Amidase and peptidase activities of polyclonal immunoglobulin G present in the sera of patients with rheumatoid arthritis

Polyclonal Immunoglobulin (Ig) G from patients with rheumatoid arthritis (RA) and healthy subjects hydrolyzed carbobenzoxy−Val−Gly−Arg p-nitroanilide and D−Pro−Phe−Arg p-nitroanilide. RA IgG exhibited higher activity against the former substrate, but not the latter. On the other hand, RA IgG showed reduced activity against D−Pro−Phe−Arg methylcoumarinamide, when compared with those of the healthy controls. These results suggest that RA IgGs differ from normal IgGs in the substrate specificity of amidase activity. Preliminary studies have shown that two out of three RA IgG samples cleaved a pentapeptide—Gln−Arg−Arg−Arg−Ala−Ala— which is assumed to be associated with the risk of developing RA (Gregersen, P. K. et al. (1987), Arthritis Rheum. 30, 1205–1213). By contrast, virtually no cleavage of the same peptide was observed with IgG from healthy controls. A peptide analog, Gln−Arg−Arg−Trp−Ala, was not cleaved at all by any IgGs examined either from RA patients or healthy controls.     

Catalytic DNA-and RNA-hydrolyzing antibodies from milk of healthy human mothers

Various catalytically active antibodies (Abs), or abzymes, have been detected recently in the sera of patients with autoimmune pathologies, in whom their presence is probably associated with autoimmunization. Normal humans are generally not considered to have abzymes, since no obvious immunizing factors are present. Here is shown by different methods that IgG from the milk of normal females possesses both DNase and RNase activities. The activities were also present in the IgG F(ab′)₂ and Fab fragments. Affinity modification of IgG by the chemically reactive derivative of an oligonucleotide led to preferential modification of the L chain of IgG. After separation of the subunits by sodium dodecyl sulfate electrophoresis in a gel containing DNA, an in-gel assay showed DNase activity in the L chain. The L chain separated by affinity chromatography on DNA-cellulose was catalytically active. These findings speak in favor of the generation of cat alytic Abs by the immune system of healthy mothers. It is known that the treatment of adults with DNases and RNases offers protection from viral and bacterial diseases. Since breast milk protects the infants from infec tions until the immune system is developed, this raises the possibility that catalytic Abs like nucleases, may possess a protective role.     

Fluorescence immunological determination of immunoglobulin G in human serum by high-performance liquid gel-permeation chromatography

Summary A high-performance liquid gel-permeation chromatographic method is described for the determination of human serum immunoglobulin G (IgG) by separating the fluorescent immuno complex from the free fluorescence-labeled antibody. Fluorescence-labeled antibody used in this study was fluorescein isothiocyanate (FITC)-labeled Fab fragment goat anti-human IgG (anti-IgG Fab). Immuno complexes and antibody of different molecular sizes can be separated. FITC-labeled anti-IgG Fab was added to the serum and the mixture is passed through the column. An immuno complex separates as well-delineated peak in the column void volume, and was measured by the fluorescence of the column eluate (Ex=490nm, Em=520nm). The total analysis time for a serum sample was approximately 15min. The minimum detection limit was 25 mg/dl. The relative standard deviation was below 2% (peak area). The results of the HPL-GPC analysis correlate well with those obtained by laser nephelometric assay (r=0.992).     

表面增强拉曼光谱研究以4,4''''-联吡啶为标记分子的免疫检测

通过表面增强拉曼光谱(SERS)研究了标记分子4,4'-联吡啶在金溶胶上的吸附行为,并将其与山羊抗小鼠IgG结合,获得SERS标记免疫金溶胶.在固相基底上组装抗体,两者组装得到固相抗体-抗原-标记抗体“三明治”结构.在单组分和双组分体系中借助抗体上标记金纳米粒子所带的SERS信号达到免疫检测的目的.     

Complexation Based Voltammetric Determination of Pantoprazole Sodium in Pharmaceutical Formulations and Rabbit Plasma

Square wave adsorptive stripping voltammetric (SQWASV) method has been utilized to confirm and elucidate the possible complexation reaction between pantoprazole sodium and cobalt as a transition metal in Britton‐Robinson buffer (pH=7.0). The current signal due to the oxidation process was a function of the amount of pantoprazole sodium, pH of the medium, cobalt concentration and accumulation time at the electrode surface. The oxidation peak current has varied linearly with the concentration over the range of 0.1–9.0 nM. The limit of detection was found to be 0.04 nM. The validity of the method was successfully applied for the determination of pantoprazole sodium in pharmaceutical formulations with a pharmacokinetic study in rabbit plasma. The simplicity, rapidity, sensitivity and selectivity of this method make it a very attractive alternative to the other existing methods in the quality control laboratories.     

