DESC:FIELD OF INVENTION
[0001] The present disclosure relates generally to the field of in-vitro methodologies and systems incidental to investigative biochemistry. In particular, the present disclosure pertains to a lateral flow device (LFD) for detecting and quantifying glycoproteins and method thereof.

BACKGROUND
[0002] Glycoproteins are a class of biomolecules that consist of both protein and carbohydrate (glycan) components. Examples of these biomolecules include structural glycoproteins (such as Collagens and Laminins), Transport Glycoproteins (such as Transferrin and Hemoglobin), Immunoglobulins (Antibodies), Enzymes (such as Acid / Alkaline Phosphatase), Hormones [such as EPO (Erythropoietin) and FSH (follicle-stimulating hormone)], Cell Adhesion Glycoproteins (such as Integrins and Selectins), Membrane Receptors [such as EGFR (epidermal growth factor receptor) and Insulin Receptor], Mucins, Circulating / glycoproteins in extracellular fluids [such as CRP (C-reactive protein)] and clotting factors [such as vWF (Willebrand factor)], to name a few.
[0003] These molecules play a crucial role in various biological processes, including cell adhesion, signaling, immune response, and molecular transport. Also, glycoproteins are often implicated in diseases such as cancer, neurodegenerative disorders and pregnancy-related adverse conditions such as immunoglobulins, membrane bound receptor proteins are important biomarkers of placental functions.
[0004] As can be readily appreciated therefore, due to the inherent diversity of glycoproteins and their involvement in various physiological processes, the identification and qualitative and / or quantitative determination of glycoproteins in biological samples in order to obtain a comprehensive understanding of their structure and function is an important aspect of both fundamental and applied research. Hence, there is a pressing need in the art to have some effective means of detecting glycoproteins.
[0005] Today, thanks to advancements in technology, there are several methods available in the state-of-art for the detection of glycoproteins, such as Lectin Affinity Chromatography, Western Blotting, Mass Spectrometry, ELLA (enzyme-linked lectin assay), Glycan Staining, Protein Microarray assays, Fluorescence-based Techniques, Immunoassays such as ELISA (enzyme-linked immunosorbent assay) and so on.
[0006] A common encumbrance with the aforementioned state-of-art methodologies is that they all, more or less, are expensive to implement, lacking in affinity / avidity to the specific ligands of interest and are not field-deployable, needing access to specialty high-tech laboratories and highly skilled personnel for undertaking the same. Therefore, a field-deployable, layman-implementable, yet sensitive and specific method is thus sorely needed for the accurate and precise on-the-fly in-vitro detection of glycoproteins in biological samples.
[0007] As recognized widely in the domain of the present invention, accurate resolution of analytes of interest (AoIs) is critical when working with biological samples, as AoIs are often masked by background interference from a consortium of non-target entities, including chemicals, metabolites, macromolecules, cells, virions, organisms, nucleic acid sequences, and other noise incidental to the test samples, making detection of the AoIs challenging. Additionally, the sensitivity to low titers of AoIs is important, as these analytes may be present in very small amounts amidst a large load of background interference in test samples, further complicating accurate detection.
[0008] Naturally, it is required that the assay systems are sensitive enough and have necessary resolution for discerning the AoI(s) to thus have adequate resolution (ability to distinguish between target and non-target entities). Usually, this mandates the implementation of highly sophisticated instruments and specialty detection systems which are not only marred by technical complexities, but also impeded from mass utilization due to inherent high costs and overall inapplicability without the involvement of highly skilled personnel, access to high-tech laboratory setups which makes their utility for on-field analytics an impossible proposition.
[0009] Presence of specific proteins in test samples is required for myriad purposes ranging from laboratory assays to clinical trials, diagnostics, prognostics, detection of chemical contamination, food safety, to production in-line quality monitoring/control/assurance applications in industries including pharmaceuticals, environmental testing, animal health, food and feed testing, and plant and crop health. LFAs, and their implementing LFDs are advocated for on-field rapid detection of AoI(s) in such use-cases, however their applicability for detection of specific AoI(s), proteins in particular, in test samples is by and large unattended till date and / or continue to be riddled by the aforementioned lacunae.
[0010] Therefore, there is a need for an effective solution embracing all considerations mentioned above, fulfilling the various requirements of the field comprehensively.

OBJECTS OF THE PRESENT DISCLOSURE
[0011] Some of the objects of the present disclosure, which at least one embodiment herein satisfies are as listed herein below.
[0012] An object of the present invention is to provide an on-field deployable lateral flow device to detect the presence, and if present the gradation of an analyte of interest in a test sample, specifically glycoproteins.
[0013] An object of the present invention is to provide an on-device, readily readable reference table to facilitate quantification of visual results obtained from the lateral flow device.
[0014] Another object of the present invention is to provide a method for detecting and quantifying glycoproteins from the biological test sample.

