Lateral flow test format for enzyme assays

Medicilon provides various enzyme activity assays for kinases, phosphatases, proteinases, deacetylase, peptidase, esterase, and other enzymes. Our line of well-characterized immunoassays and biochemical kits ensures accurate and reproducible

The invention relates to a lateral flow enzyme assay device and test kit for the determination of analyte in a test sample. The invention further relates to lateral flow method for the determination of analytes by directly using the analyte as an enzyme substrate.

The invention relates to a lateral flow enzyme assay device and test kit for the determination of an analyte in a test sample. The invention further relates to a lateral flow method for the detection of analytes by using the analyte as an enzyme substrate or co-substrate.

Many analyses of biochemicals are based on enzyme assays. A wide variety of assays are used in the fields of clinical chemistry, hygiene monitoring, life sciences research including molecular biology, microbiology and water testing.

Typically the analyte of interest is a substrate or co-substrate for an enzyme-catalysed reaction that, with addition of further substrate components, yields a product more easily detected and measured than the original analyte. Such assays can involve single or multiple enzyme steps. Detection methods can include an increase in absorbance, particularly a change in colour, change in fluorescence, change in luminescence, change in electrical potential at a surface, change in other optical properties such as circular dichroism or light scattering, or any other easily measured physical property.

Most laboratory based tests for qualitative or quantitative detection of an analyte use formats in which precise amounts of two or more liquid reagents are added to the sample according to a defined protocol to produce a signal more easily detectable than direct measurement of the analyte.

For a typical enzyme assay procedure, the operator needs enzyme and substrate reaction components to react with the analyte in solution in the sample. In addition, there may be a need for buffering components, agents to extract the analyte of interest from the sample matrix further enzyme components, further enzyme co-factors, additional signal components and a known standard concentration of analyte as a positive control for comparison with the unknown level in the sample.

Due to unwanted chemical cross-reactions and/or inappropriate ionic conditions and/or effects leading to instability in storage, the different assay components are often incompatible with one another. As a result, the assay components need to be formulated into two or more separate reagents. It is therefore usual to have at least separate enzyme and substrate reagents to add to the sample in order to undertake an enzyme assay.

As a consequence, experimental protocols for enzyme assays are often lengthy and complicated, with extensive preparation steps before the assay can be performed and the requirement for specialised apparatus and a high level of skill and training on the part of the operator. Liquid based assays require extra reaction vessels such as tubes, a means to accurately dispense solutions such as a micro-pipette and a means to dispose of the reaction components safely after use.

In addition it is often difficult to stabilise enzymes in solution within liquid reagents. Freeze dried reagents have improved stability but the need to supply one or more solutions for rehydration and also the extra steps involved represent a significant decrease in user convenience.

A new high-convenience format or device for enzyme assays would give significant benefits if it were to feature the following aspects:

Easy handling without the need for lengthy and complicated preparation steps.

The capability to incorporate from one to several assay reagents in stable formulations.

The potential to be read by a suitable instrument

The incorporation of a positive control within the same device.

Ease of manufacture.

Robust construction, enabling field use.

Such a device would be highly suitable for unskilled operators and for use both in and outside a laboratory environment.

A substantial improvement has been achieved in the last years in the field of immunoassays. Lateral flow assays in which reagents and samples are transported within a linear matrix containing reagents to detect the presence of one or more specific analytes, fulfil the above mentioned requirements. Lateral flow assays typically are immunoassays in which analytes are identified by the binding of specific antibodies. Most assays rely on two binding events, with analyte identification resulting in the analyte moving from the mobile to the immobile phase of the assay. The first binding reaction occurs in the mobile phase between the analyte and a specific binding molecule for the analyte with an attached label. The second binding reaction occurs between the analyte-binding molecule-label complex and a second specific binding molecule immobilised at the reaction site. The two binding reactions combined concentrate the label at the reaction site if the analyte is present, forming the basis of analyte detection.

For example U.S. Pat. №5,591,645 discloses a method and a device for determining the presence of an analyte in a liquid sample comprising an immobilised and a mobile analyte binding agent.

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