Fatty fluke recombinant protein and latex microsphere conjugate, immunochromatography test strip and application of immunochromatography test strip

Abstract

The invention belongs to the technical field of immunodetection, and particularly discloses a fluke fasciata recombinant protein and latex microsphere conjugate which comprises a recombinant protein rFhCL1A and latex microspheres which are covalently coupled and combined. The invention also discloses a preparation method of the fluke flakes recombinant protein and latex microsphere conjugate, an immunochromatography test strip coated with the fluke flakes recombinant protein rFhCL1A and latex microsphere conjugate, and an application of the fluke flakes recombinant protein and latex microsphere conjugate in preparation of a kit for detecting fluke flakes. The kit has the advantages of high sensitivity, strong specificity and good stability, has good temperature resistance and storability, is simple, convenient and rapid to operate and low in cost, does not need professional equipment, and is suitable for grassroots epidemic prevention institutions and on-site large-scale screening.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunodetection technology, and particularly relates to a Fasciola recombinant protein and latex microsphere conjugate, an immunochromatographic test strip and application thereof. BACKGROUND

[0002] Fasciolosis is a food-borne and water-borne parasitic zoonosis caused by two species of Fasciola, i.e., Fasciola hepatica and Fasciola gigantica. Clinically, fasciolosis can be divided into four stages, i.e., the hatching stage of larvae, the acute invasion stage, the chronic latent stage and the chronic obstruction stage. Most fasciolosis infections can be found and treated in the second and fourth stages. Generally, infections in summer and autumn have acute courses, while infections in winter and spring have chronic courses. Livestock in the acute infection stage of fasciolosis usually die within 3 to 5 days, with a short course of disease, and show symptoms of elevated body temperature, depression, reduced or lost appetite, anemia and visible mucosal pallor, accompanied by indigestion and occasional diarrhea. Livestock in the chronic infection stage have a course of disease of 1 to 2 months, and mainly show symptoms of body weight loss, moderate anemia, mucosal pallor, dull and lusterless hair and severe hair loss, reduced appetite, poor concentration and slow movement. Edema can be observed on the chest, abdomen, submandibular region and eyelids, and eventually leads to death, which has a serious impact on the development of the livestock breeding industry and causes huge economic losses.

[0003] The pathological symptoms of fasciolosis are mainly caused by the migration of larvae in the host body. At present, the life cycle of fasciola is complex, and has an immune escape mechanism, which poses a great challenge to the development of a fasciolosis vaccine. In addition, the drug resistance of fasciola to anthelmintics is increasingly serious, which further exacerbates the difficulty of controlling the disease. Against this background, the development of a timely and accurate diagnostic method is of great significance for the early detection, early treatment and epidemiological monitoring of fasciolosis.

[0004] The difference in parasitic developmental stages, the heterogeneity of host immune responses and the universality of mixed infections lead to significant complexity in clinical diagnosis, and the traditional detection methods (such as fecal egg examination and serological ELISA detection) are susceptible to sample matrix interference, egg morphology similarity and host immune state fluctuations, and have technical defects such as low sensitivity and insufficient specificity. At the same time, the existing methods rely on professional equipment such as microscopes and enzyme labelers and standardized laboratory environments, and have a complicated operation process (5-8 steps of treatment such as centrifugation and staining) and a detection period of 24-72 hours, which is difficult to meet the needs of on-site rapid screening.

[0005] Lateral flow immunoassay (LFIA) was first introduced in 1990 and is one of the most promising detection techniques in the field of biological analysis. The immunochromatographic test strip is mainly composed of a sample pad, a conjugate pad, a nitrocellulose membrane (NC), a polyvinyl chloride (PVC) base plate, and a water-absorbing pad. Its principle is to drop the liquid sample containing the target analyte onto the sample pad soaked with buffer salt and surfactant, which promotes better interaction between the sample and the detection system. The treated sample migrates through the conjugate release pad, which contains specific antibodies for the target analyte, and binds with the labeled material to form a ternary complex, which migrates to the detection area under the action of the capillary tube. The test line (T line) on the membrane is coated with a substance that can capture the target analyte, and when the ternary complex migrates to the test line, it will be captured and aggregated in large quantities to develop color. The control line (C line) is used to indicate whether the liquid on the test strip is flowing normally. Therefore, when the C line develops color and the T line does not, it indicates a negative test result; when both the C line and the T line develop color, it indicates a positive test result; if the C line does not develop color, it indicates that the test result is invalid.

