Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/54346—Nanoparticles
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54386—Analytical elements
  • G01N33/54387—Immunochromatographic test strips

Definitions

• DHA DHA
• a decrease in DHA in subsequent years is also linked to impaired brain function and the onset of Alzheimer’s disease.
• DHA also provides positive effects to conditions, such as arthritis, high blood pressure, type 2 diabetes, and some cancers. Therefore, the detection of omega-3 levels in general and especially of DHA becomes evident.
• EPA eicosapentaenoic acid
• EPA concentrations are highest in herring, salmon, eel, shrimp, and sturgeon.
• Grass-fed animal products like dairy and meats also contain some EPA.
• Some microalgae may also contain EPA.
• EPA is responsible for various physiological activities and reduce inflammation by using EPA to produce signaling molecules called eicosanoids.
• a chronic, low-level inflammation is known to drive several common diseases; and may also reduce symptoms of depression. It also prevents the blood from clotting easily, reduces triglyceride levels in blood thus subsiding pain and swelling.
• Extended use of EPA includes its use as a US FDA-approved prescription drug for reducing triglyceride levels, a supplement for heart disease, preventing heart attack, treating depression and for chemotherapy related side effects, diabetes, recovery after surgery, and many other purposes.
• omega-3 detection kits are available in the market. These conventional kits show the results in certain period of time ranging between hours to days altogether; thereby making it inconvenient.
• An object of the present invention is to provide a rapid test kit for determining the levels of Omega-3 fatty acid molecules such as docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) using whole blood, serum or plasma samples.
• DHA docosahexaenoic acid
• EPA Eicosapentaenoic acid
• Another object of the present invention is to provide a method for preparation of said DHA or EPA rapid test kit.
• Yet another object of the present invention is to provide a DHA or EPA rapid test kit which detects the sufficiency levels of DHA or EPA in a sample respectively.
• Yet another object of the present invention is to provide a DHA or EPA rapid test kit which allows to determine whether a given blood, serum or plasma sample has sufficient or deficient levels of DHA or EPA respectively.
• Yet another object of the present invention is to provide a DHA or EPA rapid test kit which yields results in 1 min.
• Yet another object of the present invention is to provide a DHA or EPA rapid test kit which allows the user to himself visually detect the DHA or EPA levels respectively, without the need of any technical device and personnel for detection and analysis.
• Yet another object of the present invention is to provide a DHA or EPA rapid test kit which is easy to use, fast and cost-effective.
• Yet another object of the present invention is to provide a DHA or EPA rapid test kit which eliminates the need of sending samples to a laboratory for detection and analysis.
• the present invention relates to a rapid test kit for detection of the Omega-3 fatty acid molecule selected from DHA or EPA, within one minute, wherein the kit is based on the principle of lateral flow immunoassay.
• the rapid test kit of the present invention comprises of an immuno -chromatographic strip encased in a cassette.
• the strip further comprises of a sample release pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad arranged in a sequential overlapping manner.
• the sample release pad is placed exactly below the sample port such that when a sample is loaded in the sample port, it directly travels and comes in contact with the sample release pad so as to allow the lateral flow of the sample from the sample release pad to the absorbent pad via capillary action.
• the lateral flow of sample includes loading a sample in the sample port that comes in contact with the sample release pad, allowing lateral flow to absorbent pad via capillary action, binding of gold nanoparticle conjugated detector antibody complex to DHA or EPA molecules to form a conjugate-antigen-antibody complex that moves from the conjugate pad to the nitrocellulose membrane, binding of anti-DHA or anti EPA capture antibodies to the respective DHA or EPA molecule bound to the gold nanoparticle -Detector antibody complex, capturing all the DHA or EPA bound gold nanoparticle -Detector antibody complexes on the test line, such that the DHA or EPA molecules are sandwiched between the gold nanoparticle -detector antibody complex and anti-DHA or anti EPA capture antibodies respectively, capturing unbound conjugated detector antibodies by anti-Mouse IgG antibodies to form IgG- IgG complex at the control line, viewing color indication at the control line that indicates the travelling of the sample across the nitrocellulose membrane, collecting the excess sample and gold nanoparticle
• the method of preparation of rapid detection kit include the steps of; cutting of sample release pad; cutting of blood separation pad; cutting of conjugate pad; cutting of nitrocellulose membrane; cutting of absorbent pad, coating of sample release pad, preparation of test and control antibody, preparation of gold conjugated detector antibodies, coating of conjugate pad, coating of nitrocellulose membrane, lamination of plastic pad, cutting of laminated plastic pad, assembly of cut laminates in cassettes, packing of aluminium foil pouch, sealing packed aluminium foil pouch, preparing test diluent, dispensing test diluent into dropper bottle, packing sealed pouches and diluent bottles in boxes.
