玉米赤霉烯酮单克隆抗体酶联免疫测定方法的建立及初步应用

用ZEN—BSA人工抗原免疫BALB／c鼠,经融合、筛选和克隆化得到可稳定分泌抗ZEN单克隆抗体的杂交瘤细胞株ZEN—lC6。zEN一lC6属IgG₁,纯化腹水抗体效价为10^-5,与5种衍生物的交叉反应系数为0.16～1.20％。用ZEN一1C6建立了检测食品(玉米、小麦、大米)中玉米赤霉烯酮的CIEIA法。该法检测纯毒素的线性范围为5～1000ng／ml,最低检出浓度为0．1ng／ml,平均回收率为84．0～105．5％。用该法测定了81份样品,均有ZEN毒素检出。     

黄曲霉毒素解毒酶催化AFB1-抗体复合物中抗原变构解离初步研究

用免疫学的方法对AFB₁在黄曲霉毒素解毒酶催化前后抗原表位发生改变并从抗原抗体复合物中解离的可能性进行研究探讨。结果表明 :经黄曲霉毒素解毒酶处理后产物中有AFB1表位特征产物的量下降了 2 4 8%～ 72 8% ,而灭活酶组平均只下降203% ;活性酶组AFB1的解离率达到 88% ,灭活酶组AFB1的解离率为 8 7% ,两组比较有显著性差异 (P <0 0 1 ) ,而灭活酶组与质控组的比较无显著性差异 (P >0 0 5 )。分子的模拟计算也支持以上结果。为进一步建立特异性抗体—固定化酶催化体系提供了重要的参考数据 。     

黄曲霉毒素B1的免疫检测Ⅱ．抗体的产生及应用

将黄曲霉毒素B₁肟(AFB₁O)与牛血清白蛋白(BSA)的连接物,通过多点、多次免疫法注射免疫兔子。分析了抗体的产生进程、效价以及特异性。注射抗原后的第60天开始有较明显抗体产生,第120天达到高峰,维持15天左右后开始下降;抗体的ELISA效价高达1：30000;和黄曲霉毒素B₁(AFB₁)的结构类似物的竞争ELISA表明,抗体有很好的特异性。运用该抗体,以ELISA分析检测了几种农产品及饲料中污染AFB₁,的含量,并和薄层层析法的分析结构进行了比较,结果表明当AFB₁．的含量大于等于5ng／ml时。两者间有很好的相关性。     

高特异性抗桔霉素单克隆抗体的制备及鉴定

以1, 4-丁二醇二缩水甘油醚(双环氧试剂)为偶联剂,合成桔霉素-蛋白偶联抗原CIT-BSA,将偶联抗原免疫BALB/C小鼠,取脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,应用有限稀释法进行筛选,经过克隆化后筛选到一株稳定分泌抗桔霉素抗体的杂交瘤细胞株H2-F8．该单克隆抗体经过初步鉴定,抗体类型为IgM类,抗体的相对亲和力为4.17×10⁸ L/mol,单抗与黄曲霉毒素B1、赭曲霉毒素A、脱氧雪腐镰刀菌烯醇和玉米赤霉烯酮等毒素的交叉反应率均低于0.1%,与红曲色素中的橙色素和红色素的交叉反应率均低于0.01%．在此基础上建立了间接竞争ELISA检测方法,线性范围为0.05～1.0 μg/L,IC₅₀值为0.3 μg/L．结果为快速检测桔霉素的酶联免疫检测方法的建立和检测试剂盒的研制提供技术依据．     

金针菇菌丝体深层培养工艺研究

在1．8L气升式发酵反应器中,研究了金针菇菌丝体深层培养工艺条件及其特性。结果表明：金针菇菌丝体深层培养可采用玉米、黄豆和糖蜜等廉价原料作主要碳氮源;最适需氧量为1．0—2．0×10^-7mol O₂／ml·min·atm,培养24—72h内,菌丝球生长遵循立方根规律·即x^1/3,=0．786+0．027t。在最佳发酵条件下,摇瓶培养4天,茵丝干重达24．4mg／ml在反应器培养4天,菌丝干重为25．7mg／ml。     

