Determination of Pea Proteinase Inhibitors Using ELISA Based on Monoclonal Antibodies

Abstract

An immunoassay method for analysis of trypsin and chymotrypsin inhibitor activity in seeds from different pea cultivars has been developed. This method is fast, cheap and well suited for screening of large numbers of seed samples. The technique is based on enzyme linked immunoassay (ELISA) using monoclonal antibodies produced against pea (Pisum sativum L.) proteintype trypsin inhibitors (PPI). The results obtained by ELISA have been compared with results achieved by using traditional enzymatic analyses for determination of both trypsin and chymotrypsin inhibitor contents, and these methods showed good agreement. Great differences in PPI levels have been found between various pea cultivars grown in Finland and Denmark, and these differences comprised both the total amount of PPI and the relative composition of individual PPI. Several proteins occurring in pea exhibited inhibitor activity, and at least 10 PPI inhibitors with p/ values 4.9–7.8 were detected. Pea cultivars with low PPI levels had a PPI composition different from the PPI composition found in cultivars with high PPI levels.     

食品农药残留分析的现代进展

主要介绍了农药残留分析中现代分析方法、超临界流全萃取的样品前处理方法，以及免疫分析技术法的原理、方法和现代农残分析的发展趋势。     

抗氯霉素单克隆抗体的制备及其化学发光酶免疫法的建立

以人工合成的氯霉素-牛血清白蛋白(CAP-HS-BSA)免疫Balb/c小鼠,应用杂交瘤技术获得了一株分泌抗氯霉素单克隆抗体(CAP-McAb)的杂交瘤细胞5D7,并对其进行效价、亲和力和特异性测定。结果显示,其分泌的抗体亚类为IgG1,间接ELISA检测细胞培养上清效价为1∶512,诱生的腹水经纯化后效价为1∶2×105,亲和常数为3.6×108L/mol;应用上述单抗建立的检测氯霉素的化学发光酶免疫分析法(ci-CLIA),其检测范围为0.001μg/L~10μg/L,检出限达0.0025μg/L,IC50为39.6μg/L,且与L-苯丙氨酸、L-酪氨酸、青霉素、四环素、庆大霉素等的交叉反应率均小于0.01%,可满足我国对水产品中氯霉素残留检测的要求。     

Prevalence of interfering antibodies in dogs and cats evaluated using a species‐independent assay

Background

Interfering antibodies in human serum and plasma are known to react with mammalian antibodies in immunoassays and cause false‐positive test results. Although this phenomenon was recently shown in companion animals, knowledge regarding immunoassay interference in veterinary medicine is very limited.

Objectives

The aims of this study were to set up a species‐independent immunoassay procedure to detect interference in serum samples, to screen for interference in a cross‐section of canine and feline patient samples from an animal hospital, and to determine if the detected interference could be neutralized using an immunoassay based on nonmammalian reagents.

Methods

A 2‐site sandwich‐type interference assay was set up using commercially available mouse reagents. A total of 369 serum samples from 320 dogs and 263 samples from 218 cats were analyzed using the interference assay. Multiple samples were submitted from 36 dogs and 39 cats. Nineteen samples identified as interference‐positive were analyzed in an assay using chicken antibodies.

Results

Interference was detected in samples from 28 dogs (9%) and 10 cats (5%) screened with the interference assay. Except for 1 cat, consistent results were obtained for all 75 dogs and cats that submitted more than 1 sample. The interference was eliminated when analyzed in the chicken‐based assay (P < .001).

Conclusions

Substances with reactivity toward mouse IgG can be detected in serum samples from dog and cat patients using a 2‐site interference assay. The detected substances are most likely interfering antibodies, possibly originating from immunization with other mammalian species.     

分子模拟在农药半抗原设计及其免疫识别机制中的应用

免疫半抗原的分子设计与合成是建立小分子免疫化学分析方法的关键步骤。在介绍农药半抗原设计一般策略的基础上,指出分子模拟技术在农药半抗原合理设计中的适用性在于其能通过对分子结构和动力学行为的模拟,获得能表示和解释农药分子免疫原性和生物活性的参数。重点阐述了分子模拟在免疫原和竞争原设计中的应用,揭示了采用分子模拟技术进行半抗原设计的优势在于其能够克服目前半抗原设计方法的经验性和主观随意性。运用分子模拟技术能够更好地研究抗原抗体反应中的分子间作用力,因此该技术有利于促进抗原抗体反应识别机制研究的深入进行。分子模拟技术还能够应用于解释抗体的交叉反应率,从而能应用于提高抗体宽谱性研究。     

Pregnancy status determination in mares using a rapid lateral flow test for measuring serum oestrone sulphate

AIMS: To develop a means of determining pregnancy status in horses based on measuring serum oestrone sulphate (OS) concentrations using a rapid lateral flow immunoassay, and to determine the assay's effectiveness using a visual end-point.

