Artificial macrocycle with high binding selectivity in water is often challenging but urgently needed in various research and application areas.Herein,we report a new water-soluble biomimetic tetralactam macrocycle an...Artificial macrocycle with high binding selectivity in water is often challenging but urgently needed in various research and application areas.Herein,we report a new water-soluble biomimetic tetralactam macrocycle and realize the ultra-high selectivity to nucleosides over corresponding monophosphate nucleotides by rational modification.The introduction of charged groups at the periphery of endofunctionalized cavity makes the selectivity(guanosine to guanosine 5-monophosphate)increase remarkably from 100 to 1119.Based on the ultra-high selectivity of biomimetic tetralactam macrocycle,the sensitive CD73 enzyme activity assay was then achieved through product-selective fluorescence indicator displacement assay.Furthermore,the capability of the proposed method for inhibitor screening was successfully displayed.展开更多
Sprague-Dawley male rats were divided into groups and maintained on a semipurified diet containing either 5 or 40 ppm of zinc or 2 or 8 ppm of copper. Half of the rats in each group received 1000 ppm aluminum in the d...Sprague-Dawley male rats were divided into groups and maintained on a semipurified diet containing either 5 or 40 ppm of zinc or 2 or 8 ppm of copper. Half of the rats in each group received 1000 ppm aluminum in the diet. It was found that aluminum accumulated in the pituitary glands and testes when dietary copper levels were suboptimal. The ALP activity in testes was depressed by the added aluminum when the intake of zinc or copper was suboptimal. SDH, LDH, and LDH-X activities were inhibited and GRS and GGTP activities were elevated in rats fed either the suboptimal zinc or copper diet. However, the added aluminum in the diet reversed the changes to normal levels. The testosterone levels in plasma changed very little when the zinc or copper intake was suboptimal. An increase in plasma FSH was demonstrated in groups of both suboptimal zinc and copper intake. But the plasma LH was elevated only in the group receiving the suboptimal copper diet, and the added aluminum reversed plasma LH to control levels. A lower level of testosterone was demonstrated in the group given suboptimal copper with aluminum. It was concluded that dietary aluminum influenced the pituitary-testicular axis by interacting with certain essential trace metals, particularly zinc. 1990 Academic Press. Inc.展开更多
To study the toxicity of extracts of Ginkgo biloba sarcotesta to Oncomelania hupensis,snails were exposed to 40% and 80% of 24 h LC 50 of the extract of Ginkgo bilba for 24 h,choline esterase(ChE),alanine aminotransfe...To study the toxicity of extracts of Ginkgo biloba sarcotesta to Oncomelania hupensis,snails were exposed to 40% and 80% of 24 h LC 50 of the extract of Ginkgo bilba for 24 h,choline esterase(ChE),alanine aminotransferase(ALT),alkaline phosphatase(ALP),lactate dehydrogenase(LDH),succino dehydrogenase(SDH),malic dehydrogenase(MDH)activities in cephalopodium and liver were determined by enzyme kinetic assay.Arecoline and niclosamide were used as reference molluscicides.The results showed that sarcotesta of Ginkgo biloba could inhibit ChE,ALT,ALP and MDH activities both in cephalopodium and liver;arecoline could inhibit ChE,ALP,SDH and MDH activities in cephalopodium and ChE,ALT,ALP,SDH and MDH activities in liver.Niclosamide had inhibitory effects upon ChE,ALT,ALP,SDH and MDH activities in cephalopodium,and ChE,ALT,ALP and SDH activities in liver.All three molluscicides did not inhibite LDH activity in cephalopodium and liver.These results indicate that lethal effects of extracts of sarcotesta of Ginkgo biloba are mediated via inhibition of MDH activitiy,and interference with the NADH respiratory chains.Inhibition of vital enzymic mechanisms causes snails to death.展开更多
Protein tyrosine phosphatases(PTPs) are crucial regulators of signal transduction. Among them, PTP-MEG2 is an intracellular enzyme of 593 amino acid residues with a putative lipid-binding domain at the N-terminus. I...Protein tyrosine phosphatases(PTPs) are crucial regulators of signal transduction. Among them, PTP-MEG2 is an intracellular enzyme of 593 amino acid residues with a putative lipid-binding domain at the N-terminus. In the present study, we cloned the full-length form of the enzyme and expressed it in E. coli cells as a 6xHis-tagged protein. The majority of the expressed enzyme was found in the inclusion body of E. coli cell extracts. Upon extraction with a buffer containing urea, the recombinant enzyme was purified to near homogeneity on a single Ni-NTA-agarose column. This procedure resulted in the production of over 100 mg of purified recombinant PTP-MEG2 from 1 L E. coli cell culture. The purified protein displayed a single polypeptide band with expected molecular size on SDS-polyacrylamide gel electrophoresis under reducing conditions. Isolated under denatured conditions in urea, the purified enzyme was re-natured by dialyzing against a refolding buffer. The re-natured enzyme effectively dephosphorylated the common PTP substrate para-nitrophenylphosphate with a specific activity of 2000 units/mg. Meanwhile, the denatured enzyme was used to immunize a rabbit to produce antibodies. The resulting anti- serum had extremely high sensitivity and specificity. When used for Western blot analysis, the anti-serum revealed a wide expression of PTP-MEG2 in many tissues of mice. Together, we developed a highly effective way to purify a large amount of PTP-MEG2 and generated highly sensitive antibodies that can specifically detect endogenous expression of the enzyme in tissues.展开更多
Objectives To produce an enzyme linked immunosorbant assay (ELISA) to detect antibodies against Epstein Barr virus (EBV) specified nuclear antigen 1 (EBNA 1) and the 18kD EBV matrix protein, and to determine and optim...Objectives To produce an enzyme linked immunosorbant assay (ELISA) to detect antibodies against Epstein Barr virus (EBV) specified nuclear antigen 1 (EBNA 1) and the 18kD EBV matrix protein, and to determine and optimize its sensitivity and specificity for the diagnosis of nasopharyngeal carcinoma (NPC).Methods We used a combination of highly purified glutathione transferase fusion proteins of the 40kD carboxy domain of EBNA1 and the 18kD EBV matrix protein for coating ELISA plates. In three separate studies, we tested for IgA antibodies in serum specimens from 28 EBV seronegative donors, 284 EBV seropositive donors and 160 newly diagnosed NPC patients. By comparing the sensitivity and specificity of diagnosis obtained for different cutoff values, we derived several quantitative parameters to evaluate assay performance, establish objective diagnostic criteria which optimize the intrinsic diagnostic capability of the assay and assess the significance of individual test results, respectively. Optimum cutoff optical density (OD) is defined as the cutoff OD where sensitivity of the assay equals its specificity, and resolution of the assay is indicated by the value of sensitivity (or specificity) determined at the optimum cutoff OD. Diagnosis of NPC was achieved by setting a cutoff zone at +/-20% of this value.Results All the EBV seronegative donors tested were not reactive, and most of the EBV seropositive donors were weakly reactive, while the majority of NPC patients were moderately or strongly reactive. While the assay was thus shown to be specific for EBV, there was an overlap in the level of these serum antibodies between few individuals of the two latter groups. It was shown that the assay performed equally well in two separate studies conducted under different testing conditions and using different collections of sera in that assay resolution determined on these occasions were 86% and 87% respectively. Diagnosis of NPC can be achieved at the same expected sensitivity of 89% and 83% determined at the lower and upper limits of the cutoff zones, with the corresponding values of specificity being 78% and 91%. It was further shown in the third study that resolution of the assay can be increased to 90% using an assay produced with a higher concentration of the same antigens, and that diagnosis of NPC can be achieved at a higher sensitivity ranging between 86% and 95% at a corresponding specificity of 93% and 86%.Conclusions After optimization and standardization, the ELISA can achieve a sensitivity ranging from 86% to 95%, with corresponding specificities of 93% and 86% respectively for the diagnosis of NPC.展开更多
