CN104316703B - A kind of Mycoplasma bovis test strip and its preparation method - Google Patents

Abstract

The invention discloses a test paper strip for detecting mycoplasma bovis antibody and a preparation method thereof. The test strip of the present invention is composed of a bottom plate, a sample absorption pad arranged linearly on the bottom plate, a colloidal gold pad coated with an antigen, a nitrocellulose membrane and a water absorption pad, and a quality control line is coated on the nitrocellulose membrane. And the detection line, wherein the detection line is coated with goat anti-bovine secondary antibody IgG, and the quality control line is polyantibody IgG of rabbit-derived P48 fusion protein. The test strip of the present invention can detect Mycoplasma bovis antibodies, including serum antibodies and whey antibodies, and has the advantages of low cost, simple operation, convenience and speed, no need for special equipment and instruments, high detection sensitivity, strong specificity, and wide application range, etc. advantage.

Description

A kind of mycoplasma bovis detection test strip and preparation method thereof

technical field

The invention relates to a method for preparing a biological detection reagent and the reagent. Specifically, the invention relates to a method for preparing a test strip for detecting mycoplasma bovis antibody and the prepared test strip.

Background technique

Mycoplasma bovis is a serious pathogenic pathogen that can cause various diseases such as bovine respiratory disease, dairy cow mastitis, arthritis, keratoconjunctivitis, reproductive tract inflammation, abortion and infertility, and can cause other pathogenic diseases once infected Secondary infections such as Cryptobacterium pyogenes, Pasteurella multocida, Mannella hemolytica, Haemophilus somniferus, bovine parainfluenza virus type 3, herpesvirus type 1, bovine respiratory syncytial virus, bovine viral diarrhea virus and bovine coronavirus, etc. The incidence rate of Mycoplasma bovis is 50%-100%, and the fatality rate is as high as 10%-50%, which has caused huge economic losses to my country's cattle industry. The actual prevalence of this disease in my country and accurate epidemiological information are still relatively scarce. The main reason for this phenomenon is that my country currently lacks relevant technologies for effective detection of Mycoplasma bovis.

The existing technologies for detecting Mycoplasma bovis are: 1. Pathogen isolation and identification, but the isolation of Mycoplasma bovis is often affected by the clinical application of antibiotics, and the separation is difficult, and the separation medium has high nutritional requirements, the isolation is difficult to identify, and the sensitivity is low. As a method for early and rapid diagnosis of Mycoplasma bovis infection, it is also difficult to popularize as a routine detection method (from CaswellJL, ArchambaultM. Mycoplasmabovispneumoniaincattle); 2. PCR detection method (from ThomasA, DizierI, LindenA, etal. Complicated treatment of disease samples (grinding, centrifugation, DNA extraction, etc.), although the detection method is sensitive, it is prone to false positives due to contamination and improper handling, which will affect the accuracy of the judgment results. In addition, certain experiments are required Room conditions and equipment, as well as high cost, are not suitable for clinical or grassroots use. 3. Indirect ELISA is currently the most important serological detection method. In the world, whole bacterial protein has been used to coat and express protein as a method of coating antigen (from GrandDL, CalavasD, BrankM, et al.SerologicalprevalenceofMycoplasmabovisinfectioninsucklingbeefcattleinFrance), whole bacterial protein The lack of specificity of the method has been rarely used. The ELISA method of expressing protein antigen has been commercialized, but which antigen protein it uses has not been announced. In addition, the ELISA method is only suitable for testing in the laboratory, and the operation steps are relatively complicated, requiring professionals to operate, and certain laboratory conditions and equipment are required for testing and analyzing the results. 4. The indirect hemagglutination detection method established by using P48 recombinant expression protein coated sheep red blood cells is relatively simple to operate, but it also takes 2 to 3 hours to determine the result. The sensitivity is weak and a specific constant temperature incubator is required.

Contents of the invention

The invention provides a method for preparing test strips capable of overcoming the deficiencies of the prior art and capable of simple, fast and sensitive detection of Mycoplasma bovis serum antibodies and whey antibodies, and the test strips prepared by the method.

