DIAGNOSTIC KIT AND METHODS THEREOF

Abstract

TITLE OF INVENTION: DIAGNOSTIC KIT AND METHODS THEREOF The present disclosure discloses a kit including at least one strip (100), and an assay buffer having a pH between 8 and 9. A top layer (103) of the strip (100) includes at least one sample portion (103a), at least one separation portion (103b), at least one conjugate portion (103c) provided with a pre-defined amount of a plurality of test antibodies ‘x’ conjugated to at least one reporter, and at least one detection portion (103d) including a test region ‘T’ having a plurality of the test antibodies ‘y’ immobilized thereon. The assay buffer includes 5mM to 20mM of a buffer, 0.5%(w/v) to 1.25%(w/v) of a metallic salt, a pre-defined amount of a surfactant, and 0.1%(w/v) to 0.3%(w/v) of a preservative agent. The sample portion (103a) of the strip (100) is configured to receive a sample mixed with the assay buffer. A limit of detection of the strip (100) is 1μg/mL or more for the sample mixed with the assay buffer. Fig. 3

Specification

Description:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(Section 10 and Rule 13)

1. TITLE OF THE INVENTION:
DIAGNOSTIC KIT AND METHODS THEREOF
2. APPLICANT:
Meril Corporation (I) Private Limited, an Indian company of the address Survey No. 135/139, Muktanand Marg, Bilakhia House, Pardi, Vapi, Valsad-396191 Gujarat, India.




The following specification particularly describes the invention and the manner in which it is to be performed:

 
FIELD OF INVENTION
[001] The present disclosure relates to a diagnostic kit. More particularly, the present disclosure relates to an alpha defensin test kit and methods thereof.
BACKGROUND OF INVENTION
[002] Periprosthetic joint infection (PJI) is a serious complication that often times occur after a joint replacement surgery (or total joint arthroplasty), such as hip replacement, knee replacement, etc. PJI is defined as an infection of the tissue(s) surrounding the prosthetic joint. The infection may origin during the surgery itself, or it may develop later on from another infection in the body.
[003] Diagnosing PJI is challenging because conventional diagnostic tools and methods take a lot of time and may not always be accurate, especially for low-grade infections or those involving slow-growing microorganisms. Diagnosing PJI can be further tricky because the symptoms of a few of the infections are very hard to detect. For better treatment outcome, it is imperative that the infection (i.e., PJI) be detected in its early stages so that a medical practitioner may determine an effective course of treatment. Delayed diagnosis may lead to inadequate interventions leading to sepsis and death.
[004] Alpha-defensins (also known as α-defensins proteins) are antimicrobial small peptides that are part of the innate immune response. They are produced by activated neutrophils (one of the white blood cells), in response to an infection caused by gram-positive bacteria, gram-negative bacteria, fungi and/or viruses. Presence of alpha-defensins (especially, human neutrophil peptides (HNPs) 1 to 4) in the synovial fluid, also known as joint fluid, surrounding the prosthesis indicate presence of an infection. Thus, Alpha-defensin (as a biomarker) may be tested for diagnosing PJI. However, conventional diagnosis tools fail to provide quick and accurate detection of alpha-defensins (i.e., without false positives).
[005] Conventionally, the sample drawn from the patient is optionally diluted and treated with multiple reagents and subjected to different instruments before the sample is tested for the presence of a biomarker. The use of multiple reagents and instruments to prepare the sample is time consuming and lead to more chemical usage that is financially expensive to the end user. More often than not, even after preparing the sample with multiple reagents and instruments, there is a significant risk of getting false positive and negative outcome.
[006] Therefore, there arises a need for a diagnostic kit and a method thereof that overcomes the drawbacks associated with the conventional diagnosis tools and methods.
SUMMARY OF INVENTION
[007] Particular embodiments of the present disclosure are described herein below with reference to the accompanying drawings, however, it is to be understood that the disclosed embodiments are mere examples of the disclosure, which may be embodied in various forms. Well-known functions or constructions are not described in detail to avoid obscuring the present disclosure in unnecessary detail. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present disclosure in virtually any appropriately detailed structure.
[008] In an embodiment, the present disclosure relates to a kit having at least one strip, and an assay buffer. The at least one strip includes a top layer. The top layer includes at least one sample portion, at least one separation portion disposed distal to the sample portion, at least one conjugate portion disposed distal to the separation portion, and at least one detection portion disposed distal to the conjugate portion. The conjugate portion is provided with a pre-defined amount of a plurality of test antibodies 'x' conjugated to at least one reporter. The detection portion includes a test region 'T' having a plurality of the test antibodies 'y' immobilized thereon. The assay buffer has a pH between 8 and 9. The assay buffer includes 5 mM to 20 mM of a buffer, 0.5% (w/v) to 1.25% (w/v) of a metallic salt, a pre-defined amount of a surfactant, and 0.1% (w/v) to 0.3% (w/v) of a preservative agent. The sample portion of the strip is configured to receive a sample mixed with the assay buffer. A limit of detection of the strip is 1 μg/mL or more for the sample mixed with the assay buffer.
[009] In yet another embodiment, the present disclosure relates to an ex vivo method to detect a presence of a biomarker in a sample. The method commences by diluting a sample using an assay buffer as described above to obtain a diluted sample mixture. A predefined amount of the diluted sample mixture is loaded on a sample portion of a strip as described above. A plurality of test antibodies 'x' provided in at least a portion of the strip binds with a plurality of biomarkers present in the diluted sample mixture. An antigen-antibody complex is formed by aggregating the test antibodies 'x' at a test region 'T' of a detection portion of the strip, aggregation of the test antibodies 'x' at the test region 'T' indicates a positive result.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Fig. 1 depicts a strip 100 in accordance with an embodiment of the present invention.
[0011] Fig. 1a depicts a casing 110 enclosing the strip 100 in accordance with an embodiment of the present invention.
[0012] Fig. 1b depicts a pre-filled vial 120 in accordance with an embodiment of the present invention.
[0013] Fig. 2 depicts a method 200 to prepare the strip 100 in accordance with an embodiment of the present invention.
[0014] Figs. 3 and 3a-3d depict a method 300 to use the strip 100 to test for a biomarker in a sample in accordance with an embodiment of the present invention.
[0015] Fig. 4 depicts experimental data of the strip 100 in accordance with an embodiment of the present invention.
