抗玉米赤霉烯酮单抗独特型抗体制备与鉴定

目的:研制抗玉米赤霉烯酮(zearalenone,ZEN)单抗独特型抗体并鉴定其特性,为建立ZEN无毒检测方法提供理论依据。方法:在研制抗ZEN单抗的基础上,将IgG与KLH偶联作为免疫原,免疫BALB/c小鼠,制备抗ZEN单抗独特型抗体;同时将经Protein G纯化的单抗用胃蛋白酶酶切获取Fab片段,作为检测原,并采用间接ELISA和间接竞争ELISA对抗独特型抗体特性进行鉴定。结果:所研制的抗独特型抗体能够与ZEN竞争结合ZEN单抗,与ZEN之间存在"内影像"关系。结论:成功研制抗ZEN单抗独特型抗体,可以替代ZEN标准品应用于ELISA检测。     

单克隆免疫亲和柱-高效液相色谱法测定玉米中的玉米赤霉烯酮

研究了单克隆免疫亲和柱-高效液相色谱法测定玉米中玉米赤霉烯酮的方法。样品通过乙腈-水(体积比9：1)提取，提取液用免疫亲和柱净化，Waters C18色谱柱分离，荧光检测器检测，外标法定量。对添加不同含量的玉米赤霉烯酮进行6次重复实验，平均回收率为87．8％～90．9％，变异系数(CV)为8．6％～11．3％，检测低限为001mg／kg。     

玉米赤霉烯酮生物降解研究进展

玉米赤霉烯酮是一种由镰刀菌产生的具有雌激素作用的真菌毒素,是世界上污染范围最广的一种镰刀菌素.由于物理、化学脱毒法存在较大弊端,生物去毒方法因其特异性、高效性及环境友好而日益被科学界所关注.目前玉米赤霉烯酮生物降解主要是微生物降解作用,本文紧跟国内外玉米赤霉烯酮的生物降解情况,文章介绍了降解菌株的种类,降解能力和最佳降解条件,包括降解产物毒性,降解酶基因的发掘,以及降解菌株和酶的应用方向及前景.     

玉米赤霉烯酮检测方法研究进展

玉米赤霉烯酮是一种具有雌激素作用真菌毒素,主要污染玉米、麦类、谷物等,严重影响农业经济及人类健康,因此有必要研究能准确、灵敏检测玉米赤霉烯酮方法。该文对玉米赤霉烯酮检测方法进行综述。     

玉米赤霉烯酮降解的研究进展

真菌毒素广泛存在于世界各地的谷物及其副产物当中.玉米赤霉烯酮(Zearalenone)作为镰刀霉毒素的代表,是影响食物安全的重要因素之一.传统的毒素清除方法包物理和化学方法.物理方法能部分清除毒素,但易破坏食物的营养物质,化学方法随着化学试剂的添加会引入不确定的危害因素.生物降解作为目前的研究热点,可在温和条件下微生物将毒素转化为无毒产物.报道了物理、化学和生物方法清除玉米赤霉烯酮的研究进展,重点对生物降解进行了综述.     

玉米赤霉烯酮单克隆抗体制备及免疫分析

采用活泼酯法将玉米赤霉烯酮(Zaralenone,ZEN)分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联合成免疫原ZEN-BSA和包被原ZEN-OVA,经紫外扫描和聚丙烯酰胺凝胶电泳鉴定后免疫BALB/c小鼠;挑选产生高效价和高敏感性抗体的小鼠进行抗原超强免疫,取脾细胞与SP2/0细胞融合获得1株稳定分泌抗玉米赤霉烯酮单克隆抗体的杂交瘤细胞(3D8),经鉴定,该抗体亚类为IgG1,轻链为k型。经体内诱生腹水并纯化获得效价为1∶2.048×106的单克隆抗体。建立了基于单克隆抗体的ZEN间接竞争ELISA法(ic-ELISA),该方法IC50和检出限分别为22.89pg/mL和10.07pg/mL。与α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉酮、α-玉米赤霉醇和β-玉米赤霉醇的交叉反应率分别为95%、8%、12%、6%和5%,而与黄曲霉毒素B1、呕吐毒素、伏马毒素B1和赭曲霉毒素A未见有交叉反应。在玉米、大麦、小麦和燕麦中加标的回收率在86.4%~104.8%之间。该方法灵敏特异,可作为快速检测谷物中玉米赤霉烯酮的初筛方法。     

