Kui Zhang

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.

Anti-epithelial cell adhesion molecule monoclonal antibody conjugated fluorescent nanoparticle biosensor for sensitive detection of colon cancer cells

In this paper, a sensitive and selective sensor for detecting colon cancer cells based on nanoparticle covalent modified anti-human epithelial cell adhesion molecule (EpCAM) antibody is developed. The transmission electron microscope (TEM) images showed that the nanoparticle and functionalized nanoparticle had good decentrality for application. The NaIO(4) oxidation method, which was used as oxidizing antibody for immobilization of conjugating antibody on the silica-coated fluorescent nanoparticles, maintained the activities of antibodies very well. The fluorescence microscopy imaging and flow cytometer (FCM) experiments demonstrated that the nanosensor could increase the signal intensity obviously and distinguish three kinds of target cells (colo205, sw480 and NCM460) well. The membrane and nuclear staining showed the distribution and abundance of EpCAM in cells membrane. It also provides a possibility to quantify special membrane proteins on different regions of cells surface. At the end, the result of detecting a simple sample proved that colo205 cells were selected by anti-EpCAM antibody nanosensors in this environment, and made a good foundation for subsequent research.

The establishment of a highly sensitive ELISA for detecting bovine serum albumin (BSA) based on a specific pair of monoclonal antibodies (mAb) and its application in vaccine quality control.

A highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for quantifying BSA was established, based on two mAbs that recognize different epitopes on a BSA molecule. Our ELISA system was used to detect BSA concentrations in several vaccines, such as the MMR (measles, mumps and rubella) vaccine, hepatitis A vaccine, and hepatitis B vaccine. Moreover, we compared the mAb ELISA and the present pAb ELISA by detecting BSA standards and bovine serum samples. The results showed that our ELISA system was in good accordance with the pAb ELISA system. A pair of mAbs (FMU-BSA NO.6 and FMU-BSA NO.11) from 11 murine hybridomas secreting BSA-specific mAbs was selected for the development of the sandwich ELISA. The detection limit of this quantitative assay reaches 0.38 μg/L, which is 10-fold more sensitive than those previously reported. The quantitative range of BSA concentration is from 0.5 to 40 μg/L, which is comparable to the currently used polyclonal antibody (pAb) ELISA. Intra-assay and inter-assay coefficient variations are both lower than 10% at the three concentrations used (10, 20, and 40 μg/L). Thus, the mAb sandwich ELISA developed herein may provide a stable, precise, and highly sensitive method for quantifying BSA, which is very useful in the quality control of some vaccines.

A novel immunoassay for residual bovine serum albumin (BSA) in vaccines using laser-induced fluorescence millimeter sensor array detection platform.

A highly sensitive and stable sandwich fluorescence immunoassay for the quantitative detection of residual BSA in vaccines based on the labels of the functionalized fluorescent core-shell silica nanoparticles and laser-induced fluorescence millimeter sensor array detection platform has been developed. On a glass slide with low fluorescence background, capture antibody against BSA was immobilized, after BSA was captured, another identify antibody against BSA which was labeled with the new fluorescent silica nanoparticles was used to recognize the BSA. The fluorescence issued from the fluorescent silica nanoparticles was successfully detected by the laser induced fluorescence millimeter sensor assay detection platform which was made by us. This method exhibited high performance with a linear correlation between response and amount of BSA in the range 1.0-100 ng/mL and the detection limit was 0.3 ng/mL (3σ). The relative standard deviation (R.S.D.) was 6.7% at the concentration of 20 ng/mL for 5 parallel measurements of BSA.

Establishment of a Highly Sensitive Sandwich Enzyme-Linked Immunosorbent Assay Specific for Ovomucoid from Hen’s Egg White

A highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) kit was established for quantifying ovomucoid from hens egg white, which has been considered as one of the major allergen in egg white. The detection limit reached 0.041 ng/mL, and linearity ranged from 0.1 to 6.25 ng/mL. Intra- and interassay coefficient variations were all lower than 5% at three concentrations (0.5, 2.5, and 5 ng/mL). No cross-reactivity was observed with bovine serum, horse serum, goat serum, human serum, duck egg white, goose egg white, quail egg white, and pigeon egg white, but a low level of cross-reactivity was found with chicken serum. The ELISA kit was established on the basis of two monoclonal antibodies (mAbs) recognizing different epitopes of ovomucoid. However, these mAbs were generated using commercially purified ovalbumin as immunogen. Studies on the relative allergenicity and antigenicity of egg white protein have been performed by many researchers, but there were controversial opinions reported previously because of the impurity of each egg white protein used in various studies. In the present work we measured the degree of ovomucoid contamination in commercially purified ovalbumin sample, and the value was about 11%. We also determined the ovomucoid residue in influenza vaccine samples for the first time. These data showed that the ELISA kit we established could serve as an effective method for precisely quantifying concentrations of ovomucoid in the egg industry and as a useful tool for the research of allergenicity and antigenicity of hens egg proteins.