[0006] Latex microspheres (LMs) are colored polystyrene latex microspheres that are synthesized by adding different colored dyes, including blue latex microspheres and red latex microspheres. As a new type of spherical marker material, they not only have rich colors and can achieve multi-target detection, but also have excellent sensitivity and stability due to their large surface area. The carboxyl functional groups on their surface allow them to bind with antibodies in a covalent coupling manner, making them one of the most popular materials for LFIA.

[0007] Currently, there are studies that use gold nanoparticles (GNPs) as markers and rCatL1D protein as antigens to establish a colloidal gold immunochromatographic test strip detection method (CGIA) for detecting sheep fascioliasis. In this method, gold particles are combined with staphylococcal protein A conjugates on the gold label pad, rCatL1D is coated on the T line, and sheep serum IgG is coated on the C line. After adding serum to the gold label pad, a serum antibody-protein A-GNPs ternary complex is formed, which migrates to the T line under the action of the capillary tube and is captured by rCatL1D and develops color. The remaining protein A-GNPs complex that is not combined with the serum binds with the sheep serum IgG coated on the C line and develops color.

[0008] However, the signal of gold particle labeling is relatively stable but weak, and due to its optical properties, the signal strength may not be sufficient to clearly distinguish the background at low concentrations; due to its weak detection signal, it is often not observed when detecting low concentration targets, resulting in low method sensitivity; the binding mode of gold particles and substances is electrostatic adsorption, which is a non-covalent binding mode, and its stability is poorer than that of chemical bond, and under long-term storage or high temperature and humidity changes, electrostatic adsorption may be affected, resulting in partial shedding of gold particles. In addition, the existing CGIA detection method coats recombinant protein on the T line, which is prone to denaturation and degradation or inactivation, resulting in quality problems of the test strip. SUMMARY

[0009] The purpose of the present application is to solve the above technical problems, and to provide a conjugate for detecting fasciolids with high sensitivity, strong specificity and good stability, and an immunochromatography test strip containing the same.

[0010] To achieve the above invention purposes, the present application provides the following technical solutions:

[0011] In a first aspect, the present application provides a fasciolids recombinant protein and latex microsphere conjugate, which comprises covalently coupled recombinant protein rFhCL1A and latex microspheres.

[0012] Preferably, the particle size of the latex microspheres is 200-400 nm, and most preferably 300 nm.

[0013] Preferably, the amino acid sequence of the recombinant protein rFhCL1A is shown in SEQ ID NO: 2.

[0014] Preferably, the nucleotide sequence encoding the recombinant protein rFhCL1A is shown in SEQ ID NO: 1.

[0015] In a second aspect, the present application provides a preparation method of the fasciolids recombinant protein and latex microsphere conjugate, which comprises the following steps:

[0016] S1, take 12.5 μL of 4% solid content red latex microsphere suspension, mix well with 10 times volume of 0.05M 2-morpholinoethanesulfonic acid buffer, centrifuge at 12000 r / min for 15 min, remove the supernatant, add 10 times volume of 2-morpholinoethanesulfonic acid buffer, and mix well by ultrasonic for 1 min;

[0017] S2, 5 μL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride with a concentration of 10 mg / mL and 10 μL of N-hydroxysuccinimide activated red latex microspheres with a concentration of 10 mg / mL were added, and after oscillation and mixing, the mixture was placed on a constant temperature shaker and activated at 37°C and 180 r / min for 30 min;

[0018] S3, centrifugation was performed at 12000 r / min for 15 min, the supernatant was removed, 10 times the volume of MES buffer was added for recovery and ultrasonic dissolution for 1 min;

[0019] S4, 50 μg of recombinant protein rFhCL1A was added, and after oscillation and mixing, the mixture was placed on a constant temperature shaker and coupled at 37°C and 180 r / min for 100 min;

