CN114544979A - Blood detection kit for early-stage rapid pregnancy diagnosis of yaks - Google Patents
Blood detection kit for early-stage rapid pregnancy diagnosis of yaks Download PDFInfo
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- 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
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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
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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Abstract
The invention belongs to the technical field of detection kits, and relates to a blood 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 red blood cell filter membrane, a gold label 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 blood samples, and has the advantages of accurate result and high sensitivity.
Description
Technical Field
The invention belongs to the technical field of detection kits, and relates to a blood detection kit for early and rapid diagnosis of yak pregnancy.
Background
The colloidal gold is called colloidal gold because the redox agent acts on the 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 yaks, identify the conception state of female yaks in a short time after hybridization, save the production cost, shorten the calving interval and have great significance in the actual production.
Disclosure of Invention
The invention aims to solve the technical problem that the blood detection kit for the early rapid pregnancy diagnosis of yaks is provided aiming at the defects of the prior art, the yak blood is collected and detected through the kit, the yak pregnancy can be efficiently detected, the detection time is short, and the sensitivity is high.
The invention provides a blood detection kit for early and rapid diagnosis of yak pregnancy, which comprises a test strip, an upper shell and a lower shell;
the test strip comprises a bottom plate, and a sample pad, a red blood cell filter membrane, 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.
Further, a gold-labeled progesterone monoclonal antibody is arranged on the gold-labeled pad, and the gold-labeled progesterone monoclonal antibody is a conjugate of the progesterone monoclonal antibody and colloidal gold.
Further, the red blood cell filter membrane is arranged between the sample pad and the gold label pad.
Furthermore, the aperture of the erythrocyte filter membrane is 0.2-4 μm.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention is based on the combination of a double-antibody sandwich method and a colloidal gold technology, and develops a detection kit aiming at the early-stage rapid pregnancy diagnosis of yaks by preparing a progesterone monoclonal antibody and goat anti-mouse IgG.
2. The invention can detect whether the yak is pregnant 22 days after hybridization by observing the detection line and the quality control line, and has early detection time and high sensitivity.
3. In the invention, a result observation area displays a detection line and a quality control line to indicate the yak pregnancy; the result observation area only displays a quality control line, which indicates 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, which indicates 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 blood detection kit for early rapid pregnancy diagnosis of yaks, which is 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 graph C and D indicate invalidity.
Description of reference numerals: 101-upper shell, 102-result observation area, 103-label, 104-sample adding hole, 105-lower shell, 106-buckle protrusion, 107-test strip clamping groove, 108-sample pad, 109-gold label pad, 110-detection line, 111-quality control line, 112-water absorption filter paper, 113-bottom plate, 114-erythrocyte filter membrane and 115-nitrocellulose membrane.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, 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 can be 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, and are only for convenience of describing technical solutions of the present invention and simplifying the description, but do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific 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 the gold-labeled pad, the specific progesterone monoclonal antibody is fixed on the nitrocellulose membrane in a strip shape to serve as a detection line, and the goat anti-mouse IgG is fixed on the nitrocellulose membrane in a strip shape to serve as a quality control line. When a sample to be detected is added on a sample pad at one end of the test strip, the sample moves forwards through capillary action, red blood cells in the sample to be detected can be filtered through a red blood cell filter membrane, then the red blood cells continue to move forwards through capillary action, antigens in the sample to be detected are combined with the progesterone monoclonal antibody marked by the colloidal gold on the gold pad, the sample to be detected continues to move forwards, when the sample moves to a detection line area, the substance to be detected is specifically combined with the progesterone monoclonal antibody on the detection line, a sandwich is formed and intercepted, the substance to be detected is gathered on the detection line, and a color development result can be observed through naked eyes.
As shown in fig. 1-4, a blood detection kit for rapid early pregnancy diagnosis of yaks comprises 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 red blood cell filter membrane 114, a gold label pad 109, a nitrocellulose membrane 115 and absorbent 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 with colloidal gold;
the nitrocellulose membrane 115 is characterized in that a detection line 110 and a quality control line 111 are sprayed on the nitrocellulose membrane 115 in parallel, the detection line 110 is a progesterone monoclonal antibody, and the quality control line is goat anti-mouse IgG;
the erythrocyte filter 114 is used for filtering erythrocytes in blood so that serum enters the test strip for detection;
the water-absorbing filter paper 112 is used for absorbing residual serum;
the upper shell 101 is provided with a sample adding hole 104 for facilitating sample adding;
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 whole blood is added into the sample adding hole 104, a blood sample passes through the sample pad 108, red blood cells are filtered out by the red blood cell filter membrane 114 and then flow to the gold label pad 109, a gold label progesterone monoclonal antibody exists on the gold label pad 109, progesterone in the sample is combined with the gold label progesterone to form a progesterone-gold label progesterone monoclonal antibody complex, the progesterone-gold label progesterone monoclonal antibody complex continuously flows to the nitrocellulose membrane 115, when the progesterone-gold label progesterone monoclonal antibody flows to the detection line 110, the progesterone-gold label progesterone monoclonal antibody complex is combined with a substance on the detection line 110 to form a progesterone monoclonal antibody-progesterone-gold label progesterone monoclonal antibody complex, a color reaction occurs due to the fact that colloidal gold is labeled, and other gold label 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 determination can be carried out;
the result observation area displays a detection line and a quality control line to indicate the yak pregnancy; the result observation area only displays a quality control line, which indicates 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, which indicates that the result is invalid.
