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

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

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

Convenient syntheses of phosphinic analogues of γ-aminobutyric- and glutamic acids

Three-steps, one-pot synthesis of 2-amino-4-(hydroxyphosphinyl)butyric acid from dibutyl ester of vinylphosphinic acid was carried out with an overall yield of 66%. 3-aminopropylphosphinic acid was prepared from allylamine in three steps with an overall yield of 56%. These improved protocols allowed to obtain these commercially unavailable phosphinic analogues of glutamic acid and GABA for testing on potential molecular targets

Fine Tuning of Pyrene Excimer Fluorescence in Molecular Beacons by Alteration of the Monomer Structure

Oligonucleotide probes labeled with pyrene pairs that form excimers have a number of applications in hybridization analysis of nucleic acids. A long excited state lifetime, large Stokes shift, and chemical stability make pyrene excimer an attractive fluorescent label. Here we report synthesis of chiral phosphoramidite building blocks based on (<i>R</i>)-4-amino-2,2-dimethylbutane-1,3-diol, easily available from an inexpensive d-(−)-pantolactone. 1-Pyreneacetamide, 1-pyrenecarboxamide, and DABCYL derivatives have been used in preparation of molecular beacon (MB) probes labeled with one or two pyrenes/quenchers. We observed significant difference in the excimer emission maxima (475–510 nm; Stokes shifts 125–160 nm or 7520–8960 cm^–1) and excimer/monomer ratio (from 0.5 to 5.9) in fluorescence spectra depending on the structure and position of monomers in the pyrene pair. The pyrene excimer formed by two rigid 1-pyrenecarboxamide residues showed the brightest emission. This is consistent with molecular dynamics data on excimer stability. Increase of the excimer fluorescence for MBs after hybridization with DNA was up to 24-fold