[0020] S5, centrifugation was performed at 12000 r / min for 15 min, the supernatant was removed, 10 times the volume of blocking solution was added for recovery and ultrasonic mixing for 1 min, and the mixture was placed on a constant temperature shaker and blocked at 37°C and 180 r / min for 60 min;

[0021] S6, centrifugation was performed at 12000 r / min for 15 min, the supernatant was removed, 10 times the volume of microsphere washing solution was added for recovery and ultrasonic mixing;

[0022] S7, the supernatant was removed by centrifugation, 10 times the volume of microsphere washing solution was added for recovery and ultrasonic mixing;

[0023] S8, the supernatant was removed by centrifugation, 0.1 mL of microsphere reconstitution solution was added for recovery and ultrasonic mixing, and the mixture was stored at 4°C for standby.

[0024] Preferably, the blocking solution is prepared as follows: 20 g of BSA (AS12659-100 g, Asbio) and 1 mL of 3% Proclin300 are added to a beaker, dissolved with 50 mL of ddH2O, and then diluted to 100 mL.

[0025] Preferably, the microsphere washing solution is PBS buffer.

[0026] Preferably, the microsphere reconstitution solution is prepared as follows: 20 mL of PBS, 5 mL of 3% Proclin300, 25 mL of Tween-20, 15 mL of 10% PVP, 25 g of sucrose, and 2.5 g of BSA are dissolved and mixed with 250 mL of ddH2O, and then diluted to 500 mL.

[0027] In a third aspect, the present application provides an immunochromatography test strip, which comprises a base plate, and a sample pad, a latex microsphere combination pad, a nitrocellulose membrane provided with a test line and a quality control line, and a water absorption pad are sequentially connected to the base plate; the sample pad and the latex microsphere combination pad are connected to one end of the nitrocellulose membrane close to the test line, and the water absorption pad is connected to the other end of the nitrocellulose membrane close to the quality control line; the latex microsphere conjugate of the recombinant Fasciola protein rFhCL1A is coated on the combination pad, HRP-protein G is coated on the test line, and His-tag monoclonal antibody is coated on the quality control line.

[0028] Preferably, the base plate is a PVC base plate, but is not limited thereto, and can also be a base plate made of other suitable materials.

[0029] Preferably, the coating concentration of the T line is 0.5 mg / mL.

[0030] Preferably, the coating concentration of the C line is 1 mg / mL.

[0031] In a fourth aspect, the present application provides the use of the latex microsphere conjugate of the recombinant Fasciola protein in the preparation of a kit for detecting Fasciola.

[0032] In a fifth aspect, the present application provides a preparation method of the immunochromatography test strip, which comprises the following steps:

[0033] S1, coating the latex microsphere conjugate of the recombinant Fasciola protein rFhCL1A on glass fiber, and drying at 37℃ to obtain a sample pad;

[0034] S2, treating a nitrocellulose membrane, coating His-tag monoclonal antibody on the quality control line, and coating HRP-protein G on the test line, and drying at 37℃;

[0035] S3, sequentially connecting the sample pad, the latex microsphere combination pad, the nitrocellulose membrane, and the water absorption pad on the base plate, covering the latex microsphere combination pad on one end of the nitrocellulose membrane close to the test line, and covering the water absorption pad on the other end of the nitrocellulose membrane close to the quality control line.

[0036] The present application combines red latex microspheres (LMs) with fasciola recombinant cathepsin L1 protein (rFhCL1A) and then spreads on the conjugate pad, coats horseradish peroxidase-protein G (HRP-Protein G) on the T line, and coats His-tag monoclonal antibody on the C line; when the serum added dropwise on the sample pad contains antibodies capable of specifically binding with rFhCL1A, a serum antibody-rFhCL1A-LMs ternary complex is formed, which is captured by HRP-Protein G and colored when migrating to the T line under the action of capillary force, and the remaining rFhCL1A-LMs complex not combined with the serum is colored by combining with the His-tag on the C line.