SUMMARY
[0015] This summary is provided to introduce a selection of concepts in a simplified form that is further described below in the detailed description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[0016] Aspects of the present disclosure relates to in-vitro methodologies and systems incidental to investigative biochemistry, specifically to the on-field deployable, semi-quantitative lateral flow immunoassay device that enables rapid and reliable detection of glycoproteins with minimal technical expertise. The device is designed for ease of use, robustness, and cost-effectiveness, suitable for both laboratory and remote applications in clinical, environmental, and industrial settings.
[0017] Accordingly, in an aspect, the present disclosure provides a lateral flow device (LFD) for detecting and quantifying glycoproteins from a test sample, including a lateral flow immunoassay assembly configured to detect and quantify the glycoproteins from the test sample. The lateral flow immunoassay assembly includes a sample pad positioned at a first end of the lateral flow immunoassay assembly configured to receive the test sample and provide controlled movement of the test sample through the lateral flow immunoassay assembly; a conjugate pad positioned adjacent and downstream to the sample pad, and configured to bind to glycoproteins in the test sample, wherein the conjugate pad is pre-impregnated with gold-coated magnetic nanoparticles (GcMNPs) conjugated with capture complex specific to glycoproteins; a chromatography membrane positioned adjacent and downstream to the conjugate pad, and configured to facilitate capillary driven flow of the test sample, the chromatography membrane includes one or more test lines impregnated with varying concentrations of detection antibody cocktail specific to glycoproteins and a control line impregnated with a control antibody specific to glycoproteins; and an absorbent pad positioned above the chromatography membrane and at a second end of the lateral flow immunoassay assembly, and configured to capture excess and processed test sample. The lateral flow device (LFD) includes a housing unit having a top portion and a base portion, and configured to encase the lateral flow immunoassay assembly, the housing unit includes a sample reservoir embedded within the top portion of the housing unit and aligned with the sample pad, and configured to allow loading the test sample, and an observation window embedded within the top portion of the housing unit and aligned with the one or more test lines and the control line on the chromatography membrane, and configured to allow visualization of the test result.
[0018] In various embodiments, the chromatography membrane is made of polyester backed nitrocellulose membrane having a pore size ranging from 145 to 155 µm, wherein the chromatography membrane has a length ranging from 40 to 50 mm.
[0019] In certain embodiments, the test sample is a biological fluid selected from a group consisting of blood, saliva, urine, cell lysates, cerebrospinal fluid (CSF), synovial fluid, amniotic fluid, and sputum.
[0020] In certain embodiments, the one or more test lines impregnated with varying concentrations of detection antibodies specific to glycoproteins comprises a first test line located adjacent to the conjugate pad and at a distance of 15 to 20 mm from a start of the chromatography membrane, the first line is configured to detect and quantify the glycoproteins at concentration of =10 ng/mL in the test sample; a second test line located 3 to 5 mm downstream from the first test line, and configured to detect and quantify the glycoproteins at concentration of 30 ng/mL in the test sample; and a third test line located 2 to 4 mm downstream mm from the second test line, and configured to detect and quantify the glycoproteins at concentration of <90 ng/mL in the test sample.
[0021] In certain embodiments, the first test line is impregnated with the detection antibody cocktail including a polyclonal human glycoproteins antibody and a monoclonal human glycoproteins antibody in a concentration of 5 mg/mL.
[0022] In various embodiments, the second test line is impregnated with the detection antibody cocktail including a polyclonal human glycoproteins antibody and a monoclonal human glycoproteins antibody in a concentration of 2.8 mg/mL.
[0023] In various embodiments, the third test line is impregnated with the detection antibody cocktail including a polyclonal human glycoproteins antibody and a monoclonal human glycoproteins antibody in a concentration of 1.4 mg/mL.
[0024] In certain embodiments, the control line is impregnated with control antibody specific to glycoproteins including anti-mouse Immunoglobulin G produced in goat in a concentration of 0.5 mg/mL.
[0025] In various embodiments, the capture complex comprises a combination of biotinylated peptides and glycoproteins specific monoclonal antibody; wherein the glycoproteins are pregnancy specific glycoproteins (PSGs) including endoglin (sEND).
[0026] In another aspect, the present disclosure provides a method for detecting and quantifying glycoproteins from a test sample in an in-vitro environment, comprising steps of:
a) sampling 10 to 30 µL of complex biological fluids to serve as a test sample;
b) loading the test sample into the lateral flow device (LFD) of claim 1 to initiate the lateral flow immunoassay;
c) allowing the test sample to flow through the LFD and visualizing the development of one or more test lines and a control line; and
d) determining the presence or absence of glycoproteins in the test sample, and if present, quantifying the concentration range of glycoproteins by comparing the intensity of the developed test lines with a reference table.
[0027] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

