甘草酸人工抗原的合成鉴定及免疫原性分析

目的制备中药甘草的活性成分甘草酸(GA)的人工抗原及抗血清,为制备GA的单克隆抗体、并建立快速检测GA的酶联免疫吸附测定(ELISA)提供技术基础。方法将GA与载体蛋白牛血清白蛋白(BSA)偶联起来制得完全抗原后,经基质辅助激光解吸飞行时间质谱鉴定其相对分子质量,用此抗原免疫BALB/c小鼠,制备抗血清,并通过间接ELISA法和竞争ELISA法检测其抗体效价和特异性。结果合成的人工抗原GA-BSA中GA与BSA的结合比约为7∶1;免疫小鼠得到特异针对GA的多抗血清,GA抗体的效价为1∶8000。结论成功地合成了GA的人工抗原,且该抗原有较好的免疫原性,可应用于建立GA的免疫分析方法。     

活泼酯法合成绿原酸人工抗原及其鉴定

目的人工合成绿原酸(chlorogenic acid,CGA)完全抗原并进行鉴定,为制备绿原酸单克隆抗体奠定基础。方法采用活泼酯法,将绿原酸分别与牛血清白蛋白(bovine serum albumin,BSA)和卵白蛋白(ovalbumin,OVA)进行偶联,用紫外扫描法、SDS-PAGE电泳法进行鉴定;间接ELISA法测定免疫小鼠血清抗体效价。结果经紫外扫描法和电泳法检测,CGA与载体蛋白成功偶联,免疫小鼠后获得的多抗血清效价达到1×106。结论成功合成绿原酸完全抗原,可用于绿原酸单克隆抗体的制备。     

抗脂联素单克隆抗体的制备及免疫学特性分析

目的:探讨抗脂联素(adiponectin,APN)单克隆抗体(McAb)的制备方法及其免疫学特性.方法:采用FMP有机化学同相合成法合成APN,用HPLC进行纯化,以此合成抗原免疫BALB/c小鼠,制备McAb,并对抗体的效价、特异性、亚类及位点进行测定.结果:融合后的克隆率为85.4%,阳性率3.2%,腹水效价为1.0×102,与内脂素、瘦素、牛血清白蛋白、人血清白蛋白无交叉反应,A2、A3、A4、A6、A8为lgG2a,A1、A2、A7为IgG2b.经单抗体相加试验和双抗体竞争试验证实,A1针对一个抗原位点,A3和A3针对另一个抗原位点.结论:利用合成的APN免疫动物可成功制备抗APN McAb,该抗体效价高、特异性强.     

抗脂联素单克隆抗体的制备及免疫学特性分析

目的:探讨抗脂联素(adiponectin,APN)单克隆抗体(McAb)的制备方法及其免疫学特性.方法:采用FMP有机化学同相合成法合成APN,用HPLC进行纯化,以此合成抗原免疫BALB/c小鼠,制备McAb,并对抗体的效价、特异性、亚类及位点进行测定.结果:融合后的克隆率为85.4%,阳性率3.2%,腹水效价为1.0×102,与内脂素、瘦素、牛血清白蛋白、人血清白蛋白无交叉反应,A2、A3、A4、A6、A8为lgG2a,A1、A2、A7为IgG2b.经单抗体相加试验和双抗体竞争试验证实,A1针对一个抗原位点,A3和A3针对另一个抗原位点.结论:利用合成的APN免疫动物可成功制备抗APN McAb,该抗体效价高、特异性强.     

