High sensitive ELISA for detection of atrazion / 中国应用生理学杂志

OBJECTIVES@#To set up ELISA for detection of atrazine with high precision.@*METHODS@#The reaction condition of indirect-ELISA was optimized, including atrazine-ovalbumin(AT-OVA) concentration and primary antibody concentration, organic solvent, goat anti-rat immunoglobin G-horseradish peroxidase(IgG-HRP) concentration. The actual samples were detected by the ELISA method established in our laboratory. Then the ELISA method was compared with the HPLC.@*RESULTS@#The specification curve of indirect-ELISA was set up after optimization. The relation coefficient R=0.9958. The limit of detection (LOD) was 1.972 ng/ml. The percent recovery of the actual samples was in range of 80%~120%. The ELISA detection sensitivity was higher than the HPLC in the range of 0 ng/ml~6 ng/ml atrazine.@*CONCLUSIONS@#The ELISA to detect atrazine has good specificity and high precision. And it can be applied in testing real atrazine samples replacing of the large-scale instrument.

Efficient Detection of Environmental Estrogens Bisphenol A and Estradiol By Sensing System Based on AuNP-AuNP-UCNP Triple Structure / 分析化学

A sensing system based on AuNP-AuNP-UCNP triple structure for efficient detection of dual targets was constructed. In the preparation of triple structure, the gold nanoparticles (AuNPs) and upconversion nanoparticles (NaYF4: Yb, Er, Gd, UCNPs) were synthesized and surface modified. Then the two nanoparticles and their aptamers were connected to form two kinds of optical fluorescent probes. A nucleic acid sequence that matches with two aptamers was designed, rendering the probes to get close based on the principle of complementary base pairing. On the basis of this, a sensing system with a triple structure was prepared,and its connecting effect was characterized by TEM. With this system, dual targets of bisphenol A and estradiol were efficiently and conveniently detected through quantitative determination by fluorescence and UV spectrophotometer. At reaction temperature of 30℃ and pH=7.8,this method exhibited good linear range for determination of bisphenol A and estradiol from 2 ng/mL to 200 ng/mL and from 10 ng/mL to 150 ng/mL, with limits of detection of 0.2 ng/mL and 0.5 ng/mL, respectively. This sensing system with the triple structure owned better specificity to structural and functional analogues, and showed good repeatability and stability. What's more,this sensing system was applied in actual water detection,with the recoveries between 86.1% and 107. 4%, and the relative standard deviation below 6. 8%. This method showed promising applications in other environmental estrogens in water samples.

Distribution Characteristics of Spermophilus dauricus in Manchuria City in China in 2015 through '3S' Technology / 生物医学与环境科学（英文）

Plague is a virulent infectious disease in China. In this study, '3S' technology was used to perform spatial autocorrelation analysis and spatial interpolation analysis for Spermophilus dauricus (S. Dauricus, a species of ground squirrel) captured in Manchuria City in 2015. The results were visually inspected. During the two-month (May to July) plague surveillance in 2015, 198 S. dauricus individuals were captured in the study area in Manchuria City (48 monitoring areas) by using a day-by-day catching method. Spatial autocorrelation was conducted using the ArcGIS software, and the following significantly different results were obtained: Moran's I=0.228472, Z-score=2.889126, and P<0.05. Thus, a spatial aggregation was observed. In 2015, the distribution of S. dauricus diminished from west to east and from north to south of Manchuria. Geo Detector software was used to analyze the habitat factors affecting the spatial distribution of S. dauricus. This highly clustered species mainly exists in suburban communities, construction sites, and areas surrounding factories. In future studies, plague surveillances should be performed in areas around Manchuria and Zhalainuoer.

Purification of monoclonal antibody to clenbuterol and its biology identity / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To identify the self-preparation monoclonal antibody which target to clenbuterol, and set up the standard curve to clenbuterol (CL) detection.</p><p><b>METHODS</b>The affinity constants and activity of the monoclonal antibody which target to CL were determined by ELISA. ELISA was also used to confirm whether the monoclonal antibody had any across-reaction with BSA and CL analogues. The rat ascites which contains the monoclonal antibody target to CL was purified by (NH4)2SO4 salt-out method and further by affinity column. At last, the CL detection standard curve which based on indirect competition ELISA was established.</p><p><b>RESULTS</b>The ELISA experiment showed that the antibody titer was 10(6) and the monoclonal antibody affinity constants was 2.90 x 10(10) L/mol. The result of the indirect competition ELISA confirmed that the monoclonal antibody had no cross-reaction with BSA and a few kind of CL analogue. CL detection standard curve based on indirect competition ELISA was established, which R2 was 0.9812, and the lowest detectable limit was 1.0 ng/ml.</p><p><b>CONCLUSION</b>The standard curve based on indirectly competitioning ELISA was established. The self-preparation monoclonal antibody which target to CL has high affinity and high specific to CL, which had established the foundation to the advanced development of the CL immune test paper and CL ELISA kit.</p>

Preparation of clenbuterol monoclonal antibody with subtractive immunization method / 中国应用生理学杂志

<p><b>AIM</b>To obtain Clenbuterol monoclonal antibodies.</p><p><b>METHODS</b>Clenbuterol complete antigen was prepared with diazotization method. BALB/c mice was immunized with subtractive immunization, Clenbuterol monoclonal antibody was prepared with rule hybridoma technique.</p><p><b>RESULTS</b>The mice obtained tolerance to BSA by subtractive immunization. The rate of the hybridoma cell with positive reaction which had obtained was 8.2%, and the specific clenbuterol monoclonal antibody was obtained at last.</p><p><b>CONCLUSION</b>Monoclonal antibodies to micromolecule contaminant be prepared by subtractive immunization, could decrease the workload in the bolting of monoclonal antibodies, and increase the chance to obtain the antibody of expected.</p>

Preparation and activity analysis of RGD-mSAK (K130T, K135R) / 生物工程学报

In order to construct RGD-mSAK mutant with reduced immunogenicity, and identify its biological activity after purification, mSAK gene fragment was amplified by over-lapping extension PCR. Then the gene was inserted into the prokaryotic expression vector pBV220 with P(R)P(L) promoters after confirmed by DNA sequencing; the expression plasmid pBV220-RGD-mSAK was constructed, and then was transformed into E. coli. DH5alpha. After temperature induction, the mutant Staphylokinase was over-expressed and much of protein was in the supernate of lysate, which is over 50% of total protein in the host. The protein was isolated and purified in Q-Sepharose FF, Sephacryl S-200 and SP, high purity protein was obtained and its purity was over 98%. The thrombolysis activity of the RGD-mSAK protein is 1.68 x 10(5) u/mg by fibrin plate assay, which is slightly higher than that of the wild-type, and antiserum titers raised against this protein in guinea pigs were much lower than those of wild-type SAK, determined by ELISA. In anti-platelets aggregation assay in vitro, the RGD-mSAK protein has obvious inhibition activity of platelet aggregation in low concentration comparing to the control group and wild-type SAK group. So the RGD-mSAK protein is a low immunogenicity, bi-function molecular with both thrombolysis activity and anti-embolism activity. It provided the basis for further research of RGD-SAK.