CN106929477B - 一株抗***素F2α的特异性单克隆抗体杂交瘤细胞株WXX-2及其应用 - Google Patents
一株抗***素F2α的特异性单克隆抗体杂交瘤细胞株WXX-2及其应用 Download PDFInfo
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Abstract
一株抗***素F2α的特异性单克隆抗体杂交瘤细胞株WXX‑2及其应用,属于临床检测技术领域。本发明抗***素F2α特异性单克隆抗体杂交瘤细胞株WXX‑2,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.13087。抗***素F2α特异性单克隆抗体,它由所述抗***素F2α特异性单克隆抗体杂交瘤细胞株WXX‑2分泌产生。所述杂交瘤细胞株WXX‑2可以分泌产生对***素F2α具有较好的特异性和较高灵敏度的单克隆抗体,对***素F2α的50%抑制浓度IC50为1.80μg/L,可以用于临床检测中***素F2α的特异性检测。
Description
技术领域
本发明一株抗***素F2α的特异性单克隆抗体杂交瘤细胞株WXX-2及其应用,涉及***素F2α的杂交瘤细胞株及其产生的抗特异性单克隆抗体,属于临床检测技术领域。
背景技术
***素(Prostaglandins,PGs)是一类化学组成相似,且生物活性广泛的不饱和羟基脂肪酸。PGs家族主要包括:***素D2、***素E2、***素F2α、***素I2和TXA2,***素在组织内含量甚微,但活性极强。子宫内膜是合成***素的重要部位之一,***素的E2和F2α是子宫内膜合成的主要花生四烯酸产物。
目前***素检测多采用免疫亲和色谱-气质联用测定法(Immuno-GC -MS)、液相色谱与质谱联用法(LC-MS)、气质联用法(GC-MS)等。上述检测方法可进行定量分析并具有较低的检测限,但是通常需要昂贵的仪器和复杂的操作,前处理及检测时间长,严重制约了这些检测方法的推广,无法达到现场大批量快速检测的要求。而免疫分析方法具有低成本、高通量、高灵敏、对技术人员相对要求低等特点,因此适用于大量样品的快速筛查。本发明的目的在于提供一种对***素F2α具有较高亲和力和检测灵敏度的单克隆抗体杂交瘤细胞株的制备方法,为间接竞争 ELISA 试剂盒以及胶体金试纸条的研发推广奠定基础。
发明内容
本发明的目的是提供一株抗***素F2α特异性单克隆抗体杂交瘤细胞株WXX-2,由该细胞株制备的抗体对***素F2α具有较好特异性和检测灵敏度,可以用来建立***素F2α的免疫学检测方法。
本发明的技术方案:一株抗***素F2α特异性单克隆抗体杂交瘤细胞株WXX-2,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,简称CGMCC,保藏编号为CGMCCNo.13087。
抗***素F2α特异性单克隆抗体,它由所述保藏编号为CGMCC No.13087的抗***素F2α特异性单克隆抗体杂交瘤细胞株WXX-2分泌产生。
所述抗***素F2α特异性单克隆抗体的应用,其在***素F2α临床检测中的应用。
所述抗***素F2α特异性单克隆抗体杂交瘤细胞株WXX-2的制备步骤为:
(1)免疫原的制备与鉴定:***素F2α通过碳化二亚胺法(EDC法)与蛋白载体的氨基相连,反应结束后,通过透析分离完全抗原和未偶联的小分子半抗原,完全抗原通过紫外吸收扫描方法鉴定;
(2)小鼠的免疫:将免疫原与福氏佐剂乳化完全后,通过皮下多点注射免疫BALB/c小鼠。首次免疫采用福氏完全佐剂,加强免疫使用福氏不完全佐剂,冲刺免疫时免疫剂量为前一次免疫剂量的一半,与生理盐水混合均匀后直接进行腹腔注射;各次免疫间隔为三周。第三次免疫后,间隔一周采血检测血清效价和抑制;
