CN114778859A - Urine detection kit for early-stage rapid pregnancy diagnosis of yaks - Google Patents
Urine detection kit for early-stage rapid pregnancy diagnosis of yaks Download PDFInfo
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- CN114778859A CN114778859A CN202210385131.0A CN202210385131A CN114778859A CN 114778859 A CN114778859 A CN 114778859A CN 202210385131 A CN202210385131 A CN 202210385131A CN 114778859 A CN114778859 A CN 114778859A
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- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/743—Steroid hormones
-
- 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
- G01N33/54306—Solid-phase reaction mechanisms
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- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- 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
- G01N33/577—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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
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Abstract
The invention belongs to the technical field of detection kits, and relates to a urine detection kit for early rapid pregnancy diagnosis of yaks, which comprises a test strip, an upper shell and a lower shell, wherein the test strip comprises a bottom plate, a sample pad, a gold-labeled pad, a nitrocellulose membrane and absorbent filter paper are sequentially arranged on the bottom plate from left to right, a detection line and a quality control line are arranged on the nitrocellulose membrane, the detection line is a progesterone monoclonal antibody, and the quality control line is goat anti-mouse IgG. The kit provided by the invention can be used for rapidly judging early pregnancy of yaks by detecting yak urine samples, and is accurate in result and high in sensitivity.
Description
Technical Field
The invention belongs to the technical field of detection kits, and relates to a urine detection kit for early and rapid diagnosis of yak pregnancy.
Background
The colloidal gold is called colloidal gold because the redox agent acts on chloroauric acid to reduce gold atoms, and the gold atoms are aggregated to form a hydrophobic colloidal solution with negative charges and are converted into a stable colloidal state due to electrostatic interaction. The basic principle of the colloidal gold rapid diagnosis technology is that a microporous membrane is used as a solid phase carrier, known specific antibodies or antigens are coated, the antigens or antibodies to be detected are added to form specific binding, and then the detection is completed through the color reaction of a marker of the colloidal gold.
Yak is the main economic animal in Qinghai-Tibet plateau area, and provides production data such as meat, milk, hair and the like for plateau areas. In the breeding process of yaks, due to special geographical factors in plateau areas, the breeding rate of the yaks is low. The early pregnancy diagnosis can detect the early pregnancy state of the yak, identify the pregnancy state of the female yak in a short time after hybridization, save the production cost, shorten the calving interval and have great significance in actual production.
Disclosure of Invention
The invention aims to solve the technical problem that the urine detection kit for the early rapid pregnancy diagnosis of yaks is provided aiming at the defects of the prior art, the urine of yaks is collected and detected through the urine detection kit, whether the yaks are pregnant or not can be detected efficiently, the detection time is short, and the sensitivity is high.
The invention provides a urine detection kit for early rapid pregnancy diagnosis of yaks, which comprises a test strip, an upper shell and a lower shell;
the test strip comprises a bottom plate, and a sample pad, a gold label pad, a nitrocellulose membrane and absorbent filter paper are sequentially arranged on the bottom plate from left to right;
the nitrocellulose membrane is provided with a detection line and a quality control line;
the detection line is a progesterone monoclonal antibody, and the quality control line is goat anti-mouse IgG;
and the upper shell is provided with a sample adding hole.
Further, still be provided with result observation area and label on going up the casing, it is protruding to go up the casing bottom to be provided with the buckle.
Furthermore, the sampling holes and the result observation area are both designed in a hollow manner.
Furthermore, the upper part of the lower shell is provided with a buckle groove which is adaptively connected with the buckle protrusion, and the lower shell is also provided with a test paper strip clamping groove.
Further, the bottom plate is made of PVC material.
Further, the gold-labeled pad is a solid-phase carrier membrane formed by combining the prepared progesterone monoclonal antibody and colloidal gold.
Furthermore, a gold-labeled progesterone monoclonal antibody, namely a conjugate of the progesterone monoclonal antibody and colloidal gold, is arranged on the gold-labeled pad.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention discloses a detection kit for early rapid yak pregnancy diagnosis by preparing a progesterone monoclonal antibody and goat anti-mouse IgG based on the combination of a double antibody sandwich method and a colloidal gold technology.