Cellulose is the main structural component of lignocellulosic wastes that can be converted to sugars and biofuels by cellulase.Due to wide applications of this enzyme in various industries around the world,cellulase i...Cellulose is the main structural component of lignocellulosic wastes that can be converted to sugars and biofuels by cellulase.Due to wide applications of this enzyme in various industries around the world,cellulase is considered as the third industrial enzyme.The ability of thermophilic bacteria in the production of heat-stable cellulases has made them valuable tools in biotechnology.The aim of this study was isolation and molecular identification of cellulolytic thermophile bacteria from Dig Rostam hot spring and investigating their cellulase activity.Samples were taken from water and sediments of this hot spring,and cellulolytic bacteria were enriched in media containing cellulose as the only carbon source.The bacteria were incubated at 60℃,and single colonies were then isolated on solid media.Congo red assay was used as a quick test for the qualitative screening of cellulase activity.According to these qualitative results,four colonies named CDB1,CDB2,CDB3,and CDB4 were isolated,and their growth curve and some other characteristics were determined by biochemical assays.Moreover,endoglucanase,exoglucanase,and FPase activities of the isolates were investigated quantitatively.Results indicated that CDB1 exhibited the highest endoglucanase(0.096 U/mL)and exoglucanase(0.156 U/mL)activities among other isolates.16S rDNA partial sequencing indicated that CDB1 had 99%similarity to the genus Anoxybacillus,and the other isolates showed the highest similarity to the genus Geobacillus.The cellulase gene of CDB1 isolate with the highest cellulase activity was also cloned,and its sequence is reported for the first time.Further studies on this thermophilic enzyme might be useful for industrial applications.展开更多
Objective: To investigate the serum levels of soluble macrophage colony-stimulating factor receptor (M-CSFsR) in normal subjects and patients with hematological diseases and its clinical implications in hematological ...Objective: To investigate the serum levels of soluble macrophage colony-stimulating factor receptor (M-CSFsR) in normal subjects and patients with hematological diseases and its clinical implications in hematological diseases. Methods: The concentration of M-CSFsR was determined by ELISA. The serum M-CSFsR was identified and characterized by immunoprecipitation and Western blotting. Results: The mean serum level of M-CSFsR of 123 normal individuals was 0.48 ng/ml ± 0.41 ng/ml. Immunoprecipitation and Western blotting assay revealed a ~ 90kD band of serum M-CSFsR. The mean serum M-CSFsR level of 60 patients with acute lymphoblastic leukemia (ALL), 36 patients with acute myeloblastic leukemia (AML), 13 patients with myelodysplastic syndrome (MDS) and 42 patients with aplastic anemia (AA) .were 0.22 ng/ml±0.23 ng/ml, 0.17 ng/ml±0.16 ng/ml, 0.19 ng/ml±0.16 ng/ml and 0.23 ng/ml±0.21 ng/ml, respectively, which were significantly lower than that of normal subjects (P=0.002 ,P<0.0001,P<0.0001 andP<0.0001). The mean serum M-CSFsR level of 51 idiopathic thrombocytopenic purpura (ITP) patients was significantly higher than that of normal subjects (2.05 ng/ml±2.75 ng/ml,P<0.0001). Conclusion: The serum M-CSFsR levels of patients with ALL, AML, MDS and AA were significantly lower, while the level of patients with ITP was significantly higher than that of normal individuals. Patients with severe ITP (platelet count<30×l09/L) had the highest M-CSFsR level. It suggested that the abnormal levels of serum M-CSFsR may associate with some hematological diseases and may contribute to the pathological process.展开更多
In this study, the levels of soluble delta-like ligand 1 in cerebmspinal fluid and serum of 50 patients with tuberculous meningitis, 30 patients with viral meningitis, 20 patients with purulent meningitis and 40 subje...In this study, the levels of soluble delta-like ligand 1 in cerebmspinal fluid and serum of 50 patients with tuberculous meningitis, 30 patients with viral meningitis, 20 patients with purulent meningitis and 40 subjects without central nervous system disease were determined using an enzyme-linked immunosorbent assay. The mean levels of soluble delta-like ligand 1 in both cerebrospinal fluid and serum from patients with tuberculous meningitis were significantly higher compared with those from patients with viral meningitis or purulent meningitis or from subjects without central nervous system disease. Meanwhile, the level of soluble delta-like ligand 1 gradually decreased as tuberculous meningitis patients recovered. If patients deteriorated after treatment, the level of soluble delta-like ligand 1 in cerebrospinal fluid gradually increased. There was no correlation between the level of soluble delta-like ligand 1 and the protein level/cell number in cerebrospinal fluid. Our findings indicate that the levels of soluble delta-like ligand 1 in cerebrospinal fluid and serum are reliable markers for the diagnosis of tuberculous meningitis and for monitoring treatment progress. At the same time, this index is not influenced by protein levels or cell numbers in cerebrospinal fluid.展开更多
The value of ELISA, N-PCR and RT-PCR in clinical practice for pregnant women with HCMV infection was investigated. 5581 pregnant women were screened by ELISA. Among them, 100 cases were positive for IgM (group 1). 69 ...The value of ELISA, N-PCR and RT-PCR in clinical practice for pregnant women with HCMV infection was investigated. 5581 pregnant women were screened by ELISA. Among them, 100 cases were positive for IgM (group 1). 69 for both IgM and serous DNA (group 2) and 69 for both IgM and mRNA (group 3). The infectious status, maternal-fetal transmission and pregnancy outcome were monitored. It was demonstrated that the accordance rate of group 3 and group 2 with group 1 was 56. 25 % and 43. 75 % , respectively. The maternal-fetal transmission rate in the group 1, 2 and 3 was 19. 00 % , 40. 58 % and 46. 15 %, respectively, with a significant difference found between group 2, 3 and group 1 (P<0. 01). Incidence of spontaneous abortion, fetal death, fetal abnormality and neonatal death in group 1, 2 and 3 was 10. 00 %, 15. 94 % and 30. 77 %, respectively, and that of group 3, 2 was 4 and 2 times as much as that of group 1, respectively (OR = 4. 00, P<0. 001; OR=2. 343, P<005, respectively). It was concluded that HCMV-IgM(+) can only be considered as an screening indicator for pregnant women with HCMV infection, while IgM(+) combined with serous DNA( + ) or mRNA( + ) indicates active infection and has a high incidence of maternal-fetal transmission and abnormal pregnancy outcome.展开更多
