MEASUREMENT OF CORTICOTROPIN-RELEASING FACTOR (CRF), CRF-BINDING PROTEIN (CRF-BP), AND CRF CRF-BP COMPLEX IN HUMAN PLASMA BY 2-SITE ENZYME-LINKED IMMUNOABSORBANT ASSAY/

The actions of human corticotropin-releasing factor (hCRF) in brain, p ituitary, and plasma are modulated by a 37-kDa protein [CRF-binding pr otein (CRF-BP)] that binds to hCRF and neutralizes the peptide's biolo gical activity, suggesting that only the free unbound peptide is biolo gically active. To accurately predict the biological consequences resu lting from changes in total hCRF levels, we have developed two-site en zyme-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 und ergone 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 a nd hCRF-BP/hCRF assays have a sensitivity of 0.4 fmol, with a useful r ange of detection from 0.4-40 fmol. Maternal plasma hCRF-BP levels rem ained unaltered between the 16-21 and 34-39 month gestational age grou ps. 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 gest ational age group. Bound hCRF levels dramatically rose from undetectab le at 16-21 months of gestation to 200 +/- 69 and 442 +/- 106 pmol/L i n the 28-33 and 34-39 month gestational age groups, respectively. In c omparison, free hCRF levels remained low throughout gestation, but dra matically rose to 318 +/- 120 pmol/L from 34-39 months of gestation. B inding site occupancy on the hCRF-BP decreased when bound and free hCR F levels were elevated. After treating the third trimester plasma samp le 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 aft er a bolus injection of 1 mu g/kg synthetic hCRF, and gradually return ed to preinjection levels after 120 min. Conversely, bound and free hC RF levels increased from undetectable levels before hCRF injection to 0.58 +/- 0.03 nmol/L at 15 min and then rapidly decreased to undetecta ble levels at 120 min. These data validate the ELISAs in combination w ith high affinity hCRF-BP ligands for measuring bound and free hCRF in human plasma and suggest the utility of these assays for further dete rmining alterations in peripheral CRF in conditions such as pregnancy.