Chemistry: analytical and immunological testing – Involving immune complex formed in liquid phase – Signal modification or steric inhibition
Patent
1989-04-13
1992-06-23
Wax, Robert A.
Chemistry: analytical and immunological testing
Involving immune complex formed in liquid phase
Signal modification or steric inhibition
G01N 33542
Patent
active
051242680
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a method of determining a biological substance involving labelling with a metal chelate. The determination being performed for example by fluorescence spectroscopy, luminometry or colourometry.
BACKGROUND ART
Methods are already known for the fluorescence spectroscopic determination of a biological substance provided with a marker consisting of a lanthanide chelate complex, formed of a lanthanide metal ion such as a europium ion or terbium ion coupled to the substance via a chelate-forming compound such as ethylene diamine tetraacetic acid (EDTA) or an analogue thereof, by excitation by a short radiation pulse and detection of the fluorescence of the marker when the fluorescence from any noise source has substantially ceased. These methods have application in time-resolved fluorometric assays using labelled tracer molecules such as labelled antigens or antibodies or nucleic acid probes.
U.S. Pat. No. 4,565,790 and European Patent 0,064,484 disclose an improvement in such a process in which, before the determination is carried out, a solution of low pH, which suitably brings the pH to 3.5 or below and contains a detergent and a .beta.-diketone, is added to dissociate the lanthanide ion from the chelate complex and to transfer the dissociated lanthanide ion into a fluorescent form, whereupon the determination is performed in the solution.
As described in those patents the solution added to reduce the pH necessarily contains a detergent and the determination of the fluorescence is necessarily carried out at the low pH, said to be suitably 3.5 or less. Also, the only disclosure of the addition of .beta.-diketone is as part of the low pH solution. The present invention seeks, inter alia, to improve that process.
DISCLOSURE OF THE INVENTION
We have found that none of the features mentioned in the preceding paragraph is essential to the successful determination of the fluorescence, and better results can be obtained more rapidly by operation in a different manner.
According to a first aspect of the present invention a method for the fluorescence spectroscopic determination of a biological substance provided with a marker consisting of a lanthanide chelate complex formed of a lanthanide metal ion coupled to the substance via a chelate-forming compound comprises adding to the chelate complex a solution which reduces the pH to a level at which the lanthanide metal ion becomes dissociated from the chelate complex and subsequently adding to the resulting solution a further solution which increases the pH to a level at which a complex formed between the dissociated lanthanide metal and a chelating chromophore incorporated in the solution is closer to its fluorescence peak for optimum sensitivity. The optimum concentration of the chelating chromophore employed is determined by the pH at which fluorescence measurement is made.
A more general aspect of this invention is the case where a metal ion, not necessarily a lanthanide metal ion, is carried on one chelate through a biochemical reaction to link with a biological substance to be determined and is then, for measurement purposes, released by dissociation and taken up into an appropriately formulated chelating chromophore for measurement by fluorimetry, luminometry or colourometry. Such an approach allows users the freedom to choose the best chelate for conjugation with the biological substance to be determined and with the tracer metal quite independently of the nature of the chelating chromophore used in the measurement step and independently of the optimum conditions for its use. It also does not require derivativization for attachment of the chelating chromophore of choice.
According to a further aspect of the present invention, therefore, there is provided a process for the determination of a biological substance comprising providing the substance with a marker consisting of a chelate complex composed of (i) a metal ion capable of being detected in conjunction with a chelating chromophore and (ii) a first chel
Ekins Roger P.
Tsung Fred
Wax Robert A.
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