A colloidal gold test strip for detecting porcine whole blood

Abstract

The present application relates to the technical field of in vitro detection, and more particularly to a colloidal gold test strip for detecting pig whole blood, wherein the sample pad of the colloidal gold test strip is coated with a complex antibody of anti-pig red blood cell antibody and anti-pig hemoglobin antibody, which can directly bind to red blood cells and hemoglobin in pig whole blood, eliminating the interference of hemolysis in whole blood detection on the result. The colloidal gold test strip has the advantages of simple operation, good serum separation effect, small sample dosage, fast detection speed and the like, and has a wide application prospect in the field detection of pig epidemic.

Description

Technical Field

[0001] This invention relates to the field of in vitro detection technology, specifically to a colloidal gold test strip for detecting porcine whole blood. Background Technology

[0002] For epidemiological testing of pigs, such as African swine fever virus, and for quarantine in international trade of live pigs, rapid on-site testing methods are generally preferred. Colloidal gold technology, a commonly used labeling technique for rapid testing, offers advantages such as convenience, high specificity and sensitivity, strong stability, no need for special equipment and reagents, and intuitive result interpretation. Therefore, it is particularly suitable for grassroots testing personnel, large-scale testing, and large-scale surveys. Its principle involves the polymerization effect of chloroauric acid and a reducing agent, forming gold particles of a certain size. These gold particles gradually become a stable colloidal state under the influence of electrostatics, forming a negatively charged hydrophobic colloidal solution. Because of the electrostatic effect, it becomes a stable colloidal state, hence the name colloidal gold.

[0003] Rapid clinical testing typically uses whole blood as the test sample. However, when whole blood is used as a sample for colloidal gold test strips, incomplete filtration of red blood cells or hemolysis can occur. This causes red blood cells and the hemoglobin released from them to precipitate along with the analyte into the detection area, increasing the background color of the detection window and interfering with qualitative, semi-quantitative, and quantitative detection. Existing colloidal gold test strips generally use blood filtration membranes to remove red blood cells, but these membranes are expensive, increasing testing costs. Furthermore, this method requires a large amount of whole blood sample, has a slow separation speed, and cannot eliminate the effects of hemolysis. Other colloidal gold test strips incorporate a separate hemoglobin adsorption pad, which effectively removes a large amount of hemoglobin, resulting in a clean background. However, this increases the material thickness, requiring adhesive tape to connect different parts for stability, making the preparation method cumbersome. Therefore, there is an urgent need for a more convenient, low-cost, and simple colloidal gold test strip for the direct detection of porcine whole blood. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a colloidal gold test strip for detecting porcine whole blood. The present invention provides a colloidal gold test strip that uses a composite antibody of anti-porcine erythrocyte antibody and anti-porcine hemoglobin antibody to bind porcine erythrocytes and porcine hemoglobin, which can quickly separate plasma and eliminate the interference caused by hemolysis when whole blood testing occurs.

[0005] The present invention provides a composite antibody, wherein an anti-porcine erythrocyte antibody and an anti-porcine hemoglobin antibody are covalently cross-linked; the molar ratio of the anti-porcine erythrocyte antibody and the anti-porcine hemoglobin antibody is (0.8-1.2):1.

[0006] The composite antibody provided by this invention can bind to porcine erythrocytes or porcine hemoglobin. Therefore, it can effectively capture and separate both porcine erythrocytes and hemoglobin in the event of hemolysis. Compared to the effect of simply mixing two antibodies (anti-porcine erythrocyte antibody and anti-porcine hemoglobin antibody), using the composite antibody results in a cleaner background on the test strip's detection window, indicating that the composite antibody has a stronger binding effect on porcine erythrocytes or porcine hemoglobin. Furthermore, the titer of the anti-porcine erythrocyte antibody is not less than 1 / 64kJ.

[0007] The titer of the anti-porcine hemoglobin antibody is not less than 1 / 128K;

[0008] The anti-porcine erythrocyte antibody has a heavy chain variable region as shown in SEQ ID NO.1 and a light chain variable region as shown in SEQ ID NO.2;

[0009] The anti-porcine hemoglobin antibody is the standardized anti-porcine hemoglobin antibody PLA114.