玉米赤霉烯酮降解微生物的筛选与鉴定

从全国各地的土壤、污水、受污染的玉米、家畜肠道粪便等134份样品中分离得到1 628株菌落形态各异的微生物.通过以玉米赤霉烯酮为唯一碳源的无机盐培养基高通量筛选,获得8株降解玉米赤霉烯酮效果良好的菌株,其中2株降解效果较高,分别命名为Y64-3和Y84-7,降解率分别为60.08％和54.90％.经初步鉴定两株菌株均为芽孢菌.     

玉米赤霉烯酮液相色谱法的应用与分析

详细介绍了液相色谱法测定玉米赤霉烯酮的原理、试样制备、样品提取、仪器调试等的操作过程和技术要领;对加标回收、质量控制样品验证等开展监控结果有效性控制,从而可确保玉米赤霉烯酮液相色谱法测定的准确性和可靠性.     

降解玉米赤霉烯酮的芽孢杆菌筛选

利用96孔板,通过菌株与毒素共培养的方法,从217株芽孢菌中,经初筛和复筛得到两株具有很好降解效果的菌株,即T-246和T-420,它们分别能够在6 h和9 h内将15μg/m L的玉米赤霉烯酮完全降解。通过16S r DNA序列分析鉴定,T-246和T-420分别为同温层芽孢杆菌(Bacillus stratosphericus)和短小芽孢杆菌(Bacillus pumilus)。对其降解机理的初步研究表明,这两株菌对玉米赤霉烯酮都具有一定程度的吸附作用,但主要作用源于胞外酶的酶促降解。     

玉米赤霉烯酮水解酶耐热性的分子改造

内酯水解酶ZHD101对玉米赤霉烯酮降解效果显著,是目前研究最广泛的玉米赤霉烯酮降解酶,但由于热稳定性较低,无法满足饲料生产制粒过程中的温度要求。作者利用理性设计和分子改造在其分子中引入二硫键,以提高ZHD101的热稳定性。通过在分子引入七对半胱氨酸双突变(A110C/P196C、S136C/R189C、D143C/P181C、S147C/P181C、D199C/A202C、L200C/A231C、R204C/G205C)以形成潜在的二硫键,研究其对热稳定性的影响。结果表明,突变体S136C/R189C和D143C/P181C在50℃加热处理2 min后的残余活性高于野生型,其中突变体D143C/P181C的残余活性是野生型的2倍,且室温下活力损失小于10%。在此基础上设计四突变(S136C/D143C/P181C/R189C),热稳定性和活力并不优于双突变D143C/P181C。本研究结果为提高ZHD101在饲料工业上的应用潜力打下了基础。     

抗赭曲霉素A单克隆杂交瘤细胞系的建立及特性

为快速检测食物中的赭曲霉素A ,建立了抗OchratoxinA的单克隆杂交瘤细胞株。用OA -匙孔嘁血蓝素 (OA KLH)偶联物免疫 8～ 10周龄雌性Balb c小鼠后 ,取脾细胞与小鼠骨髓瘤细胞系Sp2 0融合 ,经过 3～ 4次亚克隆建立了 3个稳定分泌抗赭曲霉素A抗体的杂交瘤细胞株 ,分别命名为 10G7、10G10和 5G11。将上述 3株杂交瘤细胞分别注入Balb c小鼠腹腔 ,获得含抗赭曲霉素A单克隆抗体的腹水。将腹水用饱和硫酸铵法纯化 ,得到 10G7、10G10和 5G11单克隆抗体。其中10G10、5G11单克隆抗体的Ig亚类为IgG1,10G7单克隆抗体的Ig亚类为IgG2a。抗体腹水的稀释度为 1∶6 4× 10 6～ 1∶1 3× 10 8,参考工作浓度为 1∶1 0× 10 6～ 1∶3 2× 10 7。纯化后 10G7抗体的IgG含量为 16 4g L ,亲和常数为 9× 10 -8mol L。 10G7抗体与其它结构类似物无交叉反应 ,具有较高的特异性。     

