DESC:IN-VITRO DIAGNOSTIC MEASUREMENT METHOD FOR DETECTING INSULIN AND ANTIBODIES AGAINST INSULIN/ INSULIN ANALOGUES AND KIT THEREFOR

FIELD OF THE INVENTION

[001] The present invention relates generally to detection of insulin and antibodies against insulin/insulin analogues, and, more particularly to in-vitro diagnostic (IVD) measurement method and kit for determining the levels of insulin and antibodies against insulin/insulin analogues in diabetic patients.

BACKGROUND OF THE INVENTION

[002] Diabetes epidemic is on the rise throughout the world. Particularly, in India, nearly 67 million people are diagnosed to be diabetic and another 30 million people are classified into pre-diabetic groups. Diabetes is a metabolic lifestyle disorder where lack of Insulin production in the body leads to insulin dependent diabetes mellitus (IDDM)/ Type 1. In Type2 diabetes/ NIDDM, the pancreas does not secrete enough insulin or the body cannot use the insulin well enough. When there is insufficient insulin or the insulin is not used as it should be, glucose (sugar) cannot get into the body's cells. When glucose builds up in the blood instead of going into cells, the body's cells are not able to generate energy and function properly.

[003] Generally, the diagnosis of diabetes is done by indirectly measuring the glucose level (using for example, fasting plasma glucose, oral glucose tolerance test, random plasma glucose, and similar tests) in blood. Overall (long term glycaemic index) control of diabetes is generally measured using the glycated haemoglobin content (HbA1c) in blood. However, routine tests are not conducted: (i) to measure directly whether or not patient has/secretes sufficient amount of endogenous insulin especially in Type 2 non-insulin dependent diabetic patients; and (ii) in the case of Type 1 insulin dependent diabetic patients whether they have developed anti-insulin antibodies to determine the loss of efficacy of the insulin/ insulin analogues in use. Doctors manage the diabetes control mostly by relying on the glucose level and HbA1c level to change the dose up or down or interchange to other brands of Insulin or switch patients to other insulin analogues on a trial and error basis.

[004] Type 2 patients eventually may require external Insulin to manage their diabetes and current method of detection and quantitation of endogenous Insulin secretion in patient blood is time consuming and expensive for routine monitoring. A simple rapid point of care method to determine the Insulin level (above or below semi-quantitative threshold) will avoid the use of excess Insulin leading to hypoglycaemia and also negate the risk of inducing the immune system in developing anti-Insulin antibodies.

[005] Recombinant Insulin are produced from human genes in Prokaryotic and Eukaryotic (Eg: E.coli, Yeast or other) expression systems. Insulin molecule is a 51 amino acid peptide hormone with approximate molecular weight of 6 kilo Dalton that is identical to the endogenous naturally produced human Insulin. It has two subunits comprising A (Alpha) & B (Beta) chain which are linked by two disulfide bonds with no repeating structures. There are various types of Insulin analogues that are classified based on how fast it acts, when it reaches peak level and how long the effect last. Insulin Lispro, Insulin Glargine and Insulin Aspart are the most common analogues that are commercially available for diabetes management which has mutation in the B chain of Insulin and in case of Glargine it has mutation in the A chain and B chain of Insulin.

[006] Endogenous secretion of inactive precursor Insulin comprises of C-Peptide which links the A-chain and B-chain of Insulin. Insulin hormone becomes functional and active upon processing by removal of the C-peptide leading to active conformation of Insulin A & B chain which are linked by two disulfide bonds with no repeating structures. C-peptide and A & B chain of Insulin are equal in mol to mol ratio, and it is not present in the recombinant Insulin drug and hence C-peptide has been used as surrogate marker for determining the endogenous secretion of Insulin levels.

[007] Type 1 Diabetic (T1D) patients depend on a daily insulin injection for their survival. If body of such patients develop anti-insulin antibodies that neutralize the injected insulin, then glucose control will be lost. Such patients will develop complications that may prove fatal.

[008] There is a substantial shortage in availability of diagnostic kits, systems and methods for timely and reliable detection of endogenous insulin secretion and neutralizing anti-insulin antibodies and can be categorized as an unmet medical need in major parts of the world. In some developed countries, expensive Radio Immunoassay for direct Insulin quantification or ELISA for indirectly quantifying the C- peptide levels and cell based NAb assay are used for such determinations. However, these procedures are laborious, time consuming and not affordable.

[009] Sensitive techniques such as RIA, RIPA and cell based-Neutralizing antibody assays are used during clinical trial for assessing the safety of biologics diabetic drug under evaluation. Such quantitative and semi-quantitative techniques involve use of radioactive material and high end complex and expensive equipments. Such equipments require well trained technicians to quantify / semi-quantify insulin and anti-insulin antibodies. Other non-radioactive quantitative techniques such as immunoassay ELISA, chemiluminescence assay are also in use; however they need minimum 4-6 hours to get test results.

[0010] Accordingly, there exists a need for diagnostic methods that provides for a simple, quick, user-friendly, cost efficient and reliable detection of insulin and antibodies against insulin/insulin analogues. Also, what is required is a companion diagnostic kit for diabetes management that is simple and affordable in line with the glucose and HbA1c level measuring kits where doctors could qualitatively detect the presence or absence of Insulin secretion and also antibodies against insulin/insulin analogues in a rapid manner, enabling doctors and patients to make an informed decision on efficiently managing the diabetes of a patient.

SUMMARY OF THE INVENTION

[0011] In view of the foregoing shortcomings inherent in the prior-arts, the general purpose of the present invention is to provide an improved, ingenious method of insulin and anti-insulin antibody detection and a measurement method/ kit therefor that is configured to include all advantages of the prior art and to overcome the drawbacks inherent in the prior art offering some added advantages.

