Design and construction of novel molecular conjugates for signal amplification (II): use of multivalent polystyrene microparticles and lysine peptide chains to generate immunoglobulin-horseradish peroxidase conjugates.
Spherical polystyrene microparticles expressing a large number of highly reactive functional groups were chemically engineered to generate antibody-enzyme conjugates as novel signal amplification systems. Chemically modified goat anti-human IgG and horseradish peroxidase (HRP) were combined in a 1:5 ratio and attached to 0.44 microm streptavidin microparticles or N-succinimidyl-S-acetylthioacetate (SATA)-activated 0.29 microm amino microparticles with highly reactive free sulfhydryl groups on their surface. The numbers of HRP molecules/microparticle were further increased by coupling HRP to primary amines on N-terminal biotinylated or bromoacetylated polypeptides containing 20 lysine residues prior to conjugation with streptavidin or sulfhydryl groups-containing microparticles. The antibody-poly-HRP immunoconjugates contained an estimated number of 10(5)HRP/streptavidin microparticle and 10(6)HRP/amino microparticle, respectively. These microparticle immunoconjugates efficiently bound to plasma anti-HIV-1 antibodies that had been captured by HIV antigens on 5 microm carboxyl magnetic microparticles and, upon reaction with orthophenyldiamine substrate, produced a detection signal with 5-8 times more sensitivity as compared to conventional HRP-conjugated goat anti-human IgG. The signal amplification technique by microparticle immunoconjugates may provide potentially novel tools for the development of highly sensitive diagnostic systems.
Dhawan
Subhash
S
Laboratory of Molecular Virology, Immunopathogenesis Section, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD 20852-1448, USA. dhawan@cber.fda.gov
D015483:HIV Antibodies / Q000032:analysis; D006735:Horseradish Peroxidase; D007124:Immunoenzyme Techniques; D007136:Immunoglobulins; D008239:Lysine; D011137:Polystyrenes
D015483:HIV Antibodies; D007136:Immunoglobulins; D011137:Polystyrenes; D006735:Horseradish Peroxidase; D008239:Lysine