Source: http://www.google.com/patents/US6194225?dq=5,758,352
Timestamp: 2017-02-25 18:54:26
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Matched Legal Cases: ['art 3', 'art 4', 'art 5', 'art 26', 'art 27', 'art 26', 'art 24', 'art 26', 'art 24', 'art 22', 'art 24', 'art 22', 'art 22', 'art 22', 'art 24', 'arts 24', 'arts 24', 'art 22', 'art 24']

Patent US6194225 - Immunochromatography-assisted device - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inPatentsAn immunochromatography-assisted device is disclosed which facilitates accurate and rapid detection of an antigen contained in a fluid sample and from which background coloring and blank coloring possibly causing a malfunction at detection are successfully eliminated. The immunochromatography-assisted...http://www.google.com/patents/US6194225?utm_source=gb-gplus-sharePatent US6194225 - Immunochromatography-assisted deviceAdvanced Patent SearchTry the new Google Patents, with machine-classified Google Scholar results, and Japanese and South Korean patents.Publication numberUS6194225 B1Publication typeGrantApplication numberUS 09/154,025Publication dateFeb 27, 2001Filing dateSep 16, 1998Priority dateSep 18, 1997Fee statusLapsedAlso published asCN1137382C, CN1215166A, DE69826082D1, DE69826082T2, EP0903584A1, EP0903584B1Publication number09154025, 154025, US 6194225 B1, US 6194225B1, US-B1-6194225, US6194225 B1, US6194225B1InventorsMiwa Oka, Nobuyuki Shigetou, Jinsei MiyazakiOriginal AssigneeMatsushita Electric Industrial Co., Ltd.Export CitationBiBTeX, EndNote, RefManPatent Citations (13), Referenced by (18), Classifications (26), Legal Events (6) External Links: USPTO, USPTO Assignment, EspacenetImmunochromatography-assisted device
US 6194225 B1Abstract
An immunochromatography-assisted device is disclosed which facilitates accurate and rapid detection of an antigen contained in a fluid sample and from which background coloring and blank coloring possibly causing a malfunction at detection are successfully eliminated. The immunochromatography-assisted device comprises a porous carrier having an additive-impregnated part between a sample introducing part and a determining part, the additive-impregnated part carrying at least one selected from the group consisting of a surfactant, a water-soluble ammonium salt and a pH buffer such that it dissolves in a sample introduced into the carrier in order to move with the movement of the sample through the carrier. The porous carrier also has a labelled part segmented into plural zones.
The present invention relates to an extracorporeal diagnostic drug or a portable diagnostic device for detecting microorganisms in water and a trace amount of markers in the humor. More specifically, the present invention is intended to improve the performance of an immunochromatography-assisted device which facilitates simple and rapid measurements of such substances at small-scale clinics with no installation of analytical equipment and by the subjects studied.
A labelled part 3 is formed by impregnating a glass filter with an aqueous solution of an anti-hCG antibody which is adsorbed on colloidal gold or colored latex, followed by freezing and drying it. A determining part 4 is produced by dropping an aqueous solution of another antibody on a nitrocellulose sheet in an arbitrary shape, followed by drying and rinsing it. The very strong non-specific adsorption property of nitrocellulose ensures tight adhesion of the antibody to the nitrocellulose sheet. After fixation of the antibody, the nitrocellulose sheet may be surface-treated with bovine serum albumin (hereinafter abbreviated to “BSA”), for example, in order to prevent coloring of the background due to the non-specific adsorption property of nitrocellulose during antigen-antibody reaction for detection. This surface treatment is called blocking. A fluid absorbing part 5 located at the other end of the porous carrier is formed with a material such as glass filter which is excellent in fluid absorption and fluid absorbing capacity, for the purpose of rapid absorption of excess sample.
In view of the above-mentioned drawbacks of the prior art device, the object of the present invention is to provide an immunochromatography-assisted device facilitating accurate and rapid detection of an antigen contained in a liquid sample by eliminating coloring of the background and blank coloring which may cause a malfunction during detection.
FIG. 1 is a perspective view illustrating a test strip of a conventional immunochromatography device.
The immunochromatography-assisted device in accordance with the present invention detects an antigen in an aqueous sample solution based on a specific antigen-antibody binding reaction, which comprises at least two different antibodies binding to an antigen as an analyte and a test strip composed of a porous carrier on which a labelled part containing one of the antibodies and a determining part containing the other of the antibodies are arranged such that a sample introduced into a sample introducing part is allowed to move toward the determining part via the labelled part, wherein one of the antibodies (the antibody in a mobile phase) is chemically visualized and contained in the labelled part of the porous carrier such that it dissolves in a sample solution introduced into the porous carrier in order to move inside the carrier with the movement of the sample solution, the other of the antibodies (the antibody in a fixed phase) is fixed to the determining part of the porous carrier to a degree not to be shifted in position by the movement of the sample, and the porous carrier has an impregnated part with an additive at a certain part between the sample introducing part and the determining part, the additive-impregnated part movably carrying thereon at least one selected from the group consisting of a surfactant, an ammonium salt and a pH buffer such that it dissolves in the sample solution and moves with the movement of the sample through the porous carrier.
