Immunochromatographic assay device and method for detection of sickle cell disease

Abstract

An immunochromatographic assay device for detection of sickle cell disease is provided. The immunochromatographic assay device includes a sample well for reception of an analyte that is obtained by mixing a whole blood sample with a testing buffer; a test strip comprising a sample pad, a gold pad, a nitrocellulose membrane, and an absorbent pad sequentially arranged along a length of the test strip, the analyte is allowed to flow through the test strip; and a set of capture monoclonal antibodies, a given capture monoclonal antibody, immobilized at a given capture zone of a set of capture zones in the test strip, has binding affinity with a specific haemoglobin variant in the analyte, and the sickle cell disease is detected based on binding of at least one haemoglobin variant with at least one capture monoclonal antibody.

Description

[0001] IMMUNOCHROMATOGRAPHIC ASSAY DEVICE AND METHOD FOR DETECTION OF SICKLE CELL DISEASE TECHNICAL FIELD

[0002] [1] The present disclosure relates to immunoassay systems for diagnosis of sickle cell diseases. Moreover, the present disclosure relates to an immunochromatographic assay device and a method for detection of sickle cell disease.

[0003] BACKGROUND

[0004] [2] Sickle cell disease is an inherited blood group disorder that causes red blood cells to become sickle shaped, due to presence of one or more abnormal haemoglobin variants in the heart. The sickle cell disease types include sickle cell trait (SCT; HbAS), sickle cell anaemia (HbSS), haemoglobin SC disease (HbSC), sickle HbC trait (HbC), and so on. For example, presence of sickle cell trait may be ascertained based on detection of haemoglobin-A (from one parent) and haemoglobin-S (from the other parent) in a blood sample of a subject. Similarly, presence of sickle cell anaemia may be ascertained based on detection of two haemoglobin-S one from each parent in the blood sample of the subject, presence of haemoglobin SC disease may be ascertained based on detection of haemoglobin-S (from one parent) and haemoglobin-C (from the other parent) in the blood sample of the subject, and presence of sickle HbC trait may be ascertained based on detection of haemoglobin-A (from one parent) and haemoglobin-C (from the other parent) in the blood sample of the subject.

[0005] [3] Typically, the sickle-shaped red blood cells are rigid, and thus may not be able to seamlessly pass through small or narrow blood vessels. An inhibition of normal passage of the sickle-shaped red blood cells may lead to blocking or obstruction of normal blood flow and, thereby, damage to various tissues. The blockage of normal blood flow may cause additional complications associated with sickle cell disease. Furthermore, the haemoglobin-S, may have a lifetime of about sixteen days as compared to one-hundred and the twenty-day lifetime of normal red blood cells. Sickle cell disease is a serious and lifelong health condition. Although treatment can help manage many symptoms indicative of sickle cell disease, there is possibility of occurrence of harmful complications in extreme conditions such as exposure to increased atmospheric pressure, exposure to higher altitudes, exposure to lower oxygen levels, intense athletic competition, dehydration, and the like. The carriers of sickle cell trait may be required be identified in order to monitor and, subsequently, prevent occurrence of such complications.[4] Early diagnosis (preferably as a newborn) of sickle cell disease may be crucial in order to ensure that life-saving health care therapies are administered at the earliest. Conventionally, such therapies may include penicillin prophylaxis, vaccination for pneumococcus bacteria, folic acid supplementation, pain management medications, blood transfusions, hydroxyurea, and the like. Additionally, genetic professional counselling and family planning may be used as means to manage medical, psychological, and familial implications of passing or inheriting sickle cell disease and avoid the passing of mutated sickle cell genes across generations.

[0006] [5] Therefore, in light of the foregoing discussion, there is a need to effectively diagnose sickle cell diseases.

[0007] SUMMARY

[0008] [6] The present disclosure provides an immunochromatographic assay device and a method for detection of sickle cell disease. The present disclosure also provides an in-vitro diagnostics immunochromatographic assay kit for qualitative detection of haemoglobins variants such as haemoglobin-A, haemoglobin-S and haemoglobin-C. The qualitative detection of the haemoglobins variants is based on an analysis of a whole blood specimen which may be collected by use of a finger prick or heel prick capillary and / or venipuncture. The immunochromatographic assay kit is intended for use by trained and competent personnel, who may be healthcare professionals, as an aid to diagnosis of sickle cell disease in either laboratory or point of care settings. An aim of the present disclosure is also to provide a testing buffer that can be used for qualitative detection of sickle cell anaemia, sickle cell trait, normal haemoglobin, and so on. The testing buffer comprises a haemolysis agent, a buffering agent, a blocking agent, a preservative agent, and a saline agent.

[0009] [7] One or more objectives of the present disclosure is achieved by the solutions provided in the enclosed independent claims. Advantageous implementations of the present disclosure are further defined in the dependent claims.

[0010] [8] In one aspect, the present disclosure provides an immunochromatographic assay device for detecting sickle cell disease in a subject, the immunochromatographic assay device comprising: a sample well for reception of a predetermined quantity of an analyte obtained by mixing of a whole blood sample with a testing buffer; a test strip comprising a sample pad, a gold pad, a nitrocellulose membrane, and an absorbent pad sequentially arranged along a length of the test strip, wherein analyte, received in the sample well, is allowed to laterally flow throughthe length of the test strip for a pre-defined period of time; and a set of capture monoclonal antibodies, wherein a given capture monoclonal antibody of the set of capture monoclonal antibodies is immobilized at a corresponding capture zone of a set of capture zones in the test strip, and wherein a given capture monoclonal antibody immobilized at a given capture zone has a binding affinity with a specific haemoglobin variant of a set of haemoglobin variants in the analyte, and wherein the sickle cell disease is detected based on binding of at least one haemoglobin variant of the set of haemoglobin variants with at least one capture monoclonal antibody of the set of capture monoclonal antibodies.

