Sensitive enzyme immunoassay of cephalexin residues in milk, hen tissues, and eggs

A sensitive enzyme immunoassay for cephalexin (CEX) was developed using the rabbit antiserum to CEX, beta-D-galactosidase-labeled CEX, and a double-antibody separation method. The immunogen of CEX was prepared by coupling the amino group of CEX to thiol groups introduced into bovine serum albumin by the use of N-(m-maleimidobenzoyloxy)succinimide as a cross-linker. Highly titered antiserum to CEX was produced in rabbits immunized with the immunogen. Enzyme labeling of CEX with beta-D-galactosidase was done by using N-(gamma-maleimidobutyryloxy)succinimide as the cross-linker. The limit of detection was 30 ng CEX/mL sample solution. Application of the method to CEX drug residues detected 30 ng/mL in milk, 60 ng/g in egg yolk, and 400 ng/g in hen tissue.     

Residues of the lampricides 3-trifluoromethyl-4-nitrophenol and niclosamide in muscle tissue of rainbow trout

Rainbow trout (Oncorhyncus mykiss) were exposed to the (14)C-labeled lampricide 3-trifluoromethyl-4-nitrophenol (TFM) (2.1 mg/L) or niclosamide (0.055 mg/L) in an aerated static water bath for 24 h. Fish were sacrificed immediately after exposure. Subsamples of skin-on muscle tissue were analyzed for residues of the lampricides. The primary residues in muscle tissue from fish exposed to TFM were parent TFM (1.08 +/- 0.82 nmol/g) and TFM-glucuronide (0.44 +/- 0.24 nmol/g). Muscle tissue from fish exposed to niclosamide contained niclosamide (1.42 +/- 0.51 nmol/g), niclosamide-glucuronide (0.0644 +/- 0.0276 nmol/g), and a metabolite not previously reported, niclosamide sulfate ester (1.12 +/- 0.33 nmol/g).     

Glyphosate tolerant canola meal is equivalent to the parental line in diets fed to rainbow trout

Two separate studies were conducted to evaluate the utility of glyphosate tolerant canola (GTC) as a feed ingredient in diets fed to rainbow trout. In the first study, two forms of GTC were compared to a parental line, Westar. In the second study, one line of GTC was reevaluated to Westar. In each study, processed canola meals were incorporated at 5, 10, 15, or 20% of the dry diet and a diet containing no canola was fed for comparison. All diets were fed to triplicate groups of fish in each study. In the first study, weight gain, feed efficiency (FE), protein efficiency ratio (PER), and protein retention (PR) were not significantly different in fish fed either Westar or GT200 at any level of substitution. Fish fed GT73 exhibited a gradual reduction in weight gain, FE, and PER as the level of GTC increased. However, the only significant reduction was in weight gain of fish fed 20% GT73 as compared to fish fed 5% GT73. Because of an error in preparing samples prior to the experiment, samples GT200 and GT73 were essentially equivalent in composition. The differences were explained by differences in processing temperatures that occurred after the sample mixing error occurred. In the second study, mean weight gain, PR, and survival were not significantly different among forms of canola. FE and PER values were significantly lower in fish fed 15% Westar as compared to fish fed 10% Westar; other FE and PER values were not significantly different. On the basis of these results, GTC processed into a toasted meal and incorporated into diets for rainbow trout is equivalent to a parental line of canola.     

Fatty acid pattern, oxidation product development, and antioxidant loss in muscle tissue of rainbow trout and Dicentrarchus labrax during growth

The levels of hydrophilic, lipophilic, and enzymatic antioxidants, the oxidative damage to lipids and proteins, and the fatty acid patterns of triglyceride and phospholipid fractions were assayed in fresh muscle tissue of rainbow trouts (Oncorhynchus mykiss) and sea basses (Dicentrarchus labrax) during aging, to investigate the correlation between oxidative stress and aging processes in fish. The present studies suggests that lipid peroxidation and accumulation of oxidized proteins during in vivo aging are most likely to be linked with an age-dependent decline of lipophilic antioxidants (CoQH(2), CoQ, and vitamin E) and vitamin C contents in muscle tissue, whereas fish aging is not linked to a decline in antioxidant enzymes and reduced glutathione levels. Lipophilic antioxidant and vitamin C levels represent a reliable marker of oxidative stress during aging, and their determination might be useful for the assessment of fish age.     

