Radioimmunoassay of progesterone and estriol in plasma using antibodies immobilized onto protein A-sepharose CL-4B

Protein A-Sepharose CL-4B was used as a solid phase for antibodies in the radioimmunoassay of progesterone and estriol. The method was fast and easily standardizable. Immobilized antibodies had the same binding capacity as free antibodies and gave good correlation curves (r = 0.996 for progesterone andr = 0.989 for estriol). Sensitivity was 12.5 pg/tube for progesterone and 8.0 pg/tube for estriol. Comparison of progesterone radioimmunoassay with chemically immobilized antibody onto Sepharose CL-4B was also carried out.     

Separation of the Hageman factor fragment from human serum albumin by chromatography on blue sepharose CL-6B

The separation of the Hageman factor fragment (HFf) activity from human serum albumin by chromatography on Blue Sepharose CL-6B is described. The complete separation cannot be achieved in a single chromatography step due to complex formation between HFf and albumin.     

An Improved Method for the Preparation of Chlorophyll by Means of Column Chromatography with Sepharose CL-6B

Abstract

An improved method for the preparation of chlorophyll “a” and chlorophyll “b” from fresh spinach leaves by means of the column chromatography with Sepharose CL-6B has been developed. A good separation of the green pigments was attained on the column (φ25 × 163 mm) with a mixed solvent program of 2, 3, 10 and 20 % 2-propanol in hexane.     

胆红素吸附剂功能基组成及其吸附特性研究

为了获得性能较佳的胆红素吸附剂,本研究考察了功能基种类、链长、偶联密度等因素对吸附剂吸附性能的影响。结果表明,当功能基具有足够的链长,与胆红素相似的疏水性及较高的偶联密度时,能够提高吸附剂的胆红素吸附能力。实验表明,链长21个原子、偶联73 mol/mL正丁胺的吸附剂对胆红素的吸附能力最强,在30℃、300mg/L的白蛋白结合胆红素溶液中,吸附1h即达到平衡,胆红素吸附量达到3.01mg/mL(90.3mg/g干重);较高的温度有利于其对胆红素的吸附。50mL材料对500mL实际病人血浆动态吸附2.5h,总胆红素(277.4mg/L)去除率达到55.2%,同时对血浆蛋白的吸附较低。所有结果显示该吸附剂具有很强的实际应用的潜力。     

琼脂糖凝胶CL-6B亲和色谱法一步纯化蚯蚓半乳凝素

环节动物的S型凝集素在结构和生化性质上都有别于一般的S型凝集素,其在抗癌等研究方面的潜在价值重大。根据环节动物S型凝集素的性质,采用惰性分子筛填料Sepharose CL-6B（琼脂糖凝胶CL-6B）作为亲和色谱介质对蚯蚓S型凝集素进行了纯化。以2 mmol/L乙二胺四乙酸（EDTA）-MEPBS (4 mmol/L β-巯基乙醇,150 mmol/L NaCl,20 mmol/L 磷酸盐,pH 7.2)溶液作为平衡液,以氨水(150 mmol/L,pH 10.5)作为洗脱液得到的蛋白质经SDS-PAGE（十二烷基硫酸钠-聚丙烯酰胺凝胶电泳）、凝血实验及荧光检测等证明了其即为蚯蚓S型凝集素。该方法只需一个步骤即可从蚯蚓提取液中分离到纯的S型凝集素,比传统方法更加快捷高效,优越性明显。该方法的应用将有利于对环节动物S型凝集素的深入研究。     

A RAPID AND EFFICIENT METHOD TO PREPARE CHLOROPHYLL A AND B FROM LEAVES

Abstract— Highly purified Chi a and b were prepared from spinach leaves in a short time by a combined use of the column chromatography with DEAE-Sepharose CL-6B and Sepharose CL-6B. The former chromatography eliminated carotenoids, phaeophytin and chlorophyllide, and the latter chromatography efficiently separated Chi a and b .     

Differential interactions of cyclodextrins with hydrophobic derivatives of sepharose CL-4B

The partition coefficients K_av between the solution phase and octyl- and phenyl-sepharose CL-4B were determined for three cyclodextrins (CDs), -CD, -CD and -CD in various aqueous solutions, as a measure of their interactions with the two hydrophobic ligands. K_av of the CDs increased in the order of -CD<-CD<-CD for octyl-sepharose CL-4B and -CD<-CD<-CD for phenyl-sepharose CL-4B. In all cases, K_av increased by increasing NaCl concentration in the aqueous solution phase and also by lowering temperature, but in the presence of NaBr and NaSCN, both chaotropic salts, K_av decreased markedly. The spontaneity of the transfer of the CDs from the aqueous solution phases to the gel phases was due to the enthalpy decrease. It was shown that discrete separation of the three CDs can be achieved by the hydrophobic chromatography on a short column (1×25cm) of octyl-sepharose CL-4B by adjusting the NaCl concentration and temperature.     

