兽药人工抗原合成的研究进展

在兽药的免疫化学残留分析中,能否合成稳定的具有良好免疫原性的人工抗原是制备抗体和建立免疫分析方法的最关键步骤.综述了国内外兽药人工抗原合成过程中载体的选择、半抗原的设计（强调偶联部位和间隔臂的引入）、半抗原与载体的偶联方法、人工抗原的纯化与鉴定等方面的研究进展.     

兽药单克隆抗体研究进展

兽药大多属于小分子物质(半抗原),没有免疫原性,必须与载体交联成人工合成抗原(完全抗原)才能获得免疫原性,用人工合成的抗原来免疫动物制备兽药单克隆抗体。对兽药单克隆抗体的研究是随着兽药残留检测技术发展而开始的,人们已经在制备兽药单克隆抗体所需技术如兽药人工抗原合成的方法、选择合成兽药人工抗原的所需载体、对兽药结构的改造、免疫动物的注射方式、免疫程序、杂交瘤细胞的筛选及培养等方面进行一些研究,并成功地制备出十几种兽药单克隆抗体,且大多已应用于兽药免疫测定法(IAs)。目前仅有一种兽药单克隆抗体已应用于兽药免疫亲和色谱(IAC)。     

单链抗体在小分子化学物质残留检测中的研究进展

兽药、农药及生物毒素等小分子化学物质在食品、饲料或水体中的过量残留对人、动物的健康及生态环境的平衡造成一定程度的危害,因此建立一种针对上述小分子化学物质残留的快速检测方法十分必要。以抗原抗体特异性结合为基础的免疫分析技术是残留检测的常用手段,该技术的关键是制备可用来检测特异性抗原的抗体。随着对抗体结构的解析及重组DNA技术的发展,单链抗体为小分子化学物质的残留检测提供了新的技术手段。单链抗体具有分子质量小、免疫原性低、可塑性强、可批量生产等优点,具有广阔的发展空间。近年来,以单链抗体为基础建立的酶联免疫吸附法检测小分子化学物质的残留检测成为最常用的分析工具,且单链抗体的筛选策略、表达策略、突变策略及与碱性磷酸酶形成的融合蛋白等进一步提高了抗体产量、灵敏度、广谱性,并使免疫分析方法简便化,从而推进单链抗体免疫分析方法在小分子化学物质残留检测的应用。作者综述了以单链抗体为基础的免疫分析方法在兽药、农药及生物毒素等小分子化学物质方面的研究进展,以期为小分子化学物质残留检测提供参考。     

单链抗体在小分子化学物质残留检测中的研究进展

兽药、农药及生物毒素等小分子化学物质在食品、饲料或水体中的过量残留对人、动物的健康及生态环境的平衡造成一定程度的危害,因此建立一种针对上述小分子化学物质残留的快速检测方法十分必要。以抗原抗体特异性结合为基础的免疫分析技术是残留检测的常用手段,该技术的关键是制备可用来检测特异性抗原的抗体。随着对抗体结构的解析及重组DNA技术的发展,单链抗体为小分子化学物质的残留检测提供了新的技术手段。单链抗体具有分子质量小、免疫原性低、可塑性强、可批量生产等优点,具有广阔的发展空间。近年来,以单链抗体为基础建立的酶联免疫吸附法检测小分子化学物质的残留检测成为最常用的分析工具,且单链抗体的筛选策略、表达策略、突变策略及与碱性磷酸酶形成的融合蛋白等进一步提高了抗体产量、灵敏度、广谱性,并使免疫分析方法简便化,从而推进单链抗体免疫分析方法在小分子化学物质残留检测的应用。作者综述了以单链抗体为基础的免疫分析方法在兽药、农药及生物毒素等小分子化学物质方面的研究进展,以期为小分子化学物质残留检测提供参考。     

