Histidine-epoxy-activated sepharose beads embedded poly (2-hydroxyethyl methacrylate) cryogels for pseudobiospecific adsorption of human immunoglobulin G

The use of highly purified immunoglobulin became among the most powerful adopted strategies in therapeutic trials nowadays. Their role as immunomodulatory and anti-inflammatory agents has widened their scope of use. A novel continuous supermacroporous monolithic cryogels embedded with histidine-epoxy-activated-sepharose beads were synthetized as a new monolithic adsorbents for the separation of immunoglobulin G from human serum. The histidine-epoxy-activated-sepharose beads were embedded into the 2-hydroxyethyl methacrylate (HEMA) cryogels present in frozen aqueous solution inside a plastic syringe. The microstructure morphology of the cryogels was characterized by swelling measurement and scanning electron microscopy. The adsorption of human IgG on the histidine-epoxy-activated-sepharose beads pHEMA cryogels appeared to follow the Langmuir–Freundlich adsorption isotherm model. The maximum IgG adsorption was observed at 4°C and pH 7.4 and was found to be 26.95 mg/g of cryogel which is close to that obtained experimentally (24.49 mg/g). The cryogels were used for several adsorption-desorption cycles without any negligible decrease in their adsorption capacity.     

Selective capturing and detection of Salmonella typhi on polycarbonate membrane using bioconjugated quantum dots

Present work demonstrates the utilization of surface modified polycarbonate (PC) membrane as solid phase and antibody conjugated CdSe/ZnS quantum dots (QDs) as fluorescent label for the sensitive and selective detection of Salmonella typhi (S. typhi) in water in a period of 2.5 h. PC membrane was surface modified with glycine and activated by EDC/NHS for immobilization of S. typhi specific IgG. Antibody immobilized porous PC membrane was incubated with bacteria contaminated water for immunocapturing of S. typhi. Antibody conjugated QDs were also prepared by using carbodiimide chemistry. Both modified PC membrane and quantum dots were characterized by using various modern analytical tools. It was estimated that 1.95 molecules of QDs were successfully bio-conjugated per unit of IgG. PC membrane with captured bacteria was incubated with prepared IgG conjugated QDs for the formation of sandwich complex. Analysis of the regions of interest (ROI) in fluorescent micrographs showed that newly developed method based on PC and fluorescent QDs has 100 times higher detection sensitivity (100 cells/mL) as compared with detection using conventional dye (FITC) based methods.     

Effects of peptide density and elution pH on affinity chromatographic purification of human immunoglobulins A and M

A family of linear hexamer peptide ligands HWRGWV, HYFKFD and HFRRHL, initially identified for their affinity to the Fc portion of human immunoglobulin G (hIgG), also have potential for use in the purification of human immunoglobulins A (hIgA) and M (hIgM). HWRGWV demonstrated the strongest binding affinity to hIgM, followed by hIgA and hIgG respectively. The effects of N-terminal acetylation of the peptide, as well as elution buffer pH, on the chromatographic elution of human IgG, IgA and IgM from HWRGWV resins at various peptide densities (0.04-0.55 meq/g) were investigated. Over 80% recovery and 90% purity were achieved for human IgG and IgA isolation from complete minimum essential medium (cMEM) using HWRGWV resin at optimum peptide densities. For human IgM, 75.7% recovery and 86.0% purity were achieved by using HWRGWV at a low peptide density of 0.04 meq/g. Although HYFKFD and HFRRHL exhibited their ability for isolation of human IgG, IgA and IgM from cMEM as well, HWRGWV is the best option among them for large-scale purification of human IgG, IgA and IgM based on conditions tested.     

Dissociation and fractionation of heavy and light chains from IgG monoclonal antibodies

A basic method for dissociation and fractionation of monoclonal IgG heavy and light chain is described. It employs less noxious and hazardous reagents than the classical mercaptoethanol/propionic acid process and replaces size exclusion chromatography with cation exchange on a monolith to improve productivity. Significant scope remains to refine the conditions. The method can be applied to other disulfide bonded proteins with significant affinity for cation exchangers.     

低血铅水平与免疫球蛋白和淋巴细胞相关性

目的：探讨低铅水平对IgA、 IgG 、 IgM、 C3、 C4、 B淋巴细胞的影响。方法用石墨炉原子吸收光谱法对39名印刷印染工龄5年以上工人进行血铅测定,另选择距污染源较远且无水系联系的,不受工业污染的,从未接触过重金属的为对照组,分别对两组用免疫比浊法进行（ IgA、IgG、 IgM、 C3和C4）5项测定,流式细胞仪检测血B淋巴细胞水平,全自动生化仪检测白细胞数、总淋巴细胞绝对数和百分比。结果高低铅两组IgA、 IgG 、 IgM、 C3水平比较差异有统计学意义（P＜0.05）,白细胞总数、淋巴细胞百分比均低于低铅组（P＜0.05）,总淋巴细胞百分比和NK淋巴细胞亚群的绝对数和百分比却低于低铅组（ P＜0.05）。结论高血铅对体液免疫功能有一定的影响。     

Luminescence and anisotropy decays ofN-3-pyrene maleimide labeling IgG proteins and cells

