Production and characterization of antibodies for the specific determination of the opioid peptide Met5-Enkephalin-Arg6-Phe7.
Endogenous opioids serve as modulators of neuroendocrine and immune system processes, the investigation of which calls for high-specificity radioimmunoassays (RIAs). This study focuses on the development and use of a specific radioimmunoassay for the opioid peptide Met5-Enkephalin-Arg6-Phe7 (MEAP), the C-terminus part of proenkephalin A. Antibodies were raised in four rabbits and investigated in terms of titre, avidity and specificity, followed by finding ideal conditions for these antibodies in RIA. MEAP concentrations were determined in crude extracts of rat hypothalamus, dorsal root ganglia, adrenals and ankle using this standardized assay after an oxidizing process. At reverse-phase high-pressure liquid chromatography (HPLC), the position of immunoreactive material from rat hypothalamus eluted as two peaks out of which one was compatible with that of synthetic MEAP. All rabbits exhibited individual differences in relative immune response and time of its onset. The avidity constant was 10 times higher on a molar basis for ab 4108 compared with ab 4182. There was no cross-reactivity for ab 4182 to related peptides, such as enkephalins and dynorphin B, and negligible background values for ab 4108. The relative levels ofimmunoreactive MEAP from the CNS versus peripheral tissues contrasted in accordance with current knowledge. It is suggested that reports with RIA results should include characterization of antibodies, extraction procedures, standard curves and compositions of buffers. Furthermore, the results should preferably be expressed in relation to total protein content.
Finn
A
A
Peptide Laboratory, Department of Surgical Sciences, Karolinska Institutet, Stockholm, Sweden. anja.finn@haninge.mail.telia.com
Agren
G
G
Bjellerup
P
P
Vedin
I
I
Lundeberg
T
T
D000818:Animals; D000906:Antibodies / Q000097:blood / Q000276:immunology*; D000918:Antibody Specificity; D002851:Chromatography, High Pressure Liquid; D004744:Enkephalin, Methionine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000276:immunology* / Q000302:isolation & purification; D005260:Female; D007031:Hypothalamus / Q000737:chemistry; D008297:Male; D018847:Opioid Peptides / Q000032:analysis / Q000276:immunology; D011817:Rabbits; D011863:Radioimmunoassay / Q000379:methods; D051381:Rats
D000906:Antibodies; D018847:Opioid Peptides; D004744:Enkephalin, Methionine; C027511:enkephalin-Met, Arg(6)-Phe(7)-