Rapid loss of lampricide from catfish and rainbow trout following routine treatment

Rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus) were exposed to 3-trifluoromethyl-4-nitrophenol (TFM) and Bayluscide (niclosamide) during a sea lamprey control treatment of the Ford River, located in the upper peninsula of Michigan. Caged fish were exposed to a nominal concentration of 0.02 mg/L of niclosamide for a period of approximately 12 h. Samples of fillet tissue were collected from each fish species before treatment and at 6, 12, 18, 24, 48, 96, and 192 h following the arrival of the block of chemical at the exposure site. The fish were dissected, homogenized, extracted, and analyzed by high-performance liquid chromatography. The major residues found in the fillet tissues were TFM and niclosamide. Niclosamide concentrations were highest 12 h after arrival of the chemical block for rainbow trout (0.0395 +/- 0.0251 microg/g) and 18 h after arrival of the chemical block for channel catfish (0.0465 +/- 0.0212 microg/g). Residues decreased rapidly after the block of lampricide had passed and were below the detection limits in fillets of rainbow trout within 24 h and channel catfish within 96 h after the arrival of the lampricide.     

Development and application of solvent-free extraction for the detection of aflatoxin M1 in dairy products by enzyme immunoassay

The official methods for the quantification of aflatoxin M1 in dairy products (cheese and yogurt) include extraction into dichloromethane or chloroform, evaporation of the solvent, partitioning of the reconstituted residue with hexane, and subsequent analysis. To secure a rapid and inexpensive screen for aflatoxin M1 contamination, a sensitive competitive ELISA, using a rabbit polyclonal antibody, was developed for measuring aflatoxin M1 in milk and used in a comparative study for measuring the extraction efficiency of aflatoxin M1 in aqueous or organic solvent buffers using yogurt samples. An aqueous sodium citrate solution was found to be suitable for extracting aflatoxin M1, thus eliminating the need for organic solvents. The citrate extraction proved to be efficient (recovery ranged from 70 to 124%) in fortified samples of very different kinds of dairy products, including yogurt and six types of cheese. Fourteen yogurt and cheese samples were extracted with citrate solution and analyzed by ELISA. A good correlation was observed (y=0.95x-0.59, r2=0.98) when the data were compared with those obtained through the official method, across a wide range of aflatoxin M1 contaminations (10-200 ng/kg).     

Physiological responses of rainbow trout Salmo gairdneri exposed to plastic lake inlet and outlet stream waters

In Plastic Lake, Ontario, stocked rainbow trout (Salmo gairdneri) have failed to survive, one endemic fish species has become extinct and annual fish kills included up to five species, but especially pumpkinseeds (Lepomis gibbosus). The potential toxicity of Plastic Lake water was assessed by holding (hatchery) rainbow trout in the major inlet stream, the outlet, and in a portion of the outlet stream acidified to the pH of inlet No. 1. Stress on rainbow trout was assessed by measuring plasma and muscle concentrations of Na ⁺ Cl^?, and K⁺, plus gill A1 concentration. Trout held in Plastic Lake inlet No. 1 showed a rapid loss of plasma Na⁺ from 138 to 85 meq.L^?1and Cl^? from 120 to 75 meq.L^?1 within 24 hr. Gill A1 concentration increased from 20 to 105 μg.g^?1 dry weight. Trout held in the outlet steam showed only slight loss of plasma Na⁺ and Cl^? and no accumulation of Al on the gills. Trout held in the acidified outlet showed a significant loss of ions with plasma Na⁺ depressed from 140 to 115 meq.L^?1 and plasma Cl^? from 125 to 95 meq.L^?1over 24 hr. Gill Al concentration increased from 18 to 30 μg.g^?1 dry weight. The differences in stress response of rainbow trout held in the inlet and acidified outlet are likely due primarily to the difference in Al species concentrations in the two waters.     

Two-dimensional gel electrophoresis detection of protein oxidation in fresh and tainted rainbow trout muscle

Protein oxidation is evaluated in rainbow trout muscle by labeling protein carbonyls with 2,4-dinitrophenyl hydrazine (DNPH) followed by immunoblotting of proteins separated by SDS-PAGE or two-dimensional gel electrophoresis (2D-GE). The carbonylation level is accessed on proteins in a whole muscle homogenate or proteins soluble in a high-salt or low-salt buffer. Spoilage-related changes in carbonylation are followed in the high-salt-protein and low-salt-protein fractions by 2D immunoblotting, which reveals increases regarding total number and intensity of carbonylation in both protein fractions for fish kept at room temperature for 48 h. The major amount of carbonylated proteins is found among the high-salt-soluble proteins, and this protein fraction is also responsible for the biggest increase in carbonylation during fish tainting. The results give an estimate of the level of protein carbonylation in rainbow trout and reveal that oxidation increases for a distinct number of proteins during tainting.     

