Facile generation and use of immunogenic polypeptide fusions to a sparingly soluble non-antigenic protein carrier.
Many researchers attempt to prepare antipeptide antibodies by immunizing animals with preparations of fusion proteins or conjugates between the target peptide and a larger protein (such as GST or KLH). Often, the immune response to the larger protein dominates. We have engineered a protein to be sparingly soluble in aqueous solution and nonantigenic, and show that fusions of this sparingly soluble non-antigenic protein (SSNAP) to target peptide sequences can be purified easily to a point suitable for immunizations. When animals are immunized with such fusion proteins, the majority of the immune response is to the target peptide. In all three cases tested, the peptide-specific immune response generated using the SSNAP carrier was greater than that obtained with peptides chemically linked to BSA or KLH, or expressed as fusion proteins to GST. The SSNAP carrier induced a very early IgG response with all classes of IgG well represented in the specific antibody response. All of the SSNAP fusion peptide-derived antibodies were capable of recognizing the full-length target protein in both ELISA and Western analysis. Based on the superior performance of the SSNAP antigens, these studies suggest this novel strategy will have broad utility for the generation of peptide antibodies.
Knuth
M W
MW
Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA. mknuth@promega.com
Okragly
A J
AJ
Lesley
S A
SA
Haak-Frendscho
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D000941:Antigens / Q000008:administration & dosage / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D002645:Chickens; D004274:DNA, Recombinant / Q000235:genetics; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D007114:Immunization / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010455:Peptides / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D012995:Solubility
D000941:Antigens; D002352:Carrier Proteins; D004274:DNA, Recombinant; D010455:Peptides; D011993:Recombinant Fusion Proteins