胰酶分散抗原间接荧光抗体法检测鸡肾型传染性支气管炎病毒及抗体的研究

本文采用胰酶分散法制备抗原，建立了间接荧光抗体法。用此法对人工感染鸡、人工感染鸡胚及自然发病鸡的抗原进行了测定，结果，人工感染鸡肾脏于７２～１６８ｈ１００％检测到特异性荧光，其余时间部分鸡检出特异性荧光；人工感染后病死鸡肾脏均１００％检测到特异性荧光；人工感染鸡胚于４８～１２０ｈ１００％检测到特异性荧光，其余时间部分检出特异性荧光；１７８例自然发病病例中检出抗原阳性病例１５３例。对抗原检测的结果与冰冻切片制备抗原建立的间接荧光抗体法完全一致。用胰酶分散法制备抗原，对已知阳性血清和待检血清进行检测，检测结果与用冰冻切片法制备抗原检测结果一致。该法将检测程序简化，检测时间明显缩短，适合于临诊应用及对鸡群进行抗体监测     

人工抗原的合成及单克隆抗体的纯化

制备单克隆抗体，建立优化ELISA方法检测兽药残留过程中，人工抗原的合成是较重要的步骤。对人工抗原合成时半抗原和载体的选择，化学反应的类型及应注意的事项进行了阐述，以期促进兽药残留分析的发展。     

胶体金免疫检测技术在奶牛养殖中的应用

正胶体金免疫检测技术是目前最为常见的快速检测技术,其基本原理是抗原抗体特异性反应。因其具有检测速度快、使用方便、成本低廉等优点在医学临床~([1])、动物诊断及食品、环境检测~([2])等领域被广泛应用。根据方法类别和检测对象的不同,可将其主要分为双抗体(抗原)夹心法和竞争法,前者主要用于病毒、细菌、蛋白质等大分子物质的检测,而后者则用于药物残留、真菌毒素及非法化学添加物等小分子物质的检测。在畜牧生产中,胶体金免疫检测技术也有大量应     

兽药残留量的酶联免疫吸附测定方法研究─磺胺二甲嘧啶人工抗原合成及其抗体制备

兽药残留量的酶联免疫吸附测定方法研究─磺胺二甲嘧啶人工抗原合成及其抗体制备陈慧珠，刘智宏，赵轶先（中国兽药监察所，北京１０００８１）随着畜牧业发展，兽药品种不断增加，食品中兽药的残留越来越受到人们重视。随着高效低毒低残留兽药的开发利用，以及人们对环境...     

多残留免疫检测技术及其在畜禽水产品质量安全检测中的应用

兽药残留超标是畜禽水产品的主要安全危害,利用免疫检测技术对兽药残留进行快速筛查是一种有效的监控手段。然后兽药残留种类多样性的现实与传统免疫检测技术检测对象单一的特点存在显著矛盾,因此研究建立多残留免疫检测成为一种新的趋势。文章介绍了实现多残留免疫检测的几种策略,并对多残留免疫检测技术在畜禽水产品质量安全检测中应用作了简要的概述。     

酶联免疫技术用于检测动物体内莱克多巴胺残留的研究进展

本文介绍了莱克多巴胺的基本性质及相关检测研究进展,并就近年来莱克多巴胺酶联免疫检测研究进展进行了充分论述。分析了莱克多巴胺分子结构中抗原改造位点、抗原决定簇及莱克多巴胺抗体制备的最新成果;讨论了莱克多巴胺残留检测时各种样本处理方法的差异及适用范围;对莱克多巴胺酶联免疫检测方法的建立进行了简单介绍。     

Analgesic efficacy of intra‐articular morphine in experimentally induced radiocarpal synovitis in horses

ObjectiveTo compare the analgesic effect of intra-articular (IA) and intravenous (IV) morphine in horses with experimentally induced synovitis.AnimalsEight adult horses.Study designRandomized, observer blinded, double dummy trial with sequential crossover design.MethodsRadiocarpal synovitis was induced by IA injection of lipopolysaccharide on two occasions separated by a 3-week washout period. In one study period horses received treatment IA; morphine IA (0.05 mg kg^?1) plus saline IV and in the other study period they received treatment IV; saline IA plus morphine IV (0.05 mg kg^?1). Lameness and pain were evaluated repeatedly by two observers throughout each of the two 168-hour study periods. Pain was evaluated by use of a visual analogue scale of pain intensity (VAS) and a composite measure pain scale (CMPS). Comparison of treatments was performed by analysis of variance with repeated measurements. Significance level was set to p ≤ 0.05. Inter-observer agreement and agreement between the VAS and CMPS was assessed by use of the Bland–Altman method.ResultsIntra-articular injection of LPS elicited a marked synovitis resulting in lameness and pain. IA morphine resulted in significantly less lameness than IV morphine (p = 0.03). CMPS (p = 0.09) and VAS (p = 0.10) pain scores did not differ significantly between treatments. Inter-observer agreement of the CMPS was classified as good, but only fair for the VAS. Agreement between the two pain scales was considered fair.Conclusions and clinical relevanceAn analgesic effect of IA morphine was demonstrated by significantly reduced lameness scores. The results support the common practice of including IA morphine in a multimodal analgesic protocol after arthroscopic surgery, although further studies in clinical cases are needed. The employed CMPS had good reproducibility, and was easy to use, but may have limited sensitivity at mild intensity pain.     