[0037] The latex microspheres have a larger surface area and can provide a stronger scattered light signal, so that a stronger reaction signal can be provided even at a lower target concentration, and the detection is easier, and the sensitivity of the detection method is improved; meanwhile, the latex microspheres are combined with the antigen and antibody by covalent coupling, which is more firm than electrostatic adsorption, has higher stability, so that the LMs are not easily disturbed in a complex sample, and non-specific binding is avoided, thereby improving the accuracy and specificity of the experimental results.

[0038] Compared with the existing technology, the detection method established by the present application has the advantages of high sensitivity, strong specificity and good stability, the efficiency and specificity of the combination with the target antibody are improved by coating HRP-Protein G on the T line, non-specific binding and background noise are reduced, HRP-Protein G is relatively stable and not easy to deform, has good temperature resistance and storage property, and can combine IgG in various mammalian sera, so that the detection method can be applied to different detection targets; the His-tag monoclonal antibody is coated on the C line, which can effectively capture the latex microspheres combined with the target protein, so that the coloring result is more stable. The present application uses latex microspheres as markers and combines an immunochromatographic test strip to detect fasciola, which has the advantages of simple operation, rapidness, low cost and no need for professional equipment. The present application is based on the principle of latex microsphere immunochromatography, and the result (latex microsphere aggregation coloration) can be judged by naked eye within 15 minutes, and no professional equipment and operation skills are needed, so it is suitable for primary epidemic prevention institutions and on-site large-scale screening. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The double enzyme digestion results of the recombinant plasmid are shown.

[0040] Figure 2 The purification results of the recombinant protein rFhCL1A are shown.

[0041] Figure 3 The structural schematic diagram of the immunochromatographic test strip according to the present application is shown.

[0042] Figure 4 The particle size of latex microspheres was observed naked eye and by a reading instrument.

[0043] Figure 5 The antibody labeling amount screening results are shown.

[0044] Figure 6 The antibody labeling time screening results are shown.

[0045] Figure 7 The T-line and C-line coating concentration determination results are shown. (A) T-line coating concentration naked eye observation and reading instrument results; (B) C-line coating concentration naked eye observation and reading instrument results.

[0046] Figure 8 The sensitivity test results of the immunochromatographic test strip according to the present application are shown.

[0047] Figure 9 The specificity test results of the immunochromatographic test strip according to the present application are shown.

[0048] Figure 10 The stability test results of the immunochromatographic test strip according to the present application are shown. DETAILED DESCRIPTION

[0049] The technical solutions of the present application are further described below in combination with the drawings and specific examples, but the scope of protection is not limited thereto.

[0050] Unless otherwise specified, the instruments or reagents used in the examples are conventional instruments or reagents in the art, which are conventional products available on the market. Unless otherwise specified, the specific experimental operations involved herein are understood or known by those skilled in the art according to their mastery of common knowledge or conventional technical means, and will not be described here.

[0051] 1. Construction and identification of recombinant plasmid

[0052] The FhCL1A (Fasciola hepatica secreted cathepsin L1) gene sequence (NCBI accession number: U62288) was downloaded, and after removing the signal peptide region, Nde I and Bam HI enzyme cutting sites were added at both ends and codon optimization was performed. The gene fragment (SEQ ID NO: 1) was synthesized by a commercial company. Subsequently, the gene fragment was connected with the pET-28a(+) expression vector, and the reaction system is shown in Table 1.

[0053] Table 1. Connection reaction system

[0054]

[0055] Ligation Mix was purchased from Boshijimo Biotechnology (Beijing) Co., Ltd. (Item No.: 6023Q).

[0056] The above plasmid was introduced into BL21 (DE3) competent cells, and the overnight culture broth was used to extract the plasmid and perform double enzyme digestion verification. The specific reaction system is shown in Table 2.

[0057] Table 2. Double enzyme digestion reaction system

[0058]

[0059] 10×SpeedyOne Buffer was purchased from Shengong Bioengineering (Shanghai) Co., Ltd. (Item No.: B600700).

[0060] Figure 1 The double enzyme digestion results of the recombinant plasmid are shown. Among them, lane M represents DL 12 000 DNA Marker, lane 1 represents the double enzyme digestion product of the recombinant plasmid, and lane 2 represents the recombinant plasmid without enzyme digestion. It can be seen from Figure 1 that the recombinant plasmid has been successfully double enzyme digested.

