CN109212184B - One-step calprotectin rapid detection kit - Google Patents
One-step calprotectin rapid detection kit Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G01N2800/00—Detection or diagnosis of diseases
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Abstract
The invention provides a one-step method calprotectin rapid detection kit which comprises a detection kit shell (2), a sampling tube (4), a filter tube (5) and detection test paper (6), wherein the sampling tube (4) can be inserted into the detection kit shell (2), the sampling tube (4) is movably connected with the detection kit shell (2), and the sampling tube (4), the filter tube (5) and the detection test paper (6) are sequentially arranged in the detection kit shell (2) from top to bottom when the sampling tube (4) is inserted into the detection kit shell (2). The detection process is convenient and quick, and the sample can be processed without a special operation platform or professional detection personnel.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to biological sample detection.
Background
Inflammatory Bowel Disease (IBD) is a group of chronic non-specific inflammatory diseases of the intestinal tract, including Ulcerative Colitis (UC) and Crohn's Disease (CD). In recent years, the number of patients with inflammatory bowel disease has increased, and the disease is a common disease of the digestive system. Elevated levels of calprotectin (FC) in the stool suggest that neutrophils and other calprotectin-secreting inflammatory cells are known to be a potent marker of intestinal inflammation by increasing the number of exudations from the inflamed mucosa into the intestinal lumen. Research shows that the accuracy of diagnosis and disease evaluation of Fecal Calprotectin (FC) on inflammatory bowel disease is higher than that of traditional assay indexes (such as C-reactive protein and blood sedimentation), the correlation with enteroscope results is strong, the disease activity condition of IBD can be judged, IBD recurrence is predicted, the treatment effect is evaluated, and the noninvasive and high-specificity fecal calprotectin has the advantages of no wound and high specificity.
At present, the detection method of calprotectin (FC) in excrement mainly comprises the following steps: enzyme-linked immunosorbent assay (ELISA) and gold-labeled. The enzyme-linked immunosorbent assay (ELISA) method for determining the content of calprotectin (FC) in excrement needs matched experimental equipment and longer detection time. The sensitivity of the gold-labeled method for determining calprotectin in excrement is not high.
The reagent kit used in the existing detection method needs the professional and the equipment to process the sample before detection, and is inconvenient to use.
Disclosure of Invention
In order to solve the problems, the invention provides a one-step calprotectin rapid detection kit which can facilitate the examination pretreatment of a sample, and the sample can be directly treated after sampling by directly inserting the sample into the calprotectin rapid detection kit. The one-step calprotectin rapid detection kit after sample treatment can be integrally inserted into an adaptive detection instrument or the detection test paper can be taken down and inserted into the existing detection instrument to complete detection, such as micro-detection of biotechnology
The series of fluorescence immunoassay analyzers have the advantages that the detection process is convenient and fast, and the sample can be processed without a special operation platform or professional detection personnel.
According to an aspect of the present invention, in some embodiments, there is provided a one-step method calprotectin rapid detection kit, comprising a detection kit housing, a sampling tube, a filter tube and detection test paper, wherein the sampling tube is insertable into the detection kit housing, the sampling tube is movably connected with the detection kit housing, and the sampling tube, the filter tube and the detection test paper are sequentially arranged in the detection kit housing from top to bottom when the sampling tube is inserted into the detection kit housing. The test paper comprises a fluorescent marker combination pad, a T line and a C line, wherein a quantum dot mark mouse anti-calprotectin monoclonal antibody A is contained in the fluorescent marker combination pad, the T line contains a coated calprotectin monoclonal antibody B, the C line contains a coated goat anti-mouse polyclonal antibody, and the antibody is from the Daiibo Biotech company Limited. The sampling tube of the invention is directly inserted into the shell of the detection kit to complete the sample treatment after sampling, and the treated sample is directly dripped on the detection test paper, so that the detection test paper can be used for optical detection.
According to an aspect of the present invention, in some embodiments, there is provided a one-step method calprotectin rapid detection kit, wherein a top surface of a detection kit housing is provided with a sample inlet hole matched with a sampling tube, a first fixing device for fixing the sampling tube is arranged in the detection kit housing, a second fixing device for fixing the filter tube is arranged in the detection kit housing, and the first fixing device is positioned above the second fixing device.
According to one aspect of the invention, in some embodiments, there is provided a one-step method rapid calprotectin detection kit, wherein the sampling tube comprises a lumen tube and a threaded rod, the threaded rod is positioned at the bottom of the lumen tube, the threaded rod is provided with threads, and the sampling tube is provided with a liquid outlet hole.
