展青霉素免疫学检测方法研究进展

展青霉素（Patulin, PAT）作为一种对人类和动物有致畸、致癌等多种危害的真菌毒素, 极易污染水果、蔬菜和谷物及其制品等。鉴于展青霉素PAT污染存在范围广、超标检出多等情况, 针对该毒素的即时检测对保障食品安全至关重要。免疫分析是快速检测真菌毒素污染情况的有效手段之一。但是, 尚未有灵敏性好、实用价值高的展青霉素PAT单克隆抗体和基因工程抗体报道。本文通过综述了前人在展青霉素PAT免疫学检测方面的研究进展方法研究现状, 并通过梳理其免疫学检测发展历程并对结果进行分析 , 认为提出高效特异性展青霉素PAT特异性单克隆抗体制备的最大阻碍可能是其主要包括较大的极性、非免疫原性、在动物体内易被降解的特性结构不稳定性以及抗体制备中选用的类似物结构偏差过大 。为应对展青霉素抗体制备面临的困难, 因此, 一方面, 我们认为筛选或制备结构稳定且更接近展青霉素PAT的类似化合物可能是特异性展青霉素抗体制备的关键途径。另一方面, 随着新型材料和技术（如电化学传感器）的发展，应用性能优良的抗体替代物构建建立新型免疫学或非免疫学检测方法的应用前景非常广阔将为展青霉素检测提供帮助。 期望本文能够为PAT快速免疫检测方法的建立和应用提供帮助。     

孔雀石绿多克隆抗体的制备与筛选

设计并成功合成了三种孔雀石绿半抗原,所有半抗原均采用活化酯法分别与血匙兰蛋白(KLH)偶联制备成免疫抗原,与卵清蛋白(OVA)偶联制备成包被抗原。利用所制备的三种免疫原免疫新西兰大耳白兔都获得了高效价的抗孔雀石绿抗体,并将每一种抗体都与三种包被抗原进行组合配对,通过间接竞争酶联免疫吸附检测(ELISA)方法筛选出最佳的抗体-包被抗原组合,筛选出的抗体采用Sepharose FF-Protein A亲和层析柱纯化,采用间接ELISA法测定效价及鉴定特异性。经测定筛选出的抗体效价达到1:12000,IC50值为0.65 ng/mL,交叉反应表明该抗体有较好的特异性,与结构类似物结晶紫的交叉反应率为18.73%,与隐性孔雀石绿及隐性结晶紫的交叉反应率低于10%。本研究为建立快速检测食品中孔雀石绿残留的免疫分析方法奠定了基础。     

展青霉素人工抗原及多克隆抗体的研制

采用戊二酸酐法合成展青霉素-半戊二酸（PAT-HG），通过活泼酯法将PAT-HG偶联到牛血清白蛋白，制备了展青霉素免疫抗原（PAT—HG—BSA），免疫3只BALB／c小鼠，经4次免疫后，1号小鼠血清抗展青霉索抗体效价迭1：1600，且小鼠抗展青霉素多克隆抗体与展青霉素检测抗原的结合能被展青霉素阻断。     

拟除虫菊酯类农药通用人工抗原的合成与鉴定

根据拟除虫菊酯公共结构设计合成一种拟除虫菊酯通用半抗原,所有中间体及半抗原经电喷雾电离质谱(ESI-MS)、红外光谱(IR)和核磁共振(1H NMR)鉴定确定结构.通过活化酯法将通用半抗原与钥孔血蓝蛋白(KLH)偶联制备出免疫原,紫外吸收法鉴定表明免疫原偶联成功,研究为建立拟除虫菊酯多残留免疫分析方法奠定了基础.     

