Protein A Research Articles

A comprehensive review on staphylococcal protein A (SpA): Its production and applications.

The Staphylococcus aureus protein A (SpA) can be obtained through the culture of wild-type S. aureus and also as a recombinant protein in safe bacterial hosts. Several methods have been used to purify SpA among which ion-exchange chromatography, affinity chromatography, gel filtration, and per aqueous liquid chromatography (PALC) are common. SpA has a wide range of biochemical, biotechnological, and medical applications and is most commonly used in test methods such as immunoprecipitation, enzyme-linked immunosorbent assay, and Western blotting. SpA has also been widely utilized in pharmaceutical applications to bind to immune complexes and serum immunoglobulins. SpA also directly binds to the B-cells preventing initiation of infectious diseases as well as having a role in the development of various autoimmune diseases. This review considers different applications of SpA in biotechnology and its novel clinical application for effective treatment of autoimmune diseases. It also discusses various strategies for expression and purification of the SpA including types of column chromatography that are commonly used in protein purification and developing SpA surface display technologies. Finally, this review highlights the potential and novel applications of SpA immobilization, SpA typing, protein engineering for further development of immunological and biochemical research, and also application of SpA as a diagnostic biosensor.

IgY antibodies of chicken do not bind staphylococcal binder of immunoglobulin (Sbi) from Staphylococcus aureus

The immunoglobulin (Ig) binding proteins of Staphylococcus aureus namely staphylococcal protein A (SpA) and staphylococcal binder of immunoglobulin (Sbi) are responsible for false positives during immunoassays. Avian IgY antibodies were reported to have no affinity to SpA and thus are safe for use in immunoassays. However, the behaviour of Sbi with IgY was not reported. The purpose of the present study is to evaluate the interactions between IgY antibodies and Sbi protein from different S. aureus strains. Initially, heterologous cloning and expression of complete sbi gene in Escherichia coli was undertaken. Recombinant Sbi protein was utilized to generate polyclonal anti-Sbi IgY and anti-Sbi antibodies in chicken and BALB/c mice respectively. Indirect ELISA and Western blotting were performed to evaluate the reactivity of anti-Sbi antibodies. Non-reducing PAGE followed by Western blotting and double-antibody sandwich dot-ELISA were performed to analyze the reactivity of IgY antibodies with recombinant Sbi and native Sbi from S. aureus strains. To avoid the possible interference of enzyme-conjugated secondary antibodies from mammalian sources, mouse anti-Sbi revealing antibodies were labeled with biotin so that streptavidin-HRP was used as developing reagent for chromogenic reaction. Sbi was highly immunogenic in chicken and mouse with antibody titers of 1:128,000 and 1:64,000 dilutions respectively. We observed that unimmunized IgY antibodies showed no affinity to either recombinant Sbi or native Sbi from S. aureus strains in Western blotting and double antibody sandwich ELISA. In view of these observations, we recommend that IgY antibodies are safe and free from false positives due to SpA and Sbi in immunoassays involving detection of S. aureus antigens/exotoxins.

Toward reducing biomaterial antigenic potential: a miniaturized Fc-binding domain for local deposition of antibodies.

A peptide derived from staphylococcal protein A (SpA) was developed as an affinity module for antibody delivery applications. The miniaturized protein consists of the first helix of the engineered SpA Z domain fused with the self-assembling peptide (SAP) AEAEAKAKAEAEAKAK, or EAK. The resulting peptide, named Z15_EAK, was shown to possess fibrillization properties and an Fc-binding function. The peptide induced a red shift in the Congo red absorbance characteristic of peptide fibrils, also evidenced in transmission electron microscopy images. The one-site binding affinity (Kd) of a gel-like coacervate generated by admixing Z15_EAK with EAK for IgG was determined to be 1.27 ± 0.14 μM based on a microplate-based titration assay. The coacervate was found to localize IgG subcutaneously in mouse footpads for 8 to 28 days. A set of in vivo data was fit to a one-compartment model for simulating the relative fractions of IgG dissociated from the materials in the depot. The model predicted that close to 27% of the antibodies injected were available unbound for the duration of the experiment. Z15_EAK did not appear to induce innate immune responses; injecting Z15_EAK into mouse footpads elicited neither interleukin-6 (IL-6) nor tumor necrosis factor-alpha (TNF-α) from splenocytes isolated from the animals one day, seven days, or eleven days afterward. The antigenic potential of Z15 was analyzed using a bioinformatic approach in predicting sequences in SpA and Z15 dually presented by class I and class II human MHC alleles covering the majority of the population. A peptide in SpA identified as a potential T cell epitope cross reacting with a known epitope in a microbial antigen was eliminated by miniaturization. These results demonstrate that Z15_EAK is a potential platform for generating antibody depots by which the impacts of Fc-based biotherapeutics can be enhanced through spatiotemporal control.