• the rapid detection kit (1) for DHA can detect the DHA levels upto a minimum detection level of 5.16 pg/ml; and the rapid detection kit (1) for EPA can detect the EPA levels upto a minimum detection level of 90.1pg/ml; through a color indication is visible (positive test) when the sample (23) contains Omega-3 fatty acid (including DHA or EPA) molecules (14) up to the said threshold level; and no indication (negative test) at test line (24) below threshold level thereby confirming the deficiency of DHA or EPA and ultimately Omega-3 levels within the sample (23).
• FIG. 1 illustrates the chemical structure of DHA.
• FIG. 2 illustrates the chemical structure of DHA
• FIG. 3 illustrates the external view of the rapid test kit of the present invention.
• FIG. 4 illustrates the 3D cross sectional schematic of the rapid test kit of the present invention.
• FIG. 5 illustrates schematic diagram of the gold conjugated detector antibodies.
• FIG. 6 illustrates schematic diagram of the gold conjugated detector antibody-DHA complex
• FIG. 7 illustrates the schematic diagram of the capture antibodies.
• FIG. 8 illustrates the schematic diagram of the DHA molecule sandwiched between the gold conjugated detector antibody and the capture antibody.
• FIG. 9 illustrates the overall schematic of the principle of lateral flow immunoassay.
• FIG. 10 illustrates the various kinds of visible results obtained using the kit of the present invention.
• Sample refers to human/animal body fluid such as but not limited to blood, serum and plasma.
• DHA refers an Omega-3 fatty acid molecule named Docosahexaenoic acid, having the chemical formula as depicted in FIG. 1.
• EPA refers an Omega-3 fatty acid molecule named eicosapentaenoic acid, having the chemical formula as depicted in FIG. 2.
• Capture antibody refers to an antibody or a part of an antibody, preferably attached to a solid phase, which is capable of retaining an antigen, for example one or more DHA or EPA molecules, present in a sample, by affinity binding.
• Detector antibody refers to an antibody or a part of an antibody which is labeled, for example conjugation with gold nanoparticles, is capable of binding to the captured antigen through affinity binding, by recognizing an epitope site which is different from that recognized by the capture antibody or identical due a repeat motif in the capsid.
• the present invention relates to a rapid test kit (1) for detection and indexing of the Omega-3 fatty acid molecules selected from DHA or EPA.
• the rapid test kit (1) is based on the principle of lateral flow immunoassay.
• the rapid test kit (1) of the present invention comprises of an immuno-chromatographic strip (hereinafter referred to as ‘strip’) (3) encased in a cassette (2).
• the strip (3) further comprises of a sample release pad (5), a conjugate pad (6), a nitrocellulose membrane (7), and an absorbent pad (8) arranged in a sequential manner.
• the length of the cassette (2) is70mm, the breadth is 20mm and the height is 5mm.
• the components of the strip (3) - the sample release pad (5), the conjugate pad (6), the nitrocellulose membrane (7), and the absorbent pad (8) are arranged in such a manner that the adjacent pads are connected at an overlapping junction.
• the sequence of the pads from the proximal end (9) of the strip (3) is as follows - sample release pad (5), followed by conjugate pad (6), followed by the nitrocellulose membrane (7), followed by the absorbent pad (8).
• the sample release pad (5) is placed towards the proximal end (9) of the strip (3) while the absorbent pad (8) is placed at the distal end (10) of the strip (3).
