MULTIPLEX RAPID DIAGNOSTIC KIT FOR SIMULTANEOUS DETECTION OF VECTOR-BORNE DISEASES

Abstract

ABSTRACT MULTIPLEX RAPID DIAGNOSTIC KIT FOR SIMULTANEOUS DETECTION OF VECTOR-BORNE DISEASES The present invention relates to a multiplex and simultaneous antigen detection kit (200) for the rapid diagnosis of Dengue, Chikungunya, and two forms of Malaria (Plasmodium vivax and Plasmodium falciparum) from whole blood samples. Utilizing vertical flow technology, the kit allows the detection of multiple antigens concurrently, offering faster results with enhanced sensitivity and specificity compared to traditional lateral flow assays. The diagnostic device employs metal nanoparticles and gold conjugates to detect the specific antigens, facilitating co-infection detection without the need for serum or plasma separation.

Specification

Description:FORM-2

THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patent Rules, 2003

COMPLETE SPECIFICATION
(See Section 10 and Rule 13)


Title:
MULTIPLEX RAPID DIAGNOSTIC KIT FOR SIMULTANEOUS
DETECTION OF VECTOR-BORNE DISEASES


Applicant Name Nationality Address
AMELIORATE BIOTECH PRIVATE LIMITED INDIAN ATAL INCUBATION CENTRE- JYOTHY INSTITUTE OF TECHNOLOGY FOUNDATION, PIPELINE ROAD, THATHAGUNI BENGALURU, KARNATAKA - 560082, INDIA




THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED.

FIELD OF INVENTION
This invention relates to a multiplex diagnostic kit for the simultaneous detection of mosquito-borne diseases such as dengue, chikungunya, malaria vivax and malaria falciparum.

BACKGROUND OF INVENTION
Malaria, Dengue, and Chikungunya are significant global health concerns, primarily transmitted through mosquito vectors. These diseases are characterized by high fever, severe body pain, and other overlapping symptoms, making it difficult to distinguish them without laboratory testing. Early and accurate diagnosis is critical not only to manage the individual disease but also to identify co-infections, which can complicate treatment.
Dengue, a viral infection transmitted by Aedes mosquitoes, causes flu-like symptoms and can lead to the potentially lethal Dengue Hemorrhagic Fever (DHF). Its global burden has increased 30-fold in the last 50 years, with up to 100 million infections occurring annually across over 100 endemic countries, putting nearly half of the world's population at risk.
Malaria is a parasitic disease transmitted by Anopheles mosquitoes, caused by Plasmodium species, with Plasmodium falciparum and Plasmodium vivax being the most common. In 2017, there were approximately 219 million cases of malaria across 87 countries, resulting in 435,000 deaths, with the WHO African Region bearing over 90% of both cases and fatalities.
However, with mosquito-borne diseases such as Dengue, Chikungunya, and malaria share common symptoms in their early stages, leading to challenges in accurate diagnosis and timely treatment. The similarity of clinical manifestations among these diseases often results in misdiagnosis or delays in appropriate treatment, especially in cases of co-infection. The ability to quickly and accurately differentiate between these diseases, particularly in resource-limited settings, is crucial for effective disease management and reducing mortality rates.
Currently available rapid diagnostic tests (RDTs) focus on antibody detection, which can only diagnose after the body has mounted an immune response, typically a week after initial infection. Furthermore, many of these RDTs fail to detect co-infections and lack the sensitivity required for early diagnosis, leading to false negatives, inappropriate treatment, and an increased economic burden on patients.
For instance, US Patent No. 6,819,408 describes a method and apparatus for analyzing a blood sample or other biological fluid in a resting state without the need for additional dilution reagents or fluid streams passing through the apparatus during the analytical process. The method and apparatus allow enumeration of particulate constituents of biological samples and inspection thereof using an optical scanning instrument. However overall cost and time of analysis is quite high, so difficult to implement in resource intensive set-up.
US Patent application publication No. US20230296600A1 discloses a diagnostic test kit including a test region for indicating a test result. The diagnostic test kit also includes a scan surface including one or more control markings. It is based on a lateral flow immunoassay test.
US Patent application publication No. US20130273528 assay cassettes and testing devices that can be used to provide rapid, accurate, affordable, laboratory-quality testing at the point of care.
US Patent application publication No. US 20130280698 discloses a multi-strip assay cartridge wherein multiple lateral flow assay strips are housed within a single unit. The test sample is introduced through an inlet in the housing into a diversion dam, which then splits the sample into multiple flow channels. Each channel leads to a separate assay chamber, enabling the detection of a distinct analyte. While this setup allows for multi-analyte detection, it requires complex mechanisms to divert and channel the test sample, adding to the complexity and cost of the device. Such devices, though efficient in multiplexing, face challenges in terms of affordability and ease of production, particularly in resource-limited settings.
There are also detection kits commercially available in market for detection of vector borne diseases. Most of these kits employ multiplex assay technique based on lateral flow immunoassay, as disclosed in WO2016022071, by MP Biomedical Asia Pacific. WO2014151865, by Ran Biotechnologies, simultaneous detection of multiple biologicals is described which could be achieved by aggregating particle, labelling particle etc. These tests do not provide rapid detection, as it requires non-handy ingredients. WO2015042593, by Assaypro, describes immunoassay using labelled fluorescent probes that provide multiplexing capability. Furthermore, in WO2016057749, by Theranos, method for detecting presence of virus by performing assay to detect antibodies through viral nucleic acid.
Some of the current laboratory diagnostic techniques used for detecting these infections-such as virus isolation, antigen detection (e.g., ELISA), and nucleic acid detection (e.g., RT-PCR)-while highly specific and sensitive, suffer from several drawbacks:
- Sample pre-processing delays: Many tests require time-consuming steps such as the separation of blood plasma or serum, which can take 1-3 days, delaying the crucial early diagnosis.
- High costs: The commercially available diagnostic kits, though effective, are often unaffordable for patients in lower-income brackets or in rural settings where healthcare infrastructure is limited.
- Limited single-disease detection: Most available kits are designed to detect only one disease at a time, which increases the time and cost associated with diagnosing co-infections, which are common in areas endemic to multiple vector-borne diseases.
- Low accessibility for small clinics / PoC: The specialized equipment, reagents, and skill required to perform these diagnostic tests make them inaccessible for doctors with small practices or in under-resourced areas, further compounding the challenge.
There is, therefore, an unmet need in the art for a rapid, affordable, multiplex diagnostic kit capable of simultaneously detecting Dengue, Chikungunya, Plasmodium vivax, and Plasmodium falciparum infections. Such a kit should provide accurate and fast results without the need for pre-enrichment of blood samples or laboratory infrastructure, making it suitable for both small clinics and point-of-care applications.