分泌抗黄曲霉毒素M1抗体的大鼠－小鼠淋巴细胞杂交瘤细胞株的建立及其特性的研究

通过大鼠、小鼠对AFM1－BSA抗体应答的比较,选用应答强的大鼠脾细胞作为亲本细 胞,与小鼠骨髓瘤P3X63一Ag8．653系细胞融合制作杂交瘤。经HAT培液选择和RIA的筛选, 克隆化,最终获得生长良好、稳定分泌抗AFMl抗体的5株大鼠－小鼠淋巴细胞杂交瘤细胞 株。以EusA、竞争结合的RIA进一步证明获得的5个单抗是针对AFMl,并与其衍生物 AFB1有明显的交叉反应。5个单抗亲和力的平衡常数K为10⁹－10¹¹l／M,这表明这些单抗 具有组建检测AF试剂盒的潜在实用价值。     

磁场处理对螺旋藻多糖生产影响的初步研究

一定磁场强度（0～0．6T）处理下培养的螺旋藻生长加快,于物质积累增多,由072mp／Inl增加到0．80mg／ml;多糖含量提高,由4．2％增加到5．1％．磁场处理使培养液电导率随磁场强度增加而降低,使NO^-₃-N含量随磁场强度增加而增加,有利于细胞吸收和新陈代谢。     

抗人IL-6单克隆抗体的制备及鉴定

用基因重组人IL-6免疫Balb/c小鼠,采用小鼠杂交瘤技术,筛选克隆到分泌抗人重组IL-6单克隆抗体的杂交瘤细胞株,并对其中2H₂、 1D₂ 和4B₄瘤细胞株进行了鉴定.其抗体类别均为IgG,亚类分别为IgG1和IgG2a.用多种细胞因子和无关蛋白的鉴别试验结果证实它们均特异地识别rhIL-6.免疫转染结果显示,该单抗识别分子质量为21 ku的IL-6单一条带.IL-6单克隆抗体的亲和常数K_aff= 1.62×10⁹ (mol/L)^-1.     

抗IBDV独特型抗体杂交瘤细胞株的建立及其产物生物学特性测定

用纯化的抗IBDV IgG免疫Balb/c小鼠,取其脾细胞与SP2/0细胞在聚乙二醇（PEG）作用下融合,ELISA法检测筛选,经有限稀释法克隆3次,获得2株（5F₄株,2B₆株）分泌抗IBDV独特型抗体的杂交瘤细胞株,其能诱生Balb/c小鼠产生ELISA抗体效价分别为1∶12 800和1∶25 600的含抗IBDV独特型抗体的腹水。用此独特型抗体与福氏完全佐剂和福氏不完全佐剂乳化制备成抗IBDV独特型抗体疫苗,免疫接种SPF鸡和普通京白公鸡,然后用IBDV强毒株(SD株)2000 ELD50攻毒,SPF鸡免疫组50只,有5只发病、2只死亡;对照组10只全部发病,8只死亡。普通京白鸡免疫组30只,有7只发病, 1只死亡;对照组10只全部发病,6只死亡。经X²检验,SPF鸡X²＝34.15,普通鸡X²＝16.68,查X²值表得X²_(1)0.01＝6.63, SPF鸡X²和普通鸡X2均大于X²_{(1) 0.01}（P＜0.01）,由此表明抗IBDV独特型抗体疫苗具有很好的免疫原性,对易感日龄的SPF鸡和普通鸡均具有极其明显的保护作用。从而证实了抗IBDV独特型抗体疫苗有潜在的研究和应用价值。     

葡萄糖氧化酶产生菌Z—l—C的分批发酵与恒化培养

本文对葡萄糖氧化酶产生菌z—I—c的分批发酵与恒化培养进行了研究。实验发现,在以葡萄糖为生长限制性基质的恒化培养中,该产生菌的维持系数为O．04 g葡萄糖／g菌体．H,生长得率系数Y^max_G=O．714 g菌体／g葡萄糖,最大比生长速率μmax=O．385h^-1,饱和常数Ks=4．76g/L,理论最适稀释度Dm=0．260h^-1,最大酶比活(E／x)max为2．16×10³μ／mg,其值较分批发酵的最大酶比活(1．5l×10³μ／mg)提高43％。当向恒化培养的补料培养基中添加0．02％的α-甲基-D-葡萄糖时,由于该葡萄糖结构类似物的诱导作用,可使(E／x)max达3．11×10³μ／mg,较分批发酵之值提高106％。     

