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The results of an in-house laboratory validation of a microbiological method for the screening of antibiotic residues in milk are presented. The sensitivity of this five-plate test, called Screening Test for Antibiotic Residues (STAR), was established by the analysis of milk samples spiked with 66 antibiotics at eight different concentrations. Ten different groups of antibiotics were studied: macrolides, aminoglycosides, cephalosporins, penicillins, quinolones, tetracyclines, sulphonamides, lincosamides, phenicolated and miscellaneous drugs. It was shown that 21 antibiotics were detected by the STAR protocol at or below the maximum residue limit (MRL), and that a further 27 drugs could be detected at levels from the MRL up to four times the MRL. The sensitivity of the STAR protocol was at or below the MRL for three macrolides, one tetracycline, two aminoglycosides, some sulphonamides, half of the beta-lactams, quinolones, lincosamides, trimethoprim and baquiloprim. Moreover, the STAR protocol was at least twice as sensitive as conventional methods for macrolides, quinolones and tetracyclines. The other antibiotics had limits of detection between four and 150 times the MRL. Each plate was preferentially sensitive for one or two families of antibacterials: the plate Bacillus cereus for tetracyclines, the plate Escherichia coli for quinolones, the plate Basillus subtilis for aminoglycosides, the plate Kocuria varians for macrolides, and the plate Bacillus stearothermophilus for sulphonamides and beta-lactams. This method has been used routinely on a day-to-day basis to direct the physicochemical confirmation towards one or two families of antibiotics. Considering the high cost of liquid chromatography coupled with tandem mass spectrometry detection analyses, the reduction of the range of antibiotics to test for confirmation is a significant gain in time and money.

cdc2 and several related genes encode the catalytic subunits of cyclin-dependent kinases, which have been implicated in a number of cellular processes, including control of cell division. As a first step in exploring their function in plants, we isolated four cdc2-related genes from Antirrhinum. Two genes, cdc2a and cdc2b, encode proteins that contain a perfectly conserved PSTAIRE motif characteristic of cdc2 homologs, whereas the products of the two remaining genes, cdc2c and cdc2d, appear to represent a new subclass of proteins that have so far only been identified in plants. Transcripts of these novel genes were localized in isolated cells dispersed throughout actively dividing regions of the inflorescence. This localization is consistent with accumulation that is specific to particular phases of the cell cycle. Correlating cell labeling with nuclear condensation and double-labeling experiments using cdc2 and histone H4 as probes indicated that cdc2c transcripts accumulate during S phase as well as during the G2 and M transition, whereas cdc2d expression was specific to the G2 and M phases. All cells labeled with cdc2d also contained cdc2c label, Indicating that expression of cdc2d completely overlapped with that of cdc2c. Transcripts of cdc2a and cdc2b were detected in all cells within actively dividing regions, but at levels that were only slightly higher than those observed in nondividing areas. These transcripts did not appear to accumulate in a cell cycle-specific fashion. The genes cdc2a and cdc2b were able to partially complement a yeast cdc2 mutation, although all four genes appeared to interfere with the sizing mechanism of yeast cells. We propose that plants contain at least two classes of cdc2-related genes that differ in structure, expression, and perhaps function.

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Biosensor immunoassays were developed recently for antibiotics with an established maximum residue limit (MRL). In this study, according to the regulatory banning of chloramphenicol (CAP) use for food producing animals, the main objectives were: the specificity of the biosensor assay and the lowest detection limit possible. The assay was based on the inhibition of the binding of polyclonal antibodies against CAP to immobilized CAP on a sensor chip by CAP in solution. The response varied inversely with the antibiotic concentration in the sample. Two different antibodies and two immobilization protocols were tested. As in ELISA tests the antibody influenced the assay performances. Moreover, we showed that particular care should be concentrated on the immobilization step because it is a critical point in the assay development. Three different protocols were developed in milk. The best assay was obtained with antibody 1 in milk on the CAP base surface because of its very low detection limit (0.1 m g l -1 ) and the decreased consumption of antibody (four times less than on the CAP surface). This assay is rapid (3 min/run), sensitive, and specific for CAP and CAP glucuronide. It could be integrated in a multi-residue screening test and applied to other matrices (bile, urine, meat).

The STAR protocol is a Five Plate Test (FPT) developed several years ago at the 24 Community Reference Laboratory (CRL) for the screening of antimicrobial residues in milk and muscle. This paper presents the validation of this method according to the European 26 Decision 2002/657/EC and to an internal guideline for validation. A validation protocol based 27 on "simulated tissues" and on a list of 16 representative antimicrobials to be validated has 28 been implemented in our laboratory during several months for the STAR protocol. The 29 performance characteristics of the method have been determined (specificity, detection 30 capabilities CCβ, applicability, ruggedness). In conclusion, the STAR protocol is applicable to 31 the broad spectrum detection of antibiotic residues in muscles of different animal species 32 (pig, cattle, sheep, poultry). The method has good specificity (false positive rate 4%). The 33 detection capabilities have been determined for 16 antibiotics from different families in 34 relation to their respective Maximum Residue Limit (MRL): beta-lactams (penicillins and 35 cephalosporins ≤ MRL), tetracyclines (≤MRL and ≤ 2.5 MRL), macrolides (2 MRL), 36 quinolones (≤ 2 MRL), some sulphonamides (≤ 3 MRL), trimethoprim (2 MRL). However, the 37 sensitivity of the STAR protocol towards aminoglycosides (> 8 MRL) and florfenicol (≤ 10 38 MRL) was unsatisfactory (>> MRL). The two objectives of this study have been met: firstly to 39 validate the STAR protocol according to the European decision 2002/657/EC, then 40 demonstrate that the validation guideline (Anonymous 2010) developed to implement this 41 decision is applicable to microbiological plate tests even for muscle. The use of "simulated 42 tissue appeared as a good compromise between spiked discs with antibiotic solutions and 43 incurred tissues. In addition, the choice of a list of representative antibiotics allowed the 44 reduction of the scope of the validation, which was already costly in time and effort.

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