2. The detection line and the quality control line are observed, so that whether yaks are pregnant or not can be detected 22 days after hybridization, the detection time is early, and the sensitivity is high; the result observation area displays a detection line and a quality control line to indicate the yak pregnancy; only displaying a quality control line in a result observation area, and indicating that the yak is not pregnant; the result observation area only displays the detection lines, which indicates that the result is invalid; the result observation area does not show any line, indicating that the result is invalid.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic structural diagram of a yak early-stage rapid pregnancy diagnosis urine detection kit provided by the invention;
FIG. 2 is a schematic structural view of the upper and lower housings of FIG. 1;
FIG. 3 is a schematic structural diagram of the test strip of FIG. 1;
FIG. 4 is a schematic diagram illustrating the determination of the detection result in embodiment 1 of the present invention;
wherein panel a shows positive, indicating pregnancy;
panel B shows negative, indicating no pregnancy;
both plot C and plot D indicate invalidity.
Description of the reference numerals: 101-upper shell, 102-result observation area, 103-label, 104-sample adding hole, 105-lower shell, 106-buckle bulge, 107-test strip clamping groove, 108-sample pad, 109-gold mark pad, 110-detection line, 111-quality control line, 112-water absorption filter paper, 113-bottom plate and 114-nitrocellulose membrane.
Detailed Description
The invention is described in detail below with reference to the drawings and specific examples, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the description of the present invention, it is to be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "axial", "radial", "circumferential", etc. indicate orientations or positional relationships based on those shown in the drawings, merely for convenience of description and simplification of the technical solution of the present invention, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the invention.
Example 1
The invention is based on the principle of a double antibody sandwich method and a colloidal gold immunochromatographic technique. The gold-labeled progesterone monoclonal antibody is adsorbed on a gold-labeled pad, the specific progesterone monoclonal antibody is fixed on the nitrocellulose membrane in a strip shape to be used as a detection line, and the goat anti-mouse IgG is fixed on the nitrocellulose membrane in a strip shape to be used as a quality control line. When the sample to be detected is added on the sample pad at one end of the test strip, the sample moves forwards through capillary action, the antigen in the sample to be detected is combined with the progesterone monoclonal antibody marked by the colloidal gold on the gold mark pad, the sample continues to move forwards, when the sample moves to the detection line area, the sample to be detected is specifically combined with the progesterone monoclonal antibody on the detection line, a sandwich is formed and trapped, the sandwich is gathered on the detection strip, and the color development result can be observed through naked eyes.
As shown in fig. 1-4, a yak early-stage rapid pregnancy diagnosis urine detection kit comprises a test strip, an upper shell 101 and a lower shell 105;
the test strip comprises a bottom plate 113, wherein a sample pad 108, a gold label pad 109, a nitrocellulose membrane 114 and water-absorbing filter paper 112 are sequentially arranged on the bottom plate 113 from left to right;
the gold-labeled pad is a solid phase carrier membrane formed by combining the prepared progesterone monoclonal antibody and colloidal gold;
the nitrocellulose membrane 114 is formed by spraying a detection line 110 and a quality control line 111 on the nitrocellulose membrane 114 in parallel, wherein the detection line 110 is a progesterone monoclonal antibody, and the quality control line 111 is goat anti-mouse IgG;
the absorbent filter paper 112 is used for absorbing residual urine;
the upper shell 101 is provided with a sample adding hole 104;
the sample adding hole 104 is hollow and faces the test strip sample pad 108, the result observation area 102 is hollow and faces the test strip nitrocellulose membrane 115, wherein T faces the test strip detection line 110, and C faces the test strip quality control line 111.