A test strip for detecting Mycoplasma bovis antibody of the present invention is composed of a bottom plate, a sample absorption pad arranged linearly in sequence on the bottom plate, a colloidal gold pad coated with an antigen, a nitrocellulose membrane and a water absorption pad. It is coated with a quality control line and a detection line. The antigen coated by the colloidal gold pad in the test strip of the present invention is Mycoplasma bovis P48 fusion protein, the detection line is coated with goat anti-bovine secondary antibody IgG, and the quality control line is rabbit Polyantibody IgG derived from P48 fusion protein.

The preparation method of the test paper strip for detecting the Mycoplasma bovis antibody of the present invention is: the colloidal gold pad is prepared by coating the colloidal gold with the P48 recombinant protein dialyzed after purification; the quality control line and the detection line are coated on the nitrocellulose membrane, wherein : The antigen used in the detection line is affinity purified goat anti-bovine secondary antibody IgG, and the antigen of the quality control line is P48 protein polyantibody IgG. The sample absorption pad, colloidal gold pad, coated with detection line and quality control line are respectively placed on the bottom plate. Lines of nitrocellulose membranes and absorbent pads are assembled in sequence.

In the preparation method of the test strip for detecting Mycoplasma bovis antibody of the present invention: the concentration of colloidal gold-coated P48 protein is 6ug/ml, the time for antigen-coating 1% gold sol is 30min, and 20%BSA is added in the process of antigen binding Solution (20gBSA added to 100ml deionized water) was blocked at 50ul/ml, and the blocking time was 30 minutes. After centrifugation, the colloidal gold particles were resuspended with the resuspension liquid to prepare a colloidal gold pad, and then dried. The resuspension liquid formula was: 0.02 M Sodium tetraborate, 0.5% BSA, 3% sucrose, 0.5% casein, 0.5% Tween 20, the resuspension will help the gold standard pad dissociate better, and the BSA in the resuspension can block non-specific site to avoid the appearance of false positives, and the casein and sucrose components can protect the antigens coated with gold particles. The concentration of affinity purified goat anti-bovine secondary antibody IgG antigen used in the detection line coated with nitrocellulose membrane is 100ug/ml, the coating volume is 1ul/cm, and the concentration of P48 protein polyanti-IgG antigen in the quality control line coated with nitrocellulose membrane It is 200ug/ml, and the coating volume is 1ul/cm.

The preparation method of the P48 protein polyanti-IgG antigen used in the present invention is: use Freund's complete adjuvant 3mL to add 1mg/mlP48 protein antigen 3mL to prepare Freund's complete adjuvant antigen, and fully mix and emulsify; Use Freund's incomplete adjuvant Add 3mL of 1mg/ml P48 protein to prepare Freund's incomplete adjuvant antigen, and fully mix and emulsify; select healthy male white rabbits to inoculate Freund's complete adjuvant subcutaneously at two hind legs, popliteal lymph nodes and back subcutaneously The total amount of antigen inoculated was 1.5mL; after 14 days, 1.5ml of antigen in Freund's complete adjuvant was still used to subcutaneously inoculate at multiple points on the back for the second immunization; after 14 days, the antigen in Freund's incomplete adjuvant Two spots on the paws of rabbits, subcutaneous multi-point vaccination on the back, the total amount of inoculation is 3mL, and it is the third immunization; after 14 days, the blood is collected from the ear vein to separate the serum, and the blood is tested by IHA test. The IHA titer of the immune rabbit serum reaches 1: 512, formal blood collection, 3000rpm centrifugation for 5 minutes to separate the serum to obtain P48 protein hyperimmune serum, the separated serum was purified by saturated ammonium sulfate precipitation to obtain P48 protein polyanti IgG.