Insulin-like growth factor- I (IGF- I ) is a mitogenic and anti-apoptotic factor. Serum IGF-I concentration is related to some cancer risk and tumor progression. The aim of this research was to study the association...Insulin-like growth factor- I (IGF- I ) is a mitogenic and anti-apoptotic factor. Serum IGF-I concentration is related to some cancer risk and tumor progression. The aim of this research was to study the association of preoperative serum IGF- I concentration with clinicopathological parameters and prognosis of non-small cell lung cancer (NSCLC). Preoperative serum IGF- I concentration was measured in 80 consecutive patients with NSCLC who underwent radical lung cancer resection, and 45 patients with benign pulmonary lesion (BPL) by Using enzyme linked immunosorbent assay (ELISA). The results showed that the serum IGF- I concentration was elevated and correlated with clinicopa- thological parameters and overall survival (OS) in NSCLC patients. Serum IGF- I concentration was significantly higher in patients with NSCLC tfian in those with BPL. The IGF- I concentrations were significantly higher in NSCLC patients with 〉T2, NI-3, and in IIIA-IV but not in those with 〈T2, NO, or I A- IIB. The increased serum IGF- I concentration was significantly correlated with poor prog- nosis. Our data show the positive correlation between IGF-I serum concentration and the tumor size for the first time. It seems that IGF-I related to the progression of lung cancer may depend on autocrine/paracrine function. In addition, our study reveals that higher serum IGF- I concentration is correlated with larger tumor size, advanced stages, local lymph node metastasis and worse prognosis, indicating that endocrine IGF- I is also important for the progression for NSCLC.展开更多
[ Objective] To develop an indirect ELISA assay for detecting antibodies against envelope glycoprotein ( E protein) of Japanese encephalitis virus (JEV). [ Method] Specific primers were designed according to JEV s...[ Objective] To develop an indirect ELISA assay for detecting antibodies against envelope glycoprotein ( E protein) of Japanese encephalitis virus (JEV). [ Method] Specific primers were designed according to JEV sequences published in the GenBank. The cDNA of JEV E gene (about 1 000 10p) was amplified by the RT-PCR with the specific primers. After sequencing analysis, the E gene was cloned into pET30a expression vector and expressed in E. coli BL21 (DE3) with the induction of IPTG. After denaturation, purification and renaturation, the recombinant protein was analyzed by the SDS-PAGE and the westem blotting. An indirect ELISA was developed to detect antibodies against JEV. [ Result] The E protein was mainly expressed in inclusion body. With the purified E protein, the indirect ELISA was developed and displayed good specificity, sensitivity and repeatability, [ Conclusion]The developed ELISA using the truncated E protein as antigen is a simple, convenient and rapid serological method for diagnosis, monitoring antibody level and epidemiological investigation of JEV.展开更多
The purpose of this study was to develop a method for detecting antibodies against porcine pseudorabies virus (PRV). According to the published genomic sequence of PRV SA strain, an approximately 1 070-bp gD gene fr...The purpose of this study was to develop a method for detecting antibodies against porcine pseudorabies virus (PRV). According to the published genomic sequence of PRV SA strain, an approximately 1 070-bp gD gene fragment was amplified by PCR. The PCR products were cloned into the prokaryotic expression vector pET30a and the positive recombinant plasmid was transformed into E. coli BL21. Through induction with IPTG, the recombinant gD protein was expressed as inclusion bodies. As analyzed by western blot assay, the purified recombinant gD protein had good antigenicity and high specificity. Using the purified gD protein as coating antigen and horseradish peroxidase labeled staphylococcal protein A (PPA) as secondary antibody, we developed a PPA-ELISA for detecting antibodies against porcine PPV. No cross-reaction with the positive sera against seven common pathogens in pigs including classical swine fever virus, porcine parvovirus, porcine reproductive and respiratory syndrome, Japanese encephalitis virus, porcine circovirus type 2, porcine epidemic diarrhea virus, transmissible gastroenteritis virus was observed. The repeatability test showed that the intra- and inter-assay coefficients of variation were lower than 5% and 10%, respectively. Compared with the ELISA gD antibody test kit produced by IDEXX, the coincidence, sensitivity and specificity of the developed PPA-ELISA were 92.0%, 95.1% and 88.1%, respectively. The developed PPA-ELISA had good repeatability, sensitivity and specificity and was a rapid and simple serological method for surveillance of PRV antibodies in pig herds as well as for rapid diagnosis and epidemiological investigation of PRV infection.展开更多
A new rapid,specific and sensitive method for assay of recombinant CFP10-ESAT6 amalgamation proteins from Mycobacterium tuberculosis was proposed.The method used streptavidincoated magnetic beads to enrich the specifi...A new rapid,specific and sensitive method for assay of recombinant CFP10-ESAT6 amalgamation proteins from Mycobacterium tuberculosis was proposed.The method used streptavidincoated magnetic beads to enrich the specific biotinylated anti-CFP10 antibody,then adopted a sandwich-type enzyme linked immunosorbent assay technology with two kinds of monoclonal antibodies:biotinylated anti-CFP10 antibody and HRP-labeled anti-CFP10 antibody to identify the target CFP10-ESAT6 proteins,and finally detected chemiluminescence intensity by a small home-made optical sensor.It was shown that,the corresponding chemiluminescence intensity had a good logarithmic linear response to the concentration of CFP10-ESAT6 proteins when ranging at 1~1000 ng/mL,and the correlation coefficient is 0.9937.The proposed method could detect the CFP10-ESAT6 proteins with low detection limit(1 ng/mL)and the detection time could be controlled within 45 min.Compared with commonly used detection methods of M.tuberculosis,this method was easy to operate,faster,and of higher sensitivity.The achievement of the quantitative detection of CFP10-ESAT6 proteins has important scientific significance and wide application prospects in tuberculosis control.展开更多
[Objective] To develop a new method for serodiagnosis of swine toxoplasmosis. [Method] With the purified recombinant microneme protein 3 (rMIC3) as coating antigens, an indirect ELISA was developed for detection of ...[Objective] To develop a new method for serodiagnosis of swine toxoplasmosis. [Method] With the purified recombinant microneme protein 3 (rMIC3) as coating antigens, an indirect ELISA was developed for detection of antibodies against Toxoplasma gondii. [ Result] The optimal working concentration of rMIC3 was 3. 40 ug/ml, and the optimal degree of dilution of sera was 1:160. Cross-reaction was not observed between the Toxoplasma gondii-positive sera and the positive sera against classical swine fever virus or some other pathogens. The developed ELISA had 92.56% coincidence rate with latex agglutination test. [ Conclusion] The developed ELISA is sensitive, rapid, specific and reproducible, and thus it can be applied in serodiagnosis and seroprevalence investigation of swine toxoplasmosis.展开更多
Objective: To study the therapeutic mechanism of Santeng Dingtong recipe (STDT) on monosodium urate crystal (MSU) induced rabbit arthritis Methods: Forty-two rabbits were randomly divided into six groups, 7 in each gr...Objective: To study the therapeutic mechanism of Santeng Dingtong recipe (STDT) on monosodium urate crystal (MSU) induced rabbit arthritis Methods: Forty-two rabbits were randomly divided into six groups, 7 in each group. Group 1 received 0.9% saline 2. 5 ml/kg per day by gastrogavage (ig) for 10 days; Group 2, 3 and 4 received STDT 0.125 g/kg, 1.0 g/kg and 8.0 g/kg per day respectively by ig for 10 days; Group 5 received colchicine 4. 5 mg/kg per day by ig for 4 days; and Group 6 was untreated. MSU crystals 10 mg /500ul containing polymyxin B 10 u/ml was injected into the knee joints of Group 1-5 to make rabbit arthritis models. Leukocytes in synovial lavage fluids was then counted and differentiated; pathological injury of synovial membranes was observed under HE staining; interleukin-1 beta (IL-1B), tumor necrosis factor alpha (TNFa) and leukotriene B4 (LTB4) content in synovial lavage fluids were determined by ELISA. Results: MSU caused a rapid leukocyte infiltration and increased production of IL-1B, TNFa and LTB4 2 hrs after intra-articular injection. STDT inhibited neutrophil infiltration in synovial fluids dose-dependently, protected the synovial membrane against pathological injury and reduced the production of IL-1B, TNFa and LTB4; while colchicine did not decrease the level of TNFa, but significantly inhibited neutrophil infiltration in synovial fluid and reduced the production of IL-11B and LTB4. Conclusion: STDT exerts an anti-inflammatory effect in rabbit model of acute MSU arthritis, its mechanism being probably due to the decrease of XL-1B, TNFa and LTB4 synthesis.展开更多