SUMMARY OF INVENTION
The present invention discloses a multiplex antigen detection kit for simultaneous detection of Dengue, Chikungunya, and malaria (Plasmodium vivax and Plasmodium falciparum) using whole blood samples. The diagnostic device utilizes metal nanoparticles, specifically gold conjugates, which bind to antigen-specific antibodies to produce a visible reddish-pink dot on a nitrocellulose membrane.
In some aspects according to the present invention the diagnostic test kit is enclosed in a plastic cassette and employs vertical flow technology, allowing for larger sample volume capacity and increased multiplexing capability compared to lateral flow devices.
This device according to present disclosure detects the presence of four specific antigens:
- LDH (Plasmodium vivax),
- HRP (Plasmodium falciparum),
- E2 (Chikungunya), and
- NS1 (Dengue).
In one aspect according to the present invention the kit disclosed herein upon application of the blood sample, if the antigen is present, it binds to the immobilized capture antibody and completes a sandwich formation with the antigen-specific gold conjugate. The turnaround time is 10 to 15 minutes and result is instantaneous. The result is visible in the form of spots which can stay up to 5 minutes.
The kit provides a solution to the limitations of existing diagnostic tools, including delayed diagnosis, inability to detect co-infections, and reduced sensitivity in early infection stages. This invention offers a single device to diagnose three mosquito-borne diseases with high specificity and sensitivity.

BRIEF DESCRIPTION OF FIGURES
The invention will be better understood and objects other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such description makes references to the annexed drawings wherein:
Figure. 1: Depicts a 3D drawing of vertical flow assay device Fig. 1(a)-(e):
- Fig. 1(a) Bottom cassette inner side.
- Fig. 1(b) Bottom cassette outer side.
- Fig. 1(c) Top cassette outer side.
- Fig. 1(d) Side perspective view of cassette.
- Fig. 1(e) Top cassette inner side view.
Figure. 1F: The unused assembled kit.
Figure. 2: Shows parts of the device:
- Fig. 2(a) Front/outer part of top cassette.
- Fig. 2(b) Inner part of top cassette.
- Fig. 2(c) Outer part of bottom cassette.
- Fig. 2(d) Absorption pad.
- Fig. 2(e) Nitrocellulose membrane.
Figure. 3: Shows a step-by-step of the device assembly:
- Fig. 3(a) Inner view of top cassette.
- Fig. 3(b) Nitrocellulose membrane fixed on inner part of top cassette.
- Fig. 3(c) Absorption pad below the nitrocellulose membrane.
- Fig. 3(d) Bottom cassette enabled for press fitting.
- Fig. 3(e) Final device with front / outer view of the top cassette.
Figure. 4: Shows the actual device images before and after testing:
- Fig. 4(a) The device before testing.
- Fig. 4(b) The device after testing with negative blood for all four diseases.
- Fig. 4(c) The device after testing with chikungunya positive blood.
- Fig. 4(d) The device after testing with blood that has been co-infected with malaria Vivax and dengue.
- Fig. 4(e) The device after testing with dengue positive blood.
- Fig. 4(f) The device after testing with malaria vivax positive blood.
- Fig. 4(g) The device after testing with malaria falciparum positive blood.
- Fig. 4(h) The device after testing with blood that has been co-infected with malaria vivax and malaria falciparum.
Figure. 5: Process flow chart for kit preparation.
Figure. 6: Pictorial images of complete kit box components.
- Fig. 6(a) Filter cup with membrane.
- Fig. 6(b) Dropper.
- Fig. 6(c) Filter cup without membrane.
- Fig. 6(d) Aluminium pouch containing device.
- Fig. 6(e) Buffer A bottle.
- Fig. 6(f) Buffer B bottle.
- Fig. 6(g) Gold Conjugate bottle.
Figure. 7: Displays the optical absorption spectrum of the colloidal gold solution.

