克诺罗杆菌检测用单克隆抗体的制备及功效评价

目的:选择克诺罗杆菌标准菌株中的多株代表菌株作为抗原,采用多抗原组合免疫的方法,筛选制备抗克诺罗杆菌属（Cronobacter spp.）检测用单克隆抗体（monoclonal antibody,mAb）,并对获得的抗体进行各项指标评价,为进一步建立克诺罗杆菌免疫学快速检测方法创造条件。方法:采用冻融裂解、超声破碎、全颗粒三种方法制备抗原,以克诺罗杆菌标准菌株及分离菌株20株,9株非克诺罗杆菌进行筛选。筛选获得的抗体鉴定特异性,进行Ig亚类分型,测定效价及相对亲和力常数。结果:获得6株针对克诺罗杆菌的杂交瘤细胞株,腹水效价均在1∶107以上;相对亲和常数均大于1.0×1010L/mol。采用\     

Improved methodology for the simultaneous detection of the trichothecene mycotoxins deoxynivalenol and nivalenol in cereals

A simple and sensitive method has been developed for the analysis of two trichothecene mycotoxins (deoxynivalenol and nivalenol) in cereals. These toxins were extracted with acetonitrile/water (3:1), defatted with n-hexane and purified by a two-step chromatographic procedure using Florisil and Sep-pak columns. The amounts of deoxynivalenol (DON) and nivalenol (NIV) in the column eluates were quantitated by gas chromatography with electron capture detector and gas chromatography-mass spectrometry (selected ion monitoring). The limits of detection of the method were 2.0 micrograms/kg for DON and NIV with recoveries of the toxins spiked into polished rice, wheat and corn at 300 micrograms/kg averaging 87% and 86% respectively.     

The coexistence of the Fusarium mycotoxins nivalenol, deoxynivalenol and zearalenone in Korean cereals harvested in 1983

A survey for the occurrence of nivalenol (NIV), deoxynivalenol (DON) and zearalenone (ZEN) in Korean cereals (totalling 53 samples) harvested in 1983, showed that 96%, 72% and 57% of the samples were contaminated with NIV, DON and ZEN, respectively. Average concentrations (micrograms/kg) in unpolished barley were 546 (NIV), 117 (DON) and 110 (ZEN), and those in polished barley were 130 (NIV) and 21 (DON). The ZEN levels were below the detection limit (1 microgram/kg). Malt, wheat and rye were also heavily contaminated with these Fusarium mycotoxins. The results of this survey show that Korean cereals harvested in 1983 were significantly contaminated with NIV, DON and ZEN, and the incidence and levels, where observed, are similar to those reported in Japan.     

Development of monoclonal antibodies for the fusarin mycotoxins

The fusarins are a group of mycotoxins produced by fungi that commonly infest cereal crops, in particular by the fungus Fusarium verticillioides. This group of compounds is characterized by a substituted 2-pyrrolidone ring attached to a 12-carbon polyunsaturated backbone. Several of the fusarins contain an epoxide substitution on the pyrrolidone ring and are highly mutagenic. This paper describes the development of seven monoclonal antibodies and immunoassays for detecting fusarins C and A. Fusarin C was isolated and conjugated to ovalbumin to produce the immunogen. Competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) were developed based upon the isolated monoclonal antibodies. The concentrations of fusarin C able to inhibit colour development by 50% (IC₅₀) in CI-ELISAs were 1.0, 2.0, 3.6, 23.4, 28.9, 31.4, and 66.7 ng ml^-1 for clones 1-38, 1-30, 1-5, 1-7, 1-43, 1-25, and 1-21, respectively. Cross-reactivity with fusarin A was 44.8, 51.4, 41.1, 174.0, 62.6, 78.2, and 98.0% for clones 1-38, 1-30, 1-5, 1-7, 1-43, 1-25, and 1-21, respectively. Given the sensitivity of these antibodies for fusarins it is expected that, with further development, they may be useful for detecting fusarins at relevant levels in foods.     