酚酞单克隆抗体的制备

目的 建立免疫学快速检测减肥类保健食品中非法添加的酚酞, 制备酚酞单克隆抗体并进行评价。方法 利用碳二亚胺(carbodiimide, EDC)法合成免疫原和包被原, 用免疫原免疫Balb/C小鼠, 取小鼠脾脏与SP2/0鼠骨髓瘤细胞融合。采用竞争结合双阳性两步筛选法, 筛选出能分泌特异性抗体的杂交瘤细胞, 利用有限稀释亚克隆方法得到单株细胞; 采用体内诱生法制备腹水型单克隆抗体。利用辛酸-饱和硫酸铵法对腹水型抗体进行纯化, 利用酶联免疫吸附法鉴定纯化后的抗体。结果 成功合成了酚酞-BSA免疫原和酚酞-OVA包被原, 筛选获得酚酞杂交瘤细胞株FT/BSA/2019, 单克隆抗体的效价1×105。结论 本研究初步建立了特异性高的酚酞单克隆抗体的制备方法。     

链霉素人工抗原及多克隆抗体的制备与鉴定

通过研究链霉素(SM)人工抗原的合成和多克隆抗体的制备,建立链霉素残留的免疫分析方法。以氧-缩甲基羟胺肟化链霉素的醛基,引入羧基,再用碳二亚胺法将半抗原与蛋白质载体连接,合成免疫原SM-cBSA。用戊二醛法合成包被原SM-OVA。用紫外扫描、SDS-PAGE分析偶联情况,计算得链霉素与cBSA、OVA的分子偶联比分别为7.6:1与17.7:1。经动物免疫获得效价为1:40000的链霉素多克隆抗体,采用间接竞争ELISA测定IC50为3.32ng/mL,与双氢链霉素的交叉反应率为105.21%,与同类的其他药物均无交叉反应。     

食品中展青霉素的研究进展

展青霉素作为一种广泛存在的、对人和动物健康有害的真菌毒素,是水果及其制品、谷物等食品中的天然污染物,尤其存在于变质的苹果及其制品中。研究表明,展青霉素具有致癌性、致畸性、致突变性、免疫毒性、生殖毒性等多种危害。目前关于展青霉素的脱除技术主要分为物理学方法、化学方法、生物学方法三类。将通过对展青霉素污染状况、生物毒性及脱除技术的研究进展进行综述,为后续深入研究提供理论依据。     

氨基甲酸酯类农药速灭威人工抗原的合成与鉴定

通过化学方法合成了两种完全突出氨基甲酸酯类农药速灭威特征结构的半抗原，并采用活化酯法与载体蛋白偶联制备成完全抗原。经动物试验鉴定合成的人工抗原可制备出高效价的抗速灭威抗体，结果表明速灭威人工抗原已成功合成，为建立速灭威残留免疫分析方法奠定了基础。     

螺虫乙酯单克隆抗体的制备及酶联免疫分析方法的建立

螺虫乙酯是一种杀虫杀螨剂,它通过抑制乙酰辅酶A羧化酶活性,阻断害虫正常的能量代谢,最终致其死亡。由于其作用高效,被广泛应用于防治果蔬中的刺吸口器类害虫和害螨。然而,螺虫乙酯对人和动物有毒性。该研究旨在通过制备螺虫乙酯的单克隆抗体并建立酶联免疫分析方法实现蔬菜、水果和环境样品中螺虫乙酯残留的快速检测。首先设计合成了一种新型的螺虫乙酯半抗原,通过活泼酯法偶联牛血清白蛋白得到免疫原,免疫小鼠筛选得到特异性识别螺虫乙酯和B-enol的单克隆抗体（mAb 3D11G7）。在优化的条件下,建立间接竞争酶联免疫分析方法（indirect homologous competitive enzyme-linked immunosorbent assay, icELISA）,其半数抑制质量浓度(half maximal inhibitory concentration, IC₅₀)值为2.1μg/L,最佳工作范围为0.5～8.6μg/L。选择河水、土壤、番茄和柑橘基质进行加标回收实验,其平均加标回收率为72.9%～110.1%。该研究建立的icELISA与超高效液相色谱-串联质谱测定...     

