Colloidal gold kit for estrus identification of sheep

Abstract

The invention provides a sheep oestrus identification colloidal gold kit, which relates to the technical field of animal breeding detection and comprises test paper and a kit body, the test paper comprises a sample pad, a colloidal gold combination pad, a chromatography membrane and water absorption filter paper; a detection line and a quality control line are arranged on the chromatography membrane; the detection line is coated with a luteinizing hormone monoclonal antibody 5F3G4C6, and the quality control line is coated with goat anti-mouse IgG. The sheep oestrus identification colloidal gold kit is used for ewe oestrus identification, the oestrus identification accuracy and detection efficiency are improved, and the labor cost is reduced. By improving the accuracy and efficiency of oestrus identification, the kit provided by the invention not only can improve the reproductive performance of livestock, but also can reduce the breeding cost and improve the economic benefit.

Description

Technical Field

[0001] The invention relates to the technical field of animal reproduction detection, in particular to a colloidal gold kit for identifying estrus in sheep. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] With the development of large-scale sheep farming, efficient and accurate estrus identification is crucial to improving reproductive efficiency. Currently, farms mainly rely on manual identification methods (such as external observation, vaginal examination, and estrus testing), which have two major drawbacks: (1) Dependence on operational experience: The accuracy of identification is affected by the professionalism and operational experience of the technicians; (2) Inefficiency: It cannot meet the needs of rapid screening of large-scale sheep flocks, and the labor cost accounts for a high proportion.

[0004] In the context of large-scale breeding, rapid, precise, and efficient estrus detection is crucial for improving ewe conception rates and optimizing reproductive management. Colloidal gold, as a specialized labeling technology, is widely used in medical testing for its tracer function in antigen-antibody reactions. However, this technology remains limited in the field of sheep estrus detection. Existing technologies lack a rapid colloidal gold detection system for sheep LH hormone, and there is a lack of antibody combinations compatible with sheep serum samples. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a colloidal gold kit for sheep estrus identification, which has the advantages of simple operation, accurate and reliable detection results and is suitable for application in large-scale farms.

[0006] The first aspect of the present invention provides a colloidal gold kit for sheep estrus identification, comprising: a test paper and a box body; The test paper comprises a sample pad, a colloidal gold binding pad, a chromatography membrane (NC membrane) and a water-absorbing filter paper; The chromatographic membrane is provided with a detection line and a quality control line; The detection line is coated with luteinizing hormone monoclonal antibody 5F3G4C6, and the quality control line is coated with goat anti-mouse IgG.

[0007] Furthermore, the box body includes a plastic bottom plate and a plastic cover plate; The plastic cover is provided with an injection port and an observation window, the observation window corresponds to the detection line position and the quality control line position of the chromatographic membrane, the detection line position is marked with a detection line identifier, and the quality control line position is marked with a quality control line identifier.

[0008] Furthermore, one side of the chromatography membrane is overlapped with the colloidal gold binding pad, and the other side of the chromatography membrane is overlapped with the absorbent filter paper; The side of the colloidal gold binding pad away from the chromatography membrane is overlapped with the sample pad.

[0009] Furthermore, the chromatography membrane is a nitrocellulose membrane.

[0010] Furthermore, the coating concentration of the test line is 1.5 mg / ml, and the coating concentration of the quality control line is 0.5 mg / ml.

[0011] Furthermore, the preparation method of the colloidal gold binding pad includes: The glass fiber paper is immersed in a solution containing a colloidal gold probe, wherein the colloidal gold probe is formed by combining colloidal gold with luteinizing hormone monoclonal antibody C1; After infiltration, the samples were dried at 37°C and the amount of colloidal gold probe used was 15 μL / cm.

[0012] Furthermore, the determination rules of the kit are: If the test line and the quality control line show positive color at the same time, it means the sheep is in estrus; If the test line shows no color and the quality control line shows negative color, it means the sheep is not in estrus; If the test line shows color but the quality control line does not, or both the test line and the quality control line do not show color, the result is invalid.

[0013] Furthermore, the detection object of the kit is the luteinizing hormone in the serum of small-tailed Han sheep, and the detection sample is the supernatant of EDTA-anticoagulated whole blood collected from the jugular vein after centrifugation.