Determination of niclosamide residues in rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus) fillet tissue by high-performance liquid chromatography

Bayluscide [the ethanolamine salt of niclosamide (NIC)] is a registered piscicide used in combination with 3-(trifluoromethyl)-4-nitrophenol (TFM) to control sea lamprey populations in streams tributary to the Great Lakes. A high-performance liquid chromatography (HPLC) method was developed for the determination of NIC residues in muscle fillet tissues of fish exposed to NIC and TFM during sea lamprey control treatments. NIC was extracted from fortified channel catfish and rainbow trout fillet tissue with a series of acetone extractions and cleaned up on C(18) solid-phase extraction cartridges. NIC concentrations were determined by HPLC with detection at 360 and 335 nm for rainbow trout and catfish, respectively. Recovery of NIC from rainbow trout (n = 7) fortified at 0.04 microg/g was 77 +/- 6.5% and from channel catfish (n = 7) fortified at 0.02 microg/g was 113 +/- 11%. NIC detection limit was 0.0107 microg/g for rainbow trout and 0.0063 microg/g for catfish. Percent recovery of incurred radioactive residues by this method from catfish exposed to [(14)C]NIC was 89.3 +/- 4.1%. Percent recoveries of NIC from fortified storage stability tissue samples for rainbow trout (n = 3) analyzed at 5 and 7.5 month periods were 78 +/- 5.1 and 68 +/- 2.4%, respectively. Percent recoveries of NIC from fortified storage stability tissue samples for channel catfish (n = 3) analyzed at 5 and 7.5 month periods were 88 +/- 13 and 76 +/- 21%, respectively.     

免疫分析在农药残留检测中的应用和发展

通过分析免疫分析技术的基础——抗原抗体制备技术,进一步论述了主要免疫方法(放射免疫分析、酶免疫分析、荧光免疫分析、化学发光免疫分析等)的发展过程及其在农药残留检测中的应用。重点介绍了近年来免疫分析的新发展,尤其突出了免疫分析和理化分析联用技术这一痕量分析的新主流。探讨了免疫分析技术存在的问题和免疫分析的未来发展趋势。     

Development of a cell culture/ELISA assay to detect anticoagulant rodenticides and its application to analysis of rodenticide treated grain

This study describes a generic biological screening assay designed to detect anticoagulant rodenticides based on their inhibitory action on the vitamin K epoxide reductase protein complex, resulting in an accumulation of under-carboxylated prothrombin or proteins induced by vitamin K antagonism (PIVKA-II). A combined cell culture/ELISA assay was optimized to measure PIVKA-II production by the human hepatoma HepG2 cell line cultured in the presence of anticoagulant rodenticides. The specificity and sensitivity of the assay was validated using 41 grain extracts containing representative concentrations of rodenticide or appropriate nonrodenticide control compounds. In all cases, PIVKA-II produced by HepG2 cells in response to grain extracts spiked with rodenticides was detected by ELISA, while PIVKA-II was not detected in supernatants collected from cells exposed to nonrodenticide controls. This represents a novel, class-specific biological assay for the detection of anticoagulant rodenticides present in contaminated grain.     

Influence of dietary genistein levels on tissue genistein deposition and on the physical, chemical, and sensory quality of rainbow trout, Oncorhynchus mykiss

Genistein, the primary isoflavone in soybean, is one of the chemical components responsible for some of the off-flavors associated with soy-based foods. The potential effects of genistein on the sensory and chemical quality of fish muscle may affect the full utilization of soybean meal as an alternative protein in aquaculture diets. Fingerling trout fed commercial diets containing 0, 500, 1000, or 3000 ppm pure genistein were analyzed after 6 and 12 months of feeding. Genistein was extracted by enzymatic digestions in Tris buffer and quantified by high-performance liquid chromatography. Moisture, fat, protein, ash, and tristimulus color of the fillets were determined. The extent of lipid oxidation occurring in fillets harvested after 12 months of feeding was studied by measurements of thiobarbituric acid reactive substances (TBARS) after 4 and 8 days of refrigerated storage at 4 degrees C. Triangle tests were performed to determine if there were any detectable sensory differences. A dietary genistein content of 3000 ppm led to the deposition of approximately 5.4 pmol of genistein/mg of fillet. Triangle test panelists were unable to detect any significant (p < or = 0.05) differences between the fillets from trout fed the 0 and 3000 ppm genistein concentrations. Moisture, ash, and protein content were influenced by time of harvest, while color was unaffected. TBARS levels on days 4 and 8 were significantly (p < 0.05) higher in the fillets from the 0 ppm genistein level than in fillets from fish fed dietary genistein.     