(3)细胞融合与细胞株建立:通过聚乙二醇(PEG 2000)法,使小鼠脾细胞和小鼠骨髓瘤细胞融合,通过HAT培养基培养,利用间接ELISA检测阳性细胞孔,并进一步利用间接竞争ELISA法测定阳性细胞孔的抑制效果,通过有限稀释法对有最好抑制的阳性细胞孔进行三次亚克隆,最终筛选获得抗***素F2α特异性单克隆抗体杂交瘤细胞株WXX-2;
(4)杂交瘤细胞株性质的鉴定:采用小鼠单抗Ig类/亚类鉴定用酶标二抗套装测定;IC50值和亲和力的测定通过ELISA法。
本发明的有益效果:本发明获得的抗***素F2α单克隆抗体杂交瘤细胞株,对***素F2α有较好的检测灵敏度和亲和力;本发明还提供了一种新的合成***素F2α免疫原的方法,半抗原的合成步骤更加简化,有效,为今后人们的研究提供了合成免疫原的思路与方法。
生物材料样品保藏:单克隆细胞株WXX-2,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,简称CGMCC,地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,保藏编号为CGMCC No.13087,保藏日期2016年10月31日。
附图说明
图1 免疫原的紫外吸收光谱表征。
图2 抗***素F2α特异性单克隆抗体对***素F2α的标准抑制曲线。
具体实施方式
本发明下面的实施例仅作为本发明内容的进一步说明,不能作为本发明的限定内容或范围。下面通过实施例对本发明作进一步说明。
本发明通过将***素F2α完全抗原免疫小鼠,通过细胞融合,HAT选择性培养基培养,通过间接ELISA和间接竞争ELISA筛选细胞上清,最终得到了对***素F2α有较好亲和力和灵敏度的单克隆抗体杂交瘤细胞株。
实施例1抗***素F2α特异性单克隆抗体杂交瘤细胞株WXX-2的制备
(1)完全抗原的合成:
完全抗原的合成:取4mg半抗原F2α,再加入5.0 mg EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)和3.7mg NHS(N-羟基琥珀酰亚胺),使用DMF(N,N-二甲基甲酰胺)溶解,室温搅拌,活化6h;另取15mg BSA(牛血清白蛋白)溶解于3mL、0.05M、pH9.6的CBS(碳酸盐缓冲溶液)溶液中;将上述活化液逐滴加入BSA溶液中,室温搅拌反应过夜后,取出免疫原PBS透析3天,-20℃分装保存。
(2)动物免疫:选择健康的6~8周龄的BALB/c小鼠进行免疫。取***素F2α完全抗原(1mg/mL)与等量福氏佐剂乳化均匀后,通过皮下多点注射免疫BALB/c小鼠,每只100μL。首次免疫采用福氏完全佐剂,加强免疫使用福氏不完全佐剂,冲刺免疫时免疫剂量为前一次免疫剂量的一半,与生理盐水混合均匀后直接进行腹腔注射;各次免疫间隔为三周。第三次免疫后,间隔一周采血检测血清效价和抑制;选择抑制最好的小鼠,在五免后18天冲刺免疫,准备融合。
(3)细胞融合:在冲刺免疫三天后,按照常规PEG(聚乙二醇,分子量为2000)方法进行细胞融合,具体步骤如下:
无菌取小鼠脾脏,研磨并通过200目细胞筛网得到脾细胞悬液,并进行细胞计数;
收集SP2/0细胞,悬浮于RPMI-1640基础培养液中,进行细胞计数;
将脾细胞和SP2/0细胞按照2-10:1的计数比例混合,离心后用PEG融合,时间1min,之后按照从慢到快,加入RPMI-1640基础培养液,离心后悬浮于含20% 胎牛血清、2%的50×HAT的RPMI-1640筛选培养液中,加到96孔细胞培养板,置于37℃、5%CO2的培养箱中培养。
(4)细胞筛选与细胞株建立:在细胞融合的第三天对融合细胞进行RPMI-1640筛选培养液半换液,第5天进行用含20% 胎牛血清、1%的100×HT的RPMI-1640过渡培养液进行全换液,在第7天取细胞上清进行筛选。筛选分两步:第一步先用间接ELISA筛选出阳性细胞孔,第二步选用***素F2α为标准品,用间接竞争ELISA对阳性细胞进行抑制效果测定。选择对***素F2α有较好抑制的细胞孔,采用有限稀释法进行亚克隆,用同样的方法进行检测。重复三次,获得抗***素F2α特异性单克隆抗体杂交瘤细胞株WXX-2。
