WO2016035099A1 - A flow through device for detection of multiple bioanalytes and a process thereof - Google Patents

Abstract

The present invention discloses a flow through device for detection and differentiation of multiple bioanalytes from a single sample and a process thereof. The device is a visual, rapid, sensitive and reliable immunoassay analyzer for the differential detection of multiple bioanalytes in biological fluids like human blood, serum or plasma by immobilizing the respective disease specific bio-molecules i.e. antigen, antibody, peptides, synthetic peptides or recombinant proteins derived from disease causing microorganisms on the immunofilteration reaction membrane.

Description

"A FLOW THROUGH DEVICE FOR DETECTION OF MULTIPLE

BIO ANALYTES AND A PROCESS THEREOF"

FIELD OF THE INVENTION

The present invention relates to a flow through device for detection and differentiation of multiple bioanalytes from a single sample and a process thereof. The device is a visual, rapid, sensitive and reliable immunoassay analyzer for the differential detection of multiple bioanalytes in biological _¾ fluids like human blood, serum or plasma by immobilizing the respective disease specific bio-molecules i.e. antigen, antibody, peptides, synthetic peptides or recombinant proteins derived from disease causing microorganisms on the immunofilteration reaction membrane.

BACKGROUND AND PRIOR ART

A variety of immunodiagnostic assay systems which work on the principle of flow through technology that are both rapid and sensitive are known in the prior art. These assays are generally referred to as "rapid diagnostic systems". These assays have been developed to detect or determine the concentration of specific bioanalyte present in the biological fluid sample. The detection or determination of bioanalyte occurs by binding of bioanalyte (an antigen, antibody or hapten) from the biological fluid to an antigen or antibody or disease related biomolecules conjugated on the substrate. These assays generally employ labeled reagent referred to as a tracer or detection reagent for the detection of the bioanalyte in the biological fluids. The tracer may be colloidal gold, silver, latex (microspheres of polystyrene) or enzymes such as peroxidases, hydrolases, phosphatases etc. suspended in suitable biological medium which are stable and suitable for long term storage. Sometimes these tracers are conjugated with biomolecules like protein A.

The Rapid Diagnostic Systems (RDTs) are simple, easy to use and patient compliant as these systems require less number of samples for conducting the test and they do not require any external equipment and technical expertise for operation. These systems offer many advantages over the immuno-chromatography tests (generally referred to as "lateral flow technology") like higher sensitivity, higher specificity, shorter test time for detection, avoiding the "hook effect" or the "prozone effect" and reducing the chances of producing false negative results.

A unique and important advantage that a test kit based on flow through technology offers is substantial reduction in the chances of producing false positive results through its very simple sample addition protocol which includes multiple washing steps.

The above mentioned diagnostic system can be used in detecting many disease causing microorganisms, more specifically viruses like Human Immunodeficiency Virus (HIV), Hepatitis C virus (HCV), Herpes Simplex Virus (HSV 1 & 2), Varicella Zoster Virus (VZV), Syphilis, Influenza, Para influenza, Cytomegalovirus (CMV), Human respiratory syncytial virus (RSV), Adeno virus and mumps. It is known that HIV-positive patients are sometimes co-infected with HBV and HCV due to shared routes of transmission like percutaneous exposure to blood, sexual intercourse and transmission from a mother to her infant (Reference: HIV/HCV co-infection: Pathogenesis, clinical complications, treatment and new therapeutic technologies. Operskalski EA et.al; Current HIV/ AIDS Report ; 2011 March 8(l):12-22 doi:10.1007/s 11904-010-0071-3). Co-infection is also found in case of Sexually Transmitted Diseases (STDs) e.g. HIV, HCV, HBV, Syphilis etc. None of the rapid diagnostic systems available in the market provides a method for an early detection of multiple bioanalytes related to more than one disease, specifically the co- infections, in a single device.