Acetabular component orientation as an indicator of implant luxation in cemented total hip arthroplasty

OBJECTIVE: To determine the sensitivity and specificity with which acetabular component angles of inclination and version could be used, alone or in combination, to predict luxation of cemented total hip arthroplasties (THA). STUDY DESIGN: Comparison of retrospectively selected cases and controls SAMPLE POPULATION: All THA performed at the University of Florida between 1991 and 1998 with the BioMedtrix system and for which at least 2 months of radiographic follow-up were available. All THA performed at the University of Georgia with the BioMedtrix system which subsequently luxated. METHODS: Acetabular component inclination angle (IA) and acetabular version angle (VA) were determined for each THA. Data were grouped according to outcome - luxation or no luxation - with the luxated cases from the 2 institutions pooled. Receiver operator characteristic (ROC) analysis was used to evaluate decision rules for using IA and VA as tests for detecting postoperative luxation. Sensitivity and specificity for luxation and 95% confidence bounds were computed with selected values of IA and VA as cut-points. RESULTS: The nonluxation group consisted of 68 THA with a median follow-up time of 5 months (range, 2-60 months). The luxation group consisted of 12 THA with a mean time to luxation of 36 days. The nonluxation group had a mean +/- standard deviation (SD) IA and VA of 40.3 degrees +/- 8.9 degrees and 71.1 +/- 13.6 degrees, respectively, whereas the luxation group had a mean +/- SD IA and VA of 34.7 degrees +/- 12.6 degrees and 72.9 degrees +/- 16.6 degrees, respectively. An IA cut-point of 37.8 degrees achieved 58.3% sensitivity and 57.4% specificity. A VA cut-point of 73 degrees achieved 75.0% sensitivity and 51.5% specificity. IA and VA considered simultaneously achieved a 50.0% sensitivity and 88.2% specificity. CONCLUSIONS AND CLINICAL RELEVANCE: ROC analysis indicated that both IA and VA considered individually or simultaneously were poor indicators of luxation. Although extreme values of IA may predict luxation with high specificity, the potential for luxation cannot be excluded based on apparently appropriate values of IA and VA. The results of this study also indicate that a successful outcome is possible with a wide range of acetabular component positions.     

Determination of synovial fluid and serum concentrations, and morphologic effects of intraarticular ceftiofur sodium in horses