Stability of the Fusarium mycotoxins nivalenol, deoxynivalenol and zearalenone in ground maize under typical cooking environments

The effects of moisture, pH and heat on the stability of nivalenol (NIV), deoxynivalenol (DON) and zearalenone (ZEN) present as natural contaminants of ground maize were measured for different periods. Standard solution tests were also performed to measure pH, salt and temperature effects on NIV and DON. The solution tests showed NIV and DON to be relatively stable in buffer solutions over the pH range 1-10. Quite harsh conditions (pH 12, high salt concentration, 80°C, prolonged exposure) were needed to give substantial breakdown. In the ground maize substrate, these toxins were further stabilized relative to the solution tests. NIV and DON were both reduced (range 60-100%) by treatment with aqueous bicarbonate solution at 10, 20 or 50% of the ground maize dry weight, and subsequent heating at 80 or 110°C for 2 and 12 days. There was no measurable reduction at lower test temperatures (20, 40°C). NIV (but not DON) also showed some reduction following addition of water and heating at 80 or 110°C for 12 days. ZEN content was not reduced even by 12 days of heating at 110°C after treatment with a sodium bicarbonate solution.     

Development of monoclonal antibodies for the fusarin mycotoxins

The fusarins are a group of mycotoxins produced by fungi that commonly infest cereal crops, in particular by the fungus Fusarium verticillioides. This group of compounds is characterized by a substituted 2-pyrrolidone ring attached to a 12-carbon polyunsaturated backbone. Several of the fusarins contain an epoxide substitution on the pyrrolidone ring and are highly mutagenic. This paper describes the development of seven monoclonal antibodies and immunoassays for detecting fusarins C and A. Fusarin C was isolated and conjugated to ovalbumin to produce the immunogen. Competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) were developed based upon the isolated monoclonal antibodies. The concentrations of fusarin C able to inhibit colour development by 50% (IC₅₀) in CI-ELISAs were 1.0, 2.0, 3.6, 23.4, 28.9, 31.4, and 66.7?ng?ml^?1 for clones 1–38, 1–30, 1–5, 1–7, 1–43, 1–25, and 1–21, respectively. Cross-reactivity with fusarin A was 44.8, 51.4, 41.1, 174.0, 62.6, 78.2, and 98.0% for clones 1–38, 1–30, 1–5, 1–7, 1–43, 1–25, and 1–21, respectively. Given the sensitivity of these antibodies for fusarins it is expected that, with further development, they may be useful for detecting fusarins at relevant levels in foods.     

Isolation and characterization of monoclonal antibody directed against bovine alpha s2-casein.

A monoclonal antibody 62-1A of isotype IgM, directed against bovine alpha s2-casein, was isolated and characterized. Monoclonal antibody 62-1A recognized bovine alpha s2-11P- and alpha s2-9P-caseins by indirect solid phase radioimmunoassay and Western blot analysis. Crossreactivity toward native genetic variants (A, B, and C) of alpha s1-casein was similar but lower (approximately 60 to 70%) than that for alpha s2-11P-casein). Little or no crossreactivity was observed for other bovine milk or serum proteins. Antibody affinity for alpha s2-11P-casein was 1.3 x 10(9)/M. As little as 25 ng/ml (.5 ng/well) of alpha s2-11P-casein was detected by solid phase radioimmunoassay.     

Survey of the presence of the Fusarium mycotoxins nivalenol, deoxynivalenol and T-2 toxin in Korean cereals of the 1989 harvest.

Twenty eight samples of rice, barley, millet, corn and Indian millet harvested in Korea in 1989 were subjected to assay for contamination of nivalenol (NIV), deoxynivalenol (DON) and T-2 toxin by using gas chromatography and gas chromatography-mass spectrometry. Seven samples were found to be positive for NIV and DON in the ranges of 189-624 micrograms/kg and 168-506 micrograms/kg, respectively. Of the contaminated samples, three samples, one barley, one Indian millet and one corn sample were contaminated simultaneously with both NIV and DON. T-2 toxin was not detected in any samples.     