The rapid diagnostic system which works on the principle of flow through technology generally relates to a device which comprises of (a) a base, (b) positioned on the said base is an absorbent pad made up of cellulosic material having a certain thickness, and (c) an immunofilteration membrane (henceforth referred to as "membrane") which is made up of cellulosic material of specific pore size coated with homogenous mixture of different antigens or antibodies or biomolecules. The membrane is mounted over the said absorbent pad and disposed on the base. The sensitivity and specificity of the detection through the device is directly proportional to the porosity of the membrane. Smaller the pore size of the membrane, higher is the sensitivity and specificity of the detection.

Indian Patent 194638 claims one such flow through device for differentially detecting two HCV antigens in a single run on the same device for early diagnosis of HCV infection. The said device comprises of an absorbent pad made up of cellulosic material having a thickness of 2.4 to 2.7 mm and an immunofilteration membrane made up of cellulosic material having a pore size of 0.8-1.5 micron.The method according to the invention teaches the device which renders 100% sensitivity and 98.9% specificity.

Further, Indian Patent 188977 claims one such flow through device for differentially detecting two HIV antigens in a single run on the same device for early diagnosis of HIV infection. However this method does not address any process of increasing the sensitivity and specificity of the device.

Also, both the above prior arts teach detection of only two disease specific bioanalytes of a single disease, representing two test 'dots' and one control 'dot' in a single device.

In addition, the above methods detect only two disease specific antigens or bioanalytes out of a range of antigens which may be present in biological fluids due to multiple infections. The above mentioned prior arts thus do not teach detection of bioanalytes related to more than one disease in a single device.

The above mentioned limitations of the prior art makes it necessary to use more than one device for screening of multiple bioanalytes which in turn leads to patient and user discomfort, increased time of diagnosis, and increased cost. Hence there is a need for a device which can detect multiple bioanalytes of one or more than one disease in a single device using flow through technology with higher sensitivity and specificity.

OBJECT OF THE INVENTION

The object of this invention is to provide a flow through test device for rapid, visual, more sensitive and specific immunodiagnosis of multiple bioanalytes specific to one or more than one disease/s in a single device using flow through technology. This detection is done by using patient's biological fluids such as whole blood, serum or plasma. The further object of the invention is to provide a method of detection with 100% sensitivity and > 99.5% specificity.

Another object of the invention is to provide a method of manufacturing the said device and its components.

A further object of the invention is to provide a protocol of performing the detection. SUMMARY OF THE INVENTION

In accordance with the objectives, the subject of the invention is to provide a flow through test device for rapid, visual, more sensitive and more specific immunodiagnosis of multiple bioanalytes specific to one or more than one disease/s in a single device using flow through technology. These objectives are achieved by providing three or more than three dots of the immobilized biomolecules on the membrane. This detection is done by using patient's biological fluids such as whole blood, serum or plasma.

The proposed invention also provides a method of detection with 100 % sensitivity and > 99.5% specificity.

The instant invention elaborates the application of the flow through technology for the detection of a number of diseases, such as Allergy including detection of IgE, HIV, Syphilis, , Hepatitis B, Hepatitis C, HSV 1 & 2, VZV, Influenza, Para influenza, CMV, RSV, Adeno virus, Mumps and specific combinations.

Through the qualitative detection of multiple bioanalytes, specific diseases can be diagnosed at an early stage.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further illustrated in the following drawings.

FIG 1 illustrates a device for detection of HIV and HCV.

FIG 2 illustrates a device for detection of two different antibodies of HIV.

FIG 3 illustrates a device for the detection of HIV.

FIG 4 illustrates a device for the detection of HCV.

DETAILED DESCRIPTION OF THE INVENTION

The word 'Bioanalyte' as referred herein in the specification means and includes disease specific biomolecules in the patient's blood sample to be analysed for diagnosis. The word 'Biomolecules' as referred in the specification means and includes biomolecules which are coated on the immunofiltration memebrane.

