AIE fluorescence immunochromatography test strip for detecting toxoplasma gondii antibody as well as preparation method and application of AIE fluorescence immunochromatography test strip

Abstract

The invention discloses an AIE fluorescence immunochromatography test strip for detecting a toxoplasma gondii antibody as well as a preparation method and application of the AIE fluorescence immunochromatography test strip, and belongs to the technical field of production of veterinary biological diagnosis products. The test strip provided by the invention comprises a bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are bonded on the bottom plate in an end-to-end connection manner; the combination pad is coated with AIE microsphere labeled protein; the nitrocellulose membrane contains a detection line T and a quality control line C; the test strip prepared by adopting a double-antigen sandwich antibody detection method can be used for detecting serum antibodies in animal bodies such as pigs, cats and the like. Compared with a colloidal gold test strip on the market, the colloidal gold test strip has the advantages that the sensitivity is improved by 32 times, the batch-to-batch difference is less than 5%, the test performed by adopting 40 parts of pig serum and 30 parts of cat serum shows that the specificity, the stability and the accuracy are excellent, the operation is simple, the cost is low, and an accurate result can be obtained only in 10 minutes; and a rapid and effective detection method is provided for epidemiological investigation and disease diagnosis.

Description

Technical Field

[0001] The invention belongs to the technical field of production of veterinary biological diagnostic products, and in particular relates to an AIE fluorescent immunochromatographic test strip for detecting Toxoplasma gondii antibodies, and a preparation method and application thereof. Background Art

[0002] Aggregation-induced emission (AIE) fluorescent molecules emit light efficiently in the aggregated state due to restricted intramolecular motion. Compared with other fluorescent microspheres, AIE molecules make great use of the aggregation process, fundamentally solving the fluorescence quenching caused by aggregation, and greatly improving the fluorescence efficiency of AIE molecules. AIE combined with immunochromatography technology has the characteristics of high quantum yield, good photostability, and strong resistance to photobleaching. Transforming the material performance advantages into test strip product advantages, AIE immunochromatography test strips have the characteristics of high sensitivity, high accuracy, and high reliability.

[0003] Toxoplasma gondii, also known as Toxoplasma gondii, is an obligate intracellular parasite. It can cause serious parasitic diseases in humans and animals and is distributed and prevalent worldwide. The final hosts of Toxoplasma gondii are cats and felines, but the intermediate hosts include many animal species, and pigs are one of the intermediate hosts. In addition, the infection rate and mortality rate of Toxoplasma gondii in pigs are also high, and they have become an important source of human infection with Toxoplasma. If Toxoplasma gondii infects people with normal immunity, the clinical symptoms are not obvious, but for people with low immunity such as AIDS patients, it may cause death. If the pathogen infects pregnant women, it can cause serious consequences such as miscarriage and deformed babies. The disease poses a certain threat to human health and the development of the pig industry, but there is currently no specific treatment drug or effective vaccine for toxoplasmosis. Therefore, it is particularly important to establish a convenient and fast method for diagnosing toxoplasmosis.

[0004] There are many methods for diagnosing toxoplasmosis, such as etiological diagnosis (accurate and reliable test results, but time-consuming and low detection rate), molecular biology diagnosis (high specificity and high sensitivity, but requires expensive instruments and strict technical requirements for diagnostic personnel), and immunological diagnosis (simple operation, sensitive and specific, intuitive result judgment, and batch testing). Therefore, there is an urgent need to provide a fast, accurate, convenient, and highly sensitive immunological early diagnosis method for toxoplasmosis. Summary of the invention

[0005] One of the purposes of the present invention is to provide a method for preparing an AIE fluorescent immunochromatographic test strip for detecting Toxoplasma gondii antibodies, wherein the AIE fluorescent immunochromatographic test strip comprises a bottom plate, a sample pad, a conjugate pad, a nitrocellulose membrane and a water absorbent pad, and the method for preparing the AIE fluorescent immunochromatographic test strip comprises the following steps:

[0006] 1) The glass fiber is soaked in a Tris buffer containing BSA, sucrose and trehalose, and then dried, and then sprayed with a dilution solution containing AIE microspheres labeled TOXO antigen and AIE microspheres labeled chicken IgY to obtain a binding pad;

[0007] 2) TOXO antigen was coated on nitrocellulose to form a test line, and goat anti-chicken IgY was coated on nitrocellulose to form a quality control line;

[0008] 3) The sample pad, the conjugate pad, the nitrocellulose membrane, the absorbent paper and the bottom plate are assembled to obtain the AIE fluorescent immunochromatography test strip for detecting Toxoplasma gondii antibodies;

[0009] The amount of TOXO antigen labeled by the 50-250 μg AIE microspheres is 10-100 μg, the amount of chicken IgY labeled by the 50-250 μg AIE microspheres is 5-100 μg, the TOXO antigen coating concentration on the nitrocellulose membrane is 0.1-2 mg / mL, and the quality control line, i.e., the sheep anti-chicken coating concentration, is 0.1-2 mg / mL.

