Preparation method and application of mycoplasma hyopneumoniae colloidal gold test strip

Abstract

According to the mycoplasma hyopneumoniae antigen colloidal gold test strip provided by the invention, the detection sensitivity and specificity of mycoplasma hyopneumoniae are remarkably improved by optimizing antibody selection and the structure of the test strip. Particularly, two different monoclonal antibodies are selected, namely, the monoclonal antibody 1 for resisting the mycoplasma hyopneumoniae P46 protein and the monoclonal antibody 2 for resisting the mycoplasma hyopneumoniae P46 protein are respectively used as a detection antibody and a capture antibody for Mph pathogen detection. The two monoclonal antibodies are optimized and screened and can be specifically combined with mycoplasma hyopneumoniae antigen, so that the detection effect is improved. When the mycoplasma hyopneumoniae colloidal gold chromatography test strip disclosed by the invention is used for detecting, a test sample only needs to be dropwise added on the sample pad of the test strip, mycoplasma hyopneumoniae pathogens in tissues, blood and cotton swab lixivium can be quickly identified and diagnosed, and convenience is provided for clinical diagnosis.

Description

Technical Field

[0001] The invention belongs to the technical field of animal pathogen detection, in particular to the preparation and application of a colloidal gold detection test paper for Mycoplasma hyopneumoniae antigen. Background Art

[0002] Mycoplasma pneumonia in pigs is a chronic pneumonia caused by Mycoplasma hyopneumoniae, also known as porcine endemic pneumonia. The pathogen was first isolated from the lung tissue of pigs with pneumonia by Mare, Switzer and Goodwin, and the disease was replicated experimentally. After the report, the pathogen was named M.hyopneumoniae. For a long time, this disease has been considered to be one of the most common, most widespread and most difficult to purify important epidemic diseases that cause significant economic losses to the pig industry. Although this disease is an old disease, in recent years, it has been frequently mixed with other pathogens such as PRRS and circovirus, causing significant economic losses and highlighting its importance. At present, the diagnostic test methods used to detect Mhp infection mainly include isolation and culture, indirect hemagglutination test (IHA), various ELISA methods and PCR methods. Although the isolation and culture of Mhp is of decisive significance for identification and diagnosis, it is difficult to culture Mhp, the detection rate is low, and the time required is very long, so it is still difficult to promote and apply it in clinical practice. Therefore, the laboratory diagnosis of Mhp mainly relies on serological tests. At present, indirect hemagglutination test is widely used in China, but this method has the problem of difficult preservation of antigens. The PCR method, which has emerged in recent years, has the advantages of simplicity, rapidity, high sensitivity and strong specificity, but its technical requirements are high, and improper control of conditions can easily lead to false negatives and false positives. In addition, the reagents are expensive and the detection cost is high.

[0003] The adhesion proteins on the surface of the membrane of M.hyopneumoniae frequently mutate. Pathogens in nature can spontaneously change the size and structure of the membrane protein antigens on their surface, and form antigen epitope shields on the surface of the protein antigen. Many reports on the membrane proteins of M.hyopneumoniae are different, indicating that M.hyopneumoniae is prone to mutation, while the gene of P46 and the protein it encodes are highly conservative. P46 is a protein with strong species specificity in M.hyopneumoniae. It does not exist in other types of mycoplasmas or other microorganisms. It is an antigen protein that causes the body's early immune response and is also the main immunodominant protein of M.hyopneumoniae. It is a commonly used candidate protein for diagnostic reagents. Many researchers use it as a target protein or target gene for M.hyopneumoniae detection. It is an important basis for distinguishing and identifying M.hyopneumoniae from other species of mycoplasmas, and is of great significance for the diagnosis of M.hyopneumoniae.

[0004] Therefore, the antigen colloidal gold test strip prepared by the monoclonal antibody 1 (detection antibody) and the monoclonal antibody 2 (capture antibody) against the P46 protein in the present invention provides convenience for the diagnosis of Mhp. Summary of the invention

[0005] The purpose of the present invention is to provide a sensitive, specific and stable colloidal gold test strip for detecting Mycoplasma hyopneumoniae antigen.

