Improved clean-up method for the enkephalins in plasma using immunoaffinity chromatography.
A rapid and selective method of sample clean-up using immunoaffinity chromatography (IAC) was developed to isolate enkephalins from plasma. The enkephalin antibodies were produced utilizing novel protein carriers. Two antibodies, LE4H8 and 33FC6, were selected because of their moderate binding affinity and different epitopes. Enkephalin-spiked plasma was loaded onto the immunoaffinity column and eluted with acidic pH buffer. The eluate was derivatized with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide (NDA-CN), and the enkephalins were separated using reversed-phase liquid chromatography (RPLC). IAC sample clean-up of enkephalin-spiked plasma was compared to the existing solid-phase extraction method. The limit of detection for IAC was 30 pmol. The recovery of the enkephalins from plasma was 90% with a variance ranging from 2 to 9%. The immunoaffinity column was used for approximately 70 samples without any deterioration in performance.
Hendrickson
T L
TL
Department of Chemistry, University of Kansas, Lawrence 66045.
Wilson
G S
GS
D000595:Amino Acid Sequence; D000906:Antibodies; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods*; D004743:Enkephalin, Leucine / Q000097:blood*; D004744:Enkephalin, Methionine / Q000097:blood*; D006801:Humans; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence
D000906:Antibodies; D004744:Enkephalin, Methionine; D004743:Enkephalin, Leucine