OBJECTIVES: To determine the serum and synovial fluid concentrations of ceftiofur sodium after intraarticular (IA) and intravenous (IV) administration and to evaluate the morphologic changes after intraarticular ceftiofur sodium administration. STUDY DESIGN: Strip plot design for the ceftiofur sodium serum and synovial fluid concentrations and a split plot design for the cytologic and histopathologic evaluation. ANIMALS: Six healthy adult horses without lameness. METHODS: Stage 1: Ceftiofur sodium (2.2 mg/kg) was administered IV. Stage 2: 150 mg (3 mL) of ceftiofur sodium (pHavg 6.57) was administered IA into 1 antebrachiocarpal joint. The ceftiofur sodium was reconstituted with sterile sodium chloride solution (pH 6.35). The contralateral joint was injected with 3 mL of 0.9% sterile sodium chloride solution (pH 6.35). Serum and synovial fluid samples were obtained from each horse during each stage. For a given stage, each type of sample (serum or synovial fluid) was collected once before injection and 12 times after injection over a 24-hour period. All horses were killed at 24 hours, and microscopic evaluation of the cartilage and synovium was performed. Serum and synovial fluid concentrations of ceftiofur sodium were measured by using a microbiologic assay, and pharmacokinetic variables were calculated. Synovial fluid was collected from the active joints treated during stage 2 at preinjection and postinjection hours (PIH) 0 (taken immediately after injection of either the ceftiofur sodium or sodium chloride), 12, and 24, and evaluated for differential cellular counts, pH, total protein concentration, and mucin precipitate quality. RESULTS: Concentrations of ceftiofur in synovial fluid after IA administration were significantly higher (P = .0001) than synovial fluid concentrations obtained after IV administration. Mean peak synovial fluid concentrations of ceftiofur after IA and IV administration were 5825.08 microg/mL at PIH .25 and 7.31 microg/mL at PIH 4, respectively. Mean synovial fluid ceftiofur concentrations at PIH 24 after IA and IV administration were 4.94 microg/mL and .12 microg/mL, respectively. Cytologic characteristics of synovial fluid after IA administration did not differ from cytologic characteristics after IA saline solution administration. White blood cell counts after IA ceftiofur administration were < or =3,400 cells/ML. The mean synovial pH of ceftiofur treated and control joints was 7.32 (range, 7.08-7.5) and 7.37 (range, 7.31-7.42), respectively. Grossly, there were minimal changes in synovium or cartilage, and no microscopic differences were detected (P = .5147) between ceftiofur-treated joints and saline-treated joints. The synovial half-life of ceftiofur sodium after IA administration joint was 5.1 hours. CONCLUSIONS: Synovial concentrations after intraarticular administration of 150 mg of ceftiofur sodium remained elevated above minimal inhibitory concentration (MIC90) over 24 hours. After 2.2 mg/kg IV, the synovial fluid ceftiofur concentration remained above MIC no longer than 8 hours. CLINICAL RELEVANCE: Ceftiofur sodium may be an acceptable broad spectrum antimicrobial to administer IA in septic arthritic equine joints.     

Effects of intra-articular injections of bufexamac suspension in healthy horses

OBJECTIVE: To evaluate the effects of intra-articular (IA) injections of bufexamac in horses, focusing particularly on the effects of bufexamac on articular cartilage. ANIMALS: 20 Standardbreds. PROCEDURE: Horses were randomly allocated into 4 groups consisting of 5 horses each, and 20, 60, or 100 mg of bufexamac or 1 ml of sterile saline (0.9% NaCl) solution (control) was injected into 1 intercarpal joint at weekly intervals for 6 treatments (days 0, 7, 14, 21, 28, and 35). Clinical signs and results of hematologic, serum biochemical, and synovial fluid (SF) analyses and radiography were used to evaluate treatment effects. On day 49, all horses were euthanatized; gross necropsy and histologic examinations of internal organs and articular tissues were performed. Glycosaminoglycan concentration of the articular cartilage was evaluated in safranin O-stained sections by use of a semiquantitative microspectrophotometric method. RESULTS: No systemic signs were observed. Temporary mild to moderate heat and effusion were the only clinical signs observed in a number of joints after IA injections and more often only in the 100 mg group, compared with controls. The 100 mg dose resulted in significant increases in SF WBC counts, with relative neutrophilia and SF total protein concentration 24 hours after injection (day 1). No lesions suggestive of toxic effects were detected at necropsy or on histologic examination. No changes in articular cartilage glycosaminoglycan concentration were detected. CONCLUSIONS AND CLINICAL RELEVANCE: Six injections of 20, 60, or 100 mg of bufexamac at weekly intervals did not cause any untoward systemic or local effects. These data suggest that bufexamac is a safe nonsteroidal anti-inflammatory drug for IA administration in horses.     

Evaluation of the inflammatory response to two intra-articular hyaluronic acid formulations in normal equine joints

