Biochemical characterization of mouse liver IBE1-amide immunoreactivity: limitations of antibody-based approaches for verification of peptide expression.
A high titer, specific antiserum, raised against a synthetic analogue of a unique peptide region within the human IGF-IB prohormone, detected specific immunoreactivity in extracts of mouse, chicken, sheep, and human liver. Specificity was confirmed by the ablation of immunoreactivity in the presence of excess synthetic immunogen. Here we report the isolation and characterization of one of the immunoreactive species from an extract of mouse liver: amino acid sequencing revealed that the purified product was 78% identical to the NH2-terminus of the alpha-subunit of mouse hemoglobin. Immunoblot analysis of a commercial preparation of mouse hemoglobin confirmed that the antiserum recognized hemoglobin. Addition of excess synthetic peptide to the antiserum eliminated the immunobinding to hemoglobin. The apparent "specificity" of even affinity-purified antiserum for hemoglobin provides a cautionary note for the interpretation of studies concluding antigen expression based solely on the presence of positive immunoreactivity.
Quinn
K A
KA
Biomarkers and Prevention Research Branch, National Cancer Institute, Rockville, MD 20850, USA.
Unsworth
E J
EJ
Miller
M J
MJ
Mulshine
J L
JL
Cuttitta
F
F
D000595:Amino Acid Sequence; D000818:Animals; D003429:Cross Reactions; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006454:Hemoglobins / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D007334:Insulin-Like Growth Factor I / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D011498:Protein Precursors / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D011817:Rabbits; D016415:Sequence Alignment; D017421:Sequence Analysis
D000939:Epitopes; D006454:Hemoglobins; D007106:Immune Sera; D010446:Peptide Fragments; D011498:Protein Precursors; D007334:Insulin-Like Growth Factor I