBOX1. (Cancer Sci 2012; 103: 645–652)

Development of highly sensitive chemiluminescence enzyme immunoassay based on the anti-recombinant HC subunit of botulinum neurotoxin type A monoclonal antibodies

Botulinum neurotoxins (BoNTs) are the most poisonous substances ever known. The early detection of these toxins could bear more time for appropriate medical intervention. The standard method for detecting BoNTs is the mouse bioassay, which is time consuming (up to 4 days) and requires a large number of laboratory animals. The immunologic detection methods could detect the toxins within a day, but most of these methods are less sensitive compared with the mouse bioassay due to the lack of high-affinity antibodies. Recently, the recombinant H(C) subunit of botulinum neurotoxin type A (rAH(C)) was expressed as an effective vaccine against botulism, indicating that the rAH(C) could be an effective immunogen that raises the monoclonal antibody (mAb) for detecting BoNT/A. After immunized BALB/c mice with rAH(C), 56 mAbs were generated. Two of these mAbs were selected to establish a highly sensitive sandwich chemiluminescence enzyme immunoassay (CLEIA), in which FMMU-BTA-49 and FMMU-BTA-22 were used as capture antibody and detection antibody, respectively. The calculated limit of detection (LOD) based on molecular weight of rAH(C) and BoNT/A reached 0.45 pg mL(-1). This CLEIA can be used in the detection of BoNT/A in matrices such as milk and beef extract. This method has 20-40 fold lower LOD than that of the mouse bioassay and takes only 3 h to complete the detection, indicating that it can be used as a valuable method to detect and quantify BoNT/A.

Enhancement of DNA vaccine efficacy by targeting the xenogeneic human chorionic gonadotropin, survivin and vascular endothelial growth factor receptor 2 combined tumor antigen to the major histocompatibility complex class II pathway

A number of strategies have been used to improve the efficacy of the DNA vaccine for the treatment of tumors. These strategies, ranging from activating CD4+ T cell, manipulating antigen presentation and/or processing to anti‐angiogenesis, focus on one certain aspect in the functioning of the vaccine. Therefore, their combination is necessary for rational DNA vaccines design by synergizing different regimens and overcoming the limitations of each strategy.

Novel Immunohistochemical Monoclonal Antibody Against Rat B Cell Receptor Associated Protein 31 (BAP31)

BAP31 is an evolutionarily conserved polytopic integral protein of the endoplasmic reticulum (ER) membrane implicated in regulating the export of selected membrane proteins from the ER to downstream compartments of the secretory pathway. BAP31 interacts with mIgD, cellubrevin, major histocompatibility complex class I, and BCL-2/BCL-X(L) and plays an important role in regulating the egress of these proteins and in apoptosis. Although BAP31 RNA is ubiquitous, the proteins anatomic localization in rat tissues has not been determined. This is partially because production of high affinity antibodies, especially monoclonal antibodies (MAbs) suitable for immunohistochemical staining, has lagged. To gain further insight into its possible functions, we generated a novel MAb specific for rat BAP31 in immunocytochemistry and immunohistochemistry and localized BAP31 in some rat tissues. Immunoreactivity of BAP31 was prominent in fundic glands, colon, pancreatic acinuses, and liver but not in skeleton muscle and lung. Thus, successful production of rat BAP31 monoclonal antibodies provides a new powerful tool for investigation of BAP31 function in the rat model.

High sensitivity ELISA for detection of botulinum neurotoxin serotype F.

Botulinum neurotoxins (BoNTs) are classified as category A biological threat agents by the Centers for Disease Control and Prevention (CDC) in the United States for its hazardous and potential bioterrorist threat to the public. About 1% naturally occurring botulisms are caused by Botulinum neurotoxin serotype F (BoNT/F). Most of the immunoassays for detecting BoNTs focus on the serotypes A and B, but few methods have been established for the detection of BoNT/F. Recently, the recombinant Hc subunit of botulinum neurotoxin type F (rFHc) was expressed as an effective vaccine against BoNT/F, indicating that this rFHc could be an effective immunogen to raise monoclonal antibodies (MAbs) for the detection and neutralization of BoNT/F. Here we present a novel sandwich enzyme-linked immunosorbent assay (ELISA) based on two MAbs against rFHc, which were FMMU-BTF-8 and FMMU-BTF-29 as capture antibody and detection antibody, respectively. The limit of detection (LOD) of this ELISA reached 12.09 pg/mL, much less than that of the other reported immunoassays. A simple, sensitive ELISA for detecting and quantifying BoNT/F was established, which can be used as a valuable method to detect and quantify BoNT/F.