[0061] 2. Induction expression of recombinant protein

[0062] The recombinant expression strain was taken and inoculated into LB liquid medium containing kanamycin (50 mg / mL) at a ratio of 1:100. Then, the inoculated medium was placed in a shaker, the shaker speed was set to 220 r / min, and the temperature was maintained at 37°C for overnight culture to recover the strain. After the strain was recovered, the recovered broth was inoculated and cultured for 2-3 hours according to the above steps. When the OD600 value of the broth reached 0.6 to 0.8, IPTG (isopropyl-β-D-thiogalactoside) inducer was added to the broth to make the final concentration reach 0.8 mM. Then, the induction culture was continued at a speed of 220 r / min and a temperature of 25°C for 6 hours. After centrifugation, the precipitate was subjected to protein extraction and purification by affinity chromatography.

[0063] Figure 2 The purification results of the recombinant protein rFhCL1A are shown. Among them, lane M represents the protein molecular weight standard, lane 1 represents the bacterial lysate, lane 2 represents the flow-through liquid, lanes 3-8 represent the denatured wash liquid, and lanes 9-12 represent the denatured eluent. It can be seen from Figure 2 that the recombinant protein is successfully purified.

[0064] The amino acid sequence of the recombinant protein rFhCL1A is shown in SEQ ID NO: 2.

[0065] 3. Preparation of Fasciola recombinant protein rFhCL1A and latex microsphere conjugate

[0066] (1) Take 12.5 μL of 4% solid content red latex microsphere suspension (Bovine Life Science (Changzhou) Co., Ltd., product number SJ1009, containing trace amount of surfactant), shake well with 10 times volume of 0.05M 2-morpholinoethanesulfonic acid (MES) buffer, centrifuge at 12000 r / min for 15 min, remove supernatant, add 10 times volume of MES buffer to restore and mix well for 1 min;

[0067] (2) Add 5 μL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (10 mg / mL) and 10 μL of N-hydroxysuccinimide (NHS) with a concentration of 10 mg / mL to activate the red latex microspheres, shake well and then place on a constant temperature shaker at 37°C and 180 r / min for 30 min;

[0068] (3) Centrifuge at 12000 r / min for 15 min, remove supernatant, add 10 times volume of MES buffer to restore and mix well for 1 min;

[0069] (4) Add 50 μg of recombinant protein rFhCL1A, shake well and then place on a constant temperature shaker at 37°C and 180 r / min for 100 min;

[0070] (5) Centrifuge at 12000 r / min for 15 min, remove supernatant, add 10 times volume of blocking solution (blocking solution preparation: add 20 g BSA (AS12659-100 g, Asbio) and 1 mL 3% Proclin300 to a beaker, dissolve with 50 mL ddH2O, and then dilute to 100 mL) to restore and mix well for 1 min, and then place on a constant temperature shaker at 37°C and 180 r / min for 60 min;

[0071] (6) Centrifuge at 12000 r / min for 15 min, remove supernatant, add 10 times volume of microsphere washing solution (PBS buffer) to restore and mix well;

[0072] (7) Centrifuge to remove supernatant, add 10 times volume of microsphere washing solution to restore and mix well;

[0073] (8) Centrifuge to remove supernatant, add 0.1 mL of microsphere reconstitution solution (microsphere reconstitution solution preparation: dissolve 20 mL PBS, 5 mL 3% Proclin300, 25 mL Tween-20, 15 mL 10% PVP, 25 g sucrose, and 2.5 g BSA in 250 mL ddH2O, and then dilute to 500 mL), mix well, and then store in a 4°C refrigerator for standby.

[0074] 4. Preparation of sample pad and conjugate pad

[0075] Preparation of sample pad: The glass fiber was soaked in sample pad treatment solution (treatment solution preparation: 20 mL PBS, 25 mL Tween-20, 15 mL 10% PVP, 5 mL 3% Proclin300, 2.5 g BSA were fully dissolved and mixed in 250 mL ddH2O, and then made up to 500 mL) for 30 min, and then dried at 37°C for 12 h for standby, which was the sample pad.

