Invented by James William Needham, Syamal Raychaudhuri, InBios International Inc

The market for Multiplexed Lateral Flow Assay Systems and Methods for Their Use Lateral flow assay systems have revolutionized the field of diagnostic testing, providing rapid and accurate results for a wide range of applications. These systems, also known as lateral flow immunoassays, are commonly used in medical diagnostics, food safety testing, environmental monitoring, and veterinary diagnostics. However, the demand for more advanced and versatile testing methods has led to the development of multiplexed lateral flow assay systems. Multiplexed lateral flow assay systems allow for the simultaneous detection of multiple analytes in a single test, offering increased efficiency and cost-effectiveness. These systems utilize multiple test lines or zones on a single strip, each specific to a different analyte. By incorporating multiple target-specific antibodies or antigens, these systems can detect and quantify multiple analytes in a single sample. The market for multiplexed lateral flow assay systems has been growing steadily in recent years, driven by the increasing demand for point-of-care testing and the need for rapid and accurate results. These systems offer several advantages over traditional single-analyte lateral flow assays, including reduced testing time, increased sensitivity, and improved specificity. One of the key factors driving the market growth is the rising prevalence of infectious diseases and the need for rapid and accurate diagnostic tools. Multiplexed lateral flow assay systems have been widely used in the detection of infectious diseases such as HIV, malaria, and tuberculosis. These systems enable healthcare professionals to simultaneously test for multiple pathogens, allowing for early diagnosis and timely treatment. Another significant factor contributing to the market growth is the increasing demand for food safety testing. Multiplexed lateral flow assay systems have been extensively used in the detection of foodborne pathogens, allergens, and contaminants. These systems enable food manufacturers and regulatory agencies to quickly and accurately assess the safety and quality of food products, ensuring consumer protection and compliance with regulations. The market for multiplexed lateral flow assay systems is also driven by the growing focus on personalized medicine and the need for companion diagnostics. These systems can be used to detect multiple biomarkers associated with specific diseases or conditions, enabling healthcare professionals to tailor treatment plans to individual patients. This personalized approach to medicine has gained significant traction in recent years, driving the demand for multiplexed lateral flow assay systems. In terms of geographical distribution, North America and Europe currently dominate the market for multiplexed lateral flow assay systems. The presence of well-established healthcare infrastructure, favorable reimbursement policies, and a strong focus on research and development are some of the factors contributing to the market growth in these regions. However, the market is expected to witness significant growth in the Asia-Pacific region, primarily due to the increasing healthcare expenditure, rising awareness about infectious diseases, and the growing demand for point-of-care testing. In conclusion, the market for multiplexed lateral flow assay systems is witnessing significant growth, driven by the increasing demand for rapid and accurate diagnostic tools. These systems offer several advantages over traditional single-analyte lateral flow assays, making them ideal for a wide range of applications. With ongoing advancements in technology and increasing focus on personalized medicine, the market for multiplexed lateral flow assay systems is expected to continue its upward trajectory in the coming years.

The InBios International Inc invention works as follows

A device is provided for performing multiplex lateral-flow immunoassays in which a sample of liquid, such as biological samples, is simultaneously tested for multiple analytes. The device can also be used to detect multiple analytes simultaneously within a liquid sample.

Background for Multiplexed Laval Flow Assay Systems and Methods for Their Use

The “lateral flow immunoassays” (also known as dipsticks, strip tests or simply strips tests) are simple, one- or two step tests for qualitative determinations of analytes in liquid samples. Striped lines or zones are applied to a test membrane. On or applied to a test membrane, the capture reagents are designed to react and bind with predefined analytes that may be present within a liquid sample. The capillary effect draws the liquid sample along the longitudinal axis when it is applied at one end of a test membrane. The capture reagents interact with the analytes present in the test sample, causing measurable and detectable differences along the striped zones of the assay. They are user-friendly, they can be done in a short amount of time and they’re stable over many climates. Strip tests are ideal for home testing, rapid point of care testing, or testing in the field. They also provide reliable testing which might not be otherwise available in developing nations.

A rapid-lateral-flow test is a system consisting of porous materials that are layered and contain the dried components required to perform the test. These membranes can be assembled into small strips that are placed in a plastic case for easy handling. The lateral flow test can be used both qualitatively, and in some cases semi-quantitatively, to detect ligands that are bound to a visually visible capture reagent attached on a solid support. The most common lateral-flow tests on the market today are for pregnancy, Chlamydia and strep. “A quantitative assay isn’t necessary for these conditions.

Figure 1 shows a typical lateral flow assay prior art format. 1. Sample application pad 10 is used to load the sample for testing, which could be a biological sample. On sample pad 10, the separation of plasma and blood cells is performed for whole blood samples or capillary-blood samples. The sample application sheet 10 is usually adhered to rigid or semi-rigid back card 11. The sample pad 10 can be laminated with a mylar backing film, which is used as the backing card. The liquid fraction is then passed through a release pad 12 that has been coated with a dried conjugate. The conjugate is composed of detection molecules directed specifically against the analyte and indicator particles such as gold sol or colloidal gold. The conjugate is redissolved and binds specifically to any analyte in the liquid sample. This forms an analyte/conjugate complex. In some formats, a conjugate is used in liquid form, for example a gold conjugate. The conjugate pad can be omitted. No. 8,399,261).