[0010] Specifically, in some embodiments, this invention experimentally investigated the detection performance of colloidal gold test strips coated with a single anti-porcine erythrocyte antibody, anti-porcine erythrocyte antibody and anti-porcine hemoglobin antibody, and a composite antibody on a sample pad when detecting whole pig blood. The results showed that when different degrees of hemolysis occurred in whole pig blood, the background color of the detection area of ​​the colloidal gold test strips coated with a single anti-porcine erythrocyte antibody, anti-porcine erythrocyte antibody and anti-porcine hemoglobin antibody, and the composite antibody was darker, interfering with the detection results. The colloidal gold test strip coated with the composite antibody had a clean background in the detection area, good chromatographic effect, and clearly visible detection results.

[0011] This invention provides a method for preparing the aforementioned composite antibody, comprising:

[0012] Step 1: Dilute the anti-porcine erythrocyte antibody and the anti-porcine hemoglobin antibody to 1-3 mg / mL with MES buffer, and dilute 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to 10-30 mg / mL.

[0013] Step II: Mix the diluted 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, anti-porcine erythrocyte antibody, and anti-porcine hemoglobin antibody from Step I, and react at 2-8°C in the dark for 10-14 hours to obtain the composite antibody.

[0014] The molar ratio of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to the anti-porcine erythrocyte antibody and the anti-porcine hemoglobin antibody is (3-12):(0.8-1.2):1.

[0015] Specifically, in some embodiments, the concentration of the MES buffer is 0.05 mol / L and the pH value is 6.2;

[0016] The anti-porcine erythrocyte antibody and the anti-porcine hemoglobin antibody were diluted to 2 mg / mL with the MES buffer and then mixed in equal volumes. One anti-porcine erythrocyte antibody and one anti-porcine hemoglobin antibody were cross-linked together by covalent bonds. The diluted 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was then added and reacted at 4°C in the dark for 12 hours. After the reaction was completed, the antibody was dialyzed with PBS buffer to obtain the treated composite antibody.

[0017] This invention provides a colloidal gold test strip for using whole pig blood as a sample, comprising a backing card, a sample pad, a colloidal gold pad, an NC membrane, and an absorbent plate;

[0018] The colloidal gold pad is coated with gold-labeled specific antibodies.

[0019] The sample pad is coated with the composite antibody or the composite antibody prepared according to the method;

[0020] The NC membrane is provided with a detection line and a control line; the detection line is provided with a capture antibody, and the control line is provided with a goat anti-mouse antibody;

[0021] The capture antibody is an antibody that specifically binds to the target antigen.

[0022] Specifically, in some embodiments, the sample pad, colloidal gold pad, NC membrane, and absorbent plate are sequentially fixed on the backing card;

[0023] The sample pad is made of nitrocellulose membrane material;

[0024] One end of the sample pad overlaps with one end of the colloidal gold pad, with the sample pad located on the upper side and the overlap length being 3-4 mm. Both ends of the NC membrane overlap with one end of the colloidal gold pad and one end of the absorbent plate, respectively, and both ends of the NC membrane are located on the lower side with the overlap length being 3-4 mm.

[0025] This invention does not limit the coating position of the antibody on the test strip; any coating position and / or spacing relationship commonly found in the art can achieve good detection. The NC membrane has a detection line on the side near the gold label pad and a control line on the side near the absorbent plate.

[0026] In this invention, the capture antibody specifically recognizes the analyte in the sample. The analyte includes, but is not limited to, pathogenic microorganisms or molecular markers. The pathogenic microorganism is a swine pathogen, and the molecular marker can be a disease diagnostic marker for swine or a physiological marker for swine; it can be an antigen or an antibody. This invention does not limit this. The swine pathogen refers to bacteria, fungi, viruses, mycoplasma, chlamydia, and / or bacteriophages that cause diseases in pigs.

[0027] For example, the bacteria that cause diseases in pigs include, but are not limited to, Streptococcus, Brucella suis, Pasteurella suis, Salmonella suis, Escherichia coli, Erysipelothrix rhusiopathiae, Actinobacillus pleuropneumoniae, Clostridium perfringens type C, Staphylococcus, Haemophilus parasuis, and Bordetella bronchiseptica.

[0028] The fungi that cause diseases in pigs include, but are not limited to, Trichophyton mentagrophytes and Microsporum.

[0029] The viruses that cause diseases in pigs include, but are not limited to, classical swine fever virus, foot-and-mouth disease virus, pseudorabies virus, Lelystad virus, transmissible gastroenteritis virus, porcine parvovirus, Japanese encephalitis virus, and swine influenza virus;

[0030] Based on the fact that the test strip of this invention is coated with antibodies capable of removing hemoglobin and red blood cells, it can be applied to the detection of whole blood samples, and thus to the inspection and quarantine of pigs. In this embodiment, African swine fever virus antibodies are used as an example to achieve accurate detection; that is, in this embodiment of the invention, the capture antibody on the detection line is an antibody against African swine fever virus.