Monoclonal antibodies to human recombinant interleukin 1 (IL 1)beta: quantitation of IL 1 beta and inhibition of biological activity

Monoclonal antibodies (McAb) were developed to the Mr 17,500 form of human recombinant interleukin 1, IL 1 beta. Four McAb have been identified that inhibit the biological activity of IL 1 beta. McAb H34 and H67, at 1 microgram/ml (6 X 10(-9) M), completely inhibit the capacity of 1 ng/ml (6 X 10(-11) M) recombinant IL 1 beta to stimulate the proliferation of murine thymocytes or human fibroblasts in vitro. McAb H6 and H21 are approximately 10-fold less potent, and completely inhibit IL 1 beta activity at 10 micrograms/ml (6 X 10(-8) M) in both assays. The McAb do not have a significant effect on the biological activity of human recombinant IL 1 alpha in either assay. These McAb block the binding of recombinant [125I]IL 1 beta to IL 1 receptors on mouse 3T3 fibroblasts and have affinity constants for IL 1 beta in the range of 10(9) to 10(10) liters/mol. Competition studies suggest that two nonoverlapping epitopes on the IL 1 beta molecule are recognized by the McAb. H6 and H34 recognize one epitope, and H21 and H67 another. McAb H6 and H67 have been used together in a two-site ELISA to detect IL 1 beta. The sensitivity of the ELISA, which is 15 pg/ml (0.86 pM), approaches the limit of sensitivity of the thymocyte proliferation assay. The ELISA and thymocyte proliferation assay were used to quantitate IL 1 beta in E. coli LPS-stimulated human monocyte culture supernatants (HMCS). The level of IL 1 beta detected by ELISA in culture supernatants from eight donors ranged from 1.7 to 5.6 ng/ml, with a mean value of approximately 3 ng/ml. By comparison, the thymocyte proliferation assay gave levels of IL 1 in HMCS that were eight fold higher when quantitated by using recombinant IL 1 beta as a standard. This discrepancy with the bioassay used was reflected by the three fold higher maximum stimulation of thymocyte proliferation by HMCS as compared with recombinant IL 1 alpha or IL 1 beta, and only 45% inhibition of HMCS IL 1 activity by McAb. Thus, factors other than IL 1 beta account for the IL 1-like activity in monocyte culture supernatant as measured by the bioassay. The ILB1 McAb and ELISA allow for the first time-sensitive, accurate, and convenient quantitation of IL 1 beta levels in biological fluids or specimens.     

A monoclonal antibody to aflatoxin B1: detection of the mycotoxin by enzyme immunoassay

A monoclonal antibody (McAb) was produced after fusion of mouse (X63.Ag8.6.5.3) myeloma cells with spleen cells isolated from female Balb-c/NZB F1 hybrid mice immunized with aflatoxin B₁ (oxine)-keyhole limpet haemocyanin conjugate. The hybridoma cell line producing antibody specific for aflatoxin B₁ (AFB₁) was grown in tissue culture and as an ascites tumour. The ascitic fluid gave suitably high dilution titres (1:800 000) by enzyme immunoassay and was conjugated to horseradish peroxidase by a two-step procedure with glutaraldehyde. The conjugate was used to develop a direct competitive enzyme-linked immunosorbent (ELISA) assay for AFB₁. The sensitivity of the ELISA was 0–2 ng/ml with a working range up to 10 ng/ml for AFB₁. The specificity of the McAb was determined and it was shown not to cross-react significantly with any of the metabolites tested. This McAb and the direct competitive ELISA described may prove of use in the detection of AFB₁ in foods and feeds.     