[0014] Furthermore, the colloidal gold is prepared by a trisodium citrate reduction method and calcined in a ratio of V chloroauric acid / V trisodium citrate=1:1.4 to obtain a colloidal gold solution.

[0015] Furthermore, the preparation method of the colloidal gold probe includes: Adjust the pH value of the colloidal gold solution to 8.5, add luteinizing hormone monoclonal antibody C1, mix well, and let it stand; 10% BSA solution was added for stabilization, and the supernatant was discarded after centrifugation at -4°C and 1400 rpm; The pellet was resuspended in a solution containing 0.1 M Tris, 10% sucrose, 1% PVP, 1% BSA, and 0.5% Tween-20.

[0016] Compared with the existing technology, the colloidal gold kit for estrus identification for sheep provided by the present invention has the following beneficial effects: by cooperating with the test paper of a specific structure and the box body, combined with the optimized coating scheme of the detection line and the quality control line, it can achieve rapid and accurate estrus identification, has the advantages of simple operation, accurate and reliable test results, and is suitable for application in large-scale farms.

[0017] Compared to existing technologies, traditional manual observation methods require continuous observation of ewes' behavioral changes for 2-3 days, while this approach can complete the determination within 10 minutes by testing serum hormone concentrations. The estrus test method requires mixing estrus rams with ewes, which poses a risk of cross-infection. This approach uses in vitro testing to avoid direct animal contact. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which constitute a part of the present disclosure, are used to provide a further understanding of the present disclosure. The exemplary embodiments of the present disclosure and their descriptions are used to explain the present disclosure and do not constitute an improper limitation to the present disclosure.

[0019] Figure 1 This is a schematic structural diagram of a colloidal gold kit for sheep estrus identification provided by the present invention; Figure 2 It is a schematic diagram of the detection results of the colloidal gold kit for identifying estrus in sheep provided by the present invention. DETAILED DESCRIPTION

[0020] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0021] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0022] In the absence of conflict, the embodiments of the present invention and the features thereof may be combined with each other.

[0023] All data in this embodiment is obtained in compliance with laws and regulations and based on the consent of the user, and is used legally.

[0024] Example 1 Please refer to the instruction manual Figure 1 , Figure 1 This is a schematic diagram of the colloidal gold kit for sheep estrus identification provided by the present invention. Figure 1It can be seen that the colloidal gold test kit for identifying estrus for sheep includes: test paper and a box body; the test paper includes a sample pad, a colloidal gold binding pad, a chromatography membrane and a water-absorbing filter paper; the chromatography membrane is provided with a detection line and a quality control line; the detection line is coated with luteinizing hormone monoclonal antibody 5F3G4C6, and the quality control line is coated with sheep anti-mouse IgG.

[0025] Specifically, during testing, the sample liquid permeates through the sample pad and enters the colloidal gold binding pad, dissolving the pre-immobilized labeled antibody to form a complex. Under the capillary action of the chromatographic membrane, the mixed liquid migrates toward the absorbent filter paper. When the sample contains luteinizing hormone, the labeled antibody-target hormone complex is captured by the fixed antibody at the test line, forming a double-antibody sandwich structure. Simultaneously, excess labeled antibody continues to migrate to the quality control line and is captured there. By observing the color development of the two lines, it can be determined whether the hormone concentration in the ewe has reached the estrus threshold.

[0026] The box body includes a plastic bottom plate and a plastic cover plate; the plastic cover plate is provided with an injection port and an observation window, the observation window corresponds to the detection line position and the quality control line position of the chromatographic membrane, the detection line position is marked with a detection line logo, and the quality control line position is marked with a quality control line logo.

[0027] Specifically, the plastic base and cover are assembled using a snap-fit ​​mechanism to form a sealed space, protecting the test strip from environmental interference. The sample inlet, located at the top of the cover and designed to fit the tip of a pipette, is injected through this aperture into the sample pad of the test strip. The observation window covers the test and control lines of the chromatographic membrane. The test line is labeled with the letter T, and the control line is labeled with the letter C. The operator can interpret the results by comparing the labels.