When the kit is used, after a proper amount of urine is added into the sample adding hole 104, a urine sample flows to the gold-labeled pad 109 through the sample pad 108, the gold-labeled progesterone monoclonal antibody exists on the gold-labeled pad 109, progesterone in the sample is combined with the progesterone to form a progesterone-gold-labeled progesterone monoclonal antibody complex, the progesterone continues to flow to the nitrocellulose membrane 114, when the progesterone-gold-labeled progesterone monoclonal antibody flows to the detection line 110, the progesterone monoclonal antibody-progesterone-gold-labeled progesterone monoclonal antibody complex is combined with a substance on the detection line 110, a color reaction occurs due to the fact that colloidal gold is labeled, other gold-labeled progesterone monoclonal antibodies flowing to the quality control line 111 are combined with goat anti-mouse IgG to generate a color reaction, and then result judgment can be carried out;
displaying a detection line and a quality control line in a result observation area, and explaining yak pregnancy; only displaying a quality control line in a result observation area, and indicating that the yak is not pregnant; only the detection line is displayed in the result observation area, which indicates that the result is invalid; the result observation area does not show any line, indicating that the result is invalid.
In this embodiment, the upper housing 101 is further provided with a result observation area 102 and a label 103, the bottom of the upper housing 101 is provided with a fastening protrusion 106, the result observation area 102 is used for observing a result, the label 103 is used for filling in a sample number, a part of the label can be torn off for standby, a blank part of the label can be filled in other sample information, and the fastening protrusion 106 is used for being fixedly connected with the lower housing 105.
The sample adding hole 104 and the result observation area 102 are both designed in a hollow-out manner, so that sample adding and result observation are facilitated.
The upper portion of the lower shell 105 is provided with a buckle groove which is connected with the buckle protrusion 106 in an adaptive manner, the lower shell 105 is further provided with a test strip clamping groove 107, the buckle groove is connected with the buckle protrusion in an adaptive manner to form a closed space, and the test strip clamping groove is used for placing and fixing a test strip.
The test strip clamping groove 107 is made of resin materials, so that the test strip is convenient to fix.
The base plate 113 is made of PVC material and is used to connect the various parts of the test strip (sample pad, gold pad, nitrocellulose membrane and absorbent filter paper).
The gold-labeled pad 109 is a solid-phase carrier membrane formed by combining the prepared progesterone monoclonal antibody with colloidal gold, and is used for combining progesterone in the sample and labeling the colloidal gold.
The gold-labeled pad 109 is provided with a gold-labeled progesterone monoclonal antibody.
In the detection kit for the early rapid pregnancy diagnosis of yaks, the preparation method of the colloidal gold comprises the following steps: colloidal gold is prepared by a trisodium citrate reduction method.
Diluting 1ml of 1% chloroauric acid solution to 100ml, heating to 100 ℃, adding 1% citric acid solution, stirring, heating until the solution turns color to wine red, stirring, cooling, fixing the volume to the original volume by using deionized water, and filtering at 4 ℃ for later use; the prepared colloidal gold solution is wine red, does not contain oil and spherical floating materials, and is not turbid. The prepared colloidal gold is identified by ultraviolet scanning spectrum, the maximum absorption peak value is 525nm, and the diameter is 30 nm.
In the detection kit for the early rapid pregnancy diagnosis of yaks, the preparation of the progesterone monoclonal antibody comprises the following steps:
using bovine serum albumin as an immune antigen carrier, coupling activated progesterone and ovalbumin, immunizing a mouse by using the prepared immune antigen for 14 days for the first immunization and 14 days for the second immunization, enhancing the immunization for 3-4 days, then taking out spleen, preparing myeloma cells, fusing the myeloma cells with spleen cells of the immunized mouse, culturing, screening positive cell strains for later use, selecting the mouse for good hybridoma cell inoculation, selecting the mouse with large abdominal cavity after 7 days, collecting and purifying ascites, and determining the progesterone monoclonal antibody through the content of the ascites protein, the subclass of the monoclonal antibody and the identification of specificity.
