Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1990-03-06
1994-05-17
Scheiner, Toni R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 792, 435 793, 435 794, 435 795, 435975, 436518, 436815, 436817, G01N 3353, G01N 33537, G01N 33543
Patent
active
053127293
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The present invention relates to assay techniques and to means for putting such techniques into effect. In particular it relates to an improved assay technique which provides a liquid calibrator for use in standard assays in circumstances where the ligand itself is unstable in solution or is rare, expensive or difficult to prepare in a sufficiently pure and/or quantifiable form.
The conventional multipoint calibration assay methodology facilitates normalisation of unknown sample values with respect to recurring variations in assay conditions. This system requires batch analysis of unknowns run with a replicate calibration or standard curve from which the unknown values are interpolated.
An improvement over this multipoint calibration assay has recently been described by Meyer and Keller in Clinical Chemistry 34 (1), 113-117 (1988) who disclose a one-point recalibration assay utilising a stored standard base curve with no end-user involvement. In practice, each individual unknown sample is assayed in parallel with a calibrator assay and the unknown value is then normalised using the ratio of the observed calibrator signal to that of the expected base curve dose. The normalised unknown sample signal is then read off the stored standard base curve and providing the calibrator, the value and the corresponding signal, for example optical density, which represents that value are selected at a point on the curve which best predicts and minimises deviations across it, due to minor variations in assay conditions, then this "single point calibration" (SPC) assay controls for variation in reagent performance while other assay conditions are controlled by the instrument.
The use of liquid calibrators in such types of assays and in kits designed for such analyses presents immediate advantages of convenience to the end-user. Unfortunately many analytes, for example prolactin, are unstable in liquid form and standard solutions may need to be reconstituted from freeze-dried preparations prior to use. Reconstitution is not only inconvenient for the end-user but, more importantly, introduces considerable room for imprecision which makes the use of freeze-dried standards, particularly with the single point calibration assay described above, potentially inaccurate and thus of very limited use. Thus, the present invention provides a method of assay which incorporates the use of an alternative calibrator which is stable as a liquid preparation.
SUMMARY OF THE INVENTION
In its broadest aspect the invention thus provides a method of assay for a ligand in a sample comprising specific binding partner to the ligand and with an aliquot of reagent X comprising a second specific binding partner to said ligand or a ligand analogue, said reagent X being bound, directly or indirectly, to a detectable label; an amount of said reagent X from the assay medium, the amount of reagent X removed being a function of the amount of said ligand; and X removed from the assay medium; assay which includes the step of incubating an aliquot of the said reagent X or an aliquot of a reagent Z (reagent Z comprising an appropriate reagent labelled analogously to reagent X) with a specific binding partner (not being said ligand) therefor.
Conventional standard assays utilize standard solutions containing known amounts of the ligand under assay. However, the standard assay according to the present invention utilises a non-ligand calibrator.
DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates an immunocomplex formed in a conventional sandwich assay.
FIG. 2 illustrates an immunocomplex formed in accordance with embodiment (A) of the present invention.
FIG. 3 illustrates a precision profile of sample values obtained using antigen calibrator compared to conventional standard curve derived values.
FIGS. 4a and 4b illustrate bias profiles of antigen calibrator (4a) compared to non-antigen calibrator (4b).
FIGS. 5a and 5b illustrate titration curves of the non-antigen calibrator.
FIG. 6 illustrates a precision profile of sample values ob
REFERENCES:
patent: 3998943 (1976-12-01), Ullman
patent: 4299916 (1981-11-01), Litman et al.
patent: 4791056 (1988-12-01), Sizto et al.
Allen Gerald J.
Denyer Madlyn D.
Robinson Grenville A.
Ares-Serono Research & Development Limited Partnership
Scheiner Toni R.
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