CN113311175A - Colloidal gold detection test strip for Sichuan Huaniu cattle, preparation method and application - Google Patents

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

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Publication number
CN113311175A
CN113311175A CN202110589884.9A CN202110589884A CN113311175A CN 113311175 A CN113311175 A CN 113311175A CN 202110589884 A CN202110589884 A CN 202110589884A CN 113311175 A CN113311175 A CN 113311175A
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colloidal gold
test strip
cattle
detection
progesterone
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CN113311175B (en
Inventor
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 HUSBANDRY SCIENCE RESEARCH INSTITUTE
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SICHUAN ANIMAL HUSBANDRY SCIENCE RESEARCH INSTITUTE
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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

Abstract

The colloidal gold test strip for the Sichuan huahuaniu comprises the following steps: establishing a main reproductive hormone change mode of the Shuxuan Hua cattle in the early gestation period, judging the oestrus cycle of the Shuxuan Hua cattle based on the main reproductive hormone change mode, and determining key reproductive hormone for developing the test strip according to the main reproductive hormone change mode in the oestrus cycle; preparing a colloidal gold solution by a reduction method; screening monoclonal antibodies based on key reproductive hormones, purifying the screened monoclonal antibodies, and determining the labeling conditions of the screened monoclonal antibodies; preparing and purifying a gold-labeled monoclonal antibody compound; and assembling the colloidal gold test strip. The method can simply and efficiently detect the early pregnancy state of the Sichuan huahua cow, shorten the cow breeding process, improve the breeding rate, facilitate the guidance of clinical hybridization work, make up the blank of early pregnancy detection of colloidal gold on a dairy and meat cow, and have extremely high practical application value.

Description

Colloidal gold detection test strip for Sichuan Huaniu cattle, preparation method and application
Technical Field
The invention relates to the field of animal breeding, in particular to a colloidal gold test strip for early pregnancy detection of Sichuan huahua cattle, a preparation method and application.
Background
The Sichuan Huaniu cattle is a dual-purpose dairy and meat cattle with resistance to moist-heat climate and rough feeding in southern areas of China, the total number of the cattle is 7 ten thousand at present, the number of basic cows is 2.4 thousand, and the number of stockpiles is large. The Shuxuanhuaniu is popularized to 21 cities of Sichuan as a main promotion variety of Sichuan province, and is popularized to 12 provinces of Guizhou, Yunnan, Tibet, Chongqing, Gansu, Jiangxi, Guangdong and the like in China.
At present, the traditional early pregnancy detection method of the Sichuan Huaniu cattle needs a special laboratory and veterinarians with good production experience, and is not suitable for popularization and use in cattle-raising cooperative society, family farms, farmers and the like. Meanwhile, the convenient and efficient colloidal gold test paper for detecting early pregnancy is developed based on the level of progesterone in milk of the dairy cow. For example, patent CN104237538B provides a milk progesterone colloidal gold test strip for dairy cows, which is developed based on milk progesterone for dairy cows, and can effectively detect pregnancy of dairy cows, and has high specificity, sensitivity and stability; patent CN 1796998A provides a test paper for diagnosing early pregnancy of dairy cows, which is developed based on milk progesterone of dairy cows, and has the characteristics of high specificity, high sensitivity and easy storage. The colloidal gold test strip for detecting early pregnancy developed based on the progesterone level in milk of the dairy cow can only be used for multiparous dairy cows but not for young cows, and the hormone level of different varieties of cows in each stage of pregnancy has great variation, so that the colloidal gold test strip suitable for the hollyhock buffalo needs to be developed to make up the blank of the early pregnancy test strip on a beef and cow, and further efficiently detect the early pregnancy state of the hollyhock buffalo, so that the clinical production of the hollyhock is guided better, the reproductive process of the cows is shortened, and the reproductive rate is improved.
Disclosure of Invention
The invention aims to provide a preparation method of a colloidal gold detection test strip for Sichuan huahuahua cattle and the test strip prepared by the preparation method.
The above purpose is realized by the following technical scheme:
the preparation method of the colloidal gold test strip for the Sichuan huahuaniu comprises the following steps:
establishing a main reproductive hormone change mode of the Shuxuan Hua cattle at the early gestation stage, judging the oestrus cycle of the Shuxuan Hua cattle based on the main reproductive hormone change mode, and determining key reproductive hormone for developing the test strip according to the main reproductive hormone change mode in the oestrus cycle;
preparing a colloidal gold solution by a reduction method;
screening monoclonal antibodies based on the key reproductive hormones, purifying the screened monoclonal antibodies, and determining the labeling conditions of the screened monoclonal antibodies;
preparing and purifying a gold-labeled monoclonal antibody compound;
and assembling the colloidal gold test strip.
In the prior art, commercially available early pregnancy detection test strips are developed based on the progesterone level in milk of dairy cows, the test strips can only be used for multiparous cows but not suitable for young cows, and the variation of the hormone level of each cow in each stage of gestation is large, so that the early pregnancy detection test strips developed aiming at the characteristic of the variation of the hormone level of holy-Xuan-Huaniu in gestation are lacked at present, the traditional B-mode ultrasonography and rectal detection are mainly used in the early pregnancy detection method of holy-Xuan-Huaniu, and the detection method needs a special laboratory and veterinarians with good production experience and is not suitable for popularization and use in cattle-raising cooperative, family farms, farmers and the like. The test paper strip makes up the technical blank of the test paper strip for detecting early pregnancy of the Shuxuan Hua cattle. The application provides a preparation method of a colloidal gold test strip for Sichuan huahuaniu.
In the technical scheme, a change mode of main reproductive hormones of the Shuxuanhua cattle in the early pregnancy is established at first. After the female livestock enters the gestation period, the organism generates special physiological changes, and a hypothalamus-pituitary-thyroid axis system is in a stress state, so that the generation and metabolism of reproduction-related hormones are changed. The oestrus cycle of the Shuxuan Huaniu can be judged by determining the change mode of the propagation related technology in the pregnancy process of the Shuxuan Huaniu, and the key reproductive hormone for developing the test strip is determined according to the change mode of the main reproductive hormone in the oestrus cycle, so that an important theoretical basis is provided for accurately controlling the propagation production process.
In some embodiments, cows of Shuxuan Hua cows with similar age, moderate fat and normal reproductive function are randomly selected for estrus synchronization, the estrus cows are identified and bred, and blood or urine samples are collected every 3 days from the day of breeding to 36 days. Combining rectal detection and B ultrasonic detection, dividing experimental groups into pregnant groups and non-pregnant groups according to pregnancy reaction conditions. The oestrus cycle of the Shuxuan Hua cattle is judged by analyzing the change modes of the reproductive hormones of the cows in a pregnant group and a non-pregnant group during oestrus, and the key reproductive hormone for developing the test strip is determined by comparing the change modes of the main reproductive hormones of the pregnant group and the non-pregnant group according to whether the difference is obvious or not.
In one or more embodiments, the primary reproductive hormones include Luteinizing Hormone (LH), Follicle Stimulating Hormone (FSH), progesterone (P4), and estrogen (E2), the oestrus cycle of a holly-announced cow is determined based on the pattern of change of luteinizing hormone, follicle stimulating hormone, progesterone, and estrogen in the early gestation period of the holly-announced cow, and progesterone with a significant difference in the level of pregnancy hormone from the level of non-progestogen is selected as the key reproductive hormone based on the pattern of change of luteinizing hormone, follicle stimulating hormone, progesterone, and estrogen in the oestrus cycle.
