Employment of electrostriction phenomenon for label-free electrochemical immunosensing of tetracycline.

The presented work describes a simple label-free electrochemical immunosensor for the determination of tetracycline (TC). The functioning of the sensor was based on the electrostriction of a antibody-terminated thiol layer self-assembled on a gold electrode surface, serving as a dielectric membrane. The intensity of electrostriction was correlated with the amount of TC captured through an affinity reaction with its specific antibody, and was followed in the form of capacitance-potential curves. The process of the immunosensor construction was optimized using electrochemical impedance spectroscopy (EIS). The chemisorption time of the thiol, the duration of the TCAb immobilization and its concentration were optimized. The developed immunosensor exhibited a linear response in two concentration ranges: from 0.95 to 10 µmol L-1 and from 10 to 140 µmol L-1, with the mean sensitivity of 6.27 nF µmol-1 L (88.67 nF µmol-1 L cm-2) and 0.56 nF µmol-1 L (7.84 nF µmol-1 L cm-2), respectively. The limit of detection was evaluated as 28 nmol L-1. The specificity of the proposed sensor toward other antibiotics, amoxicillin and ciprofloxacin, was examined. The immunosensor was successfully employed to quantify TC in a tablet form and in a matrix of river water.

Validation of the BetaStar® Advanced for Tetracyclines Test Kit for the Screening of Bulk Tank and Tanker Truck Milks for the Presence of Tetracycline Drug Residues.

A validation study was conducted for an immunochromatographic method (BetaStar® Advanced for Tetracyclines) for detection of tetracycline antibiotic residues in raw, commingled bovine milk. The assay was demonstrated to detect tetracycline, chlortetracycline, and oxytetracycline at levels below the FDA tolerance levels but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments. Results of internal and independent laboratory dose-response studies employing spiked samples were in agreement. All three drugs at the approximate 90/95% sensitivity levels were detected in milk collected from cows that had been treated with the specific drug. Selectivity of the assay was 100%, as no false-positive results were obtained in testing 881 control milk samples. Testing the estimated 90/95 sensitivity level for tetracycline (213 ppb), chlortetracycline (272 ppb), and oxytetracycline (180 ppb) and at 1000 ppb for each antibiotic resulted in 100% positive tests for each tetracycline. Results of ruggedness experiments established the operating parameter tolerances for the test. Results of cross-reactivity testing established that the assay detects certain other tetracycline drugs but does not cross-react with any of 32 drugs belonging to seven different drug classes. Abnormally high bacterial or somatic cell counts (SCC) in raw milk produced no assay interference.

Some properties and applications of cell lines and clones established from tet-responsive-SV40 tag mice and mES cell lines.

In this article, 11 cell lines established from transgenic mice and mouse embryonic stem cells (mESC) expressing SV40Tag under control of tetracycline/doxycycline (tet-off, tet-on) are described. Several cell lines were further transfected with a plasmid vector containing genes coding for a cytokine/protein under tet-regulation to obtain tet-co-regulated expression of cytokine/protein. A total of 29 clones and 234 subclones have been established so far. Partial characterization of these tet-responsive cell lines, clones and subclones was performed. Questions related to the rare frequency of establishing permanent cycling cell lines from this source, the unusual expression pattern of SV40Tag protein in the subcellular compartment and the phenotype of 'stemness' of several such cell lines are raised. Some future applications of these cells, related to immunology and transplantation, are discussed.

Computational prediction of antibody binding sites on tetracycline antibiotics: electrostatic potentials and average local ionization energies on molecular surfaces.

Enzyme linked immunosorbent assay (ELISA) was used for the analysis of tetracycline, chlortetracycline, oxytetracycline, and their transformed compounds in environmental water samples. The antibodies employed in ELISA showed high relative affinity for tetracycline, epitetracycline, chlortetracycline, and epichlortetracycline as compared to anhydrotetracycline, epianhydrotetracycline, and anhydrochlortetracycline. The specificity and crossreactivity of these antibodies are discussed in relation to the electrostatic potentials and average local ionization energies computed on the molecular surfaces of tetracycline antibiotics and their transformed compounds with an objective of identifying common features as well as differences that may be related to the experimentally observed variation in cross-reactivity values. The computations were performed at both the HF/STO-3G and HF/6-31+G* levels using the Gaussian 98 program. The results in this study are based upon molecular electrostatic potentials and local ionization energies computed on isodensity molecular surfaces. The surface electrostatic potentials are characterized in terms of a group of statistically defined quantities, which include the average deviation, the positive, negative, and total variances, positive and negative surface extrema, and a parameter indicating the degree of electrostatic balance.