Fate of the fusarium mycotoxins, deoxynivalenol, nivalenol and zearalenone, during extrusion of wholemeal wheat grain

In the European Union, deoxynivalenol in cereals and cereal products is controlled by recent legislation with the objective of minimizing consumer exposure to this mycotoxin. Relatively few studies have examined the loss of Fusarium mycotoxins during processing and whether this is accurately reflected by the processing factors. The behaviour of deoxynivalenol, nivalenol and zearalenone during extrusion of naturally contaminated wholemeal wheat flour has been examined using pilot-scale equipment. Factors examined were temperature and moisture content. Concentrations of the three mycotoxins were little changed by extrusion although the amount of deoxynivalenol decreased at the lowest moisture content. However, this effect did not appear to be temperature-dependent, suggesting that the apparent loss is either due to binding or inability to extract the residue. Under some conditions, concentrations of the mycotoxins, particularly nivalenol, were higher after extrusion.     

Fate of the fusarium mycotoxins,deoxynivalenol, nivalenol and zearalenone,during extrusion of wholemeal wheat grain

In the European Union, deoxynivalenol in cereals and cereal products is controlled by recent legislation with the objective of minimizing consumer exposure to this mycotoxin. Relatively few studies have examined the loss of Fusarium mycotoxins during processing and whether this is accurately reflected by the processing factors. The behaviour of deoxynivalenol, nivalenol and zearalenone during extrusion of naturally contaminated wholemeal wheat flour has been examined using pilot-scale equipment. Factors examined were temperature and moisture content. Concentrations of the three mycotoxins were little changed by extrusion although the amount of deoxynivalenol decreased at the lowest moisture content. However, this effect did not appear to be temperature-dependent, suggesting that the apparent loss is either due to binding or inability to extract the residue. Under some conditions, concentrations of the mycotoxins, particularly nivalenol, were higher after extrusion.     

Production of trichothecene mycotoxins by Australian Fusarium species.

Australian isolates of Fusarium species were grown on potato dextrose agar. Trichothecenes produced by these species were extracted by ethyl acetate followed by methanol and a silica gel column was used to clean-up the extract. The extracted samples were derivatized by acetylation with trifluoroacetic anhydride and the derivatives analysed by gas chromatography/mass spectrometry (GC/MS). Multiple ion detection was used to trace ions characteristic of the trichothecenes expected to be present. Quantitation of those found was based on a known mass of pentabromophenol that was added as an internal standard. Eight species of Fusarium (nineteen strains) were surveyed, of which three species, F. acuminatum, F. equiseti and F. sporotrichioides, produced the trichothecenes scirpentriol, diacetoxyscirpenol, neosolaniol, HT-2 toxin, T-2 toxin, T-2 tetraol and deoxynivalenol. Wheat samples were inoculated with four different species of Fusarium, F. acuminatum, F. equiseti, F. graminearum and F. sporotrichioides, and in these samples diacetoxyscirpenol, neosolaniol, HT-2 toxin and T-2 toxin were found.     

A limited survey of Fusarium mycotoxins nivalenol, deoxynivalenol and zearalenone in 1984 UK harvested wheat and barley

A limited survey for the occurrence of nivalenol (NIV), deoxynivalenol (DON) and zearalenone (ZEN) in 1984 UK-grown cereals (31 samples) have been carried out using a new procedure, which is a rapid and sensitive method for Fusarium mycotoxins. NIV, DON and ZEN were detected in 17 (55%), 20 (65%) and 4 (13%) out of 31 samples, and average levels in positive samples were 101 micrograms/kg, 31 micrograms/kg and 1 microgram/kg, respectively. Additional surveys on two wheat and eight barley samples harvested in Scotland have shown that 30%, 60% and 100% of the samples were contaminated with NIV, DON and ZEN, respectively. The contents averaged 391 micrograms/kg of NIV, 39 micrograms/kg of DON and 9 micrograms/kg of ZEN. The results of this survey show that UK-grown cereals were significantly contaminated with NIV, DON and ZEN in a similar way to that observed in Japan, Korea and China. This is the first evidence of the natural occurrence of NIV in UK cereals.     

