Tóth K, Vaughan M M, Slocum H K, Fredericks W J, Chen Y F, Arredondo M A, Harstrick A, Karakousis C, Baker R M, Rustum Y M
Department of Experimental Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263.
Am J Pathol. 1992 May;140(5):1009-16.
The applicability of a multilayer immunoperoxidase "sandwich" method (IpS) developed by Chan14 for the amplified detection of P-glycoprotein (Pgp) was investigated. The authors examined 15 formalin-fixed cell lines, as well as formalin-fixed, paraffin-embedded sections from single biopsies of 46 sarcomas. The cell lines included sensitive and multidrug resistant sublines (KB, A2780, MCF-7, HeLa) with various relative degrees of resistance to doxorubicin (Dox). The sarcoma biopsy specimens were selected on the basis of the results obtained in Western blot (WB) detection of Pgp (22 positive and 24 negative by WB) using C219 and C494 monoclonal antibodies to Pgp. The IpS method employed C219. The least resistant cell line in which Pgp could be detected by IpS was fivefold resistant to doxorubicin, whereas Pgp was detected by WB in cells greater than twofold resistant. Cell lines having greater than fivefold resistance to Dox were positive by both IpS and WB analyses. The less resistant cell lines contained more nonreactive cells whereas the highly resistant cell lines showed more homogeneous strong membrane reactions. Among the six cell lines determined to be Pgp negative by WB analysis, no false positive immunostaining by IpS existed. One of 22 WB positive and 7 of 24 WB-negative sarcoma biopsy specimens were positive by IpS methods. Reaction varied and was always focal (a minimum of 3-5 cells, ranging up to 3-4 high power fields) indicating pronounced heterogeneous distribution of Pgp. Thus, WB can detect low average (overall) levels of Pgp in tumor samples but such low concentrations of PgP at the single cell are not detectable by IpS methods. However, IpS can discern among many Pgp-negative cells small subpopulations of immunoreactive cells, which are not detected by WB analysis due to Pgp dilution by the membrane protein of numerous Pgp negative cells. IpS and WB used together as complementary methods can provide more complete information about Pgp distribution and content, particularly in the case of heterogeneous human tumors. The IpS method is more suitable for less drastically treated (not embedded) cell line specimens than for paraffin-embedded (routine) sections. Some modification of the present IpS protocol seems necessary to increase its sensitivity and reduce the disparity with WB results.
对Chan14开发的用于P - 糖蛋白（Pgp）扩增检测的多层免疫过氧化物酶“夹心”法（IpS）的适用性进行了研究。作者检测了15种福尔马林固定的细胞系，以及46例肉瘤单次活检的福尔马林固定、石蜡包埋切片。细胞系包括对阿霉素（Dox）具有不同相对耐药程度的敏感和多药耐药亚系（KB、A2780、MCF - 7、HeLa）。肉瘤活检标本是根据使用针对Pgp的C219和C494单克隆抗体在蛋白质免疫印迹（WB）检测Pgp中获得的结果进行选择的（WB检测22例阳性和24例阴性）。IpS方法采用C219。通过IpS可检测到Pgp的耐药性最低的细胞系对阿霉素具有5倍耐药性，而通过WB在耐药性大于2倍的细胞中检测到Pgp。对Dox耐药性大于5倍的细胞系通过IpS和WB分析均呈阳性。耐药性较低的细胞系含有更多无反应性细胞，而高耐药性细胞系显示出更均匀的强膜反应。在通过WB分析确定为Pgp阴性的6个细胞系中，IpS不存在假阳性免疫染色。22例WB阳性和24例WB阴性的肉瘤活检标本中，分别有1例和7例通过IpS方法呈阳性。反应各不相同且总是局灶性的（最少3 - 5个细胞，范围可达3 - 至4个高倍视野），表明Pgp分布明显不均一。因此，WB可以检测肿瘤样本中Pgp的低平均（总体）水平，但IpS方法无法检测单细胞中如此低浓度的PgP。然而，IpS可以在许多Pgp阴性细胞中识别出免疫反应性细胞的小亚群，由于众多Pgp阴性细胞的膜蛋白稀释了Pgp，这些小亚群无法通过WB分析检测到。IpS和WB一起作为互补方法可以提供关于Pgp分布和含量的更完整信息，特别是在异质性人类肿瘤的情况下。IpS方法更适用于处理程度较轻（未包埋）的细胞系标本，而不是石蜡包埋（常规）切片。似乎有必要对当前的IpS方案进行一些修改，以提高其灵敏度并减少与WB结果的差异。