[Objectives]This study was conducted to develop an enzyme-linked immunoassay kit that can detect the residual amount of pentachloronitrobenzene in Penaeus vannamei.[Methods]This study was conducted to develop an enzym...[Objectives]This study was conducted to develop an enzyme-linked immunoassay kit that can detect the residual amount of pentachloronitrobenzene in Penaeus vannamei.[Methods]This study was conducted to develop an enzyme-linked immunoassay kit that can detect the residual amount of pentachloronitrobenzene in P.vannamei.[Results]The standard curve range of the kit was 0-8.1μg/L;the detection limit for P.vannamei was 0.912μg/kg;the recovery was 80.6%-103.5%;and the relative standard deviation range within batches was 5.3%-10.1%,and the relative standard deviation range between batches was 6.7%-8.1%.The specificity of the pentachloronitrobenzene monoclonal antibody was relatively good,and the cross-reaction rates with pentachlorophenol,hexachlorobenzene,tetrachlorophthalide,and chlorothalonil were low,all of which did not exceed 30%.The ELISA kit could be stored at 4℃for 12 months,showing good stability.[Conclusions]The detection kit has low cost,short time and small deviation,and is an ideal preliminary screening method.展开更多
<strong>Objective:</strong> To evaluate three different serological tests [Indirect Hemaglutination (IHA), Enzyme Linked Immunosorbent Assay (ELISA) and Western Blotting (WB)] using native crude antigen fo...<strong>Objective:</strong> To evaluate three different serological tests [Indirect Hemaglutination (IHA), Enzyme Linked Immunosorbent Assay (ELISA) and Western Blotting (WB)] using native crude antigen for diagnosis of hepatic cystic echinococcosis (HCE) patients. <strong>Materials and Methods:</strong> Sheep hydatid fluid (HF) was collected from fertile cysts obtained from a slaughterhouse and used as an antigen. Forty patients who were attended the Dr. Ersin Arslan Training and Research Hospital in Gaziantep, Turkey, were investigated. Serum samples were obtained from surgically confirmed CE patients. Healthy Turkish people and 16 patients with other helminthic infections were included as a control group. <strong>Results:</strong> Of the 40 analyzed patients, 10 (25%) were men and 30 (75%) were female. The average age was 46.97 years (s.d.;18.95). The majority of the patients had a single cystic lesion situated in one lobe of the liver (usually in the right lobe) (55%), 32.5% of patients had two cystic lesions and 12.5% of patients had multiple cyst formations with various numbers. In all cases, ultrasound (US) examinations were positive and the size of cysts was between 2.1 - 12.7 cm. Twenty-three patients of the total 40 patients were classified according to the WHO classification system based on US findings. According to the results of WB analysis, molecular weights of 8 kDa (80%), 12 kDa (80%), 22 - 24 kDa (97.5%), 26 kDa (97.5%), 34 kDa (100%), 36 - 38 kDa (90%), 45 - 50 - 55 kDa (97.5%), and 60 - 75 kDa (97.5%) bands were identified. But 34, 50, and 55 kDa bands were also found in other helminthic diseases. <strong>Conclusion:</strong> The specificity and sensitivity of three serological tests (IHA, ELISA and WB) using crude antigen were compared by diagnosing hepatic cystic echinococcosis patients. IHA and ELISA showed high sensitivity but low specificity. Western blotting showed low sensitivity but high specificity.展开更多
Biosensors are widely used in immunoassay. The biosensor chip carries a receptor which is used in immunoassay and the chip properties have an important influence on the detecting sensitivity of the biosensor. This pap...Biosensors are widely used in immunoassay. The biosensor chip carries a receptor which is used in immunoassay and the chip properties have an important influence on the detecting sensitivity of the biosensor. This paper describes a polystyrene\|based biosensor chip developed and used as part of a surface plasmon resonance (SPR) biosensor. The SPR biosensor has a much higher detecting sensitivity than enzyme\|linked immunoserbent assay (ELISA).展开更多
The protein tyrosine phosphatase Src homology phosphotyrosyl phosphatase 2(SHP2) is implicated in various cancers, and targeting SHP2 has become a promising therapeutic approach. We herein described a robust cross-val...The protein tyrosine phosphatase Src homology phosphotyrosyl phosphatase 2(SHP2) is implicated in various cancers, and targeting SHP2 has become a promising therapeutic approach. We herein described a robust cross-validation high-throughput screening protocol that combined the fluorescence-based enzyme assay and the conformation-dependent thermal shift assay for the discovery of SHP2 inhibitors. The established method can effectively exclude the false positive SHP2 inhibitors with fluorescence interference and was also successfully employed to identify new protein tyrosine phosphatase domain of SHP2(SHP2-PTP) and allosteric inhibitors. Of note, this protocol showed potential for identifying SHP2 inhibitors against cancer-associated SHP2 mutation SHP2-E76 A. After initial screening of our in-house compound library(w2300 compounds), we identified 4 new SHP2-PTP inhibitors(0.17%hit rate) and 28 novel allosteric SHP2 inhibitors(1.22% hit rate), of which SYK-85 and WS-635 effectively inhibited SHP2-PTP(SYK-85: IC_(50) Z 0.32 mmol/L;WS-635: IC_(50) Z 4.13 mmol/L) and thus represent novel scaffolds for designing new SHP2-PTP inhibitors. TK-147, an allosteric inhibitor, inhibited SHP2 potently(IC_(50) Z 0.25 mmol/L). In structure, TK-147 could be regarded as a bioisostere of the well characterized SHP2 inhibitor SHP-099, highlighting the essential structural elements for allosteric inhibition of SHP2. The principle underlying the cross-validation protocol is potentially feasible to identify allosteric inhibitors or those inactivating mutants of other proteins.展开更多
A series of novel pyrano[2,3-d]trizaole compounds were synthesized and theirα-glucosidase inhibitory activities were evaluated by in vitro enzyme assay.The experimental data demonstrated that compound 10 f showed up ...A series of novel pyrano[2,3-d]trizaole compounds were synthesized and theirα-glucosidase inhibitory activities were evaluated by in vitro enzyme assay.The experimental data demonstrated that compound 10 f showed up to 10-fold higher inhibition(IC5074.0±1.3μmol·L-1)than acarbose.The molecular docking revealed that compound 10 f could bind toα-glucosidase via the hydrophobic,π-πstacking,and hydrogen bonding interactions.The results may benefit further structural modifications to find new and potentα-glucosidase inhibitors.展开更多
基金financially supported by National Natural Science Foundation of China(Nos.22174059 and 22201128)Hunan Provincial Natural Science Foundation of China(Nos.2022JJ40363,2022JJ40365 and 2022RC1230)+1 种基金the Excellent youth funding of Hunan Provincial Education Department(No.22B0460)China Postdoctoral Science Foundation(No.2022M721542)。
文摘Artificial macrocycle with high binding selectivity in water is often challenging but urgently needed in various research and application areas.Herein,we report a new water-soluble biomimetic tetralactam macrocycle and realize the ultra-high selectivity to nucleosides over corresponding monophosphate nucleotides by rational modification.The introduction of charged groups at the periphery of endofunctionalized cavity makes the selectivity(guanosine to guanosine 5-monophosphate)increase remarkably from 100 to 1119.Based on the ultra-high selectivity of biomimetic tetralactam macrocycle,the sensitive CD73 enzyme activity assay was then achieved through product-selective fluorescence indicator displacement assay.Furthermore,the capability of the proposed method for inhibitor screening was successfully displayed.