一株抗H5亚型禽流感病毒血凝素蛋白单克隆抗体的广谱中和活性

以H5N1禽流感病毒株Ck/HK/Yu22/02作为抗原,应用常规杂交瘤技术和血凝抑制实验筛选出抗H5亚型禽流感病毒血凝素蛋白的单抗8H5,单抗8H5经免疫荧光鉴定具有很好的H5特异性.选择33株2002～2006年不同地域,不同宿主中分离的不同遗传变异亚系的H5N1病毒代表株,对单抗8H5分别进行血凝抑制实验及中和试验分析,结果显示单抗8H5对所有H5亚型病毒均有较强反应,而对非H5亚型标准病毒株均不反应,说明8H5是一株广谱性抗H5特异性中和单抗,并提示单抗8H5的HA识别表位可能是一个相当保守的中和表位.并且单抗8H5双抗夹心系统的初步评价显示了其在诊断应用上的前景.     

马立克氏病毒单克隆抗体的研究

获得了4株分泌马立克氏病毒(MDV)特异性单克隆抗体(McAb)的杂交瘤细胞:4BS10对MDV所有毒株呈阳性反应;4CN8 对MDV血清1,3型毒株发生反应;2BN90和4CN24只对MDV血清1型毒株有阳性反应。3个McAb属IgG1,1个为IgG2b,均不中和MDV,免疫扩散试验也无沉淀线。对禽白血病毒(ALV)无交叉反应。 以2BN90和辣根过氧化物酶、异硫氰酸荧光素的结合物进行直接酶联免疫吸附试验和直接荧光抗体试验,均获得成功。抗体滴度前者为1/51,200,后者为1/640。对ALV无交叉反应。     

玉米赤霉烯酮单克隆抗体和免疫酶技术研究

采用蛋白质连接技术合成玉米赤霉烯酮抗原,免疫Balb/c鼠,通过淋巴细胞杂交瘤技术建立六株分泌抗玉米赤霉烯酮的单克隆抗体杂交瘤细胞株。间接酶联免疫吸附试验测定细胞上清抗体效价为1:2084(4H8)、1:256(6H9、4H3、2H5、2C8)、1:16(3F10);腹水抗体效价为10~9(4H3、4H8)、10~8(2H5)、10~7(6H9)、10~5(3H10)。竞争间接酶联免疫吸附试验测定六株单克隆抗体对玉米赤霉烯酮的敏感度为0.3—0.8ng/ml。六株抗体与玉米亦霉烯醇的交叉反应率为1.3—9.0％。六株单克隆抗体均属IgG类。细胞体外传代培养和冻存复苏后分泌抗体稳定。纯化抗体在37℃保存12天稳定,-30℃保存90天抗体滴度不变。用该抗体建立竞争间接酶联免疫吸附试验检测掺合玉米赤霉烯酮的玉米、小麦、饲料,平均回收率分别为105％、90％、103％,平均批间变异系数为5.8％、2.8％、6.8％,批内变异系数为3.8％、12.7％、15.7％。样品中玉米赤霉烯酮掺合量与竞争间接酶联免疫吸附试验检出量有良好相关性(r≥0.9996)。     

黄曲霉毒素B_1的免疫检测 Ⅱ.抗体的产生及应用

将黄曲霉毒素Ｂ１肟（ＡＦＢ１Ｏ）与牛血清白蛋白（ＢＳＡ）的连接物,通过多点、多次免疫法注射免疫兔子。分析了抗体的产生进程、效价以及特异性。注射抗原后的第６０天开始有较明显抗体产生,第１２０天达到高峰,维持１５天左右后开始下降;抗体的ＥＬＩＳＡ效价高达１：３００００;和黄曲霉毒素Ｂ１（ＡＦＢ１）的结构类似物的竞争ＥＬＩＳＡ表明,抗体有很好的特异性。运用该抗体,以ＥＬＩＳＡ分析检测了几种农产品及饲料中污染ＡＦＢ１的含量,并和薄层层析法的分析结构进行了比较,结果表明当ＡＦＢ１的含量大于等于５ｎｇ／ｍｌ时,两者间有很好的相关性。     