[0006] To achieve the above object, the technical solution provided by the present invention is: A colloidal gold test strip for Mycoplasma hyopneumoniae uses monoclonal antibody 1 and monoclonal antibody 2 against P46 protein as detection antibody and capture antibody, and the preparation of monoclonal antibody comprises the following steps: The P46 protein of Mycoplasma hyopneumoniae SY strain was used as an immune antigen and emulsified with Freund's complete adjuvant. Ten female Balb / c mice aged 8 weeks were immunized with 50ug of immune protein each. At 14d and 28d after the first immunization, the mice were boosted with the same dose, and the last boosted immunization was without adjuvant. After 31 days, spleen cells of the immunized mice were fused with myeloma cells SP2 / 0. The purified P46 protein with His tag was used as the coating antigen, and positive clones were screened in parallel by indirect ELISA. After three subclonings by limiting dilution method, two hybridoma cell lines that can stably secrete specific antibodies against P46 were obtained, named A8C2 and B7D4, respectively. The biological characteristics identification showed that the indirect ELISA titers of ascites monoclonal antibodies A8C4 and B7D4 were 1:105 and 1:106, respectively. The sandwich ELISA showed that the two monoclonal antibodies against P46 did not recognize the same antigen epitope and could be paired with each other. Western-blot analysis showed that the two monoclonal antibodies specifically reacted with the antigenic component of about 46KD of the SY strain of Mycoplasma suis.

[0007] The hybridoma cell line A8C2 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 9, 2025, with the deposit number CGMCC NO.46318, the classification name was hybridoma cell line (Musmusculus), and the deposit address was No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0008] The hybridoma cell line B7D4 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 9, 2025, with the deposit number CGMCC NO.46319, and the classification name was hybridoma cell line (Mus musculus). The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The invention provides a colloidal gold test strip for detecting Mycoplasma hyopneumoniae, which is prepared by the following preparation method.

[0010] Preparation of colloidal gold solution by trisodium citrate reduction method: Preparation of gold-labeled Mycoplasma hyopneumoniae P46 protein monoclonal antibody 1 (detection antibody): adjust the pH of the prepared colloidal gold solution to 8.5 using 0.1M potassium carbonate, dilute the labeled anti-P46 protein monoclonal antibody with double distilled water to a concentration of 0.5-2mg / ml, then add the detection protein dropwise into the stirred colloidal gold solution, keep stirring for 20-30min, finally add 10% BSA to a final concentration of 0.2%-0.5%, seal the exposed gold particles, continue stirring at 40°C for 20-30min, centrifuge at 14000r / min for 40-50min, and concentrate for later use; dilute the gold-labeled Mycoplasma hyopneumoniae P46 protein monoclonal antibody 1 with buffer solution 1, and evenly spray it on the gold-labeled conjugate pad to form a gold-labeled conjugate pad.

[0011] The monoclonal antibody 2 (capture antibody) of the P46 protein of Mycoplasma hyopneumoniae and goat anti-mouse IgG were diluted with buffer solution 2 respectively, and then the capture antibody of the P46 protein and goat anti-mouse IgG were coated on the nitrocellulose membrane to form the detection line and the quality control line respectively, and dried at 37-45°C for 12-18 hours for use; the sample pad was soaked in buffer solution 3, dried overnight at room temperature and humidity <20%, and cut for use; the test paper was assembled by adhering the sample pad, gold label conjugate pad, nitrocellulose membrane, and absorbent pad to the supporting bottom plate in order, and cut and packaged.

[0012] Preferably, the buffer solution 1 is composed of Tris-HCl, trehalose, BSA, PEG20000, and Tween-20, wherein the molar concentration of Tris-HCl is 10-100 mM, the final mass concentration of trehalose is 1%-5%, the final mass concentration of BSA is 1%-3%, the final mass concentration of PEG20000 is 0.05%-0.5%, and the final mass concentration of Tween-20 is 0.1%-0.5%.

[0013] Preferably, the buffer solution 2 is composed of Tris-HCl and methanol, wherein the molar concentration of Tris-HCl is 10-100 mM and the content of methanol is 1%-3%.

[0014] Preferably, the buffer solution 3 is composed of PBS and trehalose, wherein the molar concentration of PBS is 10-100 mM, and the final mass concentration of trehalose is 1%-5%.

