孔雀石绿间接竞争ELISA检测方法的建立

采用无色孔雀石绿(Leucomalachite green,LMG)单克隆抗体建立了水产品中孔雀石绿(Malachite green,MG)残留的间接竞争ELISA(ciELISA)检测方法。结果表明,LMG-McAb最佳稀释倍数为1∶80 000,包被抗原最佳质量浓度为0.80μg/mL;竞争反应时的LMG理想稀释液为40%乙腈水溶液;标准曲线呈线性相关,相关系数R2=0.9823,最适检测范围1 ng/mL~256 ng/mL,最低检测限为1.29 ng/mL,批内和批间变异系数分别为4.307%和4.566%;鳗鱼肉样的平均添加回收率为90%~110%;该检测方法与隐性结晶紫、孔雀石绿、结晶紫的交叉反应(CR%)分别为40.67%、13.50%和5.89%,与其他抗生素无交叉反应。     

氯霉素竞争ELISA检测方法的研究

用抗氯霉素的MAb 2G2建立了检测氯霉素的竞争ELISA,对主要影响因素进行了研究,其回归方程为y=0.3522x+0.5939,相关系数为RZ=0.9906,检测线性范围为0.098 ng/mL～25 ng/mL,半数抑制浓度(IC50)为1.848 ng/mL.检测限为0.135 ng/mL.与氯霉素琥珀酸钠交叉反应率为143%,与其它结构类似物和常见抗菌素的交叉反应率均小于0.01%.批内和批间变异系数分别为4.98%和9.42%,牛奶样的平均添加回收率为101.31%.所建立的检测氯霉素竞争ELISA法,符合检测氯霉素残留的要求,为氯霉素残留快速检测试剂盒的研制奠定了基础.     

建立水产品中己烯雌酚残留ELISA检测方法

本试验利用己烯雌酚(DES)单克隆抗体,通过优化反应条件,加标回收试验,确定ELISA检测方法相关参数,建立了己烯雌酚残留的酶联免疫吸附(ELISA)检测法。试验结果显示,包被抗原最佳浓度为2μg/mL,DES单克隆抗体最佳稀释倍数为1∶3×104;标准曲线呈线性相关,相关系数R2=0.9956,IC50=1.103 ng/mL,最适检测范围为0.125~128 ng/mL,检测敏感度为0.077 ng/g;批内和批间变异分别为6.14%和7.48%;鳝鱼肌肉组织的加标回收率为77.6%~94.6%;特异性试验结果表明,该单抗与己烯雌酚单羧丙基醚交叉反应率为112.7%,与其它水产禁用药物交叉反应率0.1%。     

玉米赤霉烯酮单克隆抗体的制备及间接竞争ELISA检测方法的初步建立

为了研究玉米赤霉烯酮的间接竞争ELISA检测方法,试验采用牛血清白蛋白与玉米赤霉烯酮的耦联物(ZEN-BSA)做包被抗原,标准玉米赤霉烯酮(ZEN)做竞争抗原,以制备的可稳定分泌抗ZEN的单克隆抗体为基础,初步建立了ZEN间接竞争ELISA检测方法。结果表明:间接竞争ELISA检测方法线性范围为0.363 2～78.985 2μg/L,最低检测限为0.231 9μg/L;曲线回归方程为y=68.711-25.666x,其中R2=0.987 1,批内平均变异系数为3.10%,批间平均变异系数为6.26%,与相似毒素的交叉反应率均小于0.01%。说明建立的检测方法可以用于ZEN的检测。     

牛奶中孕酮直接竞争ELISA检测方法的建立

采用直接竞争ELISA方法检测牛奶中孕酮含量.采用碳化二亚胺法,应用辣根过氧化物酶(HRP)标记11-α羟基孕酮琥珀酸酯(11α-OH-P4-HS),制备孕酮酶标抗原;酶标抗原与牛奶样品中的孕酮共同竞争结合固相包被的孕酮单克隆抗体,建立直接竞争ELISA反应体系.试验结果表明,最佳抗体包被浓度为1∶2 000,最佳酶标抗原工作浓度为1∶16 000,建立的标准曲线为y=-2.4598x+0.999(R2=0.996),牛奶中孕酮检测范围0.12～40 ng/mL,最低检测量为0.12 ng/mL,批内和批间变异系数分别为1.63%和2.49%.成功建立了可用于牛乳中孕酮快速检测的直接竞争ELISA方法.     

