CN113311175B - Colloidal gold detection test strip for Shuxuan cattle, preparation method and application - Google Patents

Colloidal gold detection test strip for Shuxuan cattle, preparation method and application Download PDF

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CN113311175B
CN113311175B CN202110589884.9A CN202110589884A CN113311175B CN 113311175 B CN113311175 B CN 113311175B CN 202110589884 A CN202110589884 A CN 202110589884A CN 113311175 B CN113311175 B CN 113311175B
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colloidal gold
test strip
cattle
progesterone
solution
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CN113311175A (en
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易军
王巍
方东辉
石溢
甘佳
邓小东
阿果约达
詹素琼
付茂忠
左之才
郭曦
梁小玉
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Sichuan Animal Science Academy
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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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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Abstract

Colloidal gold detection test strip for Shuxuan cattle, preparation method and application, wherein the method comprises the following steps: establishing a main reproductive hormone change mode of the Shuxuan cattle in early gestation, judging the estrus cycle of the Shuxuan cattle based on the main reproductive hormone change mode, and determining key reproductive hormones for developing the test strip according to the main reproductive hormone change mode in the estrus cycle; preparing a colloidal gold solution by a reduction method; screening monoclonal antibodies based on key reproductive hormones, purifying the monoclonal antibodies obtained by screening, and determining the marking conditions of the monoclonal antibodies; preparing and purifying a gold-labeled monoclonal antibody complex; and assembling the colloidal gold detection test strip. The invention can simply and efficiently detect the early gestation state of the Shuxuan flower cow, shortens the cow breeding process, improves the breeding rate, is beneficial to guiding clinical breeding work, makes up for the blank of detecting the early gestation of the colloidal gold on the beef dual-purpose cow, and has extremely high practical application value.

Description

Colloidal gold detection test strip for Shuxuan cattle, preparation method and application
Technical Field
The invention relates to the field of animal breeding, in particular to a colloidal gold detection early pregnancy test strip for Shuxuan cattle, a preparation method and application.
Background
The Shuxuan beef cattle are wet-heat resistant climates and coarse-feeding resistant beef cattle in the southern area of China, the total number of the current population is 7 tens of thousands, the number of basic cows is 2.4 tens of thousands, and the number of stock bases is large. The Shuxuan flower cattle is used as a main-pushing variety in Sichuan province and is promoted to 21 city states in Sichuan province, and is promoted to 12 provinces in Guizhou, yunnan, tibet, chongqing, gansu, jiangxi, guangdong and the like in China.
At present, the traditional early pregnancy detection method of the Shuxuan flower cattle requires a special laboratory and veterinary personnel with good production experience, and is not suitable for popularization and use by cattle raising cooperation, family farms, farmers and the like. Meanwhile, the colloidal gold detection early pregnancy test paper with convenience and high efficiency is developed based on the progesterone level in milk of cows. For example, patent CN104237538B provides a colloidal gold test strip for detecting progesterone in milk of a cow, which is developed based on progesterone in milk of a cow, can effectively detect pregnancy of a cow, and has high specificity, sensitivity and stability; the patent CN 1796998A provides a test paper for diagnosing early pregnancy of cows, which is also developed based on the milk progesterone of the cows, and has the characteristics of high specificity, high sensitivity and easy storage. The colloidal gold detection test strip developed based on the progesterone level in milk of cows can only be used for cows, but is not suitable for young cows, and the hormone level variation of different varieties of cows in each stage of gestation is huge, so that it is necessary to develop a colloidal gold detection test strip suitable for Shuxuan flower cows so as to make up for the blank of the early pregnancy test strip on dairy-meat dual-purpose cows, and further to efficiently detect the early pregnancy state of the Shuxuan flower cows, thereby better guiding the clinical production of the Shuxuan flower cows, shortening the cow reproduction process and improving the reproduction rate.
Disclosure of Invention
The invention aims to provide a preparation method of a colloidal gold test strip for a Shuxuan cattle and a test strip prepared by the preparation method, wherein the preparation method deduces the estrus cycle of the Shuxuan cattle by analyzing the change mode of main reproductive hormones in early gestation of the Shuxuan cattle, determines the reproductive hormones used for developing the test strip, and develops and prepares the colloidal gold test strip based on the reproductive hormones.
The above purpose is achieved by the following technical scheme:
the preparation method of the colloidal gold detection test strip for the Shuxuan cattle comprises the following steps:
establishing a main reproductive hormone change mode of the Shuxuan cattle in early gestation, judging the estrus cycle of the Shuxuan cattle based on the main reproductive hormone change mode, and determining key reproductive hormones for developing the test strip according to the main reproductive hormone change mode in the estrus cycle;
Preparing a colloidal gold solution by a reduction method;
screening monoclonal antibodies based on the key reproductive hormone, purifying the monoclonal antibodies obtained by screening, and determining the marking conditions of the monoclonal antibodies;
preparing and purifying a gold-labeled monoclonal antibody complex;
and assembling the colloidal gold detection test strip.
In the prior art, the commercial early pregnancy test strip is mainly developed based on the progesterone level in milk of cows, the test strip can only be used for cows to be produced but is not suitable for young cows, and the hormone level variation of each cow in each gestation period is large, so that the early pregnancy test strip developed aiming at the characteristic of the hormone level variation of the Shuxuan cow in the gestation period is lacking at present, the early pregnancy test strip of the Shuxuan cow is still mainly based on the traditional B ultrasonic and rectal tests, and the test method needs special laboratories and veterinary personnel with good production experience, and is not suitable for popularization and use by cattle-raising cooperation, family farms, farmers and the like. To make up for the technical blank of the test strip for early pregnancy detection of the Shuxuan Hua cattle. The application provides a preparation method of a colloidal gold detection test strip for Shuxuan cattle.
In the technical scheme, a main reproductive hormone change mode of the Shuxuan cattle in early gestation is established first. After female animals enter gestation period, organism changes in special physiology, hypothalamus-pituitary-thyroid axis system is in stress state, which results in generation and metabolism of reproduction related hormone. The estrus cycle of the Shuxuan cattle can be judged by defining the change mode of the breeding related technology in the gestation process of the Shuxuan cattle, and the key reproductive hormone for developing the test strip is determined according to the main reproductive hormone change mode in the estrus cycle, so that an important theoretical basis is provided for accurately controlling the breeding production process.
In some embodiments, cows of Althaea rosea with similar age, moderate fat condition and normal reproductive function are randomly selected for synchronous estrus, the estrus cows are determined and bred, and blood or urine samples are collected every 3 days from the day of breeding of the cows to the day 36. The experimental population is divided into a pregnant group and a non-pregnant group according to the pregnancy reaction condition by combining rectal detection and B-ultrasonic detection. Judging the estrus cycle of the Shuxuan flower cattle by analyzing the genital hormone change patterns of the cows in the pregnant group and the non-pregnant group during estrus, and determining the key genital hormone for developing the test strip according to whether the difference is obvious or not by comparing the main genital hormone change patterns of the pregnant group and the non-pregnant group.
In one or more embodiments, the primary reproductive hormones include Luteinizing Hormone (LH), follicle Stimulating Hormone (FSH), progesterone (P4), and estrogen (E2), the estrus cycle of the hollyhock cow is judged based on the patterns of changes in luteinizing hormone, follicle stimulating hormone, progesterone, and estrogen in early gestation of the hollyhock cow, and progesterone with significant differences in gestation and antiprogestin levels is selected as the key reproductive hormone according to the patterns of changes in luteinizing hormone, follicle stimulating hormone, progesterone, and estrogen in the estrus cycle.