Acceleration of decomposition of CL-20 explosive under nanoconfinement

Journal of Thermal Analysis and Calorimetry - The thermal properties of CL-20 explosive in the bulk and confined in controlled pore glass matrices to nanoscale dimensions were studied using dynamic...     

Low-mass ions observed in plasma desorption mass spectrometry of high explosives

The low-mass ions observed in both positive and negative plasma desorption mass spectrometry (PDMS) of the high explosives HMX, RDX, CL-20, NC, PETN and TNT are reported. Possible identities of the most abundant ions are suggested and their presence or absence in the different spectra is related to the properties of the explosives as matrices in PDMS. The detection of abundant NO+ and NO2- ions for HMX, RDX and CL-20, which are efficient matrices, indicates that explosive decomposition takes place in PDMS of these three substances and that a contribution from the corresponding chemical energy release is possible. The observation of abundant C2H4N+ and CH2N+ ions, which have high protonation properties, might also explain the higher protein charge states observed with these matrices. Also, the observation of NO2-, possibly formed by electron scavenging which increases the survival probability of positively charged protein molecular ions, completes the pattern. TNT does not give any of these ions and it is thereby possible to explain why it does not work as a PDMS matrix. For NC and PETN, decomposition does not seem to be as pronounced as for HMX, RDX and CL-20, and also no particularly abundant ions with high protonation properties are observed. The fact that NC works well as a matrix might be related to other properties of this compound, such as its high adsorption ability.     

Evaluation of silica- and sepharose-based immunoaffinity sorbents for sample cleanup in determination of fumonisins B1 and B2 in corn products

Anti-fumonisin B1 polyclonal antibodies were isolated from the serum of rabbits, immobilized onto the surface of glutaraldehyde-activated silica or Sepharose CL-4B particles, and placed into empty small plastic solid-phase extraction cartridges. The immobilized antibodies were evaluated for their ability to retain fumonisin B1 and fumonisin B2. Cartridge capacity and elution conditions were determined, and the results were compared to those obtained with a commercially available cartridge. The cartridges, which were tested for their effectiveness to isolate the fumonisins from extracts of corn flour and nacho chips, detected fumonisins down to levels of about 20 ng/g. However, additional cleanup was required for detection at lower concentrations. With the use of a strong anion-exchange cartridge as a preliminary cleanup before immunoaffinity chromatography, the detection limit reached 2-5 ng/g in the products tested. The silica sorbent material exhibited strong interactions with the fumonisins, requiring acidified ethanol-water mixtures for elution and resulting in an additional degree of selectivity in isolating fumonisins from sample extracts. The silica-based immunoaffinity cartridges were successfully reused more than 10 times; the Sepharose-based cartridges were less robust. Liquid chromatography with fluorescence detection was used after prechromatographic derivatization with o-phthaldialdehyde-mercaptoethanol.     

Comparative study of HMX and CL-20

This study deals with a well-known monocyclic nitramine HMX and a relatively new polycyclic strained-cage nitramine CL-20. Experimental data on the powder morphology, simultaneous thermal analysis (STA) and burning rate of binary formulations Al/HMX and Al/CL-20 are presented. Kinetic modelling for HMX and CL-20 are considered based on analysis of STA data obtained for low heating rates. The processing of STA data by the Kissinger method was shown to need to be supplemented with the construction of a thermokinetic model. The thermal decomposition of HMX is reliably described by the reaction of the first order with the autocatalysis. Obtained kinetic parameters of the HMX thermal decomposition correlate with literature-known data on kinetics of the lead stage of HMX combustion. Two types of aluminium powder, i.e. micron-sized and ultrafine, are used to investigate the interaction with both nitramines. Thermal analysis revealed the higher Al oxidation ability of the solid compounds produced at CL-20 thermolysis, than that one of HMX. Burning rate experiments show the differences in the combustion parameters between CL-20- and HMX-based formulations, specifically along with the burn rate level increase for CL-20 monopropellant as compared to HMX one, the pressure exponent and effect of the aluminium particle size variation are also distinct. Results are analyzed and compared to available literature data.     

Use of immobilized triazine dyes in the purification of DNA topoisomerase I (Topo I) and terminal deoxynucleotidyl transferase (TdT) from calf thymus.

The interaction between TdT and Topo I, and twelve various triazine dyes immobilized on Sepharose CL-6B was studied. Yellow lightproof 2KT-Sepharose and Bordeaux 4ST-Sepharose were used to purify TdT and Topo I, respectively. The principal role of copper ions, complexed to the dye molecules, in the dye-protein interaction was evaluated.     

