Preparation and characterization of monoclonal antibodies against pseudexin

Fifteen hybridoma cell lines secreting monoclonal antibodies against pseudexin were developed. The cell lines were grown as ascites tumors and the resulting antibodies were purified by Protein A affinity-chromatography. Several of the antibodies exhibited extensive ELISA cross-reactions with different phospholipase A2 toxins from various snake venoms, while other of the antibodies reacted only with the pseudexins. Three of the antibodies neutralized pseudexin A and B, but none of the 10 other phospholipase A2 toxins tested. These same three antibodies inhibited the enzymatic activity of pseudexin A and B and also that of notexin. After each antibody was labeled with biotin, competition experiments were carried out to determine the binding relationships among the antibodies and the pseudexins. Competitions were frequently observed, with a low of zero to a high of eight out of the 14 possibilities. Competition experiments were also carried out with biotin-labeled rabbit IgG against the pseudexins. Some of the monoclonal antibodies had no effect on rabbit IgG binding to pseudexin, while others blocked up to 50% of the binding.     

小鼠Metrnl单克隆抗体的制备及鉴定

目的 制备抗小鼠Metrnl单克隆抗体,并进行初步筛选鉴定。方法 分别制备小鼠Metrnl多肽片段和全长蛋白作为免疫小鼠抗原,取免疫后小鼠脾细胞与SP2/0骨髓瘤细胞融合,筛选阳性杂交瘤细胞,并亚克隆获得稳定细胞株,制备腹水。用ELISA方法检测腹水抗体效价;用Western blot方法鉴定抗体。结果 由14种多肽抗原制备的56株单克隆抗体中,未筛选出可用于Western blot识别Metrnl的抗体;由全长蛋白制备的25株抗体中,有12株可识别Metrnl蛋白。结论 本实验成功制备了12株单抗,可用于识别检测小鼠Metrnl蛋白。     

相思子毒素单克隆抗体的制备纯化及鉴定

目的制备相思子毒素单克隆抗体并鉴定其特性。方法以甲醛处理的相思子毒素毒蛋白为抗原免疫BALB/c小鼠;取免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞融合,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方法筛选出单克隆抗体杂交瘤细胞株,用杂交瘤细胞株诱生小鼠腹水,应用蛋白A亲和层析法进行单抗的纯化,并对单克隆抗体的特异性进行鉴定。结果获得了4株可稳定分泌单克隆抗体的杂交瘤细胞2D3、4E6、1C8和1E5,诱生的腹水效价分别为1∶1×107、1∶1×106、1∶1×105、1∶1×106,亚类鉴定表明2D3为IgG1,其余3株均为IgG2b;特异性鉴定显示它们与多种毒素均无交叉反应,经过亲和层析,获得了纯化的单抗。结论获得了特异性的相思子毒素单克隆抗体,为建立相思子毒素的检测及纯化方法奠定了基础,其中4E6的效价最高,可作为检测相思子毒素的核心试剂。     

人载脂蛋白A5单抗制备及临床应用的研究

目的制备抗人载脂蛋白A5（ApoA5）单克隆抗体（McAb）,建立ELISA双抗体夹心法,并进行初步的临床应用。方法利用合成的人ApoA5抗原免疫BALB/c小鼠,制备McAb,并对抗体的效价、特异性、亚类及位点进行了测定,建立了ELISA双抗体夹心法,测定了正常对照组（145例）和冠心病组（68例）血清中ApoA5的含量。结果获得了5个稳定分泌抗体的阳性杂交瘤细胞株,且均为IgG2a型。腹水效价达到1.6×10^5。冠心病组胆固醇（TC）、三酰甘油（TG）、低密度脂蛋白胆固醇（LDL-C）和ApoB水平明显高于对照组（P〈0.05）,而高密度脂蛋白-胆固醇（HDL-C）、ApoA1和ApoA5水平明显低于对照组（P〈0.05）。ApoA5与HDL-C、ApoA1水平呈正相关（r值分别为0.53和0.63,P〈0.05）,与TC、TG、LDL-C、ApoB呈负相关（r分别为-0.69、-0.63、和-0.59,P〈0.05）。结论ApoA5参与脂质代谢,对于心血管疾病的预防和治疗具有一定的临床意义。     

