抗乙型肝炎病毒表面抗原T7噬菌体抗体库的构建

构建T7噬菌体单链抗体(scFv)库筛选抗乙型肝炎病毒表面抗原抗体.从抗-HBs阳性患者外周血淋巴细胞中提取总RNA,反转录合成cDNA第1条链,PCR分别扩增抗体重链可变区基因(VH)和轻链可变区基因(VL),经重叠延伸拼接(SOE)PCR组成scFv基因,并将其与T7噬菌体载体的2个臂相连接.体外包装后,在宿主菌BLT5403中,扩增重组噬菌体抗体库.以乙型肝炎病毒表面抗原进行4轮“吸附-洗脱-扩增”的筛选,酶免疫实验检测抗体活性.所建抗体库库容为1.53×107,扩增后初级库滴度为2.42×1010pfu/mL.以乙型肝炎病毒表面抗原筛选后抗体出现特异性富集,经酶免疫实验鉴定,得到2株与HBsAg抗原特异结合的噬菌体抗体,成功构建了抗HBsAg蛋白T7噬菌体抗体库.     

Quantitative aspects of lipopolysaccharide and cytokine requirements to generate nitric oxide in macrophages from LPS-hyporesponsive (<Emphasis Type="Italic">Lps</Emphasis><Superscript>d</Superscript>) C3H/HeJ mice

Due to a gene defect (Lps(d)), C3H/HeJ mice are known to be hyporesponsive to the immunobiological potential of lipopolysaccharide (LPS). We studied dose requirements for LPS, IFN-gamma, and cytokines TNF-alpha and IL-10 to produce nitric oxide (NO) in peritoneal macrophages (Mphi) from these animals. In contrast to the Lps(n) C3H/HeN mice, high concentrations of LPS (up to 5 microg/mL) or IFN-gamma (up to 5 ng/mL) by themselves were unable to activate NO production in C3H/HeJ Mphi. The failure to produce NO could not be overcome by addition of L-arginine or tetrahydropterin. The high-output NO biosynthesis was dose-dependently stimulated by combined administration of varying concentrations of IFN-gamma (50-5000 pg/mL) and LPS (approximately 1 ng/mL) or to a lesser extent by IFN-gamma plus TNF-alpha or TNF-alpha/IL-10. Formation of NO in C3H/HeJ MCO triggered by high concentration of LPS (approximately 1 microg/mL) given together with IFN-gamma (0.2-5 ng/mL) reached the values typical for Lps(n) C3H/HeN mice. While Mphi from C3H/HeN mice secreted TNF-alpha, IL-10, and IL-10 upon contact with a low dose of LPS (1 ng/mL), C3H/HeJ Mphi required high concentration of LPS (5 microg/mL) to enhance the secretion of the cytokines. Yet, this dose remained ineffective to stimulate IFN-gamma in Mphi from C3H/HeJ mice. It can be presumed that one of the important factors influencing their deficient ability to form NO is a failure of Mphi to produce IFN-gamma upon LPS contact.     

Stimulation of the secretory IgA system in monkeys by parenteral immunization with a ribosomal Sonne dysentery vaccine

The experiment was made on 16 monkeys (rhesus macaques). Only 1 out of 12 monkeys immunized with S. sonnei ribosomal vaccine and all 4 control monkeys fell ill as the result of oral challenge with S. sonnei virulent strain. The immunized monkeys stopped excreting Shigellae earlier than the control monkeys. Antibody to lipopolysaccharide (LPS) in the serum and saliva of the monkeys were studied in the enzyme immunoassay with monospecific antibodies to human IgA, IgG and IgM. A single injection of the ribosomal vaccine in a dose of 600 micrograms was shown to lead to a considerable increase in the levels of IgA, IgG and IgM antibodies to LPS in saliva. In parenteral immunization with the ribosomal vaccine the stimulation of secretory IgA system is similar to that resulting from oral challenge with Shigella virulent strain introduced in a dose of 50 X 10(9) microbial cells. No difference in the response of monkeys to primary and booster immunization was noted.     