A novel colorimetric sensing platform for the detection of S. aureus with high sensitivity and specificity.

In this study, a novel colorimetric sensing platform was developed for the detection of S. aureus using dog immunoglobulin G (IgG) as the capture antibody and chicken anti-protein A immunoglobulin Y labeled with horseradish peroxidase (HRP-IgY) as the detection antibody. Dog IgG labeled with magnetic beads was used to capture S. aureus through the interaction between the Fc region of dog IgG and Staphylococcal protein A (SPA). HRP-IgY was introduced to recognize the residual SPA on the surface of S. aureus and to create a sandwich format, after which a soluble 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added. A stop solution was utilized to cease the enzymatic chromogenic reaction, and then optical density was read at 450 nm. Under optimal conditions, the proposed method displayed a low detection limit of 1.0 × 103 CFU mL−1 and a wide linear range of 3.1 × 103 to 2.0 × 105 CFU mL−1. This detection method exhibited high specificity against other foodborne bacteria. The recovery rates ranged from 95.2% to 129.2%. To our knowledge, this is the first report to employ dog IgG and chicken IgY as an antibody pair to detect S. aureus. This technique exhibits high application potential for S. aureus monitoring in various kinds of samples.

Rapid and Broad Immune Efficacy of a Recombinant Five-Antigen Vaccine Against <i>Staphylococcus aureus</i> Infection in Animal Models

Staphylococcus aureus (S.aureus) is a leading cause of both healthcare-and community-associated infections globally, which result in severe disease and readily developing antibiotic resistance. So developing an efficacious vaccine against S.aureus is urgently required. In the present study, we selected five conserved antigens, which were all well-characterized virulence factor of S. aureus by using a reverse vaccine strategy and developed a recombinant five-antigen S. aureus vaccine (rFSAV). Including the secreted factors α-hemolysin (Hla), staphylococcal enterotoxin B (SEB), and the three surface proteins staphylococcal protein A (SpA), iron surface determinant B N2 domain (IsdB-N2) and manganese transport protein C (MntC). rFSAV provided consistent protection in S. Aureus lethal sepsis, pneumonia and fracture fixation mouse models, and it showed broad immune protection when challenged with a panel of epidemiologically relevant S.aureus strains. Meanwhile, rFSAV immunized mice with an optimal immunization procedure (days 0, 3, and 7) were able to induce comprehensive cellular and humoral immune responses to reduce bacterial loads, inflammatory cytokine expression, inflammatory cell infiltration and decrease pathology after challenge with a sub-lethal dose of S. aureus. Moreover, the importance of specific antibodies in protection was demonstrated by passive transfer experiments. Altogether, our data demonstrate that rFSAV is a potentially promising vaccine candidate for defensing against S. aureus infection. Funding Statement: This work was supported by the National Natural Science Foundation of China [grant number 81172892 and 31370932] and the key project of innovative drug development [grant number 2015ZX09101033 and 2016ZX09J16102-002]. Declaration of Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics Approval Statement: All animal care and use protocols in this study were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of the People's Republic of China. All animal experiments in this study were approved by the Animal Ethical and Experimental Committee of the Third Military Medical University (Chongqing, Permit No. 2011- 04) in accordance with their rules and regulations.

Staphylococcus aureus Decolonization of Mice With Monoclonal Antibody Neutralizing Protein A.