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.

Functional defects in CD4 + CD25 high FoxP3 + regulatory cells in ankylosing spondylitis

Forkhead box P3 (FoxP3)-positive regulatory T cells (Tregs) play a pivotal role in the preservation of self-tolerance, and Treg dysfunction has been implicated in many autoimmune diseases. Whether and how Tregs participate in the pathogenesis of ankylosing spondylitis (AS) has not been fully elucidated. Here, we investigated Treg function and found that Tregs in peripheral blood (PB) from patients with active AS had lower FoxP3 mean fluorescence intensity (MFI) than those from healthy controls and could not fully suppress naïve T cell (Tn) proliferation. We also studied the mechanisms underlying PB Treg dysfunction in this context and found that PB Tregs failed to effectively utilize IL-2 and had relatively little STAT5 phosphorylation in active AS. Moreover, PB Tregs from patients with active AS exhibited greater CpG island methylation in the CNS2 region of the FOXP3 gene. Therefore, our findings indicate that functional defects in Tregs are present in AS. Abnormal IL-2 signalling and aberrant CNS2 epigenetic control induced functional defects in PB Tregs and represents a potential new mechanism for AS pathogenesis. These findings may aid the design of new treatment approaches for AS.

Construction, Expression, and Characterization of a Recombinant Immunotoxin Targeting EpCAM

Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein overexpressed in human epithelioma but with relatively low expression in normal epithelial tissues. To exploit this differential expression pattern for targeted cancer therapy, an EpCAM-targeted immunotoxin was developed and its antitumor activity was investigated in vitro. An immunotoxin (scFv2A9-PE or APE) was constructed by genetically fusing a truncated form (PE38KDEL) of Pseudomonas aeruginosa exotoxin with an anti-EpCAM single-chain variable fragment (scFv). ELISA and flow cytometry were performed to verify immunotoxin (scFv2A9-PE or APE) antigen-binding activity with EpCAM. Cytotoxicity was measured by MTT assay. Confocal microscopy was used to observe its cellular localization. The results of ELISA and flow cytometry revealed that the immunotoxin efficiently recognized recombinant and natural EpCAM. Its antigen-binding activity was relatively lower than 2A9. MTT assay confirmed potent reduction in EpCAM-positive HHCC (human hepatocellular carcinoma) cell viability (IC50 50u2009pM). Immunofluorescence revealed that the immunotoxin localized to endoplasmic reticulum 24u2009h later. In conclusion, we described the development of an EpCAM-targeted immunotoxin with potent activity against tumor cells, which may lay the foundation for future development of therapeutic antibody for the treatment of EpCAM-positive tumors.

Highly Sensitive Chemiluminescent Analysis of Residual Bovine Serum Albumin (BSA) Based on a Pair of Specific Monoclonal Antibodies and Peroxyoxalate–glyoxaline–PHPPA Dimer Chemiluminescent System in Vaccines

Enzyme-linked immunosorbent assay (ELISA), horseradish peroxidase (HRP)-catalyzed fluorescent reaction, and oxalate chemiluminescence analysis have been combined to develop a highly sensitive, simple, and rapid method for analysis of bovine serum albumin (BSA) based on a pair of specific monoclonal antibodies in vaccines. A typical “sandwich type” immunoassay was used. Reaction of 3-(4-hydroxyphenyl propionate) (PHPPA) with hydrogen peroxide-urea, catalyzed by HRP, produced fluorescence of 3-(4-hydroxyphenyl propionate) dimer, which was detected by chemiluminescence analysis with the bis(2,4,6-trichlorophenyl)oxalate (TCPO)–H2O2–glyoxaline–PHPPA dimer chemiluminescent system. This method exhibited high performance with a linear correlation between response and amount of bovine serum albumin (BSA) in the range 0.1 to 100.0xa0ngxa0mL−1 (ru2009=u20090.9988), and the detection limit was 0.03xa0ngxa0mL−1 (S/Nu2009=u20093). Intra- and interassay coefficient variations were all lower than 9.0% at three concentrations (1.0, 20.0, and 80.0xa0ngxa0mL−1). The proposed method has been used for successful analysis of the amount of residual BSA in vaccines. The results obtained compared well with those obtained by conventional colorimetric ELISA and luminol chemiluminescent ELISA.