Simplified conjugation chemistry for coupling peptides to F(ab′) fragments: autologous red cell agglutination assay for HIV-1 antibodies

Abstract

The rapid whole blood test, developed for the detection of circulating antibodies to human immunodeficiency virus type 1 (HIV-1), is based on agglutination of autologous red blood cells using an anti-human glycophorin antibody conjugated to the HIV-1 immunodominant epitope of gp41 (579–613). A simplified procedure for preparing antibody-peptide conjugates for use in the autologous red cell agglutination test is described. F(ab′) 2 fragments of the anti-glycophorin antibody were prepared by pepsin digestion and reduced to F(ab′) fragments with the use of tri- n-butylphosphine (TBP). This permitted the simultaneous reduction of the F(ab′) fragments and coupling of a bromoacetyl derivative of the synthetic immunodominant peptide gp41 (579–613) [Cys-Acm 598, Lys-BrAc 604] containing ϵ-bromoacetyl-lysine at residue 604 to the resultant F(ab′) fragment. Conjugation to the F(ab′) fragment resulted in a stable thio-ether linkage between the peptide Lys-604 and the inter heavy chain cysteines of the F(ab′). The resultant F(ab′)-peptide conjugate was comparable to the previously described disulfide coupled conjugate when used in the autologous red cell agglutination test. This simplified conjugation chemistry may also be useful for the development of reagents for FACS analysis as well as targetted vaccines.