METHODS: Serum samples from mares >100 days post-mating (n=701) were assayed using a nitrocellulose membrane-based lateral flow immunoassay device. The device was developed using membrane-bound 1,3,5 (10)-estratrien-3-ol-17-one conjugated to bovine serum albumin as the capture antigen, and an OS-detection monoclonal antibody coupled to colloidal gold as the visible detection reagent. Concentrations of the coating antigen and OS monoclonal antibody were optimised so that the working range would allow pregnancy status to be determined from a visual end-point. The test was run by adding 0.1 ml serum to the sample well of a plastic cassette encasing the test membrane. As the serum migrated along the membrane, a test dot and control line were generated on it within 5–10 min. The intensity of the test dot was inversely proportional to the concentration of OS in the serum sample being tested. Results were compared with those from a validated OS enzyme immunoassay (EIA) and subsequent foaling or return to oestrus of the mares.

RESULTS: Serum samples with OS concentrations <10 ng/ml, indicative of non-pregnancy in mares >100 days post-mating, generated a test end-point consisting of a highly visible test dot and control line, whereas serum OS concentrations >50 ng/ml, indicative of pregnancy, generated a control line only. The test correctly identified 384/389 (98.7%) non-pregnant mares tested, and 303/312 (97.1%) pregnant mares tested that were >100 days post-mating. The lateral flow test devices were stable for at least 12 months when stored at 4°C, sealed in aluminium pouches with desiccant.

CONCLUSION: This novel, rapid, easy-to-use, lateral flow immunoassay offers a practical alternative to traditional laboratory-based immunoassays for measuring serum OS concentra- tions in mares for determining their pregnancy status.     

准噶尔小胸鳖甲融合抗冻蛋白的免疫检测和抗冻活性分析

利用克隆的新疆荒漠昆虫准噶尔小胸鳖甲(Microderapunctipennisdzhungarica)抗冻蛋白基因MpAFP5的全长序列设计PCR引物,构建原核表达质粒pMALp2x-MpAFP5,以麦芽糖融合蛋白(MBP)方式在大肠杆菌(Escherichiacoli)TB1中进行表达,经Amylose树脂亲和层析纯化获得了较高纯度的MBP-MpAFP5融合蛋白。Westernblot结果证明,MBP-MpAFP5融合蛋白在大肠杆菌中得到特异性的正确表达。细菌抗冻实验结果表明,导入MpAFP5基因的大肠杆菌经诱导表达在低温处理时抗冻能力无显著提高,而外加纯化的MBP-MpAFP5融合抗冻蛋白对细菌有明显保护作用,并随浓度升高而保护作用增强。     

仿刺参体腔液补体类似物化学发光免疫检测

首次应用酶联化学发光免疫检测(Chemiluminesent Immunoassay,CLIA)技术测定仿刺参体腔液补体类似物AjC3和AjC4。羊抗人C3、C4抗体吸附到经过紫外线处理的聚苯乙烯管内,采用辣根过氧化物酶(HRP)标记抗体。过氧化氢和鲁米诺为辣根过氧化物酶的底物。捕获抗体包被最适浓度为1μg/ml,免疫反应20℃孵育2h达到平衡。HRP-IgC3、IgC4抗体复合物适宜稀释度为1:2000,HRP-IgC3I、gC4抗体复合物4℃下保存8d性能稳定,室温下5d内性能稳定。标准品浓度在0.1～10ng/ml范围内时与化学发光值之间具有良好的线性相关性,检测灵敏度为0.1ng/ml。结果表明应用酶联化学发光免疫检测技术能够检测到仿刺参体腔液中含有补体类似物,AjC3含量为6.58±1.4μg/ml,AjC4含量为0.67±0.3μg/ml。