In one or more embodiments, the oestrus cycle of the holly flower cattle is judged to be 21 days based on the change patterns of luteinizing hormone, follicle stimulating hormone, progesterone and estrogen in the early pregnancy of the holly flower cattle, the difference of blood progesterone of pregnant cattle and non-pregnant cattle is very significant (p <0.01) on the 21 st day, the difference of urine progesterone of pregnant cattle and non-pregnant cattle is significant (p <0.05), so that the blood and/or urine progesterone on the 21 st day is selected as a key reproductive hormone for judging whether the holly flower cattle is pregnant early, and the preparation of the colloidal gold test paper is carried out on the hormone.
Although the difference in key reproductive hormones in urine or blood of pregnant and non-pregnant cows of holhua cows is significant or very significant, it is usually less than 0.5 μmol/L, e.g. the progesterone level in the blood is 0.34 ± 0.11 μmol/L on the day of estrus (day 0 or day 21) and 0.70 ± 0.21 μmol/L when it peaks on day 12. The difference is far smaller than the difference between the non-pregnant cow and the pregnant cow of the milk progesterone of the dairy cow, so that the detection sensitivity of the colloidal gold detection test strip for the holly flower cattle is higher.
In the technical scheme, the colloidal gold test strip based on serum and/or urine is prepared by adopting a small molecule competition method according to the hormone level change rule of key reproductive hormones of the Shuxuan Huaniu.
In some embodiments, the method for preparing the colloidal gold solution by a reduction method specifically comprises the following steps: heating the chloroauric acid solution with the final concentration of 0.01% to boiling, adding 1% trisodium citrate aqueous solution into the boiling chloroauric acid solution until the reaction solution turns red, and continuing heating until the color is stable; after cooling, ultrapure water was added to the reaction solution to restore the original volume, and 0.02% NaN was added3And 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 gold colloid particles is a function of the amount of trisodium citrate added, and the addition of different volumes of 1% trisodium citrate solution to the chloroauric acid solution will correspond to the size of the gold colloid particles. In the technical scheme, the size of the colloidal gold particles is controlled to be 20-35 nm by adjusting the addition amount of trisodium citrate, so that the detection sensitivity of the test strip can be further improved, and preferably, the diameter of the colloidal gold particles is 30 nm.
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: observing the color change of the colloidal gold by naked eyes to see whether the colloidal gold is turbid or transparent or not, whether the colloidal gold is refractive or not and whether the colloidal gold is gel or not; observing the size, the shape and the density of the colloidal gold particles through an electron microscope; spectrophotometer OD400 by visible spectrumnmTo OD700nmScanning the colloidal gold solution in a spectral range, judging the relationship between the diameter of the colloidal gold particles and the maximum absorption wavelength according to the obtained numerical value, and evaluating the quality of the colloidal gold solution; the colloidal gold solution is placed at room temperature, 4 ℃ and 37 ℃ for 24h respectively, and the agglomeration condition is observed.
In the technical scheme, the monoclonal antibody is screened based on the key reproductive hormone, the monoclonal antibody obtained by screening is purified, and the marking condition of the screened monoclonal antibody is determined. In some examples, the monoclonal antibody selected by checkerboard titration was progesterone mouse monoclonalThe monoclonal antibody (50H10 cell line) is Progesterone (PROG), and the conjugate is Progesterone (PROG) -BSA (or OVA) antigen; when purifying, the bovine progesterone mouse monoclonal antibody is put into a dialysis bag, the dialysis bag is put into 0.01M PB solution, the solution is fully dialyzed overnight, and then the dialyzed bovine progesterone mouse monoclonal antibody is centrifuged to remove polymers, and supernatant is taken. In one or more embodiments, the dialysis bag is pre-treated, cut into pieces, checked for leaks, washed with distilled water, and then placed in 500mL of 2% NaHCO containing 1mmol/L EDTA-Na3Boiling the solution, taking out with clean forceps, boiling in distilled water, and rinsing.
After screening and purifying the monoclonal antibody, the marking condition of the screened monoclonal antibody is determined.
In some embodiments, the determined labeling conditions for the monoclonal antibody include determining the optimal pH for binding of the bovine progesterone mouse monoclonal antibody to a 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 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, respectively adding 10% NaCl solution into each tube, uniformly mixing, and standing for 1-2 hours at room temperature; observing the color change of the colloidal gold, and recording the lowest pH value for keeping red; then adjusting the pH value to the lowest gradient pH value of +/-0.1; the above experiment was repeated. Finally, the lowest pH at which the color remained red was recorded, i.e., the optimum pH. In one or more embodiments, the optimum pH of the combination of the bovine progesterone mouse monoclonal antibody and the colloidal gold solution is 8.0, and by determining the optimum pH, the sensitivity of the test strip can be further improved to detect holy flower cattle with a smaller difference between pregnant and non-pregnant progesterone.
In some embodiments, the determined labeling conditions for the monoclonal antibody include protein gradient methods to determine the optimal concentration of bovine progesterone mouse monoclonal antibody bound to colloidal gold solution. Specifically, adding prepared colloidal gold solution into each glass test tube, preferably, adjusting the colloidal gold solution to the optimal pH value; diluting the bovine progesterone mouse monoclonal antibody to 1mg/mL by using purified water, and sequentially adding 0, 5, 10, 20, 30, 40, 60 and 80 mu L of the bovine progesterone mouse monoclonal antibody into the small test tube to be uniformly mixed; after the mixture is placed for a period of time, adding 10% NaCl aqueous solution into each small test tube, uniformly mixing, and standing for 1-2 hours at room temperature; observing the color change of each small test tube, wherein the control tube and the test tube with the added protein with the amount not enough to stabilize the colloidal gold show the coagulation phenomenon from red to blue, while the test tube with the added protein amount reaching or exceeding the minimum stable amount keeps the red unchanged, finding out the boundary tube of the colloidal gold liquid from red to blue, and the protein amount contained in the boundary tube is the minimum protein amount required by the stable colloidal gold. In one or more embodiments, the minimum amount of antibody required for the progesterone mouse monoclonal antibody to stabilize 1mL of colloidal gold is 10 μ g/mL.
After the monoclonal antibody and the labeling conditions are determined, the gold-labeled monoclonal antibody compound can be prepared and purified.
In some embodiments, the preparing and purifying gold-labeled monoclonal antibody complexes comprises the following steps:
preparation: centrifuging the colloidal gold solution to remove larger polymers from the colloidal gold solution, using K2CO3The pH value of the centrifuged colloidal gold solution is adjusted to 8.0, the monoclonal antibody is slowly added into the colloidal gold solution, and after the solution is uniformly stirred at room temperature, 10% BSA is added into the colloidal gold solution until the final concentration is 0.4%.