Development of a quantum dot-based fluorescent immunoassay for progesterone determination in bovine milk

The use of semiconductor quantum dots (QDs) as fluorescent labels to develop a competitive immunoassay for sensitive detection and quantification of progesterone in cow's milk is described. Colloidal water-soluble CdSe/ZnS QDs are conjugated to an antigen derivative (progesterone-BSA conjugate) and a simple methodology is optimised to determine the antigen concentration in the final bioconjugate. The obtained QD-linked antigens were then employed together with unlabelled anti-progesterone monoclonal antibodies, as the biological recognition elements, in the development of the quantitative QDs-based fluorescent immunoassay for progesterone in bovine milk. After optimization, the developed immunoassay proved to cover a progesterone concentration range from 0.3 to 14.5 ng/mL in cow milk. Milk samples were just diluted 10-fold with deionised water and directly analysed with the proposed immunoassay, without additional sample pre-treatment or analyte extraction. The minimum detectable level (IC(10)) of the developed immunoassay turned out to be 0.1 ng/mL of progesterone in bovine milk. The sensitivity (IC(50)) achieved was 2.2 ng/mL with a reproducibility of 3.5% RSD as obtained from the results of the analysis of the triplicate of same samples but in three different days. Applicability of the proposed methodology was evaluated by analyzing cow's milk samples enriched with known concentrations of progesterone and recoveries better than 90% were achieved.     

LPS induces interleukin-6 and interleukin-8 but not tumor necrosis factor-alpha in human adipocytes

Adipose tissue-derived cytokines are presumably involved in obesity-associated pathologies including type 2 diabetes and atherosclerosis. Here we studied the lipopolysaccharide (LPS)-induced expression dynamics of tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6), IL-8 and IL-10 in human adipose tissue biopsies, in preadipocyte-derived adipocytes, and in mesenchymal stem cell (MSC)-derived adipocytes. TNFalpha, IL-6, IL-8 and IL-10 secretions by adipose tissue explants were increased 5.5-, 19.5-, 3.5- and 12.5-fold, respectively, by LPS (1 microg/mL) administration. Concordantly, IL-6 and IL-8 release was dose-dependently induced in MSC-derived adipocytes by LPS (>10 pg/mL). In contrast, TNFalpha and IL-10 remained undetectable even at the highest LPS dose (1 microg/mL) after 24h. In MSC- and preadipocyte-derived adipocytes, respectively, exposure to LPS evoked a weak and transient induction of TNFalpha mRNA whereas induction of IL-6 and IL-8 mRNA were pronounced and sustained for at least 24h. Basal glucose uptake, lipolysis and IL-6 mRNA were induced by exogenous TNFalpha (10 ng/mL) but not by IL-6 (10 ng/mL), IL-8 (100 ng/mL) and IL-10 (20 ng/mL). In this adipocyte model TNFalpha induces well known metabolic effects, but together with previous reports these data suggest that inflammation-induced TNFalpha may derive from non-adipocyte sources in adipose tissue, likely to be macrophages.     

The effect of gram-negative bacteria lipopolysaccharides on the development of Rauscher leukosis in BALB/c mice

The dose-dependent action of Shigella sonnei lipopolysaccharide (LPS) on the development of acute erythroleukocytosis, as well as Rauscher chronic myeloid and lymphoid leukosis, in BALB/c mice sensitive to Rauscher virus was shown. Bordetella pertussis LPS in the doses used in this investigation stimulated the development of both acute erythroleukosis and chronic myeloid and lymphoid leukosis in BALB/c mice infected with Rauscher virus. Lipid A isolated from B. pertussis LPS was found to produce a stimulating effect on the development of Rauscher leukosis in mice. After the treatment of B. pertussis LPS with polymyxin B blocking lipid A no stimulating effect of B. pertussis LPS on the development of Rauscher leukosis was observed. A suggestion is made that lipid A is the active principle contributing to the stimulation of the development of Rauscher leukosis in BALB/c mice.     

