Chemistry: analytical and immunological testing – Involving diffusion or migration of antigen or antibody
Reexamination Certificate
1997-07-25
2002-08-20
Swartz, Rodney P (Department: 1645)
Chemistry: analytical and immunological testing
Involving diffusion or migration of antigen or antibody
C435S007100, C435S973000, C436S098000, C436S518000, C436S534000, C436S909000
Reexamination Certificate
active
06436721
ABSTRACT:
BACKGROUND OF THE INVENTION
Immunochromatographic strip formats have become increasingly popular for qualitative and semi-quantitative assays which use visual detection schemes. This type of immunoassay involves the application of a liquid test sample suspected of containing an analyte to be detected to an application zone of an immunochromatographic test strip. The strip is comprised of a matrix material through which the test fluid and analyte suspended or dissolved therein can flow by capillarity from the application zone to a capture zone where a detectable signal, or the absence of such, reveals the presence of the analyte. Typically, the strip will include means for immunospecifically binding the analyte to be detected with its specific binding partner which bears the detectable label. In one such scheme, as disclosed in U.S. Pat. No. 4,446,232; the strip contains an enzyme labeled, mobile binding partner for the analyte which is in a zone downstream from the sample application zone. If analyte is present in the test sample, it will combine with its labeled binding partner to form a complex which will flow along the strip to a detection zone which contains a substrate for the enzyme label which is capable of providing a colored response in the presence of the enzyme label. The strip may contain a zone in which analyte is immobilized, so that labeled binding partner which does not combine with analyte, due to the absence of analyte in the sample, will be captured and thereby inhibited from reaching the detection zone. There have been published various modifications of this technique, all of which involve some competitive specific binding system in which the presence or absence of analyte in the test sample is determined by the detection or lack thereof of labeled binding partner in the capture zone.
An alternative to the above described immunometric assay which detects the free labeled antibody is the so called sandwich format in which the capture zone contains immobilized antibodies against an epitope of the analyte which is different than the epitope to which the labeled antibody is specific. In this format, there is formed a sandwich of the analyte between the immobilized and labeled antibodies and it is therefore an immunometric assay which detects the bound labeled antibody species.
Not all of the schemes for immunochromatography rely on an enzyme labeled binding partner/enzyme substrate for providing the signal for detection of the analyte. In U.S. Pat. No. 4,806,311 there is disclosed a multizone test device for the specific binding assay determination of an analyte and an immobilized binding partner therefore together with a capture zone for receiving labeled reagent which migrates thereto from the reagent zone. The capture zone contains an immobilized form of a binding substance for the labeled reagent. The labeled reagent bears a chemical group having a detectable physical property which is detectable on the basis of its own physical properties, so that it does not require a chemical reaction with another substance. Exemplary of such groups are colored species of fluorescers, phosphorescent molecules, radioisotopes and electroactive moieties.
U.S. Pat. No. 4,703,017 describes the use of visible particulate labels for the receptor. Various particulate labels such as gold sol particles and visible dye containing liposomes are mentioned.
In WO-96/34271 there is disclosed a device for determining a target analyte and creatinine in a fluid test sample which device has an assay strip for the detection of creatinine and a second assay strip for the detection of the target analyte. The creatinine concentration may be determined calorimetrically or by the specific capture of labeled creatinine binding partners. The concentration of the target analyte is corrected based on the sample's creatinine concentration which correction can either be done manually or by means of a properly programmed reflectance analyzer.
EP 0 462 376 A
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discloses an immunochromatographic procedure in which signal at the capture site and the conjugate recovery site of the strip are detected and the analyte concentration is determined by the intensity of the signal at the capture site relative to the signal at the conjugate recovery site. Also of interest in this regard is U.S. Pat. No. 5,569,608.
Immunochromatographic strip formats provide a viable system for the determination of various analytes (whether they be antigens or antibodies) but suffer from the limitation that they yield results which are at best semi-quantitative when, for some analytes, more precise, quantitative results are required. Accordingly, it would be desirable and it is an object of the present invention to provide a means for quantifying the results of analyses carried out by the use of immunochromatographic strip formats.
SUMMARY OF THE INVENTION
The present invention involves a method for determination of an analyte in a sample of body fluid which comprises the steps of:
a) providing a test strip comprising a matrix through which the fluid sample can flow by capillarity, said strip having a first region which contains mobile specific binding partner for the analyte which binding partner bears a detectable label and can react with the analyte to form an analyte/labeled binding partner complex, at least one second region which contains immobilized analyte or an immobilized binding partner which is specific for an epitope of the analyte different than that to which the labeled binding partner is specific, at least one third region which contains means for capturing the analyte/labeled specific binding partner complex which is not bound in the second region and a fourth region which contains means for producing a detectable signal the intensity of which corresponds to the level of a second analyte whose concentration is clinically related to that of the analyte whose concentration in the body fluid is being determined;
b) developing the matrix by applying a sample of a body fluid suspected of containing the first and second analytes thereto thereby allowing it to contact the labeled specific binding partner so that the analyte present in the fluid sample binds to the labeled specific binding partner to form a complex while leaving excess, unreacted labeled binding partner free to further react whereby the fluid sample carries the analyte/labeled partner complex and unreacted labeled binding partner along the matrix by capillarity to the second region containing the immobilized analyte in which region unreacted labeled binding partner is bound to the immobilized analyte in inverse relationship to the concentration of the first analyte in the fluid test sample or the analyte/labeled specific binding partner complex is bound to the immobilized specific binding partner in a direct relationship to the concentration of analyte in the fluid test sample; and the labeled specific binding partner which did not bind to the second region is carried by capillarity to the third region where it is captured by the capture means;
c) reading the second zone of the developed matrix on an instrument having a detector capable of measuring the signal from the detectable label to determine the concentration of the labeled binding partner in the second zone and reading the third zone of the developed strip in a similar manner to determine the signal from the labeled binding partner in the third zone of the matrix;
d) determining a final response signal by ratioing the signals from the labeled binding partner immobilized in the second region and the labeled binding partner captured in the third region;
e) determining the concentration of the first analyte in the fluid sample by comparing the final response signal determined in step (d) with final response signals determined in a similar manner for fluid samples containing known concentrations of the first analyte; and
f) correcting the concentration of the first analyte determined in step (e) by determining the concentration of the second analyte in the fluid test sample by measuring the inten
Kuo Hai-Hang
Miller Carol A.
Wijesuriya Dayaweera
Yip Meitak Teresa
Zimmerle Chris T.
Bayer Corporation
Jeffers Jerome L.
Swartz Rodney P
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