[0011] [9] In another aspect, the present disclosure provides a method for detecting sickle cell disease in a subject using an immunochromatographic assay device, the method comprising: receiving, in a sample well of the immunochromatographic assay device, a predetermined quantity of an analyte that is obtained by mixing a whole blood sample with a testing buffer; allowing the received predetermined quantity of the analyte to laterally flow through a length of a test strip of the immunochromatographic assay device for a pre-defined period of time, wherein the test strip comprises a sample pad, a gold pad, a nitrocellulose membrane, and an absorbent pad sequentially arranged along the length of the test strip; and allowing the analyte to interact with a set of capture monoclonal antibodies to determine presence or absence of the sickle cell disease in the subject, wherein a given capture monoclonal antibody of the set of capture monoclonal antibodies is immobilized at a corresponding capture zone of a set of capture zones in the test strip, and wherein a given capture monoclonal antibody immobilized at a given capture zone has a binding affinity with a specific haemoglobin variant of a set of haemoglobin variants in the analyte, and wherein the set of haemoglobin variants include a haemoglobin variant-A (HbA), a haemoglobin variant-S (HbS), and a haemoglobin variant-C (HbC), and wherein the sickle cell disease in the subject is detected based on binding of at least one haemoglobin variant of the set of haemoglobin variants with at least one capture monoclonal antibody of the set of capture monoclonal antibodies.

[0012]

[0010] In yet another aspect, the present disclosure provides an immunochromatographic assay kit comprising an immunochromatographic assay device, a testing buffer, a buffer container containing the testing buffer, a sample collection means, a lancet, and a swab.

[0013]

[0011] It is to be appreciated that all the aforementioned implementation forms can be combined. All steps that are performed by the various entities described in the present application, as well as the functionalities described to be performed by the various entities, areintended to mean that the respective entity is adapted to or configured to perform the respective steps and functionalities. It will be appreciated that features of the present disclosure are susceptible to being combined in various combinations without departing from the scope of the present disclosure as defined by the appended claims.

[0014]

[0012] Additional aspects, advantages, features, and objects of the present disclosure would be made apparent from the drawings and the detailed description of the illustrative implementations construed in conjunction with the appended claims that follow.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016]

[0013] The summary above, as well as the following detailed description of illustrative embodiments, is better understood when read in conjunction with the appended drawings. For the purpose of illustrating the present disclosure, exemplary constructions of the disclosure are shown in the drawings. However, the present disclosure is not limited to specific methods and instrumentalities disclosed herein. Moreover, those in the art will understand that the drawings are not to scale. Wherever possible, like elements have been indicated by identical numbers.

[0017]

[0014] Embodiments of the present disclosure will now be described, by way of example only, with reference to the following diagrams wherein:

[0018] FIG. 1 illustrates a test strip of an immunochromatographic assay device for detecting sickle cell disease in a subject, in accordance with an embodiment of the present disclosure;

[0019] FIG. 2A illustrates a process of screening a whole blood sample of a subject for diagnosing sickle cell disease, wherein the whole blood sample is obtained by means of capillary whole blood collection, in accordance with an embodiment of the present disclosure; FIG. 2B illustrates a process of screening a whole blood sample of a subject for diagnosing sickle cell disease, wherein the whole blood sample is obtained by means of venous whole blood collection, in accordance with an embodiment of the present disclosure; and FIGs. 3A-3E illustrate various outcomes of sickle cell disease tests obtained using an immunochromatographic assay device, in accordance with an embodiment of the present disclosure.

[0020]

[0015] In the accompanying drawings, an underlined number is employed to represent an item over which the underlined number is positioned or an item to which the underlined number is adjacent. A non-underlined number relates to an item identified by a line linking the nonunderlined number to the item. When a number is non-underlined and accompanied by anassociated arrow, the non-underlined number is used to identify a general item at which the arrow is pointing.

[0021] DETAILED DESCRIPTION OF EMBODIMENTS

[0022]

[0016] The following detailed description illustrates embodiments of the present disclosure and ways in which they can be implemented. Although some modes of carrying out the present disclosure have been disclosed, those skilled in the art would recognize that other embodiments for carrying out or practicing the present disclosure are also possible.

[0023]

[0017] FIG. 1 illustrates a test strip 100 of an immunochromatographic assay device for detecting sickle cell disease in a subject, in accordance with an embodiment of the present disclosure. The test strip 100 is part of an immunochromatographic assay device (not shown) that is used for detecting sickle cell disease in the subject. In this regard, the test strip 100 is arranged inside a housing (not shown here), comprising a first opening (or slot) for receiving a whole blood sample from the subject, and a second opening (or slot) for displaying the result of the assay, wherein the test strip 100 is at least partially visible from the second opening (or slot). The test strip 100 comprises a sample pad 102, a gold pad 104, a nitrocellulose membrane 106, and an absorbent pad 108. The sample pad 102, the gold pad 104, the nitrocellulose membrane 106, and the absorbent pad 108 are sequentially arranged along a length of the test strip 100.

