牛奶过敏原β-乳球蛋白的重组制备及磁微粒化学发光检测法的建立

运用原核系统表达牛奶β-乳球蛋白(Bos d5)蛋白,建立一种纳米磁微粒化学发光方法用于检测牛奶组分过敏原β-乳球蛋白特异性Ig E抗体的含量。通过优化合成牛奶β-乳球蛋白基因,与质粒重组导入Rosetta原核表达菌株中表达目标蛋白,纯化的重组蛋白采用生物素标记后,在化学发光平台上进行过敏原项目检测。获得纯度﹥85%的22.5 kD重组牛β-乳球蛋白,将生物素化的重组蛋白用于牛奶β-乳球蛋白纳米磁微粒检测试剂盒,检测临床110例血清样本,和Phadia进行方法学比对阳性符合率为88.9%,阴性符合率97.3%,总符合率94.5%,P<0.001,χ^2=84.238,Kappa=0.874,其与Phadia参照试剂盒同样具有良好的检测能力。原核表达系统中获得的重组牛奶过敏组分β-乳球蛋白具有较好的免疫活性,开发的免疫诊断试剂盒具备良好的性能,可用于临床辅助诊断。     

Chemiluminescent assay of β-D-galactosidase based on indole luminescence

We developed a novel chemiluminescent assay of β-D -galactosidase (β-gal) based on the chemiluminescence of indole. 5-Bromo-4-chloro-3-indolyl-β-D -galactopyranoside (X-gal) was used as a substrate for β-gal and also as a light emitter. X-gal was hydrolysed by β-gal to liberate free indoxyl, followed by oxidation to indigo dye, and simultaneously produces hydrogen peroxide (H₂O₂). H₂O₂ reacts with the residual X-gal in the presence of horseradish peroxidase (HRP) to emit light. The measurable range of β-gal obtained by this method was 6 × 10⁻¹⁴ mol/L to 6 × 10⁻¹¹ mol/L; the detection limit was 3 amol/assay. This chemiluminescent assay could be applied to an enzyme immunoassay of thyroxine using β-gal as the enzyme label. © 1998 John Wiley & Sons, Ltd.     

No correlation between time-linked plasma and CSF Aβ levels

Plasma β-amyloid protein (Aβ) isoforms are considered potential biomarkers for Alzheimer's disease (AD) and dementia. The relation between plasma and cerebrospinal fluid (CSF) levels of Aβ isoforms remains unclear. In order to identify possible correlations between Aβ levels in plasma and CSF we determined Aβ levels in time-linked plasma and CSF samples. Aβ concentrations in plasma (Aβ_1–42 and Aβ_N–42) and CSF (Aβ_1–42) samples from 49 AD patients, 47 non-Alzheimer's disease dementia (NONAD) patients, 39 MCI patients and 29 controls were determined using a multi-parameter fluorimetric bead-based immunoassay using xMAP^® technology (for plasma) and a conventional single-parameter ELISA (for CSF). Plasma Aβ_1–42 concentrations did not correlate with CSF Aβ_1–42 concentrations in the total study population, or in the different diagnostic groups. No correlations between plasma Aβ_N–42 and CSF Aβ_1–42 levels were found either. The CSF/serum albumin index did not show any significant differences between AD, NONAD, MCI and controls.These results suggest that the Aβ levels in plasma are independent of the Aβ levels in CSF both in dementia and controls. The fact that CSF and plasma Aβ do not correlate in patients as well as controls and no significant differences in plasma Aβ_1–42 or Aβ_N–42 between patients and controls can be detected hampers the diagnostic utility of the plasma Aβ levels as biomarkers for dementia.     

Immunological properties of the β-subunit of human chorionic gonadotropin

The β-subunit of human chorionic gonadotropin (hCG-β) has been modified to a varying degree by the cleavage of the disulfide bonds by reduction with dithioerythritol followed by S-carboxamidomethylation. The resulting derivatives of hCG-β show a preferential loss in their immunological cross-reactivity with human luteinizing hormone (hLH). The immunological specificity of the partially reduced and S-alkylated derivatives can be further enhanced by conjugation with tetanous toxoid using glutaraldehyde. Neither conjugation of hCG-β with the toxoid nor its treatment with anti-hLH immunoadsorbent affects its cross-reactivity with hLH.     

