Peptide dot immunoassay and immunoblotting: electroblotting from aluminum thin-layer chromatography plates and isoelectric focusing gels to activated nitrocellulose.
Nitrocellulose membrane was preactivated with divinyl sulfone, and a spacer of 1,6-diaminohexane was coupled to the membrane which was functionalized by glutaraldehyde, leaving a reactive carbonyl group. The peptides were coupled to the carbonyl by the side chain and terminal amino groups. The octapeptide angiotensin II (sequence: DRVYIHPF) and peptide analogs containing 6-10 amino acid residues were dotted directly onto the matrix at 45 degrees C for 15 min and detected by specific antisera, which were raised in rabbits against angiotensin I and II, respectively. They were visualized by peroxidase-coupled anti-rabbit IgG antibodies. The detection limit for synthetic angiotensin II was 500 fg per cm2 (= 500 amol per cm2) and for the decapeptide angiotensin I (sequence: DRVYIHPFHL) it was 500 pg per cm2 (= 400 fmol per cm2). Separation of synthetic angiotensin analogs by high performance thin-layer chromatography on silica coated aluminum plates was followed by electroblotting onto activated nitrocellulose and detection with specific antibodies, showing a sensitivity of 100 fg and 1 pg for angiotensin II and angiotensin I, respectively. Isoelectric focusing in agarose using Ampholine carrier ampholytes and immunoblotting with specific antisera displayed a lower sensitivity for angiotensin II and angiotensin I of 2 ng and 20 ng, respectively. The isoelectric focusing and immunoblotting technique was applied for separation of angiotensin I and II and related peptides in serum, where synthetic angiotensin I was degraded in the presence of 1 mM phenylmethylsulfonyl fluoride and 10 mM ethylenediaminetetraacetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)
Lauritzen
E
E
Department of Virology, Statens Seruminstitute, Copenhagen, Denmark.
Másson
M
M
Rubin
I
I
Bjerrum
O J
OJ
Holm
A
A
D000163:Acquired Immunodeficiency Syndrome / Q000097:blood; D000535:Aluminum; D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000032:analysis / Q000737:chemistry; D000804:Angiotensin II / Q000032:analysis / Q000737:chemistry; D000956:Antigens, Viral / Q000097:blood; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003101:Collodion; D015497:HIV-1; D015498:HIV-2; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D015151:Immunoblotting / Q000379:methods*; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D011506:Proteins / Q000032:analysis*; D014764:Viral Proteins / Q000097:blood
D000956:Antigens, Viral; D010446:Peptide Fragments; D011506:Proteins; D014764:Viral Proteins; D000804:Angiotensin II; D003101:Collodion; D000803:Angiotensin I; D000535:Aluminum