Fast and selective determination of total protein in milk powder via titration of moving reaction boundary electrophoresis.

In this paper, moving reaction boundary titration (MRBT) was developed for rapid and accurate quantification of total protein in infant milk powder, from the concept of moving reaction boundary (MRB) electrophoresis. In the method, the MRB was formed by the hydroxide ions and the acidic residues of milk proteins immobilized via cross-linked polyacrylamide gel (PAG), an acid-base indicator was used to denote the boundary motion. As a proof of concept, we chose five brands of infant milk powders to study the feasibility of MRBT method. The calibration curve of MRB velocity versus logarithmic total protein content of infant milk powder sample was established based on the visual signal of MRB motion as a function of logarithmic milk protein content. Weak influence of nonprotein nitrogen (NPN) reagents (e.g., melamine and urea) on MRBT method was observed, due to the fact that MRB was formed with hydroxide ions and the acidic residues of captured milk proteins, rather than the alkaline residues or the NPN reagents added. The total protein contents in infant milk powder samples detected via the MRBT method were in good agreement with those achieved by the classic Kjeldahl method. In addition, the developed method had much faster measuring speed compared with the Kjeldahl method.

A robust method for testing urinary iodine using a microtitre robotic system.

PROJECT: Iodine deficiency disorders are due to inadequate thyroid hormone production and 2 billion individuals worldwide are estimated to have insufficient iodine intake. Laboratory assessment methods include urinary iodine (UI) concentration, blood FT3, FT4, TSH and Thyroglobulin. The aim of this study was to set up a robust method for testing urinary iodine using a microtitre robotic system. PROCEDURE: The UI method described is based on the Sandell-Kolthoff reaction, which utilizes the catalytic role of iodine in the reduction of ceric ammonium sulphate in the presence of arsenious acid. This method was automated for use on microtitre robotic system. RESULTS: The method was compared with the currently employed manual Sandell-Kolthoff reaction method in our laboratory as reference. The two methods correlated well using weighted Deming regression analysis (slope=1.066, intercept=6.5, r=0.994; n=211). Interassay and intraassay variations were similar to the reference method, but cost analysis indicated a large reduction in costs related to increased throughput, and reduced consumables and labour. CONCLUSIONS: We have successfully adapted UI testing to an automated method, permitting cheaper, faster and robust screening of large numbers of patients and populations. The described protocol can be used on different microtitre robotic systems permitting up to 372 patient samples per run for 4 microtitre plate systems.

[Rapid immunotitration of individual toxins from Androctonus australis venom]. / Immunodosage rapide des toxines individuelles du venin d'Androctonus australis.

Passive immunotherapy against scorpion envenomations is facilitated by the preliminary titration of circulating toxins in envenomed patients. Currently, routinely used ELISA tests allow only the titration of the whole venom, without reference to the toxins which compose the venom and spread variably within the tissue. Taking as a model one of the three toxins responsible for the lethal effects of Androctonus australis hector (Aahl) venom, we developed an ELISA sandwich test based on a fragment of recombining antibody (scFv) consisting of the variable chains of the monoclonal IgG 9C2 coupled to a decapeptide showing high affinity for streptavidine. Conjugate scFvlStrep-tag was prepared by genetic engineering. It was produced in the periplasm of recombining bacteria, in a reproducible way, in a soluble form, at low cost and with an output, after purification, of 0.8 mg/L of bacterial culture. The recombinant protein, of small size (28 kDa), is bifunctional. It preserves a very high affinity for the toxin Aah I (Kd of 2.3 10(-10) M, very close to that of IgG 9C2), yet recognises streptavidine and its conjugate (streptavidine-peroxidase). The titration of the Aahl toxin used an ELISA sandwich test in which the toxin was captured in a specific way by a monoclonal antibody; the immunocomplexes were then detected by recombinant immunoconjugate, thus conferring a high specificity on titration. The test is quick (90 mn), reproducible and sensitive, with a limit of detection of 0.6 toxin (ng.ml-1). This method could be extended to two other lethal toxins of the venom of the scorpion Androctonus australis hector and to those of other species. New perspectives are thus possible for the diagnosis of the envenomations.