Comprehensive assessment of N-glycans derived from a murine monoclonal antibody: a case for multimethodological approach

Highly efficient separation techniques, laser-induced fluorescence (LIF) detection, and different mass-spectrometric (MS) measurements were combined in a multimethodological scheme to perform a comprehensive structural characterization of N-linked oligosaccharides in a murine monoclonal antibody (immunoglobulin G (IgG(kappa))). Monosaccharide compositional analysis was carried out through a capillary electrophoresis (CE)-LIF method, in which the chemically and enzymatically released sugars were fluorescently labeled. This analysis provides a preliminary assessment of certain structures, being followed by CE-LIF and matrix-assisted laser desorption/ionization (MALDI)-MS profiling of the intact glycan structures. Linkages and monosaccharide residues were confirmed by MALDI-MS in conjunction with exoglycosidase digestion. MALDI-MS and CE data were effectively combined to reveal the overall structural diversity of both acidic and neutral glycans. Finally, the sites of glycosylation and site occupancies were deduced through the measurements performed with microcolumn liquid chromatography coupled via electrospray to a quadrupole/time-of-flight instrument.     

水溶性量子点标记狂犬病P蛋白单克隆抗体的研究

利用共价偶联的方式,在水溶性缩合剂1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)和N-羟基硫代琥珀酰亚胺(Sulfo-NHS)促进作用下,将400 μL的2 g/L狂犬病P蛋白抗体与适量的聚丙烯酸修饰后的水溶性硫脲修饰ZnO掺Cd量子点进行共价偶联反应,经磷酸盐缓冲液(PBS,0.01 mol/L,pH 7.4)透析纯化得到目标偶联物,采用荧光发射光谱、生物质谱、酶联免疫法等对偶联物进行表征.结果表明:偶联后的量子点荧光最大发射波长红移了10 nm,荧光强度随着狂犬病P蛋白抗原浓度的增加而逐渐增强;量子点标记狂犬病P蛋白抗体后的分子离子峰在m/z 67580处,比狂犬病P蛋白抗体分子离子峰增大了1453.由此证实狂犬病P蛋白抗体成功偶联到水溶性量子点上,且结构未受破坏.     

放射性核素标记单克隆抗体的化学研究进展

研究用放射性核素标记单克隆抗体作为肿瘤的导向药物,是核药物化学领域的研究热点.本文从化学角度介绍了单克隆抗体标记技术发展的概况,包括核素的选择,标记的方法,抗体的偶联修饰以及提高肿瘤/正常组织比的方法。     

Effect of the total galactose content on complement-dependent cytotoxicity of the therapeutic anti-CD20 IgG1 antibodies under temperature stress conditions

The degree of monoclonal antibody galactosylation is known to affect complement-dependent cytotoxicity (CDC) activity by affecting C1q binding, suggesting that galactose is associated with CDC bioactivity. However, whether this association also exists under temperature stress conditions is not known. This study highlights the impact of variations in the terminal galactose content of an anti-CD20 monoclonal antibody on CDC bioactivity under high-temperature stress conditions compared with storage conditions at 2–8?°C. Drug product samples with a total galactose content of >38% showed stable CDC bioactivity at higher temperatures (45?°C), while those with 16% galactose content showed reduced CDC activity.     

Amperometric Immunoassay of Azinphos‐Methyl in Water and Honeybees Based on Indirect Competitive ELISA

Abstract

An electrochemical immunosensor based on indirect competitive ELISA technique has been developed and tested for the detection of azinphos‐methyl in aqueous solutions and spiked honeybee extracts. The detection of the pesticide was based on competition for binding to monoclonal antibodies with an ovalbumin (OVA) conjugate, followed by the incubation with anti‐mouse IgG labeled with horseradish peroxidase, whose activity was measured amperometrically with hydroquinone as the substrate. The sensitivity of the azinphos‐methyl assay, estimated as the IC₅₀ value, was found to be 1.2 nmol L^?1 (60 min incubation), with a linear range of 0.6–500 nmol L^?1 in optimal conditions. The matrix effect on the detection of azinphos‐methyl in honeybee extract was found negligible, with the recovery values in the range 92–105%.     

