Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving urea or urease
Reexamination Certificate
1999-11-19
2001-05-22
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving urea or urease
C435S014000, C435S025000, C435S004000, C435S007910, C435S007100, C435S287100, C205S787500, C204S175000
Reexamination Certificate
active
06235491
ABSTRACT:
The present invention relates to assay for the pressure of an analyte or for determining a biological or medical parameter and to a kit for such an assay.
When a patient is treated by a physician, it is not uncommon for the physician to take samples of body fluids to be sent on to a laboratory for analysis. Such testing inevitably gives rise to some delay in the processing of the sample. This is particularly true where samples have to be sent to a separate laboratory for testing. Even in hospitals it can often take a number of hours for the results to be communicated back to the physician. Accordingly, it is not uncommon for the physician to begin treating a patient without knowing the results of any requested testing.
It situations where the patient is seriously ill, the delay incurred in testing samples could conceivably put the well-being of that patient at risk.
One might consider that a suitable way to overcome this problem would be for the physician in charge of a particular patient to conduct the testing himself, without sending the samples away to a laboratory. However, the testing of samples is often a complex process which must be carried out by highly trained personnel if the results are to be reliable and hence of any real use to the physician.
Therefore, there is a need in the art for assays which can be quickly and reliably carried out by a user (who will sometimes be referred to as in operator), particularly for samples obtained from patients.
GB-A-2,284,890 relates to an analyte sensor in which an analyte is detected by reacting it with an enzyme or catalytic species at or very near to a polymer coated electrode. The enzyme or catalytic species directly or indirectly effects a reaction with said polymer layer wherein the polymer layer becomes porous causing a measurably change in electrical properties at the electrode surface. In one embodiment the polymer is pH-sensitive and the reactions cause a change in pH.
WO98/10788 relates a method for the measurement of clot formation times, clot dissolution time or clotting parameters by monitoring movement of magnetic particles in the sample being assayed, where the movement is induced by a magnetic field.
WO94/19690 relates to a method for performing an affinity assay and which monitors the response of an oscillating field on magnetic particles to determine the analyte concentration.
WO95/06868 also describes an oscillating particle type coagulation assay.
The present invention provides a method of testing samples, especially patient samples. Advantages of this method include the fact that the assay can use whole blood, hence avoiding a pre-assay step involving separation of blood components. The method of the present invention can be automated and will make the results of the test available quickly and thus provide an early and rapid diagnosis of a patient's condition. The method is simply and therefore minimal operator skill is required. In addition, the equipment necessary to automate the method may be readily available and therefore the method will be economic.
In its broadest aspect the present invention provides a method for carrying out an assay which has as its end point a pH change.
In a first aspect, the present invention provides an assay for the presence of an analyte or determining a biological or medical parameter comprising the following steps:
an assay component is added to a pH-sensitive material charged with magnetic particles, wherein the assay component causes a pH change which is a function of the parameter to be measured;
subjecting the magnetic particles to an oscillating magnetic field; and measuring the effect of the magnetic field on the magnetic particles.
The presence of the analyte is determined, or determination of the parameter is made, using analysis of the response of the magnetic particles to the magnetic field.
It will be appreciated that the pH material is changed as a function of the analyte or parameter and this results in a change in the movement of the magnetic particles in the oscillating magnetic field.
The sample may be aqueous, whole blood, serum, plasma, urine or saliva.
In a second aspect, there is provided a method for carrying out an assay for the presence of an analyte, comprising:
contacting a sample to be assayed for the presence of an analyte with a reactive species, the analyte reacting with the reactive species resulting in a change in pH;
directly or indirectly effecting a reaction with a pH-sensitive material, which is charged with pH-sensitive material magnetic particles;
applying a magnetic field to the magnetic particles;
monitoring a response of said magnetic particles to the magnetic field; and
determining the amount of analyte in said sample by analysis of the response of the magnetic particles.
The change in pH is a function of the presence of the analyte.
The interaction of the analyte with the reactive species gives rise to an assay component and the assay component results in the change in pH.
The analyte and reactive species interact to form an assay component such that the pH change is a function of the presence of the analyte. This function may be allow a qualitative or quantitative determination of the amount of analyte present in the sample.
Preferably the assay employs binding pairs. A non-exclusive list of commonly used binding pairs includes avidin/biotin, antibody/antigen, haptens and nucleic acid (DNA and RNA). Generally when the binding pair is antibody/antigen the assay is referred to as an immunoassay. Other biosubstances capable of molecular recognition include lectins for saccarides, hormone receptors for hormones and drug receptors for drugs and active drug metabolites.
In a preferred aspect the method is used for performing an immunoassay.
In a third aspect, the present invention relates to an enzyme immunoassay in which the pH produced by the enzyme reaction is a function of the amount of analyte, and wherein liberation of magnetic particles from a pH-sensitive material is detected.
As changes in pH are associated with many enzyme reactions the present invention provides a flexible assay with wide applicability. Generally, in enzyme immunoassays, the enzyme is used as a label or marker which is bound to one member of the antigen-antibody pair identical to that in the sample to be measured. The enzyme bound antigen/antibody then competes with the sample antigen/antibody for the binding site on a limited supply of its complement antibody/antigen.
Classical methods for immunoassay include:
(i) A capture antibody on a solid phase, such as a plastic microtitre plate, exposure to the biological sample to attach the antigen of interest, washing and then exposure to a second labelled antibody. The label on the antibody may be an enzyme for example. Further washing is followed by detection of the label (and hence the amount of antigen in the original sample). This is known as a sandwich assay or two-site assay, or
(ii) A capture antibody on the solid phase followed by exposure to the biological sample containing antigen and an added amount of labelled antigen. Labelled and unlabelled antigen compete on the solid phase for the antibody sites. The amount of label revealed after washing is inversely proportional to the amount of true antigen in the biological sample. This is known as a competitive assay.
Other immunoassays methods which are known, or become known, to a skilled worker may also be used. For example, the assay may use direct molecular recognition. In this approach, one of the binding pair, e.g. an antibody, which is immobilised binds with its binding pair present in the sample, causing a pH change. The advantage of direct-recognition assays is simplicity.
In enzyme immunoassays the label is measured by adding the enzyme substrate and monitoring the product by a suitable method. In the present case, the enzyme reaction gives rise to a pH change and this monitored using the effect of a magnetic field on magnetic particles captured in a pH-sensitive material. The signal is then processed and a result calculated.
In another prefer
Byk Gulden Italia S.p.A.
Jacobson Price Holman & Stern PLLC
Leary Louise N.
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