The Py.M (N-3-Pyrene Maleimide) is a dye that covalently binds to reactive amino or sulfhycryl groups to give highly fluorescent protein conjugates. Measurements of luminescence lifetimes and anisotropy decays have been performed with a Phase and Modulation Fluorometer. Complexes of Py.M-antibody (IgG antimouse) and tumoral cells C6 labeled with Py.M have been investigated. The Py.M fluorescence in buffer solution and the protein and cells natural fluorescence have been checked. For Py.M-IgG and labeled cells, the fluorescence decays present interesting behaviours. The least-squares analysis of the experimental results on Py.M-IgG complex points out two lorentzian distributions centered at 74 ns and 11 ns, on the contrary, for the labeled cells, a discrete component at 100 ns and a lorentzian distribution centered at 5 ns are shown. In both systems a weak component lower than 1 ns is observed. The fluorescence decays, mainly the long lifetime one, are very sensitive to oxygen quenching, showing the high efficiency of O₂ quenching. For samples N₂ bubbled, the lifetime experimental resuits show a decrease of the oxygen accessibility from free probe in solution to Py.M-IgG complex and to labeled cells, compatible with a more compact packing of the probe binding site. The experimental results of anisotropy decays of degassed samples show for Py.M-IgG complexes a long rotation correlation time of about 200 ns at T=5°C, assigned to overall rotation of the protein, besides shorter correlation times attributable to inner protein motions. For labeled cells, the long rotation correlation time becomes of the order of 580 ns confirming a progressive increase of the stabilization of the binding site.     

基于可控相转变温度热敏高分子的人IgG酶联荧光免疫分析

以温度敏感高分子聚N－异丙基丙烯酰胺－丙烯酰胺[P(NIP-AA)]作为载体,建立了酶联荧光免疫分析人IgG的新方法。AA摩尔含量为8％的高分子P(NIP-AA)其临界溶解温度为37 °C。竞争型免疫测定中,被固定的IgG和标准溶液（或样品）在33 °C均相条件下竞争性地与辣根过氧化物酶标记抗体反应,升高温度分离出高分子免疫复合物,沉淀重新溶解后通过偶联过氧化氢与对羟基苯乙酸的荧光反应进行定量,线性范围为100-1000 ng/mL,检测限为2.0 ng/mL。方法灵敏、快速操作简便,且提高了免疫反应效率。此外,灵敏度与用传统微孔板做载体相似,但测定时间更快（从100-120分钟减少到30分钟）。该法用于测定人血清中IgG的含量,结果满意。     

吸附型“人工肝”辅助材料的制备及其性能研究Ⅱ.免疫吸附剂的制备及其吸附性能

本文介绍以醋酸乙烯酯为单体,二乙烯苯为交联剂制得大孔共聚物;经皂化、活化后,偶联IgG制得免疫吸附剂。讨论了不同活化剂对偶联配体的影响,观察其对人血清中的HBsAg的吸附性能。     

Surface modification of polyacrylonitrile fiber for immobilization of antibodies and detection of analyte

Pendent nitrile groups of multifilamentous polyacrylonitrile (PAN) fibers were reduced to amino groups using lithium aluminum hydride for different time of reduction and amine content was estimated by performing acid-base titrations. Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) and Differential Scanning Calorimetry (DSC) were used for the characterization of the generated amino groups and thermal properties of the reduced fibers, respectively. The surface morphology of the fibers after reduction and immobilization was characterized using Scanning Electron Microscope (SEM). The newly formed amino groups of the fibers were activated by using glutaraldehyde for the covalent linking of Goat anti-Rabbit IgG-HRP (GAR-HRP) antibody enzyme conjugate. Modified PAN fibers were evaluated as a matrix for sandwich ELISA by using Goat anti-Rabbit antibody (GAR-IgG), Rabbit anti-Goat (RAG-IgG) as analyte and enzyme conjugate GAR-HRP. The fibers reduced for 24 h were able to detect the analyte RAG-IgG at a concentration as low as 3.75 ng mL⁻¹ with 12% skimmed milk as blocking reagent for the optimized concentration of primary antibody GAR-IgG 3 μg mL⁻¹ and peroxidase conjugate GAR-HRP dilution of 8000 fold. The sensitivity, specificity and reproducibility of the developed immunoassay was further established with antibodies present in human blood using Rabbit anti-Human (RAH-IgG) antibody and the corresponding HRP enzyme conjugate. As low as 0.1 μL of human blood was sufficient to perform the assay with the modified fibers.     

Two-dimensional fluorescence difference gel electrophoresis for comparison of affinity and non-affinity based downstream processing of recombinant monoclonal antibody

Although Staphylococcus Protein A (SpA) affinity chromatography is the state of the art capture step for antibody purification, non-affinity methods are more economical. We used two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) to evaluate the purification of a recombinant IgG₁ antibody from cultured cells, with two different processes: (1) SpA capture followed by cation-exchange chromatography (CEX); and (2) CEX capture, followed by anion exchanger, then hydrophobic interaction chromatography. Efficiencies were similar in sodium dodecylsulphate polyacrylamide gel electrophoresis and size-exclusion chromatography; however, 2-D DIGE revealed higher efficiency with SpA than with CEX capture. Thus, 2-D DIGE is a valuable tool for downstream process development.