Expression of Shigella sonnei lipopolysaccharide in Vibrio cholerae

Making use of a newly designed mobilizable suicide vector, the genetic determinants encoding Shigella sonnei lipopolysaccharide (LPS) were stably integrated into the chromosome of the live attenuated Vibrio cholerae vaccine strain CVD103-HgR. Expression studies showed that the production of complete S. sonnei O-polysaccharide (O-PS)-bearing LPS was limited in bivalent recombinant strains that were also proficient in the synthesis of the host-encoded Inaba O-PS. Conversely, high amounts of LPS carrying S. sonnei O-PS are produced in monovalent Inaba-deficient derivatives, even in those strains which do not co-express the compatible R1 LPS core. Thus, the non-enterobacterial V. cholerae LPS core efficiently acts as a receptor for covalent binding of S. sonnei O-PS provided that competition with the host O-PS is avoided. Expression of the R1 core interferes with cell division in recombinant V. cholerae without affecting other physiological properties of vaccine strain CVD103-HgR. Both monovalent and bivalent strains stimulated high serum-antibody titres specific for their respective O-serotype(s) when administered to rabbits. The potential of V. cholerae as an expression carrier for heterologous O-serotypes is discussed.     

Flow injection fluorescence immunoassay for gentamicin using sol-gel-derived mesoporous biomaterial

Sol-gel-derived mesoporous biomaterials were used for the first time in the flow-injection fluorescence immunoassay system. Anti-gentamicin antibody was immobilized in a mesoporous sol-gel material using tetramethoxysilane as a precursor and poly(ethylene glycol) as a template. The sol-gel glass was used to develop an immunoaffinity column for the flow-injection immunoassay of gentamicin. Little unspecific adsorption of gentamicin on the sol-gel and no antibody leaching under harsh elution conditions were found. The immunoassay is based on the competition between gentamicin and fluorescein isothiocyanate-labeled gentamicin for a limited number of encapsulated antibody binding sites. NaOH solution of 5 x 10(-3)mol/L is used for the regeneration of encapsulated antibody binding sites after each measurement, which allows the immunoreactor to be used for up to 20 times without any loss of reactivity. Sample preconcentration is not needed and a single assay can be performed within 10 min. The calibration for gentamicin has a working range of 250-5000 ng/mL with a detection limit of 200 ng/mL, which is close to that of the fluorescence immunoassay and fluorescence polarization immunoassay using the same reactants. Comparison of the results from this method with that obtained from HPLC showed an excellent correlation.     

Simultaneous detection of multiple chemical residues in milk using broad-specificity antibodies in a hybrid immunosorbent assay

In this study, a novel immunoassay using 2 types of sensors (QDs and an enzyme) were simultaneously used for detecting multiple structurally different molecules in milk. The method integrates the fluorescence-linked immunosorbent assay (FLISA) using QD605 and QD655 as probes and an enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase (HRP) labeled secondary antibody. The FLISA was produced by anti-sulfonamide and anti-quinolone broad-specificity monoclonal antibodies (MAbs) for simultaneously detecting 6 sulfonamides and 11 quinolones. Combined with the FLISA, an ELISA was utilized for detecting melamine from the same milk samples. The cross-reactivity of the MAbs was retained while binding the QDs by using avidin and a secondary antibody as bridges. Milk samples were detected using this hybrid immunoassay, with limits of detection (LOD) of the quinolones (0.18 ng mL(-1)), sulfonamides (0.17 ng mL(-1)) and melamine (7.5 ng mL(-1)), respectively. The results demonstrated that the detection limits of the integrated methods were better than required and simplified the sample pretreatment process. The developed immunoassay is suitable for high-throughput screening of low-molecular weight contaminants.     