The present invention provides a flow through test device for rapid, visual, more sensitive and more specific immunodiagnosis of multiple bioanalytes specific to one or more than one disease/s in a single device using flow through technology. This detection is done by using patient's biological fluids such as whole blood, serum or plasma.

The said device works on the principle of immunofilteration which involves flow of biological fluid through a porous membrane into an absorbent pad. The bioanalytes in the biological fluid sample irreversibly bind to the antigen or antibodies or disease related biomolecules that are immobilized on the surface of the porous membrane. The bound bioanalytes are then visualized by the addition of an indicator, a tracer buffer or a reagent which induces the color change by specific labeled molecule.

The diseases which can be detected using the device of the instant invention include Allergy, HIV, Syphilis, Hepatitis B, Hepatitis C, HSV 1 & 2, VZV, Influenza, Para influenza, CMV, Adeno virus, RSV and Mumps or a combination thereof.

The device of the instant invention can be tailor made to detect specific combination of diseases based on specific requirements.

THE METHOD OF MANUFACTURE

The device of the current invention comprises a top cover, a plastic base, an absorbent pad, and a porous membrane. The absorbent pad is made of cellulosic material which is 1.5 to 2.3 mm thick, applied in 1-3 layers. The function of this pad is to retain the fluids used in the detection as per the test protocol. The membrane cut from a cellulosic material is mounted on the absorbent pad. The absorbent pad and the membrane are housed in the casing comprising of a top cover and a plastic base tightly pressed together to make the connection airtight. A hole is provided in the top cover at its center through which the membrane mounted on the absorbent pad is exposed.

In a preferred embodiment of the invention, the membrane and absorption pad are made of material selected from the group of cellulosic materials like nitrocellulose or cellulose acetate or polymeric material like poly vinyl difluoride or nylon. In a more preferred embodiment the membrane and absorption pad are made of cellulosic material preferably nitrocellulose.

In another preferred embodiment, the absorption pad is made of nitrocellulose which may be laminated or non-laminated. The thickness of this pad varies from 1.5 to 2.0 mm.

In yet another preferred embodiment, pore size of the porous membrane made of nitrocellulose is of thickness varying from 0.2 to 0.5 mm and has pore size varies from 0.4 microns to 0.7 microns which increases the sensitivity and specificity of the detection. This will render the device with 100% sensitivity and > 99.5 specificity.

In yet another embodiment, the disease specific biomolecules are immobilized on the porous membrane as dots. These dots can be antibodies which can be in any of the forms such as (a) monoclonal or polyclonal antibodies (b) recombinant or synthetic or natural peptides (c) biotinylated or non biotinylated antibodies, (d) natural or synthetic antibodies (e) renatured or ^* denatured peptides,(f) lysate antigens, (f) recombinant antigens (h) synthetic antigens or a mixtures thereof are immobilized in the form of two or more test dots and one control dot.

In yet another embodiment of the invention, the disease specific biomolecules immobilized on the porous membrane can be antigens which can be in the form of lysate or recombinant or synthetic antigens immobilized in the form of two or more test dots and one control dot.

The disease specific biomolecules mentioned in the above embodiments can be used in combination thereof.

In all the embodiments described herein the device contains three or more than three dots of the immobilized biomolecules on the immunofilteration membrane. In an embodiment of the invention if the device has three dots, referred as "3 DOT" device, where two out of three dots will be test dots and one dot will be a control dot. Similarly, if the device has four dots, it is referred to as "4 DOT" device, where three dots out of four dots are "test dots" and one dot is "control dot". Similarly if the device has five dots, referred as "5 DOT" or "Penta-DOT" device, where four out of five dots are "test dots" and one dot is "control dot". Similarly if the device has six dots, referred to as "6 DOT" or "Hexa-Dot" device, where five out of six dots are "test dots" and one dot is "control dot".

