Conjugation to preadsorbed preactivated proteins and efficient generation of anti peptide antibodies.
A solid phase conjugation method is described based on the preadsorption of proteins to aluminium hydroxide adjuvant followed by activation of the adsorbed carrier proteins with iodoacetic acid N-hydroxysuccinimidester or other conjugation reagents. Cysteine-containing peptides were coupled to the iodoacetic acid-activated carrier-adjuvant particles through their SH groups. No dialysis is required since the reaction product is isolated at each step of the procedure by a simple centrifugation and can easily be extensively washed between individual manipulations. The method generates peptide-carrier-adjuvant particles with sterically defined presentation of the peptides at the surface of the particles. When used for immunization of mice and rabbits the conjugates elicited high-titered specific anti-peptide sera, which reacted well with the parent protein in ELISA. The strongest reactions were with the denatured form of the parent protein. On immunoblots antisera to the N- and C-terminus of calreticulin recognized the same M, 52,000 protein.
Houen
G
G
Department of Autoimmunology, Statens Serum Institut, Copenhagen, Denmark.
Jakobsen
M H
MH
Svaerke
C
C
Koch
C
C
Barkholt
V
V
D000327:Adsorption; D000536:Aluminum Hydroxide; D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D002135:Calcium-Binding Proteins / Q000008:administration & dosage / Q000276:immunology / Q000378:metabolism; D037282:Calreticulin; D002352:Carrier Proteins / Q000008:administration & dosage / Q000276:immunology* / Q000378:metabolism*; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000008:administration & dosage / Q000276:immunology / Q000378:metabolism; D010446:Peptide Fragments / Q000008:administration & dosage / Q000276:immunology*; D011817:Rabbits; D012261:Ribonucleoproteins / Q000008:administration & dosage / Q000276:immunology / Q000378:metabolism; D014373:Tuberculin / Q000008:administration & dosage / Q000276:immunology / Q000378:metabolism; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000276:immunology / Q000378:metabolism
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D002352:Carrier Proteins; D010446:Peptide Fragments; D012261:Ribonucleoproteins; D014373:Tuberculin; D014614:Vaccines, Synthetic; D000536:Aluminum Hydroxide; D010047:Ovalbumin