Enzyme-linked immunosorbent assay for the determination of 20(S)-protopanaxatriol

A highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) method has been developed for the determination of 20(S)-protopanaxatriol (PPT), one of the major aglycones of dammarane-type ginseng saponins. Polyclonal antibodies raised against ginsenoside F₁ (GF₁)-bovine serum albumin showed high reactivities to PPT and GF₁, whereas they exhibited minor or even no cross-reactivities to other ginsenosides and protopanaxadiol (0.19%). The working range of this method extends from 50 pg ml⁻¹ to 20 ng ml⁻¹ of PPT. The assay reported here has been validated against an HPLC technique using PPT-containing samples and was shown to correlate closely (γ=0.993). This ELISA could be a useful tool for the determination of PPT contained in biological fluids and plant materials.     

Rapid high-performance isoelectric focusing of monoclonal antibodies in uncoated fused-silica capillaries

Rapid (<5 min) high-performance isoelectric focusing was performed in uncoated fused-silica capillaries to resolve isoforms of monoclonal antibodies and to determine their isoelectric points (pI). The methodology involved the use of a 32 cm (effective length 9 cm)×50 μm I.D. uncoated capillary. (Hydroxypropyl)methyl cellulose was used as an additive to suppress analyte–wall interaction and to precisely control electroosmotic flow so that focusing and mobilization of focused zones past detector occur simultaneously. Urea was included in the separation medium to solubilize the antibodies that precipitated at their point of focusing. The methods with and without urea used ampholytes pH 5–8 to generate a demonstrable linear gradient between pH 5.4 and pH 7.2, based on the separation of various protein standards. Reproducibility [<2% (R.S.D.)] of the migration times (corresponding to the detectable isoforms of the antibodies) was obtained by using two sets of reagents and capillaries on three consecutive days. pI values determined from day-to-day with a reference standard were shown to vary by only 0.01 pH unit. The described capillary isoelectric focusing methods provided a rapid, simple and reproducible way of monitoring micro-heterogeneity and pI of the murine monoclonal antibodies investigated.     

Biocatalysis and biorecognition in nonaqueous media. Some perspectives in analytical biochemistry

Biocatalysis and, to a lesser extent, biorecognition in non-aqueous media (including organic solvents as well as supercritical fluids and gases) constitute at present an exciting research area which has already demonstrated its biotechnological potential in numerous, varied applications. Less attention, however, has been paid to its analytical possibilities, even though many advantages have been postulated and a wide range of poorly water-soluble analytes are present in samples (or waste materials) from food and drink, petrochemical, pharmaceutical, military and other industries. The main approaches, developed in recent years to exploit the use of enzymes, antibodies or antibody mimics in water-restricted environments for analytical purposes, as well as possible future directions are briefly discussed.     

Novel functional activities of anti-dna autoantibodies from sera of patients with lymphoproliferative and autoimmune diseases

DNA-hydrolyzing activity of IgG autoantibodies from sera of patients with various types of lymphoproliferative diseases was investigated. The association of DNA-hydrolyzing activity with the antibody (Ab) fraction has been proved by newly developed affinity-capture assay. Study of abzyme incidence in blood tumors and systemic lupus erythematosis (SLE) revealed linkage of anti-DNA Ab catalysts to mature B-cell tumors, and increased probability of DNA-abzymes formation on the background of autoimmune manifestations. These data suggest possible similarity between mechanisms of abzyme formation in SLE and B-cell lymphomas. A new mechanism of formation of DNA-specific catalytic Abs has been proposed based on the increased crossreactivity of polyclonal DNA-abzymes to DNA-depleted nuclear matrix proteins. The possibility of the abzyme production as Ab to the energetically destabilized ground state of the antigen has been discussed. Preliminary results were obtained that indicate the complement-independent cytotoxicity of anti-DNA autoantibodies isolated from blood of patients with SLE and chronic lymphocytic leukemia.     

Application of recombinant phage display antibody system in study of Codakia orbicularis gill proteins

We used the recombinant phage display antibody system (RPAS) to obtain chimeric single-chain fragment variable (ScFv) antibodies to gill proteins of the white clam Codakia orbicularis (Linné, 1758). After three rounds of selection on immunotubes loaded with total gill protein extract, recombinant phages exhibiting antibodies to gill proteins were isolated and tested by enzyme-linked immunosorbent assay (ELISA). Clones exhibiting a high affinity for the mollusk proteins were selected for production of soluble ScFv antibodies, which were purified for subsequent analysis. ScFv antibodies exhibited a reaction specific for a protein whose molecular mass was about 15,000 Daltons and that was detected by the antigen capture technique followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting.     