Larger polymers formed during the preparation of the colloidal gold solution can be removed by centrifugation of the colloidal gold solution so that they do not affect the adsorption of the antibody protein to the colloidal gold particles during the labeling process, preferably, the colloidal gold solution is centrifuged at 3000r/min for 20 min. 10% BSA is used as a stabilizer, in one or more embodiments, 10% polyethylene glycol (MW20000) can also be added, the mixture is stirred for 10min at room temperature, centrifuged for 40-60min at 9000-11000r/min, the supernatant is discarded, the precipitate is dissolved in the colloidal gold-antibody preservation solution and filtered by a 0.45um filter membrane, and the obtained colloidal gold-antibody conjugate stock solution is obtained.
And (3) purification: centrifuging the colloidal gold solution at a first rotation speed, sucking the supernatant, centrifuging the supernatant at a second rotation speed, discarding the supernatant, and dissolving the precipitate in the original substanceIn the TBS solution, the pH of the TBS solution was 8.0, and the TBS solution contained 1% BSA and 0.02% NaN3And repeating centrifugal washing for 2-3 times to obtain the gold-labeled monoclonal antibody compound.
And the purification step adopts a low-temperature ultracentrifugation method, firstly, the gold-labeled antibody is subjected to low-speed centrifugation at 1500r/min at 4 ℃, supernatant is sucked, the precipitate is discarded, then, the supernatant is subjected to ultracentrifugation at 13000r/min at 4 ℃, the supernatant is discarded, finally, the precipitate is dissolved by TBS buffer solution with the original volume, the centrifugal washing is repeated for 2-3 times, 1/10 with the original volume is subjected to heavy suspension precipitation, and the precipitate is stored at 4 ℃ for later use.
Further, assembling the colloidal gold test strip comprises the following steps:
an antibody solid phase NC membrane, namely a glass fiber membrane perfused with gold-labeled antibodies, is adhered to 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 membrane, 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;
adhering a probe strip to one end, close to the detection line, of the bottom plate, wherein the probe strip is at least partially overlapped with the antibody solid-phase NC membrane, and adhering a sample pad to the lower end of the bottom plate, and the sample pad is at least partially overlapped with the probe strip;
and adhering absorbent paper on one end of the bottom plate close to the control line, wherein the absorbent paper is at least partially overlapped with the antibody solid-phase NC membrane.
Preferably, the test strip for detecting blood progesterone has a T-line working concentration of 1.0mg/mL, and the test strip for detecting urine progesterone has a T-line working concentration of 1.5 mg/mL.
In some embodiments, the colloidal gold-antibody conjugate stock solution is diluted by working solution according to the ratio of 1:2, 1:4, 1:8 and 1:16, the colloidal gold-antibody conjugate diluent is uniformly added on a glass fiber membrane, the glass fiber membrane is baked at 37 ℃ to obtain a gold label 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 gold colloidal-antibody conjugate stock solution is 1:4, which ratio can further improve the detection effect of the gold-labeled pad.
In some embodiments, after the assembly is completed, the substrate is cut into 2.8mm wide strips and placed into blank card shells to form the colloidal gold test strips. In one or more embodiments, a piece of tinfoil paper is attached to the upper surface of the assembled test strip to protect the detection line, the control line and the detection hole from contamination, and the tinfoil paper is removed during detection.
The detection principle of the test strip prepared by the preparation method is as follows: the test strip contains antigen pre-immobilized on the test area (T) of the nitrocellulose membrane and II antibody of the control area (C) and gold-labeled antibody immobilized on the conjugate pad. After the sample to be detected is added, the antigen of the envelope solid phase of the T area competes for the gold-labeled antibody with the 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 antigen of the T area is reduced or completely eliminated, so that a mauve strip of the T area becomes light or is directly eliminated; if the sample is negative, a purple red band appears in the T area. No matter whether progesterone exists in the sample or not, a mauve strip appears in the C area, and if the mauve strip does not exist in the C area, the test result of the test strip is invalid.
The preparation method judges the oestrus cycle of the Sichuan huahua cattle based on the change mode of the main reproductive hormone of the Sichuan hua cattle in the early pregnancy, 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, constructs a standardized preparation method and a standardized process for detecting the Sichuan hua cattle early pregnancy detection test strip, can simply and efficiently detect the early pregnancy state of the Sichuan hua cattle, shortens the breeding process of the cows, improves the breeding rate, is beneficial to guiding clinical hybridization work, makes up the blank of early pregnancy detection of the colloidal gold on the cow with milk and meat, 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 labeling conditions such as the most suitable pH value and the minimum antibody amount, and the working concentrations of a detection line and a control line are established, so that the test strip has higher sensitivity and can detect the hormone difference value of less than 0.5 mu mol/L of pregnant cows and non-pregnant cows of the holzhou hua cows.
The invention also aims to provide a colloidal gold test strip for Sichuan huahuaniu, which is prepared by any one of the test strip preparation methods. The detection sensitivity of the detection test strip can reach 5ng/mL, so that whether the progesterone difference in urine or blood of the Sichuan huahua cattle exceeds a threshold value or not can be detected, the sensitivity and the specificity are high, whether the Sichuan hua cattle is pregnant or not can be detected conveniently and efficiently, and then the early pregnancy state of the Sichuan hua cattle can be detected efficiently, so that the clinical production of the Sichuan hua cattle can be guided better, the breeding process of the cattle can be shortened, and the breeding rate can be improved.
The invention further aims to provide application of any one of the test strips in the detection device for early pregnancy of the hollyhock propaganda cattle.
In the prior art, when the early pregnancy of the cattle is detected, a worker stores the marked sequence number of a sampling tube to be detected on a sampling tube frame, then takes out the sampling tube in sequence, and detects the sampling tube on a test strip corresponding to the sequence number. However, when the sample detection amount is large, the mode is not only easy to cause the inconsistency of the serial numbers of the sampling tube to be detected and the test strip due to misoperation, the test data is 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 not accurate.
In order to solve the problem, among 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 that detects the chamber is provided with logical groove, logical inslot is provided with the piece of loading that is used for loading the sampling pipe, the piece of loading can be at logical inslot upset, and the piece of loading is connected with the push pedal that is located the detection intracavity through the stay cord, when the test paper strip inserts to detecting the chamber, the push pedal can be moved at the detection intracavity by the promotion of test paper strip to at the removal in-process through the upset of stay cord pulling piece of loading, the piece of loading of upset drives the sampling pipe upset, and the liquid that awaits measuring in the sampling pipe can fall into to the measuring hole of the test paper strip of detection intracavity through leading to the groove.
Specifically, the detection device comprises a shell, at least one end of the shell is an open end so that the test strip can be placed in the shell. In one or more embodiments, the bottom surface of the housing is provided with a plurality of partitions, and the partitions divide the internal space of the housing into a plurality of mutually independent detection units. A detection cavity is arranged in the detection unit, and the test strip can be put into the detection cavity through the opening end of the shell.
The top in detection chamber is provided with logical groove, leads to the inside and the exterior space in groove intercommunication detection chamber. What set up in the detection chamber can be provided with the loading groove that can joint the sampling pipe for the loading piece that leads to the groove upset on this loading piece, the size and the sampling pipe phase-match in loading groove. Drive the sampling pipe upset of loading during the upset of loading piece, order about the urine or the blood that wait to detect in the sampling pipe and pour into to the detection intracavity portion that corresponds through leading to the 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 overturn when the push plate moves. The driving force of the push plate is the pushing force of a worker on the test strip card shell.