• All the above components of the strip (3) are assembled on a backing material (4) such as but not limited to polystyrene or any other plastic material coated with a medium to high tack adhesive. All the components are laminated to the backing material (4) to provide rigidity and easy handling of the strip (3).
• the backing material (4) is coated with a pressure-sensitive adhesive to hold the various components in place.
• the sample release pad (5) is made up of glass fiber which is a hydrophobic material, with a good absorption capacity and is the first pad to come in contact with the sample (23).
• the sample (23) which can be used to detect the Omega- 3 fatty acid, including either DHA or EPA levels by means of the kit (1) of the present invention include whole blood and serum. A few drops of either of these samples (23) can be loaded in the sample port (11) of the kit (1).
• the sample release pad (5) is placed exactly below the sample port (11) such that when a sample (23) is loaded in the sample port (11), it directly travels and comes in contact with the sample release pad (5).
• All the pads of the strip (3) are made up of such materials and arranged in such a manner so as to allow the lateral flow of the sample (23) from the sample release pad (5) to the absorbent pad (8) via capillary action.
• the sample release pad (5) essentially serves as receiving medium for the sample (23) and allows the movement of the sample (23) in a lateral direction towards the conjugate pad (6).
• the conjugate pad (6) is made up of a hydrophobic, synthetic release matrix with good absorption capacity and is the second pad to come in contact with the sample (23).
• the conjugate pad (6) is coated with detector antibodies (16) conjugated with gold nanoparticles (21) (hereinafter referred to as ‘conjugated detector antibodies’) antibodies or anti-EPA polyclonal antibodies raised in rabbit (IgG); that detects DHA and EPA respectively.
• the detector antibodies (16) can also be raised in horses, sheep, goats, chickens or any other suitable host animals.
• the antibodies used in the invention may include but are not limited to IgG, IgY, IgA, IgD, IgE and IgM, and/or recombinantly expressed antibodies that may be single chain antibodies, double chain antibodies, or other.
• concentration of conjugated detector antibodies (18) coated/immobilized on the conjugate pad (6) ranges from 5 pg to 10 pg with a preferential concentration of 5 pg.
• DHA Omega-3 fatty acids
• EPA Omega-3 fatty acids
• the anti-DHA polyclonal conjugated detector antibodies (16) bind to DHA molecules (14) and form a gold nanoparti cle-Detector antibody-DHA complex
• the anti-EPA polyclonal conjugated detector antibodies (16) bind to EPA molecules (14) and form a gold nanoparticle -Detector antibody-EPA complex (hereinafter referred to as ‘conjugateantigen-antibody complex’ (19)).
• conjugate-antigen-antibody complex (19) along with the unbound conjugate-antibodies (20) travel towards the nitrocellulose membrane (7) via capillary movement.
• the nitrocellulose membrane (7) comprises of two indicator lines namely one control line (25) and test line (24).
• the control line (24) is coated with antimouse IgG antibody (17) raised in mouse at a concentration of 2-5mg/ml.
• the test line (25) is coated either with monoclonal anti-DHA or anti-EPA capture antibodies (15) raised in rabbit (IgG); based on the Omega-3 fatty acid molecule to be detected.
• the detector antibodies (16) can also be raised in horses, sheep, goats, chickens or any other suitable host animals.
• the antibodies used in the invention may include but are not limited to IgG, IgY, IgA, IgD, IgE and IgM, and/or recombinantly expressed antibodies that may be single chain antibodies or other.
• concentration of capture antibodies (15) coated/immobilized on the test lines range from 1 mg/ml to2 mg/ml with a preferential concentration of 1 mg/m.
• the monoclonal anti-DHA capture antibodies (15) are highly specific and are able to bind specifically to regions of DHA molecules (14); and the monoclonal anti-EPA capture antibodies (15) are highly specific and are able to bind specifically to regions of EPA molecules.
• the conjugate-antigen-antibody complex (19) moves from the conjugate pad (6) to the nitrocellulose membrane (7), the anti-DHA capture antibodies (15) bind to the DHA molecule (14) already bound to the gold nanoparticle-Detector antibody complex (18); and the anti -EPA capture antibodies (15) bind to EPA molecule .