[0024]

[0018] In accordance with an embodiment, the gold pad 104 comprises colloidal gold nanoparticles. The colloidal gold nanoparticles are conjugated with a detector antibody. Furthermore, the nitrocellulose membrane 106 may include a set of capture zones. The set of capture zones may include a first capture zone 110, a second capture zone 112, a third capture zone 114, and a fourth capture zone 116. The immunochromatographic assay device further includes a set of capture monoclonal antibodies (not shown). A given / particular capture monoclonal antibody of the set of capture monoclonal antibodies is immobilized at a corresponding capture zone of the set of capture zones in the test strip 100. The set of capture monoclonal antibodies may include four capture monoclonal antibodies, viz., a first capture monoclonal antibody, a second capture monoclonal antibody, a third capture monoclonal antibody, and a fourth capture monoclonal antibody. In some embodiments, the first capture monoclonal antibody is mouse monoclonal haemoglobin type-A antibody, the second capture monoclonal antibody is mouse monoclonal haemoglobin type-S antibody, the third capture monoclonal antibody is mouse monoclonal haemoglobin type-C antibody, and the fourth capture monoclonal antibody is goat anti-chicken immunoglobulin-Y or chicken immunoglobulin- Y.

[0019] Specifically, the first capture monoclonal antibody is immobilized at the second capture zone 112 of the set of capture zones in the test strip 100. The second capture monoclonal antibody is immobilized at the third capture zone 114, the third capture monoclonal antibody is immobilized at the fourth capture zone 116, and the fourth capture monoclonal antibody is immobilized at the first capture zone 110. In accordance with an embodiment, the capture zones of the set of capture zones within the nitrocellulose membrane 106 of the test strip 100 are equidistant from each other. For instance, a distance between the first capture zone 110 and the second capture zone 112 is equal to a distance between the second capture zone 112 and the third capture zone 114. In addition, a distance between the second capture zone 112 and the third capture zone 114 is equal to a distance between the third capture zone 114 and the fourth capture zone 116.

[0025]

[0020] In accordance with an embodiment, different portions of the test strip 100, i.e., the sample pad 102, the gold pad 104, the nitrocellulose membrane 106, and the absorbent pad 108, are arranged in a linear array in the test strip 100. The arrangement may allow an analyte to flow in a direction 118. Thus, the analyte may initially flow through a length of the sample pad 102. Optionally, the length of the sample pad is 19 millimeters (mm). Thereafter, the analyte may flow through a length of the gold pad 104. Optionally, the length of the gold pad is 7 mm. The analyte, having flown through both the lengths of the sample pad 102 and the gold pad 104, may flow through a length of the nitrocellulose membrane 106. Optionally, the length of the nitrocellulose membrane is 25 mm. Herein, the analyte may interact with the set of capture monoclonal antibodies as the analyte passes through the set of capture zones. The interaction between the analyte and the set of capture monoclonal antibodies takes place as the set of capture monoclonal antibodies are immobilized at the set of capture zones in the nitrocellulose membrane 106. Based on the interaction, presence or absence of sickle cell disease in the subject may be determined. Once the analyte has flowed through a length of the nitrocellulose membrane 106, the analyte, having interacted with one or more capture monoclonal antibodies of the set of capture monoclonal antibodies, may be collected at the absorbent pad 108. Optionally, the length of the absorbent pad is 20 mm. Furthermore, optionally, a width of the test strip 100 is in the range 3.5-3.7 mm. Optionally, the width of each of the sample pad 102, the gold pad 104, the nitrocellulose membrane 106, and the absorbent pad 108 are identical.

[0026]

[0021] Furthermore, overlaps may exist between various portions of the test strip 100 (i.e., the sample pad 102, the gold pad 104, the nitrocellulose membrane 106, and the absorbent pad 108). Specifically, the sample pad 102 and the gold pad 104 may have a 1 mm overlap, the gold pad104 and the nitrocellulose membrane 106 may have a 2 mm overlap, and the nitrocellulose membrane 106 and the absorbent pad 108 may have a 5 mm overlap. It may be appreciated that the dimensions of overlapping between different portions of the test strip 100 is critical to ensure that an outcome of a sickle cell test performed using the immunochromatographic assay device is accurate. For instance, if the overlap between the gold pad 104 and the nitrocellulose membrane 106 is more than 2 mm, then the colloidal gold nanoparticles, which are conjugated with the detector antibody, pad may come in contact with the nitrocellulose membrane 106 and negatively influence the outcome of a sickle cell test. On the other hand, if the overlap is less than 2 mm, then the colloidal gold nanoparticles, conjugated with the detector antibody, will bind properly with the analyte. Furthermore, the overlap between the nitrocellulose membrane 106 and the absorbent pad 108 facilitates absorption of an excess amount of a mixture of a testing buffer and nanoparticles coming in contact with the test strip 100. If the overlap is less than 5 mm, the excess amount of the mixture may not move towards the absorbent pad 108. This may also negatively influence the outcome of the sickle cell test performed using the immunochromatographic assay device.

[0027]

[0022] The immunochromatographic assay device includes a sample well (see FIG. 3) into which a predetermined quantity of an analyte is dropped. For example, the quantity of the analyte that is required to be dropped may be 5 microliters (pL). The predetermined quantity of the analyte dropped maybe four drops of the analyte dropped into the sample well. The analyte is obtained by mixing a whole blood sample and a testing buffer. The whole blood sample may be collected from the subject by use of either venipuncture or finger / heel prick capillary. The testing buffer comprises various agents such as a hemolysis agent, a buffering agent, a blocking agent, a saline agent, and a preservative agent. The mixing of the whole blood sample with the various agents of the testing buffer may result in the obtaining of the analyte. Once the predetermined quantity, i.e., four drops, of the analyte is dropped onto the sample well, the analyte comes in contact with the test strip 100. Specifically, the dropped analyte contacts the sample pad 102 and laterally flows along the direction 118 from the sample pad 102 to the absorbent pad 108.