[0012] The present invention provides an in-vitro diagnostic (IVD) measurement method /kit for detecting insulin and antibodies against insulin/insulin analogues in a simple, quick, user-friendly, cost efficient and reliable manner. The IVD method of the present invention works on the principle of specific recognition of an (re)agent by a sample containing insulin and/or antibodies against insulin/insulin analogues. The IVD method of the present invention is developed on the principle of antigen and antibody interaction to monitor the presence or absence of insulin and antibodies against insulin and insulin analogues.

[0013] Further, the IVD method can also be employed as a single use stand-alone kit. Alternatively, the IVD method can be multiplexed with other existing diagnostic kits/biomarker detection kits. For example, the IVD kit of the present invention can be multiplexed as a subcomponent of an existing diagnostic kit that uses similar assay format.

[0014] In one aspect, the present invention provides an in-vitro diagnostic (IVD) kit for detecting insulin, in a serum/plasma sample. The kit comprises a sample holder having a well for holding the serum/plasma sample. Further, the kit comprises a nitrocellulose/PVDF membrane immobilized with anti-insulin monoclonal /polyclonal antibodies thereon, and arranged within or in-range of the sample holder such that when sample containing insulin is loaded in the well of the sample holder, the nitrocellulose / PVDF membrane is exposed to the sample. The unbound region of the nitrocellulose/PVDF membrane is blocked with blocking buffer containing 2% bovine serum albumin (BSA) or other blocking agents to prevent other proteins of the serum sample binding to the nitrocellulose/PVDF membrane. Furthermore, the kit comprises of specific detection reagent such as monoclonal /polyclonal Insulin specific antibodies which are conjugated to enzymes (Horseradish peroxidase, Alkaline phosphatase), dyes (Fluorescence, Chemiluminescence) or some elements such as gold, silver or copper .Upon binding of the specific detection reagents to the antigen and antibody complex signal amplification was possible by the enzyme substrates colorimetric reaction to form visible product as precipitate, emit light upon excitation of fluorescent / Luminol /ruthenium dyes, become visible to naked eye upon concentration of metals/ Quantum dots in the membrane. Specifically, the intensity of the colour developed of the conjugate reaction/activity is directly proportional to the concentration of the insulin.

[0015] In another aspect, the present invention provides an in-vitro diagnostic (IVD) kit for detecting anti-insulin antibodies, in a serum/plasma sample. The kit comprises a sample holder having a well for holding the serum/plasma sample. Further, the kit comprises a nitrocellulose/PVDF membrane immobilized with insulin (antigen) and its analogues (Lispro, Aspart & Glargine) thereon, and arranged within or in-range of the sample holder such that when sample containing anti-insulin antibodies is loaded in the well of the sample holder, the nitrocellulose / PVDF membrane is exposed to the sample. The unbound region of the nitrocellulose/PVDF membrane is blocked with blocking buffer containing 2% bovine serum albumin (BSA) or other blocking agents to prevent other proteins of the serum sample binding to the nitrocellulose/PVDF membrane. Furthermore, the kit comprises of broad detection reagents such as anti-human Immunoglobulins and protein A, G, L which are conjugated to enzymes (Horseradish peroxidase, Alkaline phosphatase), dyes (Fluorescence, Chemiluminescence, Ruthenium) or some elements such as gold, silver or copper .Upon binding of the detection reagents to the antigen and antibody complex signal amplification was possible by the enzyme substrates colorimetric reaction to form visible product as precipitate, emit light upon excitation of fluorescent / Luminol /ruthenium dyes, become visible to naked eye upon concentration of metals / Quantum dots in the membrane. . Specifically, the intensity of the colour developed of the conjugate reaction/activity is directly proportional to the concentration of the anti-insulin antibodies.

[0016] In another aspect, the present invention provides an in-vitro diagnostic (IVD) method for detecting analogue specific B-chain antibodies in a serum/plasma sample. The method includes immobilizing a nitrocellulose/PVDF membrane with any one of analogues (Lispro, Aspart & Glargine) thereon, and arranged within or in-range of the sample holder such that when sample containing anti-insulin and analogue specific antibodies is loaded in the well of the sample holder, the nitrocellulose / PVDF membrane is exposed to Immobilized Insulin antigens which acts as a trap in capturing anti-Insulin antibodies (mainly A—chain specific) and allow the analogue specific B-chain antibodies to migrate and bind to respective analogues. The unbound region of the nitrocellulose/PVDF membrane is blocked with blocking buffer containing 2% bovine serum albumin (BSA) or other blocking agents to prevent other proteins of the serum sample binding to the nitrocellulose/PVDF membrane. Furthermore, the kit comprises of broad detection reagents such as anti-human Immunoglobulins and protein A, G, L that recognize human IgG’s which are conjugated to enzymes (Horseradish peroxidase, Alkaline phosphatase), dyes (Fluorescence, Chemiluminescence, Ruthenium) or some elements such as gold, silver ,copper and quantum dots .Upon binding of the detection reagents to the antigen and antibody complex signal amplification was possible by the enzyme substrates reaction to form visible product as precipitate, emit light upon excitation of fluorescent / Luminol /Ruthenium dyes, become visible to naked eye upon concentration of metals / Quantum dots in the membrane. . Specifically, the intensity of the colour developed of the conjugate is directly proportional to the concentration of the anti-insulin antibodies.

