Design and construction of novel molecular conjugates for signal amplification (I): conjugation of multiple horseradish peroxidase molecules to immunoglobulin via primary amines on lysine peptide chains.
Immunoconjugates are widely used for indirect detection of analytes (such as antibodies or antigens) in a variety of immunoassays. However, the availability of functional groups such as primary amines or free sulfhydryls in an immunoglobulin molecule is the limiting factor for optimal conjugation and, therefore, determines the sensitivity of an assay. In the present study, an N-terminal bromoacetylated 20 amino acid peptide containing 20 lysine residues was conjugated to N-succinimidyl-S-acetylthioacetate (SATA)-modified IgG or free sulfhydryl groups on 2-mercaptoethylamine (2-MEA)-reduced IgG molecules via a thioether (S[bond]CH(2)CONH) linkage to introduce multiple reactive primary amines per IgG. These primary amines were then covalently coupled with maleimide-activated horseradish peroxidase (HRP). The poly-HRP-antibody conjugates thus generated demonstrated greater than 15-fold signal amplification upon reaction with orthophenyldiamine substrate. The poly-HRP-antibody conjugates efficiently detected human immunodeficiency virus (HIV)-1 antibodies in plasma specimens with significantly higher sensitivity than conventionally prepared HRP-antibody conjugates in an HIV-1 solid-phase enzyme immunoassay and Western blot analysis. The signal amplification techniques reported here could have the potential for development of highly sensitive immunodiagnostic assay 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
D015488:HIV Antigens / Q000032:analysis; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D007136:Immunoglobulins / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*
D015488:HIV Antigens; D007136:Immunoglobulins; D006735:Horseradish Peroxidase; D008239:Lysine