STUDY ON PREPARATION AND APPLICATION OF MONOCLONAL ANTIBODIES TO Anabaena azollae

Forty hybridoma cell lines secreting monoclonal antibodies against Anabaena azollae have been established by fusing SP_2/O myeloma cells with spleen cells from BALB/c mice immunized with purified antigen Anabaena azollae from different strains of azolla.According to the fluorescent antibody test, 40 McAbs were divided into 8 types. They may differentiate the Anabaena azollae in 195 azolla strains which are collected from different places in the world, into 8 antigenic groups.The identifying result of Anabaena azollae isolated from reconstituted azolla and sexual hybrid azolla showed that the Anabaena azollae in the reconstituted azolla retains its antigenicity from the donor azolla, and Anabacna azollae in hybrid azolla maintains its antigenicity from the female parent plant.     

基于纳升电喷雾-轨道阱超高分辨质谱对完整单克隆抗体药物进行分子量测定及糖型鉴定的条件探究

整体分子量测定和糖型鉴定是抗体药物研发及生产中不可或缺的表征内容,而在完整蛋白层面进行测定可获得最为直接的测定结果。实现此类测定有赖于可提供较高分辨能力及质量精度的仪器;测定中涉及的相对复杂的参数设置可能受多种条件的干扰而产生误导性结果。该研究考察了利用纳升电喷雾离子源-轨道阱质谱仪以非变性质谱方式测定完整单克隆抗体药时,仪器分辨率、源内裂解、碰撞诱导碎裂、溶液组成等条件对单克隆抗体药物分子量及糖型测定结果的影响,以期为此类质谱分析提供有益的借鉴。     

Purification and Properties of Acetylcholinesterase From Human Brain

Acetylcholinesterase from human caudate nucleus and partial thalamus was purified by using Con A-Sepharose, short-arm and long-armligand Sepharose affinity chromatographies. SDS-PAGE of the purified AChE under the reduced condition showed one main band, corresponding to a molecular weight of 66 kD. The purified AChE witha specific activity of 3384 U/mg protein represented 20% activity of the homogenate supernatant. Analysis of purified AChE by gradient slab PAGE and DISC-PAGE with activity staining revealed the existence of monomer, dimer, tetramer, hexamer and octomer of the enzyme. The isoelectric point of AChE ranged between pH 5.6 and 6.0. Con A-Sepharose affinity chromatography retained most of the applied AChE activity implying that the enzyme is a kind of glycoprotein. The isolated human brain AChE had no cross-immunoreactivity with 3F3 and weak cross-immunoreactivity with 2G8 and 1H11 anti-Torpedo AChE antibodies. Balb/c mice were immunized with human cerebellum AChE purified with Con A and shor     

Preparation of Europium Induced Conformation—specific anti—calmodulin Monoclonal Antibody

Monoclonal antibody technique was employed to detect the conformational difference of CaM induced by metal ions. A trivalent europium ion induced conformation-specific anti-calmodulin monoclonal antibody was successfully prepared with europium-saturated calmodulin as antigen.     

The Synthesis of N-Morphine Hapten and Production of Monoclonal Antibody

The drug overdose and addiction is a serious international problem. Therefore developing a rapid, accurate, convenient and cheap method for detecting morphine in urea is useful and necessary, especially for investigation of epidemiology, identification of medical jurisprudence and determination of drug addict etc. There are many compounds that are similar to morphine in chemical structure and tend to have cross-reaction with the anti-morphine antibody. In order to reduce the cross reaction a…     

Solvent Effects on the Radical Copolymerizabilities of Styrene with p-Substituted N,N-Diethylcinnamamides and of p-Substituted Styrenes with Methyl Vinyl Sulfoxide

Radical copolymerizations of styrene (St) with p-substituted-N, N-diethylcinnamamides (I) and also of p-substituted styrenes (II) with methyl vinyl sulfoxide (MVSO) have been carried out in benzene, acetic acid or acetonitrile. The monomer reactivity ratios (r₁) were found to be affected by the solvents, p values obtained by using the modified Hammett equation, i. e., log(1/r₁) = ρσ + γ E_R, were also found to be altered by the solvents. The results are discussed in terms of the solvent effect in the transition state of the propagation reaction.