Algammulin:一种新的疫苗佐剂的佐剂活性

新的疫苗佐剂Algammulin是由嵌入明矾的免疫刺激物γ菊粉(g-IN)所组成,其大小为1～2μm颗粒。g-IN是一种较强活性的佐剂,明矾具有蛋白载体作用,本文以钥孔蜮血蓝蛋白(KLH)作为抗原,在小鼠中分析Algammulin的佐剂活性。g-IN与明矾的比率为10:1(W/W)。选择5～8周龄的Balb/c小鼠,随机分成几个实验组(每组8～20只),经腹腔分别注射含不同量的佐剂Algammulin、g-IN、明矾的KLH盐水溶液(KLH剂量为0.1、1、10μg),     

罗汉果甜苷V特异性抗血清的制备与应用

目的制备罗汉果甜苷V(MogV)特异性抗血清,建立MogV含量竞争性抑制酶联免疫检测法。方法用罗汉果甜苷V-丁二酸酯-牛血清蛋白(MogV-HS-BSA)结合比约为44∶1为免疫抗原制备抗血清,用罗汉果甜苷V-丁二酸酯-人血清蛋白(MogV-HS-HSA)结合比约为64∶1为包被抗原,建立MogV含量竞争性抑制酶联免疫检测法。结果包被抗原工作浓度为1μg.mL 1,血清最佳稀释倍数为1∶8 000,在此工作浓度下,MogV浓度在1×10-4~1μg.mL 1内回归方程Y=0.158X+0.298,r=0.991 0。结论成功制备了MogV抗血清,MogV浓度在1×10-4~1μg.mL 1内对抗血清的OD(405 nm)值抑制作用呈良好的线性关系。     

小儿氨酚黄那敏片中人工牛黄鉴别方法的改进

目的:建立小儿氨酚黄那敏片中人工牛黄的分光光度鉴别方法。方法采用分光光度法测定人工牛黄中的胆红素的吸收峰。结果小儿氨酚黄那敏片中的胆红素在453±2nm有最大吸收,而乙酰氨基酚、马来酸氯苯那敏和空白辅料,在453±2nm波长处无吸收。结论用最大吸收波长方法可以鉴别小儿氨酚黄那敏片中人工牛黄,方法灵敏、准确,重现性好,可以作为鉴别方法之一。     

含两种不同肽段的血吸虫多抗原肽疫苗的合成及其对BALB/c小鼠的免疫保护作用

目的　设计合成由曼氏血吸虫 2 8KDaGST抗原肽段 2 6 43(P2 6) ,116 131(P116) ,141 15 3(P14 1)和日本血吸虫 2 6KDaGST抗原肽段 187 2 0 2 (J187)中的两种不同肽段组成的血吸虫混合多抗原肽疫苗 ,通过活性试验考察其抗原性、免疫原性及对BALB/c小鼠的保护性免疫效果。方法　用Boc化学和Fmoc化学相结合的策略合成含两种不同抗原肽的多抗原肽疫苗 ,产物经斑点酶联免疫吸附试验测定抗原性 ,在无免疫佐剂存在下接种小鼠 ,ELISA试验检测血清抗体 ,并用日本血吸虫尾蚴攻击感染 ,6周后剖杀小鼠进行体内成虫和肝内虫卵计数。结果和结论　合成的混合多抗原肽能够与感染日本血吸虫的病人或病兔血清结合 ,并能诱导BALB/c小鼠产生对日本血吸虫天然抗原特异的抗体应答。且合成的混合多抗原肽疫苗能够诱导BALB/c小鼠产生显著的抗日本血吸虫感染的保护性免疫力 ,这种免疫力不仅降低成虫负荷数 ,还能显著减少肝组织内虫卵检获数 ,其中 (P116) 4(P2 6) 4 MAP的减虫率和减卵率均达 70 %以上 ,因此有希望发展成为有效的抗血吸虫疫苗     

