化学发光免疫分析技术及其在动物领域中应用的研究进展

化学发光免疫分析技术是酶联免疫分析方法和化学发光技术互相融合的一种免疫检测分析新技术。该技术通过免疫反应和发光反应,对目标物进行检测,具有高灵敏度、高特异性等特点,被陆续运用于疾病检验、药物分析及食品安全等方面。笔者对化学发光免疫分析的原理、种类、特点、质量控制及其在动物领域中的应用和发展趋势进行了介绍,为今后进一步开展化学发光免疫分析技术在动物领域中的研究与应用提供理论参考。     

化学发光免疫分析技术在动物疫病检测中的应用

化学发光免疫分析(chemiluminesecence immunoassay,CLIA)是用于疫病检测的一种免疫检测技术,是基于免疫反应原理,结合化学发光检测技术而建立的。与放射免疫分析、酶免疫分析等标记免疫技术相比,具有无放射性污染、灵敏度高和特异性强的特点,已被广泛应用于疫病流行病学调查、诊断、药物分析以及微生物检测等方面。本文简要介绍了CLIA技术的基本原理及发展,从细菌、病毒和寄生虫检测方面,综述了近年来该技术在动物疫病检测中的应用,提出该技术正向高特异性、高灵敏度、高通量、快速和自动化检测的方向发展,这为今后CLIA在动物疫病检测中的应用研究提供参考。     

免疫组织化学技术在兽医诊断中的应用

免疫组织化学技术是通过利用抗原抗体特异结合的原理,把相关的物质标记在抗体上,来表示复合物的存在和状态。伴随着我国科学技术的全面发展,免疫组织化学技术已经广泛应用到兽医的诊断过程中。免疫组织化学技术应用的范围非常广泛,主要包括对病原体的检测、定位、动态分布,以及新病原体的发现和对固定组织的研究,是现代兽医诊断中的主要技术手段之一。该文通过查询大量的中外文献,就免疫组织化学技术在兽医诊断中的应用进行分析。     

免疫亲和色谱及其在兽药残留检测中的应用

随着对兽药残留检测领域研究的不断深入，免疫亲和色谱技术得到了越来越多的广泛应用。本文详细阐述了免疫亲和色谱的原理和关键技术，介绍了其在动物性食品中兽药及饲料药物添加剂残留检测中的应用与进展。     

兽药残留检测有效净化技术--免疫亲和层析

免疫亲和层析是一种有效的残留检测净化技术,在农药和兽药中得到了广泛应用。作者对免疫亲和层析的原理、关键技术作了详细的阐述,并且介绍了其在兽药及饲料药物添加剂残留中的应用与进展。     

免疫胶体金技术在动物疫病诊断领域中的专利进展

免疫胶体金技术(GICT)现已广泛应用于各检测领域。本文通过检索我国国家知识产权局专利数据库,介绍了该技术的原理和特点,分析了其在病毒、细菌和寄生虫检测等方面的应用,并对我国免疫胶体金技术在动物疫病检测领域中的专利进展进行了综述。     

线路故障指示器的分类及应用

该文对目前国内的故障指示器的功能和性能进行了分析,详细介绍了实现各种功能的主要原理和方法,尤其是对各种单相接地故障检测技术的原理进行了分析和对比。     

猪繁殖与呼吸综合征化学发光检测技术研究

根据双抗原夹心ELISA检测方法,结合化学发光检测体系建立了检测血清中PRRSV抗体的化学发光免疫分析方法,确定了检测体系中所用各组成部分的浓度,并组装为试剂盒,同时对试剂盒的灵敏度、特异性、精密度、准确性、稳定性等技术参数与酶联免疫分析进行了对比,结果表明,二者的检测值显著相关,相关系数为0.9486.该试剂盒在2～8℃下可保存1年,并且灵敏度高、特异性强、精密度好,操作简便、耗时短,能准确检测出血清中抗体的浓度.     