BRIEF DESCRIPTION OF DRAWINGS
[0028] The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.
[0029] FIG. 1 illustrates an exemplary lateral flow device (LFD), in accordance with an embodiment of the present disclosure.
[0030] FIG. 2 illustrates an exemplary lateral flow immunoassay assembly (LFIA), in accordance with an embodiment of the present disclosure.
[0031] FIG. 3 illustrates an exemplary housing unit Aa) outside view of a top portion, Ab) inside view of the top portion, Ba) outside view of a base portion, Bb) inside view of the base portion, and C) assembled housing unit, in accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention.
[0033] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0034] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0035] In some embodiments, numbers have been used for quantifying weight percentages, ratios, and so forth, to describe and claim certain embodiments of the invention and are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[0036] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0037] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0038] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0039] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
[0040] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0041] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.
[0042] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present invention. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
[0043] It should also be appreciated that the present invention can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.
[0044] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0045] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements a, b, and c, and a second embodiment comprises elements b and d, then the inventive subject matter is also considered to include other remaining combinations of a, b, c, or d, even if not explicitly disclosed.
[0046] The terms “lateral flow device” or “lateral flow immunoassay device” or “LFD” or “device” are used herein interchangeably with same meaning throughout the specification.
[0047] The terms “test sample” or “biological test sample” or “biological sample” or “sample” are used herein interchangeably with same meaning throughout the specification.
[0048] The terms “glycoproteins” or “analyte” or “analyte of interest” are used herein interchangeably with same meaning throughout the specification
[0049] Aspects of the present disclosure relates to in-vitro methodologies and systems incidental to investigative biochemistry, specifically to the on-field deployable, semi-quantitative lateral flow device (LFD) that enables rapid and reliable detection of glycoproteins with minimal technical expertise.
[0050] Referring to FIG. 1, the present disclosure provides a lateral flow device (LFD) 100 for detecting and quantifying glycoproteins from a test sample. The LFD hereof is a visual lateral flow immunoassay system provided in a sandwich format in a compact and portable housing. The device is designed for ease of use, robustness, and cost-effectiveness, suitable for both laboratory and remote applications in clinical, environmental, and industrial settings.
[0051] Referring to FIG. 2, the LFD 100 includes a lateral flow immunoassay assembly (LFIA) 102 configured to detect and quantify the glycoproteins from the test sample. The LFIA 102 includes a sample pad 104 positioned at a first end of the lateral flow immunoassay assembly 102 configured to receive the test sample and provide controlled movement of the test sample through the lateral flow immunoassay assembly 102. In one embodiment, the sample pad 104 is made of glass fiber and polysulfone for allowing loading of complex biological sample, to be separated based on molecular weight, viscosity and then assayed for presence of an analyte of interest (AOI).
[0052] The LFIA 102 also includes a conjugate pad 106 positioned adjacent and downstream to the sample pad 104, and configured to bind to glycoproteins in the test sample. The conjugate pad 106 is pre-impregnated with gold-coated magnetic nanoparticles (GcMNPs) conjugated with capture complex specific to glycoproteins and stabilizers such as sorbitol, hydrolysed gelatin, trehalose to improve shelf life, bioavailability of the conjugate matrix. In one embodiment, the conjugation pad is made of polyester. The conjugate pad 106 is pre-impregnated via passive adsorption technique. The gold-coated magnetic nanoparticles provides stability to the LFD 100 and makes the reaction more convenient by increasing the flow rate.
[0053] The LFIA 102 also includes a chromatography membrane 108 positioned adjacent and downstream to the conjugate pad 106, and configured to facilitate capillary driven flow of the test sample. The chromatography membrane 108 includes one or more test lines 112, 114, and 116 impregnated with varying concentrations of detection antibody cocktail specific to glycoproteins and a control line 118 impregnated with a control antibody specific to glycoproteins.
[0054] The LFIA 102 includes an absorbent pad 110 positioned above the chromatography membrane 108 and at a second end of the lateral flow immunoassay assembly 102, and configured to capture excess and processed test sample. The absorbent pad 110 is made of cellulose fiber for absorbing / resting the amount of test sample which flows out of the chromatography membrane 108 before the LFD 100 is discarded.
[0055] Referring to FIG. 3, the lateral flow device (LFD) 100 further includes a housing unit 300 having a top portion 302 (shown in figure 3Aa and 3Ab) and a base portion 304 (shown in figure 3Ba and 3Bb), and configured to encase the lateral flow immunoassay assembly 102. The housing unit 300 includes a sample reservoir 306 embedded within the top portion 302 of the housing unit 300 and aligned with the sample pad 104, and configured to allow loading the test sample. The housing unit 300 also includes an observation window 308 embedded within the top portion 302 of the housing unit 300 and aligned with the one or more test lines 112, 114, and 116 and the control line 118 on the chromatography membrane 108, and configured to allow visualization of the test result. The observation window 308 includes markings for corresponding one or more test lines 112, 114, and 116.
[0056] In one embodiment, the housing unit 300 is made of a polymer selected from a group consisting of plastic, polyvinyl chloride and a combination thereof. The housing unit 300 is hollow to enclose the LFIA 102 and has the dimensions including a length of 7 cm, breadth of 2 cm and a height of 0.5 cm. The LFIA 102 is assembled on the base portion 304 of the housing unit 300 and the base portion 304 is closed with the top portion 302 of the housing unit 300 (shown in figure 3C).
[0057] In an embodiment, the sample pad 104 is placed directly below the sample reservoir 306 of the housing unit 300 and the chromatography membrane 108 runs under the observation window 308 of the housing unit 300. The conjugation pad 104 and the absorbent pad 110 are hidden from external view once the housing unit 300 is closed.
[0058] In an exemplary embodiment, the LFD 100 allows the test sample, once introduced through the sample reservoir 306 using a dropper or similar device, to be absorbed by the sample pad 104. Driven by capillary action, the sample flows from the sample pad 104, through the conjugation pad, across the chromatography membrane 108, and ultimately reaches and is absorbed by the absorbent pad 110.
[0059] In various embodiments, the chromatography membrane 108 is made of polyester backed nitrocellulose membrane having a pore size ranging from 145 to 155 µm, wherein the chromatography membrane 108 has a thickness ranging from 100 to 120 µm. This specific thickness of the chromatography membrane 108 improves flow rate of the test sample on the chromatography membrane 108, and helps in increasing the reaction time between impregnated antibodies and analyte of interest (glycoproteins), providing higher binding capacity of antibodies to chromatography membrane 108.
[0060] In an exemplary embodiment, the chromatography membrane 108 has the pore size of 150 µm. The chromatography membrane 108 has the thickness of 26 mm.
[0061] In certain embodiments, the test sample (306) is a biological fluid selected from a group consisting of blood, saliva, urine, cell lysates, cerebrospinal fluid (CSF), synovial fluid, amniotic fluid, and sputum.