In one or more embodiments, the estrus cycle of the Shuxuan cattle is judged to be 21 days based on the change modes of luteinizing hormone, follicle stimulating hormone, progesterone and estrogen in early gestation of the Shuxuan cattle, and the difference of the progesterone in blood of the pregnant cattle and the progesterone in the urine of the non-pregnant cattle is extremely remarkable (p < 0.01) on the 21 st day, and the difference of the progesterone in urine of the pregnant cattle and the non-pregnant cattle is remarkable (p < 0.05), so that the progesterone in the blood and/or the urine on the 21 st day is selected as a key reproductive hormone for judging whether the Shuxuan cattle is pregnant early or not, and the colloidal gold test paper is prepared according to the hormone.
Although pregnant and non-pregnant cows of the hollyhock flower have significant or very significant differences in their urine or blood key reproductive hormones, the differences are typically less than 0.5 μmol/L, e.g., progesterone levels in the blood are 0.34±0.11 μmol/L on the day of estrus (day 0 or day 21) and 0.70±0.21 μmol/L when peaks are reached on day 12. The difference is far smaller than the difference of cow milk progesterone between an unggested cow and a pregnant cow, so that the detection sensitivity of the test strip for detecting the flower Niu Jiaoti gold of the hollyhock is higher.
According to the technical scheme, a small molecule competition method is adopted to prepare a colloidal gold test strip based on serum and/or urine according to the hormone level change rule of key reproductive hormone of the Shuxuan cattle.
In some embodiments, when preparing the colloidal gold solution by using the reduction method, the method specifically includes the following steps: heating chloroauric acid solution with final concentration of 0.01% to boil, and adding to the boiled chloroauric acid solutionAdding 1% trisodium citrate aqueous solution until the reaction solution turns red, and continuing heating until the color is stable and unchanged; after cooling, ultra-pure water was added to the reaction solution to restore the original volume, and 0.02% NaN was added 3 Filtering and sterilizing to obtain a colloidal gold solution, wherein the diameter of colloidal gold particles of the colloidal gold solution is 20-35 nm. The size of the colloidal gold particles is a function of the addition amount of trisodium citrate, and the colloidal gold particles with a certain particle size can be corresponding to the different volumes of 1% trisodium citrate solution added into chloroauric acid solution. In the technical scheme, the size of the colloidal gold particles is controlled between 20 and 35nm by adjusting the addition amount of the trisodium citrate, so that the detection sensitivity of the test strip can be further improved, and the diameter of the colloidal gold particles is preferably 30nm.
In one or more embodiments, after the colloidal gold solution is prepared, the quality of the colloidal gold solution can be comprehensively identified by the following method: the color change of the colloidal gold is observed by naked eyes to see whether turbidity exists, whether transparency exists, whether refraction exists and whether gel exists; observing the size, shape and density of the colloidal gold particles by an electron microscope; in a spectrophotometer OD400 by visible light spectrum nm To OD700 nm Scanning the colloidal gold solution in a spectrum range, judging the relation between the diameter of the colloidal gold particles and the maximum absorption wavelength according to the obtained value, and evaluating the quality of the colloidal gold solution; the colloidal gold solution was left at room temperature, 4℃and 37℃for 24 hours, respectively, and the aggregation was observed.
In the technical scheme, monoclonal antibodies are screened based on the key reproductive hormone, the screened monoclonal antibodies are purified, and the labeling conditions of the screened monoclonal antibodies are determined. In some embodiments, the monoclonal antibody obtained by checkerboard titration screening is a bovine progesterone mouse monoclonal antibody, a Progesterone (PROG) monoclonal antibody (50H 10 cell line), and the conjugate is a Progesterone (PROG) -BSA (or OVA) antigen; during purification, the bovine progesterone mouse monoclonal antibody is placed in a dialysis bag, the dialysis bag is placed in 0.01M PB liquid, the dialysis is carried out fully overnight, and then the polymer of the dialyzed bovine progesterone mouse monoclonal antibody is removed by centrifugation, and the supernatant is taken. In one or more embodiments, the dialysis bag is pre-conditionedCutting the dialysis bag into multiple small sections, strictly checking if there is a leak, washing with distilled water, placing in 500mL of 2% NaHCO containing 1mmol/L EDTA-Na 3 Boiling the solution, taking out with clean tweezers, boiling in distilled water, and rinsing.
After screening and purifying the monoclonal antibodies, the labeling conditions of the screened monoclonal antibodies are determined.
In some embodiments, the determined labeling conditions of the monoclonal antibodies include determining the optimal pH for binding of the bovine progesterone mouse monoclonal antibody to the colloidal gold solution using a colloidal gold gradient method. Specifically, the prepared colloidal gold solution is respectively added into each glass test tube, and the pH value of the colloidal gold solution is respectively adjusted to 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5 by using potassium carbonate solution; adding the bovine progesterone mouse monoclonal antibody into the colloidal gold tube, uniformly mixing and standing at room temperature; then adding 10% NaCl solution into each tube respectively, mixing uniformly, and standing at room temperature for 1-2 hours; observing the color change of the colloidal gold, and recording the lowest pH value for keeping red; then the pH value is adjusted to the lowest gradient pH value of +/-0.1; the above test was repeated. Finally, the lowest pH of the red color is recorded, namely the optimal pH. In one or more embodiments, the optimum pH for binding of the progesterone-in-bovine mouse monoclonal antibody to the colloidal gold solution is 8.0, and by determining the optimum pH, the sensitivity of the test strip can be further increased to detect a less pregnant and non-pregnant progesterone difference in the cow.
In some embodiments, the determined labeling conditions for the monoclonal antibodies include determining the optimal concentration of bovine progesterone mouse monoclonal antibody to bind to the colloidal gold solution using a protein gradient method. Specifically, the prepared colloidal gold solution is respectively added into each glass test tube, and preferably, the colloidal gold solution is adjusted to the optimal pH value; diluting the bovine progesterone mouse monoclonal antibody into 1mg/mL with purified water, and sequentially adding 0, 5, 10, 20, 30, 40, 60 and 80 mu L of each into the small test tube and uniformly mixing; after a period of time, adding 10% of NaCl aqueous solution into each small test tube, uniformly mixing, and standing at room temperature for 1-2 hours; observing the color change of each small test tube, the control tube and the test tube added with insufficient protein to stabilize the colloidal gold, showing the coagulation phenomenon from red to blue, and keeping the red unchanged when the added protein reaches or exceeds the minimum stabilizing amount, and finding out the boundary tube of the colloidal gold liquid from red to blue, wherein the protein content is the minimum protein content required for stabilizing the colloidal gold. In one or more embodiments, the minimum amount of antibody required for a progesterone mouse monoclonal antibody to stabilize 1mL of colloidal gold is 10 μg/mL.
After determining the monoclonal antibody and the labeling conditions, the gold-labeled monoclonal antibody complex can be prepared and purified.
In some embodiments, the preparing and purifying the gold-labeled monoclonal antibody complex comprises the steps of:
preparation: centrifuging the colloidal gold solution to remove larger polymer in the colloidal gold solution, using K 2 CO 3 The pH value of the centrifuged colloidal gold solution is regulated to 8.0, monoclonal antibody is slowly added into the colloidal gold solution, and after the solution is stirred uniformly at room temperature, 10% BSA is added into the colloidal gold solution to a final concentration of 0.4%.
The larger polymers formed during the preparation of the colloidal gold solution can be removed by centrifuging the colloidal gold solution so that they do not affect the adsorption of the colloidal gold particles by the antibody protein during the labeling process, preferably, the colloidal gold solution is centrifuged at 3000r/min for 20min.10% BSA as stabilizer, in one or more embodiments, 10% polyethylene glycol (MW 20000) may be added, stirred at room temperature for 10min, centrifuged at 9000-11000r/min for 40-60min, the supernatant discarded, the precipitate dissolved in the colloidal gold-antibody preservation solution, and filtered with 0.45um filter membrane, thus obtaining the colloidal gold-antibody conjugate stock solution.
Purifying: centrifuging colloidal gold solution at first rotational speed, collecting supernatant, centrifuging supernatant at second rotational speed, discarding supernatant, dissolving precipitate in TBS solution with pH of 8.0 containing 1% BSA and 0.02% NaN 3 And repeating centrifugal washing for 2-3 times to obtain the gold-labeled monoclonal antibody complex.