均相光激化学发光免疫分析技术的研究进展

均相光激化学发光免疫分析技术是一种基于纳米微珠的化学发光的新型技术,其具有更高的敏感度、均一性、背景低,且不用洗涤及样本需求量少等特点。该技术可用于生物标志物、激酶及抗原抗体的检测,蛋白：蛋白相互作用的检测,高通量分析的研究及疾病诊断等方面。优化和发展均相光激化学发光免疫分析技术将会在更多研究领域中得到应用。     

瘦肉精的毒害作用及其试纸快速检测技术

文章综述了"瘦肉精"的非法使用及其综合治理,介绍了"瘦肉精"的毒害作用和检测技术,从设计原理,定性、半定量、定量检测等方面详细阐述了免疫试纸快速检测技术及其在"瘦肉精"检测中的应用。     

化学发光直接竞争免疫分析法检测猪尿中莱克多巴胺残留

本试验研究建立了猪尿中莱克多巴胺（Ractopamine, RAC）残留的直接竞争化学发光免疫检测方法。合成了莱克多巴胺和辣根过氧化物酶的偶联物（RAC-HRP）,方法的检测限为0.09 ng/mL,空白猪尿中添加浓度为0.5、10和100 ng/mL RAC时,检测范围为0.3～157.0 ng/mL;回收率为76.3%～87.6%,批内变异系数为10.0%～12.2%,批间变异系数为14.2%～15.2%。     

Comparing the values of progesterone in the blood of bitches as measured with a chemiluminescence immunoassay and a radioimmunoassay

A ¹²⁵I radioimmunoassay (RIA) has long been used to determine the value of progesterone in serum or plasma of bitches but was discontinued in 2014. A chemiluminescence immunoassay (CLIA) gained prominence since 2003 to determine the value of progesterone in serum of bitches but the assay changed in 2012. This study assessed the agreement between progesterone values obtained with RIA in plasma (progRIA) and with the post‐2012 CLIA (progCLIA) in the serum of bitches. ProgCLIA was determined in 110 serum samples from 40 bitches in pro‐oestrus or early oestrus and compared to progRIA in plasma samples collected from the same bitches at the same time, where progRIA had a uniform distribution between 0.5 and 25 nmol/L. Two replicate analyses of each serum or plasma sample were simultaneously done in the same assay. For RIA and CLIA, the intra‐assay CVs were 5.85% and 6.70% and the interassay CVs 8.45% and 9.16%. For RIA and CLIA the progesterone values obtained with replicate analyses differed by as much as 11%–31% in 25% of samples. On average, the value of progCLIA was 85% of that of progRIA (95% CI 58%–112%, n = 110), with 88% of progCLIAs being lower than the progRIAs. This study shows that RIA and CLIA may yield replicate values that differ by as much as 11%–30% in about a quarter of samples analysed, necessitating replicate analyses if precise values are required. The study provides an equation by which to estimate progCLIA from progRIA.     

化学发光免疫分析法检测猪尿中莱克多巴胺残留

本试验运用化学发光免疫分析法（chemiluminescence immunoassay,CLIA）检测猪尿中莱克多巴胺（Ractopamine,RAC）的残留。检测限为0.01 ng/mL,检测范围为0.036～32.2 ng/mL。添加浓度分别为0.1、1、10 ng/mL,平均回收率为73.2%,批内平均变异系数为9.7%,批间平均变异系数为11.3%。而本研究所建立的化学发光免疫检测方法(CLISA)可以达到检测RAC残留的要求。     

Detection of antibodies to S. enteritidis in broilers by means of indirect ELISA and chemiluminescent immunoassay (CLIA)

This study was conducted to develop a serological detection system for the monitoring of broiler flocks for Salmonella enteritidis infections. A specific S. enteritidis antigen (FG-Antigen) was used to compare the sensitivity and the specificity of the chemiluminescent immunoassay (CLIA) with those of the indirect ELISA. This comparison was performed using a total of 578 sera, which, depending on the microbiological and vaccination history, were categorized into groups. Most of the serum samples which were classified as positive showed higher titers in CLIA than in ELISA. Using the prevalence of positive reactors, significant differences between Groups were additionally demonstrated. The absorbance values of the passively immunized group showed the highest and those of the Salmonella-negative group the lowest correlation-coefficient. Using the mean net absorbance of the prevalence group, the ELISA system exhibited a sensitivity of 100% and a specificity of 96.2%, while CLIA had a sensitivity and a specificity of 85.7% and 96.2%, respectively. ELISA and CLIA can be used in the examination of non vaccinated flocks for S. enteritidis-infections as alternative to the bacteriological culture method. CLIA is distinguished for its fast and convenient procedure as well as for its wider measurement spectrum, while the indirect ELISA is almost as efficient as CLIA and requires less investment.     

