Botryoid-shaped nanoparticles-enhanced ELISA for ochratoxin A

Botryoid-shaped Au/Ag nanoparticles (BSNPs) were prepared with a tailored galvanic reaction. Ag nanoprisms–BSA complex were employed as a template to react with HAuCl₄ in the presence of ascorbic acid. The BSNPs–HRP–IgG about 40?nm were used as the carriers of HRP–IgG for amplifying the detection signal of an indirect competitive ELISA against ochratoxin A. This BSNPs-enhanced ELISA method achieved an IC₂₀ of 0.016?ng/mL and an IC₅₀ of 0.05?ng/mL, which were 30 times more sensitive than conventional ELISA. The application of BSNPs to the enhanced ELISA showed acceptable reproducibility, stability and could be applied to determine other harmful small molecules in food.     

Expression of recombinant parvalbumin from wolf-herring fish and determination of its IgE-binding capability

In this study, we produced the recombinant form of parvalbumin from wolf-herring fish and determined its IgE reactivity. Parvalbumin cDNA was sub-cloned into pET28 and expressed in Escherichia coli BL-21. The immunoreactivities of the recombinant and native parvalbumins were compared, and the effect of calcium binding was determined by sera from 25 fish-allergic patients. ELISA and Western blotting confirmed similar IgE-reactivities of the recombinant and native proteins and confirmed that this phenomenon is highly dependent on calcium binding. The recombinant protein was 94.5% similar to carp parvalbumin (Cyp c1). Approximately 72% of patients reacted strongly with recombinant parvalbumin, 80% of them reacted with the native form and only 56% showed IgE reactivity with crude extract. Because the IgE-binding capacity of recombinant wolf-herring parvalbumin is retained and is highly similar to Cyp c1, the wild and hypoallergenic forms of this allergen could be used for diagnosis and immunotherapy of fish allergy, respectively.     

神经生长因子在食管鳞癌细胞中的表达及分泌

目的 探讨神经生长因子（NGF)与食管鳞癌细胞分化的相关性。方法 采用有限稀释法分选出圆形和梭形单细胞克隆,采用无血清悬浮培养获得细胞球细胞, 贴壁的Eca109细胞分别在含血清和不含血清培养基中培养48h;分别采用反转录-聚合酶链式反应（RT-PCR)技术和免疫印迹法,检测NGF在食管鳞癌细胞中mRNA水平和蛋白水平的表达;免疫荧光技术检测NGF在食管鳞癌细胞中的表达定位;免疫组织化学法检测食管鳞癌组织中NGF的表达定位;用酶联免疫吸附法（ELISA）检测NGF在Eca109细胞培养基中的分泌情况。结果 在Eca109细胞中能检测到NGF的mRNA水平和蛋白水平的表达,其中NGF在细胞球细胞中的mRNA水平和蛋白水平的表达量最高。食管癌组织中检测到NGF表达于细胞质。Eca109细胞在无血清培养条件下能够分泌NGF,且明显高于含血清培养基中的含量。结论 食管癌细胞系Eca109表达并分泌NGF,并且食管癌组织中表达NGF,NGF可能在维持食管鳞状细胞癌的干细胞特性发挥了重要作用。     

Inhibition of adhesion of Clostridium difficile to human intestinal cells after treatment with serum and intestinal fluid isolated from mice immunized with nontoxigenic C. difficile membrane fraction

Diarrhea and pseudomembrane colitis caused by Clostridium difficile infection is a global health concern because of the high recurrence rate after standard antibiotic therapy. Vaccination presents a powerful countermeasure against disease recurrence. In this study, mice vaccinated with the nontoxigenic C. difficile membrane fraction generated a marked immune response to the antigen, as demonstrated by the serum IgG and intestinal fluid IgA levels. Significantly, pretreatment with harvested IgG- and IgA-containing fluids was sufficient to prevent in vitro adhesion of C. difficile to human Caco-2 intestinal cells. These results highlight the potential of nontoxigenic C. difficile membrane fraction as a vaccine candidate for C. difficile infection.     

