Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-13
2003-07-15
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007500, C435S007900, C435S007920, C435S007930, C435S007940, C435S007950, C436S512000, C436S513000, C436S514000, C436S518000, C436S528000, C436S532000, C436S540000, C436S541000, C436S823000
Reexamination Certificate
active
06593085
ABSTRACT:
FIELD OF THE INVENTION
THIS INVENTION relates to an assay method and apparatus suitable for determination of analyte wherein assay apparatus is provided having one or multiple capture zones incorporating a receptor ligand for detection of analyte.
BACKGROUND OF THE INVENTION
Lateral flow membranes for immunoassays are well known and reference may be made, for example, to U.S. Pat. No. 4,943,522 which refers to a porous membrane capable of non-bibulous lateral flow which is used as an assay substrate. A member of a binding pair is affixed in an indicator zone defined in the substrate. A sample is applied at a position distant from the indicator zone and permitted to flow laterally through the zone. Any analyte in the sample is complexed by the affixed binding partner and detected. A method of detection employs entrapment of observable particles in the complex.
Another lateral flow assay is described in U.S. Pat. No. 5,798,273 wherein-the presence or amount of an analyte in a sample may be determined by contacting a sample with anti-analyte antibodies which bind to the analyte or to both analyte and an analyte analog and an assay tracer prior to application to a lateral flow device. A first member of a binding pair is incorporated with the tracer. The lateral flow device comprises a solid support having a sample addition area, a capture area containing analyte or analyte analog which is immobilized thereon and a read out area comprising one or a plurality of zones having a second member for the binding pair immobilized thereon which binds with the first member to detect the presence or amount of analyte in the sample.
Lateral flow type assays are also described in European Patent No. EP0291194 which refers to a casing having a dry porous carrier. A liquid test sample may be applied to the dry porous carrier which has a labelled specific binding reagent for an analyte which reagent is freely movable within the porous carrier when in the moist state and unlabelled specific binding reagent for the same analyte which is immobilized in a detection zone on the porous carrier. The liquid sample may pick up labelled reagent and thereafter permeate to the detection zone. The label used is a particulate direct label.
Reference may also be made to lateral flow assay devices which are described in U.S. Pat. Nos. 5,607,863, 5,648,274 and 5,468,648 which each refer to devices having a first opposable component and a second opposable component wherein at least one of the opposable components comprises capture and detection zones and at least one of the opposable components includes an absorber for contacting the other of the opposable components for detection of analyte. Each of these references describe capture zones which incorporate specific binding partners for the analyte. U.S. Pat. No. 5,468,648 describes provision of a test strip that can carry out multiple assays simultaneously.
Reference may also be made to U.S. Pat. Nos. 5,401,667, 4,855,240, 5,602,037, 5,712,170 and 4,727,019 which all refer to complicated forms of lateral flow assay devices and each of which incorporates capture zones for capture reagent or receptor to bind directly to analyte in a sample being tested.
Reference may also be made to U.S. Pat. No. 5,185,127 which refers to a vertical filter stack in an enclosure having a base portion and a lid. The filter stack has an initial hydrophilic membrane having a binder for analyte thereon, a hydrophobic membrane under the hydrophilic membrane and a pad of absorbent material under the hydrophobic membrane.
Reference may also be made to the following references which use a lateral flow device described above in U.S. Pat. No. 5,468,648, i.e.:
(i) Chew et al., 1998, Clin. Diagn. Lab. Immunol. 5 407-409;
(ii) Vaughn et al., 1998, J. Clin. Micro. 36 234-238;
(iii) Lam et al., 1998, J. Clin. Virol. 10 75-81;
(iv) Cuzzubbo et al.., Jan/Mar 1998, Singapore Microbiologist, 16-17; and
(v) Devine et al., 1998, Analytical Testing Technology 15 16.
As is clear from the prior art referred to above, a feature of such prior art is the provision of lateral flow devices or filter stacks which include one or a plurality of capture zones for direct binding of analyte for detection of the analyte.
However, it has now been realised that such prior art devices have several disadvantages, the most notable of which is the following:
(i) by employing capture zones having a specific binding agent for the analyte, it will be appreciated that such binding agents, which are usually proteins, may in some cases comprise unstable biological material that is usually incapable of being stored at room temperature;
(ii) by virtue of (i), it is usual that such prior art devices can only be used once and therefore must be discarded;
(iii) only relatively small volumes of sample and accompanying reagents can be accommodated due to the necessity for controlling the flow of sample and accompanying reagents through the prior art device to relatively slow speeds;
(iv) often accumulation of complexes occur both in the capture zones and non-capture zones restricting the flow of sample and reagents through the prior art device;
(v) often non-specific reactions and aggregation of reactants may cause non-specific binding; and
(vi) serious limitations apply to processing detection of multiple analytes in prior art devices. For example, when detection of both multiple specificities of IgG and IgM classes of antibodies is being attempted, it is not possible to use anti-IgG or anti-IgM receptors in the capture zones or antigens specific to IgG or IgM antibodies because of the danger of cross reactions occurring, preventing the differentiation between antibody specificity and immunoglobulin class.
SUMMARY OF THE INVENTION
It is therefore an object of the invention to provide an assay method to reduce the affect of the abovementioned disadvantages.
It is a further object of the invention to provide an assay apparatus which is of simple construction and which can be used for assaying a number of different analytes simultaneously.
In accordance with the invention, there is provided a method of assaying for an analyte including the steps of:
(i) mixing a liquid sample suspected of containing an analyte and reagents comprising a target ligand analyte receptor conjugate and a detectable tracer containing a label for the analyte whereby if the analyte is present, there is formed in solution a complex of analyte coupled to the conjugate after incubation of the mixture;
(ii) passing the mixture through filter apparatus containing a plurality of discrete flow zones wherein at least one zone functions as a capture zone having bonded thereto a receptor ligand for the target ligand;
(iii) detecting the presence of analyte in the sample by activation of the label in said at least one zone after binding of the complex to the receptor ligand; and
(iv) continuing the passage of the mixture past said at least one zone to a wicking zone for liquid entrapment.
Thus, one of the principal features of the present invention is that any analyte complex(es) involving the target ligand-analyte receptor conjugate are formed in a liquid or fluid phase prior to retention on a capture zone when the complex(es) bind to associated receptor ligand(s).
The above method is especially adapted for assaying a number of different analytes simultaneously. To this end, the assay apparatus may have a plurality of capture zones which have a different receptor ligand bonded thereto. The sample in step (i) may also be combined with a target ligand-analyte receptor conjugate specific for each analyte being assayed and a detectable tracer incorporating a label for each analyte. In this embodiment, step (ii) facilitates the activation of each analyte by detection of the label in each zone after bonding of each complex to its associated receptor ligand.
The method of the invention may be used for detection of a wide variety of different analytes. In this regard, it will be appreciated that the term “analyte” refers to any compound or composition to be detected that is capable
Barnett Graeme Ross
Manns Roy L.
Knobbe Martens Olson & Bear LLP
Nguyen Bao-Thuy L.
Panbio Pty Ltd
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