The purification step adopts a low-temperature ultracentrifugation method, firstly, gold-labeled antibody is centrifuged at a low speed of 1500r/min at 4 ℃, the supernatant is sucked and then the sediment is discarded, then the supernatant is ultracentrifuged at 13000r/min at 4 ℃, the supernatant is discarded, finally, the sediment is dissolved by the TBS buffer solution with the original volume, the centrifugal washing is repeated for 2-3 times, the sediment is resuspended at 1/10 of the original volume, and the sediment is preserved at 4 ℃ for standby.
Further, assembling the colloidal gold test strip comprises the following steps:
an antibody solid-phase NC film, namely a glass fiber film filled with a gold-labeled antibody, is stuck in the middle of the bottom plate, a detection line (T line) and a control line (C line) are arranged on the antibody solid-phase NC film, the working concentration of progesterone antigen of the detection line is 1.0-1.5 mg/mL, and the working concentration of goat anti-mouse IgG antibody of the control line is 1.0-1.5 mg/mL;
attaching a probe strip to one end, close to a detection line, of the base plate, wherein the probe strip at least partially overlaps with the antibody solid-phase NC film, and attaching a sample pad to the lower end of the base plate, and the sample pad at least partially overlaps with the probe strip;
and a piece of water absorbing paper is stuck on one end, close to the control line, of the bottom plate, and the water absorbing paper is at least partially overlapped with the antibody solid-phase NC film.
Preferably, the T-line working concentration of the test strip for detecting the blood progesterone is 1.0mg/mL, and the T-line working concentration of the test strip for detecting the urine progesterone is 1.5mg/mL.
In some embodiments, the stock solution of the colloidal gold-antibody conjugate is diluted with a working solution according to the ratio of 1:2, 1:4, 1:8 and 1:16, the diluted stock solution of the colloidal gold-antibody conjugate is uniformly added on a glass fiber membrane, and the glass fiber membrane is baked at 37 ℃ to obtain a gold-labeled pad, and the optimal working concentration of the colloidal gold-labeled antibody is determined after testing. In one or more embodiments, the working concentration of the colloidal gold-antibody conjugate stock solution is 1:4, which can further enhance the detection effect of the gold-labeled pad.
In some embodiments, after the assembly, the bottom plate is cut into test strips 2.8mm wide and placed in a blank cartridge to form a colloidal gold test strip. In one or more embodiments, a tinfoil is attached to the upper surface of the assembled test strip, so as to protect the detection line, the control line and the detection hole from contamination, and the tinfoil is removed during detection.
The detection principle of the test strip prepared by the preparation method is as follows: the test strip contains an antigen immobilized in advance in the nitrocellulose membrane test region (T) and an antibody II in the control region (C) and a gold-labeled antibody immobilized on a binding pad. After a sample to be detected is added, antigen of a T region envelope solid phase competes with a target antigen in the sample, if the sample is positive, the antibody is combined with the target antigen in the sample, and at the moment, the antibody combined with the T region antigen is reduced or completely absent, so that a mauve strip of the T region is lightened or directly eliminated; if the sample is negative, the T region will appear as a purple band. Whether progesterone exists in the sample or not, a mauve band appears in the C region, and if the mauve band does not exist in the C region, the detection result of the test strip is invalid.
The preparation method is based on the primary reproductive hormone change mode of the Shuxuan cattle in early gestation, judges the estrus cycle of the Shuxuan cattle, determines the key reproductive hormone for developing the early pregnancy detection test strip, prepares the colloidal gold detection test strip around the key reproductive hormone, and constructs the standardized preparation method and flow of the test strip capable of being used for detecting the early pregnancy detection of the Shuxuan cattle, the obtained colloidal gold test strip can simply and efficiently detect the early gestation state of the Shuxuan cattle, shortens the breeding process of the cattle, improves the breeding rate, is beneficial to guiding clinical mating work, makes up for the blank of detecting the early gestation of the colloidal gold on the dairy-meat dual-purpose cattle, and has extremely high practical application value. In addition, the working concentration of the colloidal gold-antibody conjugate stock solution is determined by determining the optimum pH value, the minimum antibody amount and other marking conditions, and the working concentrations of a detection line and a control line are established, so that the test strip has higher sensitivity, and the hormone difference value of less than 0.5 mu mol/L of the pregnant cows of the Shuxuan cattle and the non-pregnant cows can be detected.
The invention further aims to provide a colloidal gold detection test strip for the Shuxuan cattle, and the test strip is prepared by adopting any one of the preparation methods. The detection sensitivity of the detection test strip can reach 5ng/mL, so that whether the progesterone difference value in urine or blood of the Shuxuan cattle exceeds a threshold value or not can be detected, the sensitivity and the specificity are high, whether the Shuxuan cattle is pregnant or not can be detected conveniently and efficiently, and the early pregnancy state of the Shuxuan cattle can be detected efficiently, thereby better guiding the clinical production of the Shuxuan cattle, shortening the breeding process of the cattle and improving the breeding rate.
The invention also aims to provide the application of any one of the test strips in the device for detecting early pregnancy of the Shuxuan cattle.
In the prior art, when early pregnancy detection of cattle is carried out, a worker marks a serial number of a sampling tube to be detected and then stores the sampling tube on a sample tube rack, and then sequentially takes out the sampling tube to detect on a test strip with a corresponding serial number. However, when the sample detection amount is large, the mode is not only easy to cause incorrect operation, so that the serial numbers of the sampling tube to be detected and the test strip are inconsistent, test data are wrong, and the test strips have influence on each other, so that the detection result which is not caused by the sensitivity and the specificity of the test strip is inaccurate, and meanwhile, the traditional detection mode is inconvenient to operate, is inconvenient to manage the sampling tube and the test strip, and has low detection efficiency.
In order to solve the above-mentioned problem, in this technical scheme, detection device includes the casing, be provided with a plurality of detecting element in the casing, detecting element is including being used for holding the detection chamber of test paper strip, the top in detection chamber is provided with logical groove, be provided with the loading piece that is used for loading the sampling pipe in the logical groove, loading piece can overturn in logical groove, and loading piece is connected with the push pedal that is located the detection intracavity through the stay cord, when the test paper strip inserts in the detection chamber, the push pedal can receive the promotion of test paper strip in detection chamber to through stay cord pulling loading piece upset, the loading piece of upset drives the sampling pipe upset, and the liquid that awaits measuring in the sampling pipe can fall into the detection hole of the test paper strip of detection intracavity through logical groove.
Specifically, the detection device includes a housing having at least one end that is open to enable the test strip to be placed into the housing. In one or more embodiments, a plurality of partitions are provided on the bottom surface of the housing, the partitions dividing the interior space of the housing into a plurality of mutually independent detection units. The detection unit is internally provided with a detection cavity, and the test strip can be put into the detection cavity through the opening end of the shell.
The top of detecting the chamber is provided with logical groove, leads to the inside and the external space in groove intercommunication detection chamber. The detection cavity is internally provided with a loading piece capable of overturning relative to the through groove, the loading piece is provided with a loading groove capable of clamping the sampling tube, and the size of the loading groove is matched with that of the sampling tube. When the loading part is turned over, the loaded sampling tube is driven to turn over, and urine or blood to be detected in the sampling tube is driven to be poured into the corresponding detection cavity through the through groove.
The detection cavity is internally provided with a push plate, and a pull rope arranged between the push plate and the loading piece is used for driving the loading piece to turn over when the push plate moves. The driving force of the push plate is the thrust force of the staff on the test strip card shell.