Analysis of CL-20 in environmental matrices: water and soil

Analytical techniques for the detection of 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazatetracyclo(5.5.0.05,9.03,11)dodecane (CL-20) in water and soil are developed by adapting methods traditionally used for the analysis of nitroaromatics. CL-20 (a new explosives compound) is thermally labile, exhibits high polarity, and has low solubility in water. These constraints make the use of specialized sample handling, preparation, extraction, and analysis necessary. The ability to determine the concentrations of this new explosive compound in environmental matrices is helpful in understanding the environmental fate and effects of CL-20; understanding the physical, chemical, and biological fate of CL-20; and can be used in developing remediation technologies and determining their efficiency. The toxicity and mobility of new explosives in soil and groundwater are also of interest, and analytical techniques for quantitating CL-20 and its degradation products in soil and natural waters make these investigations possible.     

New ligand, N-(2-pyridylmethyl)aminoacetate, for use in the immobilised metal ion affinity chromatographic separation of proteins.

A new chelating compound has been developed for use in the immobilised metal ion affinity chromatographic separation of proteins. The tridentate ligand, sodium N-(2-pyridylmethyl)aminoacetate (carbpyr), 1, was prepared via a one-step synthesis from 2-picolylamine, 3 and then immobilised onto Sepharose CL-4B through the epoxide coupling procedure. The binding behaviour of the resulting IMAC sorbent, following chelation with Cu2+ ions to a density of 152 micromol Cu2+ ions/g gel was characterised by frontal analysis experiments using horse heart myoglobin (HMYO) at pH 7.0 and pH 9.0. From the derived isotherms, the adsorption capacity, q(m), for the binding of HMYO to immobilised Cu2+-N-(2-pyridylmethyl)aminoacetate (im-Cu2+-carbpyr)-Sepharose CL-4B at these pH values was found to be 1.92 and 1.91 micromol/g sorbent, respectively, whilst the dissociation constants K(D) were 0.0092 x 10(-6) M and 0.0062 x 10(-6) M at pH 7.0 and pH 9.0, respectively, indicating that the HMYO-im-Cu2+-N-(2-pyridylmethyl)aminoacetate complex was more stable under alkaline conditions, although the binding capacity in terms of micromol protein/g gel remained essentially unchanged. The selectivity features of the im-Cu2+-carbpyr-Sepharose CL-4B sorbent were further characterised in terms of the binding properties with several human serum proteins at pH 5.0, pH 7.0 and pH 9.0.     

High-performance size-exclusion chromatography of porcine colonic mucins. Comparison of Bio-Gel TSK 40XL and Sepharose 4B columns

A high-performance size-exclusion chromatography (HPSEC) method was developed for the separation of porcine colonic mucins using a Bio-Gel TSK 40XL HPSEC column (300 mm x 75 mm). In addition, porcine gastric and bovine submaxillary mucin preparations were used to describe more fully the separation characteristics of the HPSEC column. For comparison, the same preparations were also separated using a Sepharose 4B column (100 cm x 2.6 cm). The colonic and gastric mucins eluted in the void volume (V0) of both columns. Bovine submaxillary mucin was in the elution volume (Ve) of both columns. Analytical HPSEC of fractions (V0 and Ve) of the various preparations obtained by Sepharose 4B chromatography exhibited retention times identical to those for fractions obtained by HPSEC. After separation by both methods, purified mucins were obtained by CsCl2 density gradient ultracentrifugation; analytical HPSEC profiles, protein contents, and monosaccharide compositions of both gastric and colonic mucins from either column were similar. The HPSEC method, however, is ideally suited to separate microgram to milligram quantities of colonic mucin preparations quickly: 2 to 4 h, compared with 24 to 30 h for the Sepharose 4B method.     

The effect of crystal structure on the thermal reactivity of CL-20 and its C4-bonded explosives