According to one aspect of the invention, in some embodiments, there is provided a one-step method calprotectin rapid detection kit, wherein a first fixing device is provided with a liquid storage tank, one inward side of the liquid storage tank is an inclined plane, the inclined plane is provided with an opening and is plastically packaged with a membrane, such as a tin paper membrane, the liquid storage tank is connected with the first fixing device in a sliding manner, the liquid storage tank is connected with a detection kit shell through a spring, the side wall of a cavity tube is provided with puncture rods, the puncture rods can be one or more, the cavity tube is provided with a liquid inlet opening, the puncture rods are opposite to the liquid inlet opening, a guide column is arranged outside the cavity tube, a guide through hole matched with the guide column is arranged beside the sample inlet hole, and the position where a sampling tube is inserted can be fixed, so that the liquid storage tank can be inserted into the sampling tube smoothly. After sampling, the sampling tube is inserted into the sampling hole, the spring is compressed until the liquid storage tank at the liquid inlet opening is punctured into the cavity tube, the puncturing rod punctures the liquid storage tank, liquid in the liquid storage tank flows downwards, and the excrement sample stained by the liquid outlet hole is washed.
According to one aspect of the invention, in some embodiments, there is provided a one-step calprotectin rapid test kit comprising a stool dilution of 100 μ L10 mM PBS (PH 7.4), 0.02% NaN 3 . And diluting the sample by the excrement diluent so that the concentration of the substance to be detected is within the detection range of the kit.
According to an aspect of the present invention, in some embodiments, there is provided a one-step method calprotectin rapid detection kit, wherein the filter tube has an upper and lower opening structure, and a glass fiber membrane is disposed in the middle of the filter tube. When the sampling device is a sampling tube for sampling excrement, the sampling tube has the function of filtering samples. The sample liquid after being washed by the liquid in the liquid storage tank can pass through the glass fiber membrane, so that the aim of indirectly mixing the sample liquid can be fulfilled.
According to an aspect of the present invention, in some embodiments, there is provided a one-step fecal hemoglobin rapid test kit, wherein a test hole is formed on an outer wall of one side of a test kit housing, and a barcode or a two-dimensional code is further formed on the outer wall of the test kit housing for identifying a type of the test kit.
In some embodiments, the detection kit of the present invention can be integrally placed in a detection instrument for detection, and only a slight modification of the existing detection instrument (the detection instrument body adapted to the kit of the present invention) is required, that is, a cavity adapted to the detection kit is formed on the original detection instrument, and the detection head extends out of the adapted detection hole.
According to an aspect of the present invention, in some embodiments, there is provided a one-step calprotectin rapid detection kit, wherein the outer wall of one side of the detection kit housing is provided with a detection hole, and the outer wall of the detection kit housing is further provided with a bar code or a two-dimensional code. The detection kit can be integrally placed in a detection instrument for detection, and the construction scheme can be realized by only slightly modifying the existing detection instrument, namely, a cavity matched with the detection kit is formed on the original detection instrument, and the detection head extends out of the position of the matched detection hole.
According to one aspect of the present invention, in some embodiments there is provided a one-step rapid calprotectin test kit, wherein the test strip is further provided with a support plate, and the test strip and the support plate form a test plate.
According to one aspect of the present invention, in some embodiments, there is provided a one-step method rapid calprotectin test kit, wherein the test strip is connected to the test kit housing by a buckle, and the test strip is detachable from the test kit housing. After being detached, the utility model can be directly inserted into the existing detecting instrument to complete the detection.
According to one aspect of the invention, in some embodiments there is provided a one-step rapid calprotectin detection kit, wherein the exit hole sites are located at the junction of the lumen tube and the threaded rod; or the screw thread is a hollow structure and is communicated with the cavity tube, and the liquid outlet holes are distributed on the screw thread.
The whole idea of the invention is to process the sample before detection in one kit box to complete the treatment in one step, and the treatment kit can be integrally placed in a modified detection instrument or the detection test paper is taken down to be directly detected by the existing detection instrument.
The kit provided by the invention is also an integrated detection kit, and can meet the calprotectin detection requirements of rapidness, sensitivity, economy, quantification and simple operation. In some embodiments the integrated test kit (test device): the quantitative sampling tube obtains a sample and directly inserts the sample into the sample adding hole of the detection device, the detection device is placed into a matched detector, and the detector automatically detects the sample after 5-10 minutes to obtain a detection item result.
