Paloheimo L I, Rehfeld J F
Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen.
Clin Chim Acta. 1994 Sep;229(1-2):49-65. doi: 10.1016/0009-8981(94)90228-3.
In order to develop a processing-independent analysis for procholecystokinin (proCCK) and its products, antibodies were raised against the synthetic fragment 62-71 of human proCCK. All rabbits (n = 8) responded to the immunization. One (No. 89,009) produced antibodies of particularly high titer (1:350,000), homogeneity (Sips' index approximately 1.0) and binding affinity (K0 eff approximately 0.88 x 10(12) l/mol). A radioimmunoassay using this antiserum and [125I]tyrosine-extended fragment 62-71 measured the total CCK mRNA product after tryptic cleavage at Lys61 in normal and neoplastic tissue independent of the degree of precursor processing. In addition to previously known CCK producing tumors, CCK was found also in a thoracic round-cell tumor (Askin tumor) and in brain tumors (gliomas and astrocytomas). These tumors processed proCCK poorly. Thus, they contained 11 and 23 (mean n = 5) pmol/g of proCCK and its products before, versus 71 and 99 (mean) pmol/g after tryptic cleavage, respectively. Accordingly, gel chromatography revealed significant amounts of unprocessed proCCK, large molecular forms of glycine-extended CCKs and the well-known carboxyamidated and tyrosine O-sulfated bioactive CCK-83, -58, -33, -22 and -8. We conclude that monospecific antibodies directed against the N-terminus of sequence 62-71 of human proCCK are suitable for processing-independent analysis (PIA) for proCCK and its products. Moreover, we suggest that such PIA should be used for quantitation of CCK gene expression at peptide level in normal tissue and tumors.
为了开发一种不依赖加工过程的前胆囊收缩素（proCCK）及其产物分析方法，制备了针对人proCCK合成片段62 - 71的抗体。所有兔子（n = 8）对免疫均有反应。其中一只（编号89,009）产生的抗体具有特别高的效价（1:350,000）、均一性（西普斯指数约为1.0）和结合亲和力（K0 eff约为0.88×10(12) l/mol）。使用这种抗血清和[125I]酪氨酸延伸片段62 - 71进行的放射免疫分析，可独立于前体加工程度，测量正常组织和肿瘤组织中在赖氨酸61处经胰蛋白酶切割后的总CCK mRNA产物。除了先前已知的产生CCK的肿瘤外，在胸壁圆形细胞瘤（Askin瘤）和脑肿瘤（神经胶质瘤和星形细胞瘤）中也发现了CCK。这些肿瘤对proCCK的加工能力较差。因此，它们在胰蛋白酶切割前每克分别含有11和23（平均n = 5）pmol的proCCK及其产物，而切割后分别为71和99（平均）pmol/g。相应地，凝胶色谱显示有大量未加工的proCCK、甘氨酸延伸的CCK大分子形式以及众所周知的酰胺化和酪氨酸O - 硫酸化的生物活性CCK - 83、- 58、- 33、- 22和- 8。我们得出结论，针对人proCCK序列62 - 71 N端的单特异性抗体适用于对proCCK及其产物进行不依赖加工过程的分析（PIA）。此外，我们建议这种PIA应用于在正常组织和肿瘤的肽水平定量CCK基因表达。