• the DHA or EPA molecules (14) are sandwiched between the gold nanoparticle-Detector antibody complex (18) and anti-DHA or anti -EPA capture antibodies (15) respectively, showing a color indication at the test line (24) that indicates the presence of DHA or EPA molecules (14) in the blood sample (23).
• the unbound conjugated detector antibodies (20) travel further and are captured at the control line (25) by anti -mouse IgG antibodies (17) to form IgG- IgG complex (22).
• the color indication at the control line (25) indicates the travelling of the sample (23) across the nitrocellulose membrane (7).
• the excess sample (23) and gold nanoparticles conjugated detector antibodies (18) are collected at the absorbent pad (8) made up of cellulose filters.
• the color indication at the test line (24) confirms the presence of DHA or EPA molecules within the blood sample (23); whereas the color indication at the control line (25) indicates the travelling of the sample (23) across the nitrocellulose membrane (7).
• the said color detection test using the lateral flow immunoassay is completed within a range of 0-5 minutes; preferably within 1 minute.
• the rapid detection kit (1) for DHA can detect the DHA levels upto a minimum detection level of 5. 16 pg/ml; and the rapid detection kit (1) for EPA can detect the EPA levels upto a minimum detection level of 90. Ipg/ml; such that a color indication is visible (positive test) when the sample (23) contains Omega-3 fatty acid (including DHA or EPA) molecules (14) up to the said threshold level; whereas Omega-2 fatty acid (DHA or EPA) level in a sample (23) lower than the threshold level does not show color indication (negative test) at test line (24); which confirms the deficiency of DHA or EPA and ultimately Omega-3 levels within the sample (23).
• kits are used for detection of DHA and EPA; wherein the conjugate pad (6) of the DHA detection kit is coated with anti-DHA polyclonal conjugated detector antibodies (16) that bind to DHA molecules (14) and the test line (24) on nitrocellulose pad (7) is coated with monoclonal anti-DHA capture antibodies (15) which are highly specific and are able to bind specifically to regions of DHA molecules (14).
• the conjugate pad (6) of the EPA detection kit is coated with anti -EPA polyclonal conjugated detector antibodies (16) bind to EPA molecules and the test line (24) on nitrocellulose membrane (7) is coated with monoclonal anti-EPA capture antibodies (15) which are highly specific and are able to bind specifically to regions of EPA molecules.
• All the above components of the strip are assembled on a backing material such as but not limited to polystyrene or any other plastic material coated with a medium to high tack adhesive. All the components are laminated to the backing material to provide rigidity and easy handling of the strip.
• the backing material is coated with a pressure-sensitive adhesive to hold the various components in place.
• the sample release pad partly extends for 2mm over the conjugate pad.
• 2mm of the proximal end of the nitrocellulose membrane is placed under the conjugate pad.
• the absorbent pad is placed at the distal end of the strip. The absorbent pad extends over the nitrocellulose membrane for 2mm.
• the sample release pad is made from glass fiber matrix.
• the glass fiber matrix is cut into small pieces of 20mm x 3.5mm and stored at RT less than 30 Degree Celsius in a locking bag until further use.
• an additional small pad named ‘blood separation pad’ is located near the sample window.
• the blood separation pad is made from cellulose.
• the cellulose is cut into small pieces of 5mm x 3.5mm and stored in a locking bag until further use.
• the conjugate pad is made from synthetic release matrix.
• the synthetic release matrix is cut into small pieces of 5mm x 3.5 and stored in a locking bag until further use.
• the nitrocellulose membrane is cut in to small pieces of 25mm x 3.5mm and stored in a locking bag until further use.
• the absorbent pad is made from cellulose.
• the cellulose is cut into small pieces of 20 mm x 3.5 mm and stored in a locking bag until further use.
• the cut sample release pad(s) is kept on a mesh tray. Each (if multiple) cut sample release pad is coated with 2.5 mL of sample release pad buffer. The tray containing the cut sample release pad(s) is kept for drying in an incubator at 37 + 2°C for 2 hours. The dried sample release pad is stored in a locking bag with silica gel until further use.