DETAILED DESCRIPTION OF THE INVENTION
The following detailed description is merely exemplary in nature and is not intended to limit the described embodiments or the application and uses of the described embodiments. This description is not intended to be a detailed catalogue of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the scope of the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations, and variations thereof.
The terms "for example" and "such as," and grammatical equivalences thereof, the phrase "and without limitation" is understood to follow unless explicitly stated otherwise.
As used herein, the term "about" is meant to account for variations due to any experimental errors which may be commonly accepted in the field for a numeric value, for example such a variation can be considered as a ±10% of the said numeric value. All measurements reported herein are understood to be modified by the term "about," whether or not the term is explicitly used, unless explicitly stated otherwise. Further for the purposes of the present invention, ranges may be expressed as from "about" one particular value to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value to the other particular value. The recitation of numerical ranges by endpoints includes all the numeric values subsumed within that range.
As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in the present disclosure; other suitable methods and materials known in the art can also be used. The materials, methods and examples are illustrative only and not intended to be limiting by any means. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict, the present specification, including definitions, will control.
Throughout this specification, unless the context requires otherwise the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.
The term "including" is used to mean "including but not limited to", "including" and "including but not limited to" are used interchangeably.
As used herein the terms "antibody" or "antibodies" include the entire antibody and antibody fragments containing functional portions thereof. The term "antibody" includes any monospecific or bispecific compound comprised of a sufficient portion of the light chain variable region and/or the heavy chain variable region to effect binding to the epitope to which the whole antibody has binding specificity. The fragments can include the variable region of at least one heavy or light chain immunoglobulin polypeptide, and include, but are not limited to, Fab fragments, F(ab') 2 fragments, and Fc fragments.
The term "biological sample" refers to any samples which have been obtained from a human subject and which might contain antibodies or antigen. In a preferred embodiment, said biological sample is chosen from whole blood, serum, plasma, urine, seminal fluid, cerebrospinal fluid and saliva. A biological sample may also be modified prior to use, such as by centrifugation, dilution, and the like. Accordingly, a biological sample may refer to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof.
The term "Chikungunya" refers to a febrile disease that caused by a Togavirus (species Chikungunya virus of the genus Alphavirus) transmitted especially by female Aedes mosquitoes. This is a small, black and white, highly domesticated tropical mosquito that prefers to lay its eggs in artificial containers found in and around homes that may hold water such as buckets, flower vases and other water containers. Following the bite of an infective female mosquito, the virus undergoes an intrinsic incubation period of 3 to 12 days (average 4 to 7 days) after which the person may experience acute onset of fever accompanied by other non-specific signs and symptoms. During this viraemic period (which may be between 2 to 7 days) the virus circulates in the blood of infected humans. The genome of Chikungunya virus is 11.8 kbs and consists two open reading frames (ORF), one contains four non-structural proteins (nsp1-4) and the other contains five structural proteins (capsid, E3, E2, 6he conclK and E1). The virus contains three structural proteins, glycosylated E1 and E2 are embedded in the viral envelope and a non-glycosylated capsid protein E3 associates with E2 during budding and formation of mature virions. Infection with other alphaviruses and CHIKV has revealed that the neutralizing antibody response is primarily directed against E2 and to a lesser extent to E1. In the case of CHIKV, E2, E3 glycoprotein, capsid and nsP3 proteins are targets of the anti-CHIKV antibody response. Both E1 and E2 have been major targets for the development of recombinant subunit vaccine and IgM or IgG based diagnostic assays.
The term "Dengue" refers to an acute infectious disease caused by a flavivirus (species Dengue virus of the genus Flavivirus), transmitted by female Aedes mosquitoes. Dengue virus infection causes a spectrum of illness in humans depending on the infecting virus, the host's age and immunological conditions. It may result in asymptomatic illness or ranges from an undifferentiated flu-like illness (Viral syndrome) to dengue fever (DF), to dengue haemorrhagic fever (DHF), and the severe and fatal dengue shock syndrome (DSS). Dengue virus has four serotypes (DENV1-4), all of which are responsible for the spectrum of disease ranging from benign dengue fever to severe DHF/DSS. The DENV genome contains three structural proteins (capsid, pre-membrane and envelope) and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5). Amongst them, NS1 is a 46-50 kDa glycoprotein expressed in infected mammalian cells in membrane-associated (mNS1) and secreted (sNS1) forms. DENV NS1 is known to be a major target of humoral immunity in DENV infection.
The term 'immunogenic component" refers to any component derived from cells or otherwise that has been infected with the dengue virus or any antigen or antibody against such antigen etc.
The term "Malaria" refers to a human disease that is caused by sporozoan parasites (genus Plasmodium) in the red blood cells, is transmitted by the bite of Anopheles mosquitoes remains a significant global health challenge. Rapid and accurate diagnosis is crucial for effective treatment and control of malaria, and two key biomarkers utilized in diagnostic tests are lactate dehydrogenase (LDH) and histidine-rich protein 2 (HRP-2). Lactate dehydrogenase (LDH) is an enzyme involved in the anaerobic conversion of pyruvate to lactate, LDH is released from infected red blood cells during the life cycle of the Plasmodium parasite so it serves as a useful biomarker for diagnosing malaria. Histidine-rich protein 2 (HRP-2) is a protein produced exclusively by Plasmodium falciparum. HRP-2 is released into the bloodstream as P. falciparum parasites invade red blood cells. Its presence is a clear indicator of infection with this particular species, enabling specific diagnosis of P. falciparum malaria.
The term "Multiplex Technology" refers to a multiplex immunoassay which is a type of assay used in research to simultaneously measure multiple analytes (dozens or more) in a single run/cycle of the assay. It is distinguished from procedures that measure one analyte at a time.
As used herein the terms "device" or "kit" are used interchangeably to refer to the multiplex diagnostic kit for the simultaneous detection of mosquito-borne diseases according to the present invention. The kit according to the present invention is named as "ASSURED", the same has been reflected in the actual images of the kit according to the present invention.
The present invention through its various embodiments not only highlights the advancement of multi-analyte detection but also underscore the increased complexity and higher production costs associated with these technologies. The present invention addresses these challenges by utilizing a vertical flow design for antigen detection, which simplifies the sample processing while providing simultaneous detection of multiple diseases in a single device. This innovation maintains high accuracy and sensitivity without the need for complex fluid diversion mechanisms or multiple flow channels, making it a cost-effective and field-ready alternative for point-of-care diagnostics.
In one embodiment the present invention discloses a diagnostic kit focusing on a unique flow design, multiplexing capabilities, and whole blood usage, for simultaneous detection of multiple mosquito-borne diseases.
In one key embodiment the present invention provides a multiplex diagnostic kit for the simultaneous detection of mosquito-borne diseases. Specifically, the invention pertains to an antigen detection device for the simultaneous identification of Dengue, Chikungunya, and two forms of malaria (Plasmodium vivax and Plasmodium falciparum) using whole blood.
In one aspect of the above embodiment, the present invention utilizes metal nanoparticles and vertical flow technology to enhance sensitivity and specificity, enabling early and accurate diagnosis from day one of symptoms.
In one embodiment according to the present invention the kit disclosed herein is designed to provide a cost-effective, time-efficient solution, particularly useful in resource-limited settings. The unique vertical flow design enables higher multiplexing capabilities while ensuring accurate results within minutes, making it the first of its kind in India to detect Chikungunya antigen. The invention addresses key shortcomings of existing diagnostic methods, offering affordable, reliable, and rapid detection of multiple vector-borne diseases in a single test.
In some embodiments the diagnostic device of the present invention comprises a nitrocellulose membrane encased in a plastic cassette. The membrane is pre-coated with capture antibodies that are specific to the target antigens selected from:
- LDH for Plasmodium vivax,
- HRP for Plasmodium falciparum,
- E2 for Chikungunya, and
- NS1 for Dengue.
In one aspect of the above embodiment the detection method is based on antigen-antibody interactions, forming a sandwich complex that becomes visible as a coloured dot due to the binding of a gold-conjugated antibody.
In yet another aspect of the above embodiment on application of a whole blood sample to the diagnostic device, the sample flows vertically through the membrane. If the specific antigens are present, they bind to the corresponding immobilized capture antibodies. A subsequent application of gold-conjugated antibodies forms a complete sandwich complex with the antigen in the middle, producing a reddish-pink spot visible to the naked eye. The test is completed in less than 15 minutes and provides simultaneous results for all four target antigens.
In some embodiments according to the present invention the device/kit employs vertical flow technology, which increases the capacity for multiplexing while using a single blood sample loading point. This vertical flow design allows for the detection of multiple diseases in one step, unlike traditional lateral flow methods that require separate test strips for each pathogen.
In one aspect of the above embodiment the use of whole blood as the sample eliminates the need for centrifugation or other pre-processing steps, making the device user-friendly and suitable for point-of-care testing in remote or resource-constrained settings.
In yet another embodiment according to the present invention, the advantages of the disclosed multiplex diagnostic kit include its ability to detect co-infections, faster and more accurate diagnosis from day one of symptoms, reduced false positives due to the avoidance of the Hook effect, and the convenience of a single test for multiple diseases. Additionally, it reduces medical waste and lowers costs for both patients and healthcare providers by replacing multiple individual tests with a single device.
The present invention is distinct from the prior known devices, with a focus on the unique aspects of the disclosed multiplex diagnostic kit, such as vertical flow technology, single sample loading, the use of whole blood, specific antigen detection, and higher multiplexing capabilities. Some of the key aspects of the present invention diagnostic kit includes:
- The device utilizes vertical flow technology, which allows for increased sample volume capacity and simultaneous detection of multiple diseases from a single sample loading point. The device contains a vertically arranged membrane with immobilized antibodies specific to four target antigens: LDH for Plasmodium vivax, HRP for Plasmodium falciparum, NS1 for Dengue, and E2 for Chikungunya.
- Unlike the prior existing devices, which focuses on colloidal gold-conjugated antibodies for detecting antigens, the present invention leverages whole blood antigen detection, eliminating the need for serum separation. This feature is crucial for rapid, point-of-care testing in remote settings.
- The device can detect co-infections with a single application of the whole blood sample. The test results are produced in less than 15 minutes, with no cross-reactivity between the antigens for Dengue, Chikungunya, Plasmodium vivax, and Plasmodium falciparum.
- The device is encased in a plastic cassette and includes a sample loading port, vertical flow nitrocellulose membrane, and gold-conjugated detection system. A unique folding mechanism ensures that activation occurs through a single unfolding, preventing accidental activation or contamination.
- Instrument-Free Testing - Unlike the conventional kits, that require specialized instruments, this innovative method allows for antigen detection directly from whole blood without the need for serum separation. This simplification of the testing process enhances its suitability for practical applications.
In yet another embodiment the present invention provides a method for the diagnostic kit preparation for simultaneous detection of Dengue, Chikungunya, and malaria (Plasmodium vivax and Plasmodium falciparum) using whole blood samples, the said method comprises of:
A. Kit Component Preparation:
a) Nitrocellulose membranes are cut and antibody solutions for each pathogen (Dengue, Chikungunya, Plasmodium Vivax, and Plasmodium Falciparum) are prepared at specified concentrations.
b) Conjugation of antibodies with colloidal gold is carried out, ensuring proper particle size and optimal binding.
c) The assay buffer is formulated and packed alongside the cassettes and consumables.
B. Preparation of the Detection Kit:
a) Assemble a triangular diagnostic device with designated spots for the detection of Chikungunya, Plasmodium Vivax, Plasmodium Falciparum, and Dengue.
b) Coat the nitrocellulose membrane with respective antibodies and place them in flow-through cassettes.
c) Prepare the assay buffer solution with adjusted percentages of sodium azide, polyvinylpyrrolidone-30, Tween 20, and sodium dodecyl sulfate.
d) Pack the kit with pre-assembled devices, assay buffer, droppers, and filter cups.
In another embodiment the present invention provides a method for the diagnostic kit preparation for simultaneous detection of Dengue, Chikungunya, and malaria (Plasmodium vivax and Plasmodium falciparum) using whole blood samples, the said method comprises of steps:
a) Prepare the nitrocellulose membrane with a suitable pore size and cutting it into suitable size squares and placing them in flow-through cassettes.
b) Immobilize antibodies for Dengue, Plasmodium Vivax, Plasmodium Falciparum, and Chikungunya onto the nitrocellulose membrane.
The concentrations of coating antibodies are in ranges as follows:
- Dengue - about 1.0 mg/mL to about 3.0 mg/mL,
- Chikungunya - about 2.0 mg/mL to about 4.0 mg/mL,
- Plasmodium Vivax - about 1.0 mg/mL to about 2.0 mg/mL,
- Plasmodium Falciparum - about 1.0 mg/mL to about 2.0 mg/mL,
- Control - about 0.5 mg/mL to about 1.0 mg/mL.
c) Use potassium phosphate buffer (pH 7.4 ± 0.2) mixed with NaCl and sucrose as the coating buffer.
d) Dry the antibody-coated devices under laminar airflow for suitable time, followed by incubation in an incubator at about 37°C to about 40°C for sufficient time.
e) Assemble the devices by layering the absorbent pad on the cassette.
The present invention offers a groundbreaking solution to the major shortcomings seen in current diagnostic technologies by introducing a multiplex antigen detection card capable of simultaneously detecting Dengue, Chikungunya, and two forms of malaria (Plasmodium vivax and Plasmodium falciparum) from a single whole blood sample. This diagnostic kit is designed to offer significant advantages over existing methods in the following ways:
(i) Rapid, Multiplexed Detection:
Unlike most available rapid diagnostic tests (RDTs), which can detect only one disease at a time, the present invention uses vertical flow technology that enables simultaneous detection of multiple antigens-specifically targeting Dengue (NS1 antigen), Chikungunya (E2 antigen), Plasmodium vivax (LDH), and Plasmodium falciparum (HRP). This drastically reduces the time required for diagnosis, providing results in less than 15 minutes from the application of a single whole blood sample.
(ii) Whole Blood Antigen Detection Without Pre-Enrichment:
A key advancement of the present invention is its ability to detect antigens directly from whole blood, eliminating the need for sample pre-processing such as plasma or serum separation. This feature significantly reduces diagnostic delays, enabling early detection on the first day of symptom onset. This direct, rapid method is essential for healthcare providers in time-critical situations.
(iii) Cost-Effective and Field-Ready:
By using gold nanoparticles and colloidal conjugates to detect antigens in whole blood, the present kit provides a more cost-effective solution compared to current lateral flow devices and ELISA-based assays, which require multiple expensive reagents and infrastructure. The simplicity of the vertical flow format, combined with its affordable components, ensures that the test is accessible to both urban and rural healthcare facilities, even in resource-limited settings.
(iv) Detection of Co-Infections:
In endemic regions, co-infection with two or more pathogens is not uncommon. The present invention is the first multiplex kit in India capable of detecting co-infections between Dengue, Chikungunya, and two different types of Malaria in a single test, an essential feature that existing kits lack. This ability to differentiate and diagnose co-infections in real-time offers healthcare providers critical insights for timely and accurate treatment interventions.
(v) High Sensitivity and Specificity:
The vertical flow design and antigen-specific conjugates ensure that the test maintains high sensitivity and specificity for each target antigen. The antigens are immobilized in separate regions on the nitrocellulose membrane, reducing the risk of cross-reactivity and false positives, which are common issues in lateral flow tests.
The present invention provides a highly versatile, rapid, and cost-effective multiplex diagnostic kit that addresses the significant shortcomings of existing technologies. By allowing for simultaneous detection of multiple diseases using whole blood samples and eliminating the need for laboratory infrastructure or expensive reagents, this diagnostic kit fills a critical unmet need in public health, particularly in resource-limited settings where rapid diagnosis is essential for controlling the spread of vector-borne diseases like Dengue, Chikungunya, and Malaria.