[0062] In certain embodiments, the one or more test lines 112, 114, and 116 impregnated with varying concentrations of detection antibodies specific to glycoproteins includes a first test line located adjacent to the conjugate pad 106 and at a distance of 15 to 20 mm from a start of the chromatography membrane 108. The first line is configured to detect and quantify the glycoproteins at concentration of =10 ng/mL in the test sample. The one or more test lines 112, 114, and 116 includes a second test line located 3 to 5 mm downstream from the first test line, and configured to detect and quantify the glycoproteins at concentration of 30 ng/mL in the test sample. The one or more test lines 112, 114, and 116 includes a third test line located 2 to 4 mm downstream from the second test line, and configured to detect and quantify the glycoproteins at concentration of <90 ng/mL in the test sample.
[0063] In one embodiment, the one or more test lines 112, 114, and 116 and the control line 118 are printed using an Easy printer LPM -02. The first test line is printed at a distance of 19 mm from the start of the chromatography membrane 108. The second test line printed 4 mm downstream from the first test line. The third test line is printed 3 mm downstream from the second test line. The distance between the third test line and the control line 118 is 8 mm.
[0064] In certain embodiments, the first test line 112 is impregnated with the detection antibody cocktail including a polyclonal human glycoproteins antibody and a monoclonal human glycoproteins antibody in a concentration of 5 mg/mL.
[0065] In various embodiments, the second test line 114 is impregnated with the detection antibody cocktail including a polyclonal human glycoproteins antibody and a monoclonal human glycoproteins antibody in a concentration of 2.8 mg/mL.
[0066] In various embodiments, the third test line 116 is impregnated with the detection antibody cocktail including a polyclonal human glycoproteins antibody and a monoclonal human glycoproteins antibody in a concentration of 1.4 mg/mL.
[0067] The different concentrations of the test lines 112, 114, and 116 and change of flow rate increase the sensitivity of the assay by increasing interaction time, and maximize the antigen-antibody reaction. The varying concentrations of the detection antibody in the test lines 112, 114, and 116 create a concentration gradient on the membrane. This gradient is crucial as it enables the assay to detect a broad range of analyte concentrations with high sensitivity. Specifically, the higher concentration (5 mg/mL) provides a strong signal for higher levels of circulating analyte. The graduated approach improves the assay’s dynamic range and enhances assay’s ability to detect and quantify the analyte at various levels. The control line 118 is printed with a higher concentration of control antibody (0.5 mg/mL) to serve as a reference for assay performance and ensure the correct functioning of the test. Markings on the observation window 308 align with the test and control lines to facilitate accurate result interpretation. The differential signal intensities across the test lines 112, 114, and 116, combined with the control line’s 118 reference, contribute to the assay’s enhanced sensitivity and specificity and more precise categorization of samples into negligible, low, mild and high risk and also reduce LFD 100 test kit’s cost. In one embodiment, the distance between test lines 112, 114, and 116 is dissimilar, to thus help to indicate the manner in which results of the lateral flow assay (LFA) are to be visually read / interpreted by the user.
[0068] In one embodiment, the polyclonal human glycoproteins antibody and the monoclonal human glycoproteins antibody are in a ratio of 1:3. In an exemplary embodiment, the one or more test lines 112, 114, and 116 and the control line 118 are otherwise invisible to the naked eye, and can be seen only when the LFD 100 is used successfully, with a positive determination of glycoprotein being present in the test sample. However, the corresponding markings on the observation window 308 are always visible, and serve to indicate the positions on the nitrocellulose membrane, where the lateral flow immunoassay outcome, i.e. lines, should develop / appear when the LFD 100 is used successfully, with a positive determination of glycoprotein being present in the test sample.
[0069] In certain embodiments, the control line 118 is impregnated with control antibody specific to glycoproteins including anti-mouse Immunoglobulin G produced in goat in a concentration of 0.5 mg/mL.
[0070] In various embodiments, the capture complex includes a combination of biotinylated peptides and glycoproteins specific monoclonal antibody. The glycoproteins are pregnancy specific glycoproteins (PSGs) including endoglin (sEND).
[0071] In one embodiment, the lateral flow device (LFD) 100 incorporates a reference table (table 3) integrated into the device’s design to facilitate semi-quantitative analysis of glycoproteins directly at the point of care. The table 3, either embedded on the device’s 100 surface or provided as a detachable card, contains a series of color or intensity reference standards corresponding to different concentrations of glycoproteins. After loading the test sample, the user observes the development of test lines 112, 114, and 116 and compares their intensities with the calibrated standards on the reference table 3. This comparison enables the user to determine the concentration range of glycoproteins in the sample without relying on external laboratory equipment or sophisticated interpretation methods. The reference table 3 allows users, including laypersons, to perform tests in varied environments and obtain actionable results on-site. This feature enhances the device’s 100 usability in low-resource settings and supports applications in clinical diagnostics, field testing, and research.
[0072] In another aspect, the present disclosure provides a method for detecting and quantifying glycoproteins from a test sample in an in-vitro environment, comprising steps of:
a) sampling 10 to 30 µL of complex biological fluids to serve as a test sample;
b) loading the test sample into the lateral flow device (LFD) 100 of claim 1 to initiate the lateral flow immunoassay;
c) allowing the test sample to flow through the LFD 100 and visualizing the development of one or more test lines 112, 114, and 116 and a control line 118; and
d) determining the presence or absence of glycoproteins in the test sample, and if present, quantifying the concentration range of glycoproteins by comparing the intensity of the developed test lines with a reference table.
[0073] In one embodiment, the device 100 and method disclosed in present disclosure are implementable at temperatures below 40oC. The device 100 and method provides the results within 20 minutes.
[0074] The device 100 has applications across multiple fields, including drug development, medical diagnostics, biopharmaceutical quality control, and fundamental research. The LFD 100 is designed to provide rapid, sensitive, and specific analysis, facilitating insights in both clinical and laboratory settings.
[0075] For drug development, the device 100 can be employed to identify and characterize glycoprotein targets that are critical for therapeutic intervention, allowing researchers to screen for specific glycoproteins and their concentrations within test samples. This application is particularly valuable for determining the role of glycoproteins in disease pathways and assessing therapeutic potentials. In medical diagnostics, the LFD 100 supports the detection of disease-associated glycoproteins and biomarkers, making it a useful tool for diagnosing conditions linked to specific glycoprotein profiles. The device’s 100 ease of use and rapid results facilitate diagnostic workflows and patient management.
[0076] The disclosure is also highly relevant in biopharmaceutical quality control, where it can verify proper glycosylation patterns in therapeutic proteins. Ensuring accurate glycosylation is essential for the safety and efficacy of protein-based therapies, and the device 100 provides a convenient, point-of-care testing solution for monitoring glycoprotein integrity in manufacturing processes. Also, in fundamental research, the LFD 100 offers researchers a valuable tool for studying glycoprotein functions and their roles in various cellular processes, advancing knowledge of glycoprotein-mediated mechanisms in health and disease.
[0077] While the foregoing description discloses various embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope of the disclosure. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
EXAMPLES
[0078] The present invention is further explained in the form of the following examples. However, it is to be understood that the foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.