Before detection, each test strip is placed at the inlet of each detection unit, each sampling tube to be detected is placed in the loading piece of each detection unit, and the sampling tube is started, so that the test strip does not exert force on the push plate, and a pull rope between the push plate and the loading piece is in a natural loose state; during detection, the test strips are sequentially pushed inwards, the test strips moving into the detection cavity drive the push plate to move, the moving push plate drives the loading piece to overturn through the pull rope, and when the push plate moves to a preset position, the loading piece overturns by a preset angle, so that liquid in the sampling tube can fall into the detection hole of the test strip in the detection cavity through the through groove, and the sampling is completed; after sampling is completed for a period of time, each test strip is taken out for sample analysis.
In one or more embodiments, the sample to be detected may be filtered using a semipermeable membrane or a dialysis membrane before detection, and a certain amount of filtrate is taken and added into the sampling tube to be detected. In one embodiment, the amount of sample in the sample tube to be tested is 150 to 200. Mu.L.
Through the arrangement, the test strips and the sampling tubes are in one-to-one correspondence and checked before detection, so that the consistency of the serial numbers of the test strips and the sampling tubes can be ensured, and the error rate is effectively reduced; meanwhile, each test strip is positioned in an independent detection unit, so that the influence of each test strip on each other is avoided, and the accuracy of a detection result is greatly improved; in addition, the liquid in the sampling tube to be detected is driven to pour out while the test strip is pushed into the detection cavity, so that the operation is convenient, standard and quick, the detection efficiency and accuracy can be remarkably improved, and the large-scale popularization and application are facilitated.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the preparation method for the Shuxuan cattle provided by the invention is based on the primary reproductive hormone change mode of the Shuxuan cattle in early gestation, judges the estrus cycle of the Shuxuan cattle, determines the key reproductive hormone used for developing the early pregnancy detection test strip, prepares the colloidal gold detection test strip around the key reproductive hormone, and constructs the standardized preparation method and flow of the test strip capable of being used for detecting the early pregnancy detection of the Shuxuan cattle, and the obtained colloidal gold test strip can simply and efficiently detect the early gestation state of the Shuxuan cattle, shortens the breeding process of the cattle, improves the breeding rate, is beneficial to guiding clinical breeding work, fills up the blank of detecting the early gestation of the colloidal gold on the dairy-beef dual-purpose cattle and has extremely high practical application value;
2. According to the preparation method, the working concentration of the colloidal gold-antibody conjugate stock solution is determined by determining the marking conditions such as the optimal pH value, the minimum antibody amount and the like, and the working concentrations of a detection line and a control line are established, so that the test strip has higher sensitivity, and the hormone difference value of less than 0.5 mu mol/L of the pregnant cows and the non-pregnant cows of the Shuxuan cattle can be detected;
3. according to the application, through improvement of the detection device, the test strips and the sampling pipes are in one-to-one correspondence and checked before detection, so that the consistency of the serial numbers of the test strips and the sampling pipes can be ensured, and the error rate is effectively reduced; meanwhile, each test strip is positioned in an independent detection unit, so that the influence of each test strip on each other is avoided, and the accuracy of a detection result is greatly improved; in addition, the liquid in the sampling tube to be detected is driven to pour out while the test strip is pushed into the detection cavity, so that the operation is convenient, standard and quick, the detection efficiency and accuracy can be remarkably improved, and the large-scale popularization and application are facilitated.
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The accompanying drawings, which are included to provide a further understanding of embodiments of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the principles of the application. In the drawings:
FIG. 1 is a schematic flow chart of a preparation method according to an embodiment of the present invention;
FIG. 2 shows the pattern of changes in major reproductive hormones in early gestation in Shuxuan cattle in accordance with an embodiment of the invention, wherein FIG. 2 (a) shows the changes in lutein in the serum; FIG. 2 (b) shows changes in follitropin in serum; FIG. 2 (c) shows the change in serum estrogen; FIG. 2 (d) shows the change in progesterone in serum; fig. 2 (e) shows the change in progesterone in urine;
FIG. 3 is a graph showing the results of an experiment for determining the optimal pH of an antibody label in an embodiment of the present invention;
FIG. 4 is a graph showing the results of an experiment for determining the optimum amount of an antibody in an embodiment of the present invention;
FIG. 5 is a schematic diagram of different determination results of a test strip according to an embodiment of the present invention;
FIG. 6 is a schematic diagram of a detection device for mounting a test strip according to an embodiment of the present invention;
FIG. 7 is a schematic diagram of a detection unit in a ready state according to an embodiment of the present invention;
FIG. 8 is a schematic diagram of a detecting unit in a detecting state according to an embodiment of the present invention;
FIG. 9 is a schematic view of a retaining member triggered by a push plate according to an embodiment of the present invention;
FIG. 10 is a schematic view of the locking member locking the pusher plate in accordance with an embodiment of the present invention;
Fig. 11 is a schematic view of the structure of the splash shield in an embodiment of the present invention.
In the drawings, the reference numerals and corresponding part names:
the test strip comprises a 1-box body, a 2-partition plate, a 3-through groove, a 4-loading part, a 5-baffle plate, a 6-push plate, a 61-clamping groove, a 7-first spring, an 8-splash guard, an 81-opening, an 82-accommodating cavity, an 83-converging surface, a 9-locking part, a 91-connecting plate, a 92-second spring, a 93-clamping protrusion, a 10-pull rope, an 11-rotating shaft, a 12-sampling pipe, a 13-test strip and a 14-detection hole.
Detailed Description
For the purpose of making apparent the objects, technical solutions and advantages of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, wherein the exemplary embodiments of the present invention and the descriptions thereof are for illustrating the present invention only and are not to be construed as limiting the present invention.
All the raw materials of the present invention are not particularly limited in their sources, and can be commercially available or prepared according to conventional methods well known to those skilled in the art.
The purity of all the raw materials of the invention is not particularly limited, and the invention preferably adopts the conventional purity requirements in the field of analytical pure or colloidal gold detection test strips.
All raw materials of the invention, the brands and abbreviations of which belong to the conventional brands and abbreviations in the field of the related application are clear and definite, and the person skilled in the art can purchase from the market or prepare by the conventional method according to the brands, abbreviations and the corresponding application.
In the description of the present invention, it should be understood that the terms "front", "rear", "left", "right", "upper", "lower", "vertical", "horizontal", "high", "low", "inner", "outer", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are merely for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the scope of the present invention.
Example 1:
the preparation method of the colloidal gold test strip for the Shuxuan cattle shown in fig. 1 comprises the following steps:
establishing a main reproductive hormone change mode of the Shuxuan cattle in early gestation, judging the estrus cycle of the Shuxuan cattle based on the main reproductive hormone change mode, and determining key reproductive hormones for developing the test strip according to the main reproductive hormone change mode in the estrus cycle;
preparing a colloidal gold solution by a reduction method;
screening monoclonal antibodies based on the key reproductive hormone, purifying the monoclonal antibodies obtained by screening, and determining the marking conditions of the monoclonal antibodies;
Preparing and purifying a gold-labeled monoclonal antibody complex;
and assembling the colloidal gold detection test strip.
In some embodiments, the primary reproductive hormones include lutein, follicle stimulating hormone, progesterone and estrogen, the estrus cycle of the hollyhock cow is determined based on the patterns of changes in lutein, follicle stimulating hormone, progesterone and estrogen in early gestation of the hollyhock cow, and progesterone with significant differences in gestation hormone levels from the antiprogestin levels is selected as the key reproductive hormone based on the patterns of changes in lutein, follicle stimulating hormone, progesterone and estrogen in the estrus cycle.
In some embodiments, preparing the colloidal gold solution using the reduction method includes the steps of: heating chloroauric acid solution to boiling, adding trisodium citrate aqueous solution into the boiling chloroauric acid solution until the reaction solution turns red, and continuing heating until the color is stable and unchanged; after cooling, adding ultra-pure water into the reaction solution to restore the original volume, adding NaN 3 Filtering and sterilizing to obtain a colloidal gold solution, wherein the diameter of colloidal gold particles of the colloidal gold solution is 20-35 nm.