[0017] These together with other aspects of the invention, along with the various features of novelty that characterize the invention, are pointed out with particularity in the claims annexed hereto and forming a part of this disclosure. For a better understanding of the invention, its operating advantages and the specific objects attained by its uses, reference had to the accompanying drawings and descriptive matter in which there are illustrated exemplary embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] While the specification concludes with claims that particularly point out and distinctly claim the invention, it is believed that the advantages and features of the present invention will become better understood with reference to the following more detailed description of expressly disclosed exemplary embodiments taken in conjunction with the accompanying drawings. The drawings and detailed description which follow are intended to be merely illustrative of the expressly disclosed exemplary embodiments and are not intended to limit the scope of the present invention as set forth in the appended claims. In the drawings:

[0019] FIG.1 illustrates sequence of human insulin and insulin analogues with receptor binding amino acids highlighted in orange and mutation of the analogues boxed in the C terminus of A and B chain;

[0020] FIG.2 illustrates Insulin and insulin receptor complex formation through specific amino acid residues highlighted in blue in Insulin and antibody complexed to insulin potentially cause steric hinderance in binding of the insulin ligand to its insulin receptor effectively;

[0021] FIG.3 illustrates an embodiment of the present invention showing a sandwich immunoassay based method for detection of insulin;

[0022] FIGS. 4 and 6 illustrates another embodiment of the present invention showing a kit for detection of antibodies against insulin;

[0023] FIGS. 5 and 7 illustrates yet another embodiment of the present invention showing a kit for specific detection of antibodies against insulin analogues especially to the B chain C-terminal region which differ from parent Insulin sequence;

[0024] FIG. 8 illustrates an analogue specific antibody purification method;

[0025] FIGS. 9 and 10 illustrate indirect ELISA used for demonstrating the specificity and sensitivity of the Insulin and analogue specific antibodies; and

[0026] FIG. 11 illustrates double immunodiffusion demonstrating the specificity of the antibodies to the respective antigen.

[0027] FIG 12 illustrates the immunoassay format used in detecting the presence or absence of anti-Insulin antibodies in Type 1 diabetic patients samples both in ELISA as well as in the dot blot technique; and

[0028] FIG. 13 Illustrates the immunoassay format used in determining the level of insulin in Type 1 diabetic patient samples both in ELISA as well as in the dot blot technique.

[0029] Like reference numerals refer to like parts throughout the several views of the drawings.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The exemplary embodiments described herein detail for illustrative purposes are subject to many variations and designs. It should be emphasized, however that the present invention is in development phase and not limited to an in-vitro diagnostic (IVD) kit and method for detecting anti-insulin antibodies as shown and described. Rather, the principles of the present invention can be used with a variety of antigen and antibody detection system. It is understood that various omissions, substitutions of equivalents are contemplated as circumstances may suggest or render expedient, but the present invention is intended to cover the application or implementation without departing from the spirit or scope of the its claims.

[0031] In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced without these specific details.

[0032] As used herein, the term ‘plurality’ refers to the presence of more than one of the referenced item and the terms ‘a’, ‘an’, and ‘at least’ do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. The term ‘device’ and ‘test, kit, cassette or assay’ has been used interchangeably and refer to mean the same thing.

[0033] The present invention provides an in-vitro diagnostic (IVD) measurement method for detecting insulin and antibodies against insulin/insulin analogues. The present invention provides simple, quick, user-friendly, cost efficient and reliable detection of insulin and antibodies against insulin/insulin analogues that can be used along with the routine Glucose test and HbA1c measurement in diabetes management. The IVD measurement method of the present invention provides for detecting insulin and antibodies against insulin/insulin analogues, thereby allowing doctors and patients to make informed decision to change the dosing regimen or switch the patients to other insulin analogues. Specifically, the IVD measurement method aids in determining whether the loss of efficacy of insulin or its analogue in patients (for example, T1DM patients) is possibly due to presence of neutralizing antibodies in patient’s serum. Accordingly, such a detection of presence or absence of antibodies against insulin/insulin analogues enable doctors to prescribe personalized medicine that will be more efficacious in controlling glucose levels.

[0034] As used herein, the IVD measurement method can be employed as an IVD kit, and accordingly the terms “IVD measurement method” and “IVD kit” are used interchangeably to refer to the method and its employment by the present invention.

[0035] The IVD kit can reveal the presence or absence of endogenous insulin and anti-insulin antibodies in a broad sense (i.e., antibodies against insulin and insulin analogues) in a very specific manner within a couple of minutes. Instead of only relying on the surrogate biomarkers (glucose/ HbA1c) for patient management, early diagnosis for the presence of insulin, can prevent the over dosing of insulin over and above the endogenous insulin which may lead to hypoglycemia and prevent the risk inducing adverse immune response to insulin.

[0036] The IVD kit of the present invention is developed on the principle of antigen and antibody interaction to reveal the endogenous insulin secretion and to monitor the presence or absence of antibodies against insulin and insulin analogues. The IVD kit function both as a qualitative and quantitative kit.

[0037] Further, the IVD kit can be used as single use stand-alone kit. Alternatively, the IVD kit can be multiplexed with other existing diagnostic kits/biomarker detection kits. For example, the IVD kit can be multiplexed as a subcomponent of an existing diagnostic kit that uses similar assay format.

[0038] For the purposes of description herein, insulin and insulin analogues are defined with regard to FIGS. 1 and 2. Although it will be evident to a person skilled in the art that all related definitions and literature on insulin and insulin analogues are applicable wherever appropriate. Recombinant human Insulin are produced from human genes in prokaryotic and eukaryotic (for example, E.coli, Yeast or other) expression systems. Insulin molecule is a 51 amino acid peptide hormone with a molecular weight of about 6 kilo Dalton that is identical to the endogenous naturally produced human Insulin. Insulin has two subunits comprising A & B chain which are linked by two disulfide bonds with no repeating structures. There are various types of insulin analogues that are classified based on how fast the analogue acts, when it reaches peak level and how long the effect lasts. As illustrated in FIG. 1, insulin lispro, insulin glargine and insulin aspart are the most common analogues that are commercially available for diabetes management.