[0015] Preferably, when preparing the gold-labeled Mycoplasma hyopneumoniae P46 protein monoclonal antibody 1, the pH of the colloidal gold solution is 8.5, and the buffer solution 2 dilutes the Mycoplasma hyopneumoniae P46 protein monoclonal antibody 2 to 2.0 mg / mL. The buffer solution 2 dilutes the goat anti-mouse IgG to 1.0 mg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Schematic diagram, schematic diagram of test strip result determination (A: positive; B: negative; C: invalid); Figure 2 : Test strip structure diagram, gold pad: gold-linked anti-P46 protein monoclonal antibody 1; detection line: NC membrane coated with anti-P46 protein monoclonal antibody 2. DETAILED DESCRIPTION

[0017] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively with reference to the relevant drawings. In order to overcome the difficulty of relatively backward diagnosis of swine mycoplasma, the present invention provides a colloidal gold test strip for swine pneumonia mycoplasma, and monoclonal antibody 1 and monoclonal antibody 2 against P46 protein are selected as detection antibody and capture antibody respectively. Its main characteristics are sensitivity, rapidity, specificity and stability. The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0018] Preparation of monoclonal antibody against P46 protein of Mycoplasma hyopneumoniae Preparation of purified P46 protein Preparation of primary production seeds: The recombinant E. coli basic strain expressing His-tagged P46 protein is reconstituted with 1 ml of sterile physiological saline, and an appropriate amount of the basic strain is streaked on a LB solid medium plate containing 50 µg / ml kanamycin using an inoculation loop, and cultured at 37°C for 16-24 hours. At least 5 typical colonies are selected and streaked onto several agar slants containing 50 µg / ml kanamycin, and cultured at 37°C for 12-16 hours. All should be qualified after inspection according to the standard. As primary seeds, they should be stored at 2-8°C for no more than 14 days, and the subculture should not exceed 5 generations.

[0019] Preparation of secondary production seeds: scrape the primary production seeds with an inoculation loop and inoculate them into 50 ml of LB liquid culture medium containing 50 µg / ml kanamycin. Shake and culture at 180 r / min and 37°C for 12 hours. The harvested seeds are the secondary seeds and stored at 2-8°C for no more than 7 days.

[0020] Antigen preparation and testing Induce expression: Inoculate the secondary production seeds at a ratio of 4% into LB liquid culture medium containing 50µg / ml kanamycin, culture at 180r / min and 37°C until OD600nm reaches 0.6-0.8, then add IPTG with a final concentration of 0.8mmol / L to induce expression for 4-6 hours.

[0021] Purity test: The test should be carried out according to the three appendices of the current "Chinese Veterinary Pharmacopoeia" and should be pure.

[0022] Bacterial disruption: Centrifuge the induced bacterial solution at 12000r / min for 10 minutes at 2-8℃, harvest the bacterial slurry, weigh it, resuspend it with 10 times (W / V) volume of sterile saline, break the bacteria with a high-pressure homogenizer, and harvest the supernatant by centrifugation at 12000r / min for 10 minutes.

[0023] Protein purification: Purify according to the instructions of Ni column purification method. Concentrate by ultrafiltration tube. Quantitative packaging.

[0024] Balb / c mouse immunization program P46 protein of Mycoplasma hyopneumoniae SY strain was used as an immune antigen and emulsified with Freund's complete adjuvant. Ten female Balb / c mice aged 8 weeks were immunized with 50ug of immune protein each. Booster immunizations were performed with the same dose at 14d and 28d after the first immunization. No adjuvant was added for the last booster immunization. After 31 days, spleen cells of the immunized mice were fused with myeloma cells SP2 / 0.

[0025] Preparation of SP2 / 0 cells: Myeloma SP2 / 0 cells were cultured in incomplete medium. On the day of fusion, SP2 / 0 cells with good growth were taken and washed three times with PBS buffer. Cells just in the logarithmic growth phase were collected into a centrifuge tube and centrifuged at 900 rpm / min for 10 minutes. The nutrient solution was discarded, and the cells were washed once with basic culture medium and resuspended. Trypan blue staining was performed, and the cells were counted. The cells were diluted to 106 and placed in a centrifuge tube for later use.

[0026] Preparation of spleen cells: The mice were killed by cervical dislocation, disinfected with 75% alcohol, moved to a clean bench, fixed on a dissection board, and the skin and peritoneum were cut open with sterile scissors and forceps, and the spleen was removed aseptically, placed in a sterile plate containing basal culture medium for cleaning, and the connective tissue on the capsule was peeled off. The spleen was transferred to another plate with a copper mesh containing 10ml of basal culture medium, and then gently ground with a grinder. The spleen cell suspension in the plate was transferred to a 50mL centrifuge tube and centrifuged at 900r / min for 10min. The cell pellet was washed once with basal culture medium by centrifugation in the same way, and then the cells were resuspended to 10~20mL, mixed, and the live cells were counted after trypan blue staining for later use.