Purification of alginate and feasible production of monoclonal antibodies by the alginate-immobilized hybridoma cells

Alginate has an extensive usage in the immobilization of many cell types. Although they have high biocompatibility, commercial alginates contain various degrees of contaminants such as polyphenols, endotoxins and proteins. Thus, these alginates show cytotoxicity against sensitive cell types such as hybridoma cells. In the studies so far, owing to this fact, commercially purchased high-priced ultrapure alginates have been used in the immobilization of hybridoma cells for monoclonal antibody production. However in this study, as a novelty, low-priced commercial alginate was purified, and then the cultivation of alginate-immobilized hybridoma cells was performed for feasible monoclonal antibody production. Low-priced commercial alginate was purified with a profitability ratio of 40%. Then, an optimized immobilization procedure was conducted effectively by using the purified alginate. During more than 25 days of cultivation, serum concentration was kept low, and approximately 2 times greater monoclonal antibody production was achieved, in comparison with its free suspended counterpart. The results showed that the efficiency of monoclonal antibody production via alginate-immobilized hybridoma cultivation can be increased by performing a proved in-house purification method. By shedding light on the efficiency of the in-house purification method, the results also indicated a feasible way of monoclonal antibody production.     

FTB单克隆抗体的研制与特性鉴定

为建立测定ＦＴＢ的ＩＣ－ＥＬＩＳＡ方法，用复合抗原ＦＴＢＳ－ＢＳＡ和ＦＴＢＳ－ＨＳＡ作为免疫原免疫ＢＡＬＢ／ｃ小鼠，取免疫小鼠脾细胞与鼠ＳＰ２／Ｏ－Ａｇ１４骨髓瘤细胞融合，经反复筛选，获得２株稳定分泌抗ＦＴＢ抗体的单克隆杂交瘤细胞株。免疫球蛋白亚型鉴定，１株为ＩｇＭ，１株为ＩｇＧ１。两株杂交瘤腹水的ＥＬＩＳＡ效价分别为１×１０－６和１×１０－８，用ＰＥＧ沉淀法和辛酸－饱和硫酸铵法对抗体腹水进行了纯化，并分别测定了２种单抗与ＦＴＢＳ－ＩｇＧ的亲和力常数以及与其它１１种真菌毒素的相对交叉反应，建立了测定ＦＴＢ的ＩＣ－ＥＬＩＳＡ法。     

海藻糖对单克隆抗体热稳定性保护作用的研究

为研究海藻糖在保护单克隆抗体热稳定性方面的作用,将海藻糖化单克隆抗体置于室温、37℃、56℃条件下存放,采用倍比稀释法对抗体活性进行检测,同时还比较不同浓度的海藻糖对单克隆抗体热稳定性的影响。实验结果表明：海藻糖的浓度为0.250mol/L,且在室温存放的条件下,抗体可以存放一年而具有活性,因此,海藻糖可以作为一种天然保藏剂,有效地提高单克隆抗全的热稳定性,相对延长其在室温条件下的存放时间。     

Immunological characteristics of monoclonal antibodies against shellfish major allergen tropomyosin

