Development and characterization of new rat monoclonal antibodies for procalcitonin

The development of selective and sensitive biological recognition elements, e.g., antibodies, for the detection of relevant blood markers is a great challenge in the field of biosensors. In this context, five new rat monoclonal antibodies (mAbs) for procalcitonin (PCT), a marker for bacterial infection and sepsis, were developed and characterized. One mAb, PROC1 3G3, was used as capture antibody. Four mAbs, PROC4 6C6, PROC4 6B2, PROC4 1G3, and PROC4 1D6, were used as detection mAbs, either as Protein G-purified or as biotinylated mAbs. A surface plasmon resonance (SPR) biosensor was used to characterize the antigen-antibody biomolecular interactions. The capture mAb (PROC1 3G3) has an equilibrium dissociation constant (K (D)) of 3.42 x 10(-8) M. All four detection mAbs (PROC4 6C6, PROC4 6B2, PROC4 1G3, and PROC4 1D6) are of high affinity (K (A) = 2.81-6.11 x 10(8) M(-1); K (D) = 1.64-3.56 x 10(-9) M) and have moderate dissociation rate constants (k (d) = 1.70-2.40 x 10(-3) s(-1)). Four different sandwich enzyme-linked immunosorbent assays (ELISAs) with standards of human recombinant (hr) PCT, using PROC1 3G3 as capture mAb and PROC4 mAbs as detection mAbs, respectively, led to highly specific determinations of PCT without cross-reactivities to calcitonin and katacalcin. The lower limits of quantification (LLOQ) for hrPCT (in 40 mM phosphate-buffered saline (PBS), pH 7.6) with these assays ranged from 2.3 to 12.8 microg L(-1). In addition, sandwich ELISAs were set up with biotinylated PROC4 mAbs, and with hrPCT in 4% human serum albumin (diluted 1:10 in 40 mM PBS, including 1:5 (v/v) LowCross Buffer(R)). The LLOQs of these sandwich assays ranged from 4.1 to 6.0 microg L(-1) and were thus much closer together for the different assays. With the latter assay setup (PROC1 3G3 as capture mAb, PROC4 6C6-biotin as detection mAb) a first collection of five serum samples was determined (healthy volunteers, unspiked, and spiked). Recovery rates for the spiked samples ranged from 98.3 to 115.7%. The newly developed anti-PCT mAbs should find broad applications in immunosensors for point-of-care diagnostics of sepsis and systemic inflammation processes.     

Comparative surface plasmon resonance and enzyme-linked immunosorbent assay characterisation of a monoclonal antibody with N-acyl homoserine lactones

This study shows the detection of (N-acyl) homoserine lactones (AHLs or HSL) with monoclonal antibodies via a surface plasmon resonance (SPR)-based immunosensor in comparison to conventional microtiter plate-based enzyme-linked immunosorbent assay (ELISA). An HSL derivative, named HSL2 (Table 1), was attached to bovine serum albumin (BSA) and the conjugate (HSL2-BSA-r2) was either covalently immobilised on the SPR sensor chip surface via free amino groups or via adsorption on the ELISA polystyrene plate surface. With a newly developed rat monoclonal antibody (mAb HSL1/2 2C10), AHLs were detected sensitively in a competitive format with SPR and ELISA. Well comparable experiments between SPR and ELISA could be obtained in buffers. Moreover, the SPR sensor surface with the immobilised conjugate HSL2-BSA-r2 could be regenerated at least 340 times (regeneration cycles) without loss of activity. The measurement time per cycle was approximately 15 min. The competitive detection format for SPR and ELISA allowed the detection in the μg L⁻¹ range.     

Conformational isomers in the photocyclodimerization of N-acylated dibenz[b,f]azepine derivatives

The benzophenone-sensitised photodimerizations of N-acetyl- and N-propionyldibenz[e,f]azepine were investigated in acetone as the solvent. In both the systems, the ¹H NMR analysis of the products revealed two isomeric photodimers differing in the chemical shifts and coupling constants of the cyclobutane protons, aromatic protons and the protons of the acetyl or propionyl group. Upon raising the temperature to ca. 70 °C the signals merge. The findings can be ascribed to a single thermally restricted conformational process such as the rotation about the C–N amide bond. The process exhibits free activation energies: ΔG^#=(74±2) kJ mol⁻¹ (N-acetyl) and ΔG^#=(70±2) kJ mol⁻¹ (N-propionyl).     