蛇毒降纤酶多克隆抗体的制备及其某些性质

为制备长白山白眉蝮蛇蛇毒降纤酶的多克隆抗体 ,以期获得酶免疫测定所需的反应试剂 ,供药剂学的研究应用。经免疫兔获得高效价的抗长白山白眉蝮蛇蛇毒降纤酶的抗血清 ,以环状沉淀反应、琼脂免疫双扩散 ,以及酶联免疫吸附法 ,验证了产生的抗体。免疫双扩散及ELISA测定的结果表明 ,长白山白眉蝮蛇蛇毒降纤酶与日本克栓酶有着不同的抗原决定簇结构。 3只兔的抗血清以 1∶7 2万的倍数稀释时 ,其在ELISA测定中的光密度 (OD4 90nm)仍比空白对照高出 0 2以上 ,具有较高效价 ;该抗体与精制蝮蛇抗栓酶有一定的交叉反应 (比率为 0 0 2 4) ,与江浙蝮蛇蛇毒粗品及日本克栓酶间的交叉反应微小 (比率小于 0 0 0 1) ,具有较好特异性     

抗紫杉醇单克隆抗体的制备及ELISA检测方法的建立

目的:制备抗紫杉醇单克隆抗体并建立间接ELISA检测方法。方法:用琥珀酸酐法将紫杉醇(taxol)与牛血清白蛋白(BSA)偶联制成全抗原taxol-BSA,采用紫外波长扫描法和薄层层析检测法对合成抗原进行鉴定;以taxol-BSA免疫Blab/c小鼠,取免疫小鼠脾细胞与SP2/0进行融合,经间接ELISA法筛选、融合细胞有限稀释法克隆、克隆化杂交瘤细胞株的亚类鉴定等筛选出单克隆抗体杂交瘤细胞株,并对单克隆抗体的特异性进行鉴定;用杂交瘤细胞株诱生小鼠腹水,应用辛酸-饱和硫酸铵沉淀法纯化腹水。同时,对间接ELISA法反应条件进行了优化。结果:获得了3株能稳定分泌抗紫杉醇单克隆抗体杂交瘤细胞株,分别命名为taxol-A1,taxol-A2和taxol-A3;taxol-A3株腹水效价为1∶128 000,属于IgG2b亚型;建立的间接ELISA法检测条件为15μg.mL-1抗原、4℃包被过夜、1%BSA封闭1 h,PBS做抗体稀释液,单抗反应时间为30 min,酶标二抗作用时间1 h,10%浓硫酸终止反应。结论:获得了特异性的抗紫杉醇单克隆抗体并建立了间接ELISA检测方法。本研究制备的抗紫杉醇单克隆抗体及建立的间接ELISA检测方法,为从内生真菌生物合成紫杉醇发酵液中迅速、高效地分离纯化和检测紫杉醇奠定了基础。     

Production and characterization of high affinity monoclonal antibodies to cyclic anti-depressant molecules

A procedure is reported by which high levels of the tricyclic molecule desipramine was modified and conjugated at high density to the carrier molecules keyhole limpet hemocyanin and bovine serum albumin so that these could be used as immunogens in Balb/c mice. Such conjugates generated immune responses with high levels of antibody with specificity for the tricyclic. B cell hybridomas generated by fusion of immune Balb/c splenocytes to NS-1 cells which secreted monoclonal antibodies with specificity for the tricyclic were selected in a standard ELISA. In this report, we show that the binding constants of these monoclonal antibodies with various haptens can be assessed accurately by measuring fluorescence polarization, that a high degree of cross-reactivity between the monoclonals and various tricyclics exists, and that this procedure can be used to generate monoclonal antibodies of high binding constants.     

Monoclonal antibody-based enzyme-linked immunoassays for the measurement of palytoxin in biological samples.