Nucleophilic proteolytic antibodies

Proteolytic antibodies appear to utilizecatalytic mechanisms akin to nonantibody serine proteases, assessed from mutagenesis and protease-inhibitor studies. The catalytic efficiency derives substantially from the ability to recognize the ground state with high affinity. Because the proteolytic activity is germline-encoded, catalysts with specificity for virtually any target polypeptide could potentially be developed by applying appropriate immunogens and selection strategies. Analysis of transition-state stabilizing interactions suggests that chemical reactivity ofactive-site serine residues is an important contributor to catalysis. A prototype antigen analog capable of reacting covalently with nucleophilic serine residues permitted enrichment of the catalysts from a phage-displayed lupus light-chain library. Further mechanistic developments in understanding proteolytic antibodies may lead to the isolation of catalysts suitable for passive immunotherapy of major diseases, and elicitation of catalytic immunity as a component of prophylactic vaccination.     

Development of an Enzyme Immunoassay for the Determination of the Herbicide Metsulfuron-Methyl Based on Chicken Egg Yolk Antibodies

Abstract

The development of an indirect competitive enzyme immunoassay for the sulfonylurea herbicide metsulfuron-methyl (MSM) is described. In contrast to traditional antibody generation in mammals, this extremely sensitive method is based on chicken egg yolk antibodies (IgY). They were raised in laying hens using an MSM-derivative-BSA hapten as immunogen. With a 1:10000 dilution of the antibody solution and a coating antigen (MSM-derivative-KLH) concentration of 10 μg L^?1 the IC₅₀ value achieved for the target analyte was 0.4 μg L^?1. The least detectable dose was established at 13 ng L^?1. Cross-reactivity was tested with 5 structurally related compounds, where only sulfometuron showed a significant binding. The ELISA was tested with spiked tap and surface water samples. This paper, for the first time, demonstrates the production of high-affinity IgY antibodies for a herbicide compound.     

Tuning Tetrazole Photochemistry for Protein Ligation and Molecular Imaging

Photochemistry provides a wide range of alternative reagents that hold potential for use in bimolecular functionalisation of proteins. Here, we report the synthesis and characterisation of metal ion binding chelates derivatised with disubstituted tetrazoles for the photoradiochemical labelling of monoclonal antibodies (mAbs). The photophysical properties of tetrazoles featuring extended aromatic systems and auxochromic substituents to tune excitation toward longer wavelengths (365 and 395 nm) were studied. Two photoactivatable chelates based on desferrioxamine B (DFO) and the aza-macrocycle NODAGA were functionalised with a tetrazole and developed for protein labelling with ⁸⁹Zr, ⁶⁴Cu and ⁶⁸Ga radionuclides. DFO-tetrazole ( 1 ) was assessed by direct conjugation to formulated trastuzumab and subsequent radiolabelling with ⁸⁹Zr. Radiochemical studies and cellular-based binding assays demonstrated that the radiotracer remained stable in vitro retained high immunoreactivity. Positron emission tomography (PET) imaging and biodistribution studies were used to measure the tumour specific uptake and pharmacokinetic profile in mice bearing SK-OV-3 xenografts. Experiments demonstrate that tetrazole-based photochemistry is a viable approach for the light-induced synthesis of PET radiotracers.     

Partitioning in Aqueous Two-Phase Systems: Fundamentals,Applications and Trends

Over the last years, aqueous two-phase systems (ATPS) regained an increasing interest due to their potential in the downstream processing of biomolecules. After many years with only a few articles published, a lot of effort and work has been put into studying these systems for the partitioning of a range of compounds including proteins, organic low-molecular weight molecules or metal ions. Although several research and review articles appeared, a background review on ATPS partitioning fundamentals is needed. In this article, partitioning theories and main effects of several important factors for partitioning, such as molecular weight of the polymer, effect of added salts, pH, electrical charges, and temperature on phase diagrams, tie-line lengths, interfacial tension and settling time of the two aqueous phases are extensively reviewed. The trend in ATPS research is given compiling the recent 2008–2013 research articles published in the field.     

Tailored Mobility in a Zeolite Imidazolate Framework (ZIF) Antibody Conjugate**

Zeolitic imidazolate framework (ZIF) hybrid fluorescent nanoparticles and ZIF antibody conjugates have been synthesized, characterized, and employed in lateral-flow immunoassay (LFIA). The bright fluorescence of the conjugates and the possibility to tailor their mobility gives a huge potential for diagnostic assays. An enzyme-linked immunosorbent assay (ELISA) with horseradish peroxidase (HRP) as label, proved the integrity, stability, and dispersibility of the antibody conjugates, LC-MS/MS provided evidence that a covalent link was established between these metal-organic frameworks and lysine residues in IgG antibodies.