In some embodiments, the monoclonal antibody obtained by the screening is a bovine progesterone mouse monoclonal antibody, and the conjugate is a Progesterone (PROG) -BSA (or OVA) antigen; during purification, the bovine progesterone mouse monoclonal antibody is placed into a dialysis bag, the dialysis bag is placed into a phosphate buffer solution, the dialysis is carried out fully overnight, and then the polymer is removed by centrifugation of the dialyzed bovine progesterone mouse monoclonal antibody, and the supernatant is taken.
In some embodiments, the preparing and purifying the gold-labeled monoclonal antibody complex comprises the steps of:
preparation: centrifuging the colloidal gold solution to remove larger polymer in the colloidal gold solution, using K 2 CO 3 The pH value of the centrifuged colloidal gold solution is regulated to 8.0, and the solution is added to the colloidal goldSlowly adding the monoclonal antibody into the solution, stirring uniformly at room temperature, and adding 10% BSA into the colloidal gold solution to a final concentration of 0.4%;
purifying: centrifuging colloidal gold solution at first rotational speed, collecting supernatant, centrifuging supernatant at second rotational speed, discarding supernatant, dissolving precipitate in TBS solution with pH of 8.0 containing 1% BSA and 0.02% NaN 3 And repeating centrifugal washing for 2-3 times to obtain the gold-labeled monoclonal antibody complex.
In some embodiments, assembling the colloidal gold test strip includes the steps of:
an antibody solid-phase NC film is stuck in the middle of the bottom plate, a detection line and a control line are arranged on the antibody solid-phase NC film, the working concentration of a T line of a test strip for detecting the progesterone in blood is 1.0mg/mL, the working concentration of a T line of a test strip for detecting the progesterone in urine is 1.5mg/mL, and the working concentration of a goat anti-mouse IgG antibody of the control line is 1.0-1.5 mg/mL;
Attaching a probe strip to one end, close to a detection line, of the base plate, wherein the probe strip at least partially overlaps with the antibody solid-phase NC film, and attaching a sample pad to the lower end of the base plate, and the sample pad at least partially overlaps with the probe strip;
and a piece of water absorbing paper is stuck on one end, close to the control line, of the bottom plate, and the water absorbing paper is at least partially overlapped with the antibody solid-phase NC film.
The detection principle of the test strip prepared by the preparation method is as follows: the test strip contains an antigen immobilized in advance in the nitrocellulose membrane test region (T) and an antibody II in the control region (C) and a gold-labeled antibody immobilized on a binding pad. After a sample to be detected is added, antigen of a T region envelope solid phase competes with a target antigen in the sample, if the sample is positive, the antibody is combined with the target antigen in the sample, and at the moment, the antibody combined with the T region antigen is reduced or completely absent, so that a mauve strip of the T region is lightened or directly eliminated; if the sample is negative, the T region will appear as a purple band. Whether progesterone exists in the sample or not, a mauve band appears in the C region, and if the mauve band does not exist in the C region, the detection result of the test strip is invalid.
As shown in FIG. 5, when the C line develops color, the T line does not develop color or there is a tendency to decrease the color development compared with the C line, it can be judged as positive; when the C line develops color, the T line is visible to naked eyes and the color development degree is equivalent to that of the C line, and the C line can be judged as negative; when the C line does not develop, the test strip is judged to be invalid no matter whether the T line develops or not.
The preparation method of the embodiment judges the estrus cycle of the Shuxuan cattle based on the primary reproductive hormone change mode of the Shuxuan cattle in early pregnancy, determines the key reproductive hormone used for developing the early pregnancy detection test strip, prepares the colloidal gold detection test strip around the key reproductive hormone, and constructs the standardized preparation method and flow of the test strip capable of being used for detecting the early pregnancy detection of the Shuxuan cattle.
Example 2:
based on example 1, a colloidal gold test strip for the blood or urine progesterone of the flower of hollyhock was prepared by the following method.
1) Establishing a main reproductive hormone change mode of Shuxuan cattle in early gestation period
18 cows of Shuxuan flower cattle with similar ages, moderate fat conditions and normal reproductive function are randomly selected to carry out synchronous estrus, 12 cows are determined to carry out estrus and timely breeding, blood samples or urine samples are collected every 3 days from the day of breeding of the cows to the day 36, and 10ml of blood sampling liquid or 5ml of urine sample are collected every time. The experimental group is divided into a non-pregnant group and a pregnant group according to the pregnancy reaction condition by matching with rectal detection and B ultrasonic detection, wherein 7 non-pregnant groups and 5 pregnant groups are adopted.
The non-pregnant and pregnant groups were analyzed for changes in Luteinizing Hormone (LH), follicle Stimulating Hormone (FSH), progesterone (P4) and estrogen (E2) during estrus.
The changes in the major reproductive hormones are shown in FIGS. 2 (a) to (e). Experiments show that the pregnant cow has strong LH peak on 21 days after estrus, the pregnant cow has no LH peak and has stable trend, and the pregnant cow is shown to estrus again and ovulate, and the pregnant cow does not enter a new estrus period. The LH peak of young cattle is related to the in vivo embryo developmental capacity, and the pre-ovulatory LH peak can improve embryo cytoplasmic maturation, but is not a major factor affecting oocyte nuclear maturation. In contrast, FSH in both pregnant and non-pregnant cows showed 1-2 small peaks, presumably related to promotion of follicular development in cows, but serum FSH concentrations in different time periods within the same day differed greatly, so that it was difficult to capture FSH peaks on the day of oestrus in naturally oestrus cows. Progesterone belongs to steroid hormones and is mainly produced by ovarian corpus luteum cells, and intimal cells of follicles and placenta during pregnancy can also be produced. Experiments show that the progesterone level in the blood and urine of the normal estrus of the Shuxuan flower cattle is low on the estrus day and reaches a peak value on 12 days, mainly because the infertility bezoar body reaches the maximum in the estrus cycle of 8-10 days, the progesterone concentration continuously rises to reach the peak value, the luteal degeneration occurs in 15-19 days of the estrus cycle, and the progesterone concentration also drops sharply. In pregnant cows, the corpus luteum is not degenerated, so that the progesterone concentration is steadily increased. At the same time, during the estrus cycle, an increase in E2 levels is associated with each follicle, with a continued growth of the dominant follicle, while the other follicles begin to decay, with a concomitant decrease in E2 levels. The study found that the secretion of E2 from Shuxuan flower cow was high at the beginning of oestrus, then gradually declined and was minimal at 12 days, as opposed to P4 changes, with E2 reaching the next peak when the non-pregnant cow oestrus, while the pregnant cow remained low. While P4 decreases, E2 rises and reaches a higher level in estrus, consistent with follicular development and morphological changes. The hormonal changes that cause oestrus behavior are characterized by a decrease in progesterone levels followed by a rapid increase in estrogen.
Therefore, the estrus cycle of the Shuxuan cattle is judged to be 21 days based on the change patterns of luteinizing hormone, follicle stimulating hormone, progesterone and estrogen in the early gestation period of the Shuxuan cattle. The table of the major reproductive hormone differences on day 21 for the different experimental groups of the hollyhock flower cattle is shown in table 1.
TABLE 1
Day 21 FSH (blood) LH (blood) E2 (blood) P4 (blood) P4 (urine)
Non-pregnant group 0.21 Aa ±0.12 1.13 A ±0.31 2.66 Aa ±0.53 0.34 A ±0.11 0.37 Aa ±0.16
Gestational group 0.20 Aa ±0.09 0.37 B ±0.13 1.82 Aa ±0.47 0.76 B ±0.09 0.66 Ab ±0.06
As can be seen from table 1, the difference of progesterone in blood of pregnant and non-pregnant cows is very significant at 21 days (p < 0.01), the difference of progesterone level in urine is significant (p < 0.05), and by comparing the difference of major reproductive hormone at 21 days with the subsequent trend of variation, blood and/or progesterone in urine at 21 days is selected as the key reproductive hormone for determining whether or not the cow is pregnant early, and the preparation of colloidal gold test paper is performed on the hormone.
