Girkontaite I, Frischholz S, Lammi P, Wagner K, Swoboda B, Aigner T, Von der Mark K
Institute of Experimental Medicine, Friedrich-Alexander-University of Erlangen-Nuremberg, Germany.
Matrix Biol. 1996 Sep;15(4):231-8. doi: 10.1016/s0945-053x(96)90114-6.
For studies on processing and tissue distribution of type X collagen, monoclonal antibodies were prepared against human recombinant collagen type X (hrCol X) and tested by ELISA, immunoblotting and immunohistology. Forty-two clones were obtained which were grouped into four different subsets based on their reactivity against native and denatured hrCol X, pepsin-treated hrCol X, and the C-terminal NC-1 domain. Here we present results obtained with four monoclonal antibodies: Clone X 53, a representative of group I, binds with high affinity to both native and pepsin-digested hrCol X but with low affinity to the NC-1 dimer; monoclonal antibodies of group II and III recognized native and denatured hrCol X but not NC-1; antibodies of group II, but not III, reacted to some extent with pepsin treated hrCol X; one antibody (X 34) was obtained that reacted strongly with the isolated NC-1 dimer and native hrCol X but not with the NC-1 monomer or pepsin-digested hrCol X (group IV). Antibodies of all groups stained specifically the hypertrophic zone of fetal human epiphyseal cartilage. Mab X 53 stained the peri- and extracellular matrix of hypertrophic chondrocytes in the lower hypertrophic zone and in the calcified cartilage core in endochondral bone trabecules, while clone X 34 stained intracellularly and the pericellular matrix. All other tissues or cells of the epiphysis were negative. Antibody X 53 reacted also with canine, murine and guinea pig hypertrophic cartilage in tissue sections, but not with bovine or porcine type X collagen. In sections of osteoarthritic cartilage, clusters of hypertrophic chondrocytes in the deep zone were stained, confirming previous observations on enhanced chondrocyte hypertrophy and type X collagen expression in osteoarthritic articular cartilage.
为了研究X型胶原蛋白的加工过程和组织分布，制备了针对人重组X型胶原蛋白（hrCol X）的单克隆抗体，并通过酶联免疫吸附测定（ELISA）、免疫印迹和免疫组织学进行检测。获得了42个克隆，根据它们对天然和变性hrCol X、胃蛋白酶处理的hrCol X以及C末端NC-1结构域的反应性，将其分为四个不同的亚组。在此，我们展示了用四种单克隆抗体获得的结果：克隆X 53是I组的代表，它与天然和胃蛋白酶消化的hrCol X具有高亲和力，但与NC-1二聚体的亲和力较低；II组和III组的单克隆抗体识别天然和变性的hrCol X，但不识别NC-1；II组抗体（而非III组抗体）与胃蛋白酶处理的hrCol X有一定反应；获得了一种抗体（X 34），它与分离的NC-1二聚体和天然hrCol X强烈反应，但不与NC-1单体或胃蛋白酶消化的hrCol X反应（IV组）。所有组的抗体均特异性地染色了胎儿人骨骺软骨的肥大带。单克隆抗体X 53染色了软骨内骨小梁中下部肥大带肥大软骨细胞的周围和细胞外基质，而克隆X 34染色了细胞内和细胞周围基质。骨骺的所有其他组织或细胞均为阴性。抗体X 53在组织切片中也与犬、小鼠和豚鼠的肥大软骨反应，但不与牛或猪的X型胶原蛋白反应。在骨关节炎软骨切片中，深层肥大软骨细胞簇被染色，证实了先前关于骨关节炎关节软骨中软骨细胞肥大增强和X型胶原蛋白表达增加的观察结果。