进出口食用动物、饲料中盐酸克伦特罗ELISA检测方法的建立

为了建立进出口食用动物、饲料中盐酸克伦特罗残留快速检测技术,试验建立间接竞争酶联免疫吸附试验(ELISA)方法进行检测。结果表明:包被抗原的最适质量浓度为20.0μg/mL,抗体的最佳稀释度为1∶5 000,半抑制浓度(IC50)=2.1 ng/mL;该方法的最低检测限达0.05 ng/mL,与沙丁胺醇和莱克多巴胺的交叉反应率分别为8.28%、7.75%,平均加标回收率为94.52%。该方法灵敏度高,样品前处理简便、快速,适用于进出口食用动物、饲料中盐酸克伦特罗残留的大批量现场检测。     

圆弧青霉菌毒素——青霉酸ELISA定量检测试剂盒的研制

为了建立用于定量检测饲料与食品中青霉酸的ELISA试剂盒,试验在合成青霉酸人工抗原并免疫小鼠获得特异单克隆抗体的基础上,利用间接竞争ELISA方法对试剂盒各项技术参数进行测定,最终获得特异、快速的检测试剂盒。结果表明:在优化的条件下,该试剂盒对青霉酸标准品的最低检测浓度为1.6μg/mL;线性方程为y=9.54x+5.844 4(R2=0.996 4),线性范围为0.1～25.6μg/mL;回收试验的平均回收率分别为80.5%、83.3%、85.36%,37℃可保存48 h左右,与黄曲霉毒素B1、玉米赤霉烯酮、T-2毒素、伏马毒素等的交叉反应率小于0.01%,具有良好的特异性。说明已初步研制出用于检测饲料中青霉酸的ELISA试剂盒。     

猪表皮生长因子间接竞争ELISA检测方法的建立

为建立酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测猪血清或尿液中猪表皮生长因子(porcinee pidermal growth factor,pEGF)的含量,以pEGF为包被原,人表皮生长因子(hu-mane pidermal growth factor,hEGF)为竞争抗原,两者与一定量的抗pEGF多抗反应。结果表明,理想的包被抗原浓度为0.625μg/mL,抗pEGF工作浓度为1∶64000,酶标二抗工作浓度为1∶20000,可检测的最适范围为0.4ng/mL~32ng/mL,最低检测限为1ng/mL,批内和批间变异系数分别为3.50%和5.22%。得到回归方程y=-34.53x 76.06(R2=0.993)和标准曲线,从而建立了快速定量测定pEGF含量的酶联免疫吸附试验方法。     

抗黄曲霉毒素M1单克隆抗体的制备及定量ELISA方法的建立

采用黄曲霉毒素M1与牛血清白蛋白偶联物免疫8周龄BALB/c小鼠,4次免疫后,使小鼠产生免疫应答.取其脾细胞与Sp2/0骨髓瘤细胞融合、筛选,最终获得1株能稳定分泌抗黄曲霉毒素M1的杂交瘤细胞株系.其染色体平均计数为90±10对,间接ELISA检测该株系培养上清的效价为1:6 400,纯化腹水效价为5 000×27.与黄曲霉毒素M1及其结构类似物黄曲霉毒素B1和脱氧雪腐镰刀菌烯醇的反应率分别为100%、7%、0%.经传30代后.抗体效价稳定.并在此基础上建立了间接竞争ELISA检测方法,该方法的最低检出浓度是0.08 ng/mL,校正曲线的线性范围为0.08 ng/mL～ 5 ng/mL.该方法的加标回收率为80.0%～128.3%.     

