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61.
Debbie van der Burg Hermann Wätzig Cari E. Sänger-van de Griend 《Electrophoresis》2023,44(1-2):96-106
Monoclonal antibodies (mAbs) have become an important class of biopharmaceuticals used for the treatment of various diseases. Their quantification during the manufacturing process is important. In this work, a capillary zone electrophoresis (CZE) method was developed for the monitoring of the mAb concentration during cell-culture processes. CZE method development rules are outlined, particularly discussing various capillary coatings, such as a neutral covalent polyvinyl alcohol coating, a dynamic successive multiple ionic-polymer coating, and dynamic coatings using background electrolyte additives such as triethanolamine (T-EthA) and triethylamine. The dynamic T-EthA coating resulted in most stable electro-osmotic flows and most efficient peak shapes. The method is validated over the range 0.1–10 mg/ml, with a linear range of 0.08–1.3 mg/ml and an extended range of 1–10 mg/ml by diluting samples in the latter concentration range 10-fold in water. The intraday precision and accuracy were 2%–12% and 88%–107%, respectively, and inter-day precision and accuracy were 4%–9% and 93%–104%, respectively. The precision and accuracy of the lowest concentration level (0.08 mg/ml) were slightly worse and still well in scope for monitoring purposes. The presented method proved applicable for analysing in-process cell-culture samples from different cell-culture processes and is possibly well suited as platform method. 相似文献
62.
LI Wan-nan ZHUANG Yan LI He SUN Ying FU Yao WU Xiao-xia ZHAO Zhi-zhuang FU Xue-qi . Edmond H. Fischer Signal Transduction Laboratory College of Life Sciences . Key Laboratory for Molecular Enzymology & Engineering Ministry of Education Jilin University Changchun P. R. China 《高等学校化学研究》2008,24(5):592-596
This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1(designated ΔSHP-1) and the preparation of its polyclonal antibodies. A cDNA fragment encoding ΔSHP-1 was amplified by PCR and then cloned into the pT7 expression vector. The recombinant pT7-ΔSHP-1 plasmid was used to transform Rosetta(DE3) E. coli cells. ΔSHP-1 was distributed in the exclusion body of E. coli cell extracts and was purified through a two-column chromatographic procedure. The purified enzyme exhibited an expected molecular weight on SDS-gels and HPLC gel filtration columns. It possesses robust tyrosine phosphatase activity and shows typical enzymatic characteristics of classic tyrosine phosphatases. To generate polyclonal anti-ΔSHP-1 antibodies, purified recombinant ΔSHP-1 was used to immunize a rabbit. The resultant anti-serum was subjected to purification on ΔSHP-1 antigen affinity chromatography. The purified polyclonal antibody displayed a high sensitivity and specificity toward ΔSHP-1. This study thus provides the essential materials for further investigating the biological function and pathological implication of SHP-1 and screening the inhibitors and activators of the enzyme for therapeutic drug development. 相似文献
63.
Martí S Andrés J Moliner V Silla E Tuñón I Bertrán J 《Chemistry (Weinheim an der Bergstrasse, Germany)》2008,14(2):596-602
The Diels-Alder reaction is one of the most important and versatile transformations available to organic chemists for the construction of complex natural products, therapeutics agents, and synthetic materials. Given the lack of efficient enzymes capable of catalyzing this kind of reaction, it is of interest to ask whether a biological catalyst could be designed from an antibody-combining site. In the present work, a theoretical study of the different behavior of a germline catalytic antibody (CA) and its matured form, 39 A-11, that catalyze a Diels-Alder reaction has been carried out. A free-energy perturbation technique based on a hybrid quantum-mechanics/molecular-mechanics scheme, together with internal energy minimizations, has allowed free-energy profiles to be obtained for both CAs. The profiles show a smaller barrier for the matured form, which is in agreement with the experimental observation. Free-energy profiles were obtained with this methodology, thereby avoiding the much more demanding two-dimensional calculations of the energy surfaces that are normally required to study this kind of reaction. Structural analysis and energy evaluations of substrate-protein interactions have been performed from averaged structures, which allows understanding of how the single mutations carried out during the maturation process can be responsible for the observed fourfold enhancement of the catalytic rate constant. The conclusion is that the mutation effect in this studied germline CA produces a complex indirect effect through coupled movements of the backbone of the protein and the substrate. 相似文献
64.