Although pregnant and non-pregnant cows of the hollyhock flower have significant or very significant differences in their urine or blood key reproductive hormones, the differences are typically less than 0.5 μmol/L, e.g., progesterone levels in the blood are 0.34±0.11 μmol/L on the day of estrus (day 0 or day 21) and 0.70±0.21 μmol/L when peaks are reached on day 12. The difference is far smaller than the difference of cow milk progesterone between an unggested cow and a pregnant cow, so that the detection sensitivity of the test strip for detecting the flower Niu Jiaoti gold of the hollyhock is higher.
According to the method, a small molecule competition method is adopted to prepare a colloidal gold test strip based on serum and/or urine according to the hormone level change rule of key reproductive hormones of the hollyhock flower cattle.
2) Colloidal gold solution prepared by reduction method
Heating chloroauric acid solution with final concentration of 0.01% to boiling in a microwave oven, rapidly and accurately adding 1% trisodium citrate water solution under stirring, changing golden yellow chloroauric acid water solution into red within 2min, continuously boiling for 5min until color is stable, cooling, recovering original volume with ultrapure water, and adding 0.02% NaN 3 Filtering with 0.22pm filter membrane, sterilizing, and placing into clean siliconized brown glass bottle at 4deg.C for use. In this example, the amount of trisodium citrate added was adjusted so that the size of the colloidal gold particles was 20 to 35nm, preferably 30nm.
After the preparation is finished, the quality of the colloidal gold solution can be comprehensively identified by the following method: the color change of the colloidal gold is observed by naked eyes to see whether turbidity exists, whether transparency exists, whether refraction exists and whether gel exists; observing the size, shape and density of the colloidal gold particles by an electron microscope; in a spectrophotometer OD400 by visible light spectrum nm To OD700 nm Scanning the colloidal gold solution in the spectrum range, judging the relation between the diameter of the colloidal gold particles and the maximum absorption wavelength according to the obtained value, and evaluating the colloidal gold solution Is the mass of (3); the colloidal gold solution was left at room temperature, 4℃and 37℃for 24 hours, respectively, and the aggregation was observed.
3) Screening and purifying monoclonal antibodies
The optimal monoclonal antibody developed by the chessboard titration screening test paper is a Progesterone (PROG) monoclonal antibody (50H 10 cell strain), and the conjugate is a Progesterone (PROG) -BSA (or OVA) antigen.
The dialysis bag is cut into small sections with the length of 10cm, and the dialysis bag can be used when no leak is strictly checked. Washing with distilled water, then placing in 500mL of 2% NaHCO containing 1mmol/LEDTA-Na 3 Boiling the solution for 10min, taking out with clean tweezers, boiling in distilled water for 10min, and rinsing.
Placing the monoclonal antibody to be dialyzed into a dialysis bag which is treated well, fully dialyzing overnight at 4 ℃ in PB liquid of 0.01M after bundling, centrifuging the dialyzed antibody at 10000r/min at 4 ℃ for 1h, removing the polymer, and taking supernatant.
4) Preparation and purification of gold-labeled monoclonal antibody complexes
And (3) centrifuging the colloidal gold at 3000r/min for 20min before marking, and removing large polymers formed in the preparation process so as to prevent the polymers from influencing the adsorption of antibody proteins to the colloidal gold particles in the marking process. By 0.2mol/LK 2 CO 3 And regulating the pH value of the colloidal gold solution to the optimal pH value. The optimal labelling amount of monoclonal antibody was slowly added drop-wise under magnetic stirring, and 1mg of protein was added for about 5min, and labelling was performed for 30min. Slowly dropwise adding 5% BSA to a final concentration of 0.5% serving as a stabilizer, continuously stirring for 30min, sealing, and preserving at 4 ℃ in a dark place for later use.
In one or more embodiments, 10% polyethylene glycol (MW 20000) may be added, stirred at room temperature for 10min, centrifuged at 9000-11000r/min for 40-60min, the supernatant removed, and the precipitate dissolved in the colloidal gold-antibody preservation solution, and filtered with a 0.45um filter membrane to obtain the colloidal gold-antibody conjugate stock solution.
After the preparation, the gold-labeled protein was purified by low temperature ultracentrifugation to remove unlabeled protein and insufficiently labeled colloidal gold in solution and various polymers that may be formed during the labeling process. The gold-labeled antibody was first centrifuged at 1500r/min for 15min at 4℃at low speed, the supernatant carefully aspirated, and the pellet was discarded. The supernatant was then centrifuged at 13000r/min at 4℃and the supernatant was discarded, and finally the pellet was dissolved at pH 8.0.1 mOl/L TBS (1% BSA,0.02% sodium azide) in the original volume, and the centrifugation was repeated 2-3 times to resuspend the pellet at 1/10 of the original volume. Preserving at 4 ℃ for standby.
5) Assembled colloidal gold test strip
Cutting the PVC soleplate into strips with the width of 2.8mm and the length of 6 cm;
an antibody solid-phase NC film is stuck in the middle of the PVC bottom plate, and the distance between the antibody solid-phase NC film and the upper section is 1.5cm;
pasting a glass fiber membrane probe strip at the lower end of the PVC bottom plate, namely a T end close to the NC membrane, overlapping the glass fiber membrane probe strip with the antigen solid-phase NC membrane by 0.1cm, and pasting a sample pad with a width of 1.7cm at the lower end to overlap the probe strip by 0.1-0.2cm;
A piece of water absorbing paper with the width of 1.7cm is stuck on the upper end of the PVC bottom plate, namely, the C end close to the NC film, and is overlapped with the antibody solid-phase NC film by 0.1-0.2cm;
cutting into test strips with the width of 2.8mm, and filling into blank card shells;
the working concentration of the progesterone antigen of the detection line is 1.0-1.5 mg/mL, wherein the working concentration of the T line of the test strip for detecting the progesterone in blood is 1.0mg/mL, and the working concentration of the T line of the test strip for detecting the progesterone in urine is 1.5mg/mL; the working concentration of the goat anti-mouse IgG antibody of the control line is 1.0-1.5 mg/mL.
In one or more embodiments, the stock solution of the colloidal gold-antibody conjugate is diluted with a working solution according to the ratio of 1:2, 1:4, 1:8 and 1:16, the diluted solution of the colloidal gold-antibody conjugate is evenly added on a glass fiber membrane, and the glass fiber membrane is baked at 37 ℃ to obtain a gold-labeled pad, and the optimal working concentration of the colloidal gold-labeled antibody is determined after testing. In one or more embodiments, the working concentration of the colloidal gold-antibody conjugate stock solution is 1:4, which can further enhance the detection effect of the gold-labeled pad.
In one or more embodiments, several different types of NC membranes are selected, and the most suitable type of NC membrane is determined to be a cidolis CN140 by running a plate function test, a test of colloidal gold solution fluidity, hysteresis and background residue on the different types of NC membranes, a reselection test of NC membranes, and the like.
In one or more embodiments, progesterone antigen and goat anti-mouse IgG antibodies are each diluted to the desired concentration with 0.01mol/LpH in 8.0PBS and spotted on NC membranes using a laminator. The distance between the T line and the C line is 0.5cm, the parameters are 1 mu L/cm, and the sprayed NC film is dried for more than 8 hours at 37-45 ℃.
In one or more embodiments, a tinfoil is attached to the upper surface of the assembled test strip, so as to protect the detection line, the control line and the detection hole from contamination, and the tinfoil is removed during detection.
In one or more embodiments, a standard card may also be provided to facilitate staff to compare and confirm the test results.
Example 3:
on the basis of the embodiment, the determining the labeling condition of the screened monoclonal antibody comprises determining the optimal pH value of the combination of the bovine progesterone mouse monoclonal antibody and the colloidal gold solution by adopting a colloidal gold gradient method; the labeling conditions for determining the screened monoclonal antibodies comprise determining the optimal concentration of the bovine progesterone mouse monoclonal antibodies combined with the colloidal gold solution by adopting a protein gradient method.
