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°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. The versatility of dot immunobinding on activated nitrocellulose was shown with two sera from patients infected with human immunodeficiency virus, HIV‐1 and HIV‐2, by using pH 10.2 in incubation media, resulting in a low background. These sera were bound specifically to either one of the closely related HIV‐1 and HIV‐2 peptide antigens from the two viruses.