文摘Sprague-Dawley male rats were divided into groups and maintained on a semipurified diet containing either 5 or 40 ppm of zinc or 2 or 8 ppm of copper. Half of the rats in each group received 1000 ppm aluminum in the diet. It was found that aluminum accumulated in the pituitary glands and testes when dietary copper levels were suboptimal. The ALP activity in testes was depressed by the added aluminum when the intake of zinc or copper was suboptimal. SDH, LDH, and LDH-X activities were inhibited and GRS and GGTP activities were elevated in rats fed either the suboptimal zinc or copper diet. However, the added aluminum in the diet reversed the changes to normal levels. The testosterone levels in plasma changed very little when the zinc or copper intake was suboptimal. An increase in plasma FSH was demonstrated in groups of both suboptimal zinc and copper intake. But the plasma LH was elevated only in the group receiving the suboptimal copper diet, and the added aluminum reversed plasma LH to control levels. A lower level of testosterone was demonstrated in the group given suboptimal copper with aluminum. It was concluded that dietary aluminum influenced the pituitary-testicular axis by interacting with certain essential trace metals, particularly zinc. 1990 Academic Press. Inc.
文摘To study the toxicity of extracts of Ginkgo biloba sarcotesta to Oncomelania hupensis,snails were exposed to 40% and 80% of 24 h LC 50 of the extract of Ginkgo bilba for 24 h,choline esterase(ChE),alanine aminotransferase(ALT),alkaline phosphatase(ALP),lactate dehydrogenase(LDH),succino dehydrogenase(SDH),malic dehydrogenase(MDH)activities in cephalopodium and liver were determined by enzyme kinetic assay.Arecoline and niclosamide were used as reference molluscicides.The results showed that sarcotesta of Ginkgo biloba could inhibit ChE,ALT,ALP and MDH activities both in cephalopodium and liver;arecoline could inhibit ChE,ALP,SDH and MDH activities in cephalopodium and ChE,ALT,ALP,SDH and MDH activities in liver.Niclosamide had inhibitory effects upon ChE,ALT,ALP,SDH and MDH activities in cephalopodium,and ChE,ALT,ALP and SDH activities in liver.All three molluscicides did not inhibite LDH activity in cephalopodium and liver.These results indicate that lethal effects of extracts of sarcotesta of Ginkgo biloba are mediated via inhibition of MDH activitiy,and interference with the NADH respiratory chains.Inhibition of vital enzymic mechanisms causes snails to death.
基金Supported by the Plan for Development of Science & Technology in Jilin Province China(No.20090920)+2 种基金the Boyou fund from China SOONG Ching-ling Foundationthe Fund of National Institutes of Health USA(No.HL079441)
文摘Protein tyrosine phosphatases(PTPs) are crucial regulators of signal transduction. Among them, PTP-MEG2 is an intracellular enzyme of 593 amino acid residues with a putative lipid-binding domain at the N-terminus. In the present study, we cloned the full-length form of the enzyme and expressed it in E. coli cells as a 6xHis-tagged protein. The majority of the expressed enzyme was found in the inclusion body of E. coli cell extracts. Upon extraction with a buffer containing urea, the recombinant enzyme was purified to near homogeneity on a single Ni-NTA-agarose column. This procedure resulted in the production of over 100 mg of purified recombinant PTP-MEG2 from 1 L E. coli cell culture. The purified protein displayed a single polypeptide band with expected molecular size on SDS-polyacrylamide gel electrophoresis under reducing conditions. Isolated under denatured conditions in urea, the purified enzyme was re-natured by dialyzing against a refolding buffer. The re-natured enzyme effectively dephosphorylated the common PTP substrate para-nitrophenylphosphate with a specific activity of 2000 units/mg. Meanwhile, the denatured enzyme was used to immunize a rabbit to produce antibodies. The resulting anti- serum had extremely high sensitivity and specificity. When used for Western blot analysis, the anti-serum revealed a wide expression of PTP-MEG2 in many tissues of mice. Together, we developed a highly effective way to purify a large amount of PTP-MEG2 and generated highly sensitive antibodies that can specifically detect endogenous expression of the enzyme in tissues.
