Amino acids and peptides. XXXV. Synthesis of mouse metallothionein I. (2). Synthesis of a nonacosapeptide corresponding to N-terminal sequence 1-29 (beta-fragment) of mouse metallothionein I and related peptides and examination of their heavy metal-binding properties.

A nonacosapeptide corresponding to the N-terminal sequence 1-29 (beta-fragment) of mouse metallothionein I and related peptides were synthesized by the conventional solution method and their heavy metals (Cu2+, Cu+ and Cd2+)-binding properties were examined. The Cu(2+)- or Cu(+)-binding activities of various peptides were not greatly dependent on the peptide structure, so far as examined, as in the cases of N. crassa MT and A. bisporus MTs. On the contrary, the Cd(2+)-binding activities of these peptides were fairly structure-dependent.     

Amino acids and peptides. XXVI. Synthesis of Agaricus bisporus metallothionein and related peptides and examination of their heavy metal-binding properties

A pentacosapeptide corresponding to the entire amino acid sequence of Agaricus bisporus metallothionein (MT) and related cysteine-containing peptides were prepared by the conventional solution method and their heavy metal-binding properties were examined. The Cu2(+)- or Cu(+)-binding activities of various peptides were not greatly dependent on the peptide structure, so far as examined, although the pentacosapeptide, A. bisporus MT, exhibited slightly higher binding activity than the other peptides. On the contrary, the Cd2(+)-binding activities of these peptides were fairly structure-dependent.     

Using of chicken antibodies for metallothionein detection in human blood serum and cadmium‐treated tumour cell lines after dot‐ and electroblotting

Metallothionein (MT) is a low‐molecular mass protein playing an essential role in homeostasis of heavy metal ions. Its relation with formation and progression of a tumour disease is discussed in this article. Here, we propose a new methodological approach for visualization of MT on PVDF membranes after dot‐ and electroblotting by using a commercial mouse monoclonal antibody E9 and polyclonal chicken antibodies. The optimized procedure was as follows. We dotted 1 μL sample volume on PVDF membrane and let it to dry. Then, we blocked the membrane surface with 2% BSA in PBS for 30 min. After that, the membrane was incubated in chicken primary antibody (diluted 1:500), washed, and incubated in rabbit‐anti‐chicken secondary antibody conjugated with horseradish peroxidase. To visualize the interaction, we used 3‐aminoethyl‐9‐carbazole. Under these conditions, we estimated detection limit as 3 pg of MT per 1 μL. The optimal approach was further utilized for detection of MT level in two human fibroblast cell lines and in blood serum obtained from children with medulloblastoma. The results were in good agreement with differential pulse voltammetry‐Brdicka reaction.     

Inhibition of endothelin (ET)-1-and ET-2-induced vasoconstriction by anti-ET-1 monoclonal antibody

We produced a monoclonal antibody to endothelin (ET)-1, tested cross-reactivities with the related peptides by enzyme immunoassay, and investigated the effects of the antibody on ET-1- or ET-2-induced vasoconstriction of rat isolated thoracic aorta. The antibody recognized ET-1, ET-2 and ET-3, and the immunoreactive site proved to be the N-terminal region but not the C-terminal region of ET-1. Moreover, at an approximate molar-equivalent concentration, the antibody absorbed ET-1 and ET-2, and significantly inhibited ET-1- and ET-2-induced vasoconstriction notwithstanding the presence of the endothelin receptor.     

Fluorescence-labelled antigen-binding fragments (Fab) from monoclonal antibody 5F12 detect human erythropoietin in immunoaffinity capillary electrophoresis