I. Method of producing each part
To 1 L of distilled water, 0.1 mol (12.11 g) of tris(hydroxymethyl)aminomethane (hereinafter referred to as “Tris”) was added and agitated, which was then adjusted for pH at 8.2 with 1 N HCl to make a detergent for nitrocellulose. Separately, 10 g of skimmed milk was added to the detergent thus prepared and agitated to make a blocker for nitrocellulose.
A nitrocellulose sheet (HFM SPHF04020, manufactured by Nihon Millipore Limited) was cut to a sheet of 0.9 cm×1.2 cm with a thickness of about 220 μm (including the thickness of a substrate) for use as a carrier. The sheet as the carrier was applied with a phosphate buffer solution (hereinafter abbreviated to “PBS”) containing an anti-hCGβ antibody (α-hCGβ) against hCG (5 mg/mL) at a portion 0.5 cm apart from the sample introducing part and 0.7 cm apart from the fluid absorbing part and dried for 8 hours at 40° C. in a light-tight environment. After drying, the sheet was impregnated with the above-mentioned blocker so as to make a ratio of the blocker at 70 mL per 30 sheets, and then shaken in a shaker for 30 min at 30° C. for blocking. After blocking, the sheet was rinsed with the above-mentioned detergent (70 mL/30 sheets) for 30 min at 30° C. three times, which was then placed and dried in a desiccator overnight.
To 1 L of distilled water, 1 mol (121.1 g) of Tris was added and agitated, which was then adjusted for pH at 8.2 with 1 N HCl. The mixture was further added with 1 mol (109.6 g) of tetramethylammonium chloride (hereinafter abbreviated to “TMA”) and 10 mL of a 10% aqueous solution containing a Triton surfactant Triton X-100 (hereinafter referred to as “Triton”) and agitated to make a Tris (1M)-TMA (1M)-Triton (0.1%) solution. The resultant solution was applied onto a 0.9 cm×6.0 cm sheet cut from a glass fiber filter (F075-14, manufactured by Whatman Japan Ltd.) at 0.36 mL, followed by freezing and drying.
(a) A PBS of dithiobis(sulfosuccinimidylpropionate)(hereinafter abbreviated to “DTSSP”)(4.06 mg/0.1 mL) was gently dropped on a PBS of anti-hCGα antibody (α-hCGα)(10 mg/mL) and agitated for 30 min at 35° C., which was then subjected to gel filtration using a molecular weight differential column (Sephadex G25M, manufactured by Amersham Pharmacia Biotech).
(b) Gel filtration produced a polymerized anti-hCGα antibody PBS (10 mg/6 mL). The solution thus produced was stored at 4° C. until use.
(c) A BSA PBS (110 mg/mL) was added with 77 mg of dithiothreitol (hereinafter abbreviated to “DTT”) and agitated for 30 min at room temperature to make a final concentration of 7.7 mg/mL, which was then promptly subjected to gel filtration using a molecular weight differential column (Sephadex G25M, manufactured by Amersham Pharmacia Biotech). Gel filtration produced a BSA solution with a free SH group (totally 24 mL).
(d) The BSA with a free SH group (totally 24 mL) was mixed with the polymerized anti-hCGα antibody PBS (10 mg/6 mL) which had been stored at 4° C. after preparation in the above (b) and agitated for 20 hours at 4° C.
(f) The product of (e) was gently dropped with a PBS of a cyanine dye (hereinafter abbreviated to “SLIC3”) represented by the chemical formula as shown above and then agitated for 20 hours at 4° C.
(i) A glass filter (F075-14, manufactured by Whatman Japan Ltd.) was cut to a sheet of 0.9 cm×5.0 cm and impregnated with the labelled antibody stock solution containing the two stabilizers at 0.3 mL per sheet, which was then frozen and dried.
A glass fiber filter (GF/D, manufactured by Whatman Japan Ltd.) was cut to a sheet of 0.9 cm×2.5 cm and six (6) sheets were piled for use as the fluid absorbing part.