文摘Objectives To produce an enzyme linked immunosorbant assay (ELISA) to detect antibodies against Epstein Barr virus (EBV) specified nuclear antigen 1 (EBNA 1) and the 18kD EBV matrix protein, and to determine and optimize its sensitivity and specificity for the diagnosis of nasopharyngeal carcinoma (NPC).Methods We used a combination of highly purified glutathione transferase fusion proteins of the 40kD carboxy domain of EBNA1 and the 18kD EBV matrix protein for coating ELISA plates. In three separate studies, we tested for IgA antibodies in serum specimens from 28 EBV seronegative donors, 284 EBV seropositive donors and 160 newly diagnosed NPC patients. By comparing the sensitivity and specificity of diagnosis obtained for different cutoff values, we derived several quantitative parameters to evaluate assay performance, establish objective diagnostic criteria which optimize the intrinsic diagnostic capability of the assay and assess the significance of individual test results, respectively. Optimum cutoff optical density (OD) is defined as the cutoff OD where sensitivity of the assay equals its specificity, and resolution of the assay is indicated by the value of sensitivity (or specificity) determined at the optimum cutoff OD. Diagnosis of NPC was achieved by setting a cutoff zone at +/-20% of this value.Results All the EBV seronegative donors tested were not reactive, and most of the EBV seropositive donors were weakly reactive, while the majority of NPC patients were moderately or strongly reactive. While the assay was thus shown to be specific for EBV, there was an overlap in the level of these serum antibodies between few individuals of the two latter groups. It was shown that the assay performed equally well in two separate studies conducted under different testing conditions and using different collections of sera in that assay resolution determined on these occasions were 86% and 87% respectively. Diagnosis of NPC can be achieved at the same expected sensitivity of 89% and 83% determined at the lower and upper limits of the cutoff zones, with the corresponding values of specificity being 78% and 91%. It was further shown in the third study that resolution of the assay can be increased to 90% using an assay produced with a higher concentration of the same antigens, and that diagnosis of NPC can be achieved at a higher sensitivity ranging between 86% and 95% at a corresponding specificity of 93% and 86%.Conclusions After optimization and standardization, the ELISA can achieve a sensitivity ranging from 86% to 95%, with corresponding specificities of 93% and 86% respectively for the diagnosis of NPC.
基金a grant(3/22775)from Ferdowsi University of Mashhad.
文摘Cellulose is the main structural component of lignocellulosic wastes that can be converted to sugars and biofuels by cellulase.Due to wide applications of this enzyme in various industries around the world,cellulase is considered as the third industrial enzyme.The ability of thermophilic bacteria in the production of heat-stable cellulases has made them valuable tools in biotechnology.The aim of this study was isolation and molecular identification of cellulolytic thermophile bacteria from Dig Rostam hot spring and investigating their cellulase activity.Samples were taken from water and sediments of this hot spring,and cellulolytic bacteria were enriched in media containing cellulose as the only carbon source.The bacteria were incubated at 60℃,and single colonies were then isolated on solid media.Congo red assay was used as a quick test for the qualitative screening of cellulase activity.According to these qualitative results,four colonies named CDB1,CDB2,CDB3,and CDB4 were isolated,and their growth curve and some other characteristics were determined by biochemical assays.Moreover,endoglucanase,exoglucanase,and FPase activities of the isolates were investigated quantitatively.Results indicated that CDB1 exhibited the highest endoglucanase(0.096 U/mL)and exoglucanase(0.156 U/mL)activities among other isolates.16S rDNA partial sequencing indicated that CDB1 had 99%similarity to the genus Anoxybacillus,and the other isolates showed the highest similarity to the genus Geobacillus.The cellulase gene of CDB1 isolate with the highest cellulase activity was also cloned,and its sequence is reported for the first time.Further studies on this thermophilic enzyme might be useful for industrial applications.
基金National "863" High Technology Program of China ( 102-11-01-03).
文摘Objective: To investigate the serum levels of soluble macrophage colony-stimulating factor receptor (M-CSFsR) in normal subjects and patients with hematological diseases and its clinical implications in hematological diseases. Methods: The concentration of M-CSFsR was determined by ELISA. The serum M-CSFsR was identified and characterized by immunoprecipitation and Western blotting. Results: The mean serum level of M-CSFsR of 123 normal individuals was 0.48 ng/ml ± 0.41 ng/ml. Immunoprecipitation and Western blotting assay revealed a ~ 90kD band of serum M-CSFsR. The mean serum M-CSFsR level of 60 patients with acute lymphoblastic leukemia (ALL), 36 patients with acute myeloblastic leukemia (AML), 13 patients with myelodysplastic syndrome (MDS) and 42 patients with aplastic anemia (AA) .were 0.22 ng/ml±0.23 ng/ml, 0.17 ng/ml±0.16 ng/ml, 0.19 ng/ml±0.16 ng/ml and 0.23 ng/ml±0.21 ng/ml, respectively, which were significantly lower than that of normal subjects (P=0.002 ,P<0.0001,P<0.0001 andP<0.0001). The mean serum M-CSFsR level of 51 idiopathic thrombocytopenic purpura (ITP) patients was significantly higher than that of normal subjects (2.05 ng/ml±2.75 ng/ml,P<0.0001). Conclusion: The serum M-CSFsR levels of patients with ALL, AML, MDS and AA were significantly lower, while the level of patients with ITP was significantly higher than that of normal individuals. Patients with severe ITP (platelet count<30×l09/L) had the highest M-CSFsR level. It suggested that the abnormal levels of serum M-CSFsR may associate with some hematological diseases and may contribute to the pathological process.
基金supported by the National Natural Science Foundation of China,No.30770758
文摘In this study, the levels of soluble delta-like ligand 1 in cerebmspinal fluid and serum of 50 patients with tuberculous meningitis, 30 patients with viral meningitis, 20 patients with purulent meningitis and 40 subjects without central nervous system disease were determined using an enzyme-linked immunosorbent assay. The mean levels of soluble delta-like ligand 1 in both cerebrospinal fluid and serum from patients with tuberculous meningitis were significantly higher compared with those from patients with viral meningitis or purulent meningitis or from subjects without central nervous system disease. Meanwhile, the level of soluble delta-like ligand 1 gradually decreased as tuberculous meningitis patients recovered. If patients deteriorated after treatment, the level of soluble delta-like ligand 1 in cerebrospinal fluid gradually increased. There was no correlation between the level of soluble delta-like ligand 1 and the protein level/cell number in cerebrospinal fluid. Our findings indicate that the levels of soluble delta-like ligand 1 in cerebrospinal fluid and serum are reliable markers for the diagnosis of tuberculous meningitis and for monitoring treatment progress. At the same time, this index is not influenced by protein levels or cell numbers in cerebrospinal fluid.
文摘The value of ELISA, N-PCR and RT-PCR in clinical practice for pregnant women with HCMV infection was investigated. 5581 pregnant women were screened by ELISA. Among them, 100 cases were positive for IgM (group 1). 69 for both IgM and serous DNA (group 2) and 69 for both IgM and mRNA (group 3). The infectious status, maternal-fetal transmission and pregnancy outcome were monitored. It was demonstrated that the accordance rate of group 3 and group 2 with group 1 was 56. 25 % and 43. 75 % , respectively. The maternal-fetal transmission rate in the group 1, 2 and 3 was 19. 00 % , 40. 58 % and 46. 15 %, respectively, with a significant difference found between group 2, 3 and group 1 (P<0. 01). Incidence of spontaneous abortion, fetal death, fetal abnormality and neonatal death in group 1, 2 and 3 was 10. 00 %, 15. 94 % and 30. 77 %, respectively, and that of group 3, 2 was 4 and 2 times as much as that of group 1, respectively (OR = 4. 00, P<0. 001; OR=2. 343, P<005, respectively). It was concluded that HCMV-IgM(+) can only be considered as an screening indicator for pregnant women with HCMV infection, while IgM(+) combined with serous DNA( + ) or mRNA( + ) indicates active infection and has a high incidence of maternal-fetal transmission and abnormal pregnancy outcome.