The present invention has following advantage:

(1) The antigen P48 protein coated with colloidal gold in the present invention is a membrane protein of Mycoplasma bovis, which has strong specificity and can truly detect Mycoplasma bovis antibodies, including serum antibodies and whey antibodies;

(2) The P48 protein in the present invention is a prokaryotic soluble protein with high purity, high activity, high sensitivity and specificity of the detection antibody, and a wide range of applications;

(3) Low cost, simple operation, convenient and fast, no special equipment and instruments are required, and the whole process can be completed within 15 minutes, which saves cost compared with indirect hemagglutination test and ELISA test. Operators, it can be conveniently used in the epidemiological investigation of Mycoplasma bovis and promoted at the grassroots level;

(4) The marker is stable, and the labeled sample is stored at 4°C for more than two years without signal attenuation;

(5) Colloidal gold itself is red and does not need to add chromogenic reagents, which saves the steps of enzyme-labeled carcinogenic substrates and stop solutions in the ELISA detection method, and is non-toxic to the human body.

Description of drawings

Fig. 1 is the schematic diagram of test strip of the present invention, among the figure: 1 sample absorbent pad, 2 colloidal gold pads, 3 testing line T, 4 quality control line C, 5 water-absorbing pads, 6 bottom plate, 7 nitrocellulose membranes.

Figure 2 is the SDS-PAGE electrophoresis figure of the purified P48 recombinant protein, wherein: M. protein molecular mass standard; 1 lane is the protein before purification; 2 lanes are the protein of the first column volume eluted after purification; 3 lanes are purified The protein in the second column volume eluted after purification; Lane 4 is the protein in the third column volume eluted after purification; Lane 5 is the protein in the fourth column volume eluted after purification; Lane 6 is the protein after purification Protein eluted in the fifth column volume; 7. Lane is the protein eluted in the sixth column volume after purification.

Fig. 3 is test strip of the present invention to standard positive serum and negative serum detection result figure, wherein: 1 is the blank control of sample diluent; 2 is negative serum; 3 is the positive standard serum of Mycoplasma bovis; (Note: due to serum stickiness Thick, the negative serum and positive serum were diluted 10 times for detection).

Fig. 4 is the detection result figure of test strip of the present invention for the positive standard serum of different gradient dilutions, and 1 is the blank control of sample diluent; 2 is negative serum; 3 is the 1:10 dilution of Mycoplasma bovis positive standard serum; 4. 1:100 dilution of Mycoplasma bovis positive standard serum; 5 is 1:1000 dilution of Mycoplasma bovis positive standard serum; 6 is 1:10000 dilution of Mycoplasma bovis positive standard serum; 7 is 1:100000 dilution of Mycoplasma bovis positive standard serum; 8 is Mycoplasma bovis positive standard serum The positive standard serum was diluted 1:200,000.

Fig. 5 is that test strip specificity test of the present invention detects, and 1. is Mycoplasma bovis positive standard serum; 2. Mycoplasma mycoplasma goat subspecies positive standard serum; 3. Mycoplasma hyopneumoniae positive standard serum; 4. Pasteurella bovis positive Standard serum; 5. Positive standard serum for bovine pneumonia; 6. Positive standard serum for Mycoplasma genitalium; 7. Positive standard serum for Mycoplasma ovis; 8. Positive standard serum for Mycoplasma goat pneumonia; dilution).

detailed description

The present invention is illustrated below in conjunction with examples.

1. Expression and purification of P48 protein

1.1 Sequence synthesis and recombinant plasmid construction

Mycoplasma bovis p48 gene (Genebank: NC_014760.1), the present invention optimizes the codons of this sequence, and optimizes the codons according to the preference of codons in Escherichia coli. TGA (UGA) was optimized to TGG (UGG), because this codon is a stop codon in Escherichia coli, and restriction sites BamHI and XhoI were added at both ends of it, and its gene sequence is SEQID№1;

The optimized p48 gene sequence was synthesized into the pet32a expression vector plasmid by Invitrogen Company. After the synthesis, it was identified by sequencing and enzyme digestion, and the positive recombinant plasmid after successful identification was named Pet32-a(+)-p48.