DETAILED DESCRIPTION OF FIGURES:
Figure 1 is a 3D Drawing of the Vertical Flow Assay Device (200).
Fig. 1(a) - Bottom Cassette (202) Inner Side: This figure shows the inner surface of the bottom cassette, where various internal components are positioned during assembly. This side has a recessed area specifically designed to house components. It also includes structural features that allow for secure interlocking with the top cassette, ensuring the components remain in place during testing
Fig. 1(b) - Bottom Cassette (202) Outer Side: The outer side of the bottom cassette, depicted here, is designed to offer a stable base for the device.
Fig. 1(c) - Top Cassette (202) Outer Side: This outer surface of the top cassette displays the front-facing part of the device (200), where user interaction occurs, such as sample application and test result reading. This side often includes visible markers or windows to facilitate viewing of the test outcomes.
Fig. 1(d) - Side Perspective View of Cassette (202): A side view showing the alignment and locking mechanism between the top and bottom cassettes, illustrating how the device is securely closed to maintain component stability and prevent contamination during use.
Fig. 1(e) - Top Cassette (202) Inner Side View: This inner side of the top cassette (202) includes slots or guides that align the nitrocellulose membrane, absorption pad and other critical testing components, ensuring precise placement and consistent fluid flow through the device.
Figure 1F - Unused Assembled Kit (200): Depicts the diagnostic kit in its unused, fully assembled form with the immobilized nitrocellulose membrane (201), with absorption pad below it, biological sample loading port (203), visual signal production system (204). The complete device is shown here, ready for testing, with all internal components securely enclosed within the cassette (202) to maintain sterility.
Figure 2: Shows un-assembled parts of the device (200.)
Fig. 2(a) - Front/Outer Part of Top Cassette (202): Shows the external surface of the top cassette's front portion. It includes the spacing for sample application area where users apply the sample to be tested, along with any viewing window that allows users to observe results without disassembling the device. It also features visible codes and their positioning for easy interpretation of the various antigen (C, MV, MF, D) to be tested on the face of it.
Fig. 2(b) - Inner Part of Top Cassette (202): The inner side of the top cassette, which holds the nitrocellulose membrane. This membrane is essential for directing fluid flow and facilitating the interaction between the sample and detection reagents, enabling the multiplex testing capabilities.
Fig. 2(c) - Outer Part of Bottom Cassette (202): The outer surface of the bottom cassette provides a base that stabilizes the device when placed on a flat surface. Its design supports the overall structural integrity of the kit, ensuring a consistent user experience. and includes interactive images and codes for various antigens and controls (C - Chikungunya, D - Dengue, MV - Malaria Vivax, MF -Malaria falciparum, O - Control) for user information and ease of interpretation
Fig. 2(d) - Absorption Pad: A critical component positioned at the end of the fluid flow path, the absorption pad collects and retains excess fluid from the sample, preventing overflow and ensuring accurate, uninterrupted movement of the sample across the nitrocellulose membrane.
Fig. 2(e) - Nitrocellulose Membrane (201): The core of the testing mechanism, this membrane acts as the medium for antigen-antibody reactions. It enables multiplex detection by presenting distinct areas for the binding of specific antibodies or reagents corresponding to each target disease.
Figure 3: Depicts step-by-step assembly of the device (200).
Fig. 3(a) - Inner View of Top Cassette (202): This view shows the placement of the nitrocellulose membrane (201) within the top cassette, ensuring proper alignment and securing the membrane to prevent shifting during handling or testing.
Fig. 3(b) - Nitrocellulose Membrane (201) Fixed on Inner Part of Top Cassette (202): Here, the nitrocellulose membrane is securely affixed to the inner surface of the top cassette (202), establishing the testing surface where the sample will flow and reactions will take place.
Fig. 3(c) - Absorption Pad Below the Nitrocellulose Membrane (201): This assembly step places the absorption pad directly beneath the nitrocellulose membrane, positioned to capture fluid after it passes through the membrane, thereby completing the fluid flow pathway.
Fig. 3(d) - Bottom Cassette (202) enabled for press fitting: This image demonstrates the bottom cassette having press fitting for attaching it to the top cassette. The press fittings of the bottom cassette is enables for sealing the device (200) when pressed against the top cassette, maintaining the sterility of internal components and ensuring a leak-proof assembly.
Fig. 3(e) - Final Device (200) with Front/Outer View of Top Cassette: This completed assembly showcases the external front of the final device, ready for sample application and result interpretation. The structure is now stable, containing all internal components securely within the device.
Figure 4: Actual Device Images Before and After Testing
Fig. 4(a) - Device Before Testing: The unused diagnostic device, showing the initial state prior to sample application. This view allows comparison with subsequent images to assess any color changes or markings that signify test results.
Fig. 4(b) to Fig. 4(h): Device After Testing with Various Results:
4(b): Shows the result after testing with a sample negative for all target diseases, indicated by a lack of markings in the test regions.
4(c): Displays a positive result for chikungunya, identified by specific markers on the nitrocellulose membrane.
4(d): Illustrates a result for co-infection with malaria Vivax and dengue, with distinct markers in the corresponding test zones.
4(e): Shows a positive result for dengue, with a single marker in the respective test zone.
4(f): Displays a positive result for malaria Vivax, as indicated by a distinct marker.
4(g): Shows a positive result for malaria Falciparum, with a specific indicator in the test area.
4(h): Indicates co-infection with malaria Vivax and malaria Falciparum, evidenced by markers in both relevant test zones.
Figure 5: Process Flow Chart for Kit (200) Preparation: This flow chart provides a step-by-step guide for assembling the diagnostic kit, detailing each manufacturing component starting from gold colloid preparation, its conjugation and combo formulation, followed by buffer preparation and bottling, including the assembly process. From component preparation to final assembly and quality control, this chart ensures consistency and reliability in kit production.
Figure 6: Complete Kit Box Components
Fig. 6(a) - Filter Cup with Membrane: Shows the filter cup containing a membrane, designed for initial filtration of the sample to remove large particulates or contaminants.
Fig. 6(b) - Dropper: Used for accurate sample application onto the device, ensuring precise delivery to the testing area without overflow.
Fig. 6(c) - Filter Cup without Membrane: An alternative version of the filter cup, provided for users who may need sample handling flexibility based on the sample's characteristics.
Fig. 6(d) - Aluminum Pouch Containing Device: The pouch provides sterile packaging for the diagnostic device, ensuring it remains uncontaminated before use. It also shows the kit alongside the pouch.
Fig. 6(e) - Buffer A Bottle and Fig. 6(f) - Buffer B Bottle: Two buffers that stabilize and optimize the sample and reaction conditions, essential for accurate test results.
Fig. 6(g) - Gold Conjugate Bottle: Contains the gold conjugate solution used for visible signal development on the nitrocellulose membrane, enabling clear interpretation of test results.
Figure 7: Optical Absorption Spectrum of Colloidal Gold Solution: This figure displays the optical absorption spectrum of the colloidal gold solution used in the diagnostic kit. The spectrum confirms the size and stability of the gold nanoparticles, which are crucial for producing reliable colorimetric signals on the nitrocellulose membrane during testing. In the plot the x-axis represents the wavelength (in nano meters), ranging from about 400 nm to 700 nm and the y-axis typically represents the absorbance or optical density (OD).