[0027] Cell fusion: Fusion was performed according to the conventional method. Spleen cells of immune mice and myeloma cells SP2 / 0 were mixed in a cell number ratio of 5:1 to 10:1, added to a 50mL disposable sterile centrifuge tube, centrifuged at 900rpm / rnin for 10min, supernatant was discarded, and the bottom of the tube was tapped with fingers to fully mix the two cells until they became a paste; the centrifuge tube was placed in a beaker of 37℃ water, 1mL of 50% PEG solution preheated at 37℃ was slowly dripped in within 1min, and then the centrifuge tube was gently shaken in a water bath for 1min, and 1mL of basal culture solution preheated at 37℃ was slowly dripped in within 1min, while the centrifuge tube was rotated while dripping, and then 4mL of basal culture solution preheated at 37℃ was slowly dripped in within 1min, and additional basal culture solution was added to 30mL in the following 4rnin, and the centrifuge tube was gently stirred to completely dilute the PEG and lose its fusion effect. Centrifuge at 900 rpm / min for 10 min, discard the supernatant, lightly suspend the cell pellet in 40 mL of pre-warmed HAT selection culture medium containing 20% ​​fetal bovine serum, mix well, and add to a 96-well cell culture plate with feeder cells using a multichannel pipette, 100 ml per well, and move the culture plate to a 37°C, 5% CO2 incubator for culture.

[0028] Cloning and amplification (limiting dilution method): Blow up and mix the hybridoma cell colonies in the specific wells that have been detected as secretion-positive, count the cells with trypan blue staining, take the above cell suspension, dilute it with 1640 complete culture medium containing 20% ​​fetal bovine serum, and add 100µL of the diluted cell suspension to a 96-well cell culture plate pre-added with feeder cells for culture. Change the medium, screen and clone in time, and expand the remaining cloned cells for cryopreservation each time. After 2-3 cloning operations, when the positive rate of all cloned cell wells is 100%, it can be determined that the hybridoma cell line that secretes monoclonal antibodies has been obtained.

[0029] Preparation of monoclonal antibody ascites: 10 days before inoculation of hybridoma cells secreting monoclonal antibodies, 0.5 ml of liquid paraffin was injected into the abdominal cavity of 8-12 week-old female BALB / C mice. The cultured hybridoma cells were washed from the cell culture bottle, centrifuged at 1000 rpm / min for 5 minutes, and then suspended and centrifuged and washed twice with PBS buffer (pH=7.2). After trypan blue staining for live cell counting, the cell density was adjusted to 106 / mL, and 0.5 mL of cell suspension was injected into the abdominal cavity of each mouse. After inoculation, pay attention to observe the state of the mouse. When the abdomen of the mouse is obviously swollen and inconvenient to move, disinfect the skin of the lower abdomen with alcohol cotton balls, and then pierce the abdominal cavity with a syringe needle. Yellowish ascites can be seen dripping out. Collect it with a sterile centrifuge tube, place it at 4°C overnight, centrifuge it at 3000r / min for 5 minutes, remove the oil precipitate, and the supernatant is ascites. Store it in aliquots at -80°C: take it again after 2-3 days.

[0030] Determination of Hybridoma Cell Culture Supernatant and Ascites Titer The hybridoma cell culture supernatant and ascites were serially diluted, and then the OD values ​​were determined by indirect ELISA. Positive and negative serum controls were also set up. The titers of the hybridoma cell supernatant of A8C2 and B7D4 strains were both 1:1000, and the titers of the ascites were 1:10 5 and 1:10 6 .

[0031] Specificity identification of monoclonal antibodies According to the conventional method, the Mycoplasma hyopneumoniae stored in this laboratory was subjected to SDS-PAGE and then electro-transferred to the NC membrane. After transfer, it was blocked with 5% skim milk overnight, and after thorough washing, a 1:1000-fold dilution of monoclonal antibody was added, and it was allowed to act at 37℃ for 1 hour. After thorough washing, a working concentration of goat anti-mouse secondary antibody IgG was added, and it was allowed to act at room temperature for 1 hour. After thorough washing, TMB was used for color development. Color development occurred at 46KD. This shows that the monoclonal antibody has good specificity.

