Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1983-10-27
1989-06-27
Warden, Robert J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 7, 435 28, 435810, C12Q 168, C12Q 128, G01N 3353
Patent
active
048429970
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to an enhanced luminescent or luminometric assay, particularly immunoassay, and to a diagnostic kit designed to facilitate the assay.
Immunoassay is one of the most widely used analytical techniques in the clinical laboratory. At present the majority of immunoassays employ a radioactive isotope, especially iodine-125, as a label. However radioactive isotopes have a number of major disadvantages. First, the method of labelling involves the use of highly radioactive and hence potentially hazardous reagents. Second, the shelf life of the radio-actively labelled substance is often relatively short not only because by its very nature the radioactive isotope is continuously decaying but also because radioactively labelled proteins are often unstable. Third, it is often difficult to label proteins sufficiently to provide a sensitively and rapidly detectable reagent. Fourth, the disposal of radioactively labelled substances is inconvenient.
These disadvantages have stimulated a search for viable alternatives to the radio label. To be suitable as a label a substance should meet at least the following three requirements:
a. it should be detectable both rapidly and in very small quantities when attached to a ligand such as an antigen or an antibody.
b. it should be possible to attach it, without affecting its determination, to a ligand such as an antigen or an antibody, and
c. once attached, it should not significantly alter the properties of the ligand.
Some of the most promising alternative labels are either substances which can themselves take part in a reaction resulting in the emission of luminescent light or substances which, on suitable treatment, produce compounds capable of taking part in a luminescent reaction. The luminescent reaction (a chemical reaction that results in the emission of luminescent light) is generally of sufficient duration to enable the light emitted to be detected and measured, and thereby to allow the quantification of the labelled material. On the other hand the measurement of luminescence is a rapid process and may be completed in a matter of seconds rather than the several minutes generally required for the measurement of radioactivity.
Luminescence has been employed in three major luminescent or luminometric immunoassay systems:
a. Organoluminescent or organoluminometric immunoassays wherein chemiluminescent compounds which participate directly in luminescent reactions (ie which are converted to an excited state and then return to a non-excited state with the emission of a photon) have been used to label ligands such as proteins, hormones, haptens, steroids, nucleic acids, metabolites, antigens and/or antibodies. Examples of suitable compounds include luminol and isoluminol;
b. Luminescent catalyst or cofactor immunoassays wherein catalysts or cofactors of luminescent reactions have been used as labels. An example of suitable catalyst is the enzyme peroxidase, and
c. Enzyme linked immunoassays wherein luminescent reactions have been used to determine the products formed by the action of enzyme labels on suitable substrates. An example of this type of immunoassay is the determination of antibody linked glucose oxidase by reacting the enzyme/antibody reagent with glucose to form hydrogen peroxide and then measuring the amount of hydrogen peroxide produced by adding luminol under controlled conditions to initiate a luminescent reaction.
The sensitivity of the above immunoassays is determined in part by the lower limit for detection of the label or the product of the label. In the case of luminescent or luminometric immunoassays the sensitivity of the system will depend partially on the light emitted in the luminescent reaction per unit of labelled material. It is one aim of the present invention to provide a luminescent or luminometric immunoassay with an enhanced sensitivity, achieved by determining the labelled material or the product of the label via the present improved luminescent reaction.
Whilst the present improved luminescent reaction is particula
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Carter Timothy J. N.
Groucutt Carol J.
Stott Richard A. W.
Thorpe Gary H. G. H.
Whitehead Thomas P.
Deck Randall E.
National Research Development Corporation
Warden Robert J.
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