Staphylococcus aureus persistently colonizes the nasopharynx of about one-third of the human population, a key risk factor for community- and hospital-acquired invasive infections. Current strategies for S. aureus decolonization include topical and systemic administration of antibiotics, which is associated with selection for antibiotic resistance and posttreatment recolonization. Using a mouse model for S. aureus colonization, we show here that systemic administration of a recombinant monoclonal antibody neutralizing staphylococcal protein A (SpA) can stimulate antibacterial immunoglobulin G and immunoglobulin A responses and promote S. aureus decolonization. These results suggest that antibody neutralizing SpA, a B-cell superantigen, may also be useful for S. aureus decolonization in humans.

Development of sensitive polyclonal antibodies against dominant stored wheat grain fungus for its immunological detection

Fungal infestation causes deterioration of stored food grains. Most fungal species produce secondary metabolites like aflatoxins which are highly toxic to animals and humans. Aspergillus flavus has been found to be the predominant contaminant in stored wheat grains collected from the godowns of Food Corporation of India, West Bengal. The present study focuses on the development of sensitive polyclonal antibodies (PAbs) for molecular immunological detection of dominant toxigenic fungus. Pure A. flavus isolate was cultured on coconut agar media and its spores were harvested and inactivated by 4% formaldehyde. The inactivated spores were injected into a rabbit along with Freund’s complete/incomplete adjuvant for the development of PAbs. Specificity of the raised antibodies in rabbit serum was examined by enzyme-linked immunosorbent assay (ELISA) using spore proteins as antigen obtained by bead beating method. Out of several proteins (ranging from 10 to 200 kDa present in spore, only two prominent proteins of around 76 kDa and 100 kDa were detected by western blot analysis using raised polyclonal antiserum. The PAbs were purified with protein A column followed by spore proteins conjugated CNBr activated sepharose column for its use in the detection of fungal antigens. This highly purified raised antibody can be used for the development of rapid, sensitive, and accurate techniques (such as dot blot/ELISA) for the detection of toxigenic fungi present in stored wheat grains.

Fluorescence lateral flow immunoassay based point-of-care nanodiagnostics for orthopedic implant-associated infection

Detection of implant-associated infection at the earliest is necessary to prevent, treat or monitor further complications. In this work, the lateral flow immunoassay (LFIA) based on double antibody sandwich technique is developed using bright fluorescent cadmium telluride quantum dots (CdTe QDs). This point-of-care (POC) nanodiagnostics technique utilizes CdTe QDs for detection of orthopedic implant-associated infection biomarkers such as C-reactive protein (CRP) and interleukin-6 (IL-6). Staphylococcal protein A (SPA) was conjugated with CdTe QDs (SPA-QDs) and antibodies i.e. anti-CRP, anti-IL-6, and anti-rabbit IgG were bound separately on SPA-QDs complex to develop the biosensor mixture. LFIA was developed on the nitrocellulose strip with test and control spot. Color sensor based portable fluorescence strip reader (PorFloR™) was developed and calibrated. The detection range was found to be 1–300 μg/ml and 1-1000 pg/ml for CRP and IL-6, respectively. Limit of detection was found to be 0.3 μg/ml and 0.9 pg/ml for CRP and IL-6, respectively. Developed LFIA was compared with standard ELISA technique and it showed excellent accuracy, sensitivity, reproducibility, and stability. PorFloR™ device provides the quantitative approach to POC nanodiagnostics and has the potential to be utilized for early detection of implant-associated infection and assist surgeons regarding the status of an implanted device.

Essential Domain-Dependent Roles Within Soluble IgG for in vivo Superantigen Properties of Staphylococcal Protein A: Resolving the B-Cell Superantigen Paradox