[0039] Pancreas constitutively secrete insulin hormone in low amounts. During a meal, sugar (glucose) from food stimulates the pancreas to release insulin in proportion to the amount that is required by the size of that particular meal. Insulin's main role is to help move certain nutrients, especially sugar into the cells of the body's tissues. Cells use sugars and other nutrients from meals as a source of energy to function. The amount of sugar in the blood decreases once it enters the cells. Normally that signals the beta cells in the pancreas to lower the amount of insulin secreted so that patients don't develop low blood sugar levels (hypoglycaemia). But the destruction of the beta cells that occurs with type 1 diabetes throws the entire process into disarray. In people with type 1 diabetes, sugar isn't moved into the cells because insulin is not available. When sugar builds up in the blood (hyperglycaemia) instead of going into cells, the body's cells, followed by whole tissues and the organs starve for nutrients and energy leading to lack of power to maintain important bodily functions.

[0040] Insulin differs from its analogues only by few amino acid substitutions mainly in the C-terminal end of the B (beta) chain and in the case of glargine even in the C-terminus of the A (Alpha)-chain. Insulin binds to its insulin receptor with the help of couple of amino acids in A –Chain (G1 and V3) and B-chain (S9, Y16, G20). Insulin in a folded state has different structural entities (determinants) and some of the surface and buried (Cryptic) determinants have shown to elicit an immune response and produce antibodies upon chronic injection into humans. Antibodies to recombinant insulin are reported but appear to have no clinical significance. Referring to FIG. 2, in case if antibody response is mounted or if there is an epitope spread, such that either A chain or B chain receptor binding epitopes of the ligand are covered then it can potentially neutralize it from binding to its receptor.

[0041] In addition, the parenteral analogues with point mutation in the B- chain are not natural to insulin and hence body has tendency to recognize it as foreign determinants and induce an immune response. In a folded state any antibody that is against the C-terminal end of the B-chain can potentially cause steric hinderance in binding of the insulin ligand to its insulin receptor effectively which can also neutralize its effect ( fast, intermediate and slow acting) partially or completely.

[0042] In FIG. 2 Insulin receptor binding amino acids (blue) in the A chain (red) and B chain (Yellow) interact with the extracellular domain of the insulin receptor and trigger the signal cascade. If body mounts an immune response to the epitope comprising insulin amino acid sequences (Blue) that interact with the receptor or to the B- chain end of the analogues that carry the mutation, the insulin and its analogues will be blocked from its binding to the receptor and hence neutralize its function in vivo.

[0043] In one aspect, the present invention provides an in-vitro diagnostic (IVD) kit for detecting insulin, in a serum/plasma sample. The kit comprises a sample holder having a well for holding the serum/plasma sample. Further, the kit comprises a nitrocellulose/PVDF membrane immobilized with anti-insulin monoclonal /polyclonal antibodies thereon, and arranged within or in-range of the sample holder such that when sample containing insulin is loaded in the well of the sample holder, the nitrocellulose / PVDF membrane is exposed to the sample. The unbound region of the nitrocellulose/PVDF membrane is blocked with blocking buffer containing 2% bovine serum albumin (BSA) or other blocking agents to prevent other proteins of the serum sample binding to the nitrocellulose/PVDF membrane. Furthermore, the kit comprises of specific detection reagent such as monoclonal /polyclonal Insulin specific antibodies which are conjugated to enzymes (Horseradish peroxidase, Alkaline phosphatase), dyes (Fluorescence, Chemiluminescence, Ruthenium) or some elements such as gold, silver or copper. Upon binding of the specific detection reagents to the antigen and antibody complex signal amplification was possible by the enzyme substrates reaction to form visible product as precipitate, emit light upon excitation of fluorescent / Luminol /ruthenium dyes, become visible to naked eye upon concentration of metals / quantum dots in the membrane. Specifically, the intensity of the colour developed of the conjugate is directly proportional to the concentration of the insulin.

[0044] The IVD kit of the present invention work on the principle of specific recognition of an (re) agent by a sample containing insulin and/or antibodies against insulin/insulin analogues. For example, kits and methods those work on the principle of specific recognition of antigen by antibody and forming a colored perceptible complex in the presence of detection reagent (conjugate) on a nitrocellulose/PVDF membrane that is visible to the naked eye.

[0045] As illustrated in FIG. 3, in one embodiment, the present invention provides an IVD kit (100) for detecting insulin in a sample. The kit (100) includes a sample holder. In this embodiment based on dot blot assay, the sample holder is a cassette having well adapted on upper surface thereon for holding the sample, an absorbing means mounted on inner surface of a base of the cassette, and a blotting paper stacked over the absorbing means. In an embodiment, the absorbing means is any one of wick and sponge.

[0046] The kit also includes a nitrocellulose membrane dot blotted with Mouse Monoclonal or Guinea pig anti- Insulin B chain specific polyclonal antibodies and blocked the unbound region with blocking buffer containing 2 % bovine serum albumin (BSA). When serum samples containing insulin is added to the nitrocellulose membrane, the sample is exposed to the nitrocellulose membrane. The Guinea pig anti- Insulin B chain specific polyclonal antibodies dot blotted on the nitrocellulose membrane captures the insulin from the sample (if present) via B chain. Upon a quick rinse with wash buffer, the Mouse Monoclonal or Guinea pig anti- Insulin A chain specific antibody is conjugated to Horseradish peroxidase (HRP) and are immobilized by the insulin. Following a rinse with the wash buffer and upon addition of the tetramethylbenzidine (TMB) substrate, the enzyme Horseradish peroxidase (HRP) catalyze and the substrate forms a precipitable colored product on the membrane. The intensity of the color developed in the dot-blot is directly proportional to the concentration of the antigens i.e. insulin in the serum sample. The kit provides a simple, sensitive, rapid (3 min), and highly reproducible solid-phase bridge assay for the detection of insulin in human serum in the low nanogram (>5 ng).