Drawings
FIG. 1 is a schematic structural diagram of a detection kit according to an embodiment of the present invention;
FIG. 2 is a schematic top view of a detection kit according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of a test strip according to an embodiment of the present invention;
FIG. 4 is a partially enlarged schematic view of a sampling tube in accordance with an embodiment of the present invention;
FIG. 5 is a partially enlarged schematic view of a sampling tube according to another embodiment of the present invention;
FIG. 6 is a schematic view showing the external structure of the detection kit according to one embodiment of the present invention;
FIG. 7 is a schematic diagram of a chamber structure of an adapter device in a detection apparatus adapted with the detection kit of the present invention according to an embodiment of the present invention;
FIG. 8 is a schematic external view of a detection apparatus with a chamber adapted to a detection kit according to the present invention in an embodiment of the present invention;
FIG. 9 is a diagram illustrating an analysis of the test results according to an embodiment of the present invention.
Detailed Description
For the sake of understanding, the present invention will be described in detail below with reference to specific drawings and examples. It is to be expressly understood that the description is illustrative only and is not intended as a definition of the limits of the invention. Many variations and modifications of the present invention will be apparent to those skilled in the art in light of the teachings of this specification. In addition, the present invention incorporates publications which are intended to more clearly describe the invention, and which are incorporated herein by reference in their entirety as if reproduced in their entirety.
Example 1
As shown in fig. 1-4 and 6, the calprotectin rapid detection kit comprises a detection kit shell 2, a sampling tube 4, a filter tube 5 and detection test paper 6, wherein the sampling tube 4 can be inserted into the detection kit shell 2, the sampling tube 4 is movably connected with the detection kit shell 2, and the sampling tube 4, the filter tube 5 and the detection test paper 6 are sequentially arranged in the detection kit shell 2 from top to bottom when the sampling tube 4 is inserted into the detection kit shell 2. The detection test paper 6 is a calprotectin detection test paper strip, the detection test paper 6 comprises a fluorescent marker combination pad, a T line and a C line, the content of a quantum dot mark mouse anti-calprotectin monoclonal antibody A in the fluorescent marker combination pad, the T line comprises a coated calprotectin monoclonal antibody B, and the C line comprises a coated goat anti-mouse polyclonal antibody which is provided by Zhuhai Bomei biological science and technology Limited company. The sampling tube is directly inserted into the shell of the detection kit to complete sample treatment after sampling, and the treated sample is directly dripped onto the detection test paper so that the detection test paper can be used for optical detection. The top surface of the detection kit shell 2 is provided with a sample inlet hole 10 matched with the sampling tube 4, the detection kit shell 2 is internally provided with a first fixing device 11 used for stabilizing the sampling tube 4, the detection kit shell 2 is internally provided with a second fixing device 12 used for fixing the filter tube 5, and the first fixing device 11 is positioned above the second fixing device 12. Sampling tube 4 comprises a lumen 401 and a threaded rod 402, the threaded rod 402 being located at the bottom of the lumen 401. The threaded rod is stained with a fixed quantity of the fecal sample, and the threaded rod of the sampling tube is stained with 10 mu L of the fecal sample.
Be equipped with the stock solution pond 405 on first fixing device 11, stock solution pond 405 is the inclined plane on one side inwards, the inclined plane has trompil and plastic envelope to have the tinfoil membrane, stock solution pond 405 and 11 sliding connection of first fixing device, stock solution pond 405 is connected with detect reagent box casing 2 through spring 4051, chamber tube 401 lateral wall is equipped with puncture rod 406, puncture rod 406 can be one or many, chamber tube 401 is equipped with feed liquor trompil 4052, puncture rod 406 is just to feed liquor trompil 4052, the chamber tube 401 outside is equipped with guide post 407, sample hole 10 is other to be equipped with guide post 407 complex direction through-hole 408, because can fix sampling tube 4 male position messenger stock solution pond 405 and insert sampling tube 4 smoothly. After sampling, the sampling tube 4 is inserted into the sampling hole 10, then the spring 4051 is compressed until the liquid storage tank 405 at the liquid inlet opening 4052 pierces into the cavity tube 401, the piercing rod 406 pierces the liquid storage tank 405, liquid in the liquid storage tank 405 flows downwards, and the stool sample stained through the liquid outlet hole is washed.
The detection test paper 6 (detection plate) is connected with the detection kit shell 2 by a buckle, and the detection test paper 6 can be detached from the detection kit shell 2. After being detached, the utility model can be directly inserted into the existing detecting instrument to complete the detection.
The liquid outlet holes 404 are distributed at the joint of the cavity tube 401 and the threaded rod 402.
And the sample inlet hole 10 and the detection hole 7 are not sealed by tinfoil in a plastic way in the reagent detection device. Keeping the interior mainly dry and free from contamination.