Intra-articular (IA) hyaluronic acid (HA) is commonly used to treat equine arthritis. Inflammatory response or “joint flare” is a recognized potential side effect. However, the incidence and severity of inflammation following IA HA injection in horses is not well documented. This study compared the effects of two IA HA formulations of different molecular weight (MW) and a saline control on clinical signs and synovial fluid markers of inflammation in normal equine joints. Eight adult horses each had three healthy fetlock joints randomly assigned to treatment with either 1.4 mega Dalton HA, 0.8 mega Dalton HA or saline control once weekly for three weeks. Clinical evaluation and synovial fluid analysis were performed by blinded assessors. Outcomes of interest were lameness score, joint effusion score and synovial fluid white cell count and differential, total protein, viscosity and serum amyloid A. Joints injected with HA developed significant mild-to-moderate inflammatory responses often associated with lameness and joint effusion compared with saline control joints. The higher MW HA formulation elicited a significantly greater inflammatory response than the lower MW HA after the first injection. In HA injected joints, viscosity remained poor for the entire study. Both IA HA formulations in this study induced an inflammatory response in healthy equine joints. This may have implications for the use of HA in equine joints. The findings in this study are limited to the two HA formulations used. Further investigation of different HA formulations and the use of HA in normal and arthritic equine joints is warranted.     

Pharmacokinetics of tobramycin following intravenous,intramuscular, and intra‐articular administration in healthy horses

The objectives of this study were to examine the pharmacokinetics of tobramycin in the horse following intravenous (IV), intramuscular (IM), and intra‐articular (IA) administration. Six mares received 4 mg/kg tobramycin IV, IM, and IV with concurrent IA administration (IV+IA) in a randomized 3‐way crossover design. A washout period of at least 7 days was allotted between experiments. After IV administration, the volume of distribution, clearance, and half‐life were 0.18 ± 0.04 L/kg, 1.18 ± 0.32 mL·kg/min, and 4.61 ± 1.10 h, respectively. Concurrent IA administration could not be demonstrated to influence IV pharmacokinetics. The mean maximum plasma concentration (C_max) after IM administration was 18.24 ± 9.23 μg/mL at 1.0 h (range 1.0–2.0 h), with a mean bioavailability of 81.22 ± 44.05%. Intramuscular administration was well tolerated, despite the high volume of drug administered (50 mL per 500 kg horse). Trough concentrations at 24 h were below 2 μg/mL in all horses after all routes of administration. Specifically, trough concentrations at 24 h were 0.04 ± 0.01 μg/mL for the IV route, 0.04 ± 0.02 μg/mL for the IV/IA route, and 0.02 ± 0.02 for the IM route. An additional six mares received IA administration of 240 mg tobramycin. Synovial fluid concentrations were 3056.47 ± 1310.89 μg/mL at 30 min after administration, and they persisted for up to 48 h with concentrations of 14.80 ± 7.47 μg/mL. Tobramycin IA resulted in a mild chemical synovitis as evidenced by an increase in synovial fluid cell count and total protein, but appeared to be safe for administration. Monte Carlo simulations suggest that tobramycin would be effective against bacteria with a minimum inhibitory concentration (MIC) of 2 μg/mL for IV administration and 1 μg/mL for IM administration based on C_max:MIC of 10.     

Bone gentamicin concentration after intra-articular injection or regional intravenous perfusion in the horse

Objective— To compare intra-articular (IA) and bone gentamicin concentrations achieved after intra-articular administration or regional intravenous perfusion (RIP).
Study Design— Experimental study.
Animals— Twelve healthy adult horses.
Methods— Horses were assigned to 2 treatment groups (  n = 6/group  ): Group 1, 1 g gentamicin administered simultaneously in both left and right metacarpophalangeal joints and group 2, 1 g gentamicin administered simultaneously in both left and right lateral palmar veins. Serum, synovial fluid, and bone biopsy specimens were collected. Gentamicin concentrations were determined by fluorescence polarization immunoassay. Bone, synovial fluid, and serum gentamicin concentrations were compared over time and between groups using 2-way ANOVA. Significance of all tests were evaluated at   P < .05  .
Results— IA metacarpophalangeal joint administration resulted in higher concentration of gentamicin in synovial fluid than RIP administration. Synovial fluid concentration remained above minimum inhibitory concentration (MIC) for common pathogens for over 24 hours with IA and RIP administration. Bone gentamicin concentration remained above MIC for 8 hours with both methods; there was no significant difference in gentamicin concentration in bone with either method. Neither IA nor RIP administration had a significant effect on serum concentration of gentamicin.
Conclusions— In normal horses, there is no difference in bone gentamicin concentration obtained with IA or RIP administration.
Clinical Relevance— Based on MIC for common equine pathogens, administration of gentamicin intra-articularly or by regional intravenous perfusion should be useful for treatment of osteomyelitis.     