[0010] Preferably, the amount of TOXO antigen labeled with the 50-250 μg AIE microspheres is 25 to 75 μg, more preferably 25 to 50 μg; the amount of chicken IgY labeled with the 50-250 μg AIE microspheres is 5 to 20 μg, more preferably 5 to 10 μg.

[0011] More preferably, the amount of TOXO antigen labeled with the 50-250 μg AIE microspheres is 25 to 50 μg, and the amount of chicken IgY labeled with the 50-250 μg AIE microspheres is 5 to 10 μg.

[0012] Preferably, the coating concentration of TOXO antigen on nitrocellulose membrane is 0.25-0.5 mg / mL, and the coating concentration of the quality control line, i.e., goat anti-chicken, is 0.5-1 mg / mL.

[0013] Preferably, the AIE microsphere-labeled TOXO antigen and the AIE microsphere-labeled chicken IgY are mixed in a volume ratio of 1-3:0.5-1.5 to prepare a dilution, and the original concentrations of the AIE microsphere-labeled TOXO antigen and the AIE microsphere-labeled chicken IgY are both 0.5-1.5 mg / mL.

[0014] More preferably, the sample pad material is any one of glass fiber, cotton fiber, filter paper fiber or polyester fiber, and glass fiber is preferred in the present invention.

[0015] More preferably, the binding pad material is polyester fiber; and the absorbent pad is absorbent filter paper.

[0016] The second object of the present invention is to provide an AIE fluorescent immunochromatographic test strip prepared by the above preparation method.

[0017] The third object of the present invention is to provide the use of the above-mentioned AIE fluorescent immunochromatographic test strip in the preparation of Toxoplasma gondii antibody detection products.

[0018] In the present invention, the particle size of the AIE fluorescent microspheres is 50 to 500 nm, preferably 200 to 300 nm.

[0019] In the present invention, a sample diluent used in conjunction with the test strip is provided, which is a PBS buffer solution containing BSA (1-5wt%), S9 (1-5wt%), casein (0.01-0.2wt%) and a preservative (0.3-0.8%).

[0020] In the present invention, the dilution volume ratio of AIE microsphere labeled TOXO antigen to AIE microsphere labeled chicken IgY is 1-5:1, preferably 2:1; the volume ratio of microsphere to microsphere diluent is 1:1-10, preferably 1:4-6.

[0021] The experimental principle of the present invention is as follows: Figure 1 As shown, using the double antigen sandwich method, the TOXO antigen labeled with AIE microspheres is captured by the specific antibody in the positive serum. With the chromatography of the test strip, on the detection line, the labeled antigen-antibody complex is captured by the TOXO antigen on the nitrocellulose membrane to form a sandwich complex, which fluoresces under ultraviolet light; the sheep anti-chicken IgY antibody on the quality control line captures the chicken IgY antibody labeled with AIE microspheres to form a complex, which fluoresces under ultraviolet light.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The AIE fluorescent microspheres used in the AIE microsphere immunochromatographic test strip provided by the present invention have high uniformity, large Stokes shift, good photostability, high brightness, good monodispersity, and can effectively prevent fluorescence quenching.

[0024] (2) The AIE microsphere immunochromatographic test strip for detecting Toxoplasma antibodies provided by the present invention requires simple raw materials and is easy to prepare. Non-professionals can also master the operation method in a short time, and it has broad market prospects.

[0025] (3) Compared with colloidal gold test strips on the market, the test strips and sample diluent provided by the present invention have a sensitivity increased by 32 times, and the batch difference is less than 5%.

[0026] (4) The test strip and sample diluent provided by the present invention can qualitatively or quantitatively detect Toxoplasma gondii antibodies, have strong specificity and good repeatability, can realize rapid detection of samples to be tested, and can realize mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The present invention is a schematic structural diagram and a principle diagram of the immunochromatographic test strip for detecting Toxoplasma gondii antibodies.