The detection test paper 6 and the supporting plate 601 form a detection plate
Example 2
As shown in FIG. 5, on the basis of embodiment 1, the screw thread 403 is a hollow structure and is communicated with the cavity tube 401, and the liquid outlet holes 404 are distributed on the screw thread 403.
Example 3.
As shown in FIGS. 7 and 8, on the basis of embodiment 1, the detection kit housing 2 of the present invention has a detection hole 7 on one side of the outer wall, and the outer wall of the detection kit housing 2 is further provided with a bar code or two-dimensional code 14. The test kit of the present invention can be put into a suitable test apparatus 1 (as shown in FIG. 8) for testing, and the suitable test apparatus of the present invention requires only slight modification of the existing test apparatus (e.g., modification of the existing test apparatus)
Serial fluorescence immunoassay analyzer), changeThe manufacturing scheme can be that a cavity 3 of the adaptive detection kit is formed in the original detection instrument, a bar code or two-dimensional code detection device 15 is arranged on the cavity, the detection head 701 can extend out of the adaptive detection hole 7 (as shown in figure 7), and the detection head 701 is inserted into the detection kit for optical detection during detection.
Example 4
Using the kit procedure of example 1,
and detecting by a series of fluorescence immunoassay analyzers.
The detection kit comprises the following operation steps:
1. threaded rods 402 on the sampling tube 4 were used to randomly sample the fecal specimen at several different locations (note: about 50 mg).
2. The liquid storage tank 405 is pierced and then the fecal diluent flows out to wash down the fecal sample after being inserted from the sampling hole 10 of the detection kit 2.
3. The sample is filtered by the glass fiber membrane in the filter tube and indirectly mixed and dripped into the sample hole of the detection plate.
4. When the blood contains calprotectin, the quantum dot marked mouse anti-calprotectin monoclonal antibody A in the other fluorescent marker combination pads performs antigen-antibody specificity recognition to form a calprotectin-quantum dot marked mouse anti-calprotectin monoclonal antibody A combination, the combination migrates to a detection area along with the capillary action of the membrane and reacts with the mouse anti-calprotectin monoclonal antibody B on the membrane to form a mouse anti-calprotectin monoclonal antibody B-calprotectin-quantum dot marked mouse anti-calprotectin monoclonal antibody A combination, and the combination emits light under the action of ultraviolet excitation light to form a light-emitting strip. The control line (C-line) zone necessarily exhibits a luminescence band under uv excitation light as the liquid surface continues to migrate to the zone of immobilized goat anti-mouse polyclonal antibody, regardless of the presence of calprotectin in the sample. The control line (C line) area luminous strip is simultaneously used as an internal control standard of the reagent, and is helpful for judging whether the sample size is enough or whether the chromatographic process is normal.
5. And (3) when the preset detection time (about 5-10 minutes) is reached, converting the luminous intensity of the read detection line (T line) area into the amount of calprotectin contained in the sample by the detector according to the built-in standard curve.
Calprotectin assay
The sampling detection kit and the commercial detection kit are used for detecting calprotectin of 30 fecal samples in a rapid and quantitative way.
Commercial detection kit: and sampling and sample adding are carried out according to the kit specification, and the operation of reading and detecting the sample is carried out.
The data obtained were as follows:
| |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| The invention kit (mug/g) | 44.00 | 319.50 | 54.00 | 16.60 | 105.00 | 110.00 | 72.10 | 160.00 | 500.00 | 260.00 |
| Commercial kit (mu g/g) | 42.00 | 323.00 | 56.74 | 15.00 | 104.00 | 108.80 | 73.50 | 157.00 | 504.20 | 264.00 |
| |
11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| The invention kit (mug/g) | 370.40 | 200.00 | 300.70 | 220.80 | 240.15 | 420.60 | 13.54 | 270.00 | 36.30 | 290.88 |
| Commercial kit (mu g/g) | 366.00 | 198.45 | 301.00 | 222.00 | 241.00 | 425.00 | 14.86 | 273.62 | 37.50 | 288.50 |
| Sample ID | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| The invention kit (mug/g) | 310.00 | 150.60 | 450.00 | 22.20 | 410.00 | 190.50 | 361.30 | 480.00 | 570.80 | 248.00 |
| Commercial kit (mu g/g) | 315.80 | 149.00 | 448.00 | 21.68 | 409.00 | 189.80 | 365.00 | 482.50 | 566.00 | 251.00 |
The results of the assays for both kits were analyzed for Bland-Altman consistency, as shown in FIG. 9.