甲型H1N1流感病毒血凝素蛋白单抗杂交瘤细胞株的制备与初步鉴定

目的:建立具有高特异、高效价的甲型H1N1流感病毒血凝素蛋白(HA)单抗的杂交瘤细胞株。方法:以纯化的昆虫杆状病毒表达的甲型H1N1流感病毒HA蛋白为免疫原免疫BALB/c小鼠,取脾细胞与Sp2/0小鼠骨髓瘤细胞融合,通过有限稀释法筛选阳性克隆,经ELISA和Western blot分析单抗的特性和特异性。结果:获得6株甲型H1N1流感HA抗原特异单克隆抗体杂交瘤细胞株,抗原肽库ELISA检测结果表明其中3株(1E12,3F12,1C11)单抗只与甲型H1N1流感HA抗原肽库反应,不与H5N1病毒HA抗原肽库反应;Western blot分析表明,单抗1B3只特异识别甲型H1N1流感HA抗原,而与其他季节性甲流病毒(H1,H3)及人禽流感H5N1病毒不反应。结论:所获杂交瘤细胞株特异性强,效价高,分泌抗体性能稳定,为分析甲型H1N1流感病毒抗原性位点、建立诊断试剂奠定了基础。     

Monitoring of individual human exposure to aflatoxins (AF) and N-nitrosamines (NNO) by immunoassays

Highly sensitive immunoassays have been used to quantitate aflatoxins (AF) and N-nitrosamines (NNO) in human body fluids and tissues, respectively. This approach was taken in order to quantitate environmental exposure to these agents at an individual level to facilitate the investigation of their role in the etiology of human cancer. In order to analyse AF in human urine, an immunopurification step has been developed by using AF-specific antibody bound to AH-Sepharose 4B gel in a small (4-ml gel volume) affinity column prior to enzyme-linked immunosorbent assay (ELISA). The ELISA can be used to quantitate aflatoxin B1 (AFB1) over the range 0.01 ng/ml to 10 ng/ml and the assay system has been validated by using human urine samples spiked with AFB1 over this concentration range. In addition, 29 urine samples from the Philippines have been analysed and found to contain a range of levels from zero to 4.25 ng/ml AFB1 equivalent with a mean of 0.875 ng/ml. This compared with a mean of 0.066 ng/ml AFB1 equivalent in samples from France. Radioimmunoassay of O6-methyldeoxyguanosine (O6-medG) has been performed on human oesophageal and cardiac stomach mucosal DNA from tissue samples obtained during surgery in Linxian County, People's Republic of China, an area of high risk for both oesophageal and stomach cancer. Using the methodology described and having 1 mg of hydrolyzed DNA allows the detection of approximately 25 fmol O6medG per mg DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

用ELISA法测定抗人心肌肌球蛋白轻链(CMLC)单克隆抗体的K_D值

人心肌肌球蛋白由轻链（ｃａｒｄｉａｃｍｙｏｓｉｎｌｉｇｈｔｃｈａｉｎｓ,ＣＭＬＣ）和重链（ｃａｒｄｉａｃｍｙｏｓｉｎｈｅａｖｙｃｈａｉｎｓ,ＣＭＨＣ）组成．当心肌细胞遭到破坏时,ＣＭＬＣ释放入血,可致血中ＣＭＬＣ浓度增高．血中ＣＭＬＣ的测定是一种高度...     

抗人血栓调节蛋白单克隆抗体的制备与鉴定

为了制备特异性抗人血栓调节蛋白(human thrombomodulin,hTM)的单克隆抗体(monoclonal antibody,McAb),利用脂质体Lipofectamine 2000将包含hTM全长cDNA序列的重组表达质粒pThr402转染CHO细胞,经G418筛选及相关鉴定后获得高效稳定表达hTM的CHO-TM5细胞株。将CHO-TM5细胞直接免疫Balb/c小鼠,应用杂交瘤技术,通过细胞ELISA (cellular enzyme-linked immunoabsorbent assay,CELISA)筛选出阳性克隆后,将杂交瘤细胞株腹腔注射Balb/c小鼠诱生腹水。用CELISA、流式细胞术、免疫组织化学染色法及免疫印迹法对所获McAb的特异性进行鉴定。我们获得了1株可稳定分泌抗hTM的McAb的杂交瘤细胞株NH-1,其亚型为IgGl,McAb腹水效价为1×10~(-6),腹水抗体含量为20 mg/ml。NH-1对相应抗原具有较高的组织特异性,在体内与正常组织的交叉反应少,对人脐静脉内皮细胞、CHO-TM5有特异性结合反应,说明NH-1可特异性识别天然的hTM分子,为进一步应用此McAb进行hTM生物学功能及临床意义研究提供了基础。