In the detection kit for the early rapid pregnancy diagnosis of yaks, the preparation method of the gold-labeled progesterone monoclonal antibody comprises the following steps:
adjusting the pH value of a colloidal gold solution to 8.6 by using hydrochloric acid, dropwise adding a progesterone monoclonal antibody, stirring, then adding a 5% PEG-2000 solution to make the final concentration of the solution be 1%, stirring for 30min, centrifuging at 4 ℃ for 1 hour at 2000r/min, taking a supernatant, centrifuging at 15000r/min for 1 hour, removing the supernatant, centrifuging at 4 ℃ for 1 hour at 18000r/min, repeatedly centrifuging for 2 times, and diluting the precipitate with a gold-labeled antibody diluent for later use.
Example 2
Example 2 detection of sensitivity of detection kit for early rapid diagnosis of yak pregnancy: selecting estrus female yaks for artificial insemination and mating, and determining the yaks in pregnancy by combining rectal detection and B-ultrasonic method after mating, wherein the total number of the yaks is 29. Meanwhile, urine is collected, the rapid detection is carried out by using the kit provided by the invention, and progesterone in the urine is detected by using the ELISA kit, and the result is shown in table 1. Meanwhile, 10 un-bred and un-pregnant female yaks are randomly selected, and the kit is applied to pregnancy detection. The result shows that all 29 pregnant female yaks are detected to be positive, and the detection sensitivity of the kit can reach 100%. The detection results of the female yaks which are not pregnant are negative, which indicates that the sensitivity of the kit to the negative detection can reach 100%.
TABLE 1 Positive detection sensitivity of kit for rapid early pregnancy diagnosis of yaks
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (8)
1. A yak early-stage rapid pregnancy diagnosis urine detection kit is characterized by comprising a test strip, an upper shell (101) and a lower shell (105);
the test strip comprises a bottom plate (113), and a sample pad (108), a gold label pad (109), a nitrocellulose membrane (114) and absorbent filter paper (112) are sequentially arranged on the bottom plate (113) from left to right;
the nitrocellulose membrane (114) is provided with a detection line (110) and a quality control line (111);
the detection line (110) is a progesterone monoclonal antibody, and the quality control line (111) is goat anti-mouse IgG;
and the upper shell (101) is provided with a sample adding hole (104).
2. The urine detection kit according to claim 1, wherein the upper case (101) is further provided with a result observation area (102) and a label (103), and the bottom of the upper case (101) is provided with a snap projection (106).
3. The urine detection kit according to claim 2, wherein the sample application hole (104) and the result observation area (102) are both hollow.
4. The urine detection kit according to claim 2, wherein a snap groove adapted to be connected with the snap protrusion (106) is formed on the upper portion of the lower housing (105), and a test strip slot (107) is further formed on the lower housing (105).
5. The urine detection kit according to claim 4, wherein the material of the dipstick groove (107) is a resin material.
6. The urine test kit according to claim 1, wherein the bottom plate (113) is a bottom plate of PVC material.
7. The urine detection kit according to claim 1, wherein the gold-labeled pad (109) is a solid-phase carrier membrane made of a prepared progesterone monoclonal antibody combined with colloidal gold.
8. The urine detection kit according to claim 1, wherein the gold-labeled pad (109) is provided with a gold-labeled progesterone monoclonal antibody, i.e. a conjugate of progesterone monoclonal antibody and colloidal gold.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210385131.0A CN114778859A (en) | 2022-04-13 | 2022-04-13 | Urine detection kit for early-stage rapid pregnancy diagnosis of yaks |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210385131.0A CN114778859A (en) | 2022-04-13 | 2022-04-13 | Urine detection kit for early-stage rapid pregnancy diagnosis of yaks |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030073248A1 (en) * | 2001-09-28 | 2003-04-17 | J.W. Roth | Bovine pregnancy test |
| CN103336120A (en) * | 2013-05-10 | 2013-10-02 | 上海阿趣生物科技有限公司 | Milk cow pregnancy colloidal gold detection test strip |
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- 2022-04-13 CN CN202210385131.0A patent/CN114778859A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030073248A1 (en) * | 2001-09-28 | 2003-04-17 | J.W. Roth | Bovine pregnancy test |
| CN103336120A (en) * | 2013-05-10 | 2013-10-02 | 上海阿趣生物科技有限公司 | Milk cow pregnancy colloidal gold detection test strip |
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