摘要
应用鼠－鼠杂交瘤细胞技术建立了三系稳定分泌抗重组人α２ａ型干扰素（ｒＨｕ－ＩＦＮ－α２ａ）单克隆抗体细胞系１Ａ５、１Ｂ５和２７Ａ７。细胞连续传代和在液氮中冻存后复苏，分泌抗体能力不变，并且维持在较高水平，细胞培养上清效价１∶２５６～１∶４０９６，腹水１∶２６×１０５～１∶２７×１０８。Ｉｇ亚类测定，１Ａ５和１Ｂ５ＭｃＡｂ为ＩｇＧ１，２７Ａ７为ＩｇＧ２ａ。三系ＭｃＡｂ均识别ｒＨｕ－ＩＦＮ－α２ａ和－α２ｂ，与ｒＨｕ－ＩＦＮ－α１和正常大肠菌体裂解液无反应。竞争ＥＬＩＳＡ试验，三系ＭｃＡｂ分别针对ｒＨｕ－ＩＦＮ－α２ａ上三个不同表位。同ＭｃＡｂ建立双抗体夹心ＥＬＩＳＡ对ｒＨｕ－ＩＦＮ－α２ａ和－α２ｂ均可检测到１５０ｐｇ／ｍｌ，约１０ＩＵ／ｍｌ的敏感度。用提纯的单抗制备亲和层析柱，单抗偶联率９５％以上。三系单抗亲和层析柱均可将粗制ｒＨｕ－ＩＦＮ－α２ａ提纯到９７％以上纯度。平均回收率为：１Ａ５单抗柱９０２％，１Ｂ５单抗柱９５３％，２７Ａ７单抗柱９４７％。比活性平均值依次为１９４×１０８ＩＵ／ｍｇ，１９７×１０８ＩＵ／ｍｇ和１６４×１０８ＩＵ／ｍｇ，残余鼠ＩｇＧ量均符合规程要求。纯化ｒＨｕ－ＩＦＮ?
Three hybridoma cell Lines(1A5,1B5 and 27A7) to secrete McAbs steadily against recombinant human interferon-α2a(rHu-IFN-α2a) were established by fusion of Ns-1 myeloma cells with spleencells of immunized with rHu-IFN-α2a.Cells function to secrete McAb is steady,when cultivated in vitro succesively and reactivated after freezing in liguid nitrogen.McAbs dilution of cells culture supernatant is 1∶4 4～1∶4 6,ascites,1∶4 9～1∶4 14 .1A5 and 1B5 belong to IgG1 and 27A7 belong to IgG2a.All of three McAbs discern rHu-IFN-α2a and -α2b but don't react with rHu-IFN-α 1 or normol E.coli lysate.Three McAbs discern three different antigenic epitops respectively by competitive ELISA.rHu-IFN-α2a and-α2b can be determined to 150 pg/ml(approx.10 IU/ml)by sandwich ELISA.To prepare the affinity Chromatography column by use of the purity McAbs,rate of conjugation of McAbs was more than 95%.Every of 3 McAbs affinity Chromatography column can purify the raw rHu-IFN-α2a to electropheropurity.Average recovery rate of IFN was:1A5 column,90.2%;1B5 95.3%;27A7,94.7%,and their average specific activity was 1.94×10 8IU/mg, 1.97×10 8IU/mg and 1.64×10 8IU/mg,respectively.The amount of residual mouse IgG could meet the national reguirement.Result of purified rHu-IFN-α2b was as well as rHu-IFN-α2a.
出处
《微生物学免疫学进展》
1998年第4期19-24,共6页
Progress In Microbiology and Immunology
关键词
重组人干扰素
单克隆抗体
亲和层析柱
纯化
Recombinant human interferon Monoclonal antibody Affinity chromatography column Purification