脑外伤合并骨折患者骨折愈合过程中TGF-β1的作用

目的研究脑外伤合并骨折患者血清及骨痂中转化生长因子-β1(TGF-β1)表达变化。方法将57例骨折分为两组,单纯骨折组31例和脑外伤合并骨折组26例:①在伤后1、2、3、4周时取空腹静脉血,采用ELISA方法进行血清中TGF-β1含量的测定;②根据手术距受伤时间分为3~7 d、8~14 d、15~21 d 3个时间段,于术中取骨痂,采用免疫组化染色,进行TGF-β1阳性细胞表达率测定;③在伤后2、4、8周行X线摄片。结果①ELISA:脑外伤合并骨折组血清中TGF-β1的含量在1~3周时均显著高于单纯骨折组,4周时两组比较差异无统计学意义;②免疫组织化学染色:脑外伤合并骨折组在各时间段TGF-β1表达阳性细胞率均显著高于单纯骨折组;③X线观察:脑外伤合并骨折组较单纯骨折组生成骨痂加速、量多、骨折线模糊。结论脑外伤可能主要是通过促进体液因素中TGF-β1等生长因子的表达来加速骨折愈合过程的。     

Cytomegalovirus Localization in Atherosclerotic Plaques is Associated with Acute Coronary Syndromes: Report of 105 Patients

It has been shown that cytomegalovirus (CMV) is present in coronary atherosclerotic plaques, but the clinical rele-vance of this presence remains to be elucidated. In this study we sought to examine CMV infection in atherosclerosis patients defined by different methods and to identify the clinical significance of CMV replication in the atherosclerotic plaques. The study included 105 consecutive patients who were admitted to our department and underwent coronary artery bypass grafting (CABG) surgical interventions. Coronary atherosclerotic specimens as well as 53 specimens from the mamillary artery of these same patients were analyzed. Enzyme-linked immunosorbent assay (ELISA) and poly-merase chain reaction (PCR) methods were used for evaluations. The CMV PCR test result was positive for 28 (26.7%) of patients with coronary artery atherosclerosis. After adjusting for other risk factors, coronary artery disease patients with a history of acute coronary syndrome were more likely to be positive for CMV PCR test (P=0.027; odds ratio: 4.2; 95% CI: 1.18-15.0). They were also more likely to have a positive family history for cardiovascular diseases (CVD). This study confirms previous evidence about the replication of CMV virus in the atherosclerotic plaques of coronary arteries and brings clinical significance to this observation by showing a higher prevalence of acute coronary syndromes in those patients with CMV-infected plaques. Our study also suggests a familial vulnerability to CMV replication in the coronary artery walls.     

A new simple approach for the determination of pyrimidine 5'-nucleotidase activity in human erythrocytes using an ELISA reader

Introduction: Pyrimidine 5′ nucleotidase type I (P5′N‐1) deficiency is the most frequent abnormality of cell nucleotide metabolism causing hereditary non spherocytic hemolytic anemia (HNSHA). The aim of this study was to develop a simple method of determination of P5′N‐1 activity in human erythrocytes using an ELISA reader Methods: Determination of P5′N‐1 activity is based on the liberation of inorganic phosphorus (Pi) after incubation with uridine monophosphate/cytidine monophosphate. Inorganic phosphorus (Pi), a product of the enzymatic reaction is directly quantitated from its ultraviolet absorbance. Purine/Pyrimidine nucleotides ratio (OD 260: OD 280) was also measured Results: P5′N‐1 deficient patients showed reduction in P5′N‐1 activity (Mean ± SD; 4.06 ± 0.66 using an ELISA reader & 6.25 ± 1.37 using a spectrophotometer) as compared to the normal control group (ELISA reader: 13.24 ± 3.42 & Spectrophotometer: 18.25 ± 3.20). Heterozygotes showed intermediate activity (ELISA reader: 6.06 ± 0.48 & Spectrophotometer: 8.06 ± 1.28), however they would have been missed on screening using the Purine/Pyrimidine nucleotides ratio Conclusion: Determination of P5′N‐1 activity by using an ELISA reader is a new, simple, less time consuming and reliable method. It also avoids the use of radioactive material or HPLC which is a significant advantage.