抗重组人骨唾液酸蛋白单克隆抗体的制备及鉴定

目的研制重组人骨唾液酸蛋白(rhBSP)单克隆抗体(mAb),并鉴定其特性。方法以纯化的rhBSP免疫Balb/c小鼠,采用杂交瘤技术制备抗rhBSP mAb;用亚型鉴定试剂条鉴定IgG亚类;ELISA鉴定mAb的特异性和效价。结果获得2株能稳定分泌特异性mAb的抗rhBSP的杂交瘤细胞系AHB1和AHB5,Ig亚类分别为IgG2a和IgG1,轻链均为κ型,其效价分别为1×10-3和1×10-7。腹水mAb经Protein A亲和色谱柱纯化后,纯度达92%以上。结论获得抗rhBSP的mAb,为进一步研究BSP的生物学功能和用于临床诊断实验研究创造了条件。     

氯霉素单克隆抗体的制备与纯化

目的制备氯霉素(CAP)单克隆抗体。方法用人工合成抗原氯霉素-牛血清白蛋白(CAP-BSA)免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0按5∶1的比例融合,间接竞争ELISA法和有限稀释法进行单克隆杂交瘤细胞的筛选;制备腹水抗体;采用HiTrap rProtein A FF亲和色谱柱纯化抗体,用间接竞争ELISA法和间接ELISA测定抗体特异性。结果得到两株能稳定分泌单克隆抗体的杂交瘤细胞株,细胞培养上清中抗体效价达10-4以上,腹水抗体效价达10-7以上,纯化后的单克隆抗体纯度达98%,回收率达80%,抗体活性好并且与BSA、甲砜霉素、磺二甲基嘧啶等无交叉反应。结论成功获得两株能稳定分泌单克隆抗体的杂交瘤细胞,对动物性食品中CAP的检测具有较大的价值。     

抗金霉素单克隆抗体的制备与鉴定

目的制备抗金霉素(CTC)的单克隆抗体(mAb)。方法采用甲醛作为连接基,将CTC与牛血清白蛋白偶联制备免疫原,免疫BALB/c小鼠,取免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞融合,经筛选克隆,以ELISA法对杂交瘤细胞的稳定性和mAb的特性进行鉴定。结果获得1株可稳定分泌mAb的杂交瘤细胞1H4-C4-C10,间接ELISA方法测定,细胞上清抗体效价为1∶1000,腹水效价为1∶5×105,为IgG1,与四环素交叉反应率为44.4%,与土霉素的交叉反应为8.2%。体外传代培养和冻存复苏后抗体分泌稳定。结论成功制备了针对CTC的mAb,为进一步研制检测CTC的ELISA试剂盒奠定了基础。     

Production and characterization of monoclonal antibodies against Staphylococcus aureus alpha-toxin

Murine monoclonal antibodies against staphylococcal alpha-toxin were produced using a well-characterized alpha-toxin fragment preparation as immunizing agent. Three monoclonal antibodies were selected for anti-alpha-toxin activity in an ELISA using alpha-toxin as antigen. The monoclonal antibodies (MAbs) belonged to different immunoglobulin classes/subclasses and showed different abilities to neutralize the hemolytic, cell-membrane-damaging, dermonecrotizing and lethal action of alpha-toxin. One MAb was superior to mouse polyclonal antiserum in all test systems except for hemolysis, whereas another MAb neutralized essentially as the polyclonal serum. The third MAb did not neutralize the hemolytic or dermonecrotic effect but still inhibited the lethal and membrane-damaging effect of alpha-toxin. These results indicate that the three MAbs recognize different epitopes on the toxin molecule and that different biological activities might correspond to these epitopes.     

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.     