The disease specific biomolecules (i.e. proteins or antibodies or antigens) are dissolved in a biological buffer to form a homogenous solution. The biological buffer is selected from the group of Phosphate, Tris, Borate, Carbonate, HEPES, or MOPS. Bio- preservative such as glycerol is added to the buffer to preserve the biomolecules . Surfactants such as Triton-xlOO^®, Tween-20^® or Brij-35™ may be added to the buffer in order to minimize non-specific binding of antibodies and to remove unbound moieties. Denaturing agents selected from the group consisting of dithiouretheriol, beta mercaptoethanol & urea may be added optionally. These agents denature the proteins because antibodies are not accessible to epitopes of native proteins.

The homogenous solution may be formed either for individual disease specific biomolecules or all the disease specific biomolecules together.

The test dots are formed on the membrane at specific distance from each other using 0.01 to 10 microliters of the above homogenous solution.

In addition to above test dots, a "control dot" is provided. The function of the control dot is to validate the test procedure. This control dot is comprised of biomolecules selected from the group consisting of Goat Anti-Human IgG, Protein-A or Protein-L. These biomolecules are dissolved in same biological buffer as described above to form homogenous solution. This control dot is formed on the membrane at specific distance from test dots using 0.01 to 10 microliters of this homogenous solution.

Other components of the diagnostic system of the instant invention are "assay diluent" or "assay buffer" and "signal or tracer reagent". Assay diluent is a buffer composed of the following ingredients.

1. Biological buffer such as Phosphate, Tris, HEPES or MOPS in a concentration ranging from 10 to 100 millimolar. 2. Surfactants selected from the group comprising of Tween 20, or Triton X 405 or Triton X 100 in a concentration ranging from 0.001 to 4% w/w depending on the antigen used.

3. Bovine serum albumin or casein or Non-fat dry milk or Gelatin

4. Sodium chloride or potassium chloride to maintain the ionic strength of the buffer.

The signal reagent consists of colloidal gold conjugated with Protein A. The colloidal gold particles size range is between 5 and 20 nm. Protein A is a purified cellular membrane protein of Staphylococcus aureus bacteria produced by recombinant technology. The primary function of the assay diluent is to block the non-reacting surfaces of the membrane and to activate the biomolecules immobilized on the membrane for the reaction. The assay diluent soaks through the membrane and reaches the absorbent pad. The patient sample i.e. the biological fluid which has disease specific bioanalytes is added at the center of the membrane by a dropper or pipette. The sample spreads on the membrane and bioanalytes present in the patient sample comes in contact with the disease specific biomolecule immobilized as dots on the membrane. The disease specific biomolecule immobilized on the membrane will covalently bind with the disease specific bioanalyte in the patient's sample.

The non-disease specific bioanalytes present in the patient's sample will not bind with the biomolecules immobilized on the membrane. Hence, all these non-disease specific bioanalytes will get soaked through the membrane and reach the absorbent pad underneath the membrane.

When the patient's sample is completely soaked over the membrane, the assay diluent is added once again to remove any unbound bioanalytes along with those weakly bound or bound with low affinity and other particulates from the membrane which in turn get soaked through the membrane and settle into the absorbent pad.

The assay diluent completely soaks the membrane and reaches the absorbent pad by capillary action and gravity.

After the assay diluent completely soaks the membrane, the "signal reagent" is added. Functionally protein A in the signal reagent binds efficiently with Fc region of the IgG antibody (5+ reaction) along with the IgM weak binding (2+) reaction. Protein A gets directly coupled to the colloidal gold particles and the suspension becomes pinkish red in color.

After adding the signal or tracer reagent, color of the control dot will always change which confirms that the detection is done without any error. After the completion of test if there is no appearance of color in the control dot and if the background is colorless, pinkish or purplish, the test indicates error. This may either be due to procedural error or deterioration of specimen/reagents or particulate matter in the specimen. In such a case, the test is required to be repeated.