Miniaturized amperometric flow immunoassay system using a glass fiber membrane modified with anion

This paper describes a miniaturized amperometric flow immunoassay system using a glass fiber membrane modified with anion. The glass fiber membrane was functionally modified with gamma-glycidoxypropyltrimethoxysilane and sodium thiosulfate and was used for separation of protein. Anti-human chorionic gonadotrophin (HCG) immunoglobulin G (IgG) antibody conjugated with ferrocenemonocarboxylic acid (Fc), namely, Fc-conjugated IgG (Fc-IgG), was used as a novel analytical reagent. HCG and Fc-IgG complexes were separated from free Fc-IgG based on differences in isoelectric point (pI) using the glass fiber membrane modified with a thiosulfonyl acid functional group. The assay yields a linear relationship between current and HCG concentration in the range of 0-2000 mIU/mL. This simple technique enables the assay of HCG within 2 min. The modified glass fiber membrane was regenerated by occasional elution with malonate buffer (pH 6.0) containing 0.5 M NaCl, to remove free Fc-IgG. Free Fc-IgG recovered in this manner could be reused up to eight times without significant decreases in sensitivity. This miniaturized amperometric flow immunoassay requires only minute quantities of serum and generates highly reproducible results.     

On the origin of the stronger binding of PIB over thioflavin T to protofibrils of the Alzheimer amyloid-β peptide: a molecular dynamics study

Pittsburgh compound B (PIB) is a neutral derivative of the fluorescent dye Thioflavin T (ThT), which displays enhanced hydrophobicity and binding affinity to amyloid fibrils. We present molecular dynamics simulations of binding of PIB and ThT to a common cross-β-subunit of the Alzheimer Amyloid-β peptide (Aβ). Our simulations of binding to Aβ(9-40) protofibrils show that PIB, like ThT, selectively binds to the hydrophobic or aromatic surface grooves on the β-sheet surface along the fibril axis. The lack of two methyl groups and charge in PIB not only improves its hydrophobicity but also leads to a deeper insertion of PIB compared to ThT into the surface grooves. This significantly increases the steric, aromatic, and hydrophobic interactions, and hence leads to stronger binding. Simulations on protofibrils consisting of the more-toxic Aβ(17-42) revealed an additional binding mode in which PIB and ThT insert into the channel that forms in the loop region of the protofibril, sandwiched between two sheet layers. Our simulations indicate that the rotation between the two ring parts of the dyes is significantly more restricted when the dyes are bound to the surface of the cross-β-subunits or to the channel inside the Aβ(17-42) cross-β-subunit, compared with free solution. The specific conformations of the dyes are influenced by small chemical modifications (ThT versus PIB) and by the environment in which the dye is placed.     

Construction of electrochemical flow immunoassay system using capillary columns and ferrocene conjugated immunoglobulin G for detection of human chorionic gonadotrophin

In this paper is reported a miniaturized flow immunoassay system. Ferrocenecarboxylic acid (Fc) conjugated with anti-HCG immunoglobulin G (IgG) antibody (Fc–IgG) was prepared, and used as a novel analytical reagent. The system consists of the immunoreaction section, the capillary column packed with cation exchange resin, and the flow cell for electrochemical detection of Fc–IgG. Antibody–antigen complexes were separated from their free conjugate on the basis of differences in isoelectric point (pI) using a cation exchange capillary column. The assay yielded a linear relationship between signal and HCG concentration in the range 0–2000 mIU/ml. This simple technique enables the assay of HCG within 2 min. The cation exchange capillary column was regenerated by occasional elution with malonate buffer (pH 6.0) containing 0.5 M NaCl, to remove free conjugate. Free conjugate recovered in this manner could be reused up to eight times without significant decreases in the sensitivity of the immunoassay. This electrochemical flow immunoassay requires only minute quantities of serum and generates highly reproducible results.     

人绒毛膜促性腺激素β亚基酶解片段特性的研究

以嗜热菌蛋白酶水解人绒毛膜促性腺激素,亚基(hCG_β),用HPLC分离制备酶解产物。酶解片段经竞爭性放射分析,不能与LH/hCG受体结合,但保留近乎全部hCG_β的免疫反应性.园二色谱分析,与hCG_β比较,酶解片段所含α螺旋减少,β折叠与无规卷曲含量增多。表明hCG_β可能有几个肽段和/或糖链参与hCG受体相互作用;酶解片段中仍含有hCG_β的主要决定簇,该决定簇与hCG_β结合受体的结构有区别。     

