Electrochemical magneto immunosensing of antibiotic residues in milk

A novel electrochemical immunosensing strategy for the detection of sulfonamide antibiotics in milk based on magnetic beads is presented. Among the different strategies for immobilizing the class-specific anti-sulfonamide antibody to the magnetic beads--such as those based on the use of Protein A or carboxylate modified magnetic beads - ,the best strategy was found to be the covalent bonding on tosyl-activated magnetic beads. The immunological reaction for the detection of sulfonamide antibiotics performed on the magnetic bead is based on a direct competitive assay using a tracer with HRP peroxidase for the enzymatic labelling. After the immunochemical reactions, the modified magnetic beads can be easily captured by a magneto sensor made of graphite-epoxy composite (m-GEC), which is also used as the transducer for the electrochemical immunosensing. The electrochemical detection is thus achieved through a suitable substrate for the enzyme HRP and an electrochemical mediator. The electrochemical approach is also compared with a novel magneto-ELISA with optical detection. The performance of the electrochemical immunosensing strategy based on magnetic beads was successfully evaluated using spiked milk samples, and the detection limit was found to be 1.44 microg L(-1) (5.92 nmol L(-1)) for raw full cream milk. This strategy offers great promise for rapid, simple, cost-effective and on-site analysis of biological, food and environmental samples.     

Spore germination based assay for monitoring antibiotic residues in milk at dairy farm

Spore germination based assay involves the transformation of dormant spores of Bacillus stearothermophilus 953 into active vegetative cells. The inhibition of germination process specifically in presence of antibiotic residues was used as a novel approach for monitoring target contaminants in milk. The indicator organism i.e., B. stearothermophilus 953 was initially allowed to sporulate by seeding in sporulation medium and incubating at 55 °C for 18 ± 2 h. The spores exhibited a typical chain behavior as revealed through phase contrast microscopy. The minimal medium inoculated with activated spores was incubated at 64 °C for 2-3 h for germination and outgrowth in presence of specific germinant mixture containing dextrose, whey powder and skimmed milk powder added in specific ratio along with reconstituted milk as negative control and test milk samples. The change in color of the medium from purple to yellow was used as criteria for detection of antibiotic residues in milk. The efficiency of the developed assay was evaluated through a surveillance study on 228 samples of raw, pasteurized and dried milks and results were compared with AOAC approved microbial receptor assay. The presence of antibiotic level was 10.08 % at Codex maximum residual limit having false positive result only in 0.43 % of the samples. The results of the present investigation suggest that developed spore based assay can be a practical solution to dairy industry for its application at farm level, milk processing units, independent testing and R & D centres in order to comply with the legal requirements set by Codex.     

High-performance thin-layer chromatography method for inositol phosphate analysis

A simple and inexpensive high-performance thin-layer chromatography (HPTLC) method for the analysis of inositol mono- to hexakisphosphates on cellulose precoated plates is described. Plates were developed in 1-propanol–25% ammonia solution–water (5:4:1) and substance quantities as low as 100–200 pmol were detected by molybdate staining. Chromatographic mobilities of nucleotides and phosphorylated carbohydrates were also characterized. Charcoal treatment was employed to separate nucleotides from inositol phosphates with similar R_F values prior to HPTLC analysis. Practical application of the HPTLC system is demonstrated by analysis of grain extracts from wild type and low-phytate mutant barley as well as phytate degradation products resulting from barley phytase activity.     

Detection of Helicobacter pylori in cow's milk

AIMS: To investigate the existence of Helicobacter pylori in cow's milk as one of the foods which most Japanese children eat. METHODS AND RESULTS: Detection of H. pylori was demonstrated by the semi-nested polymerase chain reaction (PCR), a culture method and electron microscopy. Semi-nested PCR demonstrated the ureA gene of H. pylori in 13 of 18 (72.2%) raw milk samples and in 11 of 20 (55%) commercial pasteurized milk samples. Helicobacter pylori binding immunomagnetic beads with H. pylori-specific goat anti-H. pylori antibody was shown by electron microscopy in both raw and pasteurized milk positive for the ureA gene. Helicobacter pylori was cultured in one raw milk sample, whereas it was not cultured in pasteurized milk samples. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: There is a possibility that cow's milk is a transmission vehicle in childhood H. pylori infection, although we failed to confirm the survival of H. pylori in pasteurized milk.     