The analyte conjugate complex flows through capillary membrane 14 such as a membrane made of nitrocellulose (also known as the analytical membrane), where test and control reagents are immobilized. Membrane 14 has two regions or capture lines arranged in a sequential manner and perpendicular to the direction of flow theta. Each reagent is bound to the membrane 14’s capture lines or regions, which are arranged sequentially and perpendicular to the flow direction (? The test line 16 is made up of analyte specific molecules that are capable of binding to the complex and immobilizing it, which results in a colored line. Control line 18 contains no analyte specific molecules, but it is still able to fix conjugate-containing particles that are not bound. Control line 18 forming a colored band indicates that the sample has passed test line 16. The intensity of color at test line 16, which is directly proportional with the concentration of analyte in the sample, allows for semi-quantitative interpretation. Test line 16 and control lines 18 become visible if the analyte is above the detection level. If the analyte level is below the detection threshold, only the control line 18 will be visible.

The last component in the rapid test device, also known as an absorbent pad 20, collects fluids flowing through the system to prevent any backflow. Absorbent pad 20, allows samples that are larger than the wicking capability of the nitrocellulose membrane 14.

The traditional lateral flow immunoassays were designed to detect and quantify a single analyte on each test device. Therefore, multiple analytes can only be detected sequentially in a sample. These tests, while well-validated, can be expensive, time-consuming and sample-depleting when used to measure multiple analytes in a single sample. The principle of strip tests is the same as for bead-based test, but they use beads that are uniquely identifiable. These beads allow simultaneous detection of several analytes within a single reaction or well, but they require expensive equipment for reading the results. They are therefore not suitable for use in the field or at point-of care. Alternative methods use reagents specific to multiple analytes that are placed in specific locations (for example, in the pre-designated 96-well plates) and test samples are added into each well. These methods are also less effective at the point of care or in the field than conventional dipsticks tests.

Other multiplexed flow assays align multiple test strips or lateral-flow assays into a large cassette. The liquid sample is applied to a particular location, then divided into separate channels containing agents that detect a specific analyte. US 2013/0280698, for example, discloses a multiple-strip cartridge that contains multiple lateral flow strips within a single housing. The housing has an inlet that allows a liquid sample to be introduced into the diversion dam. It is then split up between several flow channels. Each flow channel is connected to an assay chamber containing components for detecting a specific analyte. Similarly, U.S. Pat. No. The patent 8,715,590 describes a cross flow analyte array where one or more sample are introduced into at least one input port for the test sample and then distributed to multiple fluid channels in parallel rows positioned perpendicular to or transverse to longitudinal fluid flow. These devices are more complicated and expensive to manufacture than standard dipsticks.

Other descriptions of lateral-flow assay devices can be found, for example, in Sajid M. and al. Journal of Saudi Chemical Society, 2015, v. 19, pp. 689-705, and the references cited in that article;?Design considerations for Lateral Flow Test Strips?” pp. Presentation by Michael A. Mansfield on 24 June. 2015; “Rapid Lateral flow Test Strips: Considerations for Product Design” pp. 1-39, copyright 2002, 2008 by Millipore Corporation, Billerica, Mass., available at the website Also see U.S. Pat. Nos. Nos. 2015/086974. 2014/0093865, 2013,/0017561. 2013/0022969. 2012/0040336, 2012./0015350. PCT Publication Nos. “WO2014/184151” and “WO2011/051562.

There is a need for a multiplexed, lateral-flow assay system that is easy to use, stable and relatively inexpensive.

All the subject matter discussed within the Background section does not necessarily constitute prior art, and it should not be assumed that this is the case merely because the background section discusses the topic. In this vein, any problems that are identified in prior art or related to such subject matter discussed in the Background Section should not be considered prior art unless they are explicitly stated as prior art. The discussion of any topic in the Background section is to be treated as an approach taken by the inventor in solving a particular problem. This in itself can be considered inventive.

The present disclosure discloses a device that performs a multiplex immunoassay, in which a sample of liquid, such as biological material, is tested simultaneously for the presence multiple analytes. The device can also be used to detect multiple analytes simultaneously within a liquid sample.

The devices and methods described herein are capable of detecting analytes indicative of conditions or disorders such as infections, pregnancy, microbial infection, cancer, autoimmune disorders and cardiac disorders. They can also detect drug abuse. The disclosed devices and method can detect infectious diseases such as West Nile virus, Shigella and Campylobacter, as well as malaria, scrub typhus and typhoid. The disclosed device and method can detect a wide range of molecules including, but not limited to: proteins, peptides (including ligands or receptors), carbohydrates, phospholipids, nucleic acid, small molecules and other biologically interesting molecules.