Lipopolysaccharides (LPS) modulate the metabolism of deoxynivalenol (DON) in the pig

Pigs might be exposed to lipopolysaccharides (LPS) and deoxynivalenol (DON) at the same time, and both toxins are thought to interactively affect the intestinal barrier, the innate immune system, and the xenobiotics metabolism. Hence, we aimed at examining the single and combined effects of both toxins on nutrient digestibility and DON metabolism. For this purpose, barrows (26?±?4 kg) were fed restrictedly either a control diet (CON) or a diet contaminated with 3.1 mg DON/kg (DON) for 37 days. At day 37 of the experiment, pigs were infused intravenously for 60 min either with 100 μg DON/kg body weight (BW) (CON-DON), 7.5 μg LPS/kg BW (CON-LPS, DON-LPS) or a combination of both substances (CON-DON?+?LPS), or physiological saline (CON-CON, DON-CON). Blood samples were collected frequently until 3.25 h before the pigs were sacrificed for bile, liver, and kidney collection. The apparent digestibility of N-free extractives was significantly increased by 1 % when the DON-contaminated diet was fed. The total DON content in blood was significantly higher in endotoxemic pigs (34.8 ng/mL; CON-DON?+?LPS) when compared to the pigs infused with DON alone (18.8 ng/mL; CON-DON) while bile concentrations were not influenced by LPS. DON residue levels in liver and kidney closely reflected the treatment effects as described for blood. In contrast to DON infusion, the LPS challenge resulted in a significantly lower total DON concentration (13.2 vs. 7.5 ng/mL in groups DON-CON and DON-LPS, respectively) when the pigs were exposed to DON through the diet. The conjugation degree for DON in blood and bile was not influenced by treatments. In conclusion, endotoxemic pigs are characterized by higher DON residue levels in blood, liver, and kidney, probably by a compromised elimination.     

Study of the binding between bacterial lipopolysaccharides and polymyxin by a bioluminescent method

For rating the interaction of lipopolysaccharides (LPS) with polymixin B (PmB) a bacterial bioluminescence is offered to be used. Bioluminescence level of bacteria decreases under free antibiotic amounts action. It is shown, that as a result of interaction with LPS antibiotic properties of PmB are reduced, and the intensity of bacterial bioluminescence is restored. The bioluminescence level in such system characterizes the LPS quantity. Kinetic properties of bacterial light emission at the presence of LPS and PmB are investigated as well as equilibrium state of the system. Kinetic and equilibrium constants describing this reaction are determined. The conditions of quantitative bioluminescent definition of LPS in an interval of concentration 0.166-10 micrograms/ml have been chosen and calibration curves are presented.     

In vivo microdialysis sampling of cytokines produced in mice given bacterial lipopolysaccharide

Cytokines are proteins that mediate communication between cells of the immune system as well as certain other non-immune host cells. These proteins are produced by many cell types and they mediate immune and inflammatory responses. However, the direct site analysis of these critical proteins is hampered by the lack of site-specific tools available for such direct measurements. In this study, both in vitro and in vivo microdialysis sampling of different cytokines (tumor necrosis factor-alpha [TNF-alpha], interferon-gamma [IFN-gamma], interleukin-6 [IL-6], IL-12p70, and macrophage chemoattractant protein-1 [MCP-1]) was performed. A mouse model of bacterial lipopolysaccharide (LPS) administration and response pattern was used for in vivo studies. Three cytokines, TNF-alpha, IL-6, and MCP-1 were quantified in the serum from mice given LPS. In vivo studies demonstrated the ability to monitor increasing levels of these cytokines (TNF-alpha, IL-6, and MCP-1) via microdialysis probes placed in the peritoneal cavity of mice given LPS. All three cytokines were quantified simultaneously in 15 muL of dialysate using a multiplexed bead-based immunoassay for flow cytometry. The detected dialysate cytokine concentrations varied between 200 pg/mL and 1500 pg/mL for TNF-alpha, between 600 pg/mL and 3000 pg/mL for MCP-1, and between 2700 pg/mL and more than 5000 pg/mL for IL-6. The detected serum cytokine concentrations ranged from 5700 pg/mL to 35,000 pg/mL for TNF-alpha, from 40,000 pg/mL to 65,000 pg/mL for MCP-1, and greater than than 100,000 pg/mL for IL-6. This work demonstrates that microdialysis sampling can be used in vivo to collect temporal profiles of cytokine production.     