ELISA测定猪尿液中β-受体激动剂残留的准确性评价

本研究建立克伦特罗间接竞争ELISA检测方法,对检测条件进行了优化确定,并利用猪尿液进行添加回收试验。结果显示:ELISA反应CLE-OVA包被抗原和CLE单抗的最适浓度分别为1:40 000和1:20 000,间接竞争ELISA的线性范围为0.016 l^8.520 0 ng/mL。该方法对克伦特罗、溴氯特罗、马布特罗和溴布特罗均有交叉反应。对冷冻储存半年以上的猪尿进行ELISA测定时,发现检测限可达58.848 3 ng/mL,药物添加回收率为85.19%~5 173.25%,变异系数为4.78%~96.79%;而经过MCX固相萃取柱净化处理后,检测限为0.076 ng/mL,添加回收率在101.21%~119.10%之间,变异系数为6.05%~18.38%。结果表明,SPE净化处理后,可有效去除尿液中的色素、蛋白等基质,提高了该法的灵敏度和准确性。     

Studies of the tolerance and disposition of ochratoxin A in young calves

Tolerance to and disposition of ochratoxin A (OA) were compared in preruminant and ruminant calves. Two preruminant calves receiving 4.0 mg OA/kg body weight by stomach tube died; one of two calves receiving 1.0 mg/kg body weight survived. At a dose of .5 mg OA/kg body weight both calves survived. The administered OA was converted mainly (80.1 to 88.9%) to ochratoxin-alpha (O alpha), which was found only in urine; the remaining OA appeared in the urine (3.2 to 3.3%) and feces (7.8 to 10.0%). In the one surviving calf of two given .25 mg OA/kg body weight i.v., nearly twice as much OA was excreted in the feces (44.5%) as in the urine (25.0%); no O alpha was found in urine or feces. All four calves with functional rumens receiving OA orally, 2.0 mg/kg body weight, survived without overt ill effects. Approximately 90% of the OA was excreted as O alpha, with approximately four to eight times more in the urine than in the feces; OA was low in the urine or feces. A plot of the serum OA concentration-time data revealed a prominent, sustained, secondary peak, which was described adequately by a four-exponential equation with two apparent absorption components. Accordingly, OA initially was absorbed rapidly by a first-order rate process (ka = .496/h), and following a considerable delay (tlag = 12.84 h) absorption appeared to resume by a second, slower, first-order rate process (ka = .127/h). The second absorption phase was best explained as being due to enterohepatic cycling of OA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of vitamin A,vitamin E,selenium, and L-lactate in dogs with and without osteoarthritis secondary to ruptured cranial cruciate ligament

This study compared vitamin A, vitamin E, selenium (Se), and L-lactate in blood and synovial fluid in 2 groups of 6 dogs; a control group (without OA) and an osteoarthritic group with spontaneous cranial cruciate ligament rupture and OA. Concentrations of vitamin E were significantly higher in serum than in synovial fluid in both OA (P = 0.006) and control (P = 0.0008) groups. Vitamin E concentration in synovial fluid was significantly higher in the OA group than in the control group (P = 0.009). Concentrations of Se were significantly higher in serum than in synovial fluid in both OA (P = 0.003) and control (P = 0.0006) groups. There were no significant differences in levels of Se, vitamin A, and L-lactate between the 2 groups. This is the first study to show an increased concentration of vitamin E in the synovial fluid of dogs with OA compared with dogs that did not have OA.     