人绒促性素单克隆抗体的制备与分离纯化

目的制备人绒促性素(hCG)单克隆抗体。方法hCG抗原免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0按5∶1融合,间接ELISA法筛选阳性孔,有限稀释法进行克隆化培养;制备腹水抗体;间接ELISA法测定抗体效价;采用HiTrap rProtein A FF亲和色谱柱纯化抗体。结果得到2株能稳定分泌单克隆抗体的杂交瘤细胞株,细胞培养上清中抗体效价达10-3以上,腹水抗体效价达10-7以上,纯化后的单抗纯度达98%以上,回收率达75%。结论成功获得两株能稳定分泌单克隆抗体的杂交瘤细胞,可用于早孕、肿瘤等诊断的研究。     

ATP柠檬酸裂解酶鼠源单克隆抗体的制备和鉴定

目的：制备ATP柠檬酸裂解酶（ACLY）鼠源单克隆抗体（monoclonal antibody,McAb）,并进行相关的特异性鉴定。方法：该研究采用原核表达的重组蛋白ACLY为抗原免疫6~8周龄Balb/c雌性小鼠,利用杂交瘤融合细胞技术构建稳定分泌ACLY McAb的杂交瘤细胞,用酶联免疫吸附测定（ELISA）、免疫荧光（IF）、Western-blot、免疫组织化学（IHC）、流式细胞术（FCM）等鉴定其生物活性,检测所得抗体的特异性。结果：复筛后获得了一株能稳定分泌ACLY McAb的杂交瘤细胞（5F8D11）,Ig亚类为IgG1,轻链为κ链;Western-blot、IHC、IF分析和ELISA检测证实该株ACLY McAb与ACLY具有较高的特异性。结论：该株抗ACLY特异性的McAb的成功制备,为检测ACLY蛋白表达水平及研究ACLY在肿瘤及心血管疾病中的致病机制提供有力的工具,将有助于对肿瘤及心血管疾病患者制定多样合理的治疗方案。     

Production and characterization of monoclonal antibodies against Naja naja atra cobrotoxin.

Twelve monoclonal antibodies against cobrotoxin from Naja naja atra venom were tested for cross-reactivity with eight different snake toxins, binding to linear epitopes, prevention of cobrotoxin binding to acetylcholine receptor (AchR) in vitro, and protection in mice concomitantly given a lethal dose of cobrotoxin. The antibodies were highly specific, as evidenced by little reactivity with other snake toxins. None of the monoclonal antibodies bound to reduced cobrotoxin or synthesized 8-mer regions spanning the whole molecule, thus suggesting the recognition of conformational epitopes. The in vitro binding of toxin to AchR was competitively inhibited (23-79%) with a 1.66:1 mole ratio of antibody:AchR. Preincubation of monoclonal antibody with toxin before adding AchR (3:1 mole ratio of AchR:antibody) inhibited the in vitro binding of toxin to AchR by 20-80%. Monoclonal antibodies added after the preincubation of toxin with AchR did not dissociate the toxin-AchR complex. An antibody:toxin mole ratio of 2.5:1, with 6 micrograms of cobrotoxin, delayed the time to death of mice 3.7-23.8-fold over control mice. The monoclonal antibodies that most effectively prevented in vitro binding of toxin to AchR also provided the longest delay in time to death in mice.     

Production and characterization of monoclonal antibodies against vaginolysin: Mapping of a region critical for its cytolytic activity

Vaginolysin (VLY) is a protein toxin released by Gardnerella vaginalis. VLY belongs to the group of cholesterol-dependent cytolysins (CDCs). We have generated a panel of novel monoclonal antibodies (MAbs) against VLY. For the generation of MAbs, we have used recombinant VLY expressed in Escherichia coli. The functional activity of recombinant VLY was confirmed by an in vitro hemolytic assay using human erythrocytes. The MAbs raised against recombinant VLY were reactive with VLY from G. vaginalis both by Western blot and ELISA. The cross-reactivity of MAbs with other CDCs was investigated. For this purpose, recombinant cytolysins perfringolysin, listeriolysin, intermedilysin, pneumolysin and streptolysin were expressed in E. coli. The MAbs were specific exclusively to VLY and did not react with other CDCs. All MAbs were studied for the ability to neutralize hemolytic activity of VLY in vitro and several neutralizing MAbs were identified. The MAb produced by clone 9B4 showed the most potent neutralizing activity. The epitope for this MAb was localized near the N-terminus of VLY, between amino acid (aa) residues 112 and 268. The region recognized by the neutralizing MAb 9B4 includes the conserved motif (VAARMQYD, aa 189-196) supposed to be involved in VLY oligomerization. Selected MAbs were employed to develop a sandwich ELISA for VLY quantification. The MAb-based immunoassay was suitable for the detection of VLY in the cultures of G. vaginalis. In conclusion, the MAbs described in the current study may be useful for structural and functional studies of VLY as well as immunodetection of VLY in biological specimens.     