[0028] Figure 2 Characterization of AIE fluorescent microspheres in Example 2. Figure 2 -A is a performance verification diagram of TOXO antigen: the excellent performance of TOXO antigen was verified by SDS-PAGE and protein immunoblotting experiments; Figure 2 -B is the scanning electron microscopy and transmission electron microscopy characterization images of AIE microspheres, which proves that the AIE microspheres have uniform particle size and contain abundant carboxyl groups, which can be used for subsequent labeling; Figure 2 -C and Figure 2 -D is the measurement of the excitation and emission spectra of AIE microspheres, which proves that AIE microspheres have stable and efficient optical properties; Figure 2 -E and Figure 2 -G is the UV irradiation photos and hydrated particle size test of fresh AIE microspheres and microspheres stored at 4°C for 28 days. There is no obvious aggregation visually and the particle size distribution of the hydrated particle size test is uniform, proving that they can be stored for a long time; Figure 2 -F is the fluorescence attenuation of AIE microspheres under continuous ultraviolet irradiation, which proves that AIE has good fluorescence performance and can be used for on-site detection in daily environments.

[0029] Figure 3 This is the verification of AIE microspheres labeling antigen and antibody in Example 2. Figure 3 -A and Figure 3 -B is the emission and excitation spectrum measurement of AIE microspheres after antigen and antibody labeling, and the results show that labeling has no effect on the fluorescence spectrum. 3-C is the Zeta potential and average particle size of AIE microspheres after antigen and antibody labeling compared with AIE microspheres, indicating that TOXO antigen-AIE fluorescent microspheres and chicken IgY-AIE fluorescent microspheres are successfully labeled.

[0030] Figure 4 This is a graph showing the test strip condition optimization results in Example 4. Figure 4 -A is the optimization of the coupling ratio of AIE microspheres and TOXO antigen. The results show that 25 μg TOXO antigen is the optimal labeling amount; Figure 4 -B and Figure 4 -C is the optimization of the test line and quality control line concentrations for nitrocellulose membrane coating. The results showed that the optimal coating concentration of TOXO antigen (test line) was 0.25 mg / mL, and the optimal coating concentration of goat anti-chicken IgY (quality control line) was 1 mg / mL. Figure 4 -D is the optimization of the best reaction time, and the results show that the best detection time is 10 minutes.

[0031] Figure 5 This is a graph showing the test strip performance measurement results in Example 5. Figure 5 -A is the establishment of the standard curve; Figure 5 -B is the repeatability determination; Figure 5 -C is a methodological comparison.

[0032] Figure 6 This is a graph showing the specificity evaluation results of the test strip in Example 5.

[0033] Figure 7 This is a graph showing the test results of the test strip in Example 6 for clinical samples from pigs.

[0034] Figure 8 The figure is a graph showing the test results of the test strip in Example 7 for clinical samples of cats. DETAILED DESCRIPTION

[0035] The TOXO antigen used in the following examples was purchased from Shanxi Jinnong Biotechnology Co., Ltd., chicken IgY antibody and goat anti-chicken IgY antibody were purchased from Hangzhou Huakui Jinpei Biotechnology Co., Ltd., AIE fluorescent microspheres (NARPC-020) were purchased from the Aggregation-Induced Emission Advanced Research Institute, and the sample pad, conjugate pad, bottom plate, and absorbent pad were all purchased from Shanghai Jieyi Biotechnology.

[0036] Example 1

[0037] This embodiment provides a Figure 1 The AIE immunochromatographic test strip for detecting toxoplasma antibodies shown in the figure comprises a base plate, a sample pad, a conjugation pad, a nitrocellulose membrane, and a water-absorbing pad; the sample pad, the conjugation pad, the nitrocellulose membrane, and the water-absorbing pad are connected end to end in sequence, overlapped by 1 to 3 mm, and adhered to the base plate. The conjugation pad is sprayed with a TOXO antigen-AIE microsphere labeled complex and a chicken IgY antibody-AIE microsphere labeled complex; the nitrocellulose membrane is provided with a detection line coated with TOXO antigen and a quality control line coated with sheep anti-chicken IgY antibody in sequence.

[0038] The preparation method of the AIE microsphere immunochromatographic test strip for detecting Toxoplasma gondii antibodies comprises the following steps:

[0039] 1. Preparation of TOXO antigen-AIE microsphere complex and chicken IgY antibody-AIE microsphere labeled complex

[0040] 1) Take 50 μL of AIE microsphere solution (1 wt%), add 500 μL of MES (0.005 M pH 6.0) buffer to wash, and centrifuge to remove the supernatant;

[0041] 2) Redissolve the microspheres in 500 μL MES pH 6.0 buffer, add EDC and NHS to make the final concentration of each 10 mM, rotate and shake at 40 rpm in the dark for 30 min, and centrifuge to remove the supernatant;

[0042] 3) Add 500 μL MES (0.005M pH 6.5) buffer to wash once. To prepare TOXO antigen-AIE microsphere complex, add quantitative TOXO antigen; to prepare chicken IgY antibody-AIE microsphere labeled complex, add quantitative chicken IgY antibody, rotate and shake at 40 rpm in the dark for 2 hours, and centrifuge to remove the supernatant;

[0043] 4) Add 500 μL of blocking solution (1% glycine, 0.05% BSA) to re-dissolve, rotate and shake at 40 rpm in the dark for 1 hour, and centrifuge to remove the supernatant;

[0044] 5) Add 500 μL preservation solution (0.02 M Tris, 0.1% Tween-20, 0.5% BSA, 0.03% Proclin 300, pH 8.0) to wash once, and centrifuge to remove the supernatant;

[0045] 6) Add 100 μL of storage solution to reconstitute and store at 2-8°C.