[0028] One side of the chromatography membrane is overlapped with the colloidal gold binding pad, and the other side of the chromatography membrane is overlapped with the absorbent filter paper; the side of the colloidal gold binding pad away from the chromatography membrane is overlapped with the sample pad.

[0029] Specifically, when the sample to be tested is applied to the sample pad, the liquid moves toward the colloidal gold binding pad through capillary action, and the marker is released from the colloidal gold binding pad and binds to the target substance in the sample. The liquid carries the complex into the chromatographic membrane, and an immune reaction occurs at the detection line and quality control line of the chromatographic membrane. The overlapping design of the chromatographic membrane and the absorbent filter paper allows the liquid to continue to flow toward the absorbent filter paper, avoiding background signal interference caused by liquid retention. The overlap of the colloidal gold binding pad and the sample pad ensures that the sample is evenly infiltrated into the marker release area, reducing sample residue or local concentration unevenness.

[0030] The chromatography membrane was nitrocellulose membrane.

[0031] Specifically, the nitrocellulose membrane serves as the core carrier of the chromatography reaction. Its porous structure allows the sample solution to diffuse evenly through capillary action. When luteinizing hormone in the sample binds to the colloidal gold-labeled antibody, the complex chromatographs along the membrane to the detection line. The antibody coated on the nitrocellulose membrane captures the marker through antigen-antibody specific binding, forming a visible color band. The hydrophilic surface of the material prevents nonspecific adsorption, and its stable chemical properties ensure that the antibody maintains its activity during storage.

[0032] The coating concentration of the test line was 1.5 mg / ml, and the coating concentration of the quality control line was 0.5 mg / ml; the streaking volume of both the test line and the quality control line was 1 μL / cm.

[0033] Specifically, the test line uses a coating concentration of 1.5 mg / ml. This concentration range ensures that the luteinizing hormone monoclonal antibody or goat anti-mouse IgG forms stable capture sites on the nitrocellulose membrane surface while avoiding nonspecific adsorption caused by excessive antibody. The quality control line uses a coating concentration of 0.5 mg / ml, which is lower than the test line to reduce background interference, while being sufficient to ensure effective binding of the luteinizing hormone monoclonal antibody C1 to the colloidal gold probe. The film volume for both the test line and the quality control line is controlled at 1 μL / cm. Through a precision spraying process, the antibody solution forms lines of uniform width on the membrane surface, ensuring that the boundaries of the color-developing area are clearly distinguishable.

[0034] The preparation method of the colloidal gold conjugate pad comprises: soaking glass fiber paper in a solution containing colloidal gold probe, wherein the colloidal gold probe is formed by combining colloidal gold and luteinizing hormone monoclonal antibody C1; drying at 37°C after soaking, wherein the amount of the colloidal gold probe is 15 μL / cm.

[0035] Specifically, after pretreatment, the glass fiber paper is completely immersed in a solution containing a colloidal gold probe, allowing the antibody-labeled colloidal gold particles to be fully adsorbed in the fiber pore structure. During the infiltration process, the probe solution evenly penetrates into the interior of the carrier through capillary action, and the antibody molecules are directionally bound to the colloidal gold surface through electrostatic action. Subsequently, gradient drying is carried out at a constant temperature of 37°C, which ensures that the water evaporates moderately to form a stable solid phase carrier and avoids the loss of antibody activity due to high temperature. The probe loading in the colloidal gold conjugate pad finally obtained is precisely controlled at the level of 15μL / cm, which not only meets the detection sensitivity requirements but also avoids non-specific binding caused by excessive probe.

[0036] The judgment rules of the test kit are as follows: (1) If the test line and the quality control line show color at the same time, it is positive, indicating that the sheep is in estrus; (2) If the test line does not show color and the quality control line shows color is negative, it means that the sheep is not in estrus; (3) If the quality control line and the test line do not show color or the quality control line does not show color, it is an invalid result.