基金supported by National Natural Science Foundation of China(No.81272590)
文摘Insulin-like growth factor- I (IGF- I ) is a mitogenic and anti-apoptotic factor. Serum IGF-I concentration is related to some cancer risk and tumor progression. The aim of this research was to study the association of preoperative serum IGF- I concentration with clinicopathological parameters and prognosis of non-small cell lung cancer (NSCLC). Preoperative serum IGF- I concentration was measured in 80 consecutive patients with NSCLC who underwent radical lung cancer resection, and 45 patients with benign pulmonary lesion (BPL) by Using enzyme linked immunosorbent assay (ELISA). The results showed that the serum IGF- I concentration was elevated and correlated with clinicopa- thological parameters and overall survival (OS) in NSCLC patients. Serum IGF- I concentration was significantly higher in patients with NSCLC tfian in those with BPL. The IGF- I concentrations were significantly higher in NSCLC patients with 〉T2, NI-3, and in IIIA-IV but not in those with 〈T2, NO, or I A- IIB. The increased serum IGF- I concentration was significantly correlated with poor prog- nosis. Our data show the positive correlation between IGF-I serum concentration and the tumor size for the first time. It seems that IGF-I related to the progression of lung cancer may depend on autocrine/paracrine function. In addition, our study reveals that higher serum IGF- I concentration is correlated with larger tumor size, advanced stages, local lymph node metastasis and worse prognosis, indicating that endocrine IGF- I is also important for the progression for NSCLC.
文摘[ Objective] To develop an indirect ELISA assay for detecting antibodies against envelope glycoprotein ( E protein) of Japanese encephalitis virus (JEV). [ Method] Specific primers were designed according to JEV sequences published in the GenBank. The cDNA of JEV E gene (about 1 000 10p) was amplified by the RT-PCR with the specific primers. After sequencing analysis, the E gene was cloned into pET30a expression vector and expressed in E. coli BL21 (DE3) with the induction of IPTG. After denaturation, purification and renaturation, the recombinant protein was analyzed by the SDS-PAGE and the westem blotting. An indirect ELISA was developed to detect antibodies against JEV. [ Result] The E protein was mainly expressed in inclusion body. With the purified E protein, the indirect ELISA was developed and displayed good specificity, sensitivity and repeatability, [ Conclusion]The developed ELISA using the truncated E protein as antigen is a simple, convenient and rapid serological method for diagnosis, monitoring antibody level and epidemiological investigation of JEV.
基金funded by the Special Fund for Research and Development of Application Technology of Binzhou City(200706)Youth Science and Technology Innovation Fund of Shandong Binzhou Animal Science & Veterinary Medicine Academy (2007-02)
文摘The purpose of this study was to develop a method for detecting antibodies against porcine pseudorabies virus (PRV). According to the published genomic sequence of PRV SA strain, an approximately 1 070-bp gD gene fragment was amplified by PCR. The PCR products were cloned into the prokaryotic expression vector pET30a and the positive recombinant plasmid was transformed into E. coli BL21. Through induction with IPTG, the recombinant gD protein was expressed as inclusion bodies. As analyzed by western blot assay, the purified recombinant gD protein had good antigenicity and high specificity. Using the purified gD protein as coating antigen and horseradish peroxidase labeled staphylococcal protein A (PPA) as secondary antibody, we developed a PPA-ELISA for detecting antibodies against porcine PPV. No cross-reaction with the positive sera against seven common pathogens in pigs including classical swine fever virus, porcine parvovirus, porcine reproductive and respiratory syndrome, Japanese encephalitis virus, porcine circovirus type 2, porcine epidemic diarrhea virus, transmissible gastroenteritis virus was observed. The repeatability test showed that the intra- and inter-assay coefficients of variation were lower than 5% and 10%, respectively. Compared with the ELISA gD antibody test kit produced by IDEXX, the coincidence, sensitivity and specificity of the developed PPA-ELISA were 92.0%, 95.1% and 88.1%, respectively. The developed PPA-ELISA had good repeatability, sensitivity and specificity and was a rapid and simple serological method for surveillance of PRV antibodies in pig herds as well as for rapid diagnosis and epidemiological investigation of PRV infection.
基金This project is supported by National Major Scientific Research Program of China(No.2011CB933202)National High Technology Research and Development Program of China(No.2009AA03Z411)+1 种基金National Natural Science Foundation of China(No.61002037,61101048)Knowledge Innovation Program of The Chinese Academy of Sciences(CXJJ-10-M31,KGCX2-YW-916).
文摘A new rapid,specific and sensitive method for assay of recombinant CFP10-ESAT6 amalgamation proteins from Mycobacterium tuberculosis was proposed.The method used streptavidincoated magnetic beads to enrich the specific biotinylated anti-CFP10 antibody,then adopted a sandwich-type enzyme linked immunosorbent assay technology with two kinds of monoclonal antibodies:biotinylated anti-CFP10 antibody and HRP-labeled anti-CFP10 antibody to identify the target CFP10-ESAT6 proteins,and finally detected chemiluminescence intensity by a small home-made optical sensor.It was shown that,the corresponding chemiluminescence intensity had a good logarithmic linear response to the concentration of CFP10-ESAT6 proteins when ranging at 1~1000 ng/mL,and the correlation coefficient is 0.9937.The proposed method could detect the CFP10-ESAT6 proteins with low detection limit(1 ng/mL)and the detection time could be controlled within 45 min.Compared with commonly used detection methods of M.tuberculosis,this method was easy to operate,faster,and of higher sensitivity.The achievement of the quantitative detection of CFP10-ESAT6 proteins has important scientific significance and wide application prospects in tuberculosis control.
基金supported by the Key Research Project of the Ministry of Education (105120)Educational Commission of Hubei Province of China (B20091203)
文摘[Objective] To develop a new method for serodiagnosis of swine toxoplasmosis. [Method] With the purified recombinant microneme protein 3 (rMIC3) as coating antigens, an indirect ELISA was developed for detection of antibodies against Toxoplasma gondii. [ Result] The optimal working concentration of rMIC3 was 3. 40 ug/ml, and the optimal degree of dilution of sera was 1:160. Cross-reaction was not observed between the Toxoplasma gondii-positive sera and the positive sera against classical swine fever virus or some other pathogens. The developed ELISA had 92.56% coincidence rate with latex agglutination test. [ Conclusion] The developed ELISA is sensitive, rapid, specific and reproducible, and thus it can be applied in serodiagnosis and seroprevalence investigation of swine toxoplasmosis.