An enzymatic immunoassay microfluidics integrated with membrane valves for microsphere retention and reagent mixing

The present study presents a new microfluidic device integrated with pneumatic microvalves and a membrane mixer for enzyme-based immunoassay of acute myocardial infarction (AMI) biomarkers, namely, myoglobin, and heart-type fatty acid binding protein (H-FABP). Superparamagnetic microspheres with carboxyl groups on their surfaces were used as antibody solid carriers. A membrane mixer consisting of four ψ-type membrane valves was assembled under the reaction chamber for on-chip performing microsphere trapping and reagent mixing. The entire immunoassay process, including microsphere capture, reagent input, mixing, and subsequent reaction, was accomplished on the device either automatically or manually. The post-reaction substrate resultant was analyzed using a microplate reader. The results show that the average absorbance value is correlated with the concentration of cardiac markers, in agreement with the results obtained using a conventional microsphere-based immunoassay; this indicated that the proposed on-chip immunoassay protocol could be used to detect both myoglobin and H-FABP. The minimum detectable concentration is 5 ng/mL for myoglobin and 1 ng/mL for H-FABP.     

Detecting low concentrations of Shigella sonnei in environmental water samples by PCR

Outbreaks of Shigella sonnei associated with contaminated water have been reported and methods for the simultaneous detection of Shigellae and enteroinvasive Escherichia coli in water samples have been developed with detection limits of 10(1)-10(2) CFU mL(-1) of water. Because 10(1)-10(2)Shigellae can cause disease, a more sensitive detection method as an addition to the existing methods for detection of Shigella sonnei in water samples is reported here. Initially, 33 Shigella sonnei and 72 non-Shigella sonnei isolates were tested and one primer pair was found capable of specifically amplifying a 369-bp insertion sequence 1 (IS1) fragment from all 33 Shigella sonnei isolates and one Shigella dysenteriae ATCC isolate by PCR. The detection method was developed, which included filtration of 50 mL of water through a membrane and application of PCR to the membrane using this primer pair. Environmental water samples with total bacterial numbers of 384-2.84 x 10(7) CFU L(-1) were collected and seeded with 13 Shigella sonnei and the Shigella dysenteriae ATCC isolates. Detection limits were determined as 1.7-24.7 and 270-8000 CFU per 50 mL of water, respectively, using this detection method.     

[AuCl4]− and Fe3+/[Fe(CN)6]3− ions-derivated immunosensing interface for electrochemical immunoassay of carcinoembryonic antigen in human serum

[AuCl₄]⁻ was initially deposited by electrochemical reduction on a glassy carbon electrode (GCE) to form porous nanogold layer, then prussian blue (PB) was electrodeposited onto the as-prepared nanogold layer, and then secondary nanogold particles were fabricated again on the PB surface by electrochemical reduction for the immobilization of anti-CEA antibodies. The presence of double-layer porous gold nanoparticles enhanced the immobilized amount of biomolecules, and improved the sensitivity of the immunoassay. PB, as a good redox probe, was facile to electrochemical analysis and measurement. Under optimal conditions, the developed immunoassay exhibited dynamic range from 3.0 to 80.0 ng/mL with a detection limit of 0.9 ng/mL CEA (S/N = 3). Moreover, the selectivity, reproducibility and stability of the immunosensor were acceptable.     

G-quadruplex DNAzyme-based microcystin-LR (toxin) determination by a novel immunosensor

In this paper, we demonstrate the application of versatile G-quadruplex-hemin DNAzymes in an immunoassay for detecting Microcystin-LR (MC-LR). Taking advantage of the high peroxidase activity of G-quadruplex-hemin complexes and the enhancement effect of gold nanoparticles (AuNPs), the method showed simple, high sensitive and selectivity detection of target toxin residues in water samples. The coated antigen, MC-LR-ovalbumin (OVA) coated on a plate, competed for MC-LR antibody with added target analyte to form antibody-antigen immune complexes. Subsequently, the immune complex reacted with G-quadruplex-labeled secondary antibodies for colorimetric detection of MC-LR. This assay specifically determined MC-LR in the linear range of 0.1-10 ng/ml, with a limit of detection (LOD) of 0.05 ng/mL for MC-LR. The results indicated that the novel immunoassay was an alternative to traditional plate-based immunoassay for MC-LR residue screening due to this method met the standard of World Health Organization (WHO) requirements for MC-LR content in drinking water (1 ng/mL).     

