The sensitivity of dot immunoassay for the peptides helodermin, histidine-isoleucinamide (PHI) and histidine-methioninamide (PHM) increases after peptide cross-linking to proteins prefixed on nitrocellulose.
Two out of three rabbit anti-helodermin antisera previously shown to be useful for radioimmunoassay failed to detect up 100 ng of the peptide helodermin spotted directly on a nitrocellulose membrane. A lesser shortcoming of dot immunoassay was encountered with porcine PHI (peptide histidine-isoleucinamide), a member of the same peptide family, and an anti-PHI antiserum. To improve antigen-antibody interactions in the solid phase, we compared five methods of prior peptide immobilization. The best result was obtained when the nitrocellulose membrane was pretreated for 1 min in 4% ovalbumin, followed by 10 min activation with 2.5% glutaraldehyde, before peptide spotting. After cross-linking, the peptide was immunodetected with a F(ab')2 fragment of an anti-rabbit IgG coupled to alkaline phosphatase. The peptide cross-linking method was capable of increasing the sensitivity of ensuing immunodetection by more than 1000-fold, i.e., made feasible the detection of 0.1 ng peptide/dot in cases when the direct spotting method was inefficient. The sensitivity of this new, reliable and simple dot immunoassay for peptides was comparable to the conventional dot assay of directly immobilized large proteins.
Gourlet
P
P
Department of Biochemistry and Nutrition, Medical School, Université Libre de Bruxelles, Belgium.
Svoboda
M
M
Cauvin
A
A
Christophe
J
J
D000595:Amino Acid Sequence; D000818:Animals; D003101:Collodion; D003432:Cross-Linking Reagents; D015151:Immunoblotting / Q000379:methods*; D007124:Immunoenzyme Techniques; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010451:Peptide PHI / Q000032:analysis*; D010455:Peptides / Q000032:analysis*; D011485:Protein Binding; D011817:Rabbits
D003432:Cross-Linking Reagents; D036341:Intercellular Signaling Peptides and Proteins; D010451:Peptide PHI; D010455:Peptides; C040442:heliodermin; D003101:Collodion