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。     

A Au nanoparticle and polydopamine co-modified biosensor: A strategy for in situ and label-free surface plasmon resonance immunoassays

This article reports a surface plasmon resonance (SPR) strategy capable of label-free yet amplified in situ immunoassays for sensitive and specific detection of human IgG (hIgG), a serum marker that is important for the diagnosis of certain diseases. Primarily, a wavelength-modulated Kretschman configuration SPR analyzer was constructed, and Au film SPR biosensor chips were fabricated. Specifically, based on Au nanoparticles (AuNPs) adsorbed on the surface of the Au film, the AuNP/Au film was coated with polydopamine (PDA) to fix streptavidin (SA), and then the biotinylated antibodies were connected to the surface of the biosensor chip. The SPR analyzer was utilized for in situ real-time monitoring of hIgG. Due to the immunological recognition between the receptor and target, the surface plasmon waves produced by the attenuated total reflection were affected by the changes in the surface of the biosensor chip. The resonance wavelength (λ_R) of the output spectra gradually redshifted, and the redshift degrees were directly related to the target concentration. The biosensor can realize the in situ detection of hIgG, displaying satisfactory sensitivity, excellent specificity and stability. Briefly, by monitoring the shift in λ_R after specific binding, a new SPR immunoassay can be customized for label-free, in situ and amplified hIgG detection. The operating principle of this research could be extended as a common protocol for many other targets of interest.     

兔主动脉冻结过程中未冻水份额的研究

利用差示扫描量热仪(DSC)技术中的分步扫描(Stepwise scanning)法,研究了不同浓度的低温保护剂对主动脉血管冻结过程中未冻水份额的影响。研究结果表明:分步扫描量热技术可以用于血管材料冻结过程中未冻水份额的定量研究,而且具有较高楠度;随着低温保护剂二甲亚砜(DMSO)浓度的增大,血管的初始冻结点温度呈线性降低,但其冻结过程的未冻水份额显著增大,这是由于高浓度DMSO具有很强水合能力所致。     

2,4-二硝基氟苯柱前衍生高效液相色谱法测定18种氨基酸

建立了测定18种氨基酸的高效液相色谱方法。以Hypersil ODS-c_(18)柱(100mm×4.6mm i.d.,5μm)为色谱柱,2,4-二硝基氟苯为衍生化试剂,梯度洗脱。流动相A:0.05mol/L乙酸钠缓冲溶液(pH=6.5,含10mL/LN,N-二甲基甲酰胺),流动相B:乙腈-水(体积比为1:1)。所测氨基酸组分的浓度在0.2～3.0mg/L范围内与峰面积之间具有良好的线性关系,回收率为90.4％～101.0％,检出限为0.120～1.225mg/L,测定结果的相时标准偏差为1.05％～5.93％。     

Determination of human IgG by solid-substrate room-temperature phosphorescence immunoassay based on an antibody labeled with nanoparticles containing rhodamine 6G luminescent molecules

Luminescent 50-nm silicon dioxide nanoparticles containing both types of rhodamine 6G (R; particles denoted R-SiO₂) were synthesized by the sol–gel method. In the presence of Pb(Ac)₂ as a heavy atom perturber the particle can emit the intense and stable room-temperature phosphorescence (RTP) signal of R on a polyamide membrane, with _ex^max/_em^max=470/635 nm for R. Our research indicates that the specific immune reaction between goat-anti-human IgG antibody labeled with R-SiO₂ and human IgG can be carried out quantitatively on a polyamide membrane, and the phosphorescence intensity was enhanced after the immunoreaction. Thus a new method for solid-substrate room-temperature phosphorescence immunoassay (SS-RTP-IA) for determination of human IgG was established on the basis of antibody labeled with the nanoparticles containing binary luminescent molecules. The linear range of this method is 0.0624–20.0 pg spot^–1 of human IgG (corresponding to a concentration range of 0.156–50.0 ng mL^–1, sample volume 0.40 L spot^–1). The regression equations of the working curves are I_p=71.27+7.208m_IgG (pg spot^–1) (r=0.9996). Detection limits calculated as 3S_b/k are 0.022 pg spot^–1. Compared with the same IA using fluorescein isothiocyanate (FITC) as the marker the new method was more sensitive and had a wider linear range. After elevenfold replicate measurement RSD are 4.5 and 3.6% for samples containing 0.156 and 50.0 ng mL^–1 IgG, respectively. This method is sensitive, accurate, and of high precision.