Fluorescence polarization immunoassay of colchicine

In this study, a fluorescence polarization immunoassay (FPIA) technique was developed to determine colchicine (COL), an alkaloid of noxious plants of the order Liliales that is used in a number of medications to treat gout. An optimal combination of the polyclonal antibody and the antigen labelled with fluorescein isothiocyanate (FITC) was selected. Conditions for the competitive interaction of the antigen in the tested samples and its fluorophore conjugate (COL–FITC) with anti-COL antibodies were optimised, and the analytical characteristics of the assay were determined. The developed FPIA was characterised by a detection limit of 1.8 ng/mL and a detectable analyte concentration range of 4.1–74.3 ng/mL. The duration of the analysis was 10 min. The applicability of the developed FPIA for quality control of ready-made drug formulations and for the estimation of COL content in various matrices (urine, milk), with recovery values ranging from 79–108%, was demonstrated

Production of polyclonal antibodies and development of fluorescence polarization immunoassay for sulfanilamide

N-sulfanil-4-aminobutyric acid (SAB), which mimics common parts of the sulfonamides' structure, was synthesized and used to produce antibodies to sulfanilamide. Rabbit polyclonal antibodies have been raised using SAB conjugates with ovalbumin (OVA) or soybean trypsin inhibitor (STI). The immunogen based on SAB-STI could yield higher affinity anitbodies against sulfanilamide. The same SAB derivative was used for synthesis of a fluorescein-labeled tracer with fluorescein-thiocarbamyl ethylendiamine. A fluorescence polarization immunoassay (FPIA) for sulfanilamide was developed. The limits of detection sulfanilamide were 0.07, 0.10, and 0.07 μg mL -1 for water, diluted milk, and precipitated milk samples, respectively. The developed FPIA exhibited sensitivities below the respective maximal residue limits (MRLs) for individual sulfonamides (0.1 μg mL -1). The coefficients of variation of results for milk samples were lower than 5%. Total time for simple sample pretreatment and measurement is about 10 min for one sample. High cross-reactivity with sulfaguanidine (96%), sulfamethoxypyridazine (75%), and sulfachloropyridazine (28%), which have planar structures, could be suitable for simultaneous detection of these sulfa drugs in milk and developed fluorescence polarization immunoassay could be classified as a group-selective assay. Copyright © Taylor & Francis, Inc

Production of polyclonal antibodies and development of fluorescence polarization immunoassay for sulfanilamide

N-sulfanil-4-aminobutyric acid (SAB), which mimics common parts of the sulfonamides' structure, was synthesized and used to produce antibodies to sulfanilamide. Rabbit polyclonal antibodies have been raised using SAB conjugates with ovalbumin (OVA) or soybean trypsin inhibitor (STI). The immunogen based on SAB-STI could yield higher affinity anitbodies against sulfanilamide. The same SAB derivative was used for synthesis of a fluorescein-labeled tracer with fluorescein-thiocarbamyl ethylendiamine. A fluorescence polarization immunoassay (FPIA) for sulfanilamide was developed. The limits of detection sulfanilamide were 0.07, 0.10, and 0.07 μg mL -1 for water, diluted milk, and precipitated milk samples, respectively. The developed FPIA exhibited sensitivities below the respective maximal residue limits (MRLs) for individual sulfonamides (0.1 μg mL -1). The coefficients of variation of results for milk samples were lower than 5%. Total time for simple sample pretreatment and measurement is about 10 min for one sample. High cross-reactivity with sulfaguanidine (96%), sulfamethoxypyridazine (75%), and sulfachloropyridazine (28%), which have planar structures, could be suitable for simultaneous detection of these sulfa drugs in milk and developed fluorescence polarization immunoassay could be classified as a group-selective assay. Copyright © Taylor & Francis, Inc

A triphenylcyclopropenylium mass tag: synthesis and application to ultrasensitive LC/MS analysis of amines

Thiol adducts of triphenylcyclopropenylium undergo efficient heterolytic dissociation in electrospray (ESI) or laser desorption ionization (LDI) mass spectrometry giving rise to a prominent signal of an aromatic C3Ph3+ cation.</p

Production of polyclonal antibodies and development of fluorescence polarization immunoassay for sulfanilamide

N-sulfanil-4-aminobutyric acid (SAB), which mimics common parts of the sulfonamides' structure, was synthesized and used to produce antibodies to sulfanilamide. Rabbit polyclonal antibodies have been raised using SAB conjugates with ovalbumin (OVA) or soybean trypsin inhibitor (STI). The immunogen based on SAB-STI could yield higher affinity anitbodies against sulfanilamide. The same SAB derivative was used for synthesis of a fluorescein-labeled tracer with fluorescein-thiocarbamyl ethylendiamine. A fluorescence polarization immunoassay (FPIA) for sulfanilamide was developed. The limits of detection sulfanilamide were 0.07, 0.10, and 0.07 μg mL -1 for water, diluted milk, and precipitated milk samples, respectively. The developed FPIA exhibited sensitivities below the respective maximal residue limits (MRLs) for individual sulfonamides (0.1 μg mL -1). The coefficients of variation of results for milk samples were lower than 5%. Total time for simple sample pretreatment and measurement is about 10 min for one sample. High cross-reactivity with sulfaguanidine (96%), sulfamethoxypyridazine (75%), and sulfachloropyridazine (28%), which have planar structures, could be suitable for simultaneous detection of these sulfa drugs in milk and developed fluorescence polarization immunoassay could be classified as a group-selective assay. Copyright © Taylor & Francis, Inc

Design of the blood group AB glycotope

Although the nature of the blood groups A and B has been comprehensively studied for a long time, it is still unclear as to what exactly is the epitope that is recognized by antibodies having AB specificity, i.e. monoclonal and polyclonal antibodies which are capable of interacting equally well with the antigens GalNAcalpha 1-3(Fucalpha 1-2)Gal (A trisaccharide) and Galalpha 1-3(Fucalpha 1-2)Gal (B trisaccharide), but do not react with their common fragment Fucalpha 1-2Gal. We have supposed that besides Fucalpha 1-2Gal, A and B antigens have one more shared epitope. The trisaccharides A and B are practically identical from the conformational point of view, the only difference being situated at position 2 of Galalpha residue, i.e. trisaccharide A has a NHAc group, whereas trisaccharide B has a hydroxyl group (see formulas). We have hypothesized that the AB-epitope should be situated in the part of the molecule that is opposite to the NHAc group of GalNAc residue. In order to test this hypothesis we have synthesized a polymeric conjugate in such a way that de-N-acetylated A-trisaccharide is attached to a polymer via the nitrogen in position C-2 of the galactosamine residue. In this conjugate the supposed AB-epitope should be maximally accessible for antibodies from the solution, whereas the discrimination site of antigens A and B by the antibodies should be maximally hidden due to the close proximity of the polymer. Interaction with several anti-AB monoclonal antibodies revealed that a part of them really interacted with the synthetic AB-glycotope, thus confirming our hypothesis. Moreover, similar antibodies were revealed in the blood of healthy blood group 0 donors. Analysis of spatial models was performed in addition to identify the hydroxyl groups of Fuc, Galalpha, and Galbeta residues, which are particularly involved in the composition of the AB-glycotope