DETAILED DESCRIPTION of accompanying drawings
[0016] Prior to describing the invention in detail, definitions of certain words or phrases used throughout this patent document will be defined: the terms "include" and "comprise", as well as derivatives thereof, mean inclusion without limitation; the term "or" is inclusive, meaning and/or; the phrases "coupled with" and "associated therewith", as well as derivatives thereof, may mean to include, be included within, interconnect with, contain, be contained within, connect to or with, couple to or with, be communicable with, cooperate with, interleave, juxtapose, be proximate to, be bound to or with, have a property of, or the like; Definitions of certain words and phrases are provided throughout this patent document, and those of ordinary skill in the art will understand that such definitions apply in many, if not most, instances to prior as well as future uses of such defined words and phrases.
[0017] Reference throughout this specification to "one embodiment," "an embodiment," or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases "in one embodiment," "in an embodiment," and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment, but mean "one or more but not all embodiments" unless expressly specified otherwise. The terms "including," "comprising," "having," and variations thereof mean "including but not limited to" unless expressly specified otherwise. An enumerated listing of items does not imply that any or all of the items are mutually exclusive and/or mutually inclusive, unless expressly specified otherwise. The terms "a," "an," and "the" also refer to "one or more" unless expressly specified otherwise.
[0018] Although the operations of exemplary embodiments of the disclosed method may be described in a particular, sequential order for convenient presentation, it should be understood that the disclosed embodiments can encompass an order of operations other than the particular, sequential order disclosed. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Further, descriptions and disclosures provided in association with one particular embodiment are not limited to that embodiment, and may be applied to any embodiment disclosed herein. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed system, method, and apparatus can be used in combination with other systems, methods, and apparatuses.
[0019] Furthermore, the described features, advantages, and characteristics of the embodiments may be combined in any suitable manner. One skilled in the relevant art will recognize that the embodiments may be practiced without one or more of the specific features or advantages of a particular embodiment. In other instances, additional features and advantages may be recognized in certain embodiments that may not be present in all embodiments. These features and advantages of the embodiments will become more fully apparent from the following description and apportioned claims, or may be learned by the practice of embodiments as set forth hereinafter.
[0020] The term proximal and distal used hereinafter corresponds to direction with respect to flow of a fluid, for example a sample optionally mixed with an assay buffer. The direction of the flow is from the proximal end/portion of a strip to the distal end/portion of the strip.
[0021] The present disclosure discloses a diagnostic kit (or kit), a method to prepare the kit and a method to use the kit. The kit of the present disclosure is used to detect at least one biomarker (for example, Alpha-defensin or variants thereof) from a bodily fluid. In an exemplary embodiment, the kit is used to detect Alpha-defensins including human neutrophil peptides (HNPs) 1 to 4 in a joint synovial fluid obtained from a patient who underwent a joint replacement surgery or the like. The kit of the present disclosure helps to diagnose Periprosthetic joint infection (PJI) basis the presence of Alpha-defensins in the sample.
[0022] The kit includes at least one strip and an assay buffer. The assay buffer helps to block non-specific binding, optimize specific interaction and maintain consistent flow rate of the diluted sample mixture across to the strip.
[0023] The strip has at least a sample portion, a separation portion, a conjugate portion and a detection portion. The strip may optionally be provided with at least one absorbent portion. The sample (diluted in the assay buffer) is loaded on the sample portion of the strip. The sample then flows from the sample portion to the separation portion, the conjugate portion and then to the detection portion. If the absorbent portion is present, it absorbs any residual sample that flows from the detection portion to prevent spills.
[0024] The separation portion of the strip helps to remove cells, debris or other large particles from the sample as the sample flows across the separation portion from the sample portion to the conjugate portion, thereby reducing potential interference between the sample and the antibodies. The separation portion absorbs the sample and then releases the sample towards the conjugate portion at a steady flow rate which helps to prevent undesirable sample saturation in the conjugate portion and the detection portion. The separation portion helps to prevent non-specific binding between the sample and the antibodies, thereby enhancing the specificity and sensitivity of the strip. The separation portion is provided with reagents including, but not limited to, buffers, detergents that helps to maintain the integrity and reliability of the test to detect presence of a biomarker in the sample using the strip.
[0025] The conjugate portion is provided with a plurality of test antibodies conjugated with a reporter. If the sample includes the biomarker (i.e., Alpha-defensins), then the test antibodies conjugated with the reporter binds with the biomarker when the sample flows through the conjugate portion.
[0026] The detection portion of the strip is provided with at least a test region. A plurality of the test antibodies (without the reporter) is immobilized on the test region of the detection portion. As the sample flows across the detection portion of the strip and if the sample includes the biomarker, the biomarkers are captured and immobilized at the test region of the strip via the test antibodies immobilized therein. The capture and immobilization of the biomarker at the test region leads to aggregation of the reporters bound to the biomarkers at the test region of the strip. The aggregation of the reporter at the test region is visually read by a medical practitioner which helps the medical practitioner confidently conclude the presence of the biomarker in the sample.
[0027] Optionally, a control region is provided on the detection region that is disposed distal to the test region. Aggregation of test antibodies that did not bind to the biomarker at the control region provides validity to the method (and result thereof) to test the presence of the biomarker at the test region of the strip.
[0028] The strip of the present disclosure can detect alpha-defensins in a sample mixed with the assay buffer having concentration of 1 μg of alpha-defensin (i.e., the biomarker) per ml of the sample-assay buffer mixture or more. In other words, the limit of detection (LOD) of the strip of the present disclosure is 1 μg/ml or more for the sample mixed with the assay buffer. The LOD is the lowest concentration of a biomarker in a sample that can be reliably detected by the test, but not necessarily quantified.
[0029] Now referring to the figures, Fig. 1 depicts a test strip (or strip) 100 of a kit of the present disclosure to test presence of a biomarker in a sample. The sample may include at least one of the bodily fluids or the like. In an exemplary embodiment, the strip 100 is used to detect the presence of Alpha-defensins in synovial fluid obtained from a patient who has underwent a joint replacement surgery or opting for a joint replacement surgery. The strip 100 may be optionally enclosed in a casing 110 as shown in Fig. 1a.
[0030] The kit includes an assay buffer to dilute the sample before adding the sample to the strip 100. The assay buffer includes a pre-defined amount of at least one of a buffer, a metallic salt, a surfactant, a preservative agent, etc. The amount of buffer ranges from 5mM to 20mM. The buffer may be one of Tris buffer, phosphate buffer, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer, etc. In an exemplary embodiment, the buffer is Tris buffer. The buffer helps to maintain the pH of the assay buffer thereby preserving the binding activity and stability of the biomolecules of the strip 100 and the assay buffer. The pH of the assay buffer is maintained between 8 to 9.