[0028]

[0023] In accordance with an embodiment, the sample pad 102 in the test strip is treated with one or more reagents. The treatment may ensure that a pH of the analyte is in a range 7.5-9.5 after the predetermined quantity of the analyte comes in contact with the sample pad 102 (after the analyte is dropped onto the sample well). Once the analyte has flowed through a length of the sample pad 102, it progresses towards the gold pad 104. At the gold pad 104, all hemoglobin variants present in the analyte may bind with the colloidal gold nanoparticles. The binding maytake place as the detector antibody, which is conjugated with the colloidal gold nanoparticles, has a binding affinity with all hemoglobin variants present in the analyte. The whole blood sample, mixed with the testing buffer includes one or more hemoglobin variants of a set of hemoglobin variants. The set of hemoglobin variants may include a hemoglobin variant-A (HbA), a hemoglobin variant-S (HbS), and a hemoglobin variant-C (HbC). Thus, one or more of HbA, HbS, or HbC may be present in the whole blood sample (or the analyte). The detector antibody has a binding affinity with HbA, HbS, and HbC. Therefore, all of the one or more hemoglobin variants present in the whole blood sample (or the analyte) bind with the detector antibody. The detector antibody may be a monoclonal anti-hemoglobin.

[0029]

[0024] Once the analyte flows through the gold pad 104, one or more antigen-antibody complexes (such as hemoglobin variant-detector antibody pairs) are formed. These antigenantibody complexes flow along the length of the nitrocellulose membrane 106. As the hemoglobin variant-detector antibody pairs flow, each of the one or more hemoglobin variants (such as HbA, HbS, or HbC that are bound with the detector antibody) present in the analyte interacts with a specific capture monoclonal antibody of the set of capture monoclonal antibodies immobilized at a capture zone of the set of capture zones within the nitrocellulose membrane 106. A given capture monoclonal antibody, immobilized at a given capture zone, has a binding affinity with a specific hemoglobin variant of the set of hemoglobin variants in the analyte. Specifically, the first capture monoclonal antibody has a binding affinity with HbA, the second capture monoclonal antibody has a binding affinity with HbS, and the third capture monoclonal antibody has a binding affinity with HbC. Thus, HbA, if present in the whole blood sample (or analyte) may bind / interact with the first capture monoclonal antibody at the second capture zone 112 of the set of capture zones. Similarly, HbS, if present in the whole blood sample (or analyte) may bind / interact with the second capture monoclonal antibody immobilized at the third capture zone 114 of the set of capture zones. Also, HbC, if present in the whole blood sample (or analyte) may bind / interact with the third capture monoclonal antibody immobilized at the fourth capture zone 116.

[0030]

[0025] However, the fourth capture polyclonal antibody, immobilized at the first capture zone 110 of the set of capture zones, has a binding affinity with chicken IgY antibody that is present as a chicken IgY gold conjugate. Therefore, the fourth capture polyclonal antibody may function as a control (Ctrl) for the immunochromatographic assay device. Thus, the chicken IgY antibody gold conjugate bind (or interact) with the fourth capture polyclonal antibody at the first capture zone 110 of the set of capture zones. The sickle cell disease is detected based on binding of atleast one haemoglobin variant of the set of haemoglobin variants with at least one capture monoclonal antibody of the set of capture monoclonal antibodies. The at least one haemoglobin variant may be present in the whole blood sample (or analyte) and may either be HbS, HbA and HbS, HbS and HbC, or HbA and HbC. Thus, sickle cell disease is detected if HbS is present in the analyte, both HbA and HbS are present in the analyte, both HbS and HbC are present in the analyte, or both HbA and HbC are present in the analyte.

[0031]

[0026] The outcome of the sickle cell disease is positive (+ve) if HbS binds with the third capture monoclonal antibody immobilized at the third capture zone 114, HbA and HbS bind with the second capture monoclonal antibody (immobilized at the second capture zone 112) and the third capture monoclonal antibody, respectively, HbS and HbC bind with the third capture monoclonal antibody and the fourth capture monoclonal antibody (immobilized at the fourth capture zone 116), respectively, or HbA and HbC bind with the second capture monoclonal antibody and the fourth capture monoclonal antibody respectively. It is to be noted that the outcome of the sickle cell test is negative, i.e., sickle cell disease in the subject is ruled out, if the at least one haemoglobin variant is HbA, i.e., only HbA binds with the first capture monoclonal antibody at the second capture zone 112.

[0032]

[0027] The binding of HbS with the third capture monoclonal antibody, the dual bindings of HbA with the second capture monoclonal antibody and HbS with the third capture monoclonal antibody, the dual bindings of HbS with the third capture monoclonal antibody and HbC with the fourth capture monoclonal antibody, or the dual bindings of HbA with the second capture monoclonal antibody and HbC with the fourth capture monoclonal antibody may be visualized. The visualization is deemed possible due to binding of the at least one haemoglobin variant of the set of haemoglobin variants with the detector antibody that is conjugated with colloidal gold nanoparticles. The at least one haemoglobin variant may bind with the detector antibody when the analyte flows through the gold pad 104. Thus, the colloidal gold nanoparticles facilitate visualization of the binding of each of the at least one haemoglobin variant of the set of haemoglobin variants with specific capture monoclonal antibody of the set of capture monoclonal antibodies.