Mouse monoclonal and rabbit polyclonal antibodies were produced against conjugates of keyhole limpet hemocyanin and chemically defined palytoxin haptens. Palytoxin haptens were produced by derivatization of the primary amino group with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate or succinimidyl 3-(2-pyridyldithio)propionate. Selected antibodies were used to develop five palytoxin-specific enzyme-linked immunoassay formats for the quantitation of palytoxin in biological matrices, including crude extracts of Palythoa tuberculosa. The formats developed include an indirect competitive inhibition enzyme-linked immunoassay, two types of direct competitive inhibition enzyme-linked immunoassays, and both indirect and direct sandwich enzyme-linked immunosorbent assays. The sandwich enzyme-linked immunosorbent assays are capable of detecting as little as 10 pg palytoxin per test, but may be subject to matrix interference. The direct competitive inhibition enzyme-linked immunoassays detect as little as 30 pg palytoxin per test with a total assay time of only 4 hr. The enzyme-linked immunoassays do not cross-react with the other marine toxins tested, but do cross-react with certain non-toxic, treated preparations of palytoxin. The enzyme-linked immunoassays were used to quantitate palytoxin in P. tuberculosa extracts and to monitor toxin isolation. These enzyme-linked immunoassay systems can substitute for the mouse bioassay of palytoxin, providing a rapid, sensitive, and accurate means of toxin detection.     

Enhanced immunogenicity of a bivalent nicotine vaccine

The efficacy of nicotine vaccines for smoking cessation is dependent upon their ability to elicit sufficiently high serum antibody concentrations. This study compared two nicotine immunogens representing different hapten presentations, 3'-aminomethyl nicotine conjugated to recombinant Pseudomonas exoprotein A (3'-AmNic-rEPA) and 6-carboxymethlureido nicotine conjugated to keyhole limpet hemocyanin (6-CMUNic-KLH), and assessed whether their concurrent administration would produce additive serum antibody concentrations in rats. Effects of vaccination on nicotine pharmacokinetics were also studied. Vaccination of rats with these immunogens produced non cross-reacting nicotine-specific antibodies (NicAb). Serum NicAb concentrations elicited by each individual immunogen were not affected by whether the immunogens were administered alone as monovalent vaccines or together as a bivalent vaccine. The total NicAb concentration in the bivalent vaccine group was additive compared to that of the monovalent vaccines alone. Higher serum NicAb concentrations, irrespective of which immunogen elicited the antibodies, were associated with greater binding of nicotine in serum, a lower unbound nicotine concentration in serum, and lower brain nicotine concentration. These results demonstrate that it is possible to design immunogens which provide distinct nicotine epitopes for immune presentation, and which produce additive serum antibody levels. The concurrent administration of these immunogens as a bivalent vaccine may provide a general strategy for enhancing the antibody response to small molecules such as nicotine.     

Generation of anti-(+)methamphetamine antibodies is not impeded by (+)methamphetamine administration during active immunization of rats

The goal of these studies was to determine if chronic (+)methamphetamine ((+)METH) administration affects the production of anti-(+)METH antibodies during active immunization of rats. Active immunization for the treatment of chronic drug abuse has been proposed for drugs such as cocaine and nicotine. However, studies have not adequately addressed whether continual drug use during treatment would affect the development of an immune response. For the current studies, male Sprague-Dawley rats were immunized with either keyhole limpet hemocyanin (KLH; control group) or a (+)METH hapten ((+)METH with a six carbon spacer group at the para position of the ring structure)-KLH conjugate. The (+)METH-KLH animals were further divided into two groups. One group was immunized with no subsequent administration of (+)METH, while the other group was immunized and repeatedly challenged (twice a week throughout the study) with an i.p. dose of 3 mg/kg (+)METH. The results showed that the two groups of (+)METH-KLH immunized rats developed and maintained anti-(+)METH antibody titers. The anti-(+)METH immune responses of the two groups were not statistically different (P < 0.05) as measured by serum titers and the relative antibody affinities. These data suggest that repeated administration of (+)METH does not affect the generation of an anti-(+)METH antibody response in actively immunized rats.     

Production and characterization of monoclonal antibodies against ochratoxin B.