1) Determining the optimal pH value of the combination of the bovine progesterone mouse monoclonal antibody and the colloidal gold solution
The Niu Yuntong mouse monoclonal antibody is marked by 0.02 percent 20nm colloidal gold, and the optimal pH value of the binding of the antibody and the colloidal gold is determined by a colloidal gold gradient method. The method comprises the following steps:
(a) Taking 7 sampling tubes, and respectively adding 1mL of prepared colloidal gold solution;
(b) The pH of the colloidal gold solution is respectively adjusted to 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5 by using 0.2mol/L potassium carbonate solution;
(c) Adding 50 mu L of 1mg/ml of bovine progesterone mouse monoclonal antibody into the sampling tube, uniformly mixing, and standing at room temperature for 20min;
(d) Then 100 mu L of 10% NaCl solution is added into each tube respectively, and the mixture is uniformly mixed and kept stand for 1 to 2 hours at room temperature;
(e) Observing the color change of the colloidal gold, and recording the lowest pH value for keeping red;
(f) Then the pH value is adjusted to the lowest gradient pH value of +/-0.1; the above test was repeated.
The lowest pH that remained red was recorded as the optimum pH.
The experimental results are shown in fig. 3, and the optimum pH value of the binding of the bovine progesterone mouse monoclonal antibody and the colloidal gold solution is determined to be 8.0. By determining the optimum pH, the sensitivity of the test strip can be further increased to detect a less pregnant and non-pregnant progesterone difference in a Xuanhua cattle.
2) Determining the optimal concentration of the bovine progesterone mouse monoclonal antibody combined with the colloidal gold solution
And determining the optimal concentration of the bovine progesterone mouse monoclonal antibody combined with colloidal gold by adopting a protein gradient method. The method comprises the following steps:
(a) Taking 8 sampling tubes, and respectively adding 1mL of colloidal gold solution with the pH value adjusted to 8.0;
(b) Diluting the bovine progesterone mouse monoclonal antibody into 1mg/mL with purified water, and sequentially adding 0, 5, 10, 20, 30, 40, 60 and 80 mu L of each into the small test tube and uniformly mixing;
(c) After being placed for 10min, 0.1mL of 10% NaCl aqueous solution is added into each sampling tube, and the mixture is uniformly mixed and kept stand for 1 to 2h at room temperature;
(d) Observing the color change of the small test tube, the control tube and the sampling tube added with insufficient protein to stabilize the colloidal gold, showing the coagulation phenomenon from red to blue, and keeping the red color unchanged when the added protein reaches or exceeds the minimum stabilizing amount of the sampling tube, and finding out the boundary tube of the colloidal gold liquid from red to blue, wherein the protein amount is the minimum protein amount required for stabilizing 1ml of colloidal gold.
The experimental results are shown in FIG. 4, from which the minimum amount of antibody required for the progesterone mouse monoclonal antibody to stabilize 1mL of colloidal gold was determined to be 10. Mu.g/mL.
In the preparation method, the working concentration of the colloidal gold-antibody conjugate stock solution is determined by determining the labeling conditions such as the optimal pH value, the minimum antibody amount and the like, and the working concentrations of a detection line and a control line are established, so that the test strip has higher sensitivity, and the hormone difference value of less than 0.5 mu mol/L of the pregnant cows and the non-pregnant cows of the Shuxuan cattle can be detected.
Example 4:
on the basis of the embodiment, an application of any one of the detection test strips in a detection device for early pregnancy of Shu Xuanhua cattle is provided, as shown in fig. 6 to 11, the detection device comprises a shell 1, a plurality of detection units are arranged in the shell 1, each detection unit comprises a detection cavity for containing the test strip 13, a through groove 3 is formed in the top of each detection cavity, a loading piece 4 for loading a sampling tube 12 is arranged in each through groove 3, the loading piece 4 can be overturned in each through groove 3, a push plate 6 positioned in each detection cavity is connected with a pull rope 10, when the test strip 13 is inserted into each detection cavity, the push plate 6 can be pushed by the corresponding test strip 13 to move in each detection cavity, the overturned loading piece 4 drives the corresponding sampling tube 12 to overturn by pulling the corresponding pull rope 10, and liquid to be detected in the sampling tube 12 can fall into a detection hole 14 of the test strip 13 in each detection cavity through the corresponding through grooves 3.
In one or more embodiments, a plurality of partitions 2 are provided on the bottom surface of the housing, and the partitions 2 divide the internal space of the housing into a plurality of mutually independent detection units. The detection unit is internally provided with a detection cavity, and the test strip can be put into the detection cavity through the opening end of the shell.
In one or more embodiments, as shown in fig. 6, a rotating shaft 11 is disposed on an outer wall of the loading member, and the rotating shaft is movably inserted into a wall surface of the through slot, so that the loading member can be turned relative to the through slot.
Before detection, each test strip is placed at the inlet of each detection unit, each sampling tube to be detected is placed in the loading piece of each detection unit, and the sampling tube is started, so that the test strip does not exert force on the push plate, and a pull rope between the push plate and the loading piece is in a natural loose state; during detection, the test strips are sequentially pushed inwards, the test strips moving into the detection cavity drive the push plate to move, the moving push plate drives the loading piece to overturn through the pull rope, and when the push plate moves to a preset position, the loading piece overturns by a preset angle, so that liquid in the sampling tube can fall into the detection hole of the test strip in the detection cavity through the through groove, and the sampling is completed; after sampling is completed for a period of time, each test strip is taken out for sample analysis.
In one or more embodiments, the sample to be detected may be filtered by using a semi-permeable membrane or a dialysis membrane before detection, and the step of standing the sample to be detected can be omitted by filtering the sample to be detected by using the dialysis membrane. In one embodiment, after filtration is complete, 150 to 200. Mu.L of the filtrate is added to the sample tube to be tested.
In one or more embodiments, as shown in fig. 6 to 8, the upper surface of the detection unit is further provided with a baffle 5, and the baffle 5 is located on one side of the through groove near the open end of the detection device, so that when the loading piece is turned over, a limit is formed on the turning angle of the loading piece, and the sampling tube is prevented from falling into the detection cavity through the through groove.
In one or more embodiments, each detection unit is provided with an identification area, where the identification area is used to display the serial number of the corresponding detection unit, so as to further reduce the error rate.
In some embodiments, as shown in fig. 7, a first spring 7 is further disposed between the push plate 6 and the detection cavity, and a torsion spring (not shown) is disposed on the rotating shaft, where when the test strip does not act on the push plate, the first spring is in a natural extension state, and the loading member is kept vertical or relatively vertical under the action of the torsion spring. When detecting, when the clamping shell of the test strip pushes the push plate, the push plate compresses the first spring and drives the loading piece to overcome the acting force of the torsion spring to turn over; after sampling is completed, the acting force on the test paper strip is removed, the push plate is reset under the acting force of the first spring, the pull rope is loosened, the loading piece losing the pull force of the pull rope is reset under the acting force of the torsion spring, and then the detection unit is reset to an initial undetected state. Through the setting of first spring and torsional spring for detection device can automatic recovery to initial position after the sampling is accomplished, further reduces the operation degree of difficulty, improves detection efficiency.
In some embodiments, as shown in fig. 6 to 10, the detection device is further provided with a self-locking mechanism. The self-locking mechanism comprises a locking piece 9, a connecting plate 91 is arranged on the outer wall of the locking piece, a second spring 92 is arranged on the connecting plate 91, the bottom end of the second spring 92 is connected to the upper surface of the detection device, the bottom end of the locking piece 9 movably penetrates through the detection cavity, a clamping protrusion 93 is arranged at the bottom end of the locking piece, a clamping groove 61 is formed in the push plate 6, and the clamping protrusion 93 is matched with the clamping groove 61 in size.