[0031] The colloidal gold test strip provided by the present invention has a sample pad coated with the composite antibody at a concentration of 54 μg / mL to 108 μg / mL.

[0032] Specifically, in some embodiments, the present invention explores the optimal concentration of the composite antibody coating on the sample pad through experiments. The results show that when the coating concentration of the composite antibody is 18 μg / mL and 36 μg / mL, the background color of the detection area is deepened, which interferes with the detection results. When the coating concentration is 54 μg / mL, 72 μg / mL, and 108 μg / mL, the background color of the detection area is clean and the results are clear.

[0033] The colloidal gold test strip provided by this invention uses TB buffer as the coating buffer for the composite antibody.

[0034] Furthermore, the concentration of the TB buffer is 0.01 mol / L, and the pH value is 7.6; the TB buffer contains 1 wt% BSA blocking protein and 0.01 wt% Triton-X-100 surfactant.

[0035] The colloidal gold test strip provided by this invention requires only 60 μL of sample to achieve the detection purpose in the process of detecting whole blood samples from pigs. Furthermore, the composite antibody coated in the sample pad can quickly bind to the red blood cells and hemoglobin in the sample. When the sample reaches the detection line, it will not contaminate the NC membrane in the detection area. Therefore, the test strip has a clean background and more accurate detection results. It is particularly suitable for on-site detection of African swine fever virus, epidemiological investigation, and quarantine of live pigs in international trade.

[0036] This invention provides the application of the colloidal gold test strip in the preparation of a kit for detecting swine epidemics.

[0037] The present invention also provides a kit for detecting swine epidemics, comprising the colloidal gold test strip and acceptable adjuvants. The acceptable adjuvants include, but are not limited to, blood anticoagulants and sample diluents.

[0038] The present invention provides a method for detecting analytes using whole pig blood as a sample, as described in the colloidal gold test strip or the reagent kit.

[0039] The method specifically includes (1) adding a whole blood sample of pig to a sample pad, where the composite antibody coated on the sample pad reacts with red blood cells and hemoglobin to remove red blood cells and hemoglobin from the sample; (2) the sample moves to the gold-labeled pad through capillary action, where the analyte in the sample binds to the specific antibody on the gold-labeled pad to form an analyte-gold-labeled antibody complex; (3) the sample continues to pass through capillary action into the NC membrane, where it binds to the specific antibody on the detection line for detection, and the detection result determines whether the sample contains the antibody / antigen to be detected.

[0040] The method provided by this invention can be for diagnostic purposes or non-diagnostic purposes. The non-diagnostic purpose does not directly aim to obtain the pig's health status, but merely provides a basis for judgment for rapid on-site testing. When obtaining whole blood samples, the pig can be either alive or dead.

[0041] The colloidal gold test strip for direct detection of whole pig blood in this invention is simple to prepare. It only requires covalent cross-linking two antibodies against pig red blood cells and pig hemoglobin and coating them onto the sample pad. This can quickly remove red blood cells and hemoglobin from the sample. The sample pad does not need to use expensive blood filtration membranes; it can use only conventional nitrocellulose membrane materials, thus reducing the detection cost.

[0042] Using the colloidal gold test strip of this invention, whole blood of pigs can be directly tested, with a small sample volume, a clean background, and more accurate test results. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the structure of the colloidal gold test strip of the present invention;

[0044] Figure 2 The chromatographic effect of different types of samples in Comparative Example 1 is shown when only the sample pad is coated with anti-porcine erythrocyte antibody. Among them, chromatographic strips 1# and 2# are non-hemolyzed porcine whole blood samples, while chromatographic strips 3# and 4# are hemolyzed porcine whole blood samples.

[0045] Figure 3 The chromatographic effect of different types of samples when the sample pads in Comparative Example 2 are simultaneously coated with anti-porcine erythrocyte antibodies and anti-porcine hemoglobin antibodies is shown. Among them, chromatographic strips 5# and 6# are non-hemolyzed porcine whole blood samples, while chromatographic strips 7# and 8# are hemolyzed porcine whole blood samples.