[0032] Determination of Antibody Secretion Stability of Hybridoma Cell Lines After the antibody secretion ability of hybridoma cell lines was tested by ELISA method at intervals of 2 to 4 months, both cell lines were able to secrete stably.

[0033] Preparation of Antigen Colloidal Gold Test Strips Preparation of colloidal gold solution using trisodium citrate reduction method: The colloidal gold particles in the prepared colloidal gold solution have a particle size of 35-45 nm. The specific method is as follows: Add 1000 ml of 0.01% chloroauric acid solution to a spherical bottle, use a magnetic stirrer, stir and heat at 250 rpm / min until boiling, and keep boiling for 5-10 minutes; quickly add 1.0-1.8 ml of 25% trisodium citrate; continue boiling for 10 minutes without color change, stop heating, continue stirring and cool; use double distilled water to make up to 1000 ml; use a spectrophotometer to measure, the highest absorption peak is at 525±5nm, and the OD value is 1.0±0.1.

[0034] Exploration of the Optimal Conditions for Gold Labeling of Monoclonal Antibody 1 to Mycoplasma hyopneumoniae P46 Protein Take 12 clean 1.5ml centrifuge tubes and place them in two groups on a centrifuge tube rack. Add the prepared colloidal gold solution to the centrifuge tube at a content of 1ml / tube, slowly add 0.1M potassium carbonate solution (K2CO3), stir quickly while adding, adjust the pH to 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, respectively, and set up two groups for each pH value; use PBS to dilute the monoclonal antibody of P46 protein to 1mg / ml, 2mg / ml, 3mg / ml, 4mg / ml, 5mg / ml, 6mg / ml, respectively, and add them to the colloidal gold solution, with two groups of the same concentration for each concentration, shake quickly for 5min, and let the solution stand at 4℃ for 1h after mixing; finally, add 10% sodium chloride solution, 100µL / well, mix thoroughly, and let stand at 4℃ for 1h, observe the changes in the solution, and use an enzyme reader to read the OD 450 nm value, select OD 450 The pH value and the amount of labeled antibody corresponding to the highest nm value were regarded as the optimal conditions for colloidal gold labeling of antibodies.

[0035] After determining the optimal amount of colloidal gold labeling, centrifuge at 3000rpm for 30min, discard the precipitate, and keep the supernatant; centrifuge at 14000rpm for 1h, discard the supernatant, add the precipitate to PBS containing 0.5% BSA at a ratio of 1:10, mix well, place at 4℃ for 30min, centrifuge at 2000rpm for 20min, and discard the precipitate. Transfer the supernatant to a clean centrifuge tube, and finally centrifuge at 14000rpm for half an hour, keep the precipitate, and finally resuspend the precipitate in PBS containing 50% glycerol at a ratio of 1:10. The gold-labeled monoclonal antibody is packaged and stored at 4℃.

[0036] Conclusion: When the concentration of labeled protein is low, the gold sol colloid is unstable and quickly aggregates, and the color changes from wine red to blue purple. As the protein concentration increases, the gold sol is stable and does not aggregate or change color. The lowest content that keeps the red color unchanged is the optimal labeling content, which is 5 mg / ml. 450 It can be seen from the data that when pH=8.50, the OD value is the highest, and the OD value gradually decreases with the increase or decrease of pH. Therefore, the optimal pH value of gold-labeled P46 protein monoclonal antibody 1 is 8.5, and the optimal protein labeling content is 5mg / ml.

[0037] Exploration of the optimal coating amount of nitrocellulose membrane coated with monoclonal antibody 2 of Mycoplasma hyopneumoniae P46 protein and goat anti-mouse IgG.

[0038] The T line (detection line) was coated with monoclonal antibody 2 of Mycoplasma hyopneumoniae P46 protein, which was diluted to 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 4 mg / mL with buffer 2; the C line (quality control line) was coated with goat anti-mouse IgG, which was diluted to 1 mg / ml. The C and T lines were drawn on the NC membrane at 2 µL / cm using a film drawing instrument and dried at room temperature overnight.