Before detection, placing each test strip at an inlet of each detection unit, placing each sampling tube to be detected in a loading piece of each detection unit, and opening the sampling tube, wherein the test strip does not generate acting force on a 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 strip moving towards the detection cavity drives the push plate to move, the moving push plate drives the loading piece to turn over through the pull rope, and when the push plate moves to a preset position, the loading piece turns over by a preset angle, so that liquid in the sampling pipe can fall into a detection hole of the test strip in the detection cavity through the through groove, and sampling is finished; and after sampling is completed for a period of time, taking out each test strip for sample analysis.
In one or more embodiments, a semi-permeable membrane or a dialysis membrane may be used to filter the sample to be tested before testing, and a certain amount of filtrate is added to the sample tube to be tested. In one embodiment, the sample amount in the sampling tube to be detected is 150-200 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, the test strips are positioned in the independent detection units, so that the test strips cannot be influenced mutually, and the accuracy of the 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 to enter the detection cavity, so that the operation is convenient, standard and quick, the detection efficiency and accuracy can be obviously 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 Sichuan huahua cattle, provided by the invention, is based on the change mode of the main reproductive hormone of the Sichuan hua cattle in the early pregnancy, judges the oestrus cycle of the Sichuan hua 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, constructs a standardized preparation method and a standardized process for detecting the early pregnancy detection of the Sichuan hua cattle, can simply and efficiently detect the early pregnancy state of the Sichuan hua cattle, shortens the breeding process of the cattle, improves the breeding rate, is beneficial to guiding clinical hybridization work, makes up the blank of the colloidal gold detection in the early pregnancy detection of the bovine serum on dairy cattle with meat, and has a very high practical application value;
2. the preparation method determines the working concentration of the colloidal gold-antibody conjugate stock solution by determining the labeling conditions such as the most suitable pH value, the minimum antibody amount and the like, and establishes the working concentrations of a detection line and a control line, so that the test strip has higher sensitivity and can detect the hormone difference value of less than 0.5 mu mol/L of pregnant cows and non-pregnant cows of hollyhock cattle;
3. according to the invention, through the improvement of the detection device, 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, the test strips are positioned in the independent detection units, so that the test strips cannot be influenced mutually, and the accuracy of the 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 to enter the detection cavity, so that the operation is convenient, standard and quick, the detection efficiency and accuracy can be obviously improved, and the large-scale popularization and application are facilitated.
Drawings
The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:
FIG. 1 is a schematic flow chart of a preparation method in an embodiment of the present invention;
fig. 2 shows the change pattern of the major reproductive hormones in early pregnancy of a holly flower cow according to an embodiment of the present invention, wherein fig. 2(a) shows the change of luteinizing hormone in serum; FIG. 2(b) is a graph showing the change in serum follitropin; FIG. 2(c) is a graph showing the change in estrogen in serum; FIG. 2(d) is a graph showing the change in progesterone in serum; FIG. 2(e) is a graph of the change in progesterone in urine;
FIG. 3 is a graph showing the results of an experiment for determining the optimum pH for labeling an antibody according to an embodiment of the present invention;
FIG. 4 is a graph showing the results of an experiment for determining the optimum labeling amount of an antibody in an embodiment of the present invention;
FIG. 5 is a schematic diagram of different determination results of the test strip according to an embodiment of the present invention;
FIG. 6 is a schematic structural diagram of a test device for mounting a test strip according to an embodiment of the present invention;
FIG. 7 is a schematic diagram of a detecting unit in a ready state according to an embodiment of the present invention;
FIG. 8 is a schematic structural diagram of a detecting unit in a detecting state according to an embodiment of the present invention;
FIG. 9 is a schematic view of the locking member being activated by the push plate in accordance with one embodiment of the present invention;
FIG. 10 is a schematic view of the locking member and push plate of the present invention;
FIG. 11 is a schematic view of a splash plate according to an embodiment of the present invention.
Reference numbers and corresponding part names in the drawings:
1-box body, 2-partition board, 3-through groove, 4-loading piece, 5-baffle, 6-push plate, 61-clamping groove, 7-first spring, 8-splash plate, 81-opening, 82-containing cavity, 83-converging surface, 9-locking piece, 91-connecting plate, 92-second spring, 93-clamping protrusion, 10-pull rope, 11-rotating shaft, 12-sampling tube, 13-test paper strip and 14-detection hole.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
All of the starting materials of the present invention, without particular limitation as to their source, are commercially available or can be prepared according to conventional methods well known to those skilled in the art.
All the raw materials of the invention are not particularly limited in purity, and the invention preferably adopts the conventional purity requirement in the field of analytical pure or colloidal gold test strips.
All the raw materials, the marks and the acronyms thereof belong to the conventional marks and the acronyms in the field, each mark and acronym is clear and definite in the field of related application, and the raw materials can be purchased from the market or prepared by the conventional method by the technical staff in the field according to the marks, the acronyms and the corresponding application.
In the description of the present invention, it is to 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 those shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed in a particular orientation, and be operated, and therefore, should not be taken as limiting the scope of the invention.
Example 1:
the preparation method of the colloidal gold test strip for the holy flower cattle shown in figure 1 comprises the following steps:
establishing a main reproductive hormone change mode of the Shuxuan Hua cattle at the early gestation stage, judging the oestrus cycle of the Shuxuan Hua cattle based on the main reproductive hormone change mode, and determining key reproductive hormone for developing the test strip according to the main reproductive hormone change mode in the oestrus cycle;
preparing a colloidal gold solution by a reduction method;
screening monoclonal antibodies based on the key reproductive hormones, purifying the screened monoclonal antibodies, and determining the labeling conditions of the screened monoclonal antibodies;
preparing and purifying a gold-labeled monoclonal antibody compound;
and assembling the colloidal gold test strip.
In some embodiments, the primary reproductive hormones include luteinizing hormone, follitropin, progesterone and estrogen, the estrus cycle of the holly flower cattle is judged based on the change pattern of luteinizing hormone, follitropin, progesterone and estrogen in the early gestation period of the holly flower cattle, and the progesterone with a significant difference between the level of the pregnant hormone and the level of the non-pregnant hormone is selected as the key reproductive hormone according to the change pattern of luteinizing hormone, follitropin, progesterone and estrogen in the estrus cycle.
In some embodiments, the preparation of the colloidal gold solution by a reduction method comprises the following steps: heating the chloroauric acid solution to boiling, adding a trisodium citrate aqueous solution into the boiling chloroauric acid solution until the reaction solution turns red, and continuing heating until the color is stable; after cooling, ultrapure water was added to the reaction solution to restore the original volume, and NaN was added thereto3And 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 screening is a bovine progesterone mouse monoclonal antibody, and the conjugate is Progesterone (PROG) -BSA (or OVA) antigen; when in purification, the bovine progesterone mouse monoclonal antibody is put into a dialysis bag, the dialysis bag is put into phosphate buffer solution for full dialysis overnight, and then the dialyzed bovine progesterone mouse monoclonal antibody is centrifuged to remove polymers, and the supernatant is taken.