Example 1: Preparation of reagents
[0079] Synthesis of gold nanoparticles (GNPs): GNPs were synthesized by standard citrate reduction method (referring to Frens G 1973, Nat Phys Sci 241(105):20–22) and streptavidin was conjugated to gold nanoparticles (referring to Hermanson GT 2008, Bioconjugate techniques. Pierce Biotechnology, Rockford). The GNPs solution is aged for 24 hours at room temperature to complete the synthesis followed which parameters like pH, and absorption spectrum is recorded. Magnetic nanoparticles (MNPs) were coated with Gold (GNPs) in 1:20 ratio and used further for conjugation process. The pH of the gold coated magnetic nanoparticles (GcMNPs) is adjusted to 5.35 – 6.50 by using 0.2M Potassium carbonate (K2CO3).
[0080] Characterization of the GcMNPs: The GcMNPs so formed were characterized via dynamic light scattering and charge analysis, in which the MNPs were observed to have an average size of 25 ± 5.00 nm and charge of 45 ± 3.00 mV while the GcMNPs were observed to have an average size of 85 ± 5.00 nm and charge of -35 ± 3.00 mV.
[0081] Surface modification: Bio conjugates of GcMNPs-streptavidin (Streptavidin at 10mg/mL and biotinylated peptides cocktails (Pep1: NWTTLSRSVNWP, Pep2: NNLPTSRTLAGN and Pep3: GNNPLHVWHHDKR in the ratio of 10:5:2), with monoclonal antibody are prepared. Briefly, the amount of biotinylated peptides or antibody required for saturation of the surface of the GcMNPs-Streptavidin conjugate was determined using the titrimetric method. The conjugation of biotinylated peptide or antibody and streptavidin-GcMNPs was performed by biotinylated using Streptavidin-Biotin conjugation kit. According to kit instructions, 1 µL of modifier reagent was mixed gently with 100 µL of antibody + peptide cocktail and 1 mL of GcMNP. The conjugate complex formation was confirmed by gel retardation assay.
[0082] Lateral flow assay (LFA), intended for implementation using the LFD 100 described in the present invention, is arranged for detection and quantification of the glycoproteins, if present, of the AoI (s) in test samples by involving the following selection of commercially-available antibodies-
i) Capture antibody – Combination of biotinylated peptides and PSG specific monoclonal antibody
ii) Detection antibody – Antibody cocktail (1 polyclonal and 1 monoclonal antibody) Human pregnancy specific glycoprotein (PSG) specific Antibody
iii) Control antibody - Anti-Mouse IgG (whole molecule) antibody produced in goat.