Chemiluminescent immunoassay as a microtiter system for the detection of Salmonella antibodies in the meat juice of slaughter pigs

Chemiluminescent immunoassay (CLIA) was applied in the screening of swine meat juice samples obtained from different laboratories in Germany, using the indirect enzyme linked immunosorbent assay (ELISA) as test for comparison. Out of the 1350 samples tested, 987 were found acceptable for validation of results. A good level of agreement between the two tests was obtained with a kappa value of 0.824 at 20% cut-off and 0.798 at 40% cut-off. At 20% and 40% cut-off levels, a sensitivity of 96.2% and 97.3%, respectively, and a specificity of 94.6% and 95.1%, respectively, were demonstrated between CLIA and ELISA. The detecting lipopolysaccharide (LPS) antigen was tested for specificity and a cross-reaction with two Escherichia coli and Yersinia strains was found when tested with ELISA. This reaction was not observed in CLIA, possibly because of the broader measurement spectrum of this test, which allows a more distinctive definition of immunologic reactions. The same explanation can be given for the increased number of meat juice samples which were positively detected only in ELISA but not in CLIA. Because of the wide detection range in CLIA, a normalization scheme was necessary to obtain reproducible results in this test system. The samples positively classified in screening were further tested for reciprocal titres in both test systems, and a higher correlation between screening and titration results was obtained for CLIA. Based on the results of this study, CLIA can be used as a reference method in detecting Salmonella antibodies in the meat juice of slaughter pigs.     

Chemiluminescent immunoassay in comparison with the indirect ELISA as reference method for detecting Salmonella antibodies in swine meat juice

The efficiency of chemiluminescent immunoassay (CLIA) in detecting Salmonella antibodies in the meat juice of slaughter swine was compared with the indirect ELISA (BgVV method). Based on the screening test results of 987 meat juice samples obtained from different laboratories in Germany, a good level of agreement between the two systems was obtained with a kappa value of 0.824 at 20% cut-off and 0.798 at 40% cut-off. At 20% and 40% cut-off levels, a sensitivity of 96.2% and 97.3%, respectively, and a specificity of 94.6% and 95.1%, respectively, were demonstrated between CLIA and ELISA. The detecting LPS antigen was tested for specificity and a cross-reaction with two E. coli and Yersinia strains was found when tested with ELISA. This reaction was not observed in CLIA, possibly because of the broader measurement spectrum of this test which allows a more distinctive definition of immunologic reactions. The same explanation can be given for the increased number of meat juice samples which were positively detected only in ELISA but not in CLIA. The positively classified samples in screening were further tested for reciprocal titers in both test systems, and a higher correlation between screening and titration results was obtained for CLIA. Towards the end of the study, a preliminary comparison of CLIA with two available commercial ELISA test kits was conducted and the same tendency was observed, namely, wider detection range of CLIA compared to the other tests. Based on the results of this study, CLIA can be used as a reference method in detecting Salmonella antibodies in the meat juice of slaughter pigs.     

吖啶酯偶联单克隆抗体建立化学发光法检测黄曲霉毒素B1

[目的] 建立快速检测黄曲霉毒素B₁（aflatoxin B₁,AFB₁）的方法。[方法] 将吖啶酯（acridinium ester,AE）与AFB₁单克隆抗体进行体外化学合成,分别合成了吖啶酯∶AFB₁单克隆抗体摩尔比为5∶1、10∶1、15∶1、20∶1、25∶1的吖啶酯标记AFB₁单克隆抗体（AFB₁-AE）,分析了不同摩尔比的检测效果,选择吖啶酯∶AFB₁单克隆抗体为25∶1的AFB₁-AE偶联物建立化学发光免疫分析法（chemiluminescence immunoassay,CLIA）。[结果] 获得对应的标准曲线回归方程为：y=-0.245Ln（x）+ 1.578 1,R²=0.985 7,IC₅₀=81.48 pg/mL;对样品进行加标回收实验,加标回收率88.4%~106.0%,变异系数0.92%~2.10%。[结论] 将吖啶酯与AFB₁单克隆抗体体外交联,可建立新的化学发光免疫分析方法。     