[0046] Figure 4 The chromatographic effects of different types of samples coated with a composite antibody of anti-porcine erythrocyte antibody and anti-porcine hemoglobin antibody are shown in Example 3. Among them, the chromatographic strips 9#-13# are non-hemolyzed porcine whole blood samples, and the amount of composite antibody coated on the sample pad is 1ug / person, 2ug / person, 3ug / person, 4ug / person, and 6ug / person, respectively; the chromatographic strips 14#-18# are hemolyzed porcine whole blood samples, and the amount of composite antibody coated on the sample pad is 1ug / person, 2ug / person, 3ug / person, 4ug / person, and 6ug / person, respectively. Detailed Implementation

[0047] This invention provides a colloidal gold test strip for detecting porcine whole blood. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0048] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0049] The standardized anti-porcine hemoglobin antibody used in this invention is anti-porcine hemoglobin antibody (PLA114) from BMABIOMEDICALS. The anti-porcine erythrocyte antibody is mouse anti-porcine erythrocyte membrane antibody 3B6 from Imeno.

[0050] The present invention will be further illustrated below with reference to the embodiments:

[0051] Example 1: Preparation and Detection Method of Colloidal Gold Test Strip for Direct Detection of Porcine Whole Blood

[0052] like Figure 1As shown, the colloidal gold test strip for direct detection of porcine whole blood includes a backing card with an adhesive backing. A sample pad, a colloidal gold pad, an NC membrane, and an absorbent plate are sequentially fixed to the upper end of the backing card. One end of the sample pad overlaps with one end of the colloidal gold pad, with the sample pad end positioned on the upper side, and the overlap length is 3–4 mm. The colloidal gold pad contains gold-labeled specific antibodies. Both ends of the NC membrane overlap with one end of the colloidal gold pad and one end of the absorbent plate, respectively, with both ends of the NC membrane positioned on the lower side, and the overlap length is 3–4 mm. The NC membrane has a detection line on the side near the gold-labeled pad and a control line on the side near the absorbent plate. Furthermore, the sample pad is coated with a composite antibody consisting of anti-porcine erythrocyte antibodies and anti-porcine hemoglobin antibodies.

[0053] Using the colloidal gold test strips described above, direct detection of porcine whole blood includes the following steps:

[0054] Step 1: Add the whole blood sample of the pig to be tested to the sample pad;

[0055] Step 2: The sample is placed into the sample pad described above. The sample pad is coated with anti-porcine erythrocyte antibodies and anti-porcine hemoglobin antibodies. The anti-porcine erythrocyte antibodies react with erythrocytes, and the anti-porcine hemoglobin antibodies react with hemoglobin, thereby removing erythrocytes and hemoglobin. The sample pad is made of nitrocellulose membrane material.

[0056] Step 3: After red blood cells and hemoglobin have been removed in step 2, the sample continues to move to the gold-labeled pad on the device through capillary action. The analyte in the sample binds to the specific antibody on the gold-labeled pad to form an analyte-gold-labeled antibody complex.

[0057] Step 4: The sample continues to pass through the capillary action into the NC membrane, where it binds specifically to the detection line for detection. The detection result determines whether the sample contains the antibody / antigen to be detected.

[0058] Example 2: Preparation of a composite antibody consisting of anti-porcine erythrocyte antibody and anti-porcine hemoglobin antibody

[0059] The steps for preparing a composite antibody consisting of anti-porcine erythrocyte antibody and anti-porcine hemoglobin antibody are as follows:

[0060] Step 1: Dilute the anti-porcine erythrocyte antibody (selecting the mouse anti-porcine erythrocyte membrane antibody with clone number 3B6, which is self-made by our company and has the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.1 and the light chain variable region of the amino acid sequence shown in SEQ ID NO.2) and the anti-porcine hemoglobin antibody (anti-porcine hemoglobin antibody PLA114 from BMA BIOMEDICALS) to 2 mg / mL using 0.05 mol / L, pH=6.2 MES buffer. Mix the diluted antibodies in equal volumes. Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to 20 mg / mL using 0.05 mol / L, pH=6.2 MES buffer.

[0061] Step 2: Calculate the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride according to the ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to antibody = 1.5:1 (molar ratio), and slowly add it to the reaction vessel containing the mixed antibody at room temperature. Incubate at 4°C in the dark for 12 hours.

[0062] Step 3: After the reaction is complete, dialyze the reaction solution with 0.01 mol / L PBS buffer (pH=7.2) at 4°C for 12 hours, changing the dialysate 3 times during the process, to obtain the treated composite antibody.

