Antisera specificities to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide adducts of proteins

Animals were immunized with either 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)-coupled peptide hapten-protein carrier conjugates or with N-acylurea EDCI derived protein carrier. Sera from all animals demonstrated antibody to N-acylurea EDCI when tested by double immunodiffusion using N-acylurea EDCI coupled to a heterologous carrier. Two rabbit antisera, one produced to EDCI-coupled peptide-24-thyroglobulin and another produced to N-acylurea EDCI derived thyroglobulin were further characterized using an enzyme-linked immunosorbent assay (ELISA). Competitive binding assays using either EDCI-coupled peptide-protein conjugates or N-acylurea EDCI derived proteins demonstrated the presence of N-acylurea EDCI reactive antibodies. Such antibodies were also shown to react with the EDCI isourea employed as inhibitor. The specificity of the anti-EDCI antibodies to portions of the EDCI molecule was demonstrated using structural analogues of EDCI. Both 3-dimethylaminopropylamine and 2-dimethylaminoethanol effectively inhibited anti-N-acylurea EDCI reactivity. These results show that when EDCI is used as a coupling agent, antibody is produced to N-acylurea EDCI-carrier, the antibody being primarily directed to the 3-dimethylaminopropyl end of the EDCI molecule.