In some embodiments, the preparing and purifying gold-labeled monoclonal antibody complexes comprises the following steps:
preparation: centrifuging the colloidal gold solution to remove larger polymers from the colloidal gold solution, using K2CO3Adjusting the pH value of the centrifuged colloidal gold solution to 8.0, slowly adding the monoclonal antibody into the colloidal gold solution, uniformly stirring at room temperature, and adding 10% BSA (bovine serum albumin) into the colloidal gold solution to a final concentration of 0.4%;
and (3) purification: centrifuging the colloidal gold solution at a first rotation speed, absorbing the supernatant, centrifuging the supernatant at a second rotation speed, removing the supernatant, dissolving the precipitate in an original volume of TBS solution, wherein the TBS solution has a pH value of 8.0 and contains 1% BSA and 0.02% NaN3And repeating centrifugal washing for 2-3 times to obtain the gold-labeled monoclonal antibody compound.
In some embodiments, assembling the colloidal gold test strip comprises the steps of:
an antibody solid phase NC membrane is adhered in the middle of the bottom plate, a detection line and a control line are arranged on the antibody solid phase NC membrane, the T line working concentration of a test strip for detecting blood progesterone is 1.0mg/mL, the T line working concentration of a test strip for detecting urine progesterone 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;
adhering a probe strip to one end, close to the detection line, of the bottom plate, wherein the probe strip is at least partially overlapped with the antibody solid-phase NC membrane, and adhering a sample pad to the lower end of the bottom plate, and the sample pad is at least partially overlapped with the probe strip;
and adhering absorbent paper on one end of the bottom plate close to the control line, wherein the absorbent paper is at least partially overlapped with the antibody solid-phase NC membrane.
The detection principle of the test strip prepared by the preparation method is as follows: the test strip contains antigen pre-immobilized on the test area (T) of the nitrocellulose membrane and II antibody of the control area (C) and gold-labeled antibody immobilized on the conjugate pad. After the sample to be detected is added, the antigen of the envelope solid phase of the T area competes for the gold-labeled antibody with the 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 antigen of the T area is reduced or completely eliminated, so that a mauve strip of the T area becomes light or is directly eliminated; if the sample is negative, a purple red band appears in the T area. No matter whether progesterone exists in the sample or not, a mauve strip appears in the C area, and if the mauve strip does not exist in the C area, the test result of the test strip is invalid.
As shown in FIG. 5, when the C line is colored, the T line is not colored, or the color development tends to be weaker than that of the C line, it is judged to be positive; when the C line is developed, the T line can be seen by naked eyes and is equivalent to the C line in color development degree, the result can be judged to be negative; when the C line is not developed, the test strip is judged to be invalid no matter whether the T line is developed or not.
The preparation method of the embodiment judges the oestrus cycle of the hollyhock pollen cow based on the change mode of the main reproductive hormone of the hollyhock pollen cow in the early pregnancy, 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, constructs a standardized preparation method and a standardized process for detecting the early pregnancy detection test strip of the hollyhock pollen cow, can simply and efficiently detect the early pregnancy state of the hollyhock pollen cow, shortens the propagation process of the cow, improves the propagation rate, is beneficial to guiding clinical hybridization work, makes up the blank of early pregnancy detection of colloidal gold on the cow with the milk cow and has high practical application value.
Example 2:
on the basis of example 1, a colloidal gold test strip for progesterone in blood or urine of a bovine dawn flower is prepared by the following method.
1) Establishing a main reproductive hormone change mode of Shuxuan Hua cattle in early pregnancy
Randomly selecting 18 cows of Shuxuanhua cows with similar ages, moderate fat and normal reproductive function for estrus synchronization, determining the estrus of 12 cows and timely breeding, and collecting blood samples or urine samples every 3 days from the breeding day of the cows to 36 days, wherein 10ml of blood samples or 5ml of urine samples are collected each time. And (3) matching with rectal detection and B ultrasonic detection, and dividing the experimental population into a non-pregnant group and a pregnant group according to the pregnancy reaction condition, wherein the non-pregnant group is 7 heads, and the pregnant group is 5 heads.
Changes in Luteinizing Hormone (LH), Follicle Stimulating Hormone (FSH), progesterone (P4), and estrogen (E2) during estrus were analyzed in the non-pregnant group and the pregnant group.
The changes of the major reproductive hormones are shown in fig. 2(a) - (e). Experiments show that the non-pregnant cattle have a strong LH peak at 21 days after estrus, but the pregnant cattle have no LH peak and have a smooth trend, which shows that the non-pregnant cattle estrate again and ovulate and the pregnant cattle do not enter a new estrus cycle. The peak LH value of young cattle is related to the developmental capacity of in vivo embryos, and the peak LH value before ovulation can improve the maturation of embryo cytoplasm but is not a main factor influencing the maturation of oocyte nuclei. On the other hand, FSH in estrus of both non-pregnant and pregnant cows shows 1-2 small peaks, which is presumed to be related to promotion of cow follicular development, but the FSH concentration in serum at different time periods in the same day is greatly different, so that the FSH peak in the estrus day is difficult to capture in naturally estrous cows. Progesterone is a steroid hormone, produced mainly by ovarian luteal cells, but also by the intimal cells of the follicles and by the placenta during pregnancy. Experiments show that the progesterone level in blood and urine of normal estrus of Shuxuanhua cattle is low on the day of estrus and reaches a peak value in 12 days, mainly because the progesterone concentration of non-pregnant cattle reaches the maximum value in 8-10 days of the estrus cycle, the progesterone concentration continuously rises and reaches the peak value in the period, the corpus luteum degeneration occurs in 15-19 days of the estrus cycle, and the progesterone concentration also drops sharply at the time. In pregnant cows, however, the corpus luteum is not degenerated, so the progesterone concentration rises steadily. Meanwhile, during the oestrus cycle, an increase in E2 levels is associated with each follicle waveform, with the dominant follicle continuing to grow while the other follicles begin to decline, with a consequent decrease in E2 levels. It was found that E2 secretion from the Sichuan Huaniu cow was high at the beginning of estrus, then declined gradually and was minimal at 12 days, contrary to the P4 change, E2 reached the next peak in estrus of the infertile cows, while the level of pregnant cows remained low. As P4 decreased, E2 increased and reached a higher level in estrus, consistent with follicular development and morphological changes. The hormonal changes that cause oestrus are therefore characterized by a drop in progesterone levels followed by a rapid rise in estrogens.
Therefore, the oestrus cycle of the Shuxuan Hua cattle is judged to be 21 days based on the change patterns of luteinizing hormone, follicle stimulating hormone, progesterone and estrogen of the Shuxuan Hua cattle at the early gestation stage. The major reproductive hormone differences at day 21 in different experimental groups of dao cows are shown in table 1.
TABLE 1
Day 21 FSH (blood) LH (blood) E2 (blood) P4 (blood) P4 (urine)
Group of non-pregnancies 0.21Aa±0.12 1.13A±0.31 2.66Aa±0.53 0.34A±0.11 0.37Aa±0.16
Pregnant group 0.20Aa±0.09 0.37B±0.13 1.82Aa±0.47 0.76B±0.09 0.66Ab±0.06
As can be seen from table 1, the difference between blood progesterone of pregnant cows and non-pregnant cows is very significant at 21 days (p <0.01), the difference between urine progesterone level is significant (p <0.05), and by comparing the difference between main reproductive hormones at 21 days and the subsequent trend of change, the blood and/or urine progesterone at 21 days is selected as a key reproductive hormone for determining whether the hollyhock propaganda cattle is pregnant early, and the colloidal gold test paper is prepared according to the hormones.