Staphylococcus aureus is a common commensal and frequent opportunistic pathogen that causes invasive infections that often recur. Co-evolution with the host has led to the development of toxins that affect diverse immune cell types. Recent reports have highlighted the contributions of staphylococcal protein A (SpA). This small oligomeric secreted protein contains 4–5 homologous domains with two distinct immunoglobulin-binding sites; one for IgG Fc domains, while a separate site binds an evolutionarily conserved surface on Fab encoded by VHIII clan related genes. The Fab-binding site has been implicated in in vivo supraclonal VHIII-BCR targeted B-cell depletion by an activation induced death pathway. Yet the concept of a superantigen for B lymphocytes poses a seeming paradox. Unlike TCR that are expressed only in a membrane-associated form, BCR are expressed in both a membrane BCR form and in secreted Ig forms, which permeate virtually every part of the body at high levels. We therefore asked, why circulating immunoglobulin do not block the superantigen properties of SpA? Herein, we show that soluble IgG molecules are not in vivo inhibitors of these B-cell superantigen effects but are instead essential for potentiating these properties. We also show that the Fc subclass of circulating IgG is an indirect critical determinant of the B-cell superantigen effect. In contrast, host FcγR and complement are not required for SpA mediated in vivo B-cell depletion. Unexpectedly, after VHIII-IgG2a pretreatment SpA challenge resulted in fatal anaphylactic reactions, which we speculate may have involved FcγR interactions with mast cells and basophils. Cumulatively, our findings illuminate a cunning and potent molecular strategy by which a bacterial toxin effectively confounds the contributions of host B-lymphocytes to immune defenses.

Corrigendum: Insulin- like Growth Factor-Binding Protein Action in Bone Tissue: A Key Role for Pregnancy- Associated Plasma Protein-A.

[This corrects the article DOI: 10.3389/fendo.2018.00031.].

The role of erlotinib in the acute lung injury and the expression of surfactant protein A

Objective To investigate the role of erlotinib in the expression of surfactant protein A (SP-A) in LPS-induced acute lung injury (ALI) of mice model. Methods C57BL/6 mice were randomly (random number)divided into control group (n=6), ER group (n=6), LPS group (n=6), and ER+LPS group (n=6). In the LPS group, 2 mg/kg LPS was instilled into trachea of mice to induce lung injury. In control group, normal saline was instilled into trachea of mice instead. In the ER+LPS group and ER group, 100 mg/kg of erlotinib was instilled into stomach of mice, and one hour later, 2 mg/kg LPS was instilled into trachea of mice in ER+PLS group to induce lung injury. Twenty-four hours later, bronchoalveolar lavage fluid (BALF) and lung tissue of mice in four groups were collected. HE staining were used for evaluating pathological changes of lung injury. Lung wet/dry weight ratio, protein concentrations and total cell numbers in the BALF were measured to determine the degree of pulmonary edema. Immunohistochemical staining and Western Blot were used for testing the protein expression of SP-A, Data of multiple groups were analyzed by one way variance (ANOVA) and inter-group comparisons were made by the least significant difference (LSD) tests. Results There was no significant difference in lung injury score (LIS) between control group (0.056±0.008) and ER (0.064±0.037) group, The LIS in LPS group (0.846±0.047) was higher than that in control group, however the LIS in ER+LPS group (0.279±0.020) was significant lower than that in LPS group (P < 0.05). Lung wet/dry weight, SP-A concentration and total cell numbers in the bronchoalveolar lavage fluid revealed that the degree of pulmonary edema in LPS group was higher than that in control group, and this pulmonary edema was reversed by erlotinib treatment. Immunohistochemical staining and Western blot showed that the expression of SP-A in LPS group was decreased compared with control group, but it was recovered after erlotinib treatment (P < 0.05). Conclusions Erlotinib could protect the LPS-induced ALI, and it may be related to the regulation of SP-A. Key words: Acute lung injury; Mice; Lipopolysaccharide; Erlotinib; Alveolar epithelial cells; Surfactant protein A; Inflammatory response; Epidermal growth factor receptor; Drug treatment

Isovitexin, a Potential Candidate Inhibitor of Sortase A of Staphylococcus aureus USA300.

Staphylococcus aureus (S. aureus) causes a broad variety of diseases. The spread of multidrugresistant S. aureus highlights the need to develop new ways to combat S. aureus infections. Sortase A (SrtA) can anchor proteins containing LPXTG binding motifs to the bacteria surface and plays a key role in S. aureus infections, making it a promising antivirulence target. In the present study, we used aSrtA activity inhibition assay to discover that isovitexin, a Chinese herbal product, can inhibit SrtA activity with an IC50 of 28.98 μg/ml. Using a fibrinogenbinding assay and a biofilm formation assay, we indirectly proved the SrtA inhibitory activity of isovitexin. Additionally, isovitexin treatment decreased the amount of staphylococcal protein A (SpA) on the surface of the cells. These data suggest that isovitexin has the potential to be an anti-infective drug against S. aureus via the inhibition of sortase activity.