Characterization of aroma-active compounds in rainbow trout (Oncorhynchus mykiss) eliciting an off-odor

The aroma-active and off-flavor compounds of cooked rainbow trout (Oncorhynchus mykiss) were analyzed by sensory and instrumental analyses. Sensory analysis shows that the aromatic extract obtained by vacuum steam distillation was representative of rainbow trout odor. To obtain more information on odorants of volatile compounds, analyses were conducted on two gas chromatography columns of different polarities (DB-5 and DB-Wax). The results of the gas chromatography-olfactometry analysis showed that 38 odorous compounds were perceived when the DB-5 column was used and 36 with the DB-Wax column. Of these, 31 with the DB-5 and 28 with the DB-Wax were identified. (E)-2-Nonenal, 2-ethyl-1-hexanol, 2-methylisoborneol, geosmin, 2-methylnaphthalene, and 8-heptadecene were described as off-flavor compounds by the sniffing assessors. The most powerful off-flavor compounds identified in the extract were 2-methylisoborneol and geosmin, which were described as strong musty and earthy odors, respectively.     

Protein and lipid oxidation during frozen storage of rainbow trout (Oncorhynchus mykiss)

This study aimed at investigating protein and lipid oxidation during frozen storage of rainbow trout. Rainbow trout fillets were stored for 13 months at -20, -30, or -80 degrees C, and samples were analyzed at regular intervals for lipid and protein oxidation markers. Lipid oxidation was followed by measuring lipid hydroperoxides (PV), as well as secondary oxidation products (volatiles) using dynamic headspace GC-MS. Free fatty acids (FFA) were measured as an estimation of lipolysis. Protein oxidation was followed using the spectrophotometric determination of protein carbonyls and immunoblotting. Significant oxidation was observed in samples stored at -20 degrees C, and at this temperature lipid and protein oxidation seemed to develop simultaneously. FFA, PV, and carbonyls increased significantly for the fish stored at -20 degrees C, whereas the fish stored at -30 and -80 degrees C did not show any increase in oxidation during the entire storage period when these methods were used. In contrast, the more sensitive GC-MS method used for measurement of the volatiles showed that the fish stored at -30 degrees C oxidized more quickly than those stored at -80 degrees C. Detection of protein oxidation using immunoblotting revealed that high molecular weight proteins were oxidized already at t = 0 and that no new protein oxidized during storage irrespective of the storage time and temperature. The results emphasize the need for the development of more sensitive and reliable methods to study protein oxidation in order to gain more explicit knowledge about the significance of protein oxidation for food quality and, especially, to correlate protein oxidation with physical and functional properties of foods.     

Measurement of triclosan in water using a magnetic particle enzyme immunoassay

A sensitive magnetic particle-based immunoassay to determine triclosan [5-chloro-2-(2,4-dichlorophenoxy)phenol] in drinking water and wastewater was developed. Rabbit antiserum was produced by immunizing the rabbit with 6-[5-chloro-2-(2,4-dichlorophenoxy)phenoxy]hexanoic acid-keyhole limpet hemocyanin. Horseradish peroxidase was conjugated with 4-[3-bromo-4-(2,4-dibromophenoxy)phenoxy]butyric acid via N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The triclosan antibody was coupled to magnetic particles via the NHS/EDC reaction. The antibodies were able to recognize some structurally related polybrominated biphenyl ethers but did not recognize various common pollutants that were less similar to the hapten. The ELISA could detect triclosan in standard solution (25% methanol/H2O v/v) at 20 ppt and its metabolite, methyl-triclosan, at 15 ppt. Water samples from different treatment stages were prepared to contain 25% methanol and analyzed directly without any sample extraction or preconcentration. The results showed that recoveries were >80% and the % CV was <10%, demonstrating the assay was both accurate and precise. Application of the triclosan ELISA to water treatment plants showed that tap water at various purification stages had low concentrations of triclosan (<20 ppt) and required an increased sample size for appropriate detection and measurement. Application of ELISA to the wastewater treatment plants (WWTP) demonstrated high concentrations of triclosan (in general, >3000 ppt in water entering the WWTP) with the levels decreasing as the water proceeded through the processing plant (<500 ppt at outflow sewage). The ELISA measurement was shown to be equivalent to the more specific GC-MS analysis on a number of wastewater treatment samples with a high degree of correlation, with the exception of a few samples with very high triclosan concentrations (>5000 ppt). Measurement of methyl-triclosan (in WWTP) using GC-MS demonstrated the levels of this compound to be low. In summary, a rapid, sensitive, accurate, and precise magnetic particle-based immunoassay has been developed for triclosan analysis, which can serve as a cost-effective monitoring tool for various water samples.     