In one embodiment, a device such as the one described herein contains at least two test paths for assays, with each test path containing an antigen or antibody specific to a particular analyte. The capture reagent is then sprayed on a single membrane of analysis, like a nitrocellulose. On the single membrane, detection agents specific for the analytes (such as antigens or antibodies) are also spotted/dried at precise locations along each of the two assay paths. Each assay path contains at least one detection agent labeled for an analyte. Each assay test pathway contains all the components required to detect the presence or lack of a specific analyte. The reporter agent can be any reporter agent that is known to those skilled in the art. For example, colloidal microparticles or latex microspheres. Quantum dots, enzymes and fluorophores are also suitable, as long as the labeled detection agent has a low membrane diffusion constant (typically Deff108 m2/sec.). The assay also ensures that the flow rate of the liquid test (i.e. the effective velocity) through the membrane will be approximately uniform along the lateral axis by ensuring the sample is evenly distributed before entering the membrane. The easiest way to achieve this is to allow the sample to be wetted through a treated glass fiber pad or sample pad. This pad quickly absorbs volume so that the liquid sample enters the membrane uniformly. In one embodiment, a sample moves the same distance from the pad to the detection agent in the first test path, as it does from the pad to detection agent in the second test path. The sample must travel the same distance (i.e. have the same path length) under the same conditions in order to simultaneously reach the detection reagents of the first and second assay test paths. The device disclosed herein can detect analytes simultaneously when the sample travels the same distance, i.e. has the same path length.

The dried labeled detection particles flow uniformly and solubilize upon addition of a test liquid. Due to the low diffusion constants of the labeled detection agents, the lateral diffusivity of the particles is limited. Therefore, due the fluid mechanics in the system, specific lanes or test paths of labeled particle detectors are created so that each test path can indicate the presence or lack of an analyte within the test sample.

One or multiple spots of labeled reagents may be present in a given test path so that the number analytes detected can be multiplied. A very large number analytes may be assessed from a small test sample. The liquid test sample is applied using a single application port. Optionally, a separate buffer port may be used to ensure the proper flow of immunoassay. In order to create physical lanes, no barrier is required, such as a physical or chemical barrier. When a line perpendicularly intersects two adjacent test paths in one embodiment, the line does not pass through a barrier. This could be a wall or chemical barrier like a region that repels water.

Multiple spots of labeled reagents may be present on any assay test path. This can have a multiplicative impact on the number analytes detected in a test sample. The present disclosure, therefore, provides a dense multiplexed test within a small footprint.

As no physical barrier is required to create multiplexed test paths for the assay, the assay may be performed using a “dipstick” format. Format (without a plastic enclosure or housing) is not necessary for the multiplexed test, reducing costs. In some embodiments the capture reagents are not always’spotted’ on the membrane. In some embodiments, the capture reagents on the membrane are not necessarily?spotted? across the membrane, significantly increasing the ease-of-manufacturing. “Despite the absence of diffusion along test paths, an array of analytes is generated when the test sample is added.

In a specific embodiment of the invention, a multiplex flow assay device is provided for simultaneous detection of at least one first analyte and another, different analyte in a liquid test specimen. The device comprises: (a), a test-sample receiving region, (b), a capture membrane having a separate first assay path and adjacent second assay path, wherein the first assay path includes a labeled first detection reagent for the analyte, There is no need for a barrier to separate the multiple test paths. The multiple assay test pathways can also be placed on one piece of capture material, which is a continuous piece. In some embodiments, a control line is positioned below the first test line and the second test line. The control line contains an immobilized control agent that binds with the first and the second labeled detection agents.

The present disclosure includes test strips and test devices with a plurality assay test pathways, but also test strips, devices, and methods of use that have a single test path. The single assay path can contain a unique solid dried labeled reagent. In another embodiment, the single assay path includes more than one unique solid-dried labeled detectors. For example, it may contain two, three, or four reagents. In one embodiment, the present disclosure discloses a lateral-flow assay device that measures an analyte with a solid support, including absorbent materials for capillary flows, comprising: (a) a sample receiving region to receive a sample, (b) a captured region with one or multiple solid dried labeled detection reagents distributed in localized areas, such as spots; and (c) a testing region containing a capture reagent corresponding to the analyt The lateral-flow assay device can have one assay path, two assay paths, three assay paths, four assay paths, more than four test paths. Each test path may independently comprise one or more dried-labeled detector reagents. The device does require the presence of an obstruction, such as a physical or chemical barrier between the assay paths, to maintain the separation, i.e. in a nonoverlapping configuration.

The device may include more than two assay test paths. For example, it could contain three, four or five assay test pathways, each containing labeled detection and capture reagents for the different analytes. This allows the device to be used for detecting the presence of up to six different analytes.

Kits for the simultaneous detection multiple analytes or components in a single liquid are also provided. These kits include a multiplex flow assay device described herein, and optionally a container containing a buffer. They come packaged with instructions on how to use the device and the buffer together to detect the presence of analytes or components in a sample.

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