Production and characterization of monoclonal antibodies against stonustoxin from Synanceja horrida

Stonustoxin (SNTX), a lethal factor purified from the venom of stonefish Synanceja horrida, is a protein (148,000 mol. wt) existing as a dimer comprising two subunits ( and β) of mol. wts 71,000 and 79,000, respectively. Its ld₅₀ (i.v.) is 17 ng/g in mice and it causes haemolysis of rat and rabbit erythrocytes in vitro. Eight monoclonal antibodies (Mabs) against SNTX have been developed using the Balb/C mouse. These Mabs have been purified by Protein G affinity membrane disc chromatography. They were all classified as IgG₁ with half of them having κ and the rest A light chains. They had affinity constants ranging from 3.75 × 10⁻⁹, to 9.74 × 10⁻⁹, M. Six were able to protect mice from a challenge of a lethal dose of SNTX. However, not all protective Mabs were able to neutralize the haemolytic effect in vitro. Only four Mabs (31A, 32B, 38A and 46A) could inhibit rat and rabbit erythrocyte haemolysis, while one Mab (43D) offered partial inhibition and another Mab (8A) did not inhibit haemolysis at all. The non-protective Mabs (43B and 44G) were also incapable of neutralizing haemolysis. Five epitopes were recognized by the eight Mabs. Four Mabs (31A, 32B, 38A and 46A) were found to have similar epitope specificity while the rest were directed at different epitopes on the SNTX molecule. Thus these results suggest that the domain on the SNTX molecule responsible for lethality is probably distinct from the domain important for in vitro haemolytic activity.     

Preparation of monoclonal antibody against ginsenoside Rf and its enzyme immunoassay

A rapid and sensitive indirect competitive enzyme immunoassay method has been developed for quantitating ginsenoside Rf (Rf) in crude total Panax ginseng saponins and in rat plasma using high titer mouse monoclonal antibody (mAb) raised against a conjugate of Rf and bovine serum albumin (BSA). The isotype of mAb against Rf was IgG3 with a K chain. The presence of Rf inhibited the binding of the mouse anti-Rf mAb to a Rf-BSA solid phase coating antigen. The working range was 0.01-10 ng/assay and detection limits were 20 pg in various ginseng extract fractions or 34 pg in rat plasma per assay. The anti-Rf mAb cross-reacted with ginsenoside Rg2 by 57.5%, but not with other ginsenosides. However, this anti-Rf mAb did not cross-react with BSA or cellubiose, which is a carbohydrate component of Rf. Using this standard curve, we could measure the amount of Rf in ginseng total extract, ginseng total saponins, protopanaxadiol saponins, and propanaxatriol saponins. We could also measure the amount of Rf in rat plasma after the oral administration of Rf and found that Rf reached a maximum level in rat plasma after 16 h. These results indicate that the anti-Rf mAb could be useful for the quantitation of Rf in crude ginseng fractions and in body fluids.     

Production,characterization and application of monoclonal antibodies against the organophosphorus nerve agent Vx

 Two monoclonal antibodies (Vx-BB8 and Vx-EA11) to the chemical warfare agent Vx were produced and characterized. A competitive inhibition enzyme immunoassay was developed to detect Vx concentrations as low as 3.7×10^-7–3.7×10^-6 mol/l in biological samples. Vx-BB8 400 μg given intravenously immediately before 1×LD₉₅ Vx or 400 μg Vx-BB8 intraperitoneally 1.5 h–3 days before 1×LD₉₅ Vx could protect all the tested mice from death. Received: 14 October 1994/Accepted: 2 February 1995     