EXAMPLES
The following examples include only exemplary embodiments to illustrate the practice of this disclosure. It will be evident to those skilled in the art that the disclosure is not limited to the details of the following illustrative examples and that the present disclosure may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive.

Example 1: Preparation of various components for diagnostic kit:
A. Method for Preparing Colloidal Gold:
a) Boil deionized water and add tri-sodium citrate.
b) Add gold chloride and continue boiling for 6-10 minutes.
c) The graph in Figure.7 displays the optical absorption spectrum of the colloidal gold solution where the x-axis represents the wavelength (in nano meters), ranging from about 400 nm to 700 nm and the y-axis typically represents the absorbance or optical density. Measure the absorbance of the colloid solution, ensuring the maximum absorbance is between 522-525 nm.
B. Colloidal Gold Conjugate preparation:
Materials:
- Colloidal gold, potassium phosphate buffer (10 mM, about pH 8.0).
- Antibodies for Chikungunya, Plasmodium Falciparum, Plasmodium Vivax, and Dengue.
- BSA, sucrose, sodium azide, centrifuge.
Procedure:
a) Antibodies are added dropwise to colloidal gold while stirring.
b) The mixture is stirred for 60 - 80 minutes, followed by the addition of BSA and further stirring for 30 - 40 minutes.
c) The conjugate is centrifuged at 15,000 - 20,000 rpm for 50 - 60 minutes, then resuspended in a buffer containing 10 mM potassium phosphate buffer, BSA, sucrose, and sodium azide.
C. Method for Preparing Combo Conjugate:
a) Prepare a mixture containing Chikungunya, Plasmodium Falciparum, Plasmodium Vivax, dengue each at 20 - 40 % concentration.
Details further given in Figure-5 flow chart.