Example 2: Implementation of the LFD (lateral flow device)
[0083] Sample application: The sample is applied to the sample pad 104. The sample pad 104 often contains substances that help absorb and filter the sample. For example, in case complex samples such as whole blood, retaining blood specific glycoproteins onto sample pad 104 allows serum to flow rapidly to achieve better sensitivity making sure it’s properly prepared for testing. The sample is introduced to the sample pad 104, which is engineered with a unique combination of advanced materials to enhance sample preparation. The sample pad 104 incorporates proprietary absorbent and filtering agents that are specifically tailored to optimize sample handling. These agents are designed to selectively bind and filter out contaminants, such as proteins and lipids, that could otherwise interfere with test accuracy. Additionally, the sample pad 104 features a gradient filtration system that ensures the even distribution of the sample across the testing area. This approach not only improves the precision of the assay but also reduces background noise, thereby enhancing the overall sensitivity and reliability of the test results.
[0084] Sample migration: After application, the sample moves through the LFD 100 via controlling capillary flow rate. The sample first passes through the conjugate pad 106, where the sample encounters labeled antibodies or other reagents that are specific to the target analyte (the substance being tested for). These reagents are usually attached to colored particles, nanoparticles or enzymes. The incorporation of salt, sugar, polymer, etc., on the chase buffer to modify flow rates and enhancing the signal amplification.
[0085] Conjugate binding: If the target analyte is present in the sample, it binds to the labeled antibodies in the conjugate pad 106. This complex (analyte-antibody complex) then moves further along with the sample as it continues to migrate.
[0086] Test line formation: As the sample moves through the nitrocellulose membrane, the analyte-antibody complex binds to specific capture antibodies that are immobilized at the test line region. This binding results in the accumulation of the colored particles, nanoparticles or enzymes at the test line, making a visible colored line if the target analyte is present.
[0087] Control line formation: Simultaneously, the sample continues to migrate to the control line 118 region. Here, another set of antibodies or reagents captures any excess labeled particles or enzymes, forming a second visible line. This control line 118 confirms that the test has been performed correctly and that the reagents are functioning properly.
[0088] Result interpretation: After a set amount of time, the presence or absence of lines at the test and control regions is assessed-
i) Positive Result: A colored line appears at both the test line 112, 114 and 116 and control line 118. The test line 112, 114 and 116 indicates that the target analyte is present.
ii) Negative Result: A colored line appears only at the control line 118, with no line at the test line 112, 114 and 116. This indicates that the target analyte is not present.
iii) Invalid Result: No line appears at the control line 118, indicating that the test did not function correctly, regardless of the presence or absence of the test line 112, 114 and 116.
Table 1 enlists the working parameters of the lateral flow device 100 discloses in the present disclosure.
Sample type: Biological sample / Biological specimen
MNP used: Gold coated
Capture antibody: Cocktail of biotinylated Peptide or human specific PSGs antibody
Sample volume: 10-30µL
Time for visualization of results: 20 minutes
Sensitivity: 90%
Specificity: 94.44%