(5)抗***素F2α特异性单克隆抗体的制备与鉴定:取8-10周龄BALB/c小鼠,每只小鼠腹腔注射石蜡油1mL;7天后每只小鼠腹腔注射1×106杂交瘤细胞WXX-2,从第七天开始收集腹水,将腹水通过辛酸-饱和硫酸铵法纯化,获得的单抗置于-20℃保存。
使用小鼠单抗亚型鉴定试剂盒对腹水纯化获得的单克隆抗体进行免疫球蛋白亚型鉴定,其亚型为IgG1型,具体如表1所示。
表1 ***素F2α单克隆抗体的亚型鉴定
使用间接竞争ELISA法,测定单克隆抗体对***素F2α的IC50为 1.8 μg/L,可用于***素F2α的特异性快速检测,具体如图2所示。
(6)抗体应用:将抗***素F2α特异性单克隆抗体杂交瘤细胞株WXX-2通过体内腹水制备的单克隆抗体应用于***素F2α样品测定试验,具体步骤如下:
6.1用碳酸盐缓冲液(CBS)稀释好的0.1μg/mL的***素F2α包被作为包被原包被96孔酶标板,每孔100μL,37℃包被2h后,用PBST洗液洗板三次,每次每孔200μL,每次3 min,拍干;
6.2用含0.2%明胶的CBS进行封闭,每孔200μL,37℃封闭2h,用PBST洗液洗板三次,每次每孔200μL,每次3 min,拍干;
6.3用磷酸盐缓冲液(PBS)分别配置0,0.01,0.02,0.05,0.1,0.2,0.5,1μg/L的***素F2α标准溶液。将标准溶液以及待检测样品提取液,分别加入到已经封闭好的酶标板中,每孔50μL,每个样品重复3个孔,再每孔加入50μL 1︰16000稀释的抗***素F2α单克隆抗体,37℃反应0.5h后,洗板拍干;
6.4每孔加入100μL 用含0.1% 明胶的PBS 1︰3000稀释的HRP标记的羊抗鼠IgG二抗,37℃反应0.5h后,洗板拍干;
6.5每孔加入100μL TMB显色液,37℃显色15min后,每孔加入50μL 2M H2SO4终止液,450 nm测吸光值。
溶液的配置:
碳酸盐缓冲液(CBS):称取Na2CO3 1.59g,NaHCO3 2.93g,分别溶于少量双蒸水后混合,加双蒸水至约800mL混匀,调pH值至9.6,加双蒸水定容至1000mL,4℃贮存备用;
磷酸盐缓冲液(PBS):8.00g NaCl,0.2g KCl,0.24g KH2PO4,3.62g Na2HPO4·12H2O,溶于800mL纯水中,用NaOH或HCl调pH到7.2~7.4,定容至1000mL;
PBST:含0.05% Tween20的PBS;
TMB显色液:A液:Na2HPO4·12H2O 18.43g,柠檬酸 9.33g,纯水定容至1000mL;B液:60mg TMB 溶于100mL乙二醇中。A、B液按体积比1︰5混合即为TMB显色液,现用现混。
综上所述仅为本发明的较佳实施例而已,并非用来限定本发明的实施范围。即凡依本发明申请专利范围的内容所作的等效变化与修饰,都应为本发明的技术范畴。
Claims (2)
1.一株抗***素F2α特异性单克隆抗体杂交瘤细胞株WXX-2,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.13087。
2.抗***素F2α特异性单克隆抗体,其特征在于:它由权利要求1所述保藏编号为CGMCC No.13087的抗***素F2α特异性单克隆抗体杂交瘤细胞株WXX-2分泌产生。
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CN103459595A (zh) * | 2010-09-29 | 2013-12-18 | 株式会社Nb健康研究所 | 针对人***素e2 受体ep4 的抗体 |
CN105658221A (zh) * | 2013-09-26 | 2016-06-08 | 高德美国际公司 | 用于治疗萎缩性皮肤瘢痕形成的***素F2α及其类似物 |
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