Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Patent
1994-07-15
1997-04-01
Saunders, David
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
436817, 436822, 436824, 436825, G01N 33543
Patent
active
056165030
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the determination of haptens. More specifically this invention relates to a method of determination of haptens which has the benefit of high specificity to reagents used in such a method and to their preparation and to kits useful in such a method.
Many methods of determining haptens exist but a frequent problem encountered with present commercially employed method relates to lack of specificity. This is often because the binding partners employed bind not only the hapten being determined but also other haptens of related chemical structure. If one had infinite time and resource it should be possible to generate a binding partner that bound only the chosen hapten and none other. In practice this desirable state is not achieved and an unwelcome degree of cross reactivity can occur. The determination would be improved if the disadvantageous effects of this cross reactivity were reduced or eliminated. One method of "cleaning up" samples has been to treat the sample with an antibody which removes at least some cross reacting substances before determining the hapten. However, this can require assays to be carried out on treated and untreated samples to provide full data and is generally inconvenient. Clearly it would be an advantage, for example, if in a determination for one hapten such as a steroid hormone the effect of other related haptens such as other steroid hormones could be reduced or eliminated, or for example, if in the determination of a drug of abuse the effect of chemically related innocuous materials could be reduced and only one assay needed to be carried out.
A method has now been found which enables the determination of haptens to be carried out with a good degree of specificity and without the need for more than one assay to be performed:
The present invention provides a method of determining a hapten which comprises:
1. Contacting the sample which contains or may contain the hapten with a first binding partner which binds the hapten.
2. Separating the hapten bound to the first binding partner from material which is not bound by the first binding partner.
3. Separating the hapten bound to the first binding partner with a second binding partner which binds the hapten.
4. Assaying the hapten bound to a binding partner.
It will be understood that the first and second binding partners are non-identical. This means that they will have different cross reactivities so that the effect of interfering (i.e. cross reacting) substances will be reduced. Clearly the greater the difference in cross reactivity profile the better since the greater the difference the more specific the determination becomes. It is preferred that these two binding partners will be against different epitopes on the hapten. Although it is generally considered that a hapten can be bound by only one epitope at a time, the hapten can in effect have more than one epitope that can be bound at different times.
This may be understood by considering a steroid such as cortisol. Although cortisol can bind to only one antibody at a time, it is able to bind to different antibodies in different ways. Thus, for example, one antibody could bind to the C or D rings and their substituents whereas another antibody could bind to the A and B rings and their substituents. Similarly although digitoxin can bind only one antibody at a time it is capable of binding one antibody at the glycoside portion and another at the steroid portion at a different time. Such an explanation with respect to cortisol or digoxin is put forward to aid visualisation only and should not be viewed as a limitation to the generality of employing two different binding partners.
The sample employed may be or be derived from blood, serum, saliva, urine, tissue homogenate or any other convenient sample depending on the nature of the hapten. Thus, for example, when determining the amount of a steroid hormone or medicament, drug of abuse or the like, present in a patient, the sample may be derived as appropriate from the patient's blood, serum, saliva
REFERENCES:
patent: 3992631 (1976-11-01), Harte
patent: 4376110 (1983-03-01), David et al.
patent: 4813924 (1989-03-01), Strahilevitz
patent: 4963468 (1990-10-01), Olson
patent: 5030558 (1991-07-01), Litman et al.
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