Evidence for the presence of several lipases in cow's milk

Skim milks containing sodium chloride (0.75m) were centrifuged at 80000g for 2hr. and portions of the supernatants were submitted to gel filtration on columns of Sephadex G-200. Enzymes in the effluent fractions were assayed titrimetrically for their hydrolytic activities towards tributyrin, triolein and milk-fat emulsions, and triacetin solution. Summation of the measurements gave ratios of activities towards the various substrates similar to those of the original skim milks. Although only partial separation was obtained, five enzymes appeared to be present. They showed some differences in substrate specificity, but all appeared to be lipases in that they hydrolysed the emulsified substrates more rapidly than the dissolved triacetin.     

High-performance thin-layer chromatographic determination of ibuprofen in plasma

A high-performance thin-layer chromatographic (HPTLC) method for quantitation of ibuprofen from plasma is described. The drug was extracted from acidified plasma with hexane-isopropanol (85:15). The mobile phase composition was n-hexane-ethyl acetate-anhydrous acetic acid (75:25:2). Densitometric analysis of ibuprofen was carried out at 222 nm. The calibration curves of ibuprofen in chloroform and in plasma were linear over the range 2–20 μg. The mean values of intercept, slope and correlation coefficient were 0.0422±0.0018, 0.0356±0.0213 and 0.9976±0.0013 for standard curves in chloroform and 0.1044±0.003, 0.8759±0.0213 and 0.9939±0.001 for standard curves in plasma, respectively. The limit of detection of ibuprofen from human plasma (assay sensitivity) was 50 ng and no interference was found from endogenous compounds. The recovery of ibuprofen from human plasma using the described extraction procedure was about 85%. The mean relative standard deviations for within-day and between-day analyses were 2.24 and 2.6% for 5 μg and 3.67 and 3.2% for 15μg ibuprofen concentration, respectively. The method was utilized to monitor the plasma concentration of ibuprofen post administration of sustained release capsules in human patient volunteers.     

High-performance thin-layer chromatographic determination of flurbiprofen in plasma

A high-performance thin-layer chromatographic (HPTLC) method for the assay of flurbiprofen in plasma is reported. The drug was extracted from acidified plasma with hexane–diethyl ether (80:20). The mobile phase composition was n-hexane–ethyl acetate–glacial acetic acid (60:30:10). Densitometric analysis of flurbiprofen was carried out at 247 nm. The calibration curves of flurbiprofen in methanol and in plasma were linear in the range 40–400 ng. The mean values of correlation coefficient, slope and intercept were 0.995±0.003, 0.075±0.002 and 4.39±0.05 for standard curves in methanol and 0.992±0.002, 0.066±0.007 and 3.40±0.72 for standard curves in plasma, respectively. The limit of quantitation for flurbiprofen in human plasma was 40 ng, and no interference was found from endogenous compounds. The recovery of flurbiprofen from human plasma using the described extraction procedure was about 87%. The coefficient of variation for within-day and between-day analyses was 2.53% and 3.96% for 200 ng and 1.76% and 2.30% for 400 ng flurbiprofen concentration, respectively. The method was utilized to monitor plasma concentration of flurbiprofen post administration of sustained release capsules in human patient volunteers.     

High-performance thin-layer chromatographic determination of lamotrigine in serum

A simple and rapid high-performance thin-layer chromatographic (HPTLC) determination of lamotrigine (LTG) in serum is reported. The method involves extraction of the drug by ethyl acetate followed by separation on TLC silica plates using a mixture of toluene-acetone-ammonia (7:3:0.5), as eluting solvent. Densitometric analysis was carried out at 312 nm with lamotrigine being detected at Rf of 0.54. The analytical method has excellent linearity (r=0.998) in the range of 20-300 ng/spot. This assay range is adequate for analyzing human serum, as it corresponds to lamotrigine concentrations measured in human serum from epileptic patients. The method was validated for sensitivity, selectivity, extraction efficiency, accuracy and intra and inter-day reproducibility. The limit of detection and limit of quantification were found to be 6.4 and 10.2 ng, respectively. Good accuracy is reported in the range of 92.06-97.12% and high precision with %CV in range of 0.53-2.59. The method was applied for determination of serum lamotrigine levels in epileptic patients and in pharmacokinetic study of lamotrigine administered orally to rabbits.     

