An improved conjugation method for controlled covalent coupling of synthetic peptides to proteins using glutaraldehyde in a dialysis method.
Controlled and efficient conjugation of synthetic peptides to proteins, for use in immunization or in assay procedures, is a prerequisite for the immunological applications of synthetic peptides. This study describes a new method of conjugating synthetic peptides to proteins in such a way that no homopolymers of synthetic peptides or proteins occur. To achieve this, the protein is first activated with glutaraldehyde and subsequently excess glutaraldehyde is removed. Then coupling of the synthetic peptide to the activated protein occurs while subsequently the surplus reactive glutaraldehyde groups on the protein are blocked with lysine. Excess free peptide and lysine is then removed by dialysis. This improvement not only results in better defined conjugates when compared to classical glutaraldehyde coupling, but also in the consumption of smaller amounts of synthetic peptide during conjugate formation. When used for immunization we obtained similar and sometimes even better responses with the glutaraldehyde based conjugates than with succinimidyl (MBS) conjugates of the same peptides. The performance of the modified conjugates in ELISA procedures, immunization and immunocytochemistry suggests that they are superior to conjugates formed by classical glutaraldehyde coupling.
Zegers
N
N
Department of Immunology, Medical Biological Laboratory TNO, Rijswijk, The Netherlands.
Gerritse
K
K
Deen
C
C
Boersma
W
W
Claassen
E
E
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D005976:Glutaral; D006433:Hemocyanins; D006801:Humans; D007106:Immune Sera; D007114:Immunization / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology*; D011944:Receptors, Androgen / Q000276:immunology; D012710:Serum Albumin, Bovine; D013388:Succinimides
D003432:Cross-Linking Reagents; D007106:Immune Sera; D010455:Peptides; D011944:Receptors, Androgen; D013388:Succinimides; D012710:Serum Albumin, Bovine; C013097:3-maleimidobenzoyl N-hydroxysuccinimide; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin; D005976:Glutaral