As mentioned above, the diseases which can be detected using the device of the instant invention include Allergy, HIV, Syphilis, Hepatitis B, Hepatitis C, HSV 1 & 2, VZV, Influenza, Para influenza, CMV, Adeno virus, RSV and Mumps or a combination thereof. For the detection of HIV antibodies in biological fluids of patient, HIV antigens are immobilized on the membrane. The HIV antigens can be selected from group of GP36, GP41 pure antigens or combinations thereof. For the detection of HCV antibodies in biological fluids of patient, HCV antigens are immobilized on the membrane. The HCV antigens used can be selected from the group of Core, NS3, NS4, NS5 or combinations thereof.

The following examples are given as an illustration to describe application of the device for early diagnosis of the various diseases mentioned above and their co -infections. It should be noted that the present disclosure is not limited to the specific details embodied in the examples.

Example- 1 :

This example illustrates "QUADRO HIV + HCV device". This device is depicted in Figure 1. This device consists of three test dots immobilized on the membrane which have antigens for HIV1, HIV2 and HCV respectively for detection of HIV antibodies and HCV antibodies. This device has one control dot which comprises of Goat Anti-human IgG. The device comprises of an absorbent pad of thickness 1.9 - 2.0 mm. The membrane used in the device has pore size of 0.45 micron which renders the device with 100% sensitivity and > 99.5% specificity.

Example-2:

This example illustrates "QUADRO HIV 1+2 device". This device is depicted in Figure 2. This device consists of the three test dots immobilized on the membrane which have antigens for HIV1, HIV2 and HIV 1+2 respectively for detection of HIV antibodies. This device has one control dot which comprises of Goat Anti-human IgG. The device comprises of an absorbent pad of thickness 1.9 - 2.0 mm. The membrane used in the device has pore size of 0.45 micron which renders the device with 100% sensitivity and > 99.5% specificity.

Example- 3:

This example illustrates a device for antibody detection of HIV. This device is depicted in Figure 3. This device consists of the two test dots immobilized on the membrane which have antigens for HIV land HIV2 respectively. This device has one control dot which comprises of Goat Anti-human IgG. The device comprises of an absorbent pad of thickness 1.9 - 2.0 mm. The membrane used in the device has pore size of 0.45 micron, which renders the device with 100% sensitivity and > 99.5% specificity.

Example-4:

This example illustrates a device for antibody detection of HCV. This device is depicted in Figure 4. This device consists of the two test dots immobilized on the membrane which have antigens for HCV. This device has one control dot which comprises of Goat Anti- human IgG. The device comprises of an absorbent pad of thickness 1.9 - 2.0 mm. The membrane used in the device has pore size of 0.45 micron which renders the device with 100% sensitivity and > 99.5% specificity.

Example-5:

This example illustrates "QUADRO HIV+ HCV+ Syphilis device". This device consists of the three test dots immobilized on the membrane which have antigens for HIV, HCV and Syphilis respectively for detection of HIV, HCV and Syphilis antibodies. This device has one control dot which comprises of Goat Anti-human IgG. The device comprises of an absorbent pad of thickness 1.9 - 2.0 mm. The membrane used in the device has pore size of 0.45 micron which renders the device with 100% sensitivity and > 99.5% specificity. The HIV antigens can be selected from group of GP36, GP41 pure antigens or combinations thereof while HCV antigens can be selected from the Core, NS3, NS4, NS5 antigens or combinations thereof. Pure or recombinant antigens of Syphilis can be used.

Example-6:

This example illustrates "QUADRO HIV+ HCV+ Syphilis device". This device consists of the three test dots immobilized on the membrane which have antigens for HIV, HSV and Syphilis respectively for detection of HIV, HSV and Syphilis antibodies. This device has one control dot which comprises of Goat Anti-human IgG. The device comprises of an absorbent pad of thickness 1.9 - 2.0 mm. The membrane used in the device has pore size of 0.45 micron which renders the device with 100% sensitivity and > 99.5% specificity. The HIV antigens can be selected from group of GP36, GP41 pure antigens or combinations thereof while pure or recombinant antigens of HSV and Syphilis can be used.