地西泮单克隆抗体的制备及其酶联免疫吸附检测方法的建立

目的制备地西泮(diazepam,DZP)单克隆抗体,并且对制备的抗体进行一系列性质鉴定。方法利用1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐[1-ethyl-3-(3-dimethyllaminopropyl)carbodi imide hydrochloride,EDC·HCl]法合成免疫原和包被原,用免疫原免疫Balb/C小鼠,当效价到1:16000以后取小鼠脾脏与SP2/0进行细胞融合。然后采用竞争结合双阳性两步筛选法,筛选出能分泌特异性抗体的杂交瘤细胞,并且利用有限稀释亚克隆方法得到单株细胞,采用体内诱生法制备腹水型单克隆抗体。利用辛酸-饱和硫酸铵法对腹水型抗体进行纯化,利用酶联免疫吸附法、表面等离子共振仪(surface plasmon resonance,SPR)等方法对纯化后的抗体进行性质鉴定。结果成功合成了地西泮免疫原和包被原,免疫Balb/C小鼠7次后效价达到1:16000,最终制备出单克隆抗体,抗体解离常数(dissociation constants,KDs)为4.0985×10-7 mol/L,且与大部分结构类似物没有明显的交叉反应。应用此抗体建立间接竞争ELISA法,抗体的IC50=10.8 ng/mL,检测范围为0.45~862.00 ng/mL。结论本研究制备出了地西泮单克隆抗体,为地西泮的免疫学检测提供了有力的支持。     

Removing and detoxifying methods of patulin: A review

Patulin is a toxic metabolite produced by molds, which is often found in many fruits and their products. It poses a serious threat to human health due to eating the foods contaminated by patulin frequently. So it is very important for people to find an ideal method for removing or detoxifying patulin in foods. Physical, chemical, and biological methods have been widely studied to remove or degrade it. This article reviews the latest development in the removal and detoxification of patulin using physical, chemical, and biological methods, points out their disadvantages, summarizes the degradation products and their safety of patulin, and draws the degradation pathway of patulin. Presently, no any singular method is ideal in removing or detoxifying patulin in foods, and a combination of various methods may be the better choice. Meanwhile, more attention should be paid to developing the advanced detoxification equipments during exploring the detoxification methods of patulin.     

Fate of patulin in the presence of the yeast Saccharomyces cerevisiae

Patulin is known to become analytically non-detectable during the production of cider from contaminated apple juice. The fate of [¹⁴C]-labelled patulin during the alcoholic fermentation of apple juice was studied. Three commercial cider strains of Saccharomyces cerevisiae degraded patulin during active fermentative growth, but not when growing aerobically. The products of patulin degradation were more polar than patulin itself and remained in the clarified fermented cider. Patulin did not appear to bind to yeast cells or apple juice sediment in these model experiments. HPLC analysis of patulin-spiked fermentations showed the appearance of two major metabolites, one of which corresponded by both TLC and HPLC to E-ascladiol prepared by the chemical reduction of patulin using sodium borohydride. Using a diode array detector, both metabolites had a λ_max = 271nm, identical to that of ascladiol. Thenmr spectrum of a crude preparation of these metabolites showed signals corresponding to those of the E-ascladiol prepared chemically and a weaker set of signals corresponding to those reported in the literature for Z-ascladiol.     

Purification of patulin from Penicillium expansum culture: high-speed counter-current chromatography (HSCCC) versus preparative high-performance liquid chromatography (prep-HPLC)

Patulin is a mycotoxin produced by species of Penicillium and Aspergillus and is toxic to a wide range of organisms, including humans and livestock. To produce large amount of pure patulin for research purposes, high-speed counter-current chromatography (HSCCC) and preparative high-performance liquid chromatography (prep-HPLC) were applied to the purification of patulin. Apple juice was inoculated with P. expansum and containing 0.5 mg patulin per ml was used as a starting material for separation. For HSCCC, a biphasic solvent system consisted of ethyl acetate–hexane–pH 4 acetic acid (7.5:2.5:10, v/v/v) was used. For prep-HPLC, the separation was carried out in a C18 reversed-phase preparative column with a mobile phase containing acetonitrile–pH 4 acetic acid (5:95, v/v). Fractions containing patulin were collected and analysed by analytical HPLC and identified by congruent retention time and ultraviolet/visible (UV–VIS) spectrum of the standard. The structure of the purified patulin was confirmed by mass spectrometry and nuclear magnetic resonance. HSCCC produced 21.9 mg of patulin from 50 ml apple juice culture whereas the prep-HPLC yielded 18.1 mg. HSCCC also produced purer patulin than the prep-HPLC (98.6 versus 96.3%) and higher recovery (86.2 versus 71.3%). In addition, the HSCCC method is advantageous for its lower cost and a simpler procedure compared with the prep-HPLC. This one-step HSCCC method can potentially provide a simple, effective and environmentally friendly tool for obtaining gram-level pure patulin for toxicology, detoxification and many other patulin-related studies.     