Identification of ectopic ovotestis in a dog with XX ovotesticular,SRY‐negative,disorder of sexual development

A 1‐year‐old, previously spayed phenotypic female Poodle/Soft‐coated Wheaten Terrier (Whoodle) cross was presented for a suspected ovarian remnant. Serum luteinizing hormone (LH) concentration was below the detection limit (<1 ng/ml Witness^® LH), and serum progesterone concentration was elevated in the chemiluminescence immunoassay (CLIA; 20 ng/ml), consistent with dioestrus and presence of ovarian tissue. Transabdominal ultrasound revealed a retroperitoneal soft tissue structure suspected to be a gonad. On exploratory laparotomy, a gonad was removed from the cranial retroperitoneum, cranial to the right kidney, after ligation of its primary blood supply. Histological examination proved the gonad to be an ovotestis. Subsequent cytogenetics revealed a 78 XX karyotype, thus confirming the diagnosis of ectopic ovotestis in a XX ovotesticular, SRY‐negative, disorder of sexual development in a dog.     

Variations among unbred heifers in the activities of polymorphonuclear leucocytes from the mammary gland and blood

The phenotypic characteristics are described for the activity of polymorphonuclear leucocytes NMN) obtained by either lavage of the cavity system of juvenile mammary glands stimulated with a synthetic muramyl dipeptide analogue or isolation from the peripheral blood. Attention was paid to the variability of characteristics and its sources, and to correlations among them. The following characteristics were investigated in 27 clinically healthy, unbred Bohemian Red Pied x Holstein heifers: migration activity in situ, number of phagocytosing PMN, phagocytotic index, bactericidal activity of PMN and unstimulated and zymosan-stimulated luminol-dependent chemiluminescence. Considerable individual variation was found in the characteristics. Significant differences between blood PMN and PMN from lavages after influx induction were found for bactericidal activity (P < 0.05) and chemiluminescence (P < 0.01). A significant correlation between blood PMN and mammary gland PMN was found only for the number of phagocytosing cells (r = 0.329; P < 0.01). Highly significant positive correlations (P < 0.01) were demonstrated between the number of phagocytosing PMN [a], phagocytotic index [b], and bactericidal activity [c] in both blood PMN (r(ab) = 0.602; r(ac) = 0.565; r(bc) = 0.529) and mammary gland PMN (r(ab) = 0.730, r(ac) = 0.618, r(bc) = 0.589). No significant correlation was demonstrated for non-stimulated (NS), zymosan-stimulated (ZS), or opsonized zymosan-stimulated (OZS) chemiluminescence with any of the other characteristics of phagocytotic activity, in either blood PMN or mammary gland PMN (P > 0.05). The animal was a highly significant source of variability for all the phagocytotic activity characteristics (P < 0.01). Udder quarter was a non-significant source of variability for all the characteristics of phagocytotic activity except for NS chemiluminescence (P < 0.05) and ZS or OZS chemiluminescence (P < 0.01). However, udder quarter was a non-significant source of variability of chemiluminescence indices ZS/NS and OZS/NS (P > 0.05). It has been demonstrated that in situ migration activity, the number of phagocytosing PMN, phagocytotic index, bactericidal activity of PMN and chemiluminescence indices of PMN collected from juvenile mammary glands of unbred heifers after influx induction can be regarded as candidate early markers of resistance to mammary infections.     

Effect of radical scavengers on canine peripheral blood mononuclear lymphocyte-mediated cytotoxicity and production of active oxygen

Production of active oxygen by canine peripheral blood mononuclear lymphocytes (PBLs) from beagle dog was examined by luminol-dependent chemiluminescence production. The canine PBLs rapidly produced the active oxygen in parallel with the number of cells when PBLs were cocultured with canine leukemia-derived CL-1 cells as target cells. Cytolysis of the target cells and active oxygen production were inhibited linearly by the addition of benzoic acid and n-propyl gallate as hydroxyl radical scavenger. However, superoxide dismutase and tiron which are scavengers of superoxide anion did not inhibit the cytotoxicity so much at low concentrations that inhibited the induction of luminol-dependent chemiluminescence. These results suggest that hydroxyl radical production by stimulated PBLs might be playing a major role of cytotoxic action in the case of canine system.