The difference of the detection results of the two kits on the same sample is counted, and the distribution of the detection results along with the change of the concentration is depicted for evaluating the consistency level of the two kits. Setting a consistency limit (Mean +/-1.96 SD) by using a 95% confidence interval, and when more than 95% of numerical values are within a positive and negative consistency limit range, indicating that the consistency of the two groups of data is good; the closer the median reference line is to the Y-axis zero value, the higher the level of consistency.
FIG. 9 shows that in 30 samples, the difference between the two measurement results is within the 95% consistency limit, indicating that the detection kit of the present invention has higher consistency with the measurement results of the commercial detection kit;
in combination with P0.3026, the difference in the measured values measured by the two kits can be considered to be not statistically significant at the α -0.05 level. The results of the two kits for measuring calprotectin are considered equivalent and can be interchanged.
Claims (7)
1. The one-step method calprotectin rapid detection kit is characterized by comprising a detection kit shell (2), a sampling tube (4), a filter tube (5) and detection test paper (6), wherein the sampling tube (4) can be inserted into the detection kit shell (2), and the sampling tube (4) is movably connected with the detection kit shell (2);
when the sampling tube (4) is inserted into the detection kit shell (2), the detection kit shell (2) is internally provided with the sampling tube (4), the filter tube (5) and the detection test paper (6) from top to bottom in sequence;
the test strip (6), the test strip (6) comprises a fluorescent marker binding pad, a T line and a C line, the fluorescent marker binding pad contains a quantum dot labeled mouse anti-calprotectin monoclonal antibody A, the T line contains a coated calprotectin monoclonal antibody B, and the C line contains a coated goat anti-mouse polyclonal antibody; a sample inlet hole (10) matched with the sampling tube (4) is formed in the top surface of the detection kit shell (2), a first fixing device (11) used for stabilizing the sampling tube (4) is arranged in the detection kit shell (2), a second fixing device (12) used for fixing the filter tube (5) is arranged in the detection kit shell (2), and the first fixing device (11) is located above the second fixing device (12);
the sampling tube (4) comprises a cavity tube (401) and a threaded rod (402), the threaded rod (402) is positioned at the bottom of the cavity tube (401), threads (403) are arranged on the threaded rod (402), and a liquid outlet hole (404) is formed in the sampling tube (4); be equipped with liquid storage tank (405) on first fixing device (11), liquid storage tank (405) and first fixing device (11) sliding connection, liquid storage tank (405) are connected with detect kit casing (2) through spring (4051), chamber pipe (401) lateral wall is equipped with puncture rod (406), chamber pipe (401) are equipped with feed liquor trompil (4052), puncture rod (406) just are to feed liquor trompil (4052), chamber pipe (401) outside is equipped with guide post (407), advance by being equipped with of appearance hole (10) with guide post (407) complex guide through hole (408).
2. The one-step calprotectin rapid detection kit according to claim 1, wherein said liquid storage tank (405) contains a stool diluent, said stool diluent is 100 μ L of 20mM PBS (PH 7.4), 0.5% BSA, 0.2% Tween20, 0.02% NaN 3 。
3. The kit for rapidly detecting calprotectin by the one-step method according to claim 2, wherein the filter tube (5) has an upper and lower opening structure, and a glass fiber membrane (13) is arranged in the middle of the filter tube (5).
4. The one-step calprotectin rapid detection kit according to claim 3, wherein a detection hole (7) is formed in the outer wall of one side of the detection kit shell (2), and a bar code or a two-dimensional code (14) is further formed in the outer wall of the detection kit shell (2).
5. The one-step calprotectin rapid detection kit according to claim 4, wherein the detection test paper (6) is further provided with a supporting plate (601).
6. The one-step calprotectin rapid test kit according to claim 5, wherein said test strip (6) is connected to the test kit housing (2) by a snap-fit, and said test strip (6) is detachable from the test kit housing (2).
7. The kit for rapid detection of calprotectin in one step according to claim 6, wherein the holes of the exit hole (404) are distributed at the joint of the lumen tube (401) and the threaded rod (402); or the screw thread (403) is a hollow structure and is the same as the cavity tube (401), and the liquid outlet holes (404) are distributed on the screw thread (403).
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| CN109813917A (en) * | 2019-02-22 | 2019-05-28 | 山东爱维德生物科技有限公司 | A kind of calprotectin detection kit and its test method |
| CN110850097A (en) * | 2019-11-07 | 2020-02-28 | 江苏宜偌维盛生物技术有限公司 | Calprotectin detection kit and detection method |
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