Srivastava A K, Bhattacharyya S, Castillo G, Miyakoshi N, Mohan S, Baylink D J
Musculoskeletal Disease Center, Jerry L. Pettis VA Medical Center and Loma Linda University, Loma Linda, CA 92357, USA.
Bone. 2000 Oct;27(4):529-33. doi: 10.1016/s8756-3282(00)00356-2.
The mouse is increasingly being used as an animal model for the study of skeletal phenotypes in humans, mainly because of the ease of genetic manipulation. Biochemical markers of bone metabolism provide a valuable parameter for the assessment of skeletal metabolism. In the mouse model, assays for bone formation have been available for a long time; however, little is known about bone resorption markers. The present study describes the development of a serum C-telopeptide enzyme-linked immunoassay (ELISA), which measures degradation products of type I collagen that are generated by osteoclastic bone resorption. The C-telopeptide ELISA uses affinity-purified antibodies generated against human sequence DFSFLPQPPQEKAHDGGR. The epitope involves an amino acid sequence, which is identical in the mouse and human C-terminal peptide of type I collagen (alpha1 chain). Sensitivity of the ELISA used was <0.1 ng/mL. The average intra- (n = 10) and interassay (n = 8) coefficient of variation for two controls was <12%. The average dilution and spike recovery rates were 98% and 97%, respectively. Application of the ELISA to measure C-telopeptide in 3-4-week postovariectomized (ovx) C57BL/6J (B6) mice (n = 9 or 10) showed a 45% higher C-telopeptide concentration than the sham-operated mice. Treatment of ovx mice with estradiol (400 microg/kg body weight) or alendronate (1.0 mg/kg body weight) resulted in a 20%-50% decrease in C-telopeptide levels compared to the vehicle-treated ovx group. In addition, B6 mice fed a calcium-deficient diet (0.01% calcium) showed a 50% higher C-telopeptide concentration compared to the B6 mice receiving a normal diet (0.6% calcium). In conclusion, the C-telopeptide ELISA exhibited acceptable analytical performance and sufficient discriminatory power to show expected directional changes in the rate of bone resorption following ovariectomy, ovx plus estradiol or alendronate treatment, and administration of a calcium-deficient diet. Therefore, the ELISA developed in this study could be used for measuring bone resorption in the mouse model.
小鼠越来越多地被用作研究人类骨骼表型的动物模型，主要是因为其易于进行基因操作。骨代谢的生化标志物为评估骨骼代谢提供了一个有价值的参数。在小鼠模型中，骨形成检测方法已经存在很长时间了；然而，对于骨吸收标志物却知之甚少。本研究描述了一种血清C-末端肽酶联免疫吸附测定法（ELISA）的开发，该方法可测量破骨细胞骨吸收产生的I型胶原降解产物。C-末端肽ELISA使用针对人序列DFSFLPQPPQEKAHDGGR产生的亲和纯化抗体。该表位涉及一个氨基酸序列，在小鼠和人类I型胶原（α1链）的C末端肽中是相同的。所使用的ELISA的灵敏度<0.1 ng/mL。两个对照的平均批内（n = 10）和批间（n = 8）变异系数<12%。平均稀释率和加标回收率分别为98%和97%。将ELISA应用于测量3-4周去卵巢（ovx）的C57BL/6J（B6）小鼠（n = 9或10）中的C-末端肽，结果显示其浓度比假手术小鼠高45%。用雌二醇（400 μg/kg体重）或阿仑膦酸钠（1.0 mg/kg体重）治疗ovx小鼠，与载体处理的ovx组相比，C-末端肽水平降低了20%-50%。此外，喂食缺钙饮食（0.01%钙）的B6小鼠与接受正常饮食（0.6%钙）的B6小鼠相比，C-末端肽浓度高50%。总之，C-末端肽ELISA表现出可接受的分析性能和足够的鉴别能力，以显示去卵巢、ovx加雌二醇或阿仑膦酸钠治疗以及给予缺钙饮食后骨吸收速率的预期方向性变化。因此，本研究中开发的ELISA可用于测量小鼠模型中的骨吸收。