A faster and more convenient method is required for the detection of recombinant erythropoietin (Epo) in human body fluids. In the present study we wanted to elucidate the principal suitability of immunoaffinity capillary electrophoresis (CE) in this respect. CE offers itself as a high-speed, high-throughput technique provided a suitable affinity reagent is available. We chose monoclonal antibody 5F12 from Amgen which binds to a conformation-independent epitope in the N-terminal region of the human Epo protein. For CE with laser-induced fluorescence detection it was necessary to produce fluorescently labelled antibody with one single antigen binding site. Monomeric antigen-binding fragments (Fab) were obtained by site-selective cleavage of the pure antibody and labelled with the fluorescent dye, Alexa Fluor 488. The mixture of labelled isomers was partially resolved by ion exchange HPLC and isoelectric focusing. The fluorescent Fab could be used to detect erythropoietin by immunoaffinity capillary isoelectric focusing and zone capillary electrophoresis via its antigen complex.Abbreviations BGE background electrolyte - CE capillary electrophoresis - Epo Erythropoietin - Fab antigen-binding fragment - FITC fluorescein isothiocyanate - IEF isoelectric focusing - mAb monoclonal antibody - PBS phosphate-buffered saline - rHuEpo recombinant human erythropoietin - scFv (recombinant) single chain variable fragment - SDS-PAGE denaturing polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - ECL enzyme-coupled chemoluminescence - v_H variable domain - c_H1–3 constant domains of an antibody's heavy chain     

Identification of cadmium containing metabolites in HepG2 cells after treatment with cadmium-selenium quantum dots

The transformation of quantum dots (QDs) by organisms has attracted broad attention but remains unclear. Understanding of the metabolites helps to reveal the transformation pathway of QDs. Cd containing-metallothionein (MT) are the main species formed by Cd released from CdSe QDs in HepG2 cells, while speciation analysis of Cd containing MTs remains a challenge because MTs has several subisoforms and can bind with several metals. Herein, we built a hyphenated platform for speciation analysis of QDs in HepG2 cells after treatment with CdSe/ZnS QDs. The Cd-containing MTs were separated in reversed phase high performance liquid chromatography (RP-HPLC) and subsequently online detected by inductively coupled plasma mass spectrometry (ICP-MS) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) parallelly. Four groups of Cd-containing metabolites were found by detecting Cd in ICP-MS. Their structures were identified in ESI-Q-TOF-MS and further confirmed with standards of four subisoforms of MT, including N-terminal acetylation MT2a, N-terminal acetylation MT1e, N-terminal acetylation MT1g and MT1m. Each group of them contains various stoichiometry of Cd/Zn. The metabolites of QDs remain same while the concentrations of each metabolite and its stoichiometry of Cd/Zn vary for different incubation concentration/time. This work provides a new parallel hyphenation technique of HPLC-ICP-MS/ESI-MS with high separation resolution and powerful detection ability, and the obtained results provide detailed metabolism information of QDs in HepG2 cells after treatment of CdSe/ZnS QDs, contributing to deep exploration of the functional mechanisms of QDs in organisms.     

Fragmentation reactions of deprotonated peptides containing proline. The proline effect

The collision-induced dissociation (CID) fragmentation reactions of a variety of deprotonated peptides containing proline have been studied in detail using MS(2) and MS(3) experiments, deuterium labelling and accurate mass measurements when necessary. The [M--H--CO(2)](-) (a(2)) ion derived from H-Pro-Xxx-OH dipeptides shows an unusual fragmentation involving loss of C(2)H(4); this fragmentation reaction is not observed for larger peptides. The primary fragmentation reactions of deprotonated tripeptides with an N-terminal proline are formation of a(3) and y(1) ions. When proline is in the central position of tripeptides, a(3), y(2) and y(1) ions are the primary fragmentation products of [M--H](-), while when the proline is in the C-terminal position, a(3)and y(1) ions are the major primary products. In the latter case, the a(3) ion fragments primarily to the 'b(2) ion; further evidence is presented that the 'b(2) ions have a deprotonated oxazolone structure. Larger deprotonated peptides having at least two amino acid residues N-terminal to proline show a distinct preference for cleavage of the amide bond N-terminal to proline to form, mainly, the appropriate y ion. This proline effect is compared and contrasted with the similar proline effect observed in the fragmentation of protonated peptides containing proline.     

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.     