[0076] Preparation of conjugate pad: The above recombinant protein rFhCL1A of fasciola was coupled with latex microspheres, and then spread on untreated glass fiber and dried in a constant temperature incubator at 37°C for standby, which was the latex microsphere conjugate pad.

[0077] 5. Treatment of quality control line and detection line

[0078] His-tag monoclonal antibody was used as the C line and HRP-protein G was used as the T line. After being diluted to the appropriate concentration with membrane coating solution, they were drawn on the nitrocellulose (NC) membrane. The distance between the C line and the T line was 0.5 cm. After drawing, the NC membrane was dried in a constant temperature incubator at 37°C for standby.

[0079] 6. Assembly of immunochromatographic test strip

[0080] The PVC base plate, NC membrane, latex microsphere conjugate pad, and sample pad were assembled in order, and the overlap between each two was 2 mm. The latex microsphere conjugate pad and the sample pad were attached to the end close to the T line, and the water absorption pad was attached to the end close to the C line, ensuring that the water absorption pad and the NC membrane had a 2 mm overlap. Then, the assembled test strip was cut into 3 mm wide with a cutting instrument and placed in a test strip card shell, which was stored in a dry environment away from light.

[0081] Figure 3 The structure of the latex microsphere immunochromatographic test strip is shown. As shown in the figure, the immunochromatographic test strip includes a PVC base plate, a sample pad, a latex microsphere conjugate pad, a nitrocellulose membrane provided with a test line and a quality control line, and a water absorption pad attached in order. The sample pad and the latex microsphere conjugate pad are attached to the end of the nitrocellulose membrane close to the test line, and the water absorption pad is attached to the end of the nitrocellulose membrane close to the quality control line. The conjugate pad is coated with fasciola recombinant protein rFhCL1A coupled with latex microspheres, the test line is coated with HRP-protein G, and the quality control line is coated with His-tag monoclonal antibody.

[0082] 7. Optimization of each condition of the detection method

[0083] (1) Screening of LMs with different particle sizes

[0084] The recombinant protein rFhCL1A protein is coupled with red latex microspheres with three particle sizes of 200 nm, 300 nm and 400 nm, respectively, and after being assembled into a test strip, positive and negative sera are added dropwise for detection.

[0085] Figure 4 The naked eye and reader results of the LMs particle size are shown. The results show that as the particle size of the latex microspheres increases, the T line color also gradually darkens, and when the particle size is 400 nm, the negative sample T line value is 144, which appears a false positive result. Therefore, the recombinant protein rFhCL1A is selected as the diagnostic antigen and coupled with the latex microspheres with a particle size of 300 nm to prepare the LMs-LFIA test strip of fasciola and apply it to detection.

[0086] (2) Antibody labeling amount determination

[0087] The 300 nm latex microspheres are activated with a MES activation buffer with a pH of 6.0, and are coupled with 40 μg, 50 μg and 60 μg of the recombinant protein rFhCL1A, respectively, and after being assembled into a test strip, detection is performed.

[0088] Figure 5 The antibody labeling amount screening results are shown. The detection results show that as the antibody labeling amount increases, the T line color also gradually darkens, and when 60 μg, a slight false positive appears. Therefore, 50 μg of the antibody labeling amount is selected for subsequent experiments.

[0089] (3) Antibody labeling time determination

[0090] The best antibody diluent (5 g of surfactant S9 is fully dissolved in 500 mL of PBS buffer, and then diluted to 1 000 mL) and the labeling amount (12.5 μL of 300 nm red latex microspheres: 50 μg of recombinant protein rFhCL1A) screened are selected for antibody labeling, and are labeled for 60 min, 100 min and 150 min, respectively, and after being assembled into a test strip, detection is performed.

[0091] Figure 6 The antibody labeling time screening results are shown. The detection results show that when the labeling time is 60 min, the positive and negative C line color development effects are poor; when the labeling time is 150 min, the negative T value is 211, which shows a false positive detection result; when the labeling time is 100 min, the C value is high and the positive T line color development effect is good, and no false positive result appears. Therefore, the labeling time is determined to be 100 min.