Production and characterization of monoclonal antibodies against urea derivatives

A panel of monoclonal antibodies was generated against the ureabased haptenN-(2-N-chloroacetylaminobenzyl)-N′-4-chlorophenylurea as a tool for building up sensitive immune assays to detect urea derivatives and to screen them for catalytic antibodies (Abs). Eleven hybridomas were obtained that produced Abs reactive to the hapten. All Abs were of IgG class. Crossreactivities of the Abs to different haptens were examined, especially to a possible transition-state analog. Only four of the hybridomas (R2-DA10/F7, R2-GE7/H2, R2-HC2/A5, R2-HD6/F7) produced Abs crossreactive with the transition-state analog. From the 11 hybridomas, hybridoma B76-BF5 was chosen for further characterization. Compared to the other Abs, B76-BF5 showed the strongest binding and had a rather restricted specificity. These Abs could be used to build up a sensitive enzyme immunoassay for the detection of the hapten. All Abs were screened for crossreactivity with the pesticides monuron and diuron. No reactivity could be detected. In addition, the nucleotide sequences of the variable light and heavy chain genes of the similarly reactive Abs B76-BF5, B76-BB3, R2-DA10/F7, and R2-GA6/G3 were determined to clarify whether structure and binding specificity of these Abs showed any correlation.     

Synthesis and characterization of complexes of lanthanide nitrates with N,N′-dinaphthyl-N,N′-diphenyl-3,6-dioxaoctanediamide

Solid complexes of lighter lanthanide nitrates with N,N′-dinaphthyl-N,N′-diphenyl-3,6-dioxaoctanediamide (DDD), Ln(NO₃)₃(DDD) (Ln = La---Nd, Sm) have been prepared in non-aqueous media. These complexes have been characterized by elemental analysis, conductivity measurements, IR spectra, electronic spectra and TG-DTA techniques. In all the complexes, DDD and NO₃⁻ are coordinated to the lanthanide ions as tetradentate and bidentate ligands, respectively. The differences in the IR and electronic spectra between these complexes and lanthanide nitrate complexes with N,N,N′,N′-tetraphenyl-3,6-dioxaoctanediamide (TDD) are discussed.     

Development of new rat monoclonal antibodies with different selectivities and sensitivities for 2,4,6-trinitrotoluene (TNT) and other nitroaromatic compounds

Five new rat monoclonal antibodies (mAbs) for 2,4,6-trinitrotoluene (TNT) and other nitroaromatic compounds, including, especially, the metabolite 2-amino-4,6-dinitrotoluene (2-ADNT), are described. Five heterogeneous, competitive enzyme-linked immunosorbent assays (ELISAs) were developed. Assay 1 uses mAb DNT4 3F6 as recognition element and gives a standard curve for TNT in 40 mmol L^–1 phosphate buffered saline (PBS) with a test midpoint (IC₅₀) of 0.26±0.08 g L^–1 (n=20). Assay 2 (mAb DNT4 4G4) has an IC₅₀ of 0.35±0.07 g L^–1 (n=18), assay 3 (mAb DNT4 1A3) has an IC₅₀ of 0.73±0.14 g L^–1 (n=15), and assay 4 (mAb DNT4 1A7) has an IC₅₀ of 2.32±0.70 g L^–1 (n=15). Assay 5 (mAb DNT2 4B4) is very selective for 2-ADNT and has an IC₅₀ of 8.5±1.7 g L^–1 (n=15) in PBS. These antibodies for nitroaromatic compounds differ not only in their sensitivity but also in their selectivity. Major cross-reactants are 1,3,5-trinitrobenzene, 2-ADNT, 4-amino-2,6-dinitrotoluene (4-ADNT), 2,4-dinitroaniline, 3,5-dinitroaniline, and 2,6-dinitroaniline. Although assay 5 is not highly sensitive, the mAb DNT2 4B4 in this assay is highly selective for 2-ADNT. Of all the compounds tested, only 2,4-dinitroaniline and 3,5-dinitroaniline had relevant cross reactivities, 18% and about 26%, respectively. Two ELISAs, using mAbs DNT4 3F6 and DNT2 4B4, were used to analyze different concentrations of TNT and 2-ADNT, respectively, in three different surface water matrices (river and lake water). Both assays were affected by the matrix, but usually performed well (recovery within the range 70–120%). In addition, these ELISAs were used to analyze mixtures of TNT, 2-ADNT, and 4-ADNT, at three different concentrations, in the same water matrices. A different recognition pattern was clearly visible with both assays and depended on the cross reactivities of the corresponding mAb.Dedicated to the memory of Wilhelm Fresenius     

