Apelin Radioimmunoassay

Trunk blood was collected after decapitation of the animal on ice (50 µl of 0.3 M EDTA (pH 7.4) for 1 ml of blood). Plasma samples (0.5 ml) were acidified with 0.175 ml of 3 M HCl and stored at −80°C until apelin RIA. After thawing the sample on ice, 0.05% BSA was added and the samples were centrifuged at 20,000 x g and 4°C for 10 min. The supernatants were collected and the pH was adjusted to 6.5 with 10 M NaOH and 2 M Tris-HCl buffer (pH 7.4). Apelin was extracted from plasma by mixing 0.3 ml of the supernatant with 0.3 ml of 1% trifluoroacetic acid (TFA)—0.1% BSA, and loading it onto a Sep-Pak C18 cartridge (Waters, Massachusetts, United States) previously washed with 2 ml 100% acetonitrile and equilibrated with 5 ml 1% TFA—0.1% BSA. The columns then were washed with 3 ml 1% TFA - 0.1% BSA and apelin was eluted with 1.5 ml 100% acetonitrile. The samples were dried and dissolved in 0.32 ml of RIA buffer (19 mM NaH₂PO₄ ·H₂O, 81 mM Na₂HPO₄ ·2H₂O, 50 mMNaCl, 0.1% TritonX-100, 0.01% NaN₃, 0.1% BSA).

Plasma apelin levels were determined by RIA on 0.1 ml of plasma, with a polyclonal K17F antiserum (0.05 ml at a dilution of 1:4,500) and ¹²⁵I-labeled pE13F (iodinated on Lys⁸ by the Bolton and Hunter method, 2,200 Ci/mmol, Perkin-Elmer, Waltham, MA; 0.05 ml, 19,000 dpm) as a tracer, incubated at 4°C overnight. Samples were mixed with ¹²⁵I-labeled pE13F and polyclonal K17F antiserum (dilution: 1:4,500) to give a total volume of 0.2 ml, and were incubated at 4°C overnight. We then added 0.5 ml of Amerlex (Amersham RPN 510), and the resulting mixture was incubated for 10 min at room temperature. The tubes were centrifuged at 2,600 x g at 4°C for 20 min. The supernatant was removed and the radioactivity of the precipitates was measured. We assessed the cross-reactivity of the apelin antiserum with various N- and C-terminally truncated fragments of K17F and several other bioactive peptides. K17F (200% cross-reactivity), the pyroglutamyl form of apelin-13 (pE13F) and apelin-36 (100% cross-reactivity) were well recognized by the antiserum, whereas the removal of the phenylalanine residue at the extreme end of the C-terminus of K17F (forming K16P) decreased recognition to barely detectable levels (<0.3% cross-reactivity). Negligible cross-reactivity was observed for angiotensin II, angiotensin III, neuropeptide Y and AVP. This antiserum identified the apelin present in the plasma of rodents as pE13F and, to a lesser extent, K17F (De Mota et al., 2004). Most of the apelin in human plasma was K17F, followed by pE13F, and, to a lesser extent, apelin-36 (Azizi et al., 2008).