[0092] (4) T line and C line coating concentration determination

[0093] The best conditions screened above (the labeling amount is 12.5 μL of 300 nm red latex microspheres: 50 μg of recombinant protein rFhCL1A; the antibody labeling time is 100 min) are selected for antibody labeling, and HRP-Protein G is diluted into three concentration gradients of 0.25 mg / mL, 0.5 mg / mL and 1.0 mg / mL using a coating solution for detection, and the best T-line coating concentration is screened.

[0094] In the same way, His-tag monoclonal antibody is diluted into three concentration gradients of 0.25 mg / mL, 0.5 mg / mL and 1.0 mg / mL for detection, and the best C-line coating concentration is screened.

[0095] Figure 7 The T-line and C-line coating concentration determination results are shown. Figure 7 A in the table is the naked eye observation and reader result of the T-line coating concentration, and B is the naked eye observation and reader result of the C-line coating concentration.

[0096] The results show that when the T-line coating concentration is 0.5 mg / mL and the C-line is 1 mg / mL, the color development effect is better, and the value is higher. Therefore, the T-line coating concentration is determined to be 0.5 mg / mL, and the C-line coating concentration is determined to be 1 mg / mL.

[0097] In summary, the best preparation conditions of the immunochromatography test strip of the application are as follows: the latex microsphere particle size is 300 nm, the labeling amount is 12.5 μL of latex microspheres for labeling 50 μg of recombinant protein rFhCL1A, the labeling time is 100 min, the T-line coating concentration is 0.5 mg / mL, and the C-line coating concentration is 1 mg / mL.

[0098] 8. Effect evaluation of detection method

[0099] (1) Sensitivity test

[0100] The test strip is prepared and assembled using the best conditions screened, the positive serum is diluted according to four concentration gradients of 1:10, 1:100, 1:1000 and 1:10000 using a sample diluent, and the sensitivity of the method is evaluated.

[0101] Figure 8 The sensitivity test results are shown. The results show that when the positive serum is diluted at a dilution ratio of 1:10000, the T-line band is weak but can still be distinguished by naked eye observation, indicating that the method established has high sensitivity.

[0102] (2) Specificity test

[0103] The test paper strip is prepared and assembled using the screened optimal conditions, and is used for detecting bovine Dicrocoelium positive serum, bovine Prosthogonimus positive serum, bovine Fasciola positive serum and negative serum respectively, so as to evaluate the specificity of the method.

[0104] Figure 9 Specificity test results are shown. From left to right, they are bovine Fasciola positive serum, bovine Dicrocoelium positive serum, bovine Prosthogonimus positive serum and negative serum.

[0105] The results show that the test paper strip only shows obvious red bands on the T line when the Fasciola positive serum is added, and does not react with positive serum of other parasites, indicating that the method has good specificity.

[0106] (3) Stability test

[0107] The test paper strip prepared using the screened optimal conditions is selected from the same batch, and is detected after being placed at room temperature for 0, 15, 30 and 60 days respectively.

[0108] Figure 10 Stability test results are shown. The results show that the T line and the C line can still show red bands after being placed at room temperature for 60 days, and the band color has no obvious change, indicating that the stability of the test paper strip is at least 60 days under room temperature conditions.

[0109] (4) Clinical sample test

[0110] The test paper strip is prepared and assembled using the screened optimal conditions, and is used for detecting 82 bovine sera with known backgrounds after dissection.

[0111] Table 3 shows the clinical sample detection results. The results show that the number of positive samples detected by the LMs-LFIA detection method established in the application is 9, and the number of negative samples is 73, that is, the positive coincidence rate is 81.8%, the negative coincidence rate is 100%, and the total coincidence rate is 97.6%.

[0112] Table 3. Clinical sample detection

[0113]

[0114] The above only describes preferred embodiments of the present application and does not limit the present application in any form. Any simple modification, equivalent change and modification of the above embodiments without departing from the technical solution of the present application, and according to the technical essence of the present application, all still belong to the scope of the technical solution of the present application.

Claims

1. A conjugate of recombinant protein from *Fragaria schistosome* and latex microspheres, characterized in that, This includes covalently coupled recombinant protein rFhCL1A and latex microspheres.