Reaction of N-vinylindazolium and N-vinylbenzotriazolium salts with nucleophiles

Reaction of N-vinylindazolium tetrafluoroborates with aqueous potassium carbonate or sodium borohydride affords 2,3-dihydroquinazolines which evolve to 1,2,3,4-tetrahydroquinazolines by intra or intermolecular nucleophilic attack. The X-ray structure of one tetrahydroquinazoline, the tricyclic compound 17a, was determined (C₁₄H₁₆N₂O₅, P2₁/n, a=6.001(4)Å, b=13.601(8)Å, c=17.452(6)Å, β=94.93(3)°, V=1419(1)ų, Z=4, R=0.078 for 1541 observed reflexions). Only open-chain compounds are obtained when these salts react with methanol. 2-Vinylindazolium and 3-vinylbenzotriazolium tetrafluoroborates react with aqueous potassium carbonate and with sodium borohydride yielding the corresponding neutral benzazoles by cleavage of the azole-vinyl bond. They add methanol to the exocyclic double bond and, in the case of indazole derivative, an expansion to a 1,2-dihydroquinazoline is observed in basic medium.A general mechanism is proposed for the reaction of vinylpyrazolium and indazolium salts with nucleophiles.     

Synthesis and characterization of temperature-sensitive and biodegradable hydrogel based on N-isopropylacrylamide

Based on a biodegradable cross-linker, N-maleyl chitosan (N-MACH), a series of Poly(N-isopropylacrylamide) (PNIPAAm) and Poly(N-isopropylacrylamide-co-acrylamide) [P(NIPAAm-co-Am)] hydrogels were prepared, and their lower critical solution temperature (LCST), swelling kinetics, equilibrium swelling ratio in NaCl solution, and enzymatic degradation behavior in simulated gastric fluids (SGF) were discussed. The LCST did not change with different cross-linker contents. By altering the NIPAAm/Am molar ratio of P(NIPAAm-co-Am) hydrogels, the LCST could be increased to 39°C. The LCST of the hydrogel was significantly influenced by the monomer ratio of the NIPAAm/Am but not by the cross-linker content. In the swelling kinetics, all the dry hydrogels exhibited fast swelling behavior, and the swelling ratios were influenced by the cross-linker content and NIPAAm/Am molar ratios. Equilibrium swelling ratio of all the hydrogels decreased with increasing NaCl solution concentration. In enzymatic degradation tests, the weight loss of hydrogels was dependent on the cross-linker contents and the enzyme concentration.      

Specific assay for homoserine and its lactone in Pisum sativum. Preparation of homoserine hydroxamic acid

The existence of homoserine lactone in Pisum sativum seedlings is demonstrated. L-Homoserine lactone reacts with hydroxylamine, at neutral or alkaline pH, to form homoserine hydroxamic acid. Procedures are described for preparing L-homoserine lactone and L-homoserine hydroxamic acid. The hydroxamic acid yields a color with maximum absorbance at 492 nm with Fe³⁺ in 0.25 N HCl. This reaction permitted assay for total homoserine and homoserine lactone. Six-day old Pisum sativum seedlings, with cotyledons removed, were extracted with 90% ethanol. Evaporation of the ethanol and addition of Na₂SO₄ solution and toluene and centrifugation removed protein lipids and esters. After clarification with activated charcoal, homoserine lactone content was estimated by reaction with NH₂OH and Fe³⁺ reagents. For total homoserine, protein precipitation was with 2 N HCl and toluene. Evaporation to dryness at 60 °C under vacuum converted all homoserine to the lactone. The values found for total homoserine (μmols/g, wet weight) and preformed lactone (%) with the various growth media used were as follows: nitrate 87.4 (14.7%), NH₂OH 75.2 (6.3%), water 70.5 (7.9%), urea 56.4 (18.9%). Acetic anhydride added to homoserine hydroxamic acid forms acetohydroxamic acid, which yields a color with maximum absorbance at 505 nm with Fe³⁺. This color reaction is seven times as sensitive as the reaction of Fe³⁺ with homoserine hydroxamic acid itself.     