[0039] Colloidal gold labeled Mycoplasma hyopneumoniae P46 protein monoclonal antibody 1 was diluted 1, 2, 3, and 5 times with buffer 1, and 20 µL / cm was evenly sprayed on the gold label pad and dried overnight at room temperature. After drying, the test strip was assembled. The most suitable dilution multiple of the gold labeled P46 protein monoclonal antibody 2 was selected according to the color depth and intensity of the test line, the color development time, and other characteristics.

[0040] After determining the above conditions, goat anti-mouse IgG was diluted to 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, and 2.5 mg / ml with buffer 2, and the color depth, stability, and color development speed of the detection line were determined according to the color development results. The condition in which the color intensity of the T line and the C line remained consistent was selected to determine the optimal dilution of IgG.

[0041] The results showed that as the dilution multiple of the gold-labeled P46 protein monoclonal antibody 1 increased, the color development of the C line became weaker and weaker, and as the content of the P46 protein monoclonal antibody 2 increased, the color development intensity of the T line became stronger and stronger, until the gold-labeled P46 protein monoclonal antibody 1 was diluted to 3 times and the P46 protein monoclonal antibody 2 was 2 mg / mL, the color development of the C and T lines was determined to be optimal. The results are shown in Table 1 below.

[0042] Table 1 Color depth of P46 protein monoclonal antibody 2 and gold-labeled P46 monoclonal antibody 1 at different dilution multiples

[0043] Note: "- " indicates negative, "±" indicates gray area, "+" indicates weak positive, "++" indicates moderate positive, and "+++" indicates strong positive.

[0044] When the concentration of goat anti-mouse IgG is higher than 2.0 mg / ml, the color of the quality control line is obviously darker than the detection line. Therefore, the coating concentration of goat anti-mouse IgG is lower than 2.0 mg / ml. The results are shown in Table 2. When the goat anti-mouse IgG is diluted to 1.0 mg / ml, the color of the quality control line is equivalent to that of the detection line.

[0045] Table 2 Color development results of goat anti-mouse IgG at different concentrations

[0046] In this embodiment, the buffer system solution involved in the preparation of the test strip of the present invention is prepared according to the following formula: Buffer solution 1: Tris-HCl molar concentration is 10-100mM, trehalose mass final concentration is 1%-5%, BSA mass final concentration is 1%-3%, PEG20000 mass final concentration is 0.05%-0.5%, Tween-20 mass final concentration is 0.1%-0.5%. Buffer solution 2: Tris-HCl molar concentration is 10-100mM, methanol mass final concentration is 1%-3%. Buffer solution 3: PBS molar concentration is 10-100mM, trehalose mass final concentration is 1%-5%.

[0047] Test strip assembly, testing and result determination The test strip consists of five parts, which are PVC plate, nitrocellulose membrane, sample pad, gold-labeled pad, sample pad and absorbent pad from left to right. The film drawing instrument draws T and C lines on the NC membrane with Mycoplasma hyopneumoniae P46 protein monoclonal antibody 2 and goat anti-mouse IgG, respectively. The gold-labeled Mycoplasma hyopneumoniae P46 protein monoclonal antibody 1 is evenly sprayed on the conjugate pad, and then tightly attached to the PVC plate after drying at 37°C overnight. The sample pad and absorbent pad are attached to both ends of the test strip, respectively, and the strip is cut into 60mmx3.0mm test strips, fixed in a plastic shell, sealed and light-proofed at room temperature for future use. The Mycoplasma pneumoniae pathogen in the tissue fluid is diluted and dripped on the test strip. It is observed for about 10 minutes whether there are red strips and how many red strips are produced.

[0048] The result is judged as follows: if both the quality control band and the test band have clearly visible red bands, it is positive; if only the quality control band has clearly visible red bands, it is negative; if a clearly visible red band appears on the quality control band and a light-colored band appears on the test band, it is questionable; if no red band appears on the quality control band, it is invalid. See principle Figure 1 .

[0049] Sensitivity test Three different buffer solutions ①, ② and ③ were prepared respectively. ① consisted of Tris-HCl (100mM), 5% trehalose, 1% BSA, 0.1% PEG20000, and 0.5% surfactant Tween-20; ② consisted of Tris-HCl (100mM), 5% sucrose, 1% BSA, 0.1% PEG20000, and 0.5% surfactant Tween-20; ③ consisted of Tris-HCl (100mM), 5% sucrose, 1% BSA, and 0.5% surfactant Tween-20. Three different P46 protein detection antibody conjugate pads were prepared; three different test strips were tested using different concentrations of quality control products, and the results are shown in Table 3.