Antigen-Bound and Free β-Amyloid Autoantibodies in Serum of Healthy Adults

Physiological β-amyloid autoantibodies (Aβ-autoantibodies) are currently investigated as potential diagnostic and therapeutic tools for Alzheimer's disease (AD). In previous studies, their determination in serum and cerebrospinal fluid (CSF) using indirect ELISA has provided controversial results, which may be due to the presence of preformed Aβ antigen-antibody immune complexes. Based on the epitope specificity of the Aβ-autoantibodies, recently elucidated in our laboratory, we developed (a) a sandwich ELISA for the determination of circulating Aβ-IgG immune complexes and (b) an indirect ELISA for the determination of free Aβ-autoantibodies. This methodology was applied to the analysis of serum samples from healthy individuals within the age range of 18 to 89 years. Neuropsychological examination of the participants in this study indicated non-pathological, age-related cognitive decline, revealed especially by tests of visual memory and executive function, as well as speed-related tasks. The ELISA serum determinations showed significantly higher levels of Aβ-IgG immune complexes compared to free Aβ-autoantibodies, while no correlation with age or cognitive performance of the participants was found.     

Immunoassay of nine serological tumor markers on a hydrogel-based microchip

A prototype test-system for simultaneous quantitative assay of nine tumor markers in blood serum was developed. The main constituent of the test-system is an OM-9 biochip containing immobilized antibodies against nine oncomarkers: α-fetoprotein (AFP), carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), cancer antigen 15-3 (CA 15-3), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA 19-9), total and free forms of prostate-specific antigen (PSA_tot and PSA_free), and neuron-specific enolase (NSE). The biochip-based two-step sandwich immunoassay procedure for carrying out simultaneous quantitative determination of nine tumor markers in patients’ blood serum was proposed. The main analytical characteristics of the method were obtained. The results suggest that the prototype of the test-system could be a promising instrument for clinical application. The test-system prototype was tested using blood serum samples of oncological patients (252 samples) and healthy donors (185 samples). Increased concentrations of one or more tumor markers above the normal level were found in 76.6% cases of oncological patients and only in 6% cases of healthy donors. For colorectal cancer patients, application of modern statistical methods of data processing in medical research, i.e., receiver operating characteristics analysis (ROC curve) and logistic regression, indicated that the simultaneous assay of nine markers on biochips showed much more diagnostic significance (area under the ROC curve, AUC, was 0.84) than a traditional assay of two tumor markers, CEA and CA 19-9 (AUC = 0.59). The developed biochip-based test-system can be recommended for both the estimation of people’s health, e.g., for standard medical examination, and tracking the tumoral process in the postsurgical period or after specific tumor treatment.     

Specific Recognition of Biologically Active Amyloid-β Oligomers by a New Surface Plasmon Resonance-based Immunoassay and an in Vivo Assay in Caenorhabditis elegans

Soluble oligomers of the amyloid-β (Aβ) peptide play a key role in the pathogenesis of Alzheimer's disease, but their elusive nature makes their detection challenging. Here we describe a novel immunoassay based on surface plasmon resonance (SPR) that specifically recognizes biologically active Aβ oligomers. As a capturing agent, we immobilized on the sensor chip the monoclonal antibody 4G8, which targets a central hydrophobic region of Aβ. This SPR assay allows specific recognition of oligomeric intermediates that rapidly appear and disappear during the incubation of synthetic Aβ(1-42), discriminating them from monomers and higher order aggregates. The species recognized by SPR generate ionic currents in artificial lipid bilayers and inhibit the physiological pharyngeal contractions in Caenorhabditis elegans, a new method for testing the toxic potential of Aβ oligomers. With these assays we found that the formation of biologically relevant Aβ oligomers is inhibited by epigallocatechin gallate and increased by the A2V mutation, previously reported to induce early onset dementia. The SPR-based immunoassay provides new opportunities for detection of toxic Aβ oligomers in biological samples and could be adapted to study misfolding proteins in other neurodegenerative disorders.     

The Swedish APP mutation alters the effect of genetically reduced BACE1 expression on the APP processing

Inhibition of β-secretase (BACE1) is a key therapeutic approach in Alzheimer's disease (AD), as BACE1 initiates amyloid-β (Aβ) cleavage from the β-amyloid precursor protein (APP). As Aβ reductions in mice lacking one BACE1 allele diverged considerably between studies we investigated the effect of BACE1 knock-out in more detail. With both BACE1 alleles the Swedish mutation (APP23 mice) increased APP processing and shifted it towards the β-secretase pathway as compared with non-mutated APP expressed at a similar level (APP51/16 mice). This effect was much smaller then observed in cell culture. An about 50% decrease in BACE1 enzyme activity resulted in a sub-proportional Aβ reduction with the Swedish mutation (-20%) and even less for non-mutated APP (-16%). In wild-type mice, the Aβ reduction may be even further diminished. Other metabolites of the β-secretase pathway decreased accordingly while the alternative α-secretase pathway increased. Complete BACE1 deletion strongly enhanced these changes. The remaining Aβ signal also described by others can be explained by assay cross-reactivity with other APP metabolites supporting BACE1 as the major β-secretase. Our data indicate that BACE1 is in excess over APP at the cleavage site(s). Alterations in APP expression or substrate properties, therefore, quantitatively change its cleavage and Aβ generation.     