[0033]

[0028] FIG. 2A illustrates a process flow of screening a whole blood sample of a subject for diagnosing sickle cell disease, wherein the whole blood sample is obtained by means of capillary whole blood collection, in accordance with an embodiment of the present disclosure. Herein, FIG. 2A is described in conjunction with the elements of FIG. 1. As shown, the screening isperformed using an immunochromatographic assay kit. The immunochromatographic assay kit includes an immunochromatographic assay device 202, a testing buffer 204, a buffer container 206 containing the testing buffer 204, a sample collection means 208, a lancet (not shown), and a swab (not shown). Based on the screening, it is determined whether the subject 200 has sickle cell disease. As shown in FIG. 2A, the whole blood sample is collected using finger prick capillary. Alternatively, the whole blood sample may be collected using heel prick capillary. The immunochromatographic assay kit at a temperature range 2-30°C (degree Celsius). If the immunochromatographic assay kit is stored in a temperature range 2-8 °C, components of the immunochromatographic assay kit (the immunochromatographic assay device 202, the testing buffer 204, the buffer container 206, the sample collection means 208, the lancet, and the swab) are required to be brought to room temperature prior to their usage (i.e., for screening). The components of the immunochromatographic assay kit should neither be frozen nor exposed to temperatures over 30°C.

[0034]

[0029] The testing buffer 204 may include a haemolysis agent, a buffering agent, a blocking agent, a saline agent, and a preservative agent. In accordance with an embodiment, the haemolysis agent may have a molar concentration in a range of 0.005-5 moles. The haemolysis agent comprises sodium dodecyl sulphate and triton x-100. Both sodium dodecyl sulphate and triton X 100 are anionic detergents that play a vital role for lysis of red blood corpuscles in the whole blood sample without denaturing of haemoglobin variants in the whole blood sample. Optionally, the haemolysis agent is in a concentration range of 0.005-10% of the testing buffer 204. The buffering agent may have a molar concentration in a range of 0.005-5 moles. The buffering agent is either a tris buffer, a borate buffer, or a HEPES buffer (4-(2-hy droxy ethyl)- 1-piperazineethanesulfonic acid buffer). The tris buffer is compatible with biological fluids (such as the whole blood sample). The pH of the tris buffer is usually stable at 8.5. The pH of the tris buffer increases probability of interaction between haemoglobin variants present in the whole blood sample and capture monoclonal antibodies.

[0035]

[0030] The blocking agent may have a concentration in a range of 0.005 to 15 %. The blocking agent is one or more of casein sodium salt, foetal bovine serum, foetal goat serum, newborn calf serum, or horse serum. Both casein and foetal bovine serum act as blocking agents that prevent non-specific binding. The blocking agent ensures that specific haemoglobin variants bind with specific capture monoclonal antibodies that have binding affinities for the specific haemoglobin variants. Optionally, the blocking agent is in a concentration range of 0.005-15% of the testing buffer 204. The saline agent may have a molar concentration in a range of 0.1-1 M. The salineagent is either sodium chloride or potassium chloride. Sodium chloride may balance an ionic strength of the testing buffer 204 by preventing non-specific interactions between haemoglobin variants and capture monoclonal antibodies. The preservative agent may have a concentration in a range of 0.001 to 0.1%. The preservative agent is either sodium azide or proclin. Sodium azide may be used as the preservative agent as sodium azide increases shelf life of the testing buffer 204 by preventing microbial growth in the testing buffer 204. Optionally, the preservative agent is in a concentration range of 0.001-0.1% of the testing buffer 204.

[0036]

[0031] As shown, the process flow begins with cleaning an area, i.e., a fingertip of the subject 200, that is to be lanced. Alternatively, the area may be a heel. The area is cleansed using the swab. In accordance with an embodiment, the swab may be an alcohol swab. Once the area (i.e., the fingertip or the heel) is thoroughly cleansed, the area may be squeezed. Thereafter, the fingertip or the heel may be pricked with the sterile lancet. The lancet is safely disposed after droplets of whole blood begin to ooze from the fingertip or the heel. A few initial drops of whole blood oozing from the fingertip or the heel may be wiped with a sterile gauze. Thereafter, the sample collection means 208, i.e., a specimen transfer device, is used for collecting a predefined quantity of whole blood from the bleeding site. For example, the predefined quantity of whole blood sample, collected using the sample collection means 208, is 5 pL, for example. The sample collection means 208 may be a capillary tube. Once 5 pL of whole blood sample is collected, the whole blood sample is transferred to the buffer container 206 (such as a buffer tube) that is prefilled with the testing buffer 204. The buffer container 206 may contain a premeasured volume of the testing buffer 204. Furthermore, the buffer container 206 may include a nozzle 210 that protects the testing buffer 204 from coming in contact with atmospheric air.

[0037]

[0032] For transferring the whole blood sample to the buffer container 206, the nozzle 210 is removed from the buffer container 206 and a tip of the sample collection means 208 is immersed into the testing buffer 204 contained in the buffer container 206. Thereafter, the collected whole blood sample (5 pL) is dispensed into the testing buffer 204. After the whole blood sample is dispensed into the testing buffer 204, the sample collection means 208 may be disposed as the sample collection means 208 is a waste that is classified in bio-hazard waste category. Subsequently, the nozzle 210 is secured on top of the buffer container 206 and the buffer container 206 is inverted at least 3-4 times to ensure that the whole blood sample has thoroughly mixed with the testing buffer 204. The mixing results in obtaining an analyte 212. The nozzle 210 may include a dropper cap 214 that may be removed after the analyte 212 is obtained (i.e., the mixing is completed).