Although the difference in key reproductive hormones in urine or blood of pregnant and non-pregnant cows of holhua cows is significant or very significant, it is usually less than 0.5 μmol/L, e.g. the progesterone level in the blood is 0.34 ± 0.11 μmol/L on the day of estrus (day 0 or day 21) and 0.70 ± 0.21 μmol/L when it peaks on day 12. The difference is far smaller than the difference between the non-pregnant cow and the pregnant cow of the milk progesterone of the dairy cow, so that the detection sensitivity of the colloidal gold detection test strip for the holly flower cattle is higher.
In this embodiment, the colloidal gold test strip based on serum and/or urine is prepared by a small molecule competition method according to the hormone level change rule of key reproductive hormones of the hollyhock.
2) Preparation of colloidal gold solution by reduction method
Heating chloroauric acid solution with final concentration of 0.01% in a microwave oven to boil, rapidly and accurately adding 1% trisodium citrate aqueous solution under stirring, allowing the gold chloroauric acid aqueous solution to turn red within 2min, continuously boiling at middle level for 5min until the color is stable, cooling, recovering to original volume with ultrapure water, adding 0.02% NaN3Filtering with 0.22pm filter membrane for sterilization, and storing in clean siliconized brown glass bottle at 4 deg.C for use. In the embodiment, the addition amount of the trisodium citrate is adjusted to enable the size of the colloidal gold particles to be 20-35 nm, preferably 30 nm.
After the preparation is finished, the quality of the colloidal gold solution can be comprehensively identified by the following method: by visual inspection of colloidal goldColor change, whether turbid, transparent, refractive and gel exist; observing the size, the shape and the density of the colloidal gold particles through an electron microscope; spectrophotometer OD400 by visible spectrumnmTo OD700nmScanning the colloidal gold solution in a spectral range, judging the relationship between the diameter of the colloidal gold particles and the maximum absorption wavelength according to the obtained numerical value, and evaluating the quality of the colloidal gold solution; the colloidal gold solution is placed at room temperature, 4 ℃ and 37 ℃ for 24h respectively, and the agglomeration condition is observed.
3) Screening and purification of monoclonal antibodies
The most suitable monoclonal antibody developed by the test paper screened by chessboard titration is Progesterone (PROG) monoclonal antibody (50H10 cell strain), and the conjugate is Progesterone (PROG) -BSA (or OVA) antigen.
The dialysis bag is cut into 10 cm-long segments, and can be used when no hole is strictly checked. Washed with distilled water and then placed in 500mL of 2% NaHCO containing 1mmol/LEDTA-Na3Boiling in the solution for 10min, taking out with clean forceps, boiling in distilled water for 10min, and rinsing.
And (3) putting the monoclonal antibody to be dialyzed into a treated dialysis bag, putting the packed monoclonal antibody into 0.01M PB liquid for full dialysis overnight at 4 ℃, then centrifuging the dialyzed monoclonal antibody for 1h at 4 ℃ at 10000r/min, removing the polymer, and taking supernatant.
4) Preparation and purification of gold-labeled monoclonal antibody complexes
Before labeling, the colloidal gold is centrifuged at 3000r/min for 20min, and large polymers formed in the preparation process are removed, so that the adsorption of antibody protein on the colloidal gold particles is prevented from being influenced by the polymers in the labeling process. With 0.2mol/LK2CO3And adjusting the pH value of the colloidal gold solution to the optimal pH value. The optimal labeling amount of monoclonal antibody was slowly added dropwise with magnetic stirring, 1mg of protein was added over about 5min, and labeled for 30 min. Slowly adding 5% BSA dropwise to final concentration of 0.5% as stabilizer, stirring for 30min, sealing, and storing at 4 deg.C in dark place.
In one or more embodiments, 10% polyethylene glycol (MW20000) may also be added, stirred at room temperature for 10min, centrifuged at 9000-.
After preparation, the gold-labeled protein is purified by low-temperature ultracentrifugation to remove unlabeled protein and insufficiently labeled colloidal gold in the solution as well as various polymers that may be formed during the labeling process. The gold-labeled antibody is firstly centrifuged at 1500r/min for 15min at 4 ℃, the supernatant is carefully absorbed, and the precipitate is discarded. The supernatant was then centrifuged again at 13000r/min at 4 ℃ and the supernatant was discarded, and finally the pellet was dissolved in the original volume of pH 8.00.1 moL/L TBS (containing 1% BSA, 0.02% sodium azide), and the centrifugation wash was repeated 2-3 times and the original volume of 1/10 was resuspended in the pellet. Storing at 4 deg.C for use.
5) Assembled colloidal gold test strip
Cutting the PVC base plate into strips with the width of 2.8mm multiplied by the length of 6 cm;
an antibody solid phase NC membrane is stuck in the middle of the PVC bottom plate and is 1.5cm away from the upper section;
sticking a glass fiber membrane probe strip at the lower end of the PVC bottom plate, namely the end of the T line close to the NC membrane, overlapping the glass fiber membrane probe strip with the antigen solid phase NC membrane by 0.1cm, and sticking a 1.7cm wide sample pad at the lower end to overlap the probe strip by 0.1-0.2 cm;
adhering 1.7cm wide absorbent paper at the upper end of the PVC bottom plate, namely the end close to the C line of the NC membrane, and overlapping 0.1-0.2cm with the antibody solid-phase NC membrane;
cutting into test strips with the width of 2.8mm, and filling into blank card shells;
the working concentration of the progesterone antigen in the detection line is 1.0-1.5 mg/mL, wherein the T-line working concentration of the test strip for detecting blood progesterone is 1.0mg/mL, and the T-line working concentration of the test strip for detecting urine progesterone is 1.5 mg/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 colloidal gold-antibody conjugate stock solution is diluted by working solution according to the ratio of 1:2, 1:4, 1:8 and 1:16, the colloidal gold-antibody conjugate diluent solution is uniformly added on a glass fiber membrane and 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 gold colloidal-antibody conjugate stock solution is 1:4, which ratio can further improve the detection effect of the gold-labeled pad.
In one or more embodiments, several different models of NC membranes are selected, and the most suitable model of NC membrane is determined to be the sartorius CN140 through running board function tests, tests of fluidity, hysteresis, and background residues of colloidal gold solution on the different models of NC membranes, and reselection tests of the NC membranes.
In one or more embodiments, progesterone antigen and goat anti-mouse IgG antibody were separately diluted to the desired concentration with 0.01mol/LpH, 8.0PBS and blotted onto NC membranes using a membrane striper. The distance between the T line and the C line is 0.5cm, the parameters are all 1 mu L/cm, and the sprayed NC film is dried at 37-45 ℃ for more than 8 h.
In one or more embodiments, a piece of tinfoil paper is attached to the upper surface of the assembled test strip to protect the detection line, the control line and the detection hole from contamination, and the tinfoil paper is removed during detection.
In one or more embodiments, a standard card may be further provided, so that the staff can compare and confirm the detection result.
Example 3:
on the basis of the above embodiment, the determining of the labeling conditions of the selected monoclonal antibody comprises determining the optimum pH value of the combination of the bovine progesterone mouse monoclonal antibody and a colloidal gold solution by a colloidal gold gradient method; the method for determining the labeling conditions of the screened monoclonal antibodies comprises the step of determining the optimal concentration of the combination of the bovine progesterone mouse monoclonal antibodies and the colloidal gold solution by a protein gradient method.
