Measurement of corticotropin-releasing factor (CRF), CRF-binding protein (CRF-BP), and CRF/CRF-BP complex in human plasma by two-site enzyme-linked immunoabsorbant assay
Measurement of corticotropin-releasing factor (CRF), CRF-binding protein (CRF-BP), and CRF/CRF-BP complex in human plasma by two-site enzyme-linked immunoabsorbant assay
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DOI：
10.1210/jc.81.7.2579
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发表时间：
1996-07-01
影响因子：
5.8
通讯作者：
deSouza, EB
中科院分区：
文献类型：
--
作者：
Behan, DP;Khongsaly, O;deSouza, EB
人促肾上腺皮质激素释放因子（hCRF）在脑、垂体和血浆中的作用受37-kDa蛋白质[CRF结合蛋白（CRF-BP）]的调节，该蛋白质结合hCRF并中和肽的生物活性，这表明只有游离的未结合肽具有生物活性。为了准确预测总hCRF水平变化所导致的生物学后果，我们开发了hCRF-BP、游离hCRF和hCRF-BP/hCRF复合物的双位点酶联免疫吸附试验（ELISA）。通过测量1）CRF和hCRF-BP水平改变时的母体血浆和2）接受hCRF刺激试验的正常老年人受试者的血浆中的每种因子，对试验进行了验证。hCRF-BP ELISA的灵敏度为2.7 fmol，检测范围为2.7-8000 fmol。hCRF和hCRF-BP/hCRF测定的灵敏度均为0.4 fmol，检测范围为0.4-40 fmol。母体血浆hCRF-BP水平在16-21和34-39个月胎龄组之间保持不变。然而，水平从16-21个月胎龄组的0.88 +/- 0.069 nmol/L升至28-33个月胎龄组的1.01 +/- 0.09 nmol/L。结合hCRF水平从妊娠16-21个月时检测不到显著升高至28-33个月和34-39个月胎龄组分别为200 +/- 69和442 +/- 106 pmol/L。相比之下，游离hCRF水平在整个妊娠期间保持较低，但从妊娠34-39个月急剧上升至318 +/- 120 pmol/L。当结合和游离hCRF水平升高时，hCRF-BP上的结合位点占有率降低。在用高亲和力hCRF-BP配体α-螺旋CRF-（9-41）处理晚期妊娠血浆样品后，所有结合的hCRF从结合蛋白中被置换，游离hCRF水平从87 pmol/L上升到284 pmol/L。正常志愿者血浆hCRF-BP水平为0.9 ± 0.08 nmol/L（10名男子和9名妇女;平均± 50岁，74.2 ± 7.7岁），在推注1 μ g/kg合成hCRF后15分钟降至基础水平的60%，并在120分钟后逐渐恢复至注射前水平。相反，结合和游离hCRF水平从hCRF注射前的不可检测水平增加到15分钟时的0.58 +/- 0.03 nmol/L，然后迅速下降到120分钟时的不可检测水平。BP配体测量结合和游离的人血浆中的hCRF，并建议这些测定进一步确定外周CRF的变化，如妊娠条件的效用。
The actions of human corticotropin-releasing factor (hCRF) in brain, pituitary, and plasma are modulated by a 37-kDa protein [CRF-binding protein (CRF-BP)] that binds to hCRF and neutralizes the peptide's biological activity, suggesting that only the free unbound peptide is biologically active. To accurately predict the biological consequences resulting from changes in total hCRF levels, we have developed two-site enzyme-linked immunosorbent assays (ELISAs) for hCRF-BP, free hCRF, and the hCRF-BP/hCRF complex. The assays were validated by measuring each factor in 1) maternal plasma at times when CRF and hCRF-BP levels are altered, and 2) plasma from normal elderly human subjects who have undergone a hCRF stimulation test. The hCRF-BP ELISA has a sensitivity of 2.7 fmol and a range of detection from 2.7-8000 fmol. Both the hCRF and hCRF-BP/hCRF assays have a sensitivity of 0.4 fmol, with a useful range of detection from 0.4-40 fmol. Maternal plasma hCRF-BP levels remained unaltered between the 16-21 and 34-39 month gestational age groups. However, levels rose from 0.88 +/- 0.069 nmol/L in the 16-21 month gestational age group to 1.01 +/- 0.09 nmol/L in the 28-33 month gestational age group. Bound hCRF levels dramatically rose from undetectable at 16-21 months of gestation to 200 +/- 69 and 442 +/- 106 pmol/L in the 28-33 and 34-39 month gestational age groups, respectively. In comparison, free hCRF levels remained low throughout gestation, but dramatically rose to 318 +/- 120 pmol/L from 34-39 months of gestation. Binding site occupancy on the hCRF-BP decreased when bound and free hCRF levels were elevated. After treating the third trimester plasma sample with the high affinity hCRF-BP ligand, a-helical CRF-(9-41), all of the bound hCRF was displaced from the binding protein, and free hCRF levels rose from 87 to 284 pmol/L. The plasma hCRF-BP level was 0.9 +/- 0.08 nmol/L in normal human volunteers (10 men and 9 women; mean +/- so age, 74.2 +/- 7.7 yr), decreased to 60% of basal levels 15 min after a bolus injection of 1 mu g/kg synthetic hCRF, and gradually returned to preinjection levels after 120 min. Conversely, bound and free hCRF levels increased from undetectable levels before hCRF injection to 0.58 +/- 0.03 nmol/L at 15 min and then rapidly decreased to undetectable levels at 120 min. These data validate the ELISAs in combination with high affinity hCRF-BP ligands for measuring bound and free hCRF in human plasma and suggest the utility of these assays for further determining alterations in peripheral CRF in conditions such as pregnancy.