Wilner OI Guidotti C Wieckowska A Gill R Willner I 《Chemistry (Weinheim an der Bergstrasse, Germany)》2008,14(26):7774-7781
Three different methods to investigate the activity of a protein kinase (casein kinase, CK2) are described. The phosphorylation of the sequence-specific peptide (1) by CK2 was monitored by electrochemical impedance spectroscopy (EIS). Phosphorylation of the peptide monolayer assembled on a Au electrode yields a negatively charged surface that electrostatically repels the negatively charged redox label [Fe(CN)6]3-/4-, thus increasing the interfacial electron-transfer resistance. The phosphorylation process by CK2 is further amplified by the association of the anti-phosphorylated peptide antibody to the monolayer. Binding of the antibody insulates the electrode surface, thus increasing the interfacial electron-transfer resistance in the presence of the redox label. This method enabled the quantitative analysis of the concentration of CK2 with a detection limit of ten units. The second method employed involved contact-angle measurements. Although the peptide 1-functionalized electrode revealed a contact angle of 67.5 degrees , phosphorylation of the peptide yielded a surface with enhanced hydrophilicity, 36.8 degrees. The biocatalyzed cleavage of the phosphate units with alkaline phosphatase regenerates the hydrophobic peptide monolayer, contact angle 55.3 degrees . The third method to characterize the CK2 system involved chemical force measurements between the phosphorylated peptide monolayer associated with the Au surface and a Au tip functionalized with the anti-phosphorylated peptide antibody. Although no significant rupture forces existed between the modified tip and the 1-functionalized surface (6+/-2 pN), significant rupture forces (multiples of 120+/-20 pN) were observed between the phosphorylated monolayer-modified surface and the antibody-functionalized tip. This rupture force is attributed to the dissociation of a simple binding event between the phosphorylated peptide and the fluorescent antibody (Fab) binding region. 相似文献
65.
Giménez E de Bolós C Belalcazar V Andreu D Borrás E De la Torre BG Barbosa J Segura J Pascual JA 《Analytical and bioanalytical chemistry》2007,388(7):1531-1538
Erythropoietin (EPO) is a hormone that regulates red blood cell production. Recombinant human EPO (rHuEPO) and NESP (novel
erythropoiesis stimulating protein) have been produced for therapeutic purposes and also to improve sports performance. The
primary sequences of rHuEPO and NESP differ by just five amino acids. Due to the high homology, no antibodies that are able
to discriminate between both molecules have been obtained until now. The aim of the present work was to design synthetic peptides
corresponding to the sequence that differs between EPO and NESP (87–90aa), that can then be used as immunogens to develop
specific rabbit polyclonal antibodies for selectively detecting EPO and NESP. Three peptides were synthesized: EPO (81–95),
NESP (81–95), and NESP (86–104), and these were coupled to KLH and OVA for immunization and screening purposes, respectively.
The sera obtained were tested by ELISA on synthetic peptide–OVA conjugates and purified by immunoaffinity chromatography against
the corresponding synthetic peptide. The specific purified antibodies were characterized by ELISA, SDS-PAGE, and isoelectric
focusing, followed by western blot. Antisera raised against EPO (81–95) recognized rHuEPO but not NESP. In contrast, anti-NESP
(84–106) sera gave a specific anti-NESP response only after immunoaffinity purification on a NESP (86–91) column. An efficient
strategy for generating specific antibodies against EPO and NESP can be achieved by selecting suitable synthetic peptides.
The antibodies obtained are able to differentiate between rHuEPO and NESP, and may be particularly useful for screening purposes
in both therapeutic and antidoping contexts. 相似文献
66.
A fiberoptic evanescent-wave sensor has been developed for the measurement of antinuclear antibodies in sera from patients
and healthy individuals. The sensor was constructed on the basis of modification of the unclad portion of an optical fiber
with self-assembled gold colloids, where the colloidal gold surface was further functionalized with extractable nuclear antigens.
Results show that detection of antinuclear antibodies by this sensor agrees quantitatively with the clinically accepted enzyme-linked
immunosorbent assay (ELISA) method. This sensing platform has the following advantages: label-free and real-time detection
capability, simple to construct and use, highly sensitive, and does not require a secondary antibody. The sensitivity of this
platform is at least an order of magnitude higher than that of the ELISA method and thus may lead to a new direction in recognition
of immune response.
