SIMPLIFIED CONJUGATION CHEMISTRY FOR COUPLING PEPTIDES TO F(AB') FRAGMENTS - AUTOLOGOUS RED-CELL AGGLUTINATION ASSAY FOR HIV-1 ANTIBODIES

The rapid whole blood test, developed for the detection of circulating antibodies to human immunodeficiency virus type 1 (HIV-1), is based o n agglutination of autologous red blood cells using an anti-human glyc ophorin antibody conjugated to the HIV-1 immunodominant epitope of gp4 1 (579-613). A simplified procedure for preparing antibody-peptide con jugates for use in the autologous red cell agglutination test is descr ibed. F(ab')(2) fragments of the anti-glycophorin antibody were prepar ed 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 t he synthetic immunodominant peptide gp41 (579-613) [Cys-Acm 598, Lys-B rAc 604] containing epsilon-bromoacetyl-lysine at residue 604 to the r esultant F(ab') fragment. Conjugation to the F(ab') fragment resulted in a stable thio-ether linkage between the peptide Lys-604 and the int er heavy chain cysteines of the F(ab'). The resultant F(ab')-peptide c onjugate was comparable to the previously described disulfide coupled conjugate when used in the autologous red cell agglutination test. Thi s simplified conjugation chemistry may also be useful for the developm ent of reagents for FACS analysis as well as targetted vaccines.