Establishment of a competitive ELISA for detection of florfenicol antibiotic in food of animal origin.

Florfenicol (FF) is a synthetic antibiotic with a broad antibacterial spectrum and the high therapeutic effectiveness that has been developed specifically for veterinary use. Obviously, FF adulterated in animal supplies is one of essential global concerns. A competitive ELISA for the detection of florfenicol in food of animal origin (swine, chicken, and fish) is described. Influence of immunoconjugate structure on the assay sensitivity and specificity was investigated. The new ELISA showed much lower than the MRPLs for FF at 100-3,000 mg kg(-1) in the European Communities and the sensitivity of our ELISA method was superior to that described in other reports. According to the test preparation record, the limit of detection of the developed ELISA performed on meat species was 0.3 µg kg(-1) (IC50 value 1.9 µg kg(-1)). The method developed permits FF concentrations to be determined in the range 0.3-24.3 µg kg(-1). A low cross-reactivity with florfenicol amine (FFA), thiamphenicol (TAP), and chloramphenicol (CAP) was displayed (16.2%, 9.5%, and 9.4%, respectively). Recovery in different food samples (swine, chicken, and fish) averages between 87-115%. The method can be applied for inspection of animal supplies for trace florfenicol residues.

Development of a competitive ELISA for the detection of a furaltadone marker residue, 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), in cultured fish samples.

This report describes an enzyme-linked immunosorbent assay (ELISA) for tissue-bound metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) and the application to residue analysis in cultured fish samples. The residue is monitored as a marker for the drug furaltadone. The assay enables the detection of protein bound AMOZ in the form of a 2-nitrophenyl derivative (2-NP-AMOZ) in sample supernatant or extract after acid hydrolysis and derivatization with o-nitrobenzaldehyde. Polyclonal rabbit antibodies were produced with a new immunogen hapten, 2-NP-HXA-AMOZ. The new ELISA had adequate analytical sensitivity (IC(50) value 0.325 µg kg(-1); limit of detection 0.1 µg kg(-1)) to determine a trace of AMOZ residue and had a high selectivity. Recoveries of AMOZ fortified at the levels of 0.1, 0.5 and 1.0 µg kg(-1) ranged from 89.8 to 112.5% with coefficients of variation of 12.4-16.2% over the range of AMOZ concentrations studied. The results obtained with the ELISA correlated well with those obtained by commercial test kits for 150 tested samples (r=0.984). The results suggest that the developed ELISA is a highly specific, accurate, and sensitive method suitable for high throughput screening for AMOZ residues.

Human monoclonal antibody GNX-8 directed to extended type 1 chain: Specific binding to human colorectal cancer.

We observed previously that two carbohydrate epitopes, extended type 1 chain Le(a)-Le(a) and Le(b)-Le(a), are expressed strongly in human gastric or colorectal cancer and cell lines derived therefrom, but their expression in human normal colorectal cells is highly limited. A monoclonal antibody, termed GNX-8, was established through immunization of "KM mice" with colonic cancer cell line Colo205, and with purified Le(b)-Le(a) glycosphingolipid, followed by screening human IgG directed to this antigen. KM mice possess human chromosome fragments and are capable of producing human immunoglobulin. GNX-8 reacted specifically with extended type 1 chain epitope Le(b)-Le(a), bound to all five colonic cancer cell lines so far tested, and displayed strong complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). The antigens defined by GNX-8, expressed in Colo205 cells, were: (i) glycosphingolipids with epitope Le(b)-Le(a), whose reactivity was abolished upon defucosylation; (ii) glycoproteins with molecular mass range from 32 to >175 kDa, which were depleted in cells cultured in the presence of benzyl-alpha-GalNAc, indicating that these epitopes are O-linked glycans.Immunohistological reactivity of GNX-8 at 1 mug/ml, applied on tissue sections from colorectal and various other types of cancer, was much stronger than that with various normal cells and tissues. GNX-8 reactivity with normal cells required a much higher concentration (150 mug/ml), and this reactivity was based on cross-reaction with non-extended, normal blood group Le(b) antigen. Growth of subcutaneous xenograft of human colonic cancer cells, Colo205 or DLD-1, in nude mice or SCID mice, was strongly inhibited by administration of GNX-8. These observations, taken together, indicate that antibody GNX-8, directed specifically to Le(b)-Le(a) antigen, provides a novel direction of immunotherapy for human colorectal cancer. (c) 2009 UICC.

Screening of salbutamol residues in swine meat and animal feed by an enzyme immunoassay in Taiwan.