Production and evaluation of monoclonal antibodies for alphafetoprotein

Human AFP was used as an antigen for the development of monoclonal antibodies by the hybridoma technique. Balb/c mice were immunized with highly purified AFP. The preparation of I¹²⁵-AFP was carried out by lactoperoxidase oxidation method, preparation of AFP standards was carried out from cord sera. The antibody titer of the serum was tested by RIA-AFP system. The spleen of the immunized Balb/c was fused with Sp₂0 mouse myeloma cells. The cells from the positive hybridomas were cloned twice using limiting dilution method. Eleven stable clones were thus established for secreting monoclonal antibodies to AFP. Cells in this growth phase were chosen for freezing.     

Development and characterization of methacrylate-based hydrazide monoliths for oriented immobilization of antibodies

Convective interaction media (CIM; BIA Separations) monoliths are attractive stationary phases for use in affinity chromatography because they enable fast affinity binding, which is a consequence of convectively enhanced mass transport. This work focuses on the development of novel CIM hydrazide (HZ) monoliths for the oriented immobilization of antibodies. Adipic acid dihydrazide (AADH) was covalently bound to CIM epoxy monoliths to gain hydrazide groups on the monolith surface. Two different antibodies were afterwards immobilized to hydrazide functionalized monolithic columns and prepared columns were tested for their selectivity. One column was further tested for the dynamic binding capacity.     

N-vinyl-2-pyrrolidone and methacrylonitrile copolymers: nuclear magnetic resonance characterization

The copolymers of N-vinyl-2-pyrrolidone and methacrylonitrile (V/N) were prepared by free radical bulk polymerisation. The copolymer composition was determined from the quantitative ¹³C{¹H} NMR spectrum. The reactivity ratios for N-vinyl-2-pyrrolidone (V) and methacrylonitrile (N) were found to be r_V=0.04, r_N=1.56. The complete spectral assignment of the overlapped and complex carbon and proton NMR spectra were done with the help of two dimensional ¹³C–¹H Heteronuclear Single Quantum Correlation Spectroscopy (HSQC) and Total Correlation Spectroscopy (TOCSY). Distortionless Enhancement by Polarization Transfer (DEPT) was used to differentiate between the methylene, the methine and the methyl carbon resonance signals of the copolymers.     

Syntheses of N-alkyl, N,N-dialkyl, and N-(4-substituted phenyl) O-ethyl thioncarbamates: a kinetic study

Abstract  

The kinetics of the syntheses of N-alkyl, N,N-dialkyl, and N-(4-substituted phenyl) O-ethyl thioncarbamates from sodium ethyl xanthogenacetate, ten alkylamines, and eight substituted anilines were studied at 25, 30, 35, and 40 °C. The reactions were found to follow second-order kinetics. The kinetic (Arrhenius) parameters, such as the activation energy and the frequency factor, as well as the Eyring parameters, such as the standard entropy, the standard Gibbs energy, and the standard enthalpy of activation, were calculated from the second-order rate constants. The mechanism of the reaction was postulated based on the kinetic studies presented and the optimization of the reaction mechanism using the MOPAC PM6 semi-empirical method.     