[0047] As illustrated in FIGS. 4 and 5, in another embodiment, the present invention provides IVD kit (200) for detecting antibodies against insulin/insulin analogues in a serum sample. Specifically, FIG. 4 illustrates another embodiment of the IVD kit (200) based on dot blot principle for detection of antibodies against insulin. Further, FIG. 5 illustrates yet another embodiment of the IVD kit (300) of the present invention as a dot blot assay for detection of antibodies against insulin analogues.

[0048] Referring to embodiments of IVD kit (200, 300) of FIGS. 4 and 5, the kit (200, 300) includes a sample holder. In this embodiment based on dot blot assay, the sample holder is a cassette having well adapted on upper surface thereon for holding the sample, an absorbing means mounted on inner surface of a base of the cassette, and a blotting paper stacked over the absorbing means. In an embodiment, the absorbing means is any one of wick and sponge.

[0049] The kit (200, 300) also includes a nitrocellulose membrane which includes antigens, for example insulin immobilized thereon as a dot. When respective antibody containing samples are added, the antibodies in the serum sample form a complex with the antigen (insulin). Protein A bound antibody antigen complex precipitate and forms a round spot with an intensity that is visible to the naked eye. FIG. 4 illustrates the kit (200) having dot blot for reactive broad spectrum antibodies that can bind to insulin. Once, it confirmed that the samples are positive for anti- insulin antibodies, to determine whether or not the sample possess antibodies that could also bind to the B chain epitopes of analogues that carry the mutation for their unique action a second dot blot assay used.

[0050] FIG. 5 illustrates a kit (300) based on dot-blot assay where in after processing the test sample by passing through an insulin affinity columns, to deplete broad reactive anti- insulin antibodies it is possible to detect the presence of analogue specific antibodies . Even though this is a qualitative technique, the presence of anti- insulin antibody can be attributed to the loss of efficacy of the drug to certain degree. With a single kit of the present invention, it is possible determine whether the analogues also bind to the insulin antibody, thereby it is possible to conclude that negative results indicate absence of antibodies to an analogue and it is the best substitute insulin analogue drug that can be used for diabetes management.

[0051] Referring to FIG. 5 again, it illustrates the potential results that are possible when three insulin analogues can be assessed for anti-insulin antibody binding. A doctor can make an appropriate conclusion for a patient based on the results. For example, being positive on the anti-insulin antibody but negative for the three analogues, the doctor can switch to one of the three analogues. In another example, the patient had been on analogue lispro, and the patient has developed anti-lispro antibody. In this case, the samples are likely to show positive for insulin and lispro; however negative for glargine and aspart. Accordingly, the doctor can prescribe one of these two analogues to the patient for diabetes management.

[0052] As illustrated in FIGS. 6 and 7, in another embodiment, the present invention provides an IVD kit (400, 500) embodied as a lateral flow device for detecting antibodies against insulin/insulin analogues in a sample. Specifically, FIG. 6 illustrates an embodiment of the IVD kit (400) of the present invention as a lateral flow device for detection of antibodies against insulin. Next, specifically, FIG. 7 illustrates another embodiment of the IVD kit (500) of the present invention as a lateral flow device for specific detection of antibodies against insulin analogues.

[0053] Referring to FIG. 6 embodiment, the IVD (400) kit reveals the presence or absence of antibodies against insulin. If found positive, then the IVD kit (500) of FIG. 7 can be used to choose the best analogue that has not shown to have developed Antibodies to its B chain. Accordingly, the lateral flow device i.e. IVD kit (500) of FIG. 7 of the present invention comprises an inbuilt insulin trapper to trap all antibodies against the common epitope shared between insulin and insulin analogue antibodies. The IVD kit (400, 500) of embodiments of figure 6 and 7 includes a sample holder. In this embodiment, the sample holder includes plastic base having is a capillary beds. The capillary bed includes a sample well configured thereon, pieces of stacked porous blotting paper, conjugate pad, followed by sintered polymer, for example, nitrocellulose, and PVDF membranes that can immobilize the proteins (antigen or antibodies) and a wicking pad arranged in a series on the plastic base. All components in the series have the capacity to transport body fluids (e.g., serum, plasma, saliva, urine) spontaneously. The sample pad acts as a sponge and holds an excess of sample fluid and when saturated the fluid migrates to the second element (i.e.) conjugate pad containing conjugates (such as gold conjugated protein A, G, L or specific detection conjugate) that can bind to antibodies in the sample. Conjugate bound antibodies migrate laterally to the nitrocellulose membrane and if there are specific antibodies against the antigens for example, insulin, lispro, aspart, glargine that is immobilized on the test line (often called stripes), it will precipitate and form a colour. Specifically, the control line typically coated with anti human IgG and when free or IgG bound conjugate migrate to the control line, it will precipitate to show the proper functioning of the kit. After passing these reaction zones the fluid enters the final porous material, the wick that simply acts as a waste container.

[0054] Upon loading the serum into sample loading well of the sample holder, the serum component move laterally with the help of buffer and all antibodies against shared epitopes of insulin and its analogues are trapped by the insulin coated on to the membrane. If there are any analogue B chain specific antibodies are present, they laterally move along with other non-specific antibodies and bind and carry the gold conjugated protein A and precipitate as antigen and antibody complex at the region in the membrane coated with the specific analogues or synthetic analogue specific peptides.

[0055] In IVD kit (400) of FIG. 6, the absence of the insulin trapper pad will allow all reactive antibodies to insulin and its analogue to migrate to conjugate pad and then the complex form a precipitate stripe in the insulin coated nitrocellulose membrane thus revealing the presence of broad reactive (analogues also) but specific anti-insulin antibodies. On the other hand, in IVD kit (500) of in FIG. 7, the trapper pad containing excess insulin is in between the sample and conjugate pad, which will selectively trap reactive antibodies to insulin and analogues and let the analogue specific antibodies to migrate to form a mix with conjugates and then the mixture form precipitate strip line only if it is specific for the respective analogues.