[0031] The amount of metallic salt ranges from 0.5% (w/v) to 1.25% (w/v). The metallic salt may be one of sodium chloride (NaCl), potassium chloride (KCl), etc. In an exemplary embodiment, the metallic salt is NaCl. The metallic salt helps to maintain the ionic strength of the assay buffer and ensure proper binding interactions.
[0032] The surfactant is at least one of 0.1% (v/v) to 1% (v/v) TritonX-100, 0.2% (v/v) to 0.5% (v/v) TritonX-305, 1% (v/v) to 3% (v/v) TritonX-705, 0.1% (v/v) to 0.5% (v/v) Tween-20, and 0.1% (w/v) to 0.5% (w/v) sodium dodecyl sulphate. The surfactant reduces surface tension, helps to block non-specific binding and to denature the proteins in the sample.
[0033] The amount of preservative agent ranges from 0.1% (w/v) to 0.3% (w/v). In an exemplary embodiment, the preservative agent is sodium azide. The preservative agent helps to prevent bacterial contamination of the sample and helps to improve the shelf life of the assay buffer.
[0034] In an exemplary embodiment, the assay buffer includes 10 mM Tris buffer, 1.05% (w/v) sodium chloride, 0.5% (w/v) sodium dodecyl sulphate, 0.5% (v/v) TritonX-100 and 0.2% (w/v) sodium azide. The assay buffer may be added to the sample or the sample may be added to the buffer. The assay buffer helps to block non-specific binding, optimize specific interaction and maintain consistent flow rate of the diluted sample mixture across to the strip 100.
[0035] In an exemplary embodiment, as shown in Fig. 1b, the assay buffer is provided in a pre-filled vial 120. A pre-defined amount of the sample is added to the pre-filled vials 120 and uniformly mixed to obtain a diluted sample mixture. In an exemplary embodiment, the pre-filled vial 120 contains 1 mL of assay buffer which is mixed with 10 µL of the sample. A pre-defined amount of the diluted sample mixture is then used with the strip 100 to test for the presence of the biomarker in the sample. The amount of assay buffer and sample may vary depending on the strip 100.
[0036] The strip 100 includes a pre-defined length ranging from 57 mm to 61 mm. The strip 100 includes a pre-defined width ranging from 3.6 mm to 3.8 mm. In an exemplary embodiment, the length and width of the strip 100 is 60 mm and 3.7 mm, respectively. The dimensions of the strip 100 may vary based on the size of the casing 110.
[0037] The strip 100 includes at least two layers, namely a bottom layer 101 and a top layer 103. The bottom layer 101 includes a pre-defined thickness ranging from 0.25 mm to 0.4 mm. In an exemplary embodiment, the thickness of the bottom layer 101 is 0.3 mm. The bottom layer 101 is made of one or materials including, but not limited to, plastic, polyvinyl chloride (PVC), etc. In an exemplary embodiment, a bottom layer 101 is made of PVC. The bottom layer 101 provides support to the top layer 103.
[0038] The top layer 103 is at least partially disposed over the bottom layer 101. In an exemplary embodiment, the top layer 103 is disposed completely over the bottom layer 101. The top layer 103 includes a plurality of portions along its length. The plurality of portions of the top layer 103 include at least one sample portion 103a, at least one separation portion 103b, at least one conjugate portion 103c, and at least one detection portion 103d. The top layer 103 may optionally be provided with at least one absorbent portion 103e. The plurality of portions of the top layer 103 may either be discrete portions of the top layer 103 or may form an integral structure of the top layer 103. Alternatively, each of the plurality of portions of the top layer 103 has an overlap with the at least one of the adjacent portions.
[0039] In an exemplary embodiment, as shown in Fig. 1, the separation portion 103b is disposed between the sample portion 103a and the conjugate portion 103c. The conjugate portion 103c is disposed between the separation portion 103b and the detection portion 103d. And, the detection portion 103d is disposed between the conjugate portion 103c and the absorbent portion 103e.
[0040] The sample portion 103a includes a pre-defined length ranging from 15 mm to 17 mm. The sample portion 103a includes a pre-defined thickness ranging from 0.3 mm to 0.45 mm. In an exemplary embodiment, the length and thickness of the sample portion 103a is 17 mm and 0.4 mm, respectively. The sample portion 103a of the top layer 103 is made of one or more materials including, but not limited to, glass fiber, polyester, cellulose fiber, etc. In an exemplary embodiment, the sample portion 103a is made of glass fiber. The sample portion 103a is configured to receive the sample, i.e., bodily fluids or the like. In an exemplary embodiment, the sample portion 103a is configured to receive synovial fluid (optionally diluted in the assay buffer) to be tested for the presence of a biomarker, i.e., alpha-defensin or variants thereof. The sample portion 103a helps to uniformly diffuse the sample loaded thereon and facilitates the sample to flow to the separation portion 103b via capillary forces.
[0041] With reference to flow of the sample, the separation portion 103b is disposed distal to the sample portion 103a. The separation portion 103b may define an overlap region o1 with the sample portion 103a. The overlap region o1 includes a pre-defined length ranging from 2 mm to 3 mm. In an exemplary embodiment, the overlap region o1 is 2 mm. The separation portion 103b includes a pre-defined length ranging from 5 mm to 7 mm. The separation portion 103b includes a pre-defined thickness ranging from 0.3 mm to 0.45 mm. In an exemplary embodiment, the length and thickness of the separation portion 103b is 7 mm and 0.45 mm, respectively. The separation portion 103b of the top layer 103 is made of one or more materials including, but not limited to, polyester, glass fiber, cellulose fiber, etc. In an exemplary embodiment, the separation portion 103b is made of glass fiber. The separation portion 103b helps to remove cells, debris or other large particles from the sample as the sample flows across the separation portion 103b from the sample portion 103a to the conjugate portion 103c, thereby reducing potential interference between the sample and the antibodies. The separation portion 103b absorbs the sample flowing from the sample portion 103a and then releases the sample towards the conjugate portion 103c at a steady flow rate which helps to prevent undesirable sample saturation in the conjugate portion 103c and the detection portion 103d.
[0042] The separation portion 103b is provided with a pre-defined amount of a plurality of Anti-RBC antibodies which helps to prevent non-specific binding within the detection portion 103d, thereby enhancing the specificity and sensitivity of the strip 100. The anti-RBC antibodies have a binding specificity towards red blood cells (RBCs) or the like, if present in the sample. Thus, the separation portion 103b substantially prevents the RBCs to flow to the conjugate portion 103c and the detection portion 103d. Other functionally equivalent antibodies may be used instead of the Anti-RBC antibodies and the same is within the scope of the teachings of the present disclosure.