Biomolecular binding of antinuclear antibodies (ANA) with extractable nuclear antigens (ENA)-functionalized gold nanoparticles
results in a change of surface plasmon absorption. When light propagates in an optical fiber by multiple total internal reflection,
such a change in signal can be significantly enhanced. 相似文献
67.
Pastor-Navarro N Gallego-Iglesias E Maquieira A Puchades R 《Analytica chimica acta》2007,583(2):377-383
Sulfasalazine is an antibiotic used in the treatment of inflammatory bowel diseases. For the assessment of sulfasalazine in several biological matrices, an Enzyme-Linked Immunosorbent Assay (ELISA) method based on polyclonal antibodies was developed and characterized.The immunoassay showed a high sensitivity (IC50 = 0.51 ng mL−1) and specificity, a detection limit of 0.02 ng mL−1 and a dynamic range of 0.06-3.75 ng mL−1 (80-20% inhibition). The immunoassay performed well when it was applied to spiked plasma samples (from 0.5 to 2.0 ng mL−1) previously cleaned up by protein precipitation with methanol. Recoveries ranged from 83 to 119%, with a mean value of 99% (CV = 13%).Since sulfasalazine remaining of a treatment reaches the systemic circulation in unchanged form, the immunoassay can be applied to the determination of this pharmaceutical in human plasma in order to facilitate the control of the patients through the application of personal doses. 相似文献
68.
艾滋病(AIDS)病毒抗体检测方法研究进展 总被引:1,自引:1,他引:0
对艾滋病(A IDS) 病毒抗体检测方法的研究进展作了较系统的评述。从抗原试剂的构成及样品的来源等方面对各种检测方法进行归纳分类。论述了病毒抗体检测方法研究发展的三个阶段, 分析比较了各种艾滋病毒抗体检测方法的优缺点。给出125 篇参考文献。 相似文献
69.
A one-step immunochromatographic assay (ICA) was developed for the detection of seven kinds of cephems in milk. Polyclonal antibodies (PcAb) with group-specific to cephems were raised in rabbits after immunization with cephalexin-keyhole limpet hemocyanin (KLH) conjugate. The specificity of anti-sera was determined by indirect competitive enzyme-linked immunosorbent assay (icELISA), and the 50% inhibitions (IC50) of cephalexin and cefadroxil were obtained at 1.5 ng mL−1; IC50 of cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were 4, 3.7, 3.2, 4.5 and 5 ng mL−1, respectively. The PcAb against cephems were conjugated to colloidal gold particles as the detection reagent for ICA strips to test for cephems. This method achieved semi-quantitative detection of cephems in <5 min, with high sensitivity to cephalexin and cefadroxil (both 0.5 ng mL−1). At the same time, cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were detected at <100 ng mL−1 in spiked processed-milk samples. This method was compared with an enzyme-linked immunosorbent assay by testing 40 milk samples, and the positive samples were validated by a high-performance liquid chromatographic method, with an agreement rate of 100% for both comparisons. In conclusion, the method was rapid and accurate for the multi-residue detection of cephems in milk. 相似文献
70.
In the biopharmaceutical industry, column chromatography residuals are routinely assessed by the direct measurement of mock eluates. In this study, we evaluated virus and other impurity carryover between protein A cycles and the feasibility of using a total organic carbon (TOC) analyzer to monitor for column impurity leakage as a correlate for actual measured carryover in mock eluates. Commercial process intermediates were used in scaled down studies of two protein A media, ProSep A (Millipore, Bedford, MA, USA) and MabSelect SuRe (GE Healthcare, Uppsala, Sweden). The chromatography system was programmed to run up to 200 normal load/elution cycles with periodic blank cycles to measure protein and phage carryover, and water flush cycles to measure TOC release. Sustained phage carryover was evident in each study. Carryover and TOC release was lowest in the case where cleaning was most stringent (50 mM NaOH/0.5 M Na2SO4 with MabSelect SuRe). The TOC analysis at this time does not appear to be a viable practical means of measuring impurity carryover; direct measurements in mock eluates appears to be more predictive of column performance. 相似文献