[0050] Table 3 Effects of different buffer solutions ①, ② and ③ on detection sensitivity

[0051] Note: "-" indicates negative, "±" indicates gray area, "+" indicates weak positive, "++" indicates moderate positive, and "+++" indicates strong positive.

[0052] The present invention screens and compares buffer solutions ①, ② and ③, wherein buffer solution ① can effectively protect protein activity, the polymer skeleton can promote the rapid release of gold-labeled protein antibodies, and the optimal sensitivity can reach 20ug / ml.

[0053] Specificity test The Mycoplasma hyopneumoniae antigen, pseudorabies virus, porcine blue ear virus, classical swine fever virus, Mycoplasma suis, Haemophilus parasuis and two sample dilutions were tested and determined.

[0054] Table 4 Test strip specificity test results

[0055] Note: "-" indicates negative, "±" indicates gray area, "+" indicates weak positive, "++" indicates moderate positive, and "+++" indicates strong positive.

[0056] Test strips prepared with monoclonal antibodies prepared from different membrane immune proteins of Mycoplasma hyopneumoniae are used for specific detection of Mycoplasma hyopneumoniae.

[0057] The colloidal gold test strips prepared by the screened monoclonal antibodies 3 and 4 against the P97 protein and the test strips of the present invention are used to detect and determine antigens of Mycoplasma hyopneumoniae of different strains.

[0058] Table 5 Detection results of different strains on test strips

[0059] Note: "-" indicates negative, "±" indicates gray area, "+" indicates weak positive, "++" indicates moderate positive, and "+++" indicates strong positive.

[0060] The present invention uses different labeling parameters and buffer solution formulas to detect the sensitivity of the test strip, so that the activity of the protein itself can be better exerted, the binding efficiency is improved, and the detection limit is improved. The test strip prepared by the process of the present invention detects and determines the Mycoplasma hyopneumoniae antigen, pseudorabies virus, porcine blue ear virus, classical swine fever virus, mycoplasma suis, Haemophilus parasuis and 2 sample dilutions, and no band is detected, and the test strip has good specificity. The test strip prepared by the present invention using anti-P46 protein monoclonal antibody 1 and monoclonal antibody 2 has a strong positive reaction to mycoplasma antigens of different strains, while the test strip prepared by using anti-P97 protein monoclonal antibody 1 and monoclonal antibody 2 has a weaker positive band than P46 protein for mycoplasma antigens of different strains, and may not be detected when the pathogen content is low.

[0061] Test strip repeatability and stability test Three different mycoplasma pathogen samples were randomly selected and diluted 0 times, 5 times, 10 times, and 100 times. The colloidal gold test strips prepared in the same batch (batch 2024001) were used for repeatability test, and each sample was repeated three times to observe the color development.

[0062] Randomly take a different mycoplasma pathogen sample, dilute 0 times and 5 times respectively, and use three different batches (2024001 batch, 2024002 batch, 2024003 batch) of colloidal gold test strips to do repeatability test, observe the color development, and the results are shown in Table 5, Table 6-1, and Table 6-2. The test results of the same sample on different batches of test strips are the same, and different samples have good repeatability in the same batch of test strips, indicating that the test strips have good repeatability between batches and within batches.

[0063] Table 6-1 Intra-batch repeatability verification results of 2024001 batch of test strips

[0064] Table 6-2 Results of batch repeatability verification of test strips

[0065] Note: "-" indicates negative, "±" indicates gray area, "+" indicates weak positive, "++" indicates moderate positive, and "+++" indicates strong positive 36 colloidal gold test strips were extracted from the three batches of test strips prepared, 2024001, 2024002 and 2024003. The first group was test strips stored at room temperature for 3 months; the second group was test strips stored at room temperature for 6 months, and the third group was test strips stored at room temperature for 12 months. Three parallel verifications were performed, and a mycoplasma pathogen sample was randomly taken, diluted 0 times, 5 times, 10 times, and 100 times, respectively, and dripped on different batches of test strips stored at room temperature for 3 months, 6 months, 12 months, and 18 months to conduct stability tests and observe the color development. The results are shown in Table 7. When the test strips were stored for 3 months, 6 months, and 12 months, the color development of the sample detection was not much different. When the test strips were stored for 18 months, the sample detection band became lighter. Therefore, the test strips can be stored at room temperature for at least 12 months.