Immunoenzyme analysis of angiogenin in cow's milk]

Competitive enzyme immunoassay based on polyclonal antibodies can be used for determining the content of angiogenin in milk. These polyclonal antibodies had no cross-reactions with ribonuclease or other milk whey proteins. Milk angiogenin levels in samples taken fvom animals of a separate population varied from 2.09 to 4.85 mg/l. Unlike cow milk productivity, the number of calvings affects the milk angiogenin content.     

Thermal inactivation of Bacillus anthracis spores in cow's milk

Decimal reduction time (time to inactivate 90% of the population) (D) values of Bacillus anthracis spores in milk ranged from 3.4 to 16.7 h at 72 degrees C and from 1.6 to 3.3 s at 112 degrees C. The calculated increase of temperature needed to reduce the D value by 90% varied from 8.7 to 11.0 degrees C, and the Arrhenius activation energies ranged from 227.4 to 291.3 kJ/mol. Six-log-unit viability reductions were achieved at 120 degrees C for 16 s. These results suggest that a thermal process similar to commercial ultrahigh-temperature pasteurization could inactivate B. anthracis spores in milk.     

Detection of a new K-casein variant in cow's milk

In German Simmental cattle a new kappa-casein variant was detected by isoelectric focusing, which cannot be distinguished from kappa-casein A by polyacrylamide and starch gel electrophoresis. It was named kappa-casein D. The frequencies of kappa-casein alleles were kappa-Cn A 0.75, kappa-Cn B 0.24, kappa-Cn D 0.01.     

High-performance thin-layer chromatography/mass spectrometry for the analysis of neutral glycosphingolipids

This mini-review summarizes the protocol we have developed for the analysis of neutral glycosphingolipids (GSLs) by high-performance thin layer chromatography (HPTLC)-mass spectrometry (MS). We also present results obtained using this glycolipidomic approach to study neutral GSLs from mouse kidney, spleen, and small intestine. Finally, we discuss what is required for further development of this method, as well as what is expected for the future of glycolipid biology.     

Procedure for simultaneous phenotyping of genetic variants in cow's milk by isoelectric focusing

A method is described which allows the simultaneous separation of all polymorphic protein fractions in cow's milk in one single run. The separation could be achieved by isoelectric focusing in ultrathin-layer polyacrylamide gels. The method is especially suitable for screening purposes because it combines low costs, high resolution and short separation time.     

High-performance thin-layer chromatographic determination of 5-methoxypsoralen in serum from patients

A simple and rapid high-performance thin-layer chromatographic (HPTLC) determination of 5-methoxypsoralen in serum is necessary for the therapeutic survey of patients treated with Puvatherapy (psoralen+UV A). The assay for this biological fluid involves an extraction with heptane-dichloromethane (4:1, v/v). The analytical method is linear from 50 to 250 ng/ml. This assay range is adequate for analysing human serum, as it corresponds to psoralen concentrations measured in serum from patients treated with psoralen and UV A against psoriasis and vitiligo. The limit of detection is 15 ng/ml. The coefficient of variation was less than 7%.     

Novel microbiological system for antibiotic detection in ovine milk

This article presents a microbiological system composed of a “BT” bioassay (Beta-lactams and Tetracyclines) and a “QS” bioassay (Quinolones and Sulfonamides). The “BT” bioassay contains spores of Geobacillus stearothermophilus, bromocresol purple and cloramphenicol in a culture medium (incubation time: 2.45 h), while the “QS” bioassay uses spores of Bacillus subtilis, trifenyltetrazolium – toluidine blue and trimethoprim in a suitable culture medium (incubation time: 5.5 h). The detection capability (CC_β) of 27 antimicrobial agents in ovine milk were determined by logistic regression models. Thus, the “BT” bioassay detects amoxycillin, ampicillin, penicillin “G”, cloxacillin, oxacillin, cephalexin, cefoperazone, ceftiofur, chlortetracycline, oxytetracycline, tetracycline, neomycin, gentamycin and tylosin, while “QS” bioassay detects: ciprofloxacin, enrofloxacin, marbofloxacin, sulfadiazine, sulfadimethoxine, sulfamerazine, sulfamethazine, sulfamethoxazole, sulfathiazole, erythromycin, lincomycin and spiramycin at levels close to their respective Maximum Residue Limits. The simultaneous use of both bioassays detects a large number of antibiotics in milk given each method's adequate complementary sensitivity.