[0037] Specifically, when the luteinizing hormone in the sample reaches the threshold, the antibody carried by the colloidal gold probe binds to the target hormone to form a complex, which migrates to the detection line under the action of chromatography and is captured by the fixed antibody, forming a colored strip. At this time, if the fixed antibody at the quality control line captures the unbound probe, color will develop synchronously, indicating that the detection system is operating effectively. If the hormone concentration in the sample is lower than the detection threshold, the probe cannot aggregate on the detection line, but the quality control line can still capture the free probe for color development. When the quality control line does not develop color, it indicates that the kit has failed to preserve or there is an operational error, and the result cannot be trusted.

[0038] like Figure 2 As shown in the experimental results, the first and second left test lines are colored and the quality control line is not colored, indicating that the two sheep are not in estrus. The first right test line and the quality control line are both colored positive, indicating that the sheep is in estrus.

[0039] Figure 2 The experimental samples were 280 healthy, well-developed Small-Tail Han sheep (all multiparous), provided by the National Standardized Demonstration Farm and the National Small-Tail Han Sheep Conservation Farm in Jiaxiang County, Shandong Province. 140 of these sheep were in estrus and 140 were in diestrus. All the Small-Tail Han sheep were housed in semi-enclosed, high-bedded barns. Daily husbandry and immunization procedures were strictly adhered to the farm's current regulations.

[0040] The sampling method for the experimental Small Tail Han sheep ewes was to collect blood from the jugular vein. 5 mL of blood was collected from the jugular vein and placed in a 5 mL ETA anticoagulant blood collection tube. The tube was shaken upside down 5-8 times to prevent blood coagulation. After standing for 15-20 minutes, it was centrifuged for 15 minutes at a speed of 3500 r / min and a temperature of 4°C. The supernatant (serum) was dripped into the sample pad. If there was any supernatant left, it was stored at -20°C.

[0041] C1 and 5F3G4C6 LH monoclonal antibodies were purchased from Beijing Wanhua Bioengineering Co., Ltd., and goat anti-mouse IgG was purchased from Weifang BioNuodi Biotechnology Co., Ltd.

[0042] The test object of the kit is the luteinizing hormone in the serum of small-tail Han sheep. The test sample is the supernatant of EDTA-anticoagulated whole blood collected from the jugular vein after centrifugation.

[0043] Specifically, the sample preparation process includes: after collecting blood through the jugular vein, it is immediately mixed with EDTA anticoagulant to prevent coagulation; the mixed solution is then transferred to a centrifuge tube and centrifuged at a specific speed to separate the supernatant. This supernatant is directly added to the sample pad of the test kit, and through chromatography, the luteinizing hormone in the sample is combined with the colloidal gold probe, and finally color is developed at the test line. The use of EDTA anticoagulant treatment can reduce the impact of blood cell rupture on the test. At the same time, the jugular vein blood collection operation is convenient and suitable for rapid screening in large-scale farming scenarios. Colloidal gold was prepared by the trisodium citrate reduction method and calcined in a ratio of V chloroauric acid / V trisodium citrate = 1:1.4 to obtain a colloidal gold solution.

[0044] Specifically, when preparing the colloidal gold solution, the chloroauric acid solution is first placed in a heating device to maintain a constant temperature, and then a pre-prepared trisodium citrate solution is added at a uniform rate through a titrator. During the addition process, the color of the solution gradually changes from light yellow to wine red, indicating that the gold ions are reduced to form nano-scale colloidal particles. After the reaction is terminated, a stable colloidal gold sol system is formed by natural cooling. The selection of a volume ratio of 1:1.4 in this preparation process can balance the reducing agent concentration and reaction activity, avoiding excessive reducing agent causing particle agglomeration or excessively wide particle size distribution.

[0045] The preparation method of the colloidal gold probe includes: adjusting the pH value of the colloidal gold solution to 8.5, adding luteinizing hormone monoclonal antibody C1, mixing and then standing; adding 10% BSA solution to stabilize, centrifuging at -4°C and 1400 rpm, and discarding the supernatant; adding a resuspension solution containing 0.1MTris, 10% sucrose, 1% PVP, 1% BSA, and 0.5% Tween-20 to the precipitate for re-dissolution.