[0043] The separation portion 103b is provided with reagents including, but not limited to, buffers, detergents that helps to maintain the integrity and reliability of the test to detect presence of the biomarker in the sample using the strip 100. In an exemplary embodiment, the separation portion 103b is treated with 5mM to 10mM sodium phosphate monobasic and disodium hydrogen phosphate, 0.45% (w/v) to 0.9% (w/v) sodium chloride, and 0.1% (w/v) sodium azide.
[0044] With reference to flow of the sample, the conjugate portion 103c is disposed distal to the separation portion 103b. The conjugate portion 103c may define an overlap region o2 with the separation portion 103b. The overlap region o2 includes a pre-defined length ranging from 2 mm to 3 mm. In an exemplary embodiment, the overlap region o2 is 2 mm. The conjugate portion 103c includes a pre-defined length ranging from 5 mm to 7 mm. The conjugate portion 103c includes a pre-defined thickness ranging from 0.3 mm to 0.45 mm. In an exemplary embodiment, the length and thickness of the conjugate portion 103c is 7 mm and 0.45 mm, respectively. The conjugate portion 103c of the top layer 103 is made of one or more materials including, but not limited to, polyester, glass fiber, cellulose fiber, etc. In an exemplary embodiment, the conjugate portion 103c is made of polyester.
[0045] The conjugate portion 103c may be provided with a pre-defined amount of a plurality of test antibodies 'x' conjugated to at least one reporter. The test antibodies 'x' may include monoclonal antibodies for the biomarker to be detected in the sample. In other words, the test antibodies 'x' has a binding affinity towards the biomarker to be tested. In an exemplary embodiment, the test antibodies 'x' include antihuman α-defensin monoclonal antibodies having binding affinity towards Alpha-defensins (an exemplary biomarker). The Alpha-defensins are encoded by at least one of SEQ ID No. 1 and SEQ ID No. 2. An antigen binding site of the test antibody 'x' corresponds to a first epitope disposed on the outer surface of the biomarker. Other functionally equivalent test antibodies 'x' corresponding to the biomarker to be tested are within the scope of the teachings of the present disclosure. While the sample flows across the conjugate portion 103c and if the sample includes the biomarker, the biomarker binds with at least one of the test antibodies 'x'.
[0046] The reporter is conjugated with each of the plurality of test antibodies 'x' via at least one of covalent bonds, ionic bonds, linker, etc. In an exemplary embodiment, the reporter is conjugated with the plurality of the test antibodies 'x' through ionic interactions. The reporter helps the medical practitioner to visualize the outcome of the test to detect the presence of the biomarker in the sample. In an exemplary embodiment, the reporter includes at least one gold nanoparticle (AuNP). Other functionally equivalent reporters are within the scope of the teachings of the present disclosure.
[0047] The AuNPs may have a pre-defined diameter ranging from 20 nm to 100 nm. According to the diameter, the color of the AuNPs may vary from pink to deep red/purple. In an exemplary embodiment, the AuNPs have a diameter of 40 nm and a color of pinkish red. The color of the AuNPs is visible to the naked eye when the AuNPs aggregate together.
[0048] With reference to flow of the sample, the detection portion 103d is disposed distal to the conjugate portion 103c. The detection portion 103d may define an overlap region o3 with the conjugate portion 103c. The overlap region o3 includes a pre-defined length ranging from 2 mm to 4 mm. In an exemplary embodiment, the overlap region o3 is 2mm. The detection portion 103d includes a pre-defined length ranging from 23 mm to 26 mm. The detection portion 103d may have a porous structure. The detection portion 103d includes a pre-defined pore size ranging from 12 μm to 15 μm. In an exemplary embodiment, the length and pore size of the detection portion 103d is 25 mm and 12 μm, respectively. The detection portion 103d of the top layer 103 is made of one or more materials including, but not limited to, nitrocellulose membrane (NCM), nylon membrane, cellulose acetate membrane, etc. In an exemplary embodiment, the detection portion 103d is made of NCM.
[0049] The detection portion 103d includes at least one coated region, namely a test region 'T'. The detection portion 103d may optionally include another coated region, namely, a control region 'C'. In an exemplary embodiment, as shown in Fig.1, the control region 'C' is disposed distal to the test region 'T'. In an alternate embodiment, the test region 'T' is disposed distal to the control region 'C'. The test region 'T' includes a coating of a plurality of test antibodies 'y' immobilized on an outer surface of the detection portion 103d via at least one of passive adsorption, hydrogen bond, vander waals force, etc. The test antibodies 'y' of the detection portion 103d is same as the test antibodies 'x' of the conjugate portion 103c, except that, the test antibodies of the detection portion 103d is not conjugated with any reporter. As the sample flows across the test region 'T' and if the sample has the biomarker, the biomarkers bind with at least one of the test antibodies 'y' of the test region 'T'.
[0050] Alternatively, the test antibodies 'y' of the test region 'T' may have binding affinity at least partially towards the biomarker and at least partially towards the test antibodies 'x' of the conjugate portion 103c bound to the biomarker and the same is within the scope of the teachings of the present disclosure.
[0051] In an exemplary embodiment, if the sample contains Alpha-defensin, the test antibodies 'y' of the test region 'T' are configured to capture and immobilize at least the Alpha-defensin biomarker bound to the antihuman α-defensin monoclonal antibodies (i.e., test antibodies 'x') that are conjugated with AuNPs reporter. In other words, the alpha-defensin biomarkers are sandwiched between the test antibodies 'y' immobilized on the test region 'T' and the test antibodies 'x' of the conjugate portion 103c. Accordingly, the AuNPs aggregate at the test region 'T' and are observed as reddish-purple color band by the medical practitioner which is indicative of the fact that the sample loaded on the sample portion 103a includes the Alpha-defensin biomarker. Alternatively, if the sample does not contain the Alpha-defensin biomarker, then AuNPs does not aggregate at the test region 'T' to form the reddish-purple color band.
[0052] The control region 'C' includes a coating of a plurality of control antibodies 'z' immobilized on an outer surface of the detection portion 103d via at least one of passive adsorption, hydrogen bond, vander waals force, etc. The control antibodies 'z' include at least one of goat anti-chicken antibodies, goat anti-mouse antibodies, goat anti-rabbit antibodies, etc. having binding affinity to the test antibodies 'x' of the conjugate portion 103c. As the sample flows across the control region 'C', the test antibodies 'x' bind with at least one of the control antibodies 'z' of the control region 'C'. Since the control region 'C' is disposed distal to the test region 'T' and the test antibodies 'x' bound to the biomarker is captured by the test antibodies 'y' of the test region 'T', only the test antibodies 'x' that are unbound to any biomarker are captured by the control antibodies 'z' of the control region 'C'.