The critical temperature and mechanism functions for thermal decomposition of ε-CL-20, RS-ε-CL-20, α-CL-20, ε-CL-20/C4, and RS-ε-CL-20/C4 were evaluated based on non-isothermal TG data. A two-step mechanism has been found for thermal decomposition of α-CL-20, ε-CL-20/C4, and RS-ε-CL-20/C4, where the initial step is partly controlled by crystal structure of CL-20. The more reasonable mean activation energies could be obtained after peak separation for each individual steps. In fact, the activation energy for the post integrated process is almost equivalent with that of the second step, indicating that the total activation energy at the main decomposition process is dominated by thermolysis of CL-20 molecular. Besides, it has been found that the decomposition of C4 matrix does not affect the decomposition of normal ε-CL-20, resulting in identical activation energy and reaction model. However, the interaction between the C4 matrix and RS-ε-CL-20 is significant especially at the initial stage, where the activation energy of RS-ε-CL-20/C4 was overestimated before peak separation, while the activation energy for the second step due to thermolysis of CL-20 molecular is underestimated. The first decomposition step for α-CL-20, ε-CL-20/C4, and RS-ε-CL-20/C4 could be considered as autocatalytic process (AC model), whereas the second as JMA model, which is also applicable to that of pure ε-CL-20 and RS-ε-CL-20. Moreover, The critical temperatures of thermal explosion (T _b) are obtained as 205.6, 205.5, 209.4, 214.4, and 227.5 °C for α-CL-20, ε-CL-20, RS-ε-CL-20, ε-CL-20/C4, and RS-ε-CL-20/C4, respectively. It proves that the C4 matrix could stabilize ε-CL-20 while the crystal form of CL-20 has little effect on its thermal stability.     

Compared stability of Sepharose-based immunoadsorbents prepared by various activation methods.

During the use of chromatographic supports for the purification of proteins or the selective removal of substances by immunoaffinity, leakage of the antibodies immobilized on the matrix is systematically observed. When the cleansing of blood plasma by extracorporeal circulation is concerned, it is of prime importance that the immunoadsorbents exhibit an extensive chemical stability over the whole range of experimental conditions. To study and minimize this leakage, a matrix, Sepharose CL-4B, was activated by various chemical reagents and coupled to goat anti-apolipoprotein B polyclonal antibodies. Immunoadsorbents thus prepared were compared with those obtained earlier by cyanogen bromide activation. It turns out that divinyl sulphone- and tresyl chloride-activated supports lead to similar results in terms of coupling yield and adsorption capacity, but to a significant reduction in released antibodies.     

三维氧化铁/石墨烯的构建及其对CL-20的热分解性能的影响

High-performance solid propellants are very important for the development of modern weapons. Aside from their high energy and high burning rate, safety performance is regarded as the most important factor that should be considered whenever a new solid propellant recipe is formulated. Therefore, exploring a new type of combustion catalyst that can improve both catalytic activity and reduce the sensitivity of the energetic component is significant. Traditionally, transition metals or metal oxides are used as a combustion catalyst for accelerating the thermal decomposition of energetic components. However, the existing problem of these catalysts is the aggregation of particles accompanied by poor surface area. Coupling metal oxides with graphene is a promising approach to obtain a binary composite with stable structure and large specific surface area. In this work, rod-like and granular Fe₂O₃ nanoparticles were synthesized using a hydrothermal method. Then, the two as-prepared Fe₂O₃ nanoparticles were coupled with graphene sheets using an interfacial self-assembly method, which can effectively prevent the aggregation of Fe₂O₃ particles and simultaneously increase the active sites that participate in the reaction. X-ray diffraction and X-ray photoelectron spectroscopy were used to identify the phase states and chemical compositions of the prepared samples. The morphology and internal structures were further demonstrated through scanning electron microscopy, transmission electron microscopy and nitrogen adsorption-desorption tests. Both phase analysis and structure identification indicate that the prepared Fe₂O₃/G has high purity and high surface area. The catalytic performance of the prepared Fe₂O₃ and Fe₂O₃/G in the thermal decomposition of hexanitrohexaazaisowurtzitane (CL-20) was evaluated based on thermal gravimetric analysis-infrared spectroscopy (TGA-IR) and differential scanning calorimetry (DSC) tests. The non-isothermal decomposition kinetics of CL-20, Fe₂O₃/CL-20, and Fe₂O₃/G/CL-20 were further studied by DSC. The results reveal the excellent catalytic activity of Fe₂O₃/G in the thermal decomposition of CL-20, which is attributed to the presence of abundant pore structure and large surface area. The reaction mechanisms of the exothermic decomposition process of CL-20, Fe₂O₃/CL-20, and Fe₂O₃/G/CL-20 were obtained by the logical choice method, and the composites all followed same mechanism function model as CL-20. Through comparison, the rod-like Fe₂O₃ coupled with graphene was found to have the best catalytic activity in the thermal decomposition of CL-20. Thus, the rod-like Fe₂O₃ and its Fe₂O₃/G composite were used to investigate their influence on the impact sensitivity of CL-20 by fall hammer apparatus. The results show that rFe₂O₃/G can effectively decrease the impact sensitivity of CL-20 compared with pure CL-20 and rFe₂O₃/CL-20. Therefore, rFe₂O₃ coupled with graphene not only promotes the thermal decomposition but also improves the safety performance of CL-20.