CAJ | 학술논문

目的制备高亲和力、高特异性的抗人骨唾液蛋白(BSP)单克隆抗体(mAb),并对其生物学特性进行鉴定,为BSP作为乳腺癌骨转移临床诊断靶点的研究奠定基础.方法重组BSP蛋白免疫BALB/c小鼠,取其脾细胞与Sp2/0细胞融合,经筛选建立可稳定分泌抗BSPmAb细胞株,制备腹腔积液,Protein G纯化.ELISA检测抗体的效价和亲和力,并用亚型鉴定试纸条鉴定mAb的亚型.应用Western blotting检测mAb的特异性,进一步利用纯化的mAb经免疫组织化学法检测乳腺癌细胞MDA-MB-231中BSP的表达情况.结果获得9株抗BSP mAb细胞株,选择其中2株(D001和D002)进一步鉴定,其上清效价分别为1∶5120和1∶10 240,腹腔积液效价分别为1∶25 600和1∶51 200.2株抗体亚型均为IgG1型,轻链均为κ型.Western blotting结果均在预期位置出现阳性条带.利用此2株细胞株所得抗体均可在乳腺癌细胞MDA-MB-231中检测到BSP的表达.结论成功制备能特异性识别BSP的mAb,为进一步研究BSP的生物学功能以及对BSP作为乳腺癌骨转移的标志物进行临床评价奠定基础.
목적 제비고친화력、고특이성적항인골타액단백(BSP)단극륭항체(mAb),병대기생물학특성진행감정,위BSP작위유선암골전이림상진단파점적연구전정기출.방법 중조BSP단백면역BALB/c소서,취기비세포여Sp2/0세포융합,경사선건립가은정분비항BSPmAb세포주,제비복강적액,Protein G순화.ELISA검측항체적효개화친화력,병용아형감정시지조감정mAb적아형.응용Western blotting검측mAb적특이성,진일보이용순화적mAb경면역조직화학법검측유선암세포MDA-MB-231중BSP적표체정황.결과 획득9주항BSP mAb세포주,선택기중2주(D001화D002)진일보감정,기상청효개분별위1∶5120화1∶10 240,복강적액효개분별위1∶25 600화1∶51 200.2주항체아형균위IgG1형,경련균위κ형.Western blotting결과 균재예기위치출현양성조대.이용차2주세포주소득항체균가재유선암세포MDA-MB-231중검측도BSP적표체.결론 성공제비능특이성식별BSP적mAb,위진일보연구BSP적생물학공능이급대BSP작위유선암골전이적표지물진행림상평개전정기출.
Objective To prepare the human bone sialoprotein (BSP) monoclonal antibodies (mAb)with high titer and specificity and identify its characterization, which is based on further studying BSP as clinical biomarker for breast cancer metastasizing to bone. Methods BALB/c mice were immunized with purified recombinant BSP protein. Cell fusion was performed between mouse splenic cells and myeloma cells (Sp2/0), and then the hybridoma cell lines secreting mAb against BSP antigen were screened and cloned. The ascites were prepared and purified with Protein G affinity chromatography. The titer and subtypes of mAb against BSP were identified and measured by ELISA and Western blotting analysis. Results Nine hybridoma cell lines that stably secreted mAb against BSP were successfully obtained. Two of them, D001 and D002,were further identified, which belonged to the subtypes of IgG1 and κ light chain. The two antibodies titers in culture supernatant were 1∶5120 and 1∶10 240, respectively, and those in the ascites fluid were 1∶25 600 and 1∶51 200, respectively. Results of Western blotting analysis and immunohistochemistry showed that the two antibodies could specifically bind with BSP derived from human breast cancer cells. Conclusion Nine mAb against BSP have been successfully prepared which can be used for further studying the biological properties of BSP and reveal its relationship with data from clinic patients.