[0053] With reference to flow of the sample, the absorbent portion 103e is optionally disposed distal to the detection portion 103d. The absorbent portion 103e may define an overlap region o4 with the detection portion 103d. The overlap region o4 includes a pre-defined length ranging from 3 mm to 5 mm. In an exemplary embodiment, the overlap region o4 is 4 mm. The absorbent portion 103e includes a pre-defined length ranging from 20 mm to 22 mm. The absorbent portion 103e includes a pre-defined thickness ranging from 1.05 mm to 1.08 mm. In an exemplary embodiment, the length and thickness of the absorbent portion 103e is 20 mm and 1.08 mm, respectively. The absorbent portion 103e of the top layer 103 is made of one or more materials including but not limited to cellulose, cotton fiber, blended material, super absorbent polymers, polyurethane foam, etc. In an exemplary embodiment, the absorbent portion 103e is made of cellulose cotton pulp. The absorbent portion 103e is configured to absorb any residual sample from the detection portion 103d and prevent spillage of the same. The absorbent portion 103e prevents backflow of the residual sample.
[0054] Additionally or optionally, as shown in Fig. 1a, the strip 100 is enclosed inside the casing 110. The casing 110 is provided with at least two openings, namely first opening 111, and a second opening 113. The first opening 111 is disposed at least partially on the sample portion 103a of the strip 100. The first opening 111 allows the user to load the sample on the sample portion 103a of the strip 100.
[0055] The second opening 113 is disposed at least partially on the detection portion 103d of the strip 100. The second opening 113 allows the medical practitioner to view the test region 'T' and the control region 'C' of the strip 100 when the strip 100 is placed inside the casing 110.
[0056] Alternatively, not shown, the casing 110 may be provide two separate openings for the test region 'T' and the control region 'C' or a single opening for the sample portion 103a, the test region 'T' and the control region 'C'. The same is within the scope of the teachings of the present disclosure.
[0057] Fig. 2 depicts an exemplary method 200 to prepare the strip 100 of the present disclosure. The method 200 commences at step 201 by preparing the reporter that is to be conjugated with the test antibodies 'x' of the conjugate portion 103c.
[0058] In an exemplary embodiment, the reporter includes AuNPs. The AuNPs are prepared by mixing 1.0% (w/v) to 2.5% (w/v) trisodium citrate with 0.5% (w/v) to 1.5% (w/v) gold chloride to obtain a first mixture. The first mixture is subjected to a pre-defined temperature for a pre-defined time period. The pre-defined temperature ranges from 350 °C to 450 °C. The pre-defined time period ranges from 8 minutes to 10 minutes. In an exemplary embodiment, the first mixture is heated at 400 °C for 10 minutes. After the first mixture is subjected to the pre-defined temperature for the pre-defined time period, the λmax of the first mixture ranges from 526 nm to 530 nm (as measured using a spectrophotometer). The optical density (O.D.) of the first mixture ranges from 2.9 to 4.0 when measured at 650 nm to 450 nm (as measured using a spectrophotometer). The first mixture containing the plurality of reporter as obtained from step 201 includes the AuNPs suspended therein.
[0059] At step 203, the reporter obtained from the step 201 is conjugated with the test antibodies 'x'.
[0060] In an exemplary embodiment, the reporter (i.e., the AuNPs) obtained from step 201 are conjugated with antihuman α-defensin monoclonal antibodies (i.e., the test antibody 'x'). The test antibody 'x' is added to the first mixture (containing the reporter) at a concentration ranging from 20 μg/mL to 30 μg/mL. The pH of the first mixture is maintained between 7.4 to 7.6. The first mixture is subjected to mixing for a pre-defined time period ranging from 10 minutes to 30 minutes. In an exemplary embodiment, the test antibodies 'x' are mixed with the first mixture using a magnetic stirrer for 30 minutes. A blocking reagent is added to the first mixture in a concentration ranging from 0.1% (v/v) to 0.5% (v/v). The blocking reagent includes at least one of a bovine serum albumin (BSA) and casein. The blocking reagent helps to prevent side reaction(s), and improves reaction efficiency. After adding the blocking reagent, the first mixture is mixed for a pre-defined time period ranging from 10 minutes to 30 minutes. In an exemplary embodiment, the blocking reagent is mixed with the first mixture using a magnetic stirrer for 30 minutes. Other functionally equivalent technique to mix the first mixture is within the scope of the teachings of the present disclosure. After mixing, the first mixture is then subjected to centrifugation at 6000 rpm to 12000 rpm at a pre-defined temperature ranging from 3 °C to 6 °C for a pre-defined time period ranging from 10 minutes to 45 minutes. In an exemplary embodiment, the first mixture is centrifuged at 10,000 rpm at 4 °C for 45 minutes. After centrifugation, a supernatant obtained from the centrifugation of the first mixture having an optical density (O.D.) ranging from 30 to 50 measured at 650 nm to 450 nm (as measured using a spectrophotometer). The λmax of the supernatant ranges from 530 nm to 545 nm (as measured using a spectrophotometer). A pellet obtained from the centrifugation of the first mixture includes the test antibodies 'x' conjugated with the AuNPs.
[0061] At step 205, the pellet of the test antibodies 'x' obtained from step 203 is resuspended in a resuspension buffer. In an exemplary embodiment, the resuspension buffer includes 10 mM to 20 mM Tris buffer to maintain the pH, 0.6% (w/v) to 1.8% (w/v) sodium chloride (NaCl) for ionic strength, 3% (w/v) to 6% (w/v) sucrose for preventing aggregation and maintaining antibody activity, 2% (w/v) to 4(w/v) D-Trehalose for stabilizing protein and reducing non-specific binding, 0.05% (w/v) to 0.3% (w/v) bovine serum albumin (BSA) to prevent non-specific binding, and 0.05% (w/v) to 0.3% (w/v) sodium azide as preservative agent. The pH of the resuspension buffer is maintained between 8 to 8.5. After resuspending the pellet in the resuspension buffer, the optical density (O.D.) of the resuspended pellet (i.e., the pellet suspended in the resuspension buffer) ranges from 30 to 60 when measured at 650 nm to 450 nm (as measured using a spectrophotometer). At the end of step 205, the test antibodies conjugated with the reporters and suspended in the resuspension buffer is obtained.