Preparation of anti-tetracycline antibodies and development of an indirect heterologous competitive enzyme-linked immunosorbent assay to detect residues of tetracycline in milk.

Tetracycline (TC) is a broad-spectrum antibiotic used increasingly in animal husbandry to treat diseases or to promote growth as feed additives. To avoid using labor-intensive instrumental methods to detect residues of TC in food and food products, a simple and convenient indirect heterologous competitive enzyme-linked immunosorbent assay (ELISA) method for TC was developed using polyclonal antibody prepared in this study. Three new immunogens, TC-o-tolidine-bovine serum albumin (BSA), TC- 4-aminobenzoic acid-cationized BSA (cBSA), and TC-1,1'-carbonyldiimidazole-cBSA, were synthesized in this research to develop anti-TC antibodies. All antibodies raised in rabbits and coating antigens synthesized were screened and characterized using homologous and heterologous ELISA formats to select the best combination. An optimized ELISA gave an IC50 value of 3.92 mug/mL toward TC in PBS buffer. The specificity of the assay was studied by measuring cross-reactivity of the antibody with the structurally closely related compounds of chlortetracycline (112%) and oxytetracycline (<2%). The recovery rates from the TC-fortified raw milk samples were in the range of 74-116%, while the intra- and interassay coefficients of variation were <14.5 and <25.0, respectively.

Raising antibodies in chickens against primaquine, pyrimethamine, dapsone, tetracycline, and doxycycline.

Antibodies against primaquine, pyrimethamine, dapsone, tetracycline, and doxycycline were raised in chickens inoculated with each drug conjugated to a rabbit albumin carrier. Antibody titres against drug and carrier were highest during week 6 postinoculation. Affinity purified anti-primaquine antibodies did not recognise other drugs, but affinity purified anti-doxycycline and anti-tetracycline antibodies recognised both tetracycline and doxycycline in addition to primaquine. Primaquine was detected in urine from 6 to 12 hours after ingestion of therapeutic doses of the drug by anti-primaquine antibodies in a competitive ELISA. Affinity purified anti-primaquine antibodies detected primaquine in the cytoplasm and localised in organelles in monocytes that had been incubated with therapeutic concentrations of the drug.

A threshold for central T cell tolerance to an inducible serum protein.

We report an inducible system of self Ag expression that examines the relationship between serum protein levels and central T cell tolerance. This transgenic approach is based on tetracycline-regulated expression of a secreted form of hen egg lysozyme, tagged with a murine hemoglobin (Hb) epitope. In the absence of the tetracycline-regulated transactivator, serum levels of the chimeric protein are extremely low (< or = 0.1 ng/ml) and the mice show partial tolerance to both Hb(64-76) and lysozyme epitopes. In the presence of the transactivator, expression increases to 1.5 ng/ml and the mice are completely tolerant. Partial tolerance was further investigated by crossing these mice to strains expressing transgenic TCRs. At the lowest Ag levels, 3.L2tg T cells (specific for Hb(64-76)/I-E(k)) escape the thymus and approximately 10% of CD4(+) splenocytes express the 3.L2 TCR. In contrast, 3A9 T cells (specific for hen egg lysozyme(46-61)/I-A(k)) are completely eliminated by negative selection. These data define a tolerogenic threshold for negative selection of Ag-specific T cells by circulating self proteins that are 100-fold more sensitive than previously demonstrated. They suggest that partial tolerance at extremely low levels of self Ag exposure is the result of a restricted repertoire of responding T cells, rather than a simple reduction in precursor frequency; tolerogenic thresholds are T cell specific.

Affinities of mAbs to Tet repressor complexed with operator or tetracycline suggest conformational changes associated with induction.