1) Determining the optimum pH value of the combination of the bovine progesterone mouse monoclonal antibody and the colloidal gold solution
The bovine progesterone mouse monoclonal antibody is marked by 0.02 percent of 20nm colloidal gold, and the optimal pH value of the combination of the antibody and the colloidal gold is determined by a colloidal gold gradient method. The method comprises the following specific steps:
(a) taking 7 sampling tubes, and respectively adding 1mL of prepared colloidal gold solution;
(b) adjusting the pH of the colloidal gold solution to 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5 by using 0.2mol/L potassium carbonate solution respectively;
(c) adding 50 mu L of 1mg/ml bovine progesterone mouse monoclonal antibody into the sampling tube, uniformly mixing, and standing at room temperature for 20 min;
(d) then, adding 100 mu L of 10% NaCl solution into each tube, mixing uniformly, and standing for 1-2h at room temperature;
(e) observing the color change of the colloidal gold, and recording the lowest pH value for keeping red;
(f) then adjusting the pH value to the lowest gradient pH value of +/-0.1; the above experiment was repeated.
The lowest pH at which the red color remained was recorded, i.e., the optimum pH.
The results are shown in FIG. 3, which confirms that the optimum pH value of the combination of the bovine progesterone mouse monoclonal antibody and the colloidal gold solution is 8.0. By determining the optimal pH value, the sensitivity of the test strip can be further improved so as to detect Sichuan huahua cattle with a smaller difference between pregnant and non-pregnant progesterone.
2) Determining the optimum concentration of the combination of the bovine progesterone mouse monoclonal antibody and the colloidal gold solution
And determining the optimal concentration of the combination of the bovine progesterone mouse monoclonal antibody and the colloidal gold by a protein gradient method. The method comprises the following specific 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 to 1mg/mL by using purified water, and sequentially adding 0, 5, 10, 20, 30, 40, 60 and 80 mu L of the bovine progesterone mouse monoclonal antibody into the small test tube to be uniformly mixed;
(c) standing for 10min, adding 0.1mL of 10% NaCl aqueous solution into each sampling tube, mixing uniformly, and standing at room temperature for 1-2 h;
(d) observing the color change of the small test tube, wherein the control tube and the sampling tube which is not added with protein in an amount enough to stabilize the colloidal gold show the coagulation phenomenon from red to blue, the sampling tube which is added with protein in an amount which reaches or exceeds the lowest stable amount keeps the red unchanged, finding out the boundary tube of the colloidal gold liquid from red to blue, and the protein amount contained in the boundary tube is the minimum protein amount required by stabilizing 1ml of the colloidal gold.
The results are shown in FIG. 4, from which it was determined that the minimum amount of antibody required for the progesterone mouse monoclonal antibody to stabilize 1mL of colloidal gold was 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 most suitable pH value, the minimum antibody amount and the like, and the working concentrations of the detection line and the control line are established, so that the test strip has higher sensitivity and can detect the hormone difference value of less than 0.5 mu mol/L of pregnant cows and non-pregnant cows of hollyhock.
Example 4:
on the basis of the above embodiments, an application of any one of the foregoing test strips in a detection device for early pregnancy of a milch buffalo is provided, as shown in fig. 6 to 11, the detection device includes a housing 1, a plurality of detection units are disposed in the housing 1, each detection unit includes a detection cavity for accommodating the test strip 13, a through groove 3 is disposed at a top of the detection cavity, a loading member 4 for loading a sampling tube 12 is disposed in the through groove 3, the loading member 4 can be turned over in the through groove 3, the loading member 4 is connected to a push plate 6 located in the detection cavity through a pull rope 10, when the test strip 13 is inserted into the detection cavity, the push plate 6 can be pushed by the test strip 13 to move in the detection cavity, and in the moving process, the loading member 4 is pulled to turn over through the pull rope 10, the turned over loading member 4 drives the sampling tube 12 to turn over, and a liquid to be detected in the sampling tube 12 can fall into a detection hole 14 of the test strip 13 in the detection cavity through the through groove 3.
In one or more embodiments, a plurality of partition plates 2 are disposed on the bottom surface of the housing, and the partition plates 2 divide the internal space of the housing into a plurality of mutually independent detection units. A detection cavity is arranged in the detection unit, 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, placing each test strip at an inlet of each detection unit, placing each sampling tube to be detected in a loading piece of each detection unit, and opening the sampling tube, wherein the test strip does not generate acting force on a 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 strip moving towards the detection cavity drives the push plate to move, the moving push plate drives the loading piece to turn over through the pull rope, and when the push plate moves to a preset position, the loading piece turns over by a preset angle, so that liquid in the sampling pipe can fall into a detection hole of the test strip in the detection cavity through the through groove, and sampling is finished; and after sampling is completed for a period of time, taking out each test strip for sample analysis.
In one or more embodiments, the semi-permeable membrane and the dialysis membrane can be used for filtering the sample to be detected before detection, and the standing step of the sample to be detected can be omitted by filtering the sample to be detected by using the dialysis membrane. In one embodiment, after the filtration is completed, 150 to 200 μ 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, a baffle 5 is further disposed on the upper surface of the detection unit, and the baffle 5 is located on one side of the through groove close to the open end of the detection device, so that when the loading member is turned over, the turning angle of the loading member is limited, 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, and the identification area is used for displaying 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, a torsion spring (not shown) is disposed on the rotating shaft, 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. During detection, when the card shell of the test strip pushes the push plate, the push plate compresses the first spring and simultaneously drives the loading piece to overcome the acting force of the torsion spring to overturn; after sampling is finished, acting force on the test strip is removed, the push plate resets under the acting force of the first spring, the pull rope is loosened, the loading piece losing pull force of the pull rope resets under the acting force of the torsion spring, and then the detection unit resets 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. Self-locking mechanism includes retaining member 9, is provided with connecting plate 91 on the outer wall of retaining member, is provided with second spring 92 on connecting plate 91, and second spring 92's bottom is connected to detection device's upper surface, and the bottom activity of retaining member 9 runs through to the inside that detects the chamber, and the bottom of retaining member is provided with the protruding 93 of card, be provided with draw-in groove 61 on the push pedal 6, the protruding 93 of card and the size phase-match of draw-in groove 61.
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 protrusion, the clamping protrusion is pushed by the push plate, the locking part vertically moves upwards, when the clamping groove of the push plate passes through the clamping protrusion, the clamping protrusion without support 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 part and the push plate are relatively fixed, and the push plate reaches a preset position; after the sampling is completed, the locking piece is lifted upwards to unlock, and the push plate is reset under the action of the first spring. This setting makes the staff need not to consider the distance that the push pedal removed in the testing process, and the inspection hole of test paper strip can align with the drop point of the liquid that awaits measuring in the sampling pipe after the locking, further simplifies the operating procedure, improves the degree of accuracy that detects.
In one or more embodiments, the side of the locking protrusion 93 facing the push plate 6 is a slope, so that when the locking protrusion is acted by the push plate, a vertical component force can be generated to drive the locking member to move vertically upward.