Structure-reactivity relationships among metallothionein three-metal domains: role of non-cysteine amino acid residues in lobster metallothionein and human metallothionein-3

Metallothionein (MT) domains of different origins, exhibiting distinct, highly conserved cysteine positions, show differences in metal-cysteine coordination and reactivity. Lobster MT, which includes two Cd3S9 beta domains, was chosen as a basic model to study the structure-function relationship among the clusters. The possible influence of (1) the position of the cysteine residues and (2) the steric and electrostatic effects of neighboring amino acids on the folding and stability of MT clusters have been examined with the native lobster beta C and beta N domains, each having nine cysteines and binding three M2+ ions, and a modified domain beta C-->N, in which the cysteines of the C-terminal domain are relocated so they are spaced as in the N-terminal domain. Each has been synthesized and characterized by UV, CD, 113Cd NMR, and 1H NMR spectroscopies. The synthetic native domains (Cd3 beta C and Cd3 beta N) displayed spectroscopic properties, metal-binding affinities, and kinetic reactivity similar to those of the holo protein. In contrast, the modified Cd3 beta C-->N domain was unusually reactive and, in the presence of Chelex, a metal-ion chelating resin, was converted to a Cd5(beta C-->N)2 dimer. These differences in structure and reactivity demonstrate that the requirements for formation of a stable type-B, Cd3S9, beta cluster are more stringent than simply the sequential positions of the cysteines along the peptide chain and include specific interactions with neighboring amino acids. Molecular mechanics calculations suggest that changes of even a single amino acid in lobster Cd3 beta N toward lobster Cd3 beta C-->N or in mammalian MT1 or MT2 toward Cd3 beta-MT3 (GIF) can destabilize their structures.     

Monoclonal-based enzyme-linked immunosorbent assay and immunochromatographic rapid assay for monensin.

Monensin, a member of the ionophoric polyether antibiotics, is used primarily as a coccidiostat. A protein conjugate of monensin was prepared and utilized to produce monoclonal antibodies in the BALB/c-P3X63Ag8U.1 fusion system. Only one hybridoma that produces monoclonal antibody against monensin was isolated from one in 329 wells. The monoclonal antibody was used to develop quantitative assays for monensin by means of an enzyme-linked immunosorbent assay (ELISA). The detection limit was 1 ng ml-1 and the relative standard deviations were 2.1-6.3% intra-assay and 5.9-12.9% inter-assay. All ELISA results for assay of chicken plasma and cattle milk were confirmed using a bioassay to be used as the official method. The ELISA and bioassay results showed close correlations for plasma (r2 = 0.98, n = 25) and milk (r2 = 0.95, n = 25). Using the anti-monensin monoclonal antibodies produced, a rapid test kit based on the immunochromatographic method was developed. Detection limits of monensin for cattle milk, cattle plasma and chicken plasma were about 40, 40 and 160 ppb. respectively.     

Sensitivity of steroid enzyme immunoassays. Comparison of four label enzymes in an assay system using a monoclonal anti-steroid antibody

The sensitivities of monoclonal antibody-based enzyme immunoassays for 11-deoxycortisol using alkaline phosphatase (AP), horseradish peroxidase (HRP), beta-galactosidase (beta-GAL) and glucose oxidase (GOD) as labels were compared. The anti-11-deoxycortisol antibody used was that produced in ascites by inoculating antibody-secreting hybridoma cells into mice. Enzyme labeling of 11-deoxycortisol was carried out by the N-succinimidyl ester method. The activated ester of 4-(2-carboxyethylthio)-11-deoxycortisol was treated with each enzyme to give a homologous enzyme-labeled antigen. In the competitive immunoassay, the bound and free enzyme-labeled antigens were separated by a double antibody method and the enzymic activity of the immune precipitate was determined by colorimetric and fluorimetric methods. The AP activity was measured in three ways, using p-nitrophenyl phosphate, nicotinamide adenine dinucleotide phosphate (NADP), and 4-methylumbelliferyl phosphate as substrates. o-Nitrophenyl beta-D-galactopyranoside and 4-methylumbelliferyl beta-D-galactopyranoside were used for beta-GAL, and 3,3',5,5'-tetramethylbenzidine (TMB) and 3-(p-hydroxyphenyl)propionic acid (HPPA) for HRP. In the case of GOD, TMB and HPPA were used in combination with HRP. A dose-response curve with a high sensitivity was obtained in each 11-deoxycortisol assay system by the use of a minimum amount of the enzyme-labeled antigen at an appropriate dilution of monoclonal anti-11-deoxycortisol antibody (Ka = 2 x 10(10) M-1). The amounts of 11-deoxycortisol needed to displace 50% of the bound label ranged from 5 to 15 pg in the colorimetric methods, and 4-9 pg in the fluorimetric methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acids and peptides. XXVII. Synthesis of phytochelatin-related peptides and examination of their heavy metal-binding properties

