Development of an Evidence biochip array kit for the multiplex screening of more than 20 anthelmintic drugs.

Anthelmintic drugs are used in clinical and veterinary practice for the treatment of infections caused by parasitic worms. Their extensive use in food-producing animals can cause the presence of residues in food. For consumer protection it is necessary to monitor the levels of anthelmintic residues to ensure that they remain within the legally permitted maximum acceptable concentrations. For this purpose, the use of multiplex screening methods is advantageous. Biochip array technology allows the simultaneous determination of multiple analytes from a single sample at a single point in time. This study reports the development of an Evidence biochip array for the multiplex screening of anthelmintic drugs. Simultaneous competitive chemiluminescent immunoassays are employed. The solid support and vessel is the biochip, which contains an array of discrete test sites. The assays were applied to the semiautomated bench-top analyser Evidence Investigator. The aminobenzimidazoles assay detected aminomebendazole, albendazole 2-aminosulphone and aminoflubendazole, the avermectins assay detected emamectin benzoate, eprinomectin, abamectin, ivermectin and doramectin, the benzimidazoles assay detected albendazole sulphone, albendazole, albendazole sulphoxide, oxibendazole, oxfendazole and flubendazole, the thiabendazole assay detected cambendazole, thiabendazole and 5-hydroxythiabendazole and the triclabendazole assay detected ketotriclabendazole, triclabendazole and triclabendazole sulphoxide. The limits of detection ranged from 0.3 ppb (aminobenzimidazoles) to 2.0 ppb (levamisole) in milk and from 0.15 ppb (aminobenzimidazoles) to 6.5 ppb (levamisole) in tissue. The average recovery range was 71-135 %. This multianalytical approach on a biochip platform is applicable to the screening of more than 20 anthelmintic drugs in different food matrices, leading to consolidation of tests and enhancement of the test result output.

The use of pulsed amperometry combined with ion-exclusion chromatography for the simultaneous analysis of ascorbic acid and sulfite.

Initial attempts to monitor ascorbic acid and sulfite, in a beer matrix, by combining ion-exclusion chromatography with a pulsed amperometric detector using a single applied voltage to the platinum working electrode, were unsuccessful. Alternatively, good chromatograms for the separation of the two antioxidants were achieved utilizing a standard, amperometric cell. However, remarkably superior results were observed when this standard cell was operated in a pulsed mode and cleaning cycles were continually applied throughout the analysis. The working electrode stability and precision have been examined. Preliminary spike recovery data indicate acceptable accuracy for the method. Comparisons of this method to standard reference methods are currently ongoing.

Hemoglobin-based blood substitutes: characterization of five pyridoxal 5'-phosphate derivatives of hemoglobin.

Six fractions (I-VI) of pyridoxal 5'-phosphate (PLP) hemoglobin (Hb), prepared by the method of De Venuto and Zegna [J. Surg. Res., 34 (1983) 205] have been isolated and purified by anion-exchange high-performance liquid chromatography (HPLC). Total phosphate analyses indicate that I is HbA, II and III are double-labelled, IV and V are tetra-labelled and VI contains 6 mol of phosphorus per mol of hemoglobin. The purified components have been resolved into their alpha and beta chains by preparative reversed-phase HPLC using a macroporous C4 support. Phosphate analyses indicate that the beta chains of II, III and IV each contain one phosphate per chain while the beta chains of V and VI each contain two phosphates. The alpha chains of IV and VI were found to be monophosphate-labelled. Reversed phase HPLC analysis of the tryptic peptides of the beta chains indicates that the label is bound exclusively to the 1-valine residue in II, III and IV while both the 1-valine and the 82-lysine are labelled in V and VI. Similarly, modification of the 1-valine residue of the alpha chains of IV and VI was detected. Components II and III have the same molecular formula. Evidence is presented which shows that they are interconvertible and that they correspond to the PLP2-Hb species and component V is Benesch's PLP4-Hb [J. Biol. Chem., 257 (1983) 1320 and references cited therein]. Component IV is III with one additional PLP per alpha chain and similarly VI is V with monolabelled alpha chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and partial characterization of pyridoxal 5'-phosphate hemoglobins by high-performance liquid chromatography as a quality-control method for hemoglobin-based blood substitutes.

Pyridoxal 5'-phosphate hemoglobin (PLP-Hb), prepared from hemoglobin and a four-fold excess of pyridoxal 5'-phosphate by the method of De Venuto and Zegna [J. Surg. Res., 34 (1983) 205], has been chromatographically resolved into six components via a quaternary ammonium monobead support. On an analytical scale, the separations have been found to be rapid (ca. 50 min) and highly reproducible. The results also indicate that the preparation of PLP-Hb yields a reproducible product ratio. The potential of the analytical method for the routine quality control of blood substitutes derived from PLP-Hb is discussed. All five of the PLP derivatives (components II-VI), isolated and purified via a combination of conventional and preparative monobead anion-exchange chromatography, gave single peaks when analyzed by high-performance liquid chromatography. Total phosphate analyses indicated that components II and III each contain two PLPs per Hb, IV and V four and VI six.

Effect of fasting on thyroxine, 3,5,3'-triiodothyronine, and cortisol concentrations in serum of dogs.

The present study was designed to compare basal and stimulated concentrations of 3,5,3'-triiodothyronine (T3), thyroxine (T4), and cortisol in serum of dogs fasted 12 or 18 hours (to represent overnight fasting) or 24 or 36 hours (to represent prolonged inappetence) with those of dogs that were not fasted. Twenty-five adult Beagle bitches were allotted to 5 experimental fasting groups (0, 12, 18, 24, and 36 hours). Blood samples for hormonal analyses were obtained 4, 3, 2, and 1 hour before food was removed; at the time of food removal; 1 hour after food was removed; and every 2 hours during experimental fasting until 0800 hours on the day fasting ended. Dogs were injected with 5 IU of thyrotropin, IV, and 2.2 IU of adrenocorticotropin/kg, IM, to evaluate thyroidal and adrenocortical endocrine reserves. Additional blood samples were collected 0.5, 1, 2, 3, and 4 hours after injections were given. Serum concentrations of T3, T4, and cortisol were determined by validated radioimmunoassays. Body weights and ages of the dogs and food consumption during a 2-hour preliminary feeding period before dogs were fasted did not differ among fasting groups. Length of fasting did not affect serum concentrations of T3 or T4 in dogs at 12, 18, 24, or 36 hours after food was removed. Mean serum concentrations of cortisol in dogs fasted 12 or 24 hours were lower than those in dogs that were not fasted. Serum concentrations of the hormones after thyrotropin and adrenocorticotropin were injected were not affected by fasting.(ABSTRACT TRUNCATED AT 250 WORDS)

Pneumatic orthosis for paraplegic patients: functional evaluation and prescription considerations.

Fourteen paraplegic patients were studied during ambulation training, 11 with pneumatic orthoses and with conventional metal orthoses, and 3 with pneumatic orthoses only. The purposes of the study were to establish whether pneumatic orthoses could be used by paraplegic patients for effective ambulation, whether these devices presented any advantages or disadvantages over conventional orthoses and how their use would affect the expected level of rehabilitation and independence. As far as application, standing up, sitting down and ambulation within a rehabilitation center are concerned, similar skills and levels of independence were obtained with both types of orthoses. Reduced tendency for orthostatic hypotension, availability, light weight and better endurance make the pneumatic orthosis especially suitable for early ambulation training, but several mechanical problems, mainly its inflation with motorized and heavy compressor units, limit its usefulness for community ambulation.