Example-7:

This example illustrates "QUADRO VZV+ CMV+ Adeno device". This device consists of the three test dots immobilized on the membrane which have antigens for VZV, CMV and Adeno respectively for detection of VZV, CMV and Adeno antibodies. This device has one control dot which comprises of Goat Anti-human IgG. The device comprises of an absorbent pad of thickness 1.9 - 2.0 mm. The membrane used in the device has pore size of 0.45 micron which renders the device with 100%) sensitivity and > 99.5% specificity. Pure or recombinant antigens of VZV, CMV and Adeno virus can be used.

Example- 8:

This example illustrates "QUADRO RSV+ Mumps+ Adeno device". This device consists of the three test dots immobilized on the membrane which have antigens for RSV, Mumps and Adeno virus respectively for detection of RSV, Mumps and Adeno virus antibodies. This device has one control dot which comprises of Goat Anti-human IgG. The device comprises of an absorbent pad of thickness 1.9 - 2.0 mm. The membrane used in the device has pore size of 0.45 micron which renders the device with 100% sensitivity and > 99.5% specificity. Pure or recombinant antigens of RSV, Mumps and Adeno virus can be used. Example-9:

This example illustrates "QUADRO Influenza+ Para influenza + Mumps device". This device consists of the three test dots immobilized on the membrane which have antigens for Influenza, Para influenza and Mumps virus respectively for detection of Influenza, Para influenza and Mumps virus antibodies. This device has one control dot which comprises of Goat Anti-human IgG. The device comprises of an absorbent pad of thickness 1.9 - 2.0 mm. The membrane used in the device has pore size of 0.45 micron which renders the device with 100% sensitivity and > 99.5% specificity.

Pure or recombinant antigens of Influenza, Para influenza and Mumps can be used.

Example- 10:

This example illustrates "QUADRO IgE+ RSV+ Mumps device". This device consists of the three test dots immobilized on the membrane which have antigens for IgE, RSV and Mumps respectively for detection of IgE, RSV and Mumps virus antibodies. This device has one control dot which comprises of Goat Anti-human IgG. The device comprises of an absorbent pad of thickness 1.9 - 2.0 mm. The membrane used in the device has pore size of 0.45 micron which renders the device with 100% sensitivity and > 99.5% specificity. Pure or recombinant antigens of IgE, RSV and Mumps can be used.

By means of this IgE detection, the allergies and allergens can be detected at a faster rate than the conventional diagnostic methods.

Example-11 :

This example illustrates a device for antibody detection of HIV and Syphilis. This device consists of the two test dots immobilized on the membrane which have antigens for HIV 1+2 and Syphilis. This device has one control dot which comprises of Goat Anti-human IgG. The device comprises of an absorbent pad of thickness 1.9 - 2.0 mm. The membrane used in the device has pore size of 0.45 micron which renders the device with 100% sensitivity and > 99.5% specificity.

The HIV antigens can be selected from group of GP36, GP41 pure antigens or combinations thereof while pure or recombinant antigens of Syphilis can be used. Example- 12:

This example illustrates a device for antibody detection of HIV and HSV. This device consists of the two test dots immobilized on the membrane which have antigens for HIV and HSV. This device has one control dot which comprises of Goat Anti-human IgG. The device comprises of an absorbent pad of thickness 1.9 - 2.0 mm. The membrane used in the device has pore size of 0.45 micron which renders the device with 100% sensitivity and > 99.5% specificity.

The HIV antigens can be selected from group of GP36, GP41 pure antigens or combinations thereof while pure or recombinant antigens of HSV can be used.