西马特罗单克隆抗体制备以及酶联免疫试剂盒的研究

通过制备西马特罗半抗原,载体蛋白与之偶联合成免疫原,免疫原用于免疫动物,获得抗西马特罗单克隆抗体,制备能够检测猪尿中西马特罗残留量的酶联免疫试剂盒。结果表明,该试剂盒对猪尿中西马特罗的检测限为0.295 μg/L,半抑制浓度（IC50）值为0.870 μg/L,回收率范围为81.5%～103.5%,标准曲线线性范围为0.1～8.1 μg/L,精密度试验结果批内、批间的相对标准偏差（RSD）均＜15%,表明该检测方法准确度和精密度高。该试剂盒特异性较好,于4 ℃条件下能够保存12个月,稳定性较好,可以用于猪尿中西马特罗德快速检测。     

Production of mycotoxins by selected Fusarium graminearum and F. crookwellense isolates.

Corn cultures (five isolates each of Fusarium graminearum Group 1 from wheat crowns, Group 2 from scabby wheat grains and from ear rot of corn and five isolates of F. crookwellense) were screened for their ability to produce deoxynivalenol (DON), nivalenol (NIV), fusarenon-x (FUS-X) and zearalenone (ZEA). Nine of the ten F. graminearum isolates from wheat produced DON (5-165 micrograms g-1) but none produced either NIV or FUS-X. Conversely, 3/5 and 2/5 of the F. graminearum isolates from corn produced NIV (5-40 micrograms g-1) and FUS-X (5-7 micrograms g-1), respectively, while none produced DON. All but one of the F. graminearum isolates produced ZEA (2-1160 micrograms g-1). None of the F. crookwellense isolates produced DON, but 5/5 and 4/5 produced NIV (6-170 micrograms g-1) and FUS-X (3-90 micrograms g-1), respectively, and all produced ZEA (605-1030 micrograms g-1). The results confirmed previous findings on the presence of two distinct F. graminearum chemotypes.     

Identification of tetrodotoxin antigens and a monoclonal antibody

Two kinds of antigens for tetrodotoxin (TTX) were made, using carrier protein ovalbumin (OVA) and bovine serum albumin (BSA), by the formaldehyde method. Nondenaturing gel electrophoresis, gel filtration and ultraviolet spectrophotometry were employed to analyze the coupling of TTX to carrier proteins. The results indicate that nondenaturing gel electrophoresis and gel filtration can be employed to analyze the coupling of TTX to BSA and OVA qualitatively, and ultraviolet spectrophotometry can be employed to analyze the molecular coupling ratio of TTX to BSA and OVA quantitatively. Spleen cells from Balb/C mice immunized with an artificial TTX-formaldehyde-ovalbumin conjugate were fused with SP2/0 myeloma cells. A hybridoma cell line (4D5), which secreted IgG₁ subclass monoclonal antibody against TTX, was produced by “limiting dilution” cloning. The molecular weight, the affinity constant and the titre of the monoclonal antibody (mAb) secreted by 4D5 were 183.69 kDa, 0.98 × 10⁸ M⁻¹ and 3.6 × 10⁵, respectively. The number of the hybridoma chromosome was 88–104.     

高有机溶剂耐受性的苯并(a)芘单克隆抗体制备及其免疫反应特性研究

目的 制备适应油脂样品中苯并(a)芘[Benzo(a)pyrene,BaP]检测需求的抗BaP单克隆抗体。方法 利用杂交瘤单克隆抗体技术制备抗BaP的抗体,在抗体筛选过程中使用高浓度的有机溶剂(50%甲醇)来选择具有较高有机溶剂耐受能力的抗体,并利用间接竞争酶联免疫吸附法考察了抗体的免疫反应特性。结果 筛选获得一株对有机溶剂有较高耐受能力的、稳定分泌BaP单克隆抗体的细胞株2H10,该抗体为IgG1亚型,可耐受高浓度的甲醇、丙酮、二甲亚砜(Dimethyl sulfoxide,DMSO)和二甲基甲酰胺(Dimethylformamide,DMF)。60%甲醇和60% DMSO作为样品稀释液时,抗体对BaP的IC₅₀分别为81.4和24.5 ng/mL,以60% DMSO作为样品稀释液,可以显著降低BaP抗体对其它多环芳烃化合物的交叉反应率。结论 本研究获得高特异性的BaP人工抗原及高有机溶剂耐受性的抗BaP单克隆抗体。     