[0033] The immunochromatographic assay device 202 includes a sample well 216 onto which a predetermined quantity of the analyte 212 is dropped. The removal of the dropper cap 214 allows dropping the predetermined quantity of the analyte 212 onto the sample well 216. For example, the predetermined quantity of the analyte 212 dropped onto the sample well 216 may be four drops. Once the predetermined quantity of the analyte is dropped, the analyte 212 contacts a test strip (such as the test strip 100) in the immunochromatographic assay device 202. The analyte 212 laterally flows along a length of the test strip. A portion of the test strip of the immunochromatographic assay device 202 is visible through a window 218 (namely the second opening of the housing as mentioned in FIG. 1) in the immunochromatographic assay device 202. The visible portion of the test strip is a nitrocellulose membrane where haemoglobin variants present in the whole blood sample interact with a set of capture monoclonal antibodies.

[0038]

[0034] A given capture monoclonal antibody of the set of capture monoclonal antibodies is immobilized at a corresponding capture zone of a set of capture zones within the nitrocellulose membrane. The set of capture zones is visible through the window 218. The interaction causes each haemoglobin variant present in the whole blood sample to bind with a specific capture monoclonal antibody of the set of capture monoclonal antibodies. The determination of whether the subject 200 has sickle cell disease is based on the binding of each haemoglobin variant present in the whole blood sample with a specific capture monoclonal antibody of the set of capture monoclonal antibodies. The binding may be visualized as one or more line, such as straight lines, that appear within the window 218 as the interaction between the haemoglobin variants present in the whole blood sample and the set of capture monoclonal antibodies takes place. Based on the visualization of the one or more straight lines, results of the screening are required to be interpreted. The results may be required to be interpreted after elapsing of ten minutes since the analyte 212 is dropped onto the sample well 216.

[0039]

[0035] FIG. 2B illustrates a process flow of screening a whole blood sample of a subject for diagnosing sickle cell disease, wherein the whole blood sample is obtained by means of venous whole blood collection, in accordance with an embodiment of the present disclosure. FIG. 2B is described in conjunction with the elements of FIG. 1 and FIG. 2A. As shown in FIG. 2B, the whole blood sample may be collected by a trained and qualified medical professional 220 using venipuncture. Alternatively, the whole blood sample may be collected by the subject using venipuncture. This involves cleaning a portion of an arm of the subject 200. The portion of the arm may refer to a region around a vein which is to be punctured for collection of a predefined quantity of whole blood sample. For example, the predefined quantity of whole blood sample is2.5 to 5 pL. After the portion of an arm is cleansed using the swab (for example, alcohol swab), the vein is punctured using the sample collection means 208. The sample collection means 208 may be a syringe. The venipuncture allows collecting the predefined quantity (for example, 2.5 to 5 pL) of whole blood sample into the syringe (i.e., the sample collection means 208). Once the whole blood sample is collected, the whole blood sample is transferred from the syringe (i.e., the sample collection means 208) to anticoagulant vial 208A, the syringe is disposed as the syringe is a waste that is classified in bio-hazard waste category. Now collect 2.5 to 5 pL whole blood from anticoagulant vial 208A to the buffer container 206 (such as the buffer tube) that is prefilled with the premeasured volume of the testing buffer 204. The transfer involves removing the nozzle 210 from the buffer container 206, immersing the specimen transfer devices into the buffer container 206, and dispensing the 2.5 to 5pL of the collected whole blood sample into the testing buffer 204. The collected whole blood sample is dispensed into the testing buffer 204 by the pressing specimen transfer devices (i.e., the sample collection means 208) such that the collected whole blood sample is forced out of a barrel of the specimen transfer devices into the testing buffer 204. After the whole blood sample is dispensed into the testing buffer 204. Subsequently, the nozzle 210 may be secured on top of the buffer container 206 and the buffer container 206 is inverted at least 3-4 times to ensure that the whole blood sample has thoroughly mixed with the testing buffer 204. The mixing results in obtaining an analyte 212. The nozzle 210 may include a dropper cap 214 that may be removed after the analyte 212 is obtained.

[0040]

[0036] Subsequent actions involved in the screening (after obtaining the analyte 212), for determination of whether the subject 200 has sickle cell disease, is identical to that discussed in FIG. 2A. For the sake of brevity, the contents of FIG. 2A have not been repeated.

[0041]

[0037] FIGs. 3A-3E illustrate various outcomes of sickle cell disease tests obtained using an immunochromatographic assay device as discussed above, in accordance with an embodiment of the present disclosure. FIGs. 3A-3E are described in conjunction with the elements of FIG. 1, FIG. 2A, and FIG. 2B. With reference to FIGs. 3A-3E, there are shown five possible outcomes of a sickle cell disease test that is performed using the immunochromatographic assay device 202. The sample well 216 of the immunochromatographic assay device 202 may receive a predetermined quantity of an analyte obtained by mixing a whole blood sample with a testing buffer. The predetermined quantity of the analyte may laterally flow through a length of a test strip of the immunochromatographic assay device 202 for a pre-defined period of time. As the analyte laterally flows through the length of the test strip, the analyte may interact with a set of capture monoclonal antibodies. Based on the interaction, presence or absence of the sickle celldisease in a subject (such as the subject 200) is determined. A particular capture monoclonal antibody of the set of capture monoclonal antibodies is immobilized at a corresponding capture zone of a set of capture zones in a nitrocellulose membrane of the test strip. The nitrocellulose membrane can be visualized through the window 218.