Example 2: Preparation of the Detection Kit
Materials:
- Antibodies for Dengue, Chikungunya, Plasmodium Vivax, Plasmodium Falciparum
- Nitrocellulose membrane (pore size 0.4 - 0.8 µm, dimensions 17 x 17 mm - 22 x 22 mm)
- Absorption pads (22 x 22 mm - 25 x 25 mm, thickness 1.7 - 2.0 mm)
- Colloidal gold solution
- Coating buffer: potassium phosphate buffer pH 7.4 with NaCl and sucrose
- Centrifuge, incubator, droppers, filter cups, and flow-through cassettes
Procedure:
a) The nitrocellulose membrane is coated with the respective antibodies (Dengue, Chikungunya, Plasmodium Vivax, and Plasmodium Falciparum) at concentrations of 2-4 mg/mL, respectively.
b) Each antibody is coated with 1 - 2 µL of antibody solution onto designated spots on the membrane.
c) The membranes are dried under laminar airflow for few minutes, followed by incubation at 37 - 40 °C for 1 - 2 hours.
d) Colloidal gold is prepared by boiling water, adding tri-sodium citrate, followed by gold colloid, ensuring an absorbance of 520 - 525 nm.
e) The antibodies are conjugated to the colloidal gold by mixing with potassium phosphate buffer, BSA, and sodium azide, and centrifuged to concentrate the conjugate.
f) The kit is assembled with the diagnostic device, assay buffer, and other consumables (Fig. 6).
This methodology prepares a rapid and multiplex diagnostic kit capable of detecting Dengue, Chikungunya, Plasmodium Vivax, and Plasmodium Falciparum simultaneously.