In some embodiments, as shown in fig. 7, 8 and 11, a splash shield is further disposed in the detection chamber below the partition plate, and the splash shield is used for guiding the liquid poured out from the sampling tube to drip to the detection hole below. In one or more embodiments, the splash guard is a hollow structure, the surface of the splash guard facing the through groove is an inclined surface, the inclined surface is provided with an opening to communicate the accommodating cavity and the external space inside the splash guard, and the bottom surface of the accommodating cavity is a converging surface, so that liquid entering the accommodating cavity through the opening can converge towards the bottom of the accommodating cavity through the converging surface and finally drops into the detection hole through the opening at the bottom of the accommodating cavity. Through the setting, empty the liquid on the splash shield and divide into two parts, partly prolong the inclined plane flow of splash shield, another part collects in holding the chamber, avoids the liquid instantaneous flow of pouring out in the sampling pipe too big and leads to partial liquid to sputter behind the contact inclined plane, leads to the sampling volume not up to standard, further improves the degree of accuracy that detects.
As used herein, "first," "second," etc. (e.g., first spring, second spring, etc.) are used only for clarity of description to distinguish between corresponding components and are not intended to limit any order or to emphasize importance, etc. Further, the term "connected" used herein may be either directly connected or indirectly connected via other components without being particularly described.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (10)

1. The preparation method of the colloidal gold test strip for the Sichuan huahuaniu is characterized by comprising the following steps of:
establishing a main reproductive hormone change mode of the Shuxuan Hua cattle at the early gestation stage, judging the oestrus cycle of the Shuxuan Hua cattle based on the main reproductive hormone change mode, and determining key reproductive hormone for developing the test strip according to the main reproductive hormone change mode in the oestrus cycle;
preparing a colloidal gold solution by a reduction method;
screening monoclonal antibodies based on the key reproductive hormones, purifying the screened monoclonal antibodies, and determining the labeling conditions of the screened monoclonal antibodies;
preparing and purifying a gold-labeled monoclonal antibody compound;
and assembling the colloidal gold test strip.
2. The method for preparing the colloidal gold test strip for the holly flower-shaped cattle according to claim 1, wherein the main reproductive hormones comprise luteinizing hormone, follicle stimulating hormone, progesterone and estrogen, the oestrus cycle of the holly flower-shaped cattle is judged based on the change patterns of the luteinizing hormone, the follicle stimulating hormone, the progesterone and the estrogen in the early gestation period of the holly flower-shaped cattle, and the progesterone with the pregnant hormone level and the non-progestational hormone level which are different significantly is selected as the key reproductive hormone according to the change patterns of the luteinizing hormone, the follicle stimulating hormone, the progesterone and the estrogen in the oestrus cycle.
3. The method for preparing the colloidal gold test strip for the holy-xuan-hua cattle according to claim 2, wherein the step of preparing the colloidal gold solution by using a reduction method comprises the following steps:
heating the chloroauric acid solution to boiling, adding a trisodium citrate aqueous solution into the boiling chloroauric acid solution until the reaction solution turns red, and continuing heating until the color is stable; after cooling, ultrapure water was added to the reaction solution to restore the original volume, and NaN was added thereto3And 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.
4. The method for preparing the colloidal gold test strip for the holy-announced-flowered cattle according to claim 2, wherein the monoclonal antibody obtained by screening is a bovine progesterone mouse monoclonal antibody, and the conjugate is Progesterone (PROG) -BSA (or OVA) antigen; when in purification, the bovine progesterone mouse monoclonal antibody is put into a dialysis bag, the dialysis bag is put into phosphate buffer solution for full dialysis overnight, and then the dialyzed bovine progesterone mouse monoclonal antibody is centrifuged to remove polymers, and the supernatant is taken.
5. The method for preparing the colloidal gold test strip for Sichuan huaniu according to claim 4, wherein the determining of the labeled conditions of the selected monoclonal antibody includes determining the optimum pH value of the combination of the bovine progesterone mouse monoclonal antibody and the colloidal gold solution by using a colloidal gold gradient method.
6. The method for preparing the colloidal gold test strip for Sichuan huaniu according to claim 4, wherein the determining the labeled conditions of the selected monoclonal antibody includes determining the optimal concentration of the bovine progesterone mouse monoclonal antibody combined with the colloidal gold solution by protein gradient method.
7. The method for preparing the colloidal gold test strip for the althuahua cattle according to claim 2, wherein the step of preparing and purifying the gold-labeled monoclonal antibody complex comprises the following steps:
preparation: centrifuging the colloidal gold solution to remove larger polymers from the colloidal gold solution, using K2CO3Adjusting the pH value of the centrifuged colloidal gold solution to 8.0, slowly adding the monoclonal antibody into the colloidal gold solution, uniformly stirring at room temperature, and adding 10% BSA (bovine serum albumin) into the colloidal gold solution to a final concentration of 0.4%;
and (3) purification: centrifuging the colloidal gold solution at a first rotation speed, absorbing the supernatant, centrifuging the supernatant at a second rotation speed, removing the supernatant, dissolving the precipitate in an original volume of TBS solution, wherein the TBS solution has a pH value of 8.0 and contains 1% BSA and 0.02% NaN3And repeating centrifugal washing for 2-3 times to obtain the gold-labeled monoclonal antibody compound.
8. The method of claim 2, wherein the assembly of the colloidal gold test strip comprises the steps of:
an antibody solid phase NC membrane is adhered to the middle of the bottom plate, a detection line and a control line are arranged on the antibody solid phase NC membrane, the working concentration of a progesterone antigen of the detection line is 1.0-1.5 mg/mL, and the working concentration of a goat anti-mouse IgG antibody of the control line is 1.0-1.5 mg/mL;
adhering a probe strip to one end, close to the detection line, of the bottom plate, wherein the probe strip is at least partially overlapped with the antibody solid-phase NC membrane, and adhering a sample pad to the lower end of the bottom plate, and the sample pad is at least partially overlapped with the probe strip;
and adhering absorbent paper on one end of the bottom plate close to the control line, wherein the absorbent paper is at least partially overlapped with the antibody solid-phase NC membrane.
9. A colloidal gold test strip for Sichuan huahua cattle, which is prepared by the preparation method of any one of claims 1-8, and is used for detecting whether the Sichuan hua cattle is pregnant or not according to the progesterone content in urine or blood of the Sichuan hua cattle.
10. The use of the test strip in the detection device for early pregnancy of Sichuan Huaniu cattle according to claim 9, wherein the detection device comprises a housing (1), a plurality of detection units are arranged in the housing (1), the detection units comprise detection chambers for accommodating the test strips (13), a through slot (3) is arranged at the top of the detection chambers, a loading member (4) for loading a sampling tube (12) is arranged in the through slot (3), the loading member (4) can be turned over in the through slot (3), the loading member (4) is connected with a push plate (6) positioned in the detection chambers through a pull rope (10), when the test strips (13) are inserted into the detection chambers, the push plate (6) can be pushed by the test strips (13) to move in the detection chambers, and the loading member (4) is pulled to turn through the pull rope (10) during the movement, the overturning loading piece (4) drives the sampling pipe (12) to overturn, and liquid to be detected in the sampling pipe (12) can fall into a detection hole (14) of a test strip (13) in the detection cavity through the through groove (3).
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