Example-13:

This example illustrates a device for antibody detection of HCV and Syphilis. This device consists of the two test dots immobilized on the membrane which have antigens for HCV and Syphilis. This device has one control dot which comprises of Goat Anti-human IgG. The device comprises of an absorbent pad of thickness 1.9 - 2.0 mm. The membrane used in the device has pore size of 0.45 micron which renders the device with 100% sensitivity and > 99.5% specificity.

The HCV antigens used can be selected from the Core, NS3, NS4, NS5 antigens or combinations thereof while pure or recombinant antigens of Syphilis can be used.

Claims

We Claim,

1) A flow through device for simultaneous detection of multiple disease specific bioanalytes from patient's single biological fluid sample with 100% sensitivity and > 99.5% specificity comprising a cellulosic immunofiltration membrane having a thickness of 0.2 to 0.5 mm and pore size of 0.4 micron to 0.7 micron, provided with two or more test dots and one in built procedural control dot formed by coating with homogeneous solutions of a disease specific biomolecules.

2) The flow through device as claimed in claim 1, wherein, the cellulosic immunofiltration membrane is mounted on an absorbent pad made of cellulosic material with thickness ranging from 1.5 to 2.3 mm, applied in 1 to 3 layers which are housed in a casing comprising of a top cover provided with a central hole exposing the membrane and a plastic base pressed together air tightly.

3) The flow through device as claimed in claim 1, wherein the test dots and one in built procedural control dot are at a precise distance formed by coating with homogeneous solution in an amount ranging from 0.01 microliter to 10 microliter.

4) The flow through device as claimed in claim 1 , wherein . the disease specific bioanalytes are selected from the group comprising of antibodies or antigens produced by the viruses of HIV, Hepatitis C, Hepatitis B, allergy, Syphilis, HSV 1 & 2, VZV, Influenza, Para influenza, CMV, RSV, Adeno virus, Mumps in the patient's sample.

5) The flow through device as claimed in claim 1 , wherein the homogeneous solution comprises ,

(i) disease specific biomolecule(s);

(ii) biological buffers selected from the group comprising of Phosphate, Tris, Borate, Carbonate, HEPES, MOPS buffers;

(iii) glycerol as a bio preservative;

(iv) surfactants selected from the group comprising of Triton-xl00®, Tween-20® and Brij-35TM ; (v) optionally with denaturing agents selected from the group comprising of di thiouretheriol, beta mercaptoethanol and urea.

6) The flow through device as claimed in claim 1, wherein the disease specific biomolecules are selected from the group comprising of (a) monoclonal or polyclonal antibodies, (b) recombinant or synthetic peptides, (c) biotinylated or non biotinylated antibodies, (d) natural or synthetic antibodies, (e) renatured or denatured antibodies (f) lysate antigens (g) recombinant antigens (h) synthetic antigens or a mixtures thereof.

7) The flow through device as claimed in claim 1, wherein the in built procedural control dot provided is coated with homogeneous solution comprising of biomolecules selected from the group consisting of goat-anti human IgG, protein-A, protein-L dissolved in the biological buffer.

8) A kit for simultaneous detection of multiple disease specific bioanalytes from patient's single biological fluid sample comprising a) flow through device as claimed in claim 1 , b) an assay diluent and c) a signal or tracer reagent.

9) The kit as claimed in claim 8, wherein the assay diluent comprises

(i) biological buffer selected from the group consisting of Phosphate buffer, Tris buffer, HEPES buffer and MOPS buffer in a concentration ranging from lOmM to lOO mM;

(ii) surfactants selected from the group consisting of Tween 20, Triton X 405, Triton X 100 in a concentration ranging from 0.001 to 4% w/w ;

(iii) bovine serum albumin or casein or Non-fat dry milk or Gelatin,

(iv) sodium chloride or potassium chloride to maintain the ionic strength of the buffer.

10) The kit as claimed in claim 8, wherein the signal or tracer reagent comprises colloidal gold particles having size ranging from 5 and 20 nm conjugated with protein A.