[0042]

[0038] A particular capture monoclonal antibody immobilized at a given capture zone has a binding affinity with a specific haemoglobin variant of a set of haemoglobin variants in the analyte (i.e., the whole blood sample). The set of haemoglobin variants include HbA, HbS, and HbC. The sickle cell disease in the subject is detected based on binding of at least one haemoglobin variant of the set of haemoglobin variants present in the analyte with at least one capture monoclonal antibody of the set of capture monoclonal antibodies during the interaction. As shown in FIG. 3A, the at least one haemoglobin variant present in the analyte is HbA. A first capture monoclonal antibody of the set of capture monoclonal antibodies, immobilized at a given capture zone, may bind with HbA. This is because the first capture monoclonal antibody has a binding affinity with HbA. In this scenario, sickle cell disease in the subject is ruled out.

[0043]

[0039] As shown in FIG. 3B, the at least one haemoglobin variant present in the analyte includes HbA and HbS. The first capture monoclonal antibody may bind with HbA. A second capture monoclonal antibody of the set of capture monoclonal antibodies, immobilized at a given capture zone, may bind with HbS. This is because the second capture monoclonal antibody has a binding affinity with HbS. As shown in FIG. 3C, the at least one haemoglobin variant present in the analyte is HbS. The second capture monoclonal antibody may bind with HbS. As shown in FIG. 3D, the at least one haemoglobin variant present in the analyte includes HbA and HbC. The first capture monoclonal antibody may bind with HbA. A third capture monoclonal antibody of the set of capture monoclonal antibodies, immobilized at a given capture zone, may bind with HbC. This is because the third capture monoclonal antibody has a binding affinity with HbC. As shown in FIG. 3E, the at least one haemoglobin variant present in the analyte includes HbS and HbC. The second capture monoclonal antibody may bind with HbS. The third capture monoclonal antibody may bind with HbC. In the scenarios illustrated in FIGs 3B-3E, the subject is diagnosed with sickle cell disease and effective measures may be suggested.

[0044]

[0040] Additionally, in FIGs. 3A-3E, a fourth capture monoclonal antibody of the set of capture polyclonal antibodies, immobilized at a given capture polyclonal antibodies, has a binding affinity with Chicken IgY gold conjugate. The fourth capture Polyclonal antibody functions as Ctrl for the immunochromatographic assay device and binds with chicken IgY gold conjugate.Furthermore, the binding of HbA with the first capture monoclonal antibody, the binding of HbS with the second capture monoclonal antibody, the binding of HbC with the third capture monoclonal antibody, and the binding of at least one of HbA, HbS, or HbC with the fourth capture monoclonal antibody, is visualized on the window 218 based on the binding between the at least one of HbA, HbS, or HbC with a detector antibody conjugated with the colloidal gold nanoparticles in a gold pad of the test strip. In accordance with an embodiment, the bindings between the at least one haemoglobin variant of the set of haemoglobin variants present in the analyte and the at least one capture monoclonal antibody of the set of capture monoclonal antibodies are visible as straight lines of reddish-purple colour.

[0045]

[0041] Modifications to embodiments of the present disclosure described in the foregoing are possible without departing from the scope of the present disclosure as defined by the accompanying claims. Expressions such as "including", "comprising", "incorporating", "have", "is" used to describe, and claim the present disclosure are intended to be construed in a nonexclusive manner, namely allowing for items, components or elements not explicitly described also to be present. Reference to the singular is also to be construed to relate to the plural. The word "exemplary" is used herein to mean "serving as an example, instance or illustration". Any embodiment described as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments and / or to exclude the incorporation of features from other embodiments. The word "optionally" is used herein to mean "is provided in some embodiments and not provided in other embodiments". It is appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable combination or as suitable in any other described embodiment of the disclosure.

[0046] EXPERIMENTAL PART

[0047]

[0042] Tables 1, 2, 3 and 4 below depicts data for sickle cell anaemia testing buffer trials 1-4. As shown, Trial 1 shows different pH concentration of Existing Buffer. Trials 2, 3 and 4 show different buffer compositions with tris buffer. As shown, Existing Buffer gave better results compared to all the trials.

[0048]

[0049]

[0050] Table 1

[0051]

[0052]

[0053] Table 2

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] Table 4

Claims

We claim:

1. An immunochromatographic assay device (202) for detecting sickle cell disease in a subject (200), the immunochromatographic assay device (202) comprising:a sample well (216) for reception of a predetermined quantity of an analyte (216) obtained by mixing of a whole blood sample with a testing buffer (204);a test strip (100) comprising a sample pad (102), a gold pad (104), a nitrocellulose membrane (106), and an absorbent pad (108) sequentially arranged along a length of the test strip (100), wherein the analyte (216), received in the sample well (216), is allowed to laterally flow through the length of the test strip (100) for a pre-defined period of time; anda set of capture monoclonal antibodies, wherein a given capture monoclonal antibody of the set of capture monoclonal antibodies is immobilized at a corresponding capture zone of a set of capture zones in the test strip (100), and wherein a given capture monoclonal antibody immobilized at a given capture zone has a binding affinity with a specific haemoglobin variant of a set of haemoglobin variants in the analyte, and wherein the sickle cell disease is detected based on binding of at least one haemoglobin variant of the set of haemoglobin variants with at least one capture monoclonal antibody of the set of capture monoclonal antibodies.

2. The immunochromatographic assay device (202) as claimed in claim 1, wherein the set of capture antibodies includes a first capture polyclonal antibody that has a binding affinity with chicken IgY gold conjugate haemoglobin variant-A (HbA), a second capture monoclonal antibody that has a binding affinity with haemoglobin variant-S (HbS), and a third capture monoclonal antibody that has a binding affinity with haemoglobin variant-C (HbC), and wherein the first capture monoclonal antibody, the second capture monoclonal antibody, and the third capture monoclonal antibody are immobilized at different equidistant capture zones of the set of capture zones within the nitrocellulose membrane (106).