[0056] In another aspect, the present invention provides an in-vitro diagnostic (IVD) method for, antibodies against insulin and insulin analogue in a serum sample. The method includes immobilizing a nitrocellulose membrane with insulin (antigen).

[0057] Further, the method includes blocking unbound region of the nitrocellulose membrane with blocking buffer containing 2% bovine serum albumin (BSA) to prevent other proteins of the serum sample from binding to the nitrocellulose membrane.

[0058] Furthermore, the method includes adding serum sample containing any one of insulin, and insulin analogue to the nitrocellulose membrane, wherein the antibodies against insulin and insulin analogue captures the insulin via B chain.

[0059] Thereafter, the method includes rinsing the nitrocellulose membrane with wash buffer, wherein the insulin antibodies and insulin analogue antibodies are immobilized by the insulin and its respective analogues.

[0060] Finally, the kit comprises of specific detection reagent such as monoclonal /polyclonal anti-human antibodies or Protein A, Protein G or Protein L which are conjugated to enzymes (Horseradish peroxidase, Alkaline phosphatase), dyes (Fluorescence, Chemiluminescence, Ruthenium, quantum dot) or some elements such as gold, silver or copper .Upon binding of the specific detection reagents to the antigen and antibody complex signal amplification was possible by the enzyme substrates reaction to form visible product as precipitate, emit light upon excitation of fluorescent / Luminol /Ruthenium dyes, become visible to naked eye upon concentration of metals in the membrane. . Specifically, the intensity of the colour developed of the conjugate is directly proportional to the concentration of the anti- insulin and anti- analogue specific antibodies.

[0061] Specifically, the insulin antibodies are any one of Guinea/human/ pig anti- insulin B chain specific antibodies and insulin analogue specific B chain antibodies.

[0062] More specifically, the insulin analogues are any one of Lispro glargine, aspart and combination thereof.

[0063] Specifically, the anti- insulin antibodies that react to Insulin and analogues and the analogue specific B chain antibodies (Insulin depleted) were differentially purified using respective analogue antigen specific affinity columns (as illustrated in FIG. 8). These anti insulin antibodies were used for the demonstration. To assess whether animals injected with Insulin(s) and its analogues mount an immune response and if so what percentage of the total IgG’s are against the common sequence of Insulin and what percentage of the antibodies are specifically against the mutation of analogues (i.e.,) B chain specific, guinea pigs were immunised with Insulin or its analogues (lispro, aspart and glargine) in separate groups using complete and incomplete adjuvants and followed a differential purification strategy to purify the antibodies from the hyperimmune serum using Insulin for negative purification and analogues for positive purification. The Immunization scheme, schedule and purification strategy is depicted below in Tables 1 and 2 and in FIG. 8.

Table 1: Immunization scheme and schedule:
Animal species and strain Guinea Pigs, Dunkin Hartley
Sex Male and Female animals
Age 4-5 Months
Body weight range 350-500 g
Total Duration of the Immunization (a+b) 79 days
a.Acclimatization period 7 days
study duration 75 days

Table 2: Immunization schedule for generation of antibodies against Insulin and analogue in Guinea pigs
Day Activity Concentration of antigen with adjuvant per animal Route of administration
Day 0 Pre bleed collection - -
Day 1 Primary Immunization 200 µg Sub cutaneous injection at 2 sites
Day 15 Booster Dose – I 150 µg Sub cutaneous injection at 2 sites
Day 30 Test Bleed I collection
Day 30 Booster Dose – II 150 µg Sub cutaneous injection at 2 sites
Day 45 Test Bleed II collection
Day 45 Booster Dose – III 150 µg Sub cutaneous injection at 2 sites
Day 60 Test Bleed III collection
Day 60 Booster Dose – IV 150 µg Sub cutaneous injection at 2 sites
Day 75 Terminal bleed (Approximately 10-12 ml serum from each animal)

[0064] The analogue specific B chains antibodies from the Guinea pig hyper immune sera were purified by passing through an Insulin column (negative /subtractive purification), to eliminate those antibodies that bind to common epitopes found in both Insulin and analogues. Unbound antibodies collected as flow through are mixture containing the B chain specific antibodies along with non-specific Guinea pig IgG was further purified by an analogue specific affinity column. The analogue specific antibodies were eluted from the affinity column and further concentrated by passing through a Protein A columns and quantified the antibody purification yield using UV 280 spectrophotometric method. In order to determine the percentage yield of anti- Insulin (A & B chain specific) and analogue (B chain) specific antibodies, the typical yield of total IgG/mL of serum ( Table 3A and 3B ) was determined by passing the hyperimmmune sera through a Protein A column.

[0065] Referring to the Tables 3A and 3B below:
Table 3A: Yield of anti- Insulin and analogue specific B chain antibodies:
Source Typical yield of Total IgG (mg/mL) Insulin A+B chain Antibodies (mg/mL) Specific B chain IgG (mg/mL)
Insulin anti sera from Guinea pig 8.6 0.16 -
Glargine anti sera from Guinea pig 8.5 0.18 0.035
Aspart anti sera from Guinea pig 9.2 0.18 0.03
Lispro anti sera from Guinea pig 8.9 0.165 0.026

Table 3B: Yield of anti- Insulin and analogue specific B chain antibodies representation in relative percentage
Source Total IgG % % of specific A+B chain antibodies % of Specific B chain IgG in total IgG % of Specific B chain IgG in A+B chain antibodies
Insulin anti sera from Guinea pig 100 1.8 - -
Glargine anti sera from Guinea pig 100 2.1 0.41 19.52
Aspart anti sera from Guinea pig 100 1.95 0.32 16.41
Lispro anti sera from Guinea pig 100 1.85 0.29 15.68