Example 3: Method for Performing the Test:
a) Pre-wet the device with 4 drops (~160 µL) of buffer A.
b) Pour 4 drops of buffer A into the filter cup containing the membrane, add two drops of antigen sample, and incubate for 2-3 minutes.
c) In the filter cup without the membrane, add one drop of conjugate (~40 µL) and 4-5 drops of blood sample, then incubate for 5 minutes.
d) Pour the sample from the filter cup onto the device.
e) Add 6 drops (~240 µL) of buffer B,
f) Add 1 drop of conjugate (~40 µL) and more 6 drops (~240 µL) of buffer B.
g) Interpret the results based on spot formation on the membrane (i.e., Chikungunya at the top, Plasmodium Falciparum on the left, Dengue on the right, and Plasmodium Vivax between Falciparum and Chikungunya).
Note: Alternate approaches may be followed for using the kit for simultaneous detection of malaria borne diseases.

Example 4: Comparison with existing kits
A comparison study of the present kit was done with market available kits to evaluate the efficiency of the present kit. The details of the study are provided in below Table:
Table - 1: Comparative study of kits:
Analysis Criteria Market available kit Present invention kit (Ex-2)
Principal Technology Lateral flow Vertical Flow
Sample Preparation Different type One type
Device Individual device for each disease One device
Testing time 30-40 min 15 mins
Antigen used Dengue - NS1
Chikungunya E2 antigen
Malaria-LDH (Vivax)
HRP (falciparum) Dengue - NS1
Chikungunya E2 antigen
Malaria-LDH (Vivax)
HRP (falciparum)
Co-infection Not detectable Detectable
Equipment requirement Required Not required
Price to patient (INR) 3000-4000 1500
Accessibility No Yes , Claims:WE CLAIM:
1. A multiplex diagnostic device (200) characterized by rapid and simultaneous detection of Dengue, Chikungunya, Plasmodium vivax, and Plasmodium falciparum from biological sample, the device comprising:
a vertical flow nitrocellulose membrane (201),
a plastic cassette (202) configured to encase the membrane and support a single biological sample loading port (203),
a visible signal production system (204) comprising gold-conjugated antibodies for each target antigen, wherein the device is configured to detect co-infections in less than 15 minutes.
2. The device (200) as claimed in Claim 1, wherein said nitrocellulose membrane (201) allows for simultaneous detection of the four target antigens using a single biological sample applied at one loading port, with results visible within 15 minutes.
3. The device (200) as claimed in Claim 1, wherein the biological sample used is whole blood, eliminating the need for serum separation or pre-processing, and wherein antigen detection occurs from day one of symptom onset.
4. The device (200) as claimed in Claim 1, wherein the device has vertical flow configuration (205), which enhances the sample volume capacity and increases multiplexing capability, improving diagnostic accuracy and speed compared to lateral flow devices.
5. The device (200) as claimed in Claim 1, wherein the immobilized antibodies on the nitrocellulose membrane (201) are spaced apart to prevent cross-reactivity, offering high specificity and sensitivity for each target antigen.
6. The device (200) as claimed in Claim 1, wherein no additional instrument is required for the antigen detection process, making the device suitable for resource-limited and remote settings.
7. A method for simultaneous detection of Dengue, Chikungunya, Plasmodium vivax, and Plasmodium falciparum antigens, wherein the method characteristically uses single biological sample, and the method comprises of the steps of:
a) Immobilizing specific antibodies for Dengue, Chikungunya, Plasmodium Vivax, Plasmodium Falciparum, and a control antibody on predefined positions of a nitrocellulose membrane;
b) Applying the biological sample to a pre-wetted diagnostic device containing the immobilized antibodies;
c) Introducing a conjugate solution comprising colloidal gold nanoparticles conjugated with antibodies specific to the aforementioned antigens;
d) Allowing antigen-antibody interaction to occur on the membrane at the immobilized antibody spots;
e) Adding a buffer solution to facilitate flow of the sample across the membrane; and
f) Detecting the presence of Dengue, Chikungunya, Plasmodium Vivax, and Plasmodium Falciparum antigens based on the development of colored spots at the respective immobilized antibody locations on the membrane.
8. The method as claimed in claim 1, wherein the specific antibodies for Dengue, Chikungunya, Plasmodium Vivax, and Plasmodium Falciparum are immobilized by applying a solution of the antibodies in a potassium phosphate buffer with sodium chloride and sucrose, followed by drying under controlled airflow conditions and incubating at 37°C.
9. The method as claimed in claim 1, wherein the colloidal gold conjugate is prepared by mixing colloidal gold nanoparticles with antibodies for Dengue, Chikungunya, Plasmodium Vivax, and Plasmodium Falciparum, and stabilizing with bovine serum albumin (BSA) and sucrose, followed by centrifugation and resuspension in a suspension buffer.
10. The method as claimed in claim 1, wherein the antigens selected for detection are Lactate Dehydrogenase (LDH) for Plasmodium vivax, Histidine Rich Protein (HRP) for Plasmodium falciparum, and specific viral proteins for Dengue and Chikungunya, with each antigen being targeted by its corresponding immobilized antibody on the nitrocellulose membrane.
Dated this 9th day of November, 2024

Biswajit Biswal
[IN/PA-2659]
Agent for the Applicant

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