Neisseria meningitidis can induce pro-inflammatory cytokine production via pathways independent from CD14 and toll-like receptor 4

Fulminant meningococcal sepsis (FMS) is considered the prototypical Gram-negative sepsis. Lipopolysaccharide (LPS) is thought to be the main toxic element that induces pro-inflammatory cytokine production after interaction with CD14 and toll-like receptor 4 (TLR4). However, there is increasing evidence that LPS is not the sole toxic element of meningococci. The aim of the present study was to determine the role of CD14 and TLR4 in pro-inflammatory cytokine induction by meningococci. To this end, cytokine induction by isolated meningoccal LPS, wild-type N. meningitidis H44/76 (LPS+-meningococci) matched for concentrations of LPS and LPS-deficient N. meningitidis H44/76lpxA (LPS - -meningococci) was studied in human PBMCs and murine peritoneal macrophages (PMs). Pre-incubation of PBMCs with WT14, a monoclonal antibody against CD14, abolished TNF-alpha and IL-1beta induction by E. coli LPS, while cytokine induction by meningococcal LPS was only partially inhibited. When LPS+- and LPS - -meningococci at higher concentrations were used as stimuli, anti-CD14 had a minimal effect. In C3H/HeJ murine PMs, devoid of a functional TLR4, minimal IL-1alpha, IL-6 and TNF-alpha production was seen after stimulation with 10 ng/mL E. coli or meningococcal LPS. However, at higher concentrations (1000 ng LPS/mL) the production of TNF-alpha, but not IL-1alpha or IL-6, occurred also independently of TLR4. The expression of a functional TLR4 in murine PMs had no effect on the cytokine induction by LPS+- or LPS - -meningococci. It is concluded that pro-inflammatory cytokine induction by N. meningitidis can occur independently of CD14 and TLR4.     

Tumor necrosis factor release from lipopolysaccharide-stimulated human monocytes: Lipopolysaccharide tolerance in vitro

Human peripheral blood monocytes secrete tumor necrosis factor (TNF) in response to stimulation with bacterial lipopolysaccharide (LPS). We have shown that isolated human monocytes pretreated with LPS for 24 h secrete lower levels of TNF on a second stimulation with LPS than monocytes that have been stimulated with a single dose of LPS either immediately after isolation or 24 h after isolation. The levels of TNF released by monocytes after the second stimulation with LPS are proportional to the LPS concentration over a range from 1 ng/mL to 10 micrograms/mL. Increasing concentrations of LPS used during the first 24-h stimulation induce greater suppression of TNF release after a second stimulation with LPS. After an initial stimulus of 10 micrograms/mL LPS, a second stimulation of monocytes even with 10 micrograms/mL LPS will result in TNF secretion similar to that of unstimulated cells. This in vitro tolerance apparently can be overcome by stimulating previously activated cells with phorbol myristate acetate. We have also shown that neither prostaglandin E2 nor dexamethasone added during the initial stimulation with LPS had an effect on the subsequent reduction in TNF release on a second stimulation of monocytes with LPS.     

痢疾结合疫苗LPS制备条件的优化研究

通过对痢疾杆菌LPS提取过程中主要制备环节的优化和改进,确立最佳提取条件和纯化过程,并应用优化后的工艺路线分别制备八批次福氏2 a痢疾杆菌和宋内氏痢疾杆菌LPS;福氏2 a痢疾杆菌LPS批平均产量为1.633g,宋内氏痢疾杆菌LPS批产量平均为1.251g,批产量相对稳定,平均产量比优化改进前提高20%以上。LPS经过酸水解、柱层析纯化获得目标O-SP,福氏2 a痢疾杆菌和宋内氏痢疾杆菌O-SP的得率分别为20%和28%。检测结果证明,LPS和O-SP各项指标均符合规程(草案)相关要求。实验为今后痢疾结合疫苗大规模制备工艺的改进和提高打下了基础。