Sensitivity enhanced SPR immunosensor based on graphene oxide and SPA co-modified photonic crystal fiber

A sensitivity enhanced surface plasmon resonance (SPR) immunosensor based on Graphene oxide (GO) composite and staphylococcal protein A (SPA) co-modified photonic crystal fiber (PCF) is developed for human IgG detection. The PCF spliced between two section of multimode fibers (MMFs) is sputtered with gold film, which was then modified with graphene oxide and SPA for further immunosensing. Graphene oxide with abundant functional groups has a large surface area for more biomolecules immobilization and can enhance the interaction between Au film and external medium, leading to the sensitivity increased greatly. SPA can specifically bind the Fc region of the antibodies and has a high degree of orientation for capturing antibodies. Experimental results indicated that the refractive index (RI) sensitivity of graphene oxide modified Au-PCF SPR sensor reaches 4649.8 nm/RIU, which is about 1888 nm/RIU higher than that without graphene oxide film and the human IgG detection limit reaches as low as 10 ng/mL. Such a sensitivity enhanced immunosensor based on graphene oxide and SPA co-modified PCF shows a great promise in biosensing.

Septal secretion of protein A in Staphylococcus aureus requires SecA and lipoteichoic acid synthesis.

Surface proteins of Staphylococcus aureus are secreted across septal membranes for assembly into the bacterial cross-wall. This localized secretion requires the YSIRK/GXXS motif signal peptide, however the mechanisms supporting precursor trafficking are not known. We show here that the signal peptide of staphylococcal protein A (SpA) is cleaved at the YSIRK/GXXS motif. A SpA signal peptide mutant defective for YSIRK/GXXS cleavage is also impaired for septal secretion and co-purifies with SecA, SecDF and LtaS. SecA depletion blocks precursor targeting to septal membranes, whereas deletion of secDF diminishes SpA secretion into the cross-wall. Depletion of LtaS blocks lipoteichoic acid synthesis and abolishes SpA precursor trafficking to septal membranes. We propose a model whereby SecA directs SpA precursors to lipoteichoic acid-rich septal membranes for YSIRK/GXXS motif cleavage and secretion into the cross-wall.

Staphylococcal Protein A Contributes to Persistent Colonization of Mice with Staphylococcus aureus.

Staphylococcus aureus persistently colonizes the nasopharynx in humans, which increases the risk for invasive diseases, such as skin infection and bacteremia. Nasal colonization triggers IgG responses against staphylococcal surface antigens; however, these antibodies cannot prevent subsequent colonization or disease. Here, we describe S. aureus WU1, a multilocus sequence type 88 (ST88) isolate that persistently colonizes the nasopharynx in mice. We report that staphylococcal protein A (SpA) is required for persistence of S. aureus WU1 in the nasopharynx. Compared to animals colonized by wild-type S. aureus, mice colonized with the Δspa variant mount increased IgG responses against staphylococcal colonization determinants. Immunization of mice with a nontoxigenic SpA variant, which cannot cross-link B cell receptors and divert antibody responses, elicits protein A-neutralizing antibodies that promote IgG responses against colonizing S. aureus and diminish pathogen persistence.IMPORTANCEStaphylococcus aureus persistently colonizes the nasopharynx in about one-third of the human population, thereby promoting community- and hospital-acquired infections. Antibiotics are currently used for decolonization of individuals at increased risk of infection. However, the efficacy of antibiotics is limited by recolonization and selection for drug-resistant strains. Here, we propose a model of how staphylococcal protein A (SpA), a B cell superantigen, modifies host immune responses during colonization to support continued persistence of S. aureus in the nasopharynx. We show that this mechanism can be thwarted by vaccine-induced anti-SpA antibodies that promote IgG responses against staphylococcal antigens and diminish colonization.