• the anti-DHA rabbit antigeneral DHA polyclonal antibody or anti-EPA rabbit anti-general EPA polyclonal antibody is diluted in buffer with a ratio of 2-3mg/ml along with stabilizer and methanol.
• the anti-mouse IgG is diluted in buffer along with stabilizer in ratio of l-2mg/ml. Both the prepared test and control antibodies are separately dispensed in aliquots of 1 mL in tubes and stored at -20- 50 degree Celsius until further use.
• a conjugate diluent is first prepared in order to prepare the gold conjugated detector antibodies.
• the conjugate diluent is prepared by dissolving 10% of Sucrose and 5% of Trehalose in Tris buffer and made up in volume upto 50-100 ml.
• the gold conjugated detector antibody including either anti-DHA rabbit anti-general DHA polyclonal antibody or anti-EPA rabbit anti-general EPA polyclonal antibody, is diluted in the conjugate diluent of optical density ranging between 5 OD to 10 OD.
• the gold conjugated detector antibodies so prepared are stored at 2 to 8 Degree Celsius until further use.
• the required number of cut conjugate pads are placed on a mesh tray. 600-900 pL of the gold conjugated detector antibodies is coated on each of the conjugate pads.
• the tray containing the conjugate pads is kept for drying in an incubator at 37 + 2°C for 1 hour.
• the dried conjugate pad is removed from the incubator and is again dried in a vacuum over at 37 + 2°C for 15 minutes.
• the dried conjugate pad so obtained is stored in a locking bag with silica gel until further use.
• the locking bag is kept in an aluminium foil pouch and is sealed, until further use. J. Coating of nitrocellulose membrane:
• the receptacles in the machine ISO Flow Dispenser for test and control antibodies are fdled with 1% Sodium Hypochlorite. They are initially washed thrice. Later, they are washed with purified water 20 times following which the water is deloaded from them.
• the receptacles for test antibody are filled with test antibody and the receptacle for control antibody are filled with 1 ml of control antibody.
• the nitrocellulose membrane is coated with 20-30 pL of test and control antibodies.
• the coated nitrocellulose membranes are placed in a steel tray and kept for drying at 37 + 2°C for 1 hour in an incubator.
• the membranes are stored at room temperature less than 30 degrees until further use.
• a plastic pad laminated with 55 mm double sided Polyester tape is placed on the working table. Marking a distance of 1.9 cm from the operator side, the coated membrane (nitrocellulose) is pasted on the upper edge of the pad marking.
• the absorbent pad is pasted on the upper edge of the membrane forming an overlapping junction of 2 mm.
• the conjugate pad is pasted on the lower edge of the membrane forming an overlapping junction of 2 mm.
• the cut blood separation pad is pasted on the lower edge of the conjugate pad.
• the sample release pad is pasted on top of the blood separation pad.
• a 12.5 mm tape is pasted on top of the sample release pad forming an overlapping junction of 2 mm on the coated nitrocellulose membrane. All the laminated pads are kept in a locking bag and stored at room temperature less than 30 Degree until further use.
• the laminated plastic pad is mounted on the strip cutter machine with pre-programmed cutting dimensions.
• the cutting process is initiated and the first two pieces are discarded.
• the third piece is checked for correctness of dimensions and if any burrs are found it is stored in process rejection tray. Similar process is adopted for further pieces being cut as well.
• the bottom of the Lateral Flow cassettes is placed on the working table.
• the sample release pad of the cut laminate is places in the closed bracket of the cassette.
• the visual sample display board is referred for identification of the devices.
• the cassette is pressed using the pressing machine to seal the unit. All the cassettes so prepared are placed in a tray.
• silica gel is dried before use in a hot air over at 90 +5 °C for 90 minutes.
• One cassette is placed inside a pouch followed by silica gel sachet and a dropper.
• the sealing machine is set at 200-300 degree Celsius . After the said temperature is achieved, the open side of the pouch is sealed using the sealing machine. The sealed pouch is stored in a box.
• test diluent 0.1 to 0.5% of detergent trition is dissolved in buffer 100 ml and the test diluent so prepared is stored at room temperature.
• test diluent is dispensed into dropper bottles and a sticker for the same is pasted on the bottle.

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