The Generation of Antibody in Mice to Tuftsin: A Naturally Occurring Phagocytosis Stimulating Tetrapeptide

Abstract

Tuftsin (Thr-Lys-Pro-Arg) is a naturally occurring tetrapeptide that stimulates most known functions of the polymorphonuclear leukocyte and macrophage cell lines. We previously reported our unsuccessful attempts to generate antituftsin antibodies by conjugating tuftsin to several carrier proteins and by polymerizing the peptide with glutaraldehyde. To render tuftsin antigenic the following modifications were made to native tuftsin: three glycine residues were added to the N terminus of tuftsin (Gly₃-tuf) and cysteine was added to the N terminus (Cys-tuf) and to the C terminus (tuf-Cys). Native tuftsin was covalently conjugated to sheep red blood cells (SRBC). In a separate experiment Balb/c mice primed with SRBC were immunized with 10⁷ SRBC peptide conjugate. Native tuftsin and Gly₃-tuf were also conjugated to keyhole limpet hemocyanin (KLH). In another experiment KLH and cationized bovine serum albumin (cBSA) were activated with sulfo-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (s-SMCC), which was used to control orientation of tuf-Cys and Cys-tuf when conjugated to each carrier protein. All conjugates were administered in complete Freund's adjuvant (CFA) except for cBSA conjugates which were administered in alum. Antibody response was determined by solid phase radioimmunoassay. Results showed that specific antituftsin antibodies were elicited only by Cys-tuf, conjugated to KLH. This study reaffirms that tuftsin is weakly antigenic and confirms the previous work by Gottlieb et al. [9] that antibody to tuftsin can only be elicited when tuftsin is conjugated to the carrier protein KLH in a manner that leaves the peptide carboxyl end free.