Molecular characteristics and antimicrobial susceptibility to Staphylococcus aureus strains isolated from children with skin and soft tissue infections

Objective To investigate the molecular characteristics, virulence genes and antimicrobial susceptibility to Staphylococcus aureus strains isolated from children with skin and soft tissue infections(SSTIs) in Beijing. Methods A total of 52 Staphylococcus aureus isolates were collected from children with SSTIs in Beijing Children′s Hospital, Capital Medical University, and the clinical data were collected and analyzed.Methicillin-resistant Staphylococcus aureus(MRSA) and methicillin-susceptible Staphylococcus aureus(MSSA) were identified by using the cefoxi-tin disc method and the detection of mecA gene.Multilocus sequence typing (MLST) and staphylococcal protein A(spa) typing were analyzed by the PCR method, and the staphylococcal chromosomal cassette mec (SCCmec) type was analyzed for the MRSA isolates.The pvl, eta, etb, tsst-1 and hlg genes were also detected by PCR.The susceptibility strains to 16 antibiotics were evaluated by using the agar dilution method. Results A total of 52 Staphylococcus aureus SSTIs patients, 30 with MRSA infections and 22 with MSSA infections were included in the study.There were 23 patients (44.2%) less than 1 year old.The most frequent infections were the newborn omphalitis (12/52 strains, 23.1%)and abscess(11/52 strains, 21.2%). ST59-MRSA-SCCmecⅣa-t437 was the most predominant clones of MRSA isolates.Among the MSSA isolates(14/30 strains, 46.7%), no significant epidemic clone was found.Ten sequence types (STs) and 14 spa types were identified in MSSA, and the most common types were ST22(6/22 strains, 27.3%)and t309(5/22 strains, 22.7%), respectively.Notably, the multidrug resistant rates of MRSA and MSSA isolates were all >85%.The percentages of the Staphylococcus aureus SSTIs strains resistant to Erythromycin, Penicillin, Chloramphenicol and Clindamycin were 100.0%, 94.2%, 69.2% and 63.5%, respectively.The tested isolates were susceptible to Trimethoprim/Sulfamethoxazole, Mupirocin, Fusidic acid, Tigecycline, Linezolid and Vancomycin.The pvl gene′s positive rate was 40.4%, and no significant difference between MRSA and MSSA was observed (P>0.05). Eta and etb genes were detected in 2 patients with staphylococcal scalded skin syndrome. Conclusions The Staphylococcus aureus SSTIs strains are most frequently isolated from newborn omphalitis and abscess in Beijing.The multidrug resistant rate is relatively high, so the erythromycin and clindamycin should not be preferred in empiric treatment of children with Staphylococcus aureus SSTIs.The prevalence of pvl gene is 40.1%.ST59-MRSA-SCCmecⅣa-t437 is the common clone of MRSA, while the MSSA isolates have a more diverse genetic background. Key words: Staphylococcus aureus; Skin and soft tissue infections; Molecular characteristics; Virulence; Antimicrobial susceptibility; Child

Backbone Dynamics and Model-Free Analysis of N-terminal Domain of Human Replication Protein A 70

Backbone Dynamics and Model-Free Analysis of N-terminal Domain of Human Replication Protein A 70

A novel bivalent fusion vaccine induces broad immunoprotection against Staphylococcus aureus infection in different murine models

With more and more drug-resistant Staphylococcus aureus strains emerging in hospitals, there is an urgent need to develop an effective vaccine to combat S. aureus infection. In this study, we constructed a novel bivalent fusion vaccine, SpA-DKKAA-FnBPA37-507 (SF), based on the D domain of staphylococcal protein A (SpA) and the A domain of fibronectin-binding protein A (FnBPA). Immunisation with SF induced a more ideal protective effect compared with the single components alone in a sepsis model. It also showed broad immunoprotection against seven FnBPA isotypes. Vaccination with SF induced strong antibodies responses and Th1/Th17 polarized cellular responses. Further we demonstrated the protective effect of antibodies by the opsonophagocytic assay (OPA) and passive immunisation. Moreover, vaccination with SF showed protective efficacy in a murine pneumonia model and skin abscess model. These results suggest that SF can be regarded as a promising vaccine candidate for the prevention of S. aureus infections.

