MONO-SUBSTITUTED CONJUGATES IN ENZYMEIMMUNOASSAYS

Project: Research project

Description

Enzyme-linked competitive binding assays are well recognized as
alternatives to radioimmunoassays for the sensitive and selective
determination of a variety of biologically important analytes
(ligands). An inherent drawback of these assays is associated with
the fact that multi-subsituted enzyme-ligand conjugates are
typically being used. This increases the affinity of the binder for
the conjugate (i.e., the binder favors the conjugate over the
analyte) and results in assays with poor (not optimum) detection
limits. In this study, we propose to develop and evaluate novel
enzyme-linked competitive binding assays for biomolecules by
taking advantage of the selective interaction between biological
binders (antibodies, binding proteins, receptors, etc.) and ligands,
as well as the sensitivity associated with enzyme-amplified
detection. Instead of multi-substituted, mono-substituted
enzyme-ligand conjugates (i.e., conjugates where one molecule of
ligand is attached per enzyme molecule) are proposed.
Theoretical models predict that the use of such conjugates could
improve the overall dose-response characteristics of the enzyme-
linked assays. Three different approaches to the synthesis of
mono-substituted conjugates will be undertaken. First, enzymes
possessing covalently attached prosthetic groups can be also
viewed as mono-substituted conjugates and will be used in their
natural state for the assay of their corresponding prosthetic
groups (e.g., biotin, lipoic acid, etc.). Second, ligand-substituted
enzymes will be prepared by incubating apoenzymes with ligand-
prosthetic group conjugates. Therefore, the resulting
holoenzymes will be, essentially, mono-substituted with the
ligand. Finally, a substrate-or coenzyme-directed photoaffinity
labeling procedure will be used to attach a ligand to the vicinity
of the active site of enzymes. We intend to study the effect that
specific biological binders have on the catalytic activity of these
enzyme-ligand conjugates. Specifically, systems in which
formation of a complex between the conjugate and its
corresponding biological binder will cause inhibition of the
enzymatic activity of the conjugate will be optimized in order to
develop simple homogeneous assays for ligands in physiological
samples. In cases where no substantial inhibition of the
enzymatic activity is observed, the development of solid phase
heterogeneous type assays will be investigated. In both cases,
basic fundamental studies will be undertaken to investigate the
nature of the observed assay characteristics and to improve the
sensitivity and detection limits of the technique.
StatusFinished
Effective start/end date7/1/886/30/94

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $103,781.00
  • National Institutes of Health

Fingerprint

Assays
Ligands
Binders
Enzymes
Prosthetics
Apoenzymes
Thioctic Acid
Molecules
Coenzymes
Biomolecules
Biotin
Catalyst activity
Carrier Proteins
Antibodies
Substrates

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)