[0046] Specifically, after the pH of the colloidal gold solution is precisely controlled, the amino groups of the luteinizing hormone monoclonal antibody C1 produce electrostatic adsorption with the colloidal gold surface to form a stable antibody-colloidal gold complex. After the addition of BSA for blocking, the active sites on the free colloidal gold surface are fully covered to prevent nonspecific binding during subsequent storage. During low-temperature centrifugation, unbound antibody molecules and small molecule impurities are effectively separated, leaving only the complete probe complex precipitate. The resuspension in the reconstitution stage works synergistically through multiple components, such as Tris buffer to maintain a physiological pH environment, sucrose to form a glassy protective layer, and PVP to inhibit particle aggregation, ultimately allowing the probe to maintain structural integrity and biological activity when stored in a liquid state.

[0047] In a specific embodiment, the specific preparation method of the kit is as follows: Using nitrocellulose membrane as the solid phase carrier, a certain concentration of LH monoclonal antibody 5F3G4C6 was used to make the test line (T), and a certain concentration of goat anti-mouse IgG secondary antibody was used to make the quality control line (C), which were coated on the nitrocellulose membrane.

[0048] Immunogold was prepared using colloidal gold and LH monoclonal antibody C1, which was dried and embedded on glass fiber paper at a certain concentration to make a colloidal gold pad.

[0049] The coating film and the gold pad are combined with the sample pad, absorbent filter paper, PVC bottom plate, etc. to make a large plate, which is then cut into test strips of a certain width using a strip cutting machine, and then carded, bagged, and packaged.

[0050] 1. Preparation of colloidal gold Colloidal gold was prepared by the "trisodium citrate reduction method" and calcined in a ratio of V chloroauric acid (4%) / V trisodium citrate (4%) = 1:1.4, with a particle size of about 40 nm.

[0051] 2. Confirm the optimal pH value (1) The optimal pH value was screened using the chessboard method. 0.5 mL of the prepared colloidal gold solution was added to each 1.5 mL centrifuge tube. (2) Add 0.2 mol / L K2CO3 solution while stirring to adjust the pH value gradient to 6, 7, 8, 9, 10, and 11.

[0052] (3) Add 20 μg of goat anti-mouse IgG to each tube, mix well, and let it stand at room temperature for 15-20 minutes. Add 100 μL of 10% NaCl solution to terminate the reaction, mix well, and let it stand at room temperature for 1 hour. The lowest pH value when the colloidal gold solution does not change in color and has no precipitation or aggregation compared with the original solution is the optimal pH value.

[0053] 3. Confirm the optimal labeling concentration (observation method, gradient verification method) (1) Take a 1.5 mL centrifuge tube, add colloidal gold solution and adjust it to the optimal pH value.

[0054] (2) Add different amounts of goat anti-mouse IgG to make the final antibody content 5μL, 10μL, 15μL, 20μL, 25μL, and 30μL, respectively. Mix well and let stand at room temperature for 30 minutes.

[0055] (3) Add 100 μL of 10% BSA solution to each tube, mix well, and let stand at room temperature for 30 minutes.

[0056] (4) Take a small amount of solution from each tube and measure the absorbance at 450-650 nm. The amount of antibody added corresponding to the maximum absorbance peak is considered the optimal labeling concentration.

[0057] 4. Preparation of colloidal gold probe (1) Take a 250 mL conical flask and add 5 mL of the prepared colloidal gold solution. Adjust the solution to the optimal pH value and the optimal labeled concentration according to the above test method and stir thoroughly at room temperature for 30 minutes.

[0058] (2) Add 10% BSA solution to stabilize the solution.

[0059] (3) Transfer the solution to a centrifuge tube and centrifuge at -4°C, 1400 rpm for 15 min. Discard the supernatant and add an equal volume of reconstitution solution to reconstitute the solution. After reconstitution, centrifuge again at -4°C, 1400 rpm for 30 min. Discard the supernatant.

[0060] (4) Prepare a resuspension solution of 0.1M Tris + 10% sucrose + 1% PVP + 1% BSA + 0.5% Tween-20, and adjust the pH to 8.5 with hydrochloric acid.

[0061] (5) Add 10% of the original volume of resuspension solution to the precipitate and resuspend the precipitate. Repeat the above centrifugation steps twice. The obtained colloidal gold probe is stored at 4°C.