COMPARISONS AMONG RADIOGRAPHY,ULTRASONOGRAPHY AND COMPUTED TOMOGRAPHY FOR EX VIVO CHARACTERIZATION OF STIFLE OSTEOARTHRITIS IN THE HORSE

A better understanding of imaging characteristics of equine stifle osteoarthritis (OA) may allow earlier detection and improve prognosis. Objectives of this ex vivo, prospective, methods comparison study were to (1) describe the location and severity of naturally acquired OA lesions in the equine stifle using ultrasound (US), radiography (XR), computed tomography (CT), and macroscopic evaluation (ME); (2) compare the diagnostic performance of each imaging modality with ME; and (3) describe subchondral bone mineral density (BMD) in equine stifle joints with OA using CT. Radiographic, CT, and US evaluations were performed on 23 equine cadaver stifles and compared with ME. Significant associations were found between osteophyte global scores for all imaging modalities (CT, P ? 0.0001; XR, P = 0.005; US, P = 0.04) vs. ME osteophyte global scores. Osteophytes were detected most frequently in the medial femorotibial (MFT) joint. A specific pattern of osteophytes was observed, with a long ridge of new bone at the insertion of the MFT joint capsule cranially on the medial femoral condyle. A novel caudo‐10°proximo‐5°lateral‐cranio‐disto‐medial oblique radiographic projection was helpful for detection of intercondylar osteophytes. Multiplanar CT reformatted images were helpful for characterizing all osteophytes. Osteophyte grades at most sites did not differ among modalities. Low sensitivity/specificity for subchondral bone sclerosis and flattening of femoral condyles suggested that these signs may not be reliable radiographic and CT indicators of equine stifle OA. Equine stifle OA was associated with a decrease in BMD and specific sites of focal subchondral bone resorption/cyst formation were found in some specimens.     

Sandwich ELISA system for cartilage oligomeric matrix protein in equine synovial fluid and serum

Reasons for performing study: Measurement of cartilage oligomeric matrix protein (COMP) in serum has potential for diagnosis of equine osteoarthritis (OA), but clinical use is currently limited by the lack of specificity of an inhibition ELISA as well as by baseline increases due to exercise. Improved methods for ELISA with increased antigen specificity and sensitivity are therefore required for reliable measurement. Hypothesis: Measurement of the serum level of COMP by sandwich ELISA allows identification of horses with OA. Methods: New monoclonal antibodies (mAbs) were elicited against equine cartilage COMP, their epitopes were determined and a sandwich ELISA was developed. The concentrations of COMP in synovial fluid (SF; n = 100) and sera (n = 100) from OA cases were measured by sandwich ELISA as well as by inhibition ELISA and compared with concentrations in normal joints (n = 95) and horses (n = 50). Results: Immunoblots of enzymatically cleaved COMP showed that the new mAbs recognised different epitopes located on a 20 kDa fragment between K⁶³ and K²³⁸ of the EGF‐like repeats. Inhibition ELISA with any mAb detected significantly increased levels of COMP in OA SF compared with normal SF, whereas no significant difference was detected between serum levels of COMP in OA and normal horses. Conversely, sandwich ELISA with the combination of unlabelled 2A11 × biotinylated 11F10 mAbs detected a significant increase in COMP levels in both serum and SF from OA cases compared with levels in normal animals. Conclusions and potential relevance: Measurement of serum COMP with sandwich ELISA may be useful in identifying horses with OA.     

Effect of graded levels of aflatoxin, ochratoxin and their combinations on the performance and immune response of broilers