降解展青霉素的乳酸菌的筛选鉴定及其降解特性研究

目的:筛选鉴定具有降解展青霉素作用的乳酸菌,并揭示其降解展青霉素的特性。方法:将从青海泡菜中分离得到的2株乳酸菌活化后与展青霉素共培养,利用高效液相色谱(HPLC)法检测共培养过程中体系中展青霉素的含量,筛选出对展青霉素具有较高降解能力的一株乳酸菌。通过对该乳酸菌形态学观察及对16S rDNA序列同源比对,确定该乳酸菌的分类进化地位。进一步准备该乳酸菌的活菌、胞内物质、胞外代谢物以及细胞壁并分别与展青霉素共培养,利用HPLC研究上述各组分对展青霉素的降解作用,探究该乳酸菌降解展青霉素的特性。结果:本研究从2株青海泡菜分离得到的乳酸菌中筛选到一株乳酸菌YZU02能够高效降解展青霉素,经微生物形态学观察及16S rDNA测序比对表明YZU02菌株为植物乳杆菌(Lactobacillus plantarum)。YZU02活菌对展青霉素降解效果较好,可在36 h内将展青霉素由10.87 μg/mL降至2.94 μg/mL。YZU02胞内物质、经展青霉素刺激后的YZU02胞内物质、YZU02胞外代谢物对展青霉素几乎没有降解作用,推测该乳酸菌去除展青霉素与生物降解无关。YZU02细胞壁可将展青霉素含量由10.84 μg/mL降低至5.10 μg/mL。结论:植物乳杆菌YZU02可通过其细胞壁的吸附作用去除溶液状态中的展青霉素,在36 h内对10.87 μg/mL的展青霉素的清除效率达到72.95%,在防治展青霉素污染方面极具应用价值。     

The mycotoxin problem: results of food monitoring

From more than 67 reports on food analysis of aflatoxin, patulin and ochratoxin, the results between 1980-1983 were evaluated and tabulated. The following number of samples were investigated: aflatoxin 15 550, patulin 356 and ochratoxin 437. The amount of Control samples for aflatoxins has reached such a high number that rapid methods for sampling, preparation and analysis are necessary. Out of the many foods investigated, only 9-10 might really be jeopardized, whereby the tendency of contamination-reduction was observed.     

食品中展青霉素的研究进展

展青霉素是由曲霉菌和青霉菌等真菌产生的次级代谢产物, 是广泛存在于水果(特别是霉变水果)、水果制品(果汁、果酱、果脯等)及谷物等食品中的天然污染物, 可以通过食物摄取的方法进入机体。展青霉素不仅具有免疫毒性和致畸性, 而且对人体多种器官都有毒害作用。展青霉素污染的食品(尤其是水果及水果制品)不仅会对人类健康造成严重的威胁, 而且会严重危害我国经济和食品加工业的发展。为了探讨食品中展青霉素的研究进展, 本文主要从展青霉素的基本性质、毒性与危害、检测方法和脱除方法等部分进行论述, 以期为展青霉素的毒性研究提供的理论参考。     

Development and characterization of a carbon-based composite material for reducing patulin levels in apple juice