Effects of hen egg yolk immunoglobulin in passive protection of rainbow trout against Yersinia ruckeri

Anti-Yersinia ruckeri egg yolk immunoglobulin (IgY) was transferred to egg yolk after immunization of White Leghorn hens with formalin-killed whole cells of serovar 1 (RS1154) and serovar 2 (RS1153)Y. ruckeri and its lipopolysaccharide (LPS). The IgY was specific for its homologous LPS in western immunoblot, whereas some protein bands were commonly recognized, even by IgY from eggs of unimmunized hens. Purified LPS from both Y. ruckeri serovar types 1 and 2 had a very poor immunogenicity. The IgY activity was stable when processed into pellet form by a microbial transglutaminase treatment and showed a considerable resistance against acid pepsin for at least 2 h. Feeding specific anti-serovar 1 Y. ruckeri IgY to fish either before or after immersion infection produced marginal reductions in mortalities and in intestine infection. The same IgY did passively protect rainbow trout against infection when administered by intraperitoneal injection 4 h before an immersion challenge.     

Gossypol and gossypolone enantiomers in tissues of rainbow trout fed low and high levels of dietary cottonseed meal

Gossypol is an antifertilizing agent in males and females. However, gossypol and its metabolite, gossypolone, have also gained interest because of their anticarcinogenic activities. This paper examines for the first time both enantiomers of tissue gossypol and gossypolone in mature rainbow trout fed two diets containing low (15%) and high (60%) levels of cottonseed meal (CM) for 9 months. The gossypol concentration was highest in liver followed by kidney, intestine, testis, blood plasma, stomach, and muscle. Gossypol was detected in muscles of fish fed low- and high-CM diets (0.31 +/- 0.03 and 1.95 +/- 0.59 microg of total gossypol/g, wet basis, respectively). The (+)-gossypol enantiomer was predominantly retained in all tissues. The ratio of (-)- to total gossypol ranged from 30 to 44% in fish fed the high-CM diet and from 23 to 30% in fish fed the low-CM diet except for muscle tissue (44%). Higher gossypolone concentrations were found in intestine than in liver. Gossypolone, however, was not detected in blood plasma, muscle, and testis of fish fed the low-CM diet. The ratio of gossypolone to gossypol was highest in muscle (1.75), followed by intestine (1.59), stomach (1.50), kidney (0.43), liver (0.34), testis (0.28), and blood plasma (0.27). This study indicated that the retention of the (-)-gossypol enantiomer is dependent on dietary concentrations and that the oxidative conversion of gossypol to gossypolone occurs more actively in the digestive tract and muscle than in other tissues in rainbow trout.     

Comparison ofDaphnia magna,rainbow trout and bacterial-based toxicity tests of Ontario Hydro aquatic effluents

Over a one year program of intensive monitoring of effluents from Ontario Hydro's nuclear, fossil and hydroelectric generating facilities, theDaphnia magna and rainbow trout,Oncorhynchus mykiss, acute toxicity tests correlated well, with 61 % of the toxic effluents toxic to both species. If the effluent was toxic to only one of the test species it was generally toxic toD. magna, with from 23 to 57% of the toxic effluents toxic toD. magna only. The greater sensitivity ofD. magna to boiler blowdown effluent likely resulted from a combination of the low conductivity of boiler blowdown effluent and the smaller size and greater surface to volume ratio ofD. magna relative to rainbow trout.D. magna were also particularly susceptible to oil/water separator samples, with the daphnids frequently observed to be caught at the surface/water interface. These observations suggest that an accumulation of organic material at the air/water interface was responsible for the mortality ofD. magna. In subsequent tests, we also examined the relationship between theD. magna acute toxicity test and a bacterial-based assay (Toxi-Chromotest®) for several toxic effluents from Ontario Hydro stations to determine if bacterial-based tests could provide similar information in less time with smaller sample volumes. TheD. magma acute toxicity test did not correlate well with the bacterial-based Toxi-chromotest®. In particular, many of the samples which were toxic toD. magna were not toxic to the Toxi-chromotest® assay. The poor correlation between theD. magna and Toxi-chromotese® likely relates to both the relatively low toxicity of many of the effluent samples, and the fact that in many cases toxicity likely resulted from relatively simple combinations of inorganic toxicants. Accordingly, the Toxi-Chromotest® assay would not seem suitable as a surrogate for theD. magna acute toxicity test for our effluents.