Coexpression and characterization of the human large-conductance Ca2+-activated K+ channel α + β1 subunits in HEK293 cells

Large-conductance Ca²⁺-activated K⁺ channel is formed by a tetramer of the pore-forming α-subunit and distinct accessory β-subunits (β1–β4) which contribute to BK_Ca channel molecular diversity. Accumulative evidences indicate that not only α-subunit alone but also the α + β subunit complex and/or β-subunit might play an important role in modulating various physiological functions in most mammalian cells. To evaluate the detailed pharmacological and biophysical properties of α + β1 subunit complex or β1-subunit in BK_Ca channel, we established an expression system that reliably coexpress hSloα + β1 subunit complex in HEK293 cells. The coexpression of hSloα + β1 subunit complex was evaluated by western blotting and immunolocalization, and then the single-channel kinetics and pharmacological properties of expressed hSloα + β1 subunit complex were investigated by cell-attached and outside-out patches, respectively. The results in this study showed that the expressed hSloα + β1 subunit complex demonstrated to be fully functional for its typical single-channel traces, Ca²⁺-sensitivity, voltage-dependency, high conductance (151 ± 7 pS), and its pharmacological activation and inhibition.     

Abnormal platelet amyloid-β precursor protein metabolism in SAMP8 mice: Evidence for peripheral marker in Alzheimer's disease

Senescence-accelerated mouse strains have proved to be an accelerated-aging model, which mimics numerous features with Alzheimer's disease (AD). Three, six, and nine-month senescence-accelerated resistant 1 and senescence-accelerated prone 8 (SAMP8) mice were used in the current study, to unravel potential mechanisms for dementia and explore new diagnostic approaches for AD. The amyloid-β (Aβ40) and Aβ42 levels were elevated in hippocampi and platelets from SAMP8, along with a reduced α-secretase expression and an enhanced β-secretase expression extent with age, compared to control mice. Furthermore, hippocampal Aβ40 and Aβ42 of SAMP8 were positively correlated with platelet of these mice with aging progression. In addition, β-γ-secretase-modulated proteolytic proceeding of amyloid precursor protein in platelet might work through the PI3K/Akt/GSK3β pathway. These results indicate that platelet could be a potential early marker in the periphery to study the age-correlative aggregation of the amyloid-β peptide in patients with AD, while still requiring the considerable study.     

Dioxin silences gonadotropin expression in perinatal pups by inducing histone deacetylases: a new insight into the mechanism for the imprinting of sexual immaturity by dioxin

Maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes the impairment of reproduction and development in the pups. Our previous studies have revealed that maternal treatment with TCDD attenuates the fetal production of pituitary gonadotropins (luteinizing hormone (LH) and follicle-stimulating hormone) at gestational day (GD) 20, leading to the impairment of sexual behavior in adulthood. However, the mechanism underlying such a reduction has remained unknown until now. When pregnant rats at GD15 were given an oral dose of TCDD (1 μg/kg), the testicular expression of steroidogenic proteins was reduced between GD20 and postnatal days (PND) 2. In accordance with this, the pituitary expression of gonadotropin β-subunit and serum gonadotropin were also attenuated from GD20 to PND0 in a pup-specific fashion. To identify the target genes linked to a fetal reduction in gonadotropin β-subunit, we performed a DNA microarray analysis using the fetal pituitary and its regulatory organ, the hypothalamus. The results obtained showed that TCDD induced histone deacetylases (HDACs) in the fetal pituitary. In support with this, TCDD markedly deacetylated histones H3 and H4 twined around the promoter of the fetal LHβ gene. This effect was fetus- and LHβ-specific, and this was not observed in the maternal pituitary or for other pituitary hormone genes. Finally, an LHβ reduction caused by TCDD was completely restored by maternal co-treatment with valproic acid, an HDAC inhibitor. These results strongly suggest that the increased deacetylation of histone owing to HDAC induction plays a critical role in the TCDD-induced reduction in LHβ in the fetal pituitary.     