Patulin, a heterocyclic lactone produced by various species of Penicillium and Aspergillus fungi, is often detected in apple juices and ciders. Previous research has shown the effectiveness of granular activated carbon for reducing patulin levels in aqueous solutions, apple juices, and ciders. In this study, ultrafine activated carbon was bonded onto granular quartz to produce a composite carbon adsorbent (CCA) with a high carbonaceous surface area, good bed porosity, and increased bulk density. CCA in fixed-bed adsorption columns was evaluated for efficacy in reducing patulin levels from aqueous solutions and apple juice. Columns containing 1.0, 0.5, and 0.25 g of CCA were continuously loaded with a patulin solution (10 microg/ml) and eluted at a flow rate of 1 ml/min. Results indicated that 50% breakthrough capacities for patulin on 1.0-, 0.5-, and 0.25-g CCA columns were 137.5, 38.5, and 19.9 microg, respectively. The effectiveness of CCA to adsorb patulin and prevent toxic effects was confirmed in vitro using adult hydra in culture. Hydra were sensitive to the effects of patulin, with a minimal affective concentration equal to 0.7 microg/ml; CCA adsorption prevented patulin toxicity until 76% breakthrough capacity was achieved. Fixed-bed adsorption with 1.0 g of CCA was also effective in reducing patulin concentrations (20 microg/liter) in a naturally contaminated apple juice, and breakthrough capacities were shown to increase with temperature. Additionally, CCA offered a higher initial breakthrough capacity than pelleted activated carbon when compared in parallel experiments. This study suggests that CCA used in fixed-bed adsorption systems effectively reduced patulin levels in both aqueous solutions and naturally contaminated apple juice; however, the appearance and taste of apple juice may be affected by the treatment process.     

Quantification of patulin in fruit leathers by ultra-high-performance liquid chromatography-photodiode array (UPLC-PDA)

Patulin is a mycotoxin commonly found in certain fruit and fruit products. For this reason many countries have established regulatory limits pertaining to, in particular, apple juice and apple products. Fruit leathers are produced by dehydrating fruit puree, leaving a sweet product that has a leathery texture. A recent report in the literature described the detection of patulin at substantial levels in fruit leathers. To investigate this further, an ultra-high-performance liquid chromatography-photodiode array (UPLC-PDA) method was developed for the sensitive detection of patulin in fruit leathers. Investigations were also made of the suitability of direct analysis in real time-mass spectrometry (DART-MS) for detection of patulin from the surface of fruit leathers. Results indicated DART-MS was insufficiently sensitive for quantification from the surface of home-style apple leathers, although patulin spiked onto the surface of leather or peel could be detected. The UPLC-PDA method was used to determine the fate of patulin during the preparation of home-made fruit leathers. Interestingly, when a home-style process was used, the patulin was not destroyed, but rather increased in concentration as the puree was dehydrated. The UPLC-PDA method was also used to screen for patulin in commercial fruit leathers. Of the 36 products tested, 14 were above the limit of detection (3.5 μg kg^–1) and nine were above the limit of quantification (12 μg kg^–1). Positive samples were confirmed by UPLC-MS/MS. Only one sample was found above the US regulatory limit for single-strength apple juice products (50 μg kg^–1). These results suggest patulin can be concentrated during preparation and can be found in fruit leathers. The limited survey suggests that patulin is fairly prevalent in such commercial products, but that the levels are usually low.     

Survival of Penicillium expansum and Patulin Production on Stored Apples after Wash Treatments

ABSTRACT: Penicillium expansum is a widespread fungus found on apples that causes fruit decay and may lead to production of a toxic secondary metabolite, patulin. This study was undertaken to evaluate the effectiveness of several chemical sanitizers against P. expansum NRRL 2304 and to establish sanitizing wash treatments that would inhibit P. expansum growth and subsequent patulin production on Empire apples destined for cider. Wash treatments included 200 ppm NaOCl, 1% StorOx®, 0.5% potassium sorbate, 300 ppm SO₂, and 0% to 5% acetic acid. Spores of P. expansum or inoculated apple slices were dipped in sanitizing wash solution for 5 min, and mold growth and patulin production was monitored on subsequent storage. It was found that 0.5% potassium sorbate and 300 ppm SO₂ did not affect mold survival or patulin production; 1% StorOx® was effective against mold spores in solution (4 log Most Probable Number destruction of spores), but there was no significant reduction in spore count when the same solution was used to sanitize mold‐inoculated apple discs. Washing with 200 ppm NaOCl delayed growth of P. expansum on inoculated apple discs but failed to completely inhibit patulin production. Acetic acid solution (2% to 5%) was the most efficient chemical against P. expansum. A wash treatment with ≥2% acetic acid for more than 1 min is recommended to completely inhibit growth of P. expansum and subsequent patulin production on apples destined for cider.