Example 3: Experimental validation of the LFD (lateral flow device)
[0089] The LFD 100 has been reduced to practice and experimentally validated. Table 2 shows specificity of test device 100 as determined by implementing the LFA using specific glycoprotein for e.g. PSG- sEND (Pregnancy Specific Glycoproteins-soluble endoglin) with their closely resemble glycoproteins.
Sl. No. Glycoproteins Concentration (ng/mL) T1 T2 T3 C Immune response or Indication
Reading 1 VEGF 20-100 N N N Y Tumor cells, Platelets,
Macrophage, and renal mesangial cells.
Reading 2 PIGF 20-100 N N N Y Placental dysfunction and IUGR
Reading 3 IGFBP-1 20-100 N N N Y Colorectal cancer, Pregnancy related disorders

Reading 4 Fibronectin 20-100 N N N Y Wound healing, fibrosis, and Tumor progression
Reading 5 Serum Albumin 20-100 N N N Y Hypoalbuminemia, Cardiovascular disease
Reading 6 CD105 >20 Y Y N Y Neoplasm, Placental Dysfunction, Preeclampsia, Eclampsia, Arteriovenous malformations (AVMs), and Telangiectases.
Reading 7 sENG <100 Y Y Y Y
Reading 8 Saline control validation N N N Y Test device performing correctly
where, T1, T2 and T3 are test line 1, test line 2, and test line 3. C is control line. VEGF is vascular endothelial growth factor; PIGF is placenta growth factor; IGFBP-1 is insulin-like growth factor-binding protein; CD105 is endoglin; and sENG is soluble endoglin.Table 3 shows detection of specific glycoprotein for early risk assessment of developing utero-placental insufficiency or vascularized tumor development in patients. Table 3 corresponds to the reference table provided with the LFD.
Sample Type T1 T2 T3 Control Concentration of specific glycoprotein determined by ELISA ng/mL Risk assessment Clinical diagnosis
1) Blood
Reading 1 Y N N Y 10 Low Low risk confirmed by clinician
Reading 2 Y Y N Y 30 Mild Mild risk confirmed by clinician
Reading 3 Y Y Y Y 90 High high risk confirmed by clinician
2) Plasma
Reading 1 Y Y N Y 18 Mild Mild risk confirmed by clinician
Reading 2 Y N N Y 5 Low Low risk confirmed by clinician
Reading 3 Y Y Y Y 60 High high risk confirmed by clinician
3) Serum
Reading 1 N N N Y 3 Very Low No risk
Reading 2 Y Y Y Y 40 Mild Mild risk confirmed by clinician
Reading 3 Y Y Y Y 70 High high risk confirmed by clinician
4) Saline control
Saline N N N Y - No risk -
5) Vaginal swab
VS N N N Y - No risK -
where, T1, T2 and T3 are test line 1, test line 2, and test line 3. C is control line.
[0090] In conclusion, the lateral flow device (LFD) 100 and associated method for detecting and quantifying glycoproteins from complex biological samples is designed to operate effectively in low-resource, point-of-care settings. The device 100 allows for rapid, accurate assessments within approximately 20 minutes, helping to preserve biomarker or analyte concentrations and enabling timely decision-making. The device 100 is crafted for non-dependency on costly analytical instruments or specialized laboratory setups, making it accessible and easy to use for personnel with minimal training.
[0091] The LFD 100 requires only a low volume of the test sample between 10 to 30 µL, making it highly suitable for testing limited sample amounts. The LFD 100 operates efficiently at ambient room temperature, ranging from 15°C to 37°C, under standard atmospheric pressure, ensuring stable performance without the need for specialized environmental controls. The device 100 utilizes visual test lines 112, 114, and 116 that can be readily interpreted by comparing with a reference table 3 embedded on the device 100, eliminating the necessity for complex ancillary equipment or electronic readers. This setup enables high-sensitivity and specificity measurements of glycoproteins, even within complex biological matrices, providing reliable diagnostic capabilities that are cost-effective and scalable for industrial manufacturing.

ADVANTAGES OF THE PRESENT DISCLOSURE
[0092] Unlike traditional analytical methods requiring expensive equipment and complex laboratory setups, the LFD is affordable and straightforward, making it accessible in low-resource settings and feasible for widespread point-of-care use.
[0093] The LFD is engineered to detect glycoproteins with high sensitivity, even in samples with low glycoprotein concentrations or high background interference.
[0094] The LFD provides quick assessments, reducing the risk of biomarker degradation and enabling faster decision-making in clinical and research applications.
[0095] The LFD requires only a small volume of the test sample (10–30 µL), preserving precious biological samples and making it ideal for cases where sample volume is limited, such as pediatric or neonatal testing.
[0096] The LFD can function effectively at ambient temperatures (15°C to 37°C) and standard atmospheric pressure, eliminating the need for controlled environmental conditions.