1.2 Expression of P48 protein

Transform the recombinant plasmid pet32a(+)-p48 with the target gene into competent cells BL21(DE3), pick two single colonies that have been transformed into BL21(DE3) and inoculate them in 5ml LB liquid medium containing ampicillin place in a shaker at 37°C, shake at 220rpm until the OD value reaches about 0.6, then inoculate 10ml of the bacterial liquid into a new 1L LB liquid medium containing 100ug/ml ampicillin, at 16°C, 0.01mmol The expression was induced for 20 h at the concentration of /lIPTG, the cells were collected, resuspended with 100 ml of PH7.2 PBS, and subjected to sonication on ice for 30 min. Then the ultrasonically treated liquid was placed in a centrifuge and centrifuged at 11,000 rpm for 30 min, the precipitate was discarded, and 100 ml of the supernatant was collected.

1.3 Purification of P48 protein by affinity chromatography and dialysis of purified protein

The P48 recombinant protein was purified by nickel ion affinity chromatography. First, put 5mL of Ni-NTAHisBindResin (purchased from Nanjing GenScript Biotechnology Co., Ltd.) into an empty chromatography column, let the liquid drip out naturally by gravity, wash the chromatography column with 20 times the column volume of equilibrium buffer, and then put the sample Add the supernatant to the chromatographic column, combine it on ice for 60min and then let it flow out; the chromatographic column washes the impurity protein with 20 times the column volume of NTA-10; and then washes it with 6 column volumes of NTA-60 elution buffer respectively Take off, collect once every 5ml, obtain the purified protein, the electrophoresis diagram of its detection is shown in accompanying drawing 2.

Combine the first volume of purified protein, the second volume of purified protein, the third and fourth volumes of purified protein, and the fifth and sixth volumes of purified protein, put them into dialysis bags respectively, and put them into a 5L large volume of dialysate. In the beaker, the dialysate was 0.1MPB buffer, put it in a 4°C refrigerator for overnight dialyzing, took it out the next day, put the dialyzed P48 protein into an EP tube and put it in a -40°C refrigerator for storage (1mL/tube).

2. Preparation process of colloidal gold test strips

2.1 Preparation process of colloidal gold

Take 1% chloroauric acid aqueous solution 1ml and add it to 100ml deionized water, heat it to boiling at the mouth of a temperature-controlled stirrer, then quickly add 1.5ml 1% trisodium citrate at one time, continue heating and boiling for 15min, and then make up to 100ml with water. Obtain a uniform and transparent colloidal gold solution.

2.2 Preparation of colloidal gold standard pad

The purified and dialyzed P48 recombinant protein obtained by the above method is coated with colloidal gold and colloidal gold pad is prepared. The pH value of antigen-binding colloidal gold is 7.4, and the optimal concentration of colloidal gold-coated P48 protein is 6ug/ml. Antigen-coated The time for the gold particles is 30 minutes. After antigen binding, add 20% BSA50ul/ml for blocking. The blocking time is 30 minutes. After centrifugation, use the resuspension solution to resuspend the colloidal gold particles to prepare the colloidal gold standard pad, and dry at 37°C for 3 hours. . The resuspension formula is: 0.02M sodium tetraborate, 0.5% BSA, 3% sucrose, 0.5% casein, 0.5% Tween 20.

2.3 Coating of quality control line and detection line on nitrocellulose membrane

The nitrocellulose membrane model of the test strip of the present invention is M135, purchased from MILLIPORE (U.S.), the antigen used for the detection line coated with the nitrocellulose membrane is 100ug/ml affinity purified goat anti-bovine secondary antibody IgG, and the detection line 1ul coated /cm, the affinity purified goat anti-bovine secondary antibody was purchased from ImmunoReagents reagent company.

Coated nitrocellulose membrane quality control line antigen is P48 protein polyantibody IgG, using its concentration of 200ug/ml, coated quality control line 1ul/cm. The IgG is obtained through purification with hyperimmune serum. The preparation method of hyperimmune serum is as follows:

The preparation of Freund's complete adjuvant antigen and Freund's incomplete adjuvant antigen used in the preparation of hyperimmune serum, wherein: Freund's complete adjuvant antigen: Freund's complete adjuvant 3mL plus 1mg/ml P48 protein antigen 3mL; Freund's incomplete adjuvant Adjuvant antigen: Freund's incomplete adjuvant 3mL plus 1mg/ml P48 protein 3mL.