The push plate is driven by the test strip clamping shell to move into the detection cavity, when the push plate moves to the clamping bulge, the push plate pushes the clamping bulge, the locking piece vertically moves upwards, when the clamping groove of the push plate passes through the clamping bulge, the clamping bulge which is not supported vertically moves downwards under the action of the second spring and is inserted into the clamping groove to form clamping connection, after the clamping connection, the locking piece and the push plate are relatively fixed, and the push plate reaches a preset position; after sampling is completed, the locking piece is lifted upwards to unlock, and the push plate is reset under the action of the first spring. The setting makes the staff need not to consider the distance that the push pedal moved in detection process, and the detection hole of test strip can align with the drop point of the liquid that awaits measuring in the sampling pipe after locking, further simplifies the operation step, improves the degree of accuracy that detects.
In one or more embodiments, the side of the latch protrusion 93 facing the push plate 6 is inclined so that when the latch protrusion is acted upon by the push plate, a vertical component force is generated to urge the locking member vertically upward.
In some embodiments, as shown in fig. 7, 8 and 11, a splash guard is further provided in the detection chamber below the baffle for guiding the liquid poured in the sampling tube to drop to the detection hole below. In one or more embodiments, the splash guard is a hollow structure, the face of the splash guard facing the through groove is an inclined plane, an opening is formed in the inclined plane to communicate with the accommodating cavity and the external space inside the splash guard, the bottom surface of the accommodating cavity is a converging face, so that liquid entering the accommodating cavity through the opening can be converged toward the bottom of the accommodating cavity through the converging face, and finally drops into the detection hole through the opening at the bottom of the accommodating cavity. Through above-mentioned setting, the liquid of pouring on the splash guard is shunted into two parts, and one part is flowed along the inclined plane of splash guard, and another part is collecting in holding the chamber, avoids pouring the liquid instantaneous flow of pouring in the sampling pipe too big and leads to part liquid to splash behind the contact inclined plane, leads to the sampling volume not up to standard, further improves the degree of accuracy that detects.
The use of "first," "second," etc. (e.g., first spring, second spring, etc.) herein is merely for clarity of description to distinguish corresponding components and is not intended to limit any order or emphasize importance, etc. In addition, the term "coupled" as used herein may be directly coupled or indirectly coupled via other components, unless otherwise indicated.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims (9)

1. The utility model provides a colloidal gold test strip for hollyhock flower cattle, its characterized in that, the test strip is arranged in detecting means according to the progesterone content in urine or the blood of hollyhock flower cattle and detects whether the hollyhock flower cattle is pregnant, detecting means includes casing (1), be provided with a plurality of detecting element in casing (1), detecting element is including being used for holding the detection chamber of test strip (13), the top in detection chamber is provided with logical groove (3), be provided with in logical groove (3) and be used for loading piece (4) of sampling pipe (12), loading piece (4) can overturn in logical groove (3), loading piece (4) are connected with push pedal (6) that are located the detection chamber through stay cord (10), when test strip (13) are inserted in the detection chamber, push pedal (6) can be moved in the detection chamber by the promotion of test strip (13) to through stay cord (10) pulling loading piece (4) upset in the removal process, loading piece (12) upset can fall into in detection chamber (14) through the detection hole (14) that test strip (12) that test sample pipe (12) was overturned.
2. A method for preparing a colloidal gold test strip for a buffalo, characterized in that it is used for preparing the test strip according to claim 1, said method comprising the steps of:
establishing a main reproductive hormone change mode of the Shuxuan cattle in early gestation, judging the estrus cycle of the Shuxuan cattle based on the main reproductive hormone change mode, and determining key reproductive hormones for developing the test strip according to the main reproductive hormone change mode in the estrus cycle;
preparing a colloidal gold solution by a reduction method;
screening monoclonal antibodies based on the key reproductive hormone, purifying the monoclonal antibodies obtained by screening, and determining the marking conditions of the monoclonal antibodies;
preparing and purifying a gold-labeled monoclonal antibody complex;
and assembling the colloidal gold detection test strip.
3. The method for preparing the colloidal gold test strip for the Shuxuan cattle according to claim 2, wherein the main reproductive hormones comprise luteinizing hormone, follicle stimulating hormone, progesterone and estrogen, the estrus cycle of the Shuxuan cattle is judged based on the change modes of the luteinizing hormone, the follicle stimulating hormone, the progesterone and the estrogen in the early gestation period of the Shuxuan cattle, and the progesterone with obvious difference between the level of the pregnancy hormone and the level of the non-pregnancy hormone is selected as the key reproductive hormone according to the change modes of the luteinizing hormone, the follicle stimulating hormone, the progesterone and the estrogen in the estrus cycle.
4. The method for preparing a colloidal gold test strip for a flower of hollyhock according to claim 2, wherein the preparation of the colloidal gold solution by the reduction method comprises the steps of:
heating chloroauric acid solution to boiling, adding trisodium citrate aqueous solution into the boiling chloroauric acid solution until the reaction solution turns red, and continuing heating until the color is stable and unchanged; after cooling, adding ultra-pure water into the reaction solution to restore the original volume, adding NaN 3 Filtering and sterilizing to obtain colloidal gold solution, wherein the colloidal gold of the colloidal gold solution isThe particle diameter is 20-35 nm.
5. The method for preparing the colloidal gold test strip for the Shuxuan cattle according to claim 2, wherein the monoclonal antibody obtained by screening is a bovine progesterone mouse monoclonal antibody, and the conjugate is progesterone-BSA or progesterone-OVA antigen; during purification, the bovine progesterone mouse monoclonal antibody is placed into a dialysis bag, the dialysis bag is placed into a phosphate buffer solution, the dialysis is carried out fully overnight, and then the polymer is removed by centrifugation of the dialyzed bovine progesterone mouse monoclonal antibody, and the supernatant is taken.
6. The method of claim 5, wherein determining the labeling conditions of the selected monoclonal antibodies comprises determining the optimal pH for binding of the bovine progesterone mouse monoclonal antibody to the colloidal gold solution using a colloidal gold gradient method.
7. The method of claim 5, wherein determining the labeling conditions of the selected monoclonal antibodies comprises determining the optimal concentration of bovine progesterone mouse monoclonal antibodies to be combined with the colloidal gold solution by a protein gradient method.
8. The method for preparing a colloidal gold test strip for use in a flower of hollyhock according to claim 3, wherein the preparation and purification of the gold monoclonal antibody complex comprises the steps of:
preparation: centrifuging the colloidal gold solution to remove larger polymer in the colloidal gold solution, using K 2 CO 3 The pH value of the centrifuged colloidal gold solution is regulated to 8.0, monoclonal antibody is slowly added into the colloidal gold solution, and after the solution is stirred uniformly at room temperature, 10% BSA is added into the colloidal gold solution to a final concentration of 0.4%;
purifying: centrifuging colloidal gold solution at first rotation speed, collecting supernatant, centrifuging supernatant at second rotation speed, discarding supernatant, dissolving precipitate in TBS solution of original volumeThe TBS solution has a pH of 8.0 and contains 1% BSA and 0.02% NaN 3 And repeating centrifugal washing for 2-3 times to obtain the gold-labeled monoclonal antibody complex.
9. The method for preparing a colloidal gold test strip for use in a flower of hollyhock according to claim 3, wherein assembling the colloidal gold test strip comprises the steps of:
an antibody solid-phase NC film is stuck in the middle of a bottom plate, a detection line and a control line are arranged on the antibody solid-phase NC film, the working concentration of progesterone antigen of the detection line is 1.0-1.5 mg/mL, and the working concentration of goat anti-mouse IgG antibody of the control line is 1.0-1.5 mg/mL;
attaching a probe strip to one end, close to a detection line, of the base plate, wherein the probe strip at least partially overlaps with the antibody solid-phase NC film, and attaching a sample pad to the lower end of the base plate, and the sample pad at least partially overlaps with the probe strip;
and a piece of water absorbing paper is stuck on one end, close to the control line, of the bottom plate, and the water absorbing paper is at least partially overlapped with the antibody solid-phase NC film.
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