Monoclonal antibodies against ochratoxin B (OTB) were generated by immunizing Balb/c mice with OTB conjugated to keyhole limpet hemocyanin (KLH) via carbodiimide reactions with CHMC and EDAC. A stable hybridoma cell line 2F1.E10 was produced by fusion of murine splenocytes and myeloma cells. The obtained antibodies were characterized using an indirect competitive ELISA. The detection limit was calculated (27+/-2 nM OTB) and 50% binding inhibition was reached at 500 nM free OTB. A low cross-reactivity to ochratoxin A (OTA) of 3.3% and no cross-reactivities to either coumarin or DL-phenylalanine were observed, suggesting a highly specific OTB antibody. The antibody type was identified as IgG class 1 with the light chain being of the kappa configuration. These antibodies can be used in an indirect competitive ELISA to detect OTB in the nanomolar to micromolar concentration range and may be useful for the analysis of contaminated food items.     

Immunochemical studies on thymosin: radioimmunoassay of thymosin beta 4

Thymosin beta 4, a peptide with hormonal-like properties first isolated from the thymus gland, can be measured in serum using a newly described radioimmunoassay. The radioimmunoassay utilizes an antibody raised in rabbits against synthetic thymosin beta 4 conjugated by glutaraldehyde to keyhole limpet hemocyanin. A 125I-tyrosine-C13 analogue of the biologically active C-terminal fragment is used as the radioactive tracer. The radioimmunoassay is sensitive in the nanogram range and no cross-reactivity with common serum proteins is demonstrable. High performance liquid chromatography of serum samples indicates that two thymosin beta 4 cross-reactive species are present in human serum. Levels in serum range from 450 to 1100 ng/ml and decline with age.     

Assessment of the antigen-specific antibody response induced by mucosal administration of a GnRH conjugate entrapped in lipid nanoparticles

Vaccines administered parenterally have been developed against gonadotrophin-releasing hormone (GnRH) for anti-fertility and anti-cancer purposes. The aim of this study was to demonstrate whether mucosal delivery using GnRH immunogens entrapped in lipid nanoparticles (LNP) could induce anti-GnRH antibody titers. Immunogens consisting of KLH (keyhole limpet hemocyanin) conjugated to either GnRH-I or GnRH-III analogues were entrapped in LNP. Loaded non-ionic surfactant vesicles (NISVs) were administered subcutaneously, while nasal delivery was achieved using NISV in xanthan gum and oral delivery using NISV containing deoxycholate (bilosomes). NISV and bilosomes had similar properties: they were spherical, in the nanometre size range, with a slightly negative zeta potential and surface properties that changed with protein loading and inclusion of xanthan gum. Following immunization in female BALB/c mice, systemic antibody responses were similar for both GnRH-I and GnRH-III immunization. Only nasal delivery proved to be successful in terms of producing systemic and mucosal antibodies.From the Clinical EditorThe main research question addressed in this study was whether mucosal delivery using gonadotrophin-releasing hormone immunogens entrapped in lipid nanoparticles could induce anti-GnRH antibody titers. Only nasal delivery proved to be successful in terms of producing systemic and mucosal antibodies with this approach.     

The optimization of ELISA for methamphetamine determination: the effect of immunogen, tracer and antibody purification method on the sensitivity

To obtain more sensitive immunoassay for methamphetamine (MA) determination, the optimum condition of enzyme-linked immunosorbent assay (ELISA) was investigated in regard to immunogens, antibody purification methods and coating tracers. Activated MA, N-(4-aminobutyl)methamphetamine (4-ABMA), was conjugated with bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) and used as immunogen. The antibodies were purified by protein G chromatography or various immunoaffinity chromatography-linked MA-protein ligands, such as MA-BSA, MA-KLH or MA-ovalbumin (OVA). Each purified antibody was characterized by means of sensitivity and cross-reactivity using the three MA-protein coating tracers, MA-BSA, MA-KLH and MA-OVA. The best sensitivity of each antibody was acquired with the MA-OVA tracer although the tracer concentration and the antibody titer level at optimum condition were varied. The antibody with high titer level did not always yield good sensitivity. At optimum condition, immunoaffinity chromatography-purified antibodies were better for sensitivity and for specificity than protein G-purified antibodies. The cross-reactivity of the purified antibodies seemed to be affected by immunogen structure and showed somewhat different patterns according to the immunoaffinity ligand utilized. These data show that the antibody purification method as well as choice of coating tracer and immunogen is essential for the sensitivity and specificity of EIA; the optimum condition for assay should be discovered using various methods and combinations.     