[0063] Example 3: Coated composite antibody

[0064] The sample pad of the colloidal gold test strip for direct detection of porcine whole blood is coated with a composite antibody consisting of anti-porcine erythrocyte antibody and anti-porcine hemoglobin antibody using the following method:

[0065] Step 1: The composite antibody obtained in Example 2 was diluted with TB coating buffer at pH 7.6 and 0.01M to 18ug / mL, 36ug / mL, 54ug / mL, 72ug / mL and 108ug / mL respectively (corresponding to 1ug / person, 2ug / person, 3ug / person, 4ug / person and 6ug / person respectively). The coating buffer contained 1% BSA blocking protein and 0.01% Triton-X-100 surfactant.

[0066] Step 2: Dilute the antibody in the coating buffer onto a nitrocellulose membrane and dry it in a drying oven at 37.0℃±2.0℃ for 10 hours. Then, assemble the membrane into chromatography strips. Seal the chromatography strips in aluminum foil bags and store them at room temperature for later use.

[0067] Collect whole blood samples from pigs with added anticoagulants and whole blood samples from pigs with varying degrees of hemolysis. Take 60 μL of each sample and drop it onto the prepared chromatographic test strip sample pad. Place the sample horizontally for 15 minutes and observe the chromatographic results.

[0068] Comparative Example 1: Single-coated anti-porcine erythrocyte antibody

[0069] The sample pad for the colloidal gold test strip used for direct detection of porcine whole blood was coated with anti-porcine erythrocyte antibodies as follows:

[0070] Step 1: Dilute the anti-porcine erythrocyte antibody (same as in Example 2) to 36ug / mL with 0.01M TB coating buffer at pH 7.6. The coating buffer contains 1% BSA blocking protein and 0.01% Triton-X-100 surfactant.

[0071] Step 2: Dilute the antibody with coating buffer onto a nitrocellulose membrane and dry it in a drying oven at 37.0℃±2.0℃ for 10 hours. Then, assemble the chromatographic strips according to the structure of the colloidal gold test strips. Seal the chromatographic strips in aluminum foil bags and store them at room temperature for later use.

[0072] Collect whole blood samples from pigs with added anticoagulants and whole blood samples from pigs with varying degrees of hemolysis. Take 60 μL of each sample and drop it onto the prepared chromatographic test strip sample pad. Place the sample horizontally for 15 minutes and observe the chromatographic results.

[0073] Comparative Example 2: Coated with anti-porcine erythrocyte antibody and anti-porcine hemoglobin antibody

[0074] The sample pad for the colloidal gold test strip used for direct detection of porcine whole blood was coated with anti-porcine erythrocyte antibodies and anti-porcine hemoglobin antibodies as follows:

[0075] Step 1: Dilute the anti-porcine erythrocyte antibody and anti-porcine hemoglobin antibody from Example 2 to 36ug / mL with 0.01M TB coating buffer at pH 7.6, mix them in equal volumes and set aside. The coating buffer contains 1% BSA blocking protein and 0.01% Triton-X-100 surfactant.

[0076] Step 2: The antibody diluted with coating buffer is applied to a nitrocellulose membrane and dried in a drying oven at 37.0℃±2.0℃ for 10 hours, then assembled into chromatography strips. The chromatography strips are sealed in aluminum foil bags and stored at room temperature for later use.

[0077] Collect whole blood samples from pigs with added anticoagulants and whole blood samples from pigs with varying degrees of hemolysis. Take 60 μL of each sample and drop it onto the prepared chromatographic test strip sample pad. Place the sample horizontally for 15 minutes and observe the chromatographic results.

[0078] Results of tests in Example 1, Example 3, and Comparative Examples

[0079] 1. The detection results of Example 3 are as follows: Figure 4 As shown:

[0080] Chromatographic strips #9-#13 were for non-hemolyzed porcine whole blood samples, with the amount of composite antibody coated on the sample pad being 1ug / person, 2ug / person, 3ug / person, 4ug / person, and 6ug / person, respectively. Chromatographic strips #14-#18 were for hemolyzed porcine whole blood samples, with the amount of composite antibody coated on the sample pad being 1ug / person, 2ug / person, 3ug / person, 4ug / person, and 6ug / person, respectively.

[0081] The results of the chromatography strips 9#-13# showed that when the antibody concentration on the sample pad was 1ug / person, 2ug / person, 3ug / person, 4ug / person, and 6ug / person, all red blood cells were adsorbed onto the sample pad, the background was clean, and the chromatography effect was good.