Production and partial characterization of monoclonal antibodies specific to cooked poultry meat

Monoclonal antibodies (MAbs) specific to cooked poultry muscle proteins have been developed for the detection of poultry adulterants in cooked mammalian meat. Saline (0.85% NaCl) extract of heat-treated (100 °C, 15min) chicken muscle proteins was used to immunize mice for MAb development. The specificity of MAbs was tested against chicken antigen and protein extracts from seven other meat species (pork, beef, lamb, deer, horse, turkey and duck) by indirect enzyme-linked immunosorbent immunoassay (ELISA). The immunogenic components in the poultry protein extracts were determined by SDS-PAGE followed by immunoblotting. A total of six hybridoma cell lines that secrete IgG class MAbs have been developed: MAbs 3E12 and 1A5 were able to distinguish between cooked poultry and mammalian meats, MAbs 9C6 and 6F7 reacted strongly with cooked chicken only, and MAbs, 5D2 and 6G8, reacted with both cooked turkey and chicken but not other species. All six MAbs demonstrated a proportional increased ELISA response to respective adulterated poultry samples in pork over a 0-100% range of aduleration.     

Production and characterization of monoclonal antibodies specific to chicken muscle soluble proteins

Three stable hybridoma cell lines (AH4, BC9 and CF2) have been produced which secrete monoclonal antibodies specific for chicken and turkey muscle proteins. Partial characterization by ELISA and SDS-PAGE immunoblotting indicated that the antibodies failed to cross-react with similar extracts of pork, beef, lamb, horse or rabbit. One of the cell lines (AH4) secreted a monoclonal antibody that was also capable of distinguishing between chicken and turkey by indirect ELISA.     

Production of a horse-specific monoclonal antibody and detection of horse meat in raw meat mixtures by an indirect ELISA

A stable hybridoma cell line (DD3) has been produced secreting a monoclonal antibody specific for horse muscle proteins. The DD3 monoclonal antibody (mAb) did not show significant cross-reactivity when tested against beef, chicken, pig and soya proteins or bovine caseins, gelatine and bovine serum albumin. The DD3 mAb was used in an indirect ELISA format for the detection of defined amounts of horse meat (10–500 g kg-¹) in beef meat mixtures. Immunorecognition of monoclonal antibodies adsorbed to horse meat adsorbed onto the ELISA plate was made with rabbit anti-mouse immunoglobulins conjugated to the enzyme horseradish peroxidase. Subsequent enzymic conversion of the substrate gave clear optical density differences when assaying mixtures of minced beef containing different amounts of horse meat.     

呋喃它酮代谢物5-甲基吗啉-3-氨基-2-恶唑烷酮单克隆抗体的制备、鉴定与胶体金免疫层析试纸条的研制

目的:制备5-甲基吗啉-3-氨基-2-恶唑烷酮（AMOZ）单克隆抗体,并建立AMOZ胶体金试纸条检测方法。方法:AMOZ与对醛基苯甲酸反应生成半抗原CP-AMOZ,将CP-AMOZ与BSA、OVA偶联制备完全抗原。免疫小鼠后制备腹水型抗体,ELISA检测抗体效价与其对CP-AMOZ半抑制浓度（IC50）和最低检测限（LOD）,并检测抗体的特异性。胶体金与单抗偶联,同时在NC膜上包被完全抗原、兔抗鼠多抗分别作为检测线和质控线,制备免疫层析试纸条,测定试纸条的灵敏度与特异性。结果:成功制备CP-AMOZ-BSA、CP-AMOZ-OVA完全抗原及抗CP-AMOZ的单抗,抗体效价为1∶1.6×105,对CP-AMOZ IC50为0.86μg/L,LOD0.07μg/L,与其他硝基呋喃类药物代谢物及其衍生物均无交叉反应。制备的胶体金试纸条灵敏度5μg/L。结论:成功合成了CP-AMOZ的完全抗原,免疫小鼠后制备了特异性强的单抗,并以此为基础研制出敏感、特异的胶体金快速检测试纸条。