AEQUORIN AS A LABEL IN BIOLUMINESCENCE IMMUNOASSAYS

Project: Research project

Project Details

Description

Although a variety of chemiluminescent and bioluminescent labels have been
used to develop highly sensitive heterogeneous immunoassays, only a small
number of homogeneous immunoassays based on these labels have been
reported. This is a result of the properties of the labels themselves
(i.e., quantum efficiency, stability, etc.) that preclude their use in the
development of homogeneous assays with the desired sensitivity. In this
study, we propose to develop and evaluate novel 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 bioluminescence
detection. The photoprotein aequorin will be used as the bioluminescent
label. Different approaches to the synthesis of aequorin-ligand conjugates
will be undertaken. Specifically, aequorin will be modified in a
conventional way by attaching ligands to free amino groups on lysine
residues of the protein. Further, mono-substituted aequorin-ligand
conjugates (i.e., conjugates where one molecule of ligand is attached to
specific locations on the protein molecule) will be prepared by genetic
engineering of aequorin. We intend to study the effect that specific
biological binders have on the luminescence activity of these aequorin-
ligand conjugates. Moreover, systems in which the interaction between the
conjugate and its corresponding biological binder causes Inhibition of the
bioluminescence activity of the conjugate will be used to develop simple
homogeneous assays for ligands in physiological samples. In cases where no
substantial inhibition of the bioluminescence signal is observed, the
development of solid-phase heterogeneous type assays will be investigated.
In both cases, 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. It is anticipated that
extremely sensitive assays for biologically important molecules will
result from these investigations.
StatusFinished
Effective start/end date1/1/9512/31/99

Funding

  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences

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