Chemiluminescence enzyme immunoassay of dehydroepiandrosterone and its sulfate using peroxidase as label

We report a highly sensitive enzyme immunoassay for dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) using horseradish peroxidase as the label enzyme. Separation of free and bound DHEA-peroxidase conjugate was by insolubilized antibody, prepared by coupling purified IgG of goat anti-rabbit IgG serum with Sepharose 4B or a polystyrene tube. The enzyme activity was measured by the chemiluminescence reaction using luminol and hydrogen peroxide as substrate. The faint chemiluminescence was measured by a photon counter. The sensitivity was 25 pg/assay tube for DHEA and 100 pg/assay tube for DHEA-S. Upon comparison, results obtained by radioimmunoassay and this method showed good agreement; r = 0.86 for free DHEA, r = 0.92 for acid-hydrolyzed DHEA-S and r = 0.91 for solvolyzed DHEA-S. The present method is applicable in the routine determination of DHEA and DHEA-S in biological fluid.     

Luminescence in the study of lipid metabolism

Using bioluminescence assays for glycerol, free fatty acids, β-hydroxybutyrate and lactate, we were able to perform complex studies of human energy and lipid metabolism both in serum samples in vivo and in isolated fat cells in vitro. These studies would have been impossible without reliable, specific and highly sensitive luminescence methods. Oxidatively modified low density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. Adaptation of a chemiluminescence assay for lipid hydroperoxides to LDL isolated by specific precipitation from serum makes it possible to measure LDL oxidation in vivo. Cell dependent chemiluminescence was used to investigate whether receptor mediated endocytosis of LDL by macrophages leads to oxygen radical production in these cells. No activation of the membrane NAD(P)H oxidase was observed.     

Homodimerization of the Src homology 3 domain of the calcium channel β-subunit drives dynamin-dependent endocytosis

Voltage-dependent calcium channels constitute the main entry pathway for calcium into excitable cells. They are heteromultimers formed by an α(1) pore-forming subunit (Ca(V)α(1)) and accessory subunits. To achieve a precise coordination of calcium signals, the expression and activity of these channels is tightly controlled. The accessory β-subunit (Ca(V)β), a membrane associated guanylate kinase containing one guanylate kinase (β-GK) and one Src homology 3 (β-SH3) domain, has antagonistic effects on calcium currents by regulating different aspects of channel function. Although β-GK binds to a conserved site within the α(1)-pore-forming subunit and facilitates channel opening, β-SH3 binds to dynamin and promotes endocytosis. Here, we investigated the molecular switch underlying the functional duality of this modular protein. We show that β-SH3 homodimerizes through a single disulfide bond. Substitution of the only cysteine residue abolishes dimerization and impairs internalization of L-type Ca(V)1.2 channels expressed in Xenopus oocytes while preserving dynamin binding. Covalent linkage of the β-SH3 dimerization-deficient mutant yields a concatamer that binds to dynamin and restores endocytosis. Moreover, using FRET analysis, we show in living cells that Ca(V)β form oligomers and that this interaction is reduced by Ca(V)α(1). Association of Ca(V)β with a polypeptide encoding the binding motif in Ca(V)α(1) inhibited endocytosis. Together, these findings reveal that β-SH3 dimerization is crucial for endocytosis and suggest that channel activation and internalization are two mutually exclusive functions of Ca(V)β. We propose that a change in the oligomeric state of Ca(V)β is the functional switch between channel activator and channel internalizer.     

血清对全反式视黄酸抑制肺癌细胞生长的影响

 研究不同浓度的血清对全反式视黄酸 (ATRA)抑制肺癌细胞生长的影响 .当细胞培养在 10 %血清中 ,ATRA不能抑制肺癌细胞生长 ,但是当细胞培养在 1%血清中 ,ATRA能够有效地抑制肺癌细胞生长 .视黄酸受体RARβ介导视黄酸的抗癌作用 .Northern印迹分析表明 ,在高浓度血清中AT RA不能诱导RARβ表达 ,但在低浓度血清中ATRA可以诱导RARβ表达 ,并且瞬时转染和CAT测定证实是通过激活RABβ启动子转录活性而诱导RARβ表达的 .孤生受体Nur77受到血清生长因子刺激后会大量表达 ,具有抗视黄酸活性的作用 .肺癌细胞培养在低浓度血清中 ,Nur77mRNA低水平表达和Nur77蛋白不表达 .然而在高浓度血清中 ,Nur77mRNA和蛋白高水平表达 .另外 ,在无血清条件下 ,EGF也可以诱导Nur77表达 .结果提示 ,血清中的生长因子可能拮抗ATRA抑制肺癌细胞生长的作用 ,其作用途径可能是通过刺激细胞中Nur77表达 ,或者通过下调RARβ启动子的转录活性而抑制RARβ的表达