We isolated five monoclonal antibodies (mAbs) made against tetracycline repressor (TetR), one against the TetR tetracycline complex (Tc) and two against the TetR-tet operator (tetO) complex. The epitopes of the anti-TetR mAbs are localized in the alpha-helix-turn-alpha-helix motif (HTH), at different sites near the Tc binding pocket and at the dimerization interface. The anti-TetR-Tc and one of the anti-TetR-tetO mAbs recognize epitopes near the Tc binding pocket. The other anti-TetR-tetO mAb binds to an epitope within the HTH. Quantitative immunoprecipitation and competitive ELISA employing TetR, TetR-Tc, or TetR-tetO revealed different affinities of the mAbs for TetR in these functional states. Binding of the two mAbs to epitopes in the HTH was identical for TetR and TetR-Tc indicating the same conformation in both forms. The epitope located in the dimerization interface is bound more strongly in TetR compared to TetR-Tc, supporting the idea of different conformations of that epitope in these forms of TetR. The greatest affinity differences were found for epitopes around the Tc binding pocket. Two anti-TetR mAbs have the highest affinities for free TetR, somewhat reduced affinity for TetR-tetO and the lowest affinities for TetR-Tc. The anti-TetR-Tc mAb has a discontinuous epitope, formed in TetR-Tc, which is less well bound in TetR and not bound in the TetR-tetO complex. One anti-TetR-tetO mAb does not recognize TetR-Tc. Since the epitopes do not overlap with the respective ligand binding sites on TetR, these results are interpreted as conformational differences of the epitopes in these forms of TetR.

Serovars and multiple drug resistant Salmonella sp. isolated from children in Rio de Janeiro-Brazil

Foram caracterizadas sorologicamente e testadas para a susceptibilidade e antimicrobianos, 116 amostras de Salmonella isoladas de coproculturas e hemoculturas efetuadas de crianças durante o período de maio de 1987-junho 1992 no Instituto Fernando Figueira, Brasil. Detectou-se seis sorogrupos (04; 07; 08; 09; 03; 10 e 035) representados por 18 serovares, sendo a S. typhimurium (62,93 por cento) a mais frequente, seguida da S. agona (7,75 por cento). Os antimicrobianos utilizados foram a ampicilina (Ap), cefalotina (Cf), cefoxitina (Cx), ceftriaxona (Cro), pefloxacin (Pf), gentamicina (Ge), amicacina (Am), sulfametoxazoletrimetropim (SxT), cloranfenicol (CL) e tetraciclina (Te). 94.52 por cento das cepas de S. typhimurium representaram determinantes de resistência. Foram encontrados 29 padröes de resistência diferentes, sendo o mais frequente para S. typhimurium o Ap, Cf, Ge, Am, Sxt, Cl, Te, Pf (40,47 por cento) e para os outros serovares o Te (20.83 por cento). A determinaçäo da concentraçäo mínima inibitória realizada para cinco dos antimicrobianos citados evidenciou altos níveis de resistência para Ap e Cl, contrapondo-se a níveis mais baixos obtidos para Ge, Sxt e Cro

[Effect of tetracycline on the immune and hematopoietic systems of intact rabbits]. / Vliiani tetratsiklina na immunnuiu i krovetvornuiu sistemy intaktnykh krolikov.

Tetracycline was administered orally in daily doses of 20 or 40 mg to 50 practically healthy male chinchilla rabbits weighing 2-2.5 kg. The thymus, appendage, spleen, ileocecal, mesenteric and axillary lymph nodes, bone marrow and peripheral blood were examined with the methods of morpho- cytometry. To verify the data the nonparametric criterion U of Wilcoxon-Mann-Whitny and the Student criterion were used. The mean curves reflecting the reaction level in the peripheral immunity system as a whole were plotted with the use of graphical analysis. It was found that tetracycline in doses of 20 and 40 mg induced a significant change in both the central and the peripheral immunity organs. Two trends in the development of the reaction in the system of immunogenesis were observed: (a) activation, transformation and differentiation of immunocompetent cells and (b) destruction of the cells, particular by transforming and proliferating cells. This gave rise to the depletion of the immunity system which was intensified if the drug was used for a prolonged period of time and in higher doses.

Immune responses in rabbits treated with tetracycline.

Cellular and humoral immunological phenomena in rabbits treated with tetracycline were investigated by lymphocyte transformation, leucocyte migration inhibition, and indirect hemagglutination tests. Cellular and/or humoral immunity against tetracycline and/or homologous or autologous rabbit gamma globulin was acquired by about two-thirds of the rabbits, with some also acquiring immunity against homologous tissue antigen after prolonged tetracycline administration. Based on these results, the significance of immune responses to autoantigens by drug administration was discussed in relation to potential agents for the induction of autoimmune diseases.

Tetracycline asthma--a case report.