[0062] At step 207, the conjugate portion 103c of the top layer 103 of the strip 100 is either dip coated or spray coated in the resuspension buffer obtained from step 205 to imbibe the test antibodies 'x' (conjugated to the reporter) to the conjugate portion 103c. The dip coated conjugate portion 103c of the strip 100 is dried at a pre-defined temperature for a pre-defined time period ranging from 60 minutes to 360 minutes in dehumidifier room or overnight drying. The pre-defined temperature ranges from 35 °C to 45 °C. In an exemplary embodiment, the conjugate portion 103c is dried at 37 °C for 14 hours to 16 hours overnight.
[0063] At step 209, the test antibodies 'y' are immobilized at the test region 'T' of the detection portion 103d of the strip 100 via, for example, stripping coating. During stripping coating, the dispense rate of a first coating buffer is maintained between 0.06 µL/mm to 0.08 µL/mm at a dispense speed of 50 mm/second. Other functionally equivalent coating techniques to immobilize the test antibodies 'y' at the test region 'T' are within the scope of the teachings of the present disclosure. The test antibodies 'y' are suspended in the first coating buffer including 0.8 mg/mL to 2 mg/mL test antibodies 'y', 5 mM to 10 mM of sodium phosphate monobasic and disodium hydrogen phosphate to maintain pH, 0.25% (w/v) to 0.9% (w/v) sodium chloride for ionic strength, 0.5% (w/v) to 1% (w/v) D-trehalose for preventing aggregation and maintaining antibody activity, and 0.05% (w/v) to 0.1% (w/v) sodium azide as preservative agent. The pH of the first coating buffer is maintained between 7.0 to 7.6. The coated test region 'T' of the detection portion 103d of the strip 100 is dried at a pre-defined temperature for a pre-defined time period ranging from 14 hours to 16 hours. The pre-defined temperature ranges from 35 °C to 37 °C. In an exemplary embodiment, the detection portion 103d is dried at 37 °C for 14 hours to 16 hours.
[0064] At step 211, the control antibodies are immobilized at the control region 'C' of the detection portion 103d of the strip 100 via, for example, stripping coating. During stripping coating, the dispense rate is maintained between 0.06 µL/mm to 0.08 µL/mm at a dispense speed of 50 mm/second. Other functionally equivalent coating techniques to immobilize the control antibodies 'z' at the control region 'C' are within the scope of the teachings of the present disclosure. The control antibodies 'z' are suspended in a second coating buffer including 0.5 mg/mL to 1.5 mg/mL control antibodies 'z', 5 mM to 10 mM of sodium phosphate monobasic and disodium hydrogen phosphate to maintain pH, 0.25% (w/v) to 0.9% (w/v) sodium chloride for ionic strength, 0.5% (w/v) to 1% (w/v) D-trehalose for preventing aggregation and maintaining antibody activity, and 0.05% (w/v) to 0.1% (w/v) sodium azide as preservative agent. The pH of the second coating buffer is maintained between 7.0 to 7.6. The coated control region 'C' of the detection portion 103d of the strip 100 is dried at a pre-defined temperature for a pre-defined time period ranging from 14 hours to 16 hours. The pre-defined temperature ranges from 35 °C to 37 °C. In an exemplary embodiment, the detection portion 103d is dried at 37 °C for 14 hours to 16 hours.
[0065] At step 213, the separation portion 103b is treated with a treatment buffer by for example, dipping technique. In an exemplary embodiment, the treatment buffer includes 0.1 mg/mL to 0.35 mg/mL of anti-RBC antibodies, 5mM to 10mM sodium phosphate monobasic and disodium hydrogen phosphate, 0.45% (w/v) to 0.9% (w/v) sodium chloride, and 0.1% (w/v) sodium azide. After dipping the separation portion 103b in the treatment buffer, the separation portion 103b is dried for at 37 °C for 14 hours to 16 hours.
[0066] At step 215, the strip 100 is assembled by placing the portions of the top layer 103 at least partially on the bottom layer 101. Optionally, the bottom layer 101 is provided with an adhesive to enable the portions of the top layer 103 to adhere to the bottom layer 101. In an exemplary embodiment, as shown in Fig. 1, the detection portion 103d is placed on a middle region of the bottom layer 101. Thereafter, the conjugate portion 103c is placed proximal to the detection portion 103d with overlap region o3, then the separation portion 103b is placed proximal to the conjugate portion 103c with the overlap region o2. The sample portion 103a is placed proximal to the separation portion 103b with the overlap region o1 and the absorbent portion 103e is placed distal to the detection portion 103d with the overlap region o4.
[0067] At an optional step 217, the strip 100 is cut longitudinally to required sizes and enclosed in the casing 110 (as shown in Fig. 1a).
[0068] Fig. 3 depicts an exemplary ex vivo method 300 for using the strip 100 to test the presence of a biomarker in a sample. The sample may include at least one of the bodily fluids or the like. In an exemplary embodiment, the strip 100 is used to detect the presence of alpha defensin biomarkers in a joint synovial fluid obtained from a patient who has underwent a joint replacement surgery or is opting for a joint replacement surgery.
[0069] The method 300 commences at an optional step 301 by diluting the sample using the assay buffer to obtain a diluted sample mixture. In an exemplary embodiment, the assay buffer includes 10 mM Tris buffer, 1.05% (w/v) sodium chloride, 0.5% (w/v) sodium dodecyl sulphate, 0.5% (v/v) TritonX-100 and 0.2% (w/v) sodium azide. The pH of the assay buffer is maintained between 8 to 9. The assay buffer may be added to the sample or the sample may be added to the assay buffer. In an exemplary embodiment, as shown in Fig. 1b, the assay buffer is provided in pre-filled vials 120. A pre-defined amount of the sample is added to the pre-filled vials 120 and uniformly mixed to obtain a diluted sample mixture. In an exemplary embodiment, the pre-filled vial 120 contains 1 mL of assay buffer which is mixed with 10 µL of the sample. The assay buffer helps to block non-specific binding, optimize specific interaction and maintain consistent flow rate of the diluted sample mixture across to the strip 100.
[0070] At step 303, as shown in Fig. 3a, a predefined amount of the diluted sample mixture 10, optionally containing a plurality of biomarkers 11, is loaded on the sample portion 103a of the strip 100. In an exemplary embodiment, three drops of the diluted sample mixture 10 are loaded on the sample portion 103a of the strip 100.