1. The effects of dietary aflatoxin (AF, 0.5, 1.0 and 2.0 mg/kg), ochratoxin (OA, 1.0, 2.0 and 4.0 mg/kg) or combinations of these on body weight gain, feed efficiency, organ weights and immune response were studied in broilers. 2. Significant growth depression, reduced food consumption and poor food conversion efficiency were recorded in broilers fed a diet containing the greater concentrations of AF (1 and 2 mg/kg) and OA (2 and 4 mg/kg). 3. The combination of 2 mg/kg AF and 4 mg/kg OA exerted the maximum adverse effect on growth, feed intake and feed efficiency, indicating a synergistic effect on performance. 4. AF at 2 mg/kg in the diet caused a significant increase in the relative weight of liver, whereas the relative weight of kidney was significantly increased at 4 mg/kg of OA. A significant decrease in the relative weight of the bursa of Fabricius was noted at the highest concentration of AF (2 mg/kg) and combinations of 1 and 2 mg/kg AF and 2 and 4 mg/kg OA. 5. Cell mediated immunity (CMI), in terms of mean skin thickness (MST) sensitive to dinitrochlorobenzene (DNCB), was significantly reduced in chicks given the combination of 2 mg/kg AF and 4 mg/kg OA. Haemagglutination (HA) titre against sheep red blood cells (SRBCs) peaked at 42 d of age. At 42 and 47 d of age, a significant decrease in HA titres was recorded in chicks given 4 mg/kg OA or a combination of AF (1 or 2 mg/kg) and OA (2 or 4 mg/kg). 6. AF at a dietary concentration of 1 mg/kg or more and OA at 2 mg/kg or more, either alone or in combination, caused severe reductions in growth and immune response.     

Long-term administration of the mycotoxin, ochratoxin A in chickens and the residues in the meat and animal feed]

A feeding trial was performed with chick broilers (cockerels). The feed with an addition of 850 micrograms ochratoxin A (OA) per kg was administered for six weeks. The feeding of the chicks stopped twelve hours before slaughter (in keeping with slaughter technology for chicks). Blood, liver and kidney samples were taken. At the end of trial the level of OA residues in the samples did not exceed 5 micrograms per kg. In other trials the dynamics of OA residues in the blood plasma of chicks was investigated after i.v. implantation at an amount of 2 and 20 micrograms per chick (1.5 kg lw.). An open two-compartment model was used to estimate toxicokinetic parameters. The half-time of elimination (t1/2(beta)) was about 3.3 hours. The high total clearance (CL) of 34.2 ml/min/kg lw. and apparent distribution volume (Vd(area)) of 9.8 l/kg lw. demonstrate rapid distribution to the tissues and rapid OA elimination. The results document that neither at a long-term intake of feed contaminated to the level of 850 micrograms OA per kg will the present hygienic limits of residues for foods be exceeded (5 and 20 micrograms per kg) if the principles of correct slaughter technology are observed. The blood of chicks used as feed is not an important source of OA in this case.     

Osteoarthritis in cats: a retrospective radiological study

OBJECTIVES: To provide basic information about natural feline osteoarthritis (OA) as part of more extensive studies. METHODS: A retrospective study of cats (greater than one year of age) radiographed for any reason at a first opinion and referral veterinary practice was performed. Cats were classified as either having or not having radiographic OA. Computerised histories were searched for records of potential causes and clinical signs of OA. The genders and ages of the affected cats were compared with a control population using chi-squared tests. RESULTS: Of 491 cats, 292 (59 per cent) had undergone a diagnostic radiograph of at least one synovial joint. Sixty-three of 292 cats (22 per cent) showed evidence of radiographic OA; 21 (33 per cent) of which also had clinically evident OA. A potential cause of OA had been recorded in only seven of 63 cats (11 per cent). The population of cats with radiographic evidence of OA was older than the control population (P<0.001). CLINICAL SIGNIFICANCE: Radiographic OA was found in 22 per cent of the test population. In many cases there was no clinical evidence of OA recorded in the history, suggesting that either there is little correlation between clinical and radiographic OA or that clinical signs of OA in cats may not have been observed or recorded. Idiopathic/primary OA was common.     