,CLAIMS:1. A lateral flow device (LFD) (100) for detecting and quantifying glycoproteins from a test sample, comprising:
a lateral flow immunoassay assembly (LFIA) (102) configured to detect and quantify the glycoproteins from the test sample, the lateral flow immunoassay assembly includes:
a sample pad (104) positioned at a first end of the lateral flow immunoassay assembly (102) configured to receive the test sample and provide controlled movement of the test sample through the lateral flow immunoassay assembly (102),
a conjugate pad (106) positioned adjacent and downstream to the sample pad (104), and configured to bind to glycoproteins in the test sample,
wherein the conjugate pad (106) is pre-impregnated with gold-coated magnetic nanoparticles (GcMNPs) conjugated with capture complex specific to glycoproteins,
a chromatography membrane (108) positioned adjacent and downstream to the conjugate pad (106), and configured to facilitate capillary driven flow of the test sample, the chromatography membrane (108) includes one or more test lines (112, 114, and 116) impregnated with varying concentrations of detection antibody cocktail specific to glycoproteins and a control line (118) impregnated with a control antibody specific to glycoproteins,
an absorbent pad (110) positioned above the chromatography membrane (108) and at a second end of the lateral flow immunoassay assembly(102), and configured to capture excess and processed test sample; and
a housing unit (300) having a top portion (302) and a base portion (304), and configured to encase the lateral flow immunoassay assembly (102), the housing unit (300) includes:
a sample reservoir (306) embedded within the top portion (302) of the housing unit (300) and aligned with the sample pad (104), and configured to allow loading the test sample,
an observation window (308) embedded within the top portion (302) of the housing unit (300) and aligned with the one or more test lines (112, 114, and 116) and the control line (118) on the chromatography membrane (108), and configured to allow visualization of the test result.

2. The LFD (100) as claimed in claim 1, wherein the chromatography membrane (108) is made of polyester backed nitrocellulose membrane having a pore size ranging from 145 to 155 µm, wherein the chromatography membrane (108) has a length ranging from 40 to 50 mm.

3. The LFD (100) as claimed in claim 1, wherein the test sample is a biological fluid selected from a group consisting of blood, saliva, urine, cell lysates, cerebrospinal fluid (CSF), synovial fluid, amniotic fluid, and sputum.

4. The LFD (100) as claimed in claim 1, wherein the one or more test lines (112, 114, and 116) impregnated with varying concentrations of detection antibodies specific to glycoproteins comprises:
a first test line (112) located adjacent to the conjugate pad (106) and at a distance of 15 to 20 mm from a start of the chromatography membrane (108), the first line (112) is configured to detect and quantify the glycoproteins at concentration of =10 ng/mL in the test sample;
a second test line (114) located 3 to 5 mm downstream from the first test line (112), and configured to detect and quantify the glycoproteins at concentration of 30 ng/mL in the test sample; and
a third test line (116) located 2 to 4 mm downstream mm from the second test line (114), and configured to detect and quantify the glycoproteins at concentration of <90 ng/mL in the test sample.
5. The LFD (100) as claimed in claim 4, wherein the first test line (112) is impregnated with the detection antibody cocktail including a polyclonal human glycoproteins antibody and a monoclonal human glycoproteins antibody in a concentration of 5 mg/mL.

6. The LFD (100) as claimed in claim 4, wherein the second test line (114) is impregnated with the detection antibody cocktail including a polyclonal human glycoproteins antibody and a monoclonal human glycoproteins antibody in a concentration of 2.8 mg/mL.

7. The LFD (100) as claimed in claim 4, wherein the third test line (116) is impregnated with the detection antibody cocktail including a polyclonal human glycoproteins antibody and a monoclonal human glycoproteins antibody in a concentration of 1.4 mg/mL.

8. The LFD (100) as claimed in claim 1, wherein the control line (118) is impregnated with control antibody specific to glycoproteins including anti-mouse Immunoglobulin G produced in goat in a concentration of 0.5 mg/mL.

9. The LFD (100) as claimed in claim 1, wherein the capture complex comprises a combination of biotinylated peptides and glycoproteins specific monoclonal antibody; wherein the glycoproteins are pregnancy specific glycoproteins (PSGs) including endoglin (sEND).

10. A method for detecting and quantifying glycoproteins from a test sample in an in-vitro environment, comprising steps of:
a) sampling 10 to 30 µL of complex biological fluids to serve as a test sample;
b) loading the test sample into the lateral flow device (LFD) (100) of claim 1 to initiate the lateral flow immunoassay;
c) allowing the test sample to flow through the LFD (100) and visualizing the development of one or more test lines (112, 114, and 116) and a control line (118); and
d) determining the presence or absence of glycoproteins in the test sample, and if present, quantifying the concentration range of glycoproteins by comparing the intensity of the developed test lines with a reference table.