2. The trematode recombinant protein and latex microsphere conjugate according to claim 1, characterized in that, The latex microspheres have a particle size of 300 nm.

3. A method for preparing the conjugate of trematode recombinant protein and latex microspheres as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Take 12.5 μL of red latex microsphere suspension with 4% solid content, mix thoroughly with 10 times the volume of 0.05M 2-morpholine ethanesulfonic acid buffer, centrifuge at 12000 r / min for 15 min, remove the supernatant, add 10 times the volume of 2-morpholine ethanesulfonic acid buffer to restore and sonicate for 1 min to mix. S2. Add 5 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride with a concentration of 10 mg / mL and 10 μL of N-hydroxysuccinimide with a concentration of 10 mg / mL to activate the red latex microspheres. After shaking and mixing, place the microspheres on a constant temperature shaker at 37°C and activate them at 180 r / min for 30 min. S3, centrifuge at 12000r / min for 15min, remove supernatant, add 10 times the volume of MES buffer to restore and sonicate for 1min; S4. Add 50 μg of recombinant protein rFhCL1A, shake to mix, and place on a constant temperature shaker at 37°C for 180 r / min for 100 min. S5, centrifuge at 12000r / min for 15min, remove supernatant, add 10 times the volume of blocking liquid to recover and sonicate for 1min, place on a constant temperature shaker and block at 180r / min for 60min at 37℃. Centrifuge at 12000 r / min for 15 min, remove the supernatant, add 10 times the volume of microsphere washing solution to restore and sonicate to mix well; S7. Centrifuge to remove supernatant, add 10 times the volume of microsphere washing solution to restore and sonicate; S8. Centrifuge to remove supernatant, add 0.1 mL of microsphere reconstitution solution to restore and sonicate to mix well, then store at 4℃ for later use.

4. The method according to claim 3, characterized in that, The blocking solution was prepared as follows: 20g BSA (AS12659-100g, Asbio) and 1mL 3% Proclin 300 were added to a beaker, dissolved thoroughly in 50mL ddH2O, and then brought to a final volume of 100mL.

5. The method according to claim 3, characterized in that, The microsphere reconstitution solution was prepared as follows: 20 mL PBS, 5 mL 3% Proclin 300, 25 mL Tween-20, 15 mL 10% PVP, 25 g sucrose, and 2.5 g BSA were thoroughly dissolved and mixed in 250 mL ddH2O, and then the volume was adjusted to 500 mL.

6. An immunochromatographic test strip, characterized in that, The immunochromatographic test strip includes a base plate, on which a sample pad, a latex microsphere conjugate pad, a nitrocellulose membrane with test lines and control lines, and an absorbent pad are sequentially overlapped. The sample pad and the latex microsphere conjugate pad overlap the nitrocellulose membrane at the end near the test line, and the absorbent pad overlaps the nitrocellulose membrane at the end near the control line. The conjugate pad is coated with the recombinant trematode protein rFhCL1A and latex microsphere conjugate as described in claim 1 or 2, the test line is coated with HRP-protein G, and the control line is coated with His-tag monoclonal antibody.

7. The immunochromatographic test strip according to claim 6, characterized in that, The coating concentration of the test line is 0.5 mg / mL.

8. The immunochromatographic test strip according to claim 6, characterized in that, The coating concentration of the quality control line is 1 mg / mL.

9. The use of the recombinant trematode protein and latex microsphere conjugate as described in claim 1 or 2 in the preparation of a kit for detecting trematodes.

10. A method for preparing an immunochromatographic test strip as described in any one of claims 6 to 8, characterized in that, Includes the following steps: S1. The recombinant protein rFhCL1A of the trematode was coated onto glass fiber with a latex microsphere conjugate and dried at 37°C to obtain a sample pad. S2. Process the nitrocellulose membrane by coating the His-tag monoclonal antibody onto the control line and the HRP-protein G onto the test line, and then drying it at 37°C. S3. Place the sample pad, latex microsphere binding pad, nitrocellulose membrane, and absorbent pad sequentially on the base plate. Cover the end of the nitrocellulose membrane near the test line with the latex microsphere binding pad, and cover the end of the nitrocellulose membrane near the quality control line with the absorbent pad.

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