In this embodiment, the upper case 101 is further provided with a result observation area 102 and a label 103, the bottom of the upper case 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 103 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 case 105.
The sample adding hole 104 and the result observation area 102 are both hollow-out designs, which is convenient for sample adding and result observation.
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 label pad 109 is provided with a gold label progesterone monoclonal antibody, and the gold label progesterone monoclonal antibody is a conjugate of a progesterone monoclonal antibody and colloidal gold.
The red blood cell filter 114 is disposed between the sample pad 108 and the gold label pad 109 for filtering red blood cells in the blood sample.
The pore diameter of the erythrocyte filter membrane 114 is 0.2-4 μm.
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 has the maximum absorption peak value of 525nm and the diameter of 30nm through ultraviolet scanning spectral identification.
In the detection kit for the early rapid diagnosis of the yak pregnancy, 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 number of the yaks is 37. Blood is collected at the same time, rapid detection is carried out by the method, and progesterone in the blood is detected by 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 only 1 yak of 37 yaks of pregnancy is not detected, and the detection sensitivity of the kit can reach 97.30%. The detection results of the female yaks which are not pregnant are all 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 (10)
1. A blood detection kit for rapid early pregnancy diagnosis of yaks 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 red blood cell filter membrane (114), a gold label pad (109), a nitrocellulose membrane (115) and absorbent filter paper (112) are sequentially arranged on the bottom plate (113) from left to right;
the nitrocellulose membrane (115) 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 is goat anti-mouse IgG;
and the upper shell (101) is provided with a sample adding hole (104).
2. The blood 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 blood test kit according to claim 2, wherein the sample application hole (104) and the result observation area (102) are both hollow.
4. The blood detection kit according to claim 2, wherein a snap groove adapted to be connected with the snap protrusion (106) is formed at an upper portion of the lower housing (105), and a test strip slot (107) is further formed on the lower housing (105).
5. The blood test kit according to claim 4, wherein the strip slot (107) is made of resin.
6. The blood test kit according to claim 1, wherein the base plate (113) is a base plate of PVC material.
7. The blood test kit according to claim 1, wherein the gold-labeled pad (109) is a solid phase support membrane made of a prepared progesterone monoclonal antibody bound to colloidal gold.
8. The blood detection kit according to claim 1, wherein the gold-labeled pad (109) is provided with a gold-labeled progesterone monoclonal antibody, and the gold-labeled progesterone monoclonal antibody is a conjugate of a progesterone monoclonal antibody and colloidal gold.
9. The blood test kit according to claim 1, wherein the red blood cell filter (114) is disposed between the sample pad (108) and the gold-labeled pad (109).
10. The blood test kit according to claim 9, wherein the red blood cell filter (114) has a pore size of 0.2-4 μm.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210384171.3A CN114544979A (en) | 2022-04-13 | 2022-04-13 | Blood 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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| CN202210384171.3A CN114544979A (en) | 2022-04-13 | 2022-04-13 | Blood detection kit for early-stage rapid pregnancy diagnosis of yaks |
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| CN114544979A true CN114544979A (en) | 2022-05-27 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118956774A (en) * | 2024-08-06 | 2024-11-15 | 甘孜藏族自治州畜牧业科学研究所(甘孜藏族自治州牦牛产业发展中心) | A hybridoma cell line secreting monoclonal antibody against yak IgG and its monoclonal antibody and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030073248A1 (en) * | 2001-09-28 | 2003-04-17 | J.W. Roth | Bovine pregnancy test |
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2022
- 2022-04-13 CN CN202210384171.3A patent/CN114544979A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030073248A1 (en) * | 2001-09-28 | 2003-04-17 | J.W. Roth | Bovine pregnancy test |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118956774A (en) * | 2024-08-06 | 2024-11-15 | 甘孜藏族自治州畜牧业科学研究所(甘孜藏族自治州牦牛产业发展中心) | A hybridoma cell line secreting monoclonal antibody against yak IgG and its monoclonal antibody and application |
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