Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-07-27
2001-02-06
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
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Reexamination Certificate
active
06183972
ABSTRACT:
BACKGROUND OF THE INVENTION
Immunochromatographic strip formats have become increasingly popular for quantitative 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 fluid test medium 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 of the strip 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 contains another zone in which analyte is immobilized, so that the labeled binding partner which does not combine with analyte, due to the absence of sufficient 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 competitive specific binding systems 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. This type of immunostrip format works well in connection with the analysis of relatively low concentrations of analyte, but can be of limited utility in the analysis of fluids containing high analyte concentration. This adverse effect is caused by the presence of excessive free analyte in the sample that competes for binding with the immobilized antibody in the strip's capture band with the analyte which has become bound to the labeled antibody by interaction therewith in a portion of the strip upstream from the capture zone. This competition can result in less of the analyte/labeled antibody conjugate being captured by the capture antibody and consequently less signal being detected in the capture zone than would be in the case if there were less analyte in the test sample. A dose-response curve prepared using this type of test strip will show increasing signal with increasing analyte up to the point where the analyte concentration begins to block the interaction between the immobilized capture antibody and the analyte/labeled antibody complex. Beyond this point, increasing analyte in the test fluid results in a decrease in the signal, so that the dose-response curve indicates decreasing signal with increasing analyte. The slope of this sort of dose-response curve somewhat resembles a hook which accounts for this phenomena being known as the hook effect. Traditionally, when the hook effect is observed or suspected, the fluid sample is diluted to several dilutions to ensure the validity of the results. The high dose hook effect may not occur if sufficient labeled or capture antibody is present in the assay medium. A complete dose-response curve (low to high analyte concentration) is usually needed to verify the existence of this effect. Accordingly, sample dilution is generally carried out whenever there is reason to expect that the assay might exhibit the hook effect. It is an object of the present invention to provide a sandwich type assay method using an immunochromatographic strip whose efficacy is not affected by high analyte concentrations in the test sample and, accordingly, does not require sample dilution or reassaying of samples containing high analyte concentrations. This method involves providing a strip with at least two capture bands and optionally a collection band in which there is immobilized a binding partner for labeled antibody which will bind labeled antibody which has not formed a complex with analyte to thereby facilitate its capture in one of the capture bands. The collection band is optional since it is not needed for the assay method to work in the sandwich format. However, by using a strip which contains a collection band, each sample measurement will provide more information thereby improving the assay's sensitivity and/or precision.
In EP 0 462 376 A
2
there is disclosed a procedure in which signal at the capture site and conjugate collection site of an immunochromatographic 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 recovery site. Also of interest in this regard is U.S. Pat. No. 5,569,608.
In co-pending application [Ser. No. 08/900,586] there is disclosed an assay using an immunochromatographic tographic strip having multiple capture and/or collection sites in which the signal from the detectable label in the capture zone(s) and collection zone(s) is determined whereupon a final response signal is determined using an algorithm and a number of signals which are chosen in a manner suited for a particular assay to provide a value for analyte concentration.
SUMMARY OF THE INVENTION
The present invention is a method for determining the concentration of an analyte in a fluid test medium. The method comprises the steps of:
a) Providing a strip of a porous material through which a test fluid suspected of containing the analyte can flow by capillarity; the strip has at least two distinct capture regions in which there are immobilized antibodies specific to a first epitope of the analyte. There are also provided antibodies specific to a second epitope of the analyte which bear a detectable label and are capable of flowing through the strip along with the fluid test medium upon applying it to the strip up stream from the first of the at least two distinct capture zones;
b) Applying the fluid test medium to the strip and allowing it to flow along the strip carrying labeled antibodies along with it to thereby contact the immobilized antibodies in the distinct capture regions. When sufficient analyte is present in the fluid test medium to partially block binding of the immobilized antibody with the first epitope of the analyte in at least the first distinct capture region with which the fluid test medium comes into contact as it flows along the strip, there is formed a sandwich of the immobilized antibody, the analyte and labeled antibody in the distinct capture regions through which the fluid test medium carries analyte the quantity of which sandwich is limited by the partial blocking of the immobilized antibody;
c) Detecting, in a quantitative manner, the signal emitted from the label on the labeled antibody in each of the distinct capture regions in which the sandwich has formed. This provides a pattern of signals which is unique to the concentration of analyte in the fluid test medium; and
d) Mathematically combining the unique set of signals to create a monotonous dose-response curve to factor out the blocking of the binding between the immobilized antibody and the first epitope of the analyte.
REFERENCES:
patent: 5569608 (1996-10-01), Sommer
Barbarakis et al., “Observation of Hook Effects in the Inhibition and
Kuo Hai-Hang
Meritt Lisa A.
Bayer Corporation
Cook Lisa V.
Jeffers Jerome L.
Le Long V.
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