The effect of serum amyloid protein A on the hepatitis development induced by concanavalin A

Objective To investigate the effect of serum amyloid A (Saa) on the hepatitis development which induced by concanavalin A (ConA) and the action mechanism. Methods To establish hepatitis mouse model by injecting ConA in Saa over-expression transgenic mice (TG) and wild-type mice (WT), and the expression of monocyte chemotactic protein 1 (MCP1), chemokine 1α (MIP1α), chemokine 1β (MIP1β), chemokine IP10 (IP10) and Eotaxin gene were detected by real-time quantitative polymerase chain reaction (Real-time PCR) in liver tissue. The concentration of alanine transaminase (ALT) and glutamic oxalacetic transaminase (AST) in serum were measured by autoanalyzer. The concentration of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6) and interleukin-8(IL-8) in serum were measured by enzyme-linked immunosorbent assay (ELISA), and the expression of Saa, p-IκBα(Ser32/36) and IκBα proteins were detected by Western blotting. The isolation and purification of CD4+ T cells in liver and spleen were performed and tested by Flow cytometry. Results The overexpression of MCP1, MIP1α, MIP1β, IP10, Eotaxin gene in TG group (0.61±0.13, 0.89±0.15, 0.76±0.12, 0.37±0.08, 0.98±0.17) were significantly higher than WT group (0.13±0.03, 0.27±0.02, 0.16±0.06, 0.07±0.02, 0.34±0.07), (P=0.001, 0.001, 0.001, 0.021, 0.001, respectively)). At 6 h, the concentration of ALT (16.2±3.1) U/L and AST (18.8±4.2) U/L were significantly higher than WT group (P=0.002, 0.001). The secretion of TNF-α, IFN-γ, IL-6 and IL-8 were higher in serum of TG mice [(813.6±114.2), (64.5±8.9), (1 045.8±138.6), (43.4±5.5) pg/ml] than in WT mice [(253.1±33.5), (10.5±6.7), (374.1±53.7), (24.8±3.4) pg/ml] (P=0.011, 0.006, 0.005, 0.014). Flow cytometry indicated that the rates of CD4+ T cell and F4/80+ CD11b+ macrophage cell in liver of TG group (60.72±3.65)% and (9.86±0.58)% were significantly higher than WT group mice (35.41±2.45)% and (2.73±0.51)%, (P=0.011, 0.014). Western blotting indicated that the levels of p-IκBα (Ser32/36) protein levels significantly increased, while decreased after CU-CPT22 treatment in TG mice liver tissue. Conclusion Saa overexpression promoted hepatitis development by inducing chemokines expression in a Toll like receptors 2 (TLR2) activation-dependent manner, and play a role by increasing the number of CD4+ T cells and macrophages Key words: Saa; Concanavalin A; Chemokine; T cell; Hepatitis

A novel immunosensor based on excessively tilted fiber grating coated with gold nanospheres improves the detection limit of Newcastle disease virus

A novel immunosensor for detecting Newcastle disease virus (NDV) was developed using excessively tilted fiber grating (Ex-TFG) coated with gold nanospheres (AuNs). AuNs were coated on the Ex-TFG surface via Au–S bonds using 3-mercaptopropyltrimethoxysilane (MPTMS), and the activated staphylococcal protein A (SPA) was linked to AuNs by covalent bonds via cysteamine. AuNs greatly enhanced the impact of the analyte on the fiber cladding mode through the local surface Plasmon resonance (LSPR) effect, thus improving the detection limit and sensitivity of the immunosensor. Meanwhile, SPA enhanced the bioactivity of anti-NDV monoclonal antibodies (MAbs), thus promoting the effectiveness of specific binding events on the fiber surface. Immunoassays were performed by monitoring the resonance wavelength shift of the proposed sensor under NDV samples containing different particle amounts. Specificity was assessed, and clinical tests for NDV were performed by contrast experiments. Experimental results showed that the detection limit for NDV was about 5~10 times improved compared to that of reference Ex-TFG without AuN treatment. Moreover, the novel biosensor was reusable and could potentially be applied in clinic.