Phytochelatin (PC)-related peptides were prepared by a conventional solution method and their heavy metal-binding properties were examined. Different from the Cu2+ and Cu+ -binding properties of metallothionein (MT)-related peptides, the Cu2+ and Cu+ -binding properties of PC-related peptides were fairly dependent on structure. It is of interest that gamma-Glu-Cys-Gly (glutathione) exhibited quite different Cu2+ and Cu+ -binding properties from those of other PC-related peptides and its binding abilities were comparable to those of MT-related peptides. The Cd2+ -binding properties of glutathione were similar to those of Cys, and the Cd2+ -binding abilities of PC-related peptides increased in proportion to the increase of gamma-Glu-Cys peptide unit.     

Ferritin and metallothionein: dangerous liaisons

Ferritin (Ft) interaction with the Zn-complexes of mammalian MT1, MT2 and MT3 metallothioneins (MT) leads to simultaneous Fe(II) and Zn(II) release.     

Two-dimensional electrophoretic analysis of immunoglobulin patterns in monoclonal gammopathies.

Two-dimensional (2-D) electrophoresis was performed on 24 serum samples from patients diagnosed with monoclonal gammopathies. These samples had been shown to have a homogeneous immunoglobulin (M component) by zone electrophoresis and immunofixation. Using 2-D electrophoresis, the nature of this aberrant protein was further analyzed. It has been presumed that the sharp, dark stained band identified by immunofixation was the production of a monoclonal immunoglobulin. The increased resolution afforded by 2-D methodology reveals several different patterns. On 2-D electrophoresis, a monoclonal antibody has a unique pattern. It consists of 3 to 6 strong, restricted heavy chain bands and a single distorted light chain spot. The 3-6 bands are microheterogeneity of the isoelectric point, attributed to posttranslational glycosylation and/or amidation/deamidation. Analysis by 2-D electrophoresis indicated only 5 samples with a true monoclonal pattern. All but 2 of the samples clearly had aberrant immunoglobulin, but interpretation of the pattern would suggest the protein is other than a fully synthesized monoclonal antibody. The samples showed the following: a monoclonal heavy chain pattern with multiple distorted light chain spots, only an aberrant light chain area, only an aberrant heavy chain, and only a polyclonal increase. Several IgG gammopathies had, in addition, concentrations of gamma heavy chain at a reduced size (34 kDa).     

Immobilization of ultra-thin layer of monoclonal antibody on glass surface

When preparing an affinity column and a biosensor, it is desirable to immobilize a unimolecular layer of pure protein on a matrix. In this work, we tried to immobilize a monoclonal antibody on a surface of a glass test-tube as a model, to confirm the stability of this ultra-thin layer by an enzyme immunoassay, and to estimate the thickness of the layer on a slide glass by Fourier transform infrared reflection spectrometry. A new test-tube was washed and dried. The tube was filled with 5% 3-aminopropyltriethoxysilane. The 3-aminopropylsilylated surface was treated with glutaraldehyde and 5.6.10(-2) mg/ml solution of a normal mouse monoclonal antibody. The Schiff base between glutaraldehyde and the antibody was further reduced with 7.9.10(-3)% NaBH4. The tube was washed with 0.05% Tween 20 to block non-specific binding. The antibody immobilized on the surface was measured by an enzyme immunoassay based on a reaction of anti-mouse immunoglobulin G labelled with alkaline phosphatase, with which p-nitrophenol was produced from p-nitrophenylphosphate as a substrate. Meanwhile, various amounts of the antibody were immobilized on slide glasses in the same manner. The antibody on each surface was measured by Fourier transform infrared reflection spectrometry. The antibody immobilized under the final conditions was detectable by the enzyme immunoassay, and stable at 4 degrees C for ten days. The antibody on the slide glass was a unimolecular layer, as judged from the Fourier transform infrared spectra referred to -CONH- band semiquantitatively. Thus, we found the optimal conditions for immobilizing an ultra-thin layer of the monoclonal antibody on the glass surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel tangential-flow countercurrent cascade ultrafiltration configuration for continuous purification of humanized monoclonal antibody