3. The immunochromatographic assay device (202) as claimed in claim 2, wherein the set of capture monoclonal antibodies further includes a fourth capture monoclonal antibody immobilized in a capture zone (110) within the nitrocellulose membrane (106), and wherein the fourth capture monoclonal antibody has a binding affinity with an antigen that is always present in an analyte (212), and wherein the fourth capture polyclonal antibody functions as a control for the immunochromatographic assay device (202).

4. The immunochromatographic assay device (202) as claimed in claim 1, wherein the gold pad(104) comprises colloidal gold nanoparticles, and wherein the colloidal gold nanoparticles facilitate visibility of the binding of at least one haemoglobin variant of the set of haemoglobin variants with at least one capture monoclonal antibody of the set of capture monoclonal antibodies.

5. The immunochromatographic assay device (202) as claimed in claim 4, wherein the colloidal gold nanoparticles are conjugated with a detector antibody, and wherein the detector antibody has a binding affinity with HbA, HbS, and HbC.

6. The immunochromatographic assay device (202) as claimed in claim 1, wherein the sample pad (102), the gold pad (104), the nitrocellulose membrane (106), and the absorbent pad (108) are arranged in a linear array in the test strip (100), and wherein a length of the sample pad (102) is 19 millimetres (mm), a length of the gold pad (104) is 7 mm, a length of the nitrocellulose membrane (106) is 25 mm, and a length of the absorbent pad (108) is 20 mm, and wherein the sample pad (102) and the gold pad (104) have a 1 mm overlap, the gold pad (104) and the nitrocellulose membrane (106) have a 2 mm overlap, and the nitrocellulose membrane (106) and the absorbent pad (108) have a 5 mm overlap, and wherein a width of the test strip (100) is in the range 3.5-3.7 mm.

7. The immunochromatographic assay device (202) as claimed in claim 1, wherein the testing buffer (204) comprises: a haemolysis agent, a buffering agent, a blocking agent, a saline agent, and a preservative agent.

8. The immunochromatographic assay device (202) as claimed in claim 7, whereinthe haemolysis agent comprises sodium dodecyl sulphate and triton x-100,the buffering agent is one of: a tris buffer, a borate buffer, or a HEPES buffer,the blocking agent is one or more of: casein sodium salt, foetal bovine serum, foetal goat serum, newborn calf serum, or horse serum,the saline agent is one of: sodium chloride or potassium chloride, andthe preservative agent is one of: sodium azide or proclin.

9. The immunochromatographic assay device (202) as claimed in claim 7, whereinthe haemolysis agent has a concentration in a range of 0.005-5 %;the buffering agent has a concentration in a range of 0.005-5 %;the blocking agent has a concentration in a range of 0.005 - 15 %;the saline agent has a molar concentration in a range of 0.1 to IM; andthe preservative agent has a concentration in a range of 0.001 - 0.1 % M.

10. A method for detecting sickle cell disease in a subject (200) using an immunochromatographic assay device (202), the method comprising:receiving, on a sample well (216) of the immunochromatographic assay device (202), a predetermined quantity of an analyte (212) that is obtained by mixing a whole blood sample with a testing buffer (204);allowing the received predetermined quantity of analyte (212) to laterally flow through a length of a test strip (100) of the immunochromatographic assay device (202) for a pre-defined period of time, wherein the test strip (100) comprises a sample pad (102), a gold pad (104), a nitrocellulose membrane (106), and an absorbent pad (108) sequentially arranged along the length of the test strip (100); andallowing the analyte (212) to interact with a set of capture monoclonal antibodies to determine presence or absence of the sickle cell disease in the subject (200), wherein a given capture monoclonal antibody of the set of capture monoclonal antibodies is immobilized at a corresponding capture zone of a set of capture zones in the test strip (100), and wherein a given capture monoclonal antibody immobilized at a given capture zone has a binding affinity with a specific haemoglobin variant of a set of haemoglobin variants in the analyte (212), and wherein the set of haemoglobin variants include a haemoglobin variant- A (HbA), a haemoglobin variant-S (HbS), and a haemoglobin variant-C (HbC), and wherein the sickle cell disease in the subject (200) is detected based on binding of at least one haemoglobin variant of the set of haemoglobin variants with at least one capture monoclonal antibody of the set of capture monoclonal antibodies.

11. The method as claimed in claim 10, wherein the lateral flow of the analyte (212) through the length of the test strip (100) facilitates:binding between the at least one haemoglobin variant and a detector antibody that is conjugated with colloidal gold nanoparticles in the gold pad (104), andbinding between the at least one haemoglobin variant and the at least one capture monoclonal antibody of the set of capture monoclonal antibodies.

12. The method as claimed in claim 11, wherein the binding of the at least one haemoglobin variant with the at least one capture monoclonal antibody is visualized on a window (218) in the immunochromatographic assay device (202), and wherein the visualization is based on the binding between the at least one haemoglobin variant and the detector antibody conjugated with the colloidal gold nanoparticles.

13. The method as claimed in claim 10, wherein the method further comprises treating thesample pad (102) in the test strip with one or more reagents to ensure a pH of the analyte (212) is in a range 7.5-9.5 after a predetermined quantity of the analyte (212) is received on the sample well (216).

14. An immunochromatographic assay kit comprising:an immunochromatographic assay device (202) of claims 1-9;a testing buffer (204) of claims 1-9;a buffer container (206) containing the testing buffer;a sample collection means (208);a lancet; anda swab.

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