Two types of murine monoclonal antibodies (MAbs) against American lobster (Homarus americanus) were generated and characterized. Three purified MAbs were characterized to be specific to the shellfish major allergen tropomyosin. MAbs 5G5E1 and 1A3A7 were reactive to tropomyosin from crustacean species only, whereas MAb 2A7H6 was reactive to both crustacean and mollusk tropomyosins. None of the antibodies reacted to vertebrate tropomyosins. Competitive ELISA indicated that the antigenic epitopes recognized by the two types of MAbs were different from each other. In addition, competitive immunoblot results showed that the binding of shellfish-allergic patient IgEs to lobster tropomyosin was inhibited by the MAb 2A7H6 only. This finding suggests that the antigenic epitope for the 2A7H6 antibody might be similar or close to the allergenic epitope shared by crustaceans and mollusks. Consequently, the MAbs recognizing the different common antigenic epiotopes obtained in the present study would not only facilitate the allergen characterization of shellfish, but may also be useful for the development of specific and sensitive immunoassays for allergen quantification or epitope mapping.     

Characterisation of monoclonal antibody against aflatoxin B1 produced in hybridoma 2C12 and its single-chain variable fragment expressed in recombinant Escherichia coli

An anti-aflatoxin B₁ monoclonal antibody (anti-AFB₁ mAb) from the hybridoma 2C12 was established and its inhibition concentration fifty (IC₅₀) for AFB₁ and relative cross-reactivities (CRs) to other mycotoxins were estimated to be 8 ng/mL and less than 4% compared with AFB₁ by a competitive direct enzyme-linked immunosorbent assay. For production of anti-AFB₁ single-chain variable fragment (anti-AFB₁ scFv) in recombinant Escherichia coli, its scFv-coding genes were cloned from the hybridoma 2C12. The anti-AFB₁ scFv formed inclusion bodies in the cytoplasm of E. coli required in vitro refolding process and hence recovered to retain binding activity successfully. Surface plasmon resonance analysis resulted that anti-AFB₁ scFv possessed 1.16 × 10⁻⁷ M of equilibrium dissociation constant (K_D), which was about 17 times higher than the parental anti-AFB₁ mAb of 6.95 × 10⁻⁹ M.     

Development of a new monoclonal antibody based direct competitive enzyme-linked immunosorbent assay for detection of brevetoxins in food samples

Brevetoxin B (PbTx-2) was covalently linked to carrier protein bovine serum albumin and human gamma globulin. A monoclonal antibody against PbTx-2, which showed high cross-reactivity values with PbTx-1, PbTx-3 and PbTx-9 (more than 89%) was obtained from ascites and some characteristics of monoclonal antibody were studied. An direct competitive enzyme-linked immunosorbent assay (ELISA) for detection of PbTxs was developed, which showed an IC50 value of 5.3 ng mL⁻¹ with a detection limit of 0.6 ng well⁻¹. The recoveries of PbTxs from cockle (88.4%–102.3%) and oyster (89.4%–104.3%) demonstrated that the matrices of cockle and oyster where PbTxs are found do not interfere with the assay. The newly developed competitive ELISA appears to be a reliable and useful method for mass monitoring of PbTxs in mollusk.     

玉米赤霉烯酮多克隆抗体的制备及特性分析

合成玉米赤霉烯酮半抗原,应用液相色谱-质谱联用法进行鉴定,并采用活泼酯法将半抗原与载体蛋白OVA或BSA偶联,分别作为免疫原或包被原,并通过SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和紫外扫描法鉴定偶联效果,免疫3只BALB/c小鼠,制备玉米赤霉烯酮多克隆抗体。结果表明:抗血清效价最高达1∶32 000,以此多抗建立的玉米赤霉烯酮间接竞争标准曲线IC50为39.8ng/mL,IC10为0.71ng/mL,多抗与玉米赤霉烯酮类似物β-zearalenol、zear-alanone、α-zearalanol、β-zearalanol交叉反应率分别为4.80%,3.07%,0.96%,0.09%。说明试验成功制备了玉米赤霉烯酮人工抗原及高特异性多克隆抗体。