[0082] Results from chromatography strips #14-#18 showed that even with 1 μg / person antibody coating, hemolyzed samples still caused a darkening of the background color on the NC membrane in the detection area, interfering with the test results. Coating with 2 μg / person antibody improved the results. When the antibody coating concentration increased to 3 μg / person or higher, all red blood cells and hemoglobin were adsorbed onto the sample pad, resulting in a clean background and good chromatography performance.

[0083] 2. The test results of Comparative Example 1 are as follows: Figure 2 As shown:

[0084] The chromatographic strips #1 and #2 are two unhemolyzed pig whole blood samples. It can be seen that the red blood cells are completely adsorbed on the sample pad, and the background is clean, indicating good chromatographic effect.

[0085] The chromatographic strips #3 and #4 are two whole blood samples from pigs that showed different degrees of hemolysis. It can be seen that the background color on the NC membrane in the detection area is darker, which interferes with the detection results.

[0086] The test results of Comparative Example 2 are as follows Figure 3 As shown:

[0087] The chromatographic strips #5 and #6 are two unhemolyzed pig whole blood samples. It can be seen that the red blood cells are completely adsorbed on the sample pad, and the background is clean, indicating good chromatographic effect.

[0088] The 7# and 8# chromatography strips are two whole blood samples from pigs that showed different degrees of hemolysis. It can be seen that even when the sample pad is coated with two untreated antibodies, the hemolyzed sample still causes the background color on the NC membrane in the detection area to be darker, which interferes with the detection results.

[0089] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A complex antibody, characterized in that, The anti-porcine erythrocyte antibody and the anti-porcine hemoglobin antibody are covalently cross-linked; the molar ratio of the anti-porcine erythrocyte antibody and the anti-porcine hemoglobin antibody is (0.8-1.2):1; The anti-porcine erythrocyte antibody has a heavy chain variable region as shown in SEQ ID NO.1 and a light chain variable region as shown in SEQ ID NO.2; The anti-porcine hemoglobin antibody is the standardized anti-porcine hemoglobin antibody PLA114.

2. The composite antibody according to claim 1, characterized in that, The titer of the anti-porcine erythrocyte antibody is not less than 1 / 64k; the titer of the anti-porcine hemoglobin antibody is not less than 1 / 128k.

3. The method for preparing the composite antibody according to claim 1 or 2, characterized in that, include: Step 1: Dilute the anti-porcine erythrocyte antibody and the anti-porcine hemoglobin antibody to 1-3 mg / mL with MES buffer, and dilute 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to 10-30 mg / mL. Step II: Mix the diluted 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, anti-porcine erythrocyte antibody, and anti-porcine hemoglobin antibody from Step I, and react at 2-8°C in the dark for 10-14 hours to obtain the composite antibody; The molar ratio of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to the anti-porcine erythrocyte antibody and the anti-porcine hemoglobin antibody is (3-12):(0.8-1.2):

1.

4. A colloidal gold test strip using whole pig blood as a sample, characterized in that, Includes backing card, sample pad, colloidal gold pad, NC membrane and absorbent plate; The colloidal gold pad is coated with gold-labeled specific antibodies. The sample pad is coated with the composite antibody as described in claim 1 or 2 or the composite antibody prepared according to the method of claim 3; The NC membrane is provided with a detection line and a control line; the detection line is provided with a capture antibody, and the control line is provided with a goat anti-mouse antibody.

5. The colloidal gold test strip according to claim 4, characterized in that, The sample pad and the colloidal gold pad have a 3-4 mm overlap in adjacent portions; the NC membrane and the colloidal gold pad and the absorbent plate each have a 3-4 mm overlap in adjacent portions.

6. The colloidal gold test strip according to claim 4, characterized in that, The sample pad is coated with the composite antibody at a concentration of 54 μg / mL to 108 μg / mL.

7. The colloidal gold test strip according to claim 4, characterized in that, The coating buffer for the composite antibody is TB buffer; The concentration of the TB buffer solution is 0.01 mol / L, and the pH value is 7.

6. The TB buffer contains 1 wt% BSA blocking protein and 0.01 wt% Triton-X-100 surfactant.

8. The use of the colloidal gold test strip according to any one of claims 4 to 7 in the preparation of a kit for detecting swine epidemics using whole blood as a sample.

9. A kit for detecting swine epidemics using whole blood as a sample, characterized in that, Includes the colloidal gold test strip as described in any one of claims 4 to 7 and other acceptable additives.

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