基金This program was supported by the National New Drugs Foundation(No.98-35-N-13)
文摘Objective: To study the therapeutic mechanism of Santeng Dingtong recipe (STDT) on monosodium urate crystal (MSU) induced rabbit arthritis Methods: Forty-two rabbits were randomly divided into six groups, 7 in each group. Group 1 received 0.9% saline 2. 5 ml/kg per day by gastrogavage (ig) for 10 days; Group 2, 3 and 4 received STDT 0.125 g/kg, 1.0 g/kg and 8.0 g/kg per day respectively by ig for 10 days; Group 5 received colchicine 4. 5 mg/kg per day by ig for 4 days; and Group 6 was untreated. MSU crystals 10 mg /500ul containing polymyxin B 10 u/ml was injected into the knee joints of Group 1-5 to make rabbit arthritis models. Leukocytes in synovial lavage fluids was then counted and differentiated; pathological injury of synovial membranes was observed under HE staining; interleukin-1 beta (IL-1B), tumor necrosis factor alpha (TNFa) and leukotriene B4 (LTB4) content in synovial lavage fluids were determined by ELISA. Results: MSU caused a rapid leukocyte infiltration and increased production of IL-1B, TNFa and LTB4 2 hrs after intra-articular injection. STDT inhibited neutrophil infiltration in synovial fluids dose-dependently, protected the synovial membrane against pathological injury and reduced the production of IL-1B, TNFa and LTB4; while colchicine did not decrease the level of TNFa, but significantly inhibited neutrophil infiltration in synovial fluid and reduced the production of IL-11B and LTB4. Conclusion: STDT exerts an anti-inflammatory effect in rabbit model of acute MSU arthritis, its mechanism being probably due to the decrease of XL-1B, TNFa and LTB4 synthesis.
基金Key R&D Program of Hebei Province:Special Project on Key Common Technologies for High-quality Agricultural Development(20327505D).
文摘[Objectives]This study was conducted to develop an enzyme-linked immunoassay kit that can detect the residual amount of pentachloronitrobenzene in Penaeus vannamei.[Methods]This study was conducted to develop an enzyme-linked immunoassay kit that can detect the residual amount of pentachloronitrobenzene in P.vannamei.[Results]The standard curve range of the kit was 0-8.1μg/L;the detection limit for P.vannamei was 0.912μg/kg;the recovery was 80.6%-103.5%;and the relative standard deviation range within batches was 5.3%-10.1%,and the relative standard deviation range between batches was 6.7%-8.1%.The specificity of the pentachloronitrobenzene monoclonal antibody was relatively good,and the cross-reaction rates with pentachlorophenol,hexachlorobenzene,tetrachlorophthalide,and chlorothalonil were low,all of which did not exceed 30%.The ELISA kit could be stored at 4℃for 12 months,showing good stability.[Conclusions]The detection kit has low cost,short time and small deviation,and is an ideal preliminary screening method.
文摘<strong>Objective:</strong> To evaluate three different serological tests [Indirect Hemaglutination (IHA), Enzyme Linked Immunosorbent Assay (ELISA) and Western Blotting (WB)] using native crude antigen for diagnosis of hepatic cystic echinococcosis (HCE) patients. <strong>Materials and Methods:</strong> Sheep hydatid fluid (HF) was collected from fertile cysts obtained from a slaughterhouse and used as an antigen. Forty patients who were attended the Dr. Ersin Arslan Training and Research Hospital in Gaziantep, Turkey, were investigated. Serum samples were obtained from surgically confirmed CE patients. Healthy Turkish people and 16 patients with other helminthic infections were included as a control group. <strong>Results:</strong> Of the 40 analyzed patients, 10 (25%) were men and 30 (75%) were female. The average age was 46.97 years (s.d.;18.95). The majority of the patients had a single cystic lesion situated in one lobe of the liver (usually in the right lobe) (55%), 32.5% of patients had two cystic lesions and 12.5% of patients had multiple cyst formations with various numbers. In all cases, ultrasound (US) examinations were positive and the size of cysts was between 2.1 - 12.7 cm. Twenty-three patients of the total 40 patients were classified according to the WHO classification system based on US findings. According to the results of WB analysis, molecular weights of 8 kDa (80%), 12 kDa (80%), 22 - 24 kDa (97.5%), 26 kDa (97.5%), 34 kDa (100%), 36 - 38 kDa (90%), 45 - 50 - 55 kDa (97.5%), and 60 - 75 kDa (97.5%) bands were identified. But 34, 50, and 55 kDa bands were also found in other helminthic diseases. <strong>Conclusion:</strong> The specificity and sensitivity of three serological tests (IHA, ELISA and WB) using crude antigen were compared by diagnosing hepatic cystic echinococcosis patients. IHA and ELISA showed high sensitivity but low specificity. Western blotting showed low sensitivity but high specificity.
文摘Biosensors are widely used in immunoassay. The biosensor chip carries a receptor which is used in immunoassay and the chip properties have an important influence on the detecting sensitivity of the biosensor. This paper describes a polystyrene\|based biosensor chip developed and used as part of a surface plasmon resonance (SPR) biosensor. The SPR biosensor has a much higher detecting sensitivity than enzyme\|linked immunoserbent assay (ELISA).
基金the financial support from the National Natural Science Foundation of China (Nos. 31900875,81773562, 81973177, and 81703326)Program for Science&Technology Innovation Talents in Universities of Henan Province (No. 21HASTIT045, China)+1 种基金China Postdoctoral Science Foundation (Nos. 2019M662518, 2018M630840, and2019T120641, China)Postdoctoral Starting Foundation of Henan Province (No. 201903007, China)。
文摘The protein tyrosine phosphatase Src homology phosphotyrosyl phosphatase 2(SHP2) is implicated in various cancers, and targeting SHP2 has become a promising therapeutic approach. We herein described a robust cross-validation high-throughput screening protocol that combined the fluorescence-based enzyme assay and the conformation-dependent thermal shift assay for the discovery of SHP2 inhibitors. The established method can effectively exclude the false positive SHP2 inhibitors with fluorescence interference and was also successfully employed to identify new protein tyrosine phosphatase domain of SHP2(SHP2-PTP) and allosteric inhibitors. Of note, this protocol showed potential for identifying SHP2 inhibitors against cancer-associated SHP2 mutation SHP2-E76 A. After initial screening of our in-house compound library(w2300 compounds), we identified 4 new SHP2-PTP inhibitors(0.17%hit rate) and 28 novel allosteric SHP2 inhibitors(1.22% hit rate), of which SYK-85 and WS-635 effectively inhibited SHP2-PTP(SYK-85: IC_(50) Z 0.32 mmol/L;WS-635: IC_(50) Z 4.13 mmol/L) and thus represent novel scaffolds for designing new SHP2-PTP inhibitors. TK-147, an allosteric inhibitor, inhibited SHP2 potently(IC_(50) Z 0.25 mmol/L). In structure, TK-147 could be regarded as a bioisostere of the well characterized SHP2 inhibitor SHP-099, highlighting the essential structural elements for allosteric inhibition of SHP2. The principle underlying the cross-validation protocol is potentially feasible to identify allosteric inhibitors or those inactivating mutants of other proteins.
基金the National Key R&D Program of China(No.2018YF-A0507602)the National Natural Science Foundation of China(Nos.81821004 and21738001)。
文摘A series of novel pyrano[2,3-d]trizaole compounds were synthesized and theirα-glucosidase inhibitory activities were evaluated by in vitro enzyme assay.The experimental data demonstrated that compound 10 f showed up to 10-fold higher inhibition(IC5074.0±1.3μmol·L-1)than acarbose.The molecular docking revealed that compound 10 f could bind toα-glucosidase via the hydrophobic,π-πstacking,and hydrogen bonding interactions.The results may benefit further structural modifications to find new and potentα-glucosidase inhibitors.