On a surface of a lining 21 with a size of 0.9×5.5 cm and a thickness of 0.5 mm (hard polyvinyl chloride (white)) entirely and thoroughly lined with a double-sided adhesive tape of the same size (TW-12SD, manufactured by Nichiban Co., Ltd.), a determining part 26, a fluid absorbing part 27 downstream from the determining part 26 and a labelled part 24 upstream from the determining part 26 were bonded, respectively. Upstream from the labelled part 24, a sheet 23 of 0.9 cm×0.5 cm cut from a glass fiber sheet (F075-14, manufactured by Whatman Japan Ltd.) and an additive-impregnated part 22 were further bonded successively in this order. Two (2) additional sheets of the same glass fiber filter cut to a size of 0.9×3.5 cm were also bonded to the entire upper part from the labelled part 24 to the additive-impregnated part 22 to serve as a porous bypass 25. The test strip 20 thus produced was accommodated in a hollow case.
For comparison, a test strip as shown in FIG. 4 was prepared in the same manner as in Example 1, except for the use of the same glass fiber filter as used in the glass fiber filter 23 in place of the additive-impregnated part 22 and an omission of the porous bypass 25 of a glass fiber filter.
In this example, a test strip was prepared in the same manner as in Example 1, except for the use of Tris (1M)-TMA (1M) solution in place of Tris (1M)-TMA (1M)-Triton (0.1%) solution in Example 1 in producing the additive-impregnated part.
In this example, another test strip was prepared in the same manner as in Example 1, except for the use of Tris (1M)-Triton (0.1%) solution in place of Tris (1M)-TMA (1M)-Triton (0.1%) solution in Example 1 in producing the additive-impregnated part.
For comparison, a test strip was prepared in the same manner as in Example 2, except for the use of the same glass fiber filter as used in the glass fiber filter 23 in place of the additive-impregnated part 22 in Example 2.
A test strip was prepared in the same manner as in Example 1, except that the labelled part was produced in the manner as described below.
k) The frozen and dried glass filter impregnated with the labelled antibody was cut to a size of 0.9 cm×0.1 cm and used as a labelled part 24 c. l) The stock solution of the labelled antibody was diluted 4-fold with PBS and impregnated into a glass fiber filter (F075-14, manufactured by Whatman Japan Ltd.) in the same manner as described previously, which was cut into two sheets of 0.9 cm×0.1 cm and used as labelled parts 24 a and 24 b. The labelled parts 24 a, 24 b and 24 c thus prepared were assembled and bonded to the porous carrier as shown in FIG. 12 by sandwiching therebetween spacers 30 made of a glass fiber filter (F075-14, manufactured by Whatman Japan Ltd.) of the same size.
A test strip was prepared in the same manner as in Example 4, except for the use of a glass fiber filter (F075-14, manufactured by Whatman Japan Ltd.) in place of the additive-impregnated part 22 of Example 4 as shown in FIG. 13.
In the experiment, an immunochromatography-assisted device was produced using the test strip prepared in Example 5 in accordance with the present invention and compared with an immunochromatography-assisted device of a comparative example produced with a test strip similar to that of Example 5, except that the labelled part was not segmented, and the part facing the determining part was positioned at the same place as the labelled part 24 c of Example 5 for comparison purpose. This mean that both devices are the same in terms of the total amount of the labelled antibody carried on the labelled part. The analyte was hCG. Reaction-induced coloring was assayed using a reflectance spectrophotometer (CS-9300, manufactured by Shimadzu Corporation). The intensity of reflected light was measured at an incident light of 550 nm (absorbance of SLIC3). The baseline value 0 was based on the absorbance of clean and non-used nitrocellulose. hCG PBS at a concentration of 0, 50, 1,000, or 10,000 IU/L was introduced into the sample introducing part of the test strip of each device. After the device was stood still horizontally at room temperature (25° C.), the reaction was analyzed.
Next, the results of a comparison test between three (3) immunochromatography-assisted devices using the test strip of Example 1, Example 4 or Example 5. The analyte was hCG. As stated previously, the viewability was derived from the measurement of the intensity of reflected light at an incident light of 550 nm. hCG PBS at a concentration of 0, 50, 100, 1,000, or 10,000 IU/L was introduced into the sample introducing part of the test strip of each device and the device was stood still horizontally at room temperature (25° C.). FIG. 16 summarizes a comparison between the devices for the absorbance (coloring intensity) at each hCG concentration 3 minutes after sample introduction. The figure indicates that the device using the test strip of Example 4 which includes an additive and is segmented for the labelled part is improved in coloring intensity, compared to the devices using the test strip of Example 1 or Example 5 from which either one has been omitted. Blank coloring at 0 IU/L as a factor for causing a malfunction could be eliminated perfectly from Example 4.
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