The above-mentioned adjuvant antigens are all prepared before immunization, and fully mixed and emulsified.

P48 protein hyperimmune serum preparation process:

a. First, select healthy male New Zealand white rabbits about 2kg and 12 months old.

b. Take the above-mentioned fully emulsified complete Freund's adjuvant antigen, and inoculate it subcutaneously on the two hind legs of each rabbit, at the popliteal lymph node and subcutaneously on the back. The total amount of inoculation is 1.5 mL; Antigen 1.5ml was subcutaneously inoculated at multiple points on the back for the second immunization; after 14 days, the above-mentioned Freund's incomplete adjuvant antigen was inoculated subcutaneously at two points on the soles of each rabbit, and the total amount of inoculation was 3mL , for the third immunization;

c. After 14 days, the blood was collected from the ear vein to separate the serum, and the blood was tested by IHA test. The IHA titer of the immune rabbit serum reached 1:512. The blood was collected formally, and the serum was separated by centrifugation at 3000rpm for 5 minutes, which was the P48 protein hyperimmune serum.

d. The separated serum was purified by saturated ammonium sulfate precipitation method to obtain polyantibody IgG to P48 protein.

2.4 Assembly of test strips

The operation is carried out as follows: assemble the nitrocellulose membrane, colloidal gold pad, sample pad, and absorbent pad including the test line and quality control line from top to bottom on the PVC backing board in order, and set the PVC backing board on the At the bottom, there is a nitrocellulose membrane in the middle of the upper part of the liner, an absorbent pad is pasted on the upper left end of the nitrocellulose membrane, a gold standard pad is placed on the upper right end of the nitrocellulose membrane, and a sample pad is placed on the upper right end of the gold standard pad, respectively. Use a spray gun to produce a detection line and a quality control line on the nitrocellulose membrane with the affinity purified goat anti-bovine secondary antibody IgG obtained above and the purified P48 polyantibody IgG. In the test strip of the present invention, the detection line and the quality control line The distance between the two lines is kept at 0.8mm. Then the test paper is cut into strips with a width of 4 mm, and the colloidal gold test paper strip schematic diagram of the detection Mycoplasma bovis antibody of the present invention is made such as the structure of accompanying drawing 1.

The principle of the test strip of the present invention is the double-antigen sandwich method for detecting antibodies, wherein the judgment standard is that under the premise of color development of the quality control line, the color development of the detection line is positive; the detection line has no color development and is negative. If the quality control line does not develop color, it is invalid and needs to be re-measured.

Colloidal gold test strip of the present invention detects positive serum and negative serum respectively, and the results are shown in accompanying drawing 3, by accompanying drawing 3 as can be seen, positive serum shows that two lines of detection line and quality control line are red; The test line does not develop color; the blank control is only the sample diluent, and the result shows that the quality control line develops color, but the test line does not develop color. The sample diluent is 0.1 MPBS buffer containing 0.5% Tween 20.

The colloidal gold test strip of the present invention detects different dilutions of positive serum, and the results are shown in accompanying drawing 4, as can be seen from accompanying drawing 4, negative serum, sample diluent blank contrast homogeneous control line develops color, and detection line does not develop color. Positive serum 1:10, 1:100, 1:1000, 1:10000, 1:100000 are both quality control line and detection line, showing positive; while positive serum 1:200000 is only coloring of quality control line, showing Negative, so the sensitivity of the pilot test paper strip of this invention can be achieved, while the previously established Mycoplasma bovis indirect hemagglutination detection method can only detect 1:4096, so this invention greatly improves the detection sensitivity.

The detection results of the test strip specificity test of the present invention are shown in accompanying drawing 5, wherein detected Mycoplasma bovis positive standard serum, Mycoplasma mycoplasma goat subspecies positive standard serum, Mycoplasma hyopneumoniae positive standard serum, Pasteurella bovis positive standard serum, cattle Pneumonia positive standard serum, bovine genital mycoplasma positive standard serum, ovine mycoplasma pneumoniae positive standard serum and capricum mycoplasma goat pneumonia subspecies positive standard serum, the results are only positive for mycoplasma bovis positive serum, other similar mycoplasma and can cause similar diseases in cattle All pathogenic antibodies were negative, which proved that the test strip had good specificity and reliable results.