[0071] At step 305, the diluted sample mixture 10 flows from the sample portion 103a to the conjugate portion 103c via the separation portion 103b by capillary action. If the sample (or the diluted sample mixture 10) has the biomarker 11, then the test antibodies 'x' (conjugated with the reporter) present in the conjugate portion 103c binds with the biomarker 11 as shown in Fig. 3b.
[0072] At step 307, the diluted sample mixture 10 flows from the conjugate portion 103c to the detection portion 103d via capillary action. Since the control region 'C' is distal to the test region 'T', the diluted sample mixture 10 first interacts with the test region 'T' and then with the control region 'C'. If the sample (or the diluted sample mixture 10) has the biomarker 11, then the test antibodies 'y' immobilized on the test region 'T' binds with the biomarker 11 (to form an antigen-antibody complex) thereby aggregating the reporter of the test antibodies 'x' at the test region 'T'. The aggregation of the reporter is observed by the medical practitioner thereby helping the medical practitioner to confirm the presence of the biomarker 11 in the sample. Thus, aggregation of the reporter at the test region 'T' indicates a positive result as shown in Fig. 3c.
[0073] Alternatively, if the sample (and the diluted sample mixture 10) did not have any biomarker 11, then the reporter does not aggregate at the test region 'T'. Thus, the absence of aggregation of the reporter at the test region 'T' indicates a negative result as shown in Fig. 3d.
[0074] At step 309, the test antibodies 'x' of the conjugate portion 103c that did not bind with the biomarker 11 binds with the control antibodies 'z' immobilized at the control region 'C'. Thus, aggregation of the reporter at the control region 'C' is indicative of the fact that the positive result (Fig. 3c) or the negative result (Fig. 3d) indicated by the test region 'T' is valid. In the absence of the aggregation of the reporter at the control region 'C', the positive result or negative result as indicated by the test region 'T' is invalid.
[0075] At step 311, the diluted sample mixture flows from the detection portion 103d to the absorbent portion 103e via capillary action. The absorbent portion 103e absorbs the diluted sample and prevents any potential spillage. The absorbent portion 103e prevents backflow of the diluted sample.
[0076] The present disclosure will now be explained with the help of the following examples:
[0077] Example 1: Preparing the assay buffer of the present disclosure
[0078] The assay buffer was prepared by mixing 10 mM Tris buffer, 1.05% (w/v) sodium chloride, 0.5% (w/v) sodium dodecyl sulphate, 0.5% (v/v) TritonX-100 and 0.2% (w/v) sodium azide. 1mL of the assay buffer was then sealed within respective pre-filled vials 120. The assay buffer was used to dilute samples drawn from the patient.
[0079] Example 2: Diagnostic sensitivity study using the kit of the present disclosure
[0080] Samples were drawn from 50 patients diagnosed with Periprosthetic joint infection (PJI). In other words, the samples had the alpha defensin biomarker. 10 µL of each of the sample were added in respective pre-filled vial 120 obtained from example 1 above to obtain diluted sample mixture. Three drops of the diluted sample mixture of the respective patients were added to the sample portion 103a of the strip 100. The test start time and test end time was recorded for analysis. The test start time corresponds to the event of adding the diluted sample mixture to the sample portion 103a of the test strip 100. The test stop time corresponds to the event of appearance of a colored band at the control region 'C' (and the test region 'T') of the detection portion 103d of the strip 100.
[0081] The test interpretation time for alpha defensin biomarker was 10 minutes. The background clearance time was within 5-7 minutes. The sensitivity was calculated using the following relation:
Sensitivity (%) = ((True positive) / (True positive + False negative)) x 100
[0082] The sensitivity was found to be 100% due to dilution of the sample in the assay buffer.
[0083] Example 3: Diagnostic specificity study using the kit of the present disclosure
[0084] Samples were drawn from 100 patients who were not diagnosed with Periprosthetic joint infection (PJI). In other words, the samples did not have the alpha defensin biomarker. 10 µL of each of the sample were added in respective pre-filled vial 120 obtained from example 1 above to obtain diluted sample mixture. Three drops of the diluted sample mixture of the respective patients were added to the sample portion 103a of the strip 100. The test start time and test end time was recorded for analysis. The test start time corresponds to the event of adding the diluted sample mixture to the sample portion 103a of the test strip 100. The test stop time corresponds to the event of appearance of a colored band only at the control region 'C' (and not at the test region 'T') of the detection portion 103d of the strip 100.
[0085] The test interpretation time for alpha defensin biomarker was 10 minutes. The background clearance time was within 5-7 minutes. The specificity was calculated using the following relation:
Specificity (%) = ((True negative) / (True negative + False positive)) x 100
[0086] The specificity was found to be 100% due to dilution of the sample in the assay buffer.
[0087] Example 4: Limit of detection study using the kit of the present disclosure
[0088] A sample was prepared by dissolving 1000 μg of alpha defensin biomarker in 1mL of the assay buffer obtained from example 1 above to obtain a stock biomarker solution. The stock biomarker solution was serially diluted 11 times to obtain the following concentration of the alpha defensin biomarker in the assay buffer:

Diluted sample mixture Concentration of Alpha defensin biomarker in the assay buffer
Dilution 1 512 μg/mL
Dilution 2 256 μg/mL
Dilution 3 128 μg/mL
Dilution 4 64 μg/mL
Dilution 5 32 μg/mL
Dilution 6 16 μg/mL
Dilution 7 8 μg/mL
Dilution 8 4 μg/mL
Dilution 9 2 μg/mL
Dilution 10 1 μg/mL
Dilution 11 0.5 μg/mL

[0089] Three drops of the above diluted sample mixtures were added to the sample portion 103a of the respective strips 100 (in triplicates). The intensities of the colored band observed at the test region 'T' and the control region 'C' of the strips 100 were recorded for analysis. The intensities was categorized as 4+, 3+, 2+, 1+, +, and 0; where 4+ represented the most intense colored band and 0 represented absence of colored band. The average intensities (of the triplicates) of the respective diluted sample mixture as observed is plotted in the table below:

Diluted sample mixture Concentration of Alpha defensin biomarker in the assay buffer Band intensity at test region 'T' Band intensity at control region 'C'
Dilution 1 512 μg/mL 4+ 4+
Dilution 2 256 μg/mL 4+ 4+
Dilution 3 128 μg/mL 4+ 4+
Dilution 4 64 μg/mL 4+ 4+
Dilution 5 32 μg/mL 4+ 4+
Dilution 6 16 μg/mL 4+ 4+
Dilution 7 8 μg/mL 3+ 4+
Dilution 8 4 μg/mL 2+ 4+
Dilution 9 2 μg/mL 1+ 4+
Dilution 10 1 μg/mL + 4+
Dilution 11 0.5 μg/mL 0 4+

[0090] The strips 100 loaded with Dilution 6 to Dilution 11 are depicted in Fig. 4. It was concluded that, due to dilution of the sample in the assay buffer, the kit of the present disclosure detects the presence of the biomarker till 1 μg/mL.