An ELISA was developed for routine examination for extensive monitoring and screening programs for the residues of salbutamol in swine serum, animal feed, meat, and meat-related products destined for human consumption in Taiwan. Objectives of the study were to investigate the use of a new immunoassay for the detection of salbutamol residues in swine meat and animal feed samples, and to compare with a commercial kit in field test screens. A fast, simple and reliable sample preparation method for the determination of salbutamol was established. Field trials with 222 swine meat and 120 animal feed samples that were taken from local meat markets, auction markets and feed mills. The application and the results of two ELISA kits (a homemade and a commercial kit) for the screening of salbutamol were presented. Adopting 2microg kg(-1) salbutamol as a cut-off value for swine meat, the commercial beta-agonist ELISA had a sensitivity of 85.3% and a specificity of 95.2% versus GC-MS at a cut-off of 2microg kg(-1). The homemade salbutamol ELISA had a sensitivity of 100% and a specificity of 90.9% and gave no false-negative rate results. Furthermore, adopting 20microg kg(-1) salbutamol as a cut-off value for animal feed, both the commercial and homemade ELISA showed 100% sensitivity and 100% specificity of the assays. In conclusion, a sensitive, specific salbutamol polyclonal antibody-based ELISA has been developed that could serve as a rapid screening assay, and the detection of positive samples at the place of sampling can result in more effective control of the illegal use of beta-agonists.

Development and residue screening of the furazolidone metabolite, 3-amino-2-oxazolidinone (AOZ), in cultured fish by an enzyme-linked immunosorbent assay.

A sensitive and specific polyclonal enzyme-linked immunosorbent assay (ELISA) for the determination of tissue-bound metabolite 3-amino-2-oxazolidinone (AOZ) is described. The procedures allow for the detection of protein-bound AOZ in the form of a 2-nitrophenyl derivative (2-NP-AOZ) in the sample supernatant or extract after acid hydrolysis and derivatization with o-nitrobenzaldehyde. The polyclonal rabbit antibodies were produced with the immunogen hapten, 2-NP-HXA-AOZ, and the 50% inhibition values (IC(50)) of 0.14 microg kg(-1) of AOZ was achieved with the most sensitive antibody A0505. The mean lower detection limit of the ELISA method is about 0.025 microg kg(-1). According to the test preparation record, the detection limit is 0.1 microg kg(-1), which is well below the minimum required performance limits (MRPLs) for tissue-bound residues of AOZ at 1 microg kg(-1) in the European Communities. In the present study, we investigated the use of homemade ELISA, a new immunoassay, to monitor the presence of the furazolidone marker residue in 370 samples of cultured fish. Adopting 0.3 microg kg(-1) AOZ as a cutoff value, the ELISA has a sensitivity of 100% and a specificity of 98.5% versus high-performance liquid chromatography-mass spectrometry (HPLC-MS) at a cutoff of 0.3 microg kg(-1) and gives no false-negative rate results. From the practical point of view, the homemade kit could be advantageously used for the screening of large groups of animal-edible tissue samples and the kit employed has good reliability even in routine application for the control of the illegal use of the drug.

Development and fast screening of salbutamol residues in swine serum by an enzyme-linked immunosorbent assay in Taiwan.

The analysis of salbutamol in swine serum is the more practical basis for large scale surveillance programs in Taiwan. Objectives of the study were to develop a new assay and to compare with a commercially available kit in field test screens. A simple and reliable enzyme-linked immunosorbent assay (ELISA) to monitor the presence of beta-agonist, salbutamol, in 1,358 field samples of swine serum that were collected from local meat markets was described. The method proved to be suitable and sensitive for the detection of beta-agonist residues caused by growth promoting dosage. The limit of detection of the developed ELISA directly performed on diluted serum was 0.25 ng/mL. The application and the results of two ELISA kits (homemade and commercially available) for the screening of salbutamol were presented. For further confirmation, all samples that showed to be ELISA positive for salbutamol residues were analyzed by GC-MS. Adopting 1 ng/mL salbutamol as a cutoff value, the commercial beta-agonist ELISA had a sensitivity of 89.2% and a specificity of 86.7% versus GC-MS at a cutoff of 1 ng/mL. The homemade salbutamol ELISA had a sensitivity of 81.1% and a specificity of 98.6% and gave a low proportion of false-positive rate results. The reliability of the developed kit in terms of the percentage of false-positive rate results is evaluated. In conclusion, a sensitive, specific salbutamol ELISA has been developed that could serve as a rapid screening assay, and the detection of positive samples at the place of sampling can result in more effective control of the illegal use of beta-agonists.

Production of antibodies for selective detection of malachite green and the related triphenylmethane dyes in fish and fishpond water.

This study provides a practical method for production of the antibodies against malachite green (MG) and its primary metabolite leucomalachite green (LMG). Two ELISA kits are constructed with the MG and LMG antibodies for detection of the residual MG and LMG in fish muscle and fishpond water. The detection limit is established at the level of 0.05 microg/L for both MG and LMG. Our ELISA kits show the advantages of good specificity, high sensitivity, and convenience in rapid screening of MG and LMG residues. The sample of fishpond water, without extraction or prior preparation, is directly assayed by the ELISA kit. More then 80 fish samples can be simultaneously tested in a kit. The toxic crystal violet and its metabolite leucocrystal violet of illegal use in aquaculture are detected by our prepared MG and LMG antibodies, whereas the antibodies do not cross-react with common antibiotics, sulfonamides, and benzene derivatives.