The imported ELISA kit was compared with the test paper of the present invention. A total of 83 negative samples (including whey and serum) with clear background were detected, and 189 positive samples (including whey and serum) were detected. Among them, the imported ELISA kit Detect 90 parts of negative sera and 182 parts of positive sera, while the test strips of the invention detect 86 parts of negative sera and 186 parts of positive sera. Compared with imported ELISA kits, the negative coincidence rate is 95.6%, and the positive coincidence rate is 97.8%. However, the cost of testing samples with imported ELISA kits is as high as 5 yuan per copy, and the operation takes about 3 hours to get the test results. The cost of the pilot test paper strip of the present invention is 0.5 yuan/part, and clear and clear results can be presented within only 15 minutes, greatly reducing the cost and improving the efficiency.

<110> Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences

<120> A test strip for detection of Mycoplasma bovis antibody and its preparation method

<160>1

<210>1

<211>1419

<212> DNA

<213> Optimized P48 gene sequence

<400>

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Claims (3)

1. a preparation method of a test strip for detecting mycoplasma bovis antibody is characterized in that: the colloidal gold pad is prepared by coating the colloidal gold with the P48 recombinant protein dialyzed after purification; on the nitrocellulose membrane, the quality control line is coated with the Detection line, wherein: the antibody used in the detection line is affinity purified goat anti-bovine secondary antibody IgG, the quality control line antibody is P48 protein polyantibody IgG, and the sample absorption pad, colloidal gold pad, and coated with detection line are sequentially placed on the bottom plate The nitrocellulose membranes and absorbent pads of the line and the quality control line were assembled in sequence. The concentration of colloidal gold-coated P48 recombinant protein was 6 μg/ml, and the coating time was 30 minutes. After antigen binding, 50 μl/ml of 20% BSA solution was added for Blocking, the blocking time is 30 minutes, after centrifugation, use the resuspension liquid to resuspend the colloidal gold to prepare a colloidal gold pad, and then dry it. The resuspension liquid formula is: 0.02M sodium tetraborate, 0.5% BSA, 3% sucrose, 0.5 % casein, 0.5% Tween 20, coated with nitrocellulose membrane, the concentration of the affinity purified goat anti-bovine secondary antibody IgG used for the detection line is 100 μg/ml, the coating volume is 1 μl/cm, coated with nitrocellulose membrane The P48 protein polyantibody IgG concentration used in the quality control line was 200 μg/ml, and the coating volume was 1 μl/cm. 2. the preparation method of a kind of test strip that detects mycoplasma bovis antibody described in claim 1 is characterized in that adding 1mg/mlP48 protein antigen 3ml with Freund's complete adjuvant 3ml prepares Freund's complete adjuvant antigen, and fully mixes Emulsify; prepare Freund's incomplete adjuvant antigen with 3ml of Freund's incomplete adjuvant plus 1mg/ml P48 protein 3ml, and fully mix and emulsify; select healthy male white rabbits to subcutaneously in the feet of the two hind legs, popliteal lymph nodes and back Inoculate Freund's complete adjuvant antigen subcutaneously at multiple points, and the total amount of inoculation is 1.5ml; 14 days later, still use 1.5ml Freund's complete adjuvant antigen to inoculate subcutaneously at multiple points on the back for the second immunization; The incomplete adjuvant antigen was inoculated at two points on the paws of each rabbit and subcutaneously at multiple points on the back. The total amount of inoculation was 3ml, which was the third immunization; after 14 days, the blood was collected from the ear vein to separate the serum, and the blood was tested by IHA. The IHA titer of the immune rabbit serum reached 1:512, blood was formally collected, and the serum was separated by centrifugation at 3000rpm for 5 minutes to obtain P48 protein hyperimmune serum. The isolated serum was purified by saturated ammonium sulfate precipitation to obtain P48 protein polyanti-IgG. 3. the test strip of the detection mycoplasma bovis antibody prepared by the method described in claim 1 or 2.