Photochemical reactions of electron-deficient olefins with N,N,N′,N′-tetramethylbenzidine via photoinduced electron-transfer

Photoinduced electron transfer reactions of several electron-deficient olefins with N,N,N′,N′-tetramethylbenzidine (TMB) in acetonitrile solution have been studied by using laser flash photolysis technique and steady-state fluorescence quenching method. Laser pulse excitation of TMB yields ³TMB* after rapid intersystem crossing from ¹TMB*. The triplet which located at 480 nm is found to undergo fast quenching with the electron acceptors fumaronitrile (FN), dimethyl fumarate (DMF), diethyl fumarate (DEF), cinnamonitrile (CN), -acetoxyacrylonitrile (AAN), crotononitrile (CrN) and 3-methoxyacrylonitrile (MAN). Substituents binding to olefin molecule own different electron-donating/withdrawing powers, which determine the electron-deficient property (π-cloud density) of olefin molecule as well as control the electron transfer rate constant directly. The detection of ion radical intermediates in the photolysis reactions confirms the proposed electron transfer mechanism, as expected from thermodynamics. The quenching rate constants of triplet TMB by these olefins have been determined at 510 nm to avoid the disturbance of formed TMB cation radical around 475 nm. All the values approach or reach to the diffusion-controlled limit. In addition, fluorescence quenching rate constants have been also obtained by calculating with Stern–Volmer equation. A correlation between experimental electron transfer rate constants and free energy changes has been explained by Marcus theory of adiabatic outer-sphere electron transfer. Disharmonic k_q values for CN and CrN in endergonic region may be the disturbance of exciplexs formation.     

Stabilizing N-tosyl-2-lithioindoles with bis(N,N′-dimethylaminoethyl) ether—a non-cryogenic procedure for lithiation of N-tosylindoles and subsequent addition to ketones

A practical procedure suitable for large scale lithiation of N-tosylindoles and subsequent addition to ketones is described. Bis(N,N′-dimethylaminoethyl) ether was found to stabilize 2-lithio-N-tosylindole 1A at −25 °C [The temperatures cited are internal temperatures unless otherwise stated]. Addition of this reagent allows the lithiation of N-tosyl indoles and subsequent addition to ketones to operate at −25 °C, a temperature suitable for large scale reactions.     

Preparation and coordination chemistry of N-diallylaminodiphenylphosphine and N-allylaminobis(diphenyl)phosphine

N-diallylaminodiphenylphosphine and N-allylaminobis(diphenyl)phosphine have been prepared by reaction of the appropriate amine with Ph₂PCl. The coordination chemistry of these phosphines has been studied with a range of metals [Pd, Pt, Rh, Ir, Au, Ru, Mo]. Whilst the majority of the complexes are simple bidentate P,P donors, in the case of N-diallylaminodiphenylphosphinecoordination can involve the CC group or the nitrogen centre.     

Enzyme-immunoassay for intact hCG using monoclonal antibodies

Conclusions The high sensitivity and specificity makes the assay a useful tool for surveillance of very early pregnancies as well as for monitoring of tumour patients.

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Enzymimmunoassay für intaktes hCG unter Verwendung monoklonaler Antikörper

     

Transition metal complexes of N-1-tosylcytosine and N-1-mesylcytosine

The syntheses as well as chemical and X-ray structural characterization of dichlorobis[1-(p-toluenesulfonyl)cytosine]copper(II) (2), its solvated pseudopolymorph containing two methanol molecules (3), dichlorobis[1-(p-toluenesulfonyl)cytosine]cadmium(II) (4), 1-methanesulfonylcytosine (6) and its copper complex dichlorobis(1-methanesulfonylcytosine)copper(II) (7) are described. In addition, spectroscopic studies of dichlorobis[1-(p-toluenesulfonyl)cytosine]cobalt(II) (5), as well as of dichlorobis(1-mesylcytosine)cadmium(II) (8) are presented. Pseudopolymorphs 2 and 3, as well as their 1-mesylcytosine analog 7, reveal square-planar coordination spheres, almost ideal in the case of 2, but considerably distorted in the case of 3 and 7. In all cases, the Cu(II) ion is coordinated by two endocyclic N3 atoms from two ligand molecules and by two chlorine atoms. The analogous coordination sphere was found in complex 4, where Cd(II) lies in the center of a slightly distorted tetrahedron formed by two endocyclic N3 atoms and by two chlorine atoms.