A novel drug interaction between the quinolone antibiotic ciprofloxacin and a chiral metabolite of pentoxifylline

Pentoxifylline (PTX), a methylxanthine derivative, is metabolized to seven compounds in vivo, with metabolites 1 and 5 possessing biologic activity. Metabolite-1 is a chiral molecule and its S-enantiomer is selectively formed during PTX metabolism in vivo. We have developed a reproducible method of synthesizing a racemic mixture of the chiral metabolite-1 (M-1) of PTX. In this study, we examined the kinetics of racemic M-1 in mice compared to PTX. An interaction between PTX and the quinolone antibiotic ciprofloxacin has been demonstrated. A goal of this study was to determine if a similar interaction occurs between ciprofloxacin and M-1 in vivo. M-1 and PTX had similar absorption and elimination rates. M-1 was rapidly converted to PTX, while very little PTX was converted to M-1 in vivo. The peak concentration of biologically active drug (PTX+M-1) was 36% higher when M-1 was administered compared to PTX. Combination of ciprofloxacin and PTX significantly increased serum concentrations of both PTX and M-1 (2-fold) compared to controls. The combination of M-1 and ciprofloxacin significantly increased serum concentration of M-1 (3-fold) and PTX (2-fold). The ciprofloxacin/M-1 combination produced a significantly higher sera concentration of bioactive drug compared to all other groups suggesting that this combination may enhance the anti-fibrogenic effect.     

Antibody-Mediated Immunotoxicity in American Kestrels (Falco sparverius) Exposed to Polychlorinated Biphenyls

Antibody-mediated immune function in adult and recently fledged (30 to 33 d old) American kestrels (Falco sparverius) was examined in birds exposed directly, or only in ovo, to polychlorinated biphenyls (PCBs). In 1998, 9 mature male and 9 female kestrels were fed PCBs, whereas 9 females and 10 males served as controls. A mixture of Aroclors 1248:1254:1260 suspended in safflower oil was injected into the kestrels' food items, while in control diets only the same volume of oil was added. The dosage of PCBs was approximately 7 mg/kg kestrel/d, beginning in March 1998 and continuing for 120 d. In 1998, the antibody-mediated immune response was stimulated by immunization and booster vaccinations of the kestrels using a nonpathogenic antigen, dinitrophenol-keyhole limpet hemocyanin (DNP-KLH). In 1999, offspring from three treatment groups based upon maternal exposure to PCBs were similarly tested for their antibody response. None of these mothers was vaccinated with DNP-KLH the previous year. The maternal groups were: (1) exposed to PCBs in 1998 for 120 d, (2) exposed in ovo in 1998 (i.e., mothers were produced by PCB-exposed parents), or (3) unexposed to PCBs. Serum antibody levels were determined using an enzyme-linked immunosorbent assay (ELISA). In 1998, PCB-exposed adult females had a significantly higher antibody response than did controls, whereas adult males exposed to PCBs had significantly suppressed antibody production. For the nestlings produced in 1999, maternal treatment significantly affected antibody response. Generally, the antibody response in the nestlings was much lower than that seen in adult kestrels. Yet both male and female offspring from mothers that had been fed PCBs the previous year had significantly higher postbooster anti-DNP-KLH titers than control and in ovo-exposed maternal groups, thus mimicking the response seen in the adult females the previous year. These sex-specific responses in PCB-exposed birds provide further evidence of the endocrine-disrupting behavior of PCBs. Both suppression and stimulation of the antibody response are undesirable because this indicates that the immune system is not able to respond normally to challenges by infectious or other disease-causing agents.