[0066] The specificity of the antibody eluate and flow through from the Insulin column were assessed for binding to Insulin and other analogues in an ELISA assay using the Indirect ELISA format (as depicted below). Further purification of analogue specific antibody was accomplished by affinity purification in their respective analogue specific affinity column. The binding specificity to Insulin and other analogues were determined by indirect ELISA (FIG. 9). The antigen and antibody binding profile is represented in the graph below, the ELISA data reveal that Guinea pig’s have developed antibodies specifically to respective analogue and most probably to the B- chain epitopes (FIG. 10). Further, the Ouchterlony technique was used to demonstrate that the purified antibodies are specific to respective analogues. Specific (analogue-B chain specific) polyclonal antibodies or complex polyclonal antibodies (Insulin A & B chain specific) were placed in a central well in an agarose slab/slide and allowed to interact with antigens (analogues and Insulin) placed in two or four peripheral wells.

[0067] Referring to FIG. 11, slide 1 demonstrate that the precipitin lines formed in a square shape touching each other indicate that the antibodies can recognize the common epitope found in insulin and all the other three analogues (lispro, aspart and glargine). Referring again to FIG. 11, slides 2, 3 and 4 precipitin lines found only close to the analogues (right well) and not to the insulin (left well) is an indication of immunological distinctness thus proving that the antibodies recognize only the linear and / or structural epitope of analogues as result of mutations introduced in the B chain C-terminal end of insulin sequence.

[0068] Further, it was interchangebly assessed the specificity and cross reactivity of the B chain specific antibodies of lispro, aspart and glargine by comparing it using the Ouchtterlony double diffusion techniques. The qualitative results of precipitin line formation as result of antigen antibody reaction in agarose slab/slide is indicated as positive(+ : reactive/binding) or negative( - : non reactive/non binding) in the table 4 below.
Table 4: Qualitative Representation of Double Immunodiffusion Data

Antigens Anti insulin antibody Anti Glargine specific antibody Anti Lispro specific antibody Anti Aspart specific antibody
Insulin + - - -
Glargine + + - -
Lispro + - + -
Aspart + - - +

[0069] The present disclosure is further elaborated to indicate the clinical utility of the inventions by way of the following examples and accompanying figures and tables herein. However, these examples should not be construed to limit the scope of the present disclosure.

[0070] For purpose of demonstration of clinical utility of IVD method, a total of 41 insulin dependent diabetic patients’ blood samples collected by accredited laboratory for diagnosis of or screening for diabetes management was used for demonstrating the detection of anti insulin antibodies and also the insulin (exogenous) in clinical samples. Residual serum samples that were collected from diabetic patients for determining the fasting blood glucose (FBG), 2 hour post load glucose (postprandial-PP), Random sugar levels have been used for demonstrating the companion diagnostic kit. Among the 41 patients, 22 are male and 19 were female and their age group ranged from 26-80 yrs. The blood glucose level at the time of testing was found to be in the range of 121-360 mg/dL, indicating that some of the patients had poor glycemic control. Blood glucose measurement is the primary means of monitoring or evaluating therapy in individuals with diabetes. Measurement of glycated proteins, primarily HbA1C used for routine monitoring of long term glycemic status in patients with diabetes was not available from the patients.

[0071] Immunoassay format as illustrated in Figure 12 was used for the detection of anti-Insulin antibodies in quantitative ELISA and qualitative dot blot technique. Standard ELISA based approach was used as an orthogonal method for determining the levels of anti Insulin antibodies and Insulin in 41 patient’s serum samples. As describes elsewhere, Dot blot assay was also performed using the 41 patient’s serum samples in parallel. Due to sample limitations, some of the samples that were true positives in the dot blot were also evaluated in the lateral flow devise.

[0072] In the indirect immunoassay format, the sample antibody is sandwiched between the antigen coated on the plate or membrane and biotin labelled anti-species globulin followed by addition of Steptavidin HRP conjugate. The addition of an enzyme substrate-chromogen reagent causes colour to develop. This colour is directly proportional to the amount of bound sample antibody. The more antibodies present in the sample, the stronger the colour development in the test sample. This format of indirect ELISA is suitable for determining total antibody level in the samples

[0073] Forty one patients serum samples with equal representation of male and female groups aged between 26-80 years, when tested for the presence of anti-Insulin antibodies (AIA), 83 % of the samples tested positive using time consuming indirect ELISA technique and quantity of the antibodies varied from patients to patients and it ranged from 250- 1000 ng/mL. Simultaneously same serum samples were also assessed using the rapid dot blot technique for the anti-Insulin antibodies; the presence of antibodies was detected visually in a qualitative manner. Nearly 39 % of the samples tested to be positive for the AIA and 88% of samples were tested to be confirmed positive for AIA in both ELISA and dot blot techniques. Summary of the findings is depicted in the table 5 as given below.

Table 5: Summary of clinical sample analysis for the detection of anti-insulin antibodies in Type 1 diabetic patient’s serum.

[0074] Forty one clinical samples from Type 1 diabetic patients were assessed for the detection of Insulin at the time of testing using conventional ELISA quantification method and the qualitative dot blot technique (Figure 13). The immunoassay format used insulin quantification by ELISA and detection by qualitative dot blot is based where, the wells of ELISA plates /membranes are coated with specific (capture) antibody followed by incubation with test solutions containing antigen. Unbound antigen is washed out and an antigen-specific antibody conjugated to biotin followed by enzyme conjugated streptavidin (i.e., developing reagent) is added, unbound conjugate is washed out and substrate is added, the degree of substrate hydrolysis is measured. The amount of substrate hydrolyzed is proportional to the amount of antigen in the test sample.