A mechanic working in the antibiotic capsuling section of a pharmaceutical company developed asthmatic attacks 1 year after starting work. His occupation involved exposure to a variety of chemical agents including tetracycline. He developed immediate weal and flare reaction to the intradermal test and an immediate (type 1) asthmatic response to intradermal, inhalation and oral challenge tests with tetracycline. On leaving the tetracycline plant he became symptom free.

[Effect of tetracycline and oleandomycin on the indices of nonspecific resistance in aseptic inflammation under conditions of changes in the body's reactivity]. / Vliianie tetratsiklina i oleandomitsina na pokazateli nespetsificheskoi rezistentnosti pri aseptichekom vospalenii v usloviiakh izmenenii reaktivnosti organizma

The effect of tetracycline and oleandomycin on the complement titer, lysozyme content, serum bactericidal properties and presence of specific antibiotic antibodies in the blood serum was studied. The latter were shown with the Hoigné reaction under conditions of aseptic inflammation caused against the background of latent tetanus intoxication. It was shown that tetracycline and oleandomycin used in treatment of the animals with aseptic inflammation developed at the background of latent tatanus intoxication induced an increased in the complement titer, lysozyme content and bactericidal properties of the serum. Reduction of the above indices was observed by the 15th-20th day after discontinuation of the drug use. The increase in the factors of non-specific immunity under the effect of tetracycline and oleandomycin in the animals with aseptic inflammation caused against the background of latent tetanus intoxication was accompanied by appearance in the blood serum on non-specific antibodies revealed with the Hoigné reaction. Changed reactivity because of latent tetanus intoxication was accompanied by a delay in the formation of the non-specific antibodies in the blood serum. However, later the rate of their accumulation became higher and as a result the maximum titers of the antibodies were 2-3 times higher than those in the control animals.

[Tetracycline distribution in the body of animals and its effect on cellular interaction in the immune response]. / Raspredelenie tetraysiklinov v organizme zhivotnykh i vliianie ikh na vzaimodeistvie kletok v immunnom otvete

Distribution of tetracyclines and their effect on interaction of the cells in the immune response were studied on pigs and rabbits non-vaccinated, immunized with formolvaccin against paratyphoid fever and experimentally infected with the paratyphoid causative agent. It was found that oxytetracycline and tetracycline administered parentally to the animals formed complexes with the proteins and especially with albumins and gamma-globulins, were rapidly adsorbed by the lymphocytes and consumed by the cells of the reticulo-macro- and micro-phage systems, epithelium of the kidney cannaculi and the cells of the liver parenchyma. Immunomorphological changes accompanied by formation of antibodies to the antibiotics were found in the bloodforming-lymphoid system after repeated parental administrations of the tetracyclines. The titers of the antibodies to oxytetracycline and tetracycline were high by the 5th--14th day after the antibiotic administration and the relatively high levels persisted for 1.5 months. The use of tetracyclines during the induction stage of immunogenesis had a pronounced inhibitory effect on development of immunity against the paratyphoid fever antigen. On the basis of the tetracycline capacity for binding with immunoglobulins and intensive adsorption by the lymphocytes it is possible to suppose that the inhibitory effect of the antibiotics on immunogenesis was connected with their blocking the receptors of T- and B-lymphocytes.

[Tetracycline and the primary immune response]. / Tetratsiklin i pervichnyi immunnyi otvet

The effect of tetracycline on proliferation of plate- and rossette-forming cells in the spleen and mesenterial lymph nodes of rats in case of the primary immune response with respect to the sheep erythrocytes was studied using the methods of Jerne, Notta and Zaalberg. It was found that tetracycline acted as an inhibitor when it was administered 48 hours before introduction of the antigen. When they were administered simultaneously its inhibiting effect decreased. When tetracycline was injected 24 hours after the antigen administration it had induced stimulation of immunogenesis. The inhibiting effect of the antibiotic was more pronounced with respect to the plate-forming cells and less pronounced with respect to the rossette-forming cells. The inhibiting effect of the antibiotic was lower in the mesenterial lymph nodes than in the spleen. The problem of independent antibody genesis in the lymph nodes is discussed.

[Effect of antibiotics on the indices on nonspecific immunity]. / Vliianie antibiotikov na pokazateli nespetsificheskogo immuniteta

Multiple (for 14 and 20 days) administrations of antibiotics, such as tetraolean, tetracycline and penicillin from the beginning of the animal immunization (rabbits and albino rats) with typhoid fever and candidosis vaccines inhibited activity of both the non-specific and the specific immunity.