This paper discusses the use of a novel tangential-flow, three-stage, countercurrent cascade ultrafiltration configuration for continuous purification of monoclonal antibody Campath-1H from simulated mammalian cell culture supernatant. The main limitation of conventional ultrafiltration based bioseparation, particularly when using a single-stage system is the trade-off between recovery and purity. The aim of this study was to try to improve both purity and recovery of the monoclonal antibody by using the novel cascade ultrafiltration system. Purification experiments were carried out with the three-stage cascade system as well as with single-stage and two-stage versions of the proposed system for comparison. The recovery and purification factor were both significantly higher with the three-stage system. The highest purity obtained using the three-stage cascade system was 85%. The experimental purity and recovery were compared with corresponding values obtained by simulation and found to be in reasonably good agreement.     

Characterisation of combinatorial libraries of mucin-2 antigen peptides by high-resolution mass spectrometry

An epitope motif, TX(1)TX(2)T, of mucin-2 glycoprotein was identified by means of a mucin-2-specific monoclonal antibody, mAb 994, raised against a synthetic mucin-derived 15-mer peptide conjugate. For determination of the epitope sequence recognised with highest affinity by mAb 994, a combinatorial approach was applied using the portioning-mixing technique excluding Cys. Antibody binding of libraries was most profound when Gln was at the X(1) position. Analytical characterisation of the TQTX(2)T library was conducted by amino acid analysis and matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and electrospray ionisation Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometric methods. Control libraries were prepared by mixing 19 individual peptides corresponding to the TQTX(2)T sequence. Thus, mixtures of 6, 10 and 19 pentapeptides were analysed and compared with the combinatorial mixture. MALDI-TOFMS was able to detect only partially the components in the 6- and 10-member mixtures, but failed to characterise a more complex 19-member mixture. In contrast, ESI-FTICRMS resolved all mixtures of higher complexity and provided direct identification at monoisotopic resolution, such as for a peptide library containing 'isobaric' lysine and glutamine (Delta m = 0.0364 Da). The results of this study suggest that ESI-FTICRMS is a powerful tool for characterisation of combinatorial peptide libraries of higher complexity.     

Poly-N-Isopropylacrylamide Hydrogel Based Fluoroimmunoassay of Methyltestosterone

IntroductionMethyltestosterone(MT)isasynthesizedsteroid.Athletesusesyntheticsteroidstoincreasetheirsportgrade.This,fromtheviewofethics,violatedtheoriginalrulesofsport.Therefore,themonitoringofthedopeabusehasbecomemoreandmoreimportantworldwide.Sofar.themonitoringiscommonlycarriedoutbychromatographicseparationfollowedbymassspectrometricanalysis.Thetargetsamplesarenormallyurinel~2.Themonitoringbasedonbloodsampleismainlyusedasacomplementarymethodtoovercometheshortcomingfromtheanalysisusingurines…     

Multivalent RGD synthetic peptides as potent alphaVbeta3 integrin ligands

We study herein the multivalency effect of a cluster of alphaVbeta3-ligands held on a cyclodecapeptide template. An array of RAFT(c[-RGDfK-])n derivatives containing from one to sixteen clustered RGD motifs were synthesized and comparatively assayed in vitro on alphaVbeta3-expressing cells. Efficient inhibition of the alphaVbeta3-specific 23C6 monoclonal antibody fixation was observed with ligands displaying three and four copies of the cyclo[-RGDfK-] peptide.     

Reversed-phase liquid chromatography/mass spectrometry analysis of reduced monoclonal antibodies in pharmaceutics

A reversed-phase LC/MS method was developed for reduced antibodies that provides efficient separation of light chain and two variants of heavy chain containing N-terminal glutamine and pyroglutamic acid. The best separation was achieved on Zorbax CN and Varian Pursuit DiPhenyl columns eluted with increasing percentage of n-propanol and acetonitrile in 0.1% trifluoroacetic acid. Although glutamine was genetically coded for the N-terminal residue of heavy chain of a monoclonal antibody used in this study, we found that most of it (70%) was converted to pyroglutamate during production. The conversion process continued in vitro and was monitored by the method. Deconvoluted electrospray ionization mass spectrum of the heavy chain revealed the glycosylation profile of a single N-linked sugar including a-, mono-, and di-galactosylated biantennary glycans and a 5-mannose sugar form.