[0062] 5. Preparation of colloidal gold pad The concentration ratio of colloidal gold complex is 120μl / ml, and the amount of colloidal gold complex is 15μl / cm. The cut colloidal gold conjugate pad is completely immersed in the reconstituted solution, then taken out and dried in an oven at 37℃. After drying, it is sealed and stored at room temperature for future use.

[0063] 6. Preparation of coated nitrocellulose membrane Detection line (T), concentration of monoclonal antibody 5F3G4C6 was 1.5 mg / ml; Quality control line (C), goat anti-mouse IgG concentration was 0.5 mg / ml; The scratch volume of the test line (T) and the quality control line (C) was 1 μl / cm.

[0064] 7. Assembly of the kit Although the present invention is disclosed as above, the scope of protection disclosed by the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the scope of protection of the present invention.

Claims

1. A colloidal gold kit for sheep estrus identification, characterized in that: Including test strips and box body; The test paper comprises a sample pad, a colloidal gold binding pad, a chromatography membrane and a water-absorbing filter paper; The chromatographic membrane is provided with a detection line and a quality control line; The detection line is coated with luteinizing hormone monoclonal antibody 5F3G4C6, and the quality control line is coated with goat anti-mouse IgG.

2. The colloidal gold kit for sheep estrus identification according to claim 1, wherein The box body includes a plastic bottom plate and a plastic cover plate; The plastic cover is provided with an injection port and an observation window, the observation window corresponds to the detection line position and the quality control line position of the chromatographic membrane, the detection line position is marked with a detection line identifier, and the quality control line position is marked with a quality control line identifier.

3. The colloidal gold kit for sheep estrus identification according to claim 1, wherein One side of the chromatography membrane is overlapped with the colloidal gold binding pad, and the other side of the chromatography membrane is overlapped with the absorbent filter paper; The side of the colloidal gold binding pad away from the chromatography membrane is overlapped with the sample pad.

4. The colloidal gold kit for sheep estrus identification according to claim 1, wherein The chromatography membrane is a nitrocellulose membrane.

5. The colloidal gold kit for sheep estrus identification according to claim 1, wherein The coating concentration of the test line was 1.5 mg / ml, and the coating concentration of the quality control line was 0.5 mg / ml; the streaking volume of both the test line and the quality control line was 1 μl / cm.

6. The colloidal gold kit for sheep estrus identification according to claim 1, wherein The preparation method of the colloidal gold binding pad comprises: The glass fiber paper is immersed in a solution containing a colloidal gold probe, wherein the colloidal gold probe is formed by combining colloidal gold with luteinizing hormone monoclonal antibody C1; After infiltration, the samples were dried at 37°C and the amount of colloidal gold probe used was 15 μl / cm.

7. The colloidal gold kit for sheep estrus identification according to claim 1, wherein The determination rules of the kit are: If the test line and the quality control line show positive color at the same time, it means the sheep is in estrus; If the test line shows no color and the quality control line shows negative color, it means the sheep is not in estrus; If the test line shows color but the quality control line does not, or both the test line and the quality control line do not show color, the result is invalid.

8. The colloidal gold kit for sheep estrus identification according to claim 1, wherein The detection object of the kit is the luteinizing hormone in the serum of small-tailed Han sheep, and the detection sample is the supernatant of EDTA-anticoagulated whole blood collected from the jugular vein after centrifugation.

9. The colloidal gold kit for sheep estrus identification according to claim 6, wherein The colloidal gold is prepared by a trisodium citrate reduction method and fired at a ratio of V chloroauric acid / V trisodium citrate=1:1.4 to obtain a colloidal gold solution.

10. The colloidal gold kit for sheep estrus identification according to claim 9, wherein The preparation method of the colloidal gold probe comprises: Adjust the pH value of the colloidal gold solution to 8.5, add luteinizing hormone monoclonal antibody C1, mix well, and let it stand; 10% BSA solution was added for stabilization, and the supernatant was discarded after centrifugation at -4°C and 1400 rpm; The pellet was resuspended in a solution containing 0.1 M Tris, 10% sucrose, 1% PVP, 1% BSA, and 0.5% Tween-20.

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