[0066] Table 7 Stability results of test strips

[0067] Note: "-" indicates negative, "±" indicates gray area, "+" indicates weak positive, "++" indicates moderate positive, and "+++" indicates strong positive.

[0068] Application of colloidal gold test strips for Mycoplasma hyopneumoniae 50 positive nasal swabs and lung tissues of sick pigs and 10 negative samples were collected, and nucleic acid was extracted and amplified respectively. The amplified products were detected by agarose gel electrophoresis. Swabs and tissue fluid were detected by test strips. The test results of the two methods were compared in parallel. The results are shown in Table 7. The two methods have a good coincidence rate, totaling 60 samples. The clinical samples had a 100% coincidence rate with the agarose gel electrophoresis detection method.

[0069] Table 8 Comparison results of the two detection methods

[0070] The colloidal gold test paper for detecting Mycoplasma hyopneumoniae pathogen prepared by the method of the present invention has good specificity, sensitivity and repeatability, can quickly detect bands, and provides convenience for clinical diagnosis.

[0071] The above-described embodiments only express a certain implementation mode of the present invention, and the description thereof is relatively specific and detailed, but it is not intended to limit the patent scope of the present invention; for ordinary technicians in this field, several improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention; therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A colloidal gold test strip for Mycoplasma hyopneumoniae antigen, characterized in that: It comprises a PVC bottom plate, on which a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water-absorbing pad are arranged in sequence, and a detection line and a quality control line are arranged on the nitrocellulose membrane; Anti-Mycoplasma hyopneumoniae P46 protein monoclonal antibody 1 was used as the detection antibody, and P46 protein monoclonal antibody 2 was used as the capture antibody; The preparation method of monoclonal antibody 1 and monoclonal antibody 2 of swine mycoplasma P46 protein comprises the following steps: using the P46 protein of swine mycoplasma pneumoniae SY strain as an immune antigen, obtaining two hybridoma cell strains capable of stably secreting specific antibodies against P46, which are named A8C2 and B7D4 respectively; The hybridoma cell line secreting the monoclonal antibody 1 of Mycoplasma suis P46 protein is the hybridoma cell line A8C2; the hybridoma cell line secreting the monoclonal antibody 2 of Mycoplasma suis P46 protein is the hybridoma cell line B7D4; The hybridoma cell line A8C2 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 9, 2025, with the deposit number CGMCC NO.46318, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; The hybridoma cell line B7D4 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 9, 2025, with the deposit number CGMCC NO.46319, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The method for preparing the colloidal gold test strip for Mycoplasma hyopneumoniae antigen according to claim 1, characterized in that: The specific steps include: The colloidal gold solution was prepared by the trisodium citrate reduction method; The monoclonal antibody 1 of gold-labeled Mycoplasma hyopneumoniae P46 protein is diluted with buffer solution 1, and evenly sprayed on the gold-labeled conjugate pad to form a gold-labeled detection protein conjugate pad, i.e., a colloidal gold pad; The monoclonal antibody 2 of the Mycoplasma hyopneumoniae P46 protein and the goat anti-mouse IgG were diluted with the buffer solution 2 respectively, and then the capture antibody of the P46 protein and the goat anti-mouse IgG were coated on the nitrocellulose membrane to form the detection line and the quality control line respectively; Soak the sample pad in buffer solution 3, dry overnight at room temperature and humidity <20%, and cut for use; adhere the sample pad, gold label conjugate pad, nitrocellulose membrane, and absorbent pad to the support base plate in order to assemble the test paper, and cut and package; The buffer solution 1: Tris-HCl molar concentration is 10-100 mM, trehalose final mass concentration is 1%-5%, BSA final mass concentration is 1%-3%, PEG20000 final mass concentration is 0.05%-0.5%, Tween-20 final mass concentration is 0.1%-0.5%; The buffer solution 2: Tris-HCl molar concentration is 10-100 mM, and methanol content is 1%-3%; The buffer solution 3: the molar concentration of PBS is 10-100 mM, and the final mass concentration of trehalose is 1%-5%.

3. Use of the colloidal gold test strip for detecting Mycoplasma hyopneumoniae as claimed in claim 1 in detecting Mycoplasma hyopneumoniae antigens in tissues, blood and cotton swab extracts.

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