Production of monoclonal antibodies against antitumor cyclohexapeptide RA-VII from Rubia cordifolia and their characterization

An immunoassay system was established for the estimation of the quantity of an antitumor cyclic hexapeptide RA-VII (1) from Rubia cordifolia L. and R. akane Nakai (Rubiaceae). First, 1 was converted into its hapten, which was then conjugated with a carrier protein to be used as an effective antigen to obtain its monoclonal antibody (MAb). In the resulting conjugate, the molecular ratio between 1 and the carrier protein as assayed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was about 5:1. Then, the splenocytes from the mouse immunized with the conjugate were fused with mouse myeloma cells to produce hybridoma, secreting MAb against 1. Two clones were isolated, one producing MAb IgG(1) and the other IgM, both having a κ light chain. The sensitivity and cross-reactivity of the thus obtained MAb were also assayed.     

SARS冠状病毒核衣壳蛋白单克隆抗体的制备及鉴定

目的:SARS 冠状病毒(SARS-CoV)核衣壳蛋白 N(nucleocapsid protein)单克隆抗体(monoclonal antibody,McAb)的制备及鉴定。方法:用基因重组的 SARS-CoV N 蛋白免疫 BALB/c 小鼠制备 McAb,并采用间接 ELISA、免疫印迹法进行筛选和鉴定。结果:筛选出2株抗 SARS-CoV N 蛋白 McAb 杂交瘤细胞株,间接 ELISA 证实这组单克隆抗体仅与 SARS-CoV 产生特异性反应,而与其他病原体无交叉反应,IgG 亚类鉴定1株为 IgG1,另1株为 IgG2b。2株抗体亲和常数分别为4.14×10~(-9)和3.19×10~(-9)。结论:获得特异性针对 SARS-CoV 的单克隆抗体,为 SARS-CoV 早期诊断试剂的研制奠定了基础。     

抗美沙酮单克隆抗体制备、鉴定和在侧向免疫层析方法中的应用

目的:制备抗美沙酮(MTD)单克隆抗体(mAb),并将筛选到的抗体应用于侧向免疫层析方法检测尿液中MTD。方法:用美沙酮BSA偶联蛋白(MTD-BSA)免疫纯系Balb/c小鼠,采用直接及间接ELISA法进行药物交叉反应,筛选获得能稳定分泌抗MTD的单克隆杂交瘤细胞株。制备腹水,纯化后得到特异性抗MTD单克隆抗体(mAb),进行效价、特异性、纯度、亚类的鉴定分析,并用筛选到的抗体建立检测MTD的侧向免疫层析方法。结果:成功获得1株稳定分泌抗体的阳性细胞株,用建立的侧向免疫层析方法检测临床样品与对照方法比较总符合率为100%。结论:成功筛选出了能稳定分泌mAb的细胞株,抗体应用于侧向免疫层析检测方法中,能快速、特异、灵敏地检测出临床样品中的MTD,为临床应用快速检测MTD指标提供了方法。     

Preparation of monoclonal antibodies against okadaic acid prepared from the sponge Halichondria okadai

, , , and . Preparation of monoclonal antibodies against okadaic acid prepared from the sponge Halichondria okadai. Toxicon 27, 1323–1330, 1989.—Three murine monoclonal antibodies, OA-1, OA-2 and OA-3, against okadaic acid were prepared from hybridoma clones obtained by fusion of mouse 653 myeloma cells with mouse immune spleen cells sensitized to okadaic acid-ovalbumin conjugate. Each antibody reacted with dinophysistoxin-1 ( = 35-methylokadaic acid) as well as okadaic acid, but did not react with the other diarrhetic shellfish poisons or related compounds, such as 7-O-palmitoyl-okadaic acid (analogue of dinophysistoxin-3), pectenotoxin-1 and yessotoxin. A competitive inhibition enzyme-linked immunosorbent assay which employed OA-3 antibody was performed and showed a sensitivity of about 10ppb (10ng/ml) for okadaic acid. This simple and time-saving ELISA assay system may be useful for the specific detection of diarrhetic shellfish poisons.