Plasma and synovial fluid concentrations of gentamicin in horses after intra-articular administration of buffered and unbuffered gentamicin

The concentration of gentamicin in plasma and synovial fluid of normal adult horses was measured periodically for 24 hours after IV (2.2 mg/kg of body weight), intra-articular (IA; 150 mg), and simultaneous IV and IA administrations. Gentamicin also was buffered with sodium bicarbonate (3 mEq) and then was administered IA and simultaneously IV and IA. Synovial fluid specimens were obtained via an indwelling catheter placed into the antebrachiocarpal joint. The peak mean plasma gentamicin concentration (8.30 micrograms/ml) after IV administration was significantly (P less than 0.05) greater than that (0.69 microgram/ml) after IA administration of gentamicin and that (0.55 microgram/ml) after administration of gentamicin buffered with sodium bicarbonate. Gentamicin concentration greater than a therapeutic concentration was not attained in the plasma after IA administration of buffered or unbuffered gentamicin. The peak mean synovial fluid concentration (1,828 micrograms/ml) after IA administration of unbuffered gentamicin was significantly (P less than 0.05) greater than that (2.53 micrograms/ml) after IV administration and significantly (P less than 0.05) less than that (5,720 micrograms/ml) after simultaneous IV and IA administration. The peak mean synovial fluid concentration after IA administration of buffered gentamicin, with and without simultaneous IV administration (2,128 and 2,680 micrograms/ml, respectively), was not significantly different than that after IA treatment with unbuffered gentamicin. Mean synovial fluid concentration did not differ significantly between groups after IA administration of gentamicin in any combination at postinjection hours 8, 12, and 24, but remained significantly (P less than 0.05) greater than that at the same times after IV administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endocrine evaluation after an intra‐articular therapeutic dosage of dexamethasone in horses

This study investigated whether a single intra‐articular administration (IA) of dexamethasone (DEX) in horses at therapeutic dosage could exert a systemic effect by influencing the hypothalamic‐pituitary‐adrenal axis activity as a consequence of (limited) absorption and systemic distribution. The results indicated that DEX was detectable in urine collected 12–48 h after IA administration and that injection was accompanied by a reduced urine excretion of cortisol, 6β‐hydroxycortisol (6βOHF) and two other metabolites of cortisol lasting up to 48 h post‐DEX administration. The systemic effects in horses treated with DEX by IA route are similar to those that typically occur with short‐term treatment including the reduction in urinary cortisol concentration.     

Furosemide continuous rate infusion in the horse: evaluation of enhanced efficacy and reduced side effects

Continuous rate infusion (CRI) of furosemide in humans is considered superior to intermittent administration (IA). This study examined whether furosemide CRI, compared with IA, would increase diuretic efficacy with decreased fluid and electrolyte fluctuations and activation of the renin-angiotensin-aldosterone system (RAAS) in the horse. Five mares were used in a crossover-design study. During a 24-hour period, each horse received a total of 3 mg/kg furosemide by either CRI (0.12 mg/kg/h preceded by a loading dose of 0.12 mg/kg IV) or IA (1 mg/kg IV q8h). There was not a statistically significant difference in urine volume over 24 hours between methods; however, urine volume was significantly greater after CRI compared with IA during the first 8 hours ([median 25th percentile, 75th percentile]: 9.6 L [8.9, 14.4] for CRI versus 5.9 L [5.3, 6.0] for IA). CRI produced a more uniform urine flow, decreased fluctuations in plasma volume, and suppressed renal concentrating ability throughout the infusion period. Potassium, Ca, and Cl excretion was greater during CRI than IA (1,133 mmol [1.110, 1,229] versus 764 mmol [709, 904], 102.7 mmol [96.0, 117.2] versus 73.3 mmol [65.0, 73.5], and 1,776 mmol [1,657, 2.378] versus 1,596 mmol [1,457, 1,767], respectively). Elimination half-lives of furosemide were 1.35 and 0.47 hours for CRI and IA, respectively. The area under the excretion rate curve was 1,285.7 and 184.2 mL x mg/mL for CRI and IA, respectively. Furosemide CRI (0.12 mg/kg/h) for 8 hours, preceded by a loading dose (0.12 mg/kg), is recommended when profound diuresis is needed acutely in horses.