[0075] The samples were collected at various time points, such as fasting, post prandial and random and it is anticipated that insulin are most likely not endogenous Insulin and likely the residual exogenous insulin that was administered for diabetic management. The assessment was to demonstrate the proof of concept using clinical samples. Nearly 44 % patients had Insulin in the range of 4.3 to 48 ng/mL by the more sensitive quantitative ELISA technique, likewise 44 % of ELISA positive patients were also qualitatively revealed the presence of Insulin by less sensitive dot blot techniques. Summary of the clinical sample analysis by two different assay formats is depicted in the table 6 below.

Table 6: Summary of clinical sample analysis for the detection of Insulin in Type 1 diabetic patient’s serum at the time of testing

Sex n=41 Age Range Insulin ELISA Insulin ELISA Insulin Dot blot
Yrs % Range ng/mL # s % +ves
Male 22 40-80 50% 4.3-48 6/11 55
Female 19 26-75 37% 4.4-32.5 2/7 29
Overall 41 26-80 44% 4.3-48 8/18 44

[0076] For the proof of concept studies, effort was not made to qualify or validate the method. Further refinement of techniques using various detections reagents such as enzyme substrates, conjugates of gold, silver and quantum dots, and the sensitivity could be further enhanced or improved.

[0077] Although a particular exemplary embodiment of the invention has been disclosed in detail for illustrative purposes, it will be recognized to those skilled in the art that variations or modifications of the disclosed invention, including the rearrangement in the configurations of the parts, changes in sizes and dimensions, variances in terms of shape may be possible. Accordingly, the invention is intended to embrace all such alternatives, modifications and variations as may fall within the spirit and scope of the present invention.

[0078] The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and its practical application, to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omissions, substitutions of equivalents are contemplated as circumstance may suggest or render expedient, but is intended to cover the application or implementation without departing from the spirit or scope of the claims of the present invention.
,CLAIMS:WE CLAIM
1. An in vitro diagnostic (IVD) measurement method for detecting and/or quantifying an amount of human endogenous insulin and antibodies developed against recombinant Insulin and insulin analogues herein after referred as analyte in normal healthy and diabetic diseased clinical matrices sample such as serum or plasma.
2. The IVD measurement method of claim 1, wherein an assay format is used for detection and quantification of analyte in normal healthy and diabetic diseased clinical samples qualitatively or semi-quantitatively using dot blot / Lateral flow devise or quantitatively by enzyme-linked immunosorbent assay (ELISA).
3. The IVD measurement method of claim 2, where the method comprises
a sample holder for holding the serum sample,
a first capture measurement reagent being immobilised on a solid surface specifically to capture the analyte,
a means to remove the excess sample and wash buffers, and blocking of the solid surface to prevent non-specific binding of serum proteins, and
a second detection measurement reagent that specifically reacts with the analyte bound to the first capture reagent and means of revealing the analyte and capture detection reagent complex.
4. The IVD measurement method of claim 3, wherein the first capture measurement reagent is immobilized to a solid phase of nitrocellulose / PVDF membranes, protein binding ELISA plates, and wherein the second detection measurement reagent is labelled with conjugates directly or indirectly.
5. The IVD measurement method of claim 4, wherein the direct labelling is with HRP/ALP enzymes, fluorescence dyes,luminescence dyes, ruthenium, quantum dots, or gold, silver, and copper metal tags.
6. The IVD measurement method of claim 4, wherein the indirect labelling is through biotin –streptavidin HRP/ALP enzymes.
7. The IVD measurement method of claim 3, wherein the conjugate reaction or activity is colorimetric, signal emission upon at least one of excitation and visual precipitation of the product of the enzyme substrate reaction, metal and quantum dots at the site of reaction.
8. The IVD measurement method of claim 2, wherein the analyte is endogenous antibodies against Insulin and or its Insulin analogues in diseased diabetic patient samples.
9. The IVD measurement method of claim 8, wherein the insulin analogues are one selected from the group consisting of Lispro Glargine, Aspart and combination thereof.
10. The IVD measurement method of claim 3, using two types of measuring reagents, wherein
i) the first capture measurement reagent immobilized to the solid surface is Insulin and or its Insulin analogues that specifically bind to anti- insulin and analogue specific antibodies.
ii) the second detection measurement reagents is labeled/conjugated Insulin antigens, monoclonal or polyclonal anti-human Immunoglobulin antibodies and recombinant Protein A, Protein G and Protein L which react with the insulin and analogue bound antibodies.
11. The IVD measurement method of claim 8, wherein the sample is to be pretreated or exposed to remove or trap the common insulin binders, thereby enabling the capture of only analogue B chain specific antibodies by respective analogues (Lispro, Aspart and Glargine) immobilized on the solid surface.
12. The IVD measurement method of claim 2, where the analyte is endogenous Insulin in normal or diseased diabetic patient samples and the capture and detection measurement reagents are antibodies and fragment thereof that are capable of reacting to human insulin at different antigenic sites in two different chains/subunits.
13. The IVD measurement method of claim 12, using two types of antibodies, wherein
i) the first capture measurement reagent binds to one of the two Insulin chain, and
ii) the second detection measurement reagent is labeled/conjugated reacts to insulin bound to the first antibody but to the other free chain of insulin antibody complex.
14. The IVD measurement method claim 13, wherein both the first capture measurement reagent and second detection measurement reagent are one of two chain specific monoclonal antibodies or polyclonal antibodies.
15. The IVD measurement method of claim 13, wherein the first capture measurement reagent is polyclonal antibodies and the second detection measurement reagent is a monoclonal antibody.
16. The IVD measurement method of claim 13, wherein the first capture measurement reagent is a monoclonal antibody and the second detection measurement reagent is polyclonal antibodies.
17. An IVD kit employing the IVD measurement method of claim 3.