[0091] Example 5: Detection of the biomarker using a strip without the assay buffer
[0092] Samples were drawn from patients suspected with Periprosthetic joint infection (PJI). In other words, the samples had to be tested for the alpha defensin biomarker. The sample was directly loaded on the strip. It was observed that the amount of sample loaded was very high relative to diluted samples described in the examples above. Further, it was observed that the strip had background clearance issue and uneven flow during the test. The outcome of the test had numerous false positives, false negatives and other hindering factors like blood cells / pus cells interference.
[0093] The scope of the invention is only limited by the appended patent claims. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. , Claims:WE CLAIM:
1. A kit comprising:
a. at least one strip (100) including a top layer (103), the top layer (103) including:
i. at least one sample portion (103a),
ii. at least one separation portion (103b) disposed distal to the sample portion (103a),
iii. at least one conjugate portion (103c) disposed distal to the separation portion (103b), the conjugate portion (103c) is provided with a pre-defined amount of a plurality of test antibodies 'x' conjugated to at least one reporter, and
iv. at least one detection portion (103d) disposed distal to the conjugate portion (103c), the detection portion (103d) includes a test region 'T' having a plurality of the test antibodies 'y' immobilized thereon; and
b. an assay buffer having a pH between 8 and 9, the assay buffer including:
i. 5mM to 20mM of a buffer,
ii. 0.5% (w/v) to 1.25% (w/v) of a metallic salt,
iii. a pre-defined amount of a surfactant, and
iv. 0.1% (w/v) to 0.3% (w/v) of a preservative agent;
wherein, the sample portion (103a) of the strip (100) is configured to receive a sample mixed with the assay buffer;
wherein, a limit of detection of the strip (100) is 1 μg/mL or more for the sample mixed with the assay buffer.
2. The kit as claimed in claim 1, wherein the buffer is at least one of Tris buffer, phosphate buffer, and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer.
3. The kit as claimed in claim 1, wherein the metallic salt is at least one of sodium chloride, potassium chloride.
4. The kit as claimed in claim 1, wherein the surfactant is at least one of 0.1% (v/v) to 1% (v/v) TritonX-100, 0.2% (v/v) to 0.5% (v/v) TritonX-305, 1% (v/v) to 3% (v/v) TritonX-705, 0.1% (v/v) to 0.5% (v/v) Tween-20, and 0.1% (w/v) to 0.5% (w/v) sodium dodecyl sulphate.
5. The kit as claimed in claim 1, wherein the preservative agent is sodium azide.
6. The kit as claimed in claim 1, wherein the assay buffer is provided in a pre-filled vial (120).
7. The kit as claimed in claim 1, wherein the strip (100) includes a bottom layer (101) disposed at least partially below the top layer (103).
8. The kit as claimed in claim 1, wherein the sample portion (103a), the separation portion (103b), and the conjugate portion (103c) of the top layer (103) is made of one or more materials including glass fiber, polyester, and cellulose fiber.
9. The kit as claimed in claim 1, wherein the separation portion (103b) is provided with a pre-defined amount of a plurality of anti-RBC antibodies.
10. The kit as claimed in claim 1, wherein the separation portion (103b) includes 5 mM to 10 mM sodium phosphate monobasic and disodium hydrogen phosphate, 0.45% (w/v) to 0.9% (w/v) sodium chloride, and 0.1% (w/v) sodium azide.
11. The kit as claimed in claim 1, wherein the conjugate portion (103c) includes the test antibodies 'x' in a resuspension buffer including 10 mM to 20 mM Tris buffer, 0.6% (w/v) to 1.8% (w/v) sodium chloride (NaCl), 3% (w/v) to 6% (w/v) sucrose, 2% (w/v) to 4(w/v) D-Trehalose, 0.05% (w/v) to 0.3% (w/v) bovine serum albumin (BSA), and 0.05% (w/v) to 0.3% (w/v) sodium azide.
12. The kit as claimed in claim 1, wherein the test antibodies 'x' and test antibodies 'y' include monoclonal antibodies for a biomarker (11) encoded by at least one of SEQ ID No. 1 and SEQ ID No. 2.
13. The kit as claimed in claim 1, wherein the reporter includes at least one gold nanoparticle (AuNP) having a pre-defined diameter ranging from 20 nm to 100 nm.
14. The kit as claimed in claim 1, wherein the detection portion (103d) is made of one or more materials including nitrocellulose membrane (NCM), nylon membrane, and cellulose acetate membrane having a pre-defined pore size ranging from 12 μm to 15 μm.
15. The kit as claimed in claim 1, wherein the detection portion (103d) includes a control region 'C' having a plurality of control antibodies 'z' immobilized thereon and disposed distal to the test region 'T'.
16. The kit as claimed in claim 1, wherein the test region 'T' of the detection portion (103d) includes 0.8 mg/mL to 2 mg/mL of the test antibodies 'y' suspended in 5 mM to 10 mM of sodium phosphate monobasic and disodium hydrogen phosphate, 0.25% (w/v) to 0.9% (w/v) sodium chloride, 0.5% (w/v) to 1% (w/v) D-trehalose, and 0.05% (w/v) to 0.1% (w/v) sodium azide.
17. The kit as claimed in claim 1, wherein the top layer (103) includes at least one absorbent portion (103e) disposed distal to the detection portion (103d) and made of one or more materials including cellulose, cotton fiber, blended material, super absorbent polymers, polyurethane foam.
18. An ex vivo method (300) to detect a presence of a biomarker in a sample, the method (300) comprising:
a. diluting a sample using an assay buffer as claimed in claim 1 to obtain a diluted sample mixture (10);
b. loading a predefined amount of the diluted sample mixture (10) on a sample portion (103a) of a strip (100) as claimed in claim 1;
c. binding a plurality of test antibodies 'x' provided in at least a portion of the strip (100) with a plurality of biomarkers (11) present in the diluted sample mixture (10); and
d. forming an antigen-antibody complex by aggregating the test antibodies 'x' at a test region 'T' of a detection portion (103d) of the strip (100), aggregation of the test antibodies 'x' at the test region 'T' indicates a positive result.

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