The Effects of Ochratoxin A on the Immune Response of Swiss Mice

The acute intraperitoneal toxicity of ochratoxin A (OA) for adult female Swiss mice is presented. The seven-day LD₅₀ was calculated to be 48 ± 3.2 mg/kg. Daily intraperitoneal administrations of 10 mg OA/kg resulted in 50% mortality by the tenth day of injection. Clinical symptoms included depression, huddling, roughened hair coats, humped backs and reduced weight gains. Mice injected intraperitoneally daily for 50 days with 5 mg OA/kg had a significantly (P<0.01) depressed antibody response to killed Brucella abortus. In contrast, oral administrations of OA at 4 ppm in feed for 50 days did not depress titre levels. Ochratoxin A also significantly (P<0.01 intraperitoneal; P<0.05 oral administrations) reduced body weight gain over the period of the trials. Neither oral nor intraperitoneal administration of OA for 50 days affected the response of mice to sheep red blood cells although both the number of antibody-forming cells and the number of cells per spleen were significantly lowered (P<0.01) by cyclophosphamide. Both spleen and body weights were significantly lowered (P<0.05) in the groups given OA. There was a significant depression of blast transformation (P<0.01) in mice treated intraperitoneally with either OA or cyclophosphamide and stimulated with concanavalin A; oral administration of OA did not depress blast transformation. It would appear that lower levels of exposure, e.g. 4 ppm OA in feed, do not cause depression of the immune response of mice. The depressive effect seen at much higher levels may be a result of a nonselective toxic effect.     

The possible role of the tibial plateau angle for the severity of osteoarthritis in dogs with cranial cruciate ligament rupture

The purpose of this study was to determine factors correlated with the severity of radiographic osteoarthritis (OA) scoring in dogs with cranial cruciate ligament rupture (CrCLR). Three radiographs of stifle joints (craniocaudal, mediolateral, and mediolateral radiograph with 90 degree flexion of the stifle and tarsal joints) were obtained from 36 dogs with CrCLR (Clinical group) and from 22 dogs without stifle joint disease (Control group). Information about these dogs was collected from the owners and from medical records. Radiographic OA scores in each dog in the clinical group were determined from radiographs using a numeric grading system previously reported. The tibial plateau angle (TPA) in each dog in both groups was measured on mediolateral radiographs with 90 degree flexion of the stifle and tarsal joints. The Mann-Whitney's U test was used for comparing variables between the clinical group and the control group, and Spearman's rank correlation test was used for evaluating correlations between radiographic OA scores and variables in the clinical group. No significant differences were detected between the clinical group and the control group for any of the variables. There were two positive correlations; one between the radiographic OA score and TPA (r=0.395, p=0.014); and the other between body weight and OA score (r=0.399, p=0.013) in the clinical group. Our results indicate that body weight and TPA could affect the severity of the radiographic OA score in dogs with CrCLR.     

Effect of a hay and a grain diet on the bioavailability of ochratoxin A in the rumen of sheep

The role of the rumen and its contents in the detoxification of ochratoxin A (OA) was studied in sheep. The first experiment established that very little conversion of OA to alpha ochratoxin (O alpha) occurs systematically; 90 to 97% of the OA and metabolites was recovered as unaltered OA in the urine. Most of the small amount of O alpha recovered was also in the urine. In this experiment, two sheep were fasted and another two fed normally, but feed intake had no significant effect. In the second experiment, two sheep fed hay and two fed grain were dosed with OA at .5 mg/kg of BW into the rumen via a cannula. Recoveries, in urine and feces, accounted for 58 to 70% of the administered OA, but almost all (greater than 97%) was in the form of O alpha. About 76 to 92% of this O alpha was in the urine. Although excretion patterns and pharmacodynamics tended to differ with different diets, one of the sheep fed grain had very low intake and the results were equivocal. In the third experiment, eight sheep (four fed hay, four fed grain) were given a single intraruminal dose of OA (.5 mg/kg of BW). The disappearance of OA from the rumen and the corresponding formation of O alpha was much faster for hay-fed than for grain-fed sheep; the half-lives were .63 and 2.7 h for OA and .9 and 1.9 h for O alpha, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)