[0046] 2. Preparation of the Conjugate Pad

[0047] 1) Soak the conjugate pad in 0.1 M Tris (pH 8.0) buffer containing 10% sucrose, 2% trehalose, 2% BSA, and 0.2% Tween-20 for 10 min, and dry in an oven.

[0048] 2) The TOXO antigen-AIE microsphere marker, chicken IgY antibody-AIE microsphere marker and microsphere preservation solution were diluted in a volume ratio of 1:0.5:19, respectively, and then sprayed using a three-dimensional synovial gold sprayer. The parameters were: spray volume: 3 μL, spacing 3.3 mm; number 2; binding pad width 12 mm. After the pad was sprayed, it was placed in an oven for drying for later use.

[0049] 3. Preparation of Sample Pad

[0050] The glass fiber was sprayed with a Tris-HCl (0.1 M pH 8.0) buffer solution containing 0.5% BSA and 0.2% Tween-20. After the spraying, the glass fiber was placed in an oven for drying.

[0051] 4. Preparation of Nitrocellulose Membrane

[0052] 1) Dilute the TOXO antigen to 0.1-1 mg / mL with PBS buffer (containing 1% trehalose) to prepare a test line T solution;

[0053] 2) Dilute the goat anti-chicken IgY antibody to 0.1-1 mg / mL with PBS buffer to prepare a quality control line C solution;

[0054] 3) Use a three-dimensional synovial gold spraying instrument to coat the sample, so that the distance between the test line T and the quality control line C is 4 to 6 mm;

[0055] 4) Dry in an oven at 37°C for 12 hours.

[0056] 5. Assembly of test strips

[0057] The treated sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are pasted on the bottom plate in sequence with an overlap of 1 to 3 mm. The strips are cut into 4.0±0.5 test strips with a CNC cutting machine. The strips are loaded into plastic card shells, capped and transferred into aluminum foil bags. After adding desiccant, they are sealed for storage.

[0058] 6. Sample diluent

[0059] The sample diluent was 0.01 M PBS buffer, pH 7.4, containing 1-5 wt % BSA and 1-5 wt % S9 surfactant.

[0060] Example 2 Characterization of AIE microspheres, TOXO antigen-AIE microspheres and chicken IgY antibody-AIE microspheres

[0061] 1. Characterization of Unlabeled Fluorescent Microspheres

[0062] Scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HRTEM) were used to observe the morphology and particle size of the AIE fluorescent microspheres and to characterize the carbon, oxygen and hydrogen elements of the microspheres. The fluorescence spectrum of the AIE fluorescent microspheres was scanned three times. The fluorescence intensity change was measured under ultraviolet irradiation for 20 minutes (measured every 5 minutes) and repeated three times. Zeta potential analysis was performed; and hydrated particle size (DLS) analysis was performed.

[0063] Result interpretation: The result is as follows: Figure 2 As shown, SEM and HRTEM characterization of AIE fluorescent microspheres are spheres with uniform particle size, good monodispersity, and a diameter of about 200nm. Elemental characterization shows that there are abundant carboxyl groups on the surface of the microspheres, indicating that the covalent binding efficiency of antigens and antibodies to the microspheres is high. From the peak of the fluorescence spectrum, it can be seen that 365nm is the optimal excitation wavelength of AIE, and 605nm is the optimal emission wavelength. At this wavelength, the AIE microspheres emit bright orange-red light. The fluorescence intensity of the fluorescent microspheres decreased under ultraviolet irradiation, but the light intensity reduction did not reach 20%, indicating that the fluorescent microspheres are relatively stable under ultraviolet irradiation. Moreover, the average hydrodynamic diameter and Zeta potential changes between fresh fluorescent microspheres and fluorescent microspheres placed for 28 days are slightly different, and the fluorescence brightness is consistent, which further proves that the microspheres are stable.

[0064] 2. Characterization of labeled TOXO antigen-AIE and chicken IgY antibody-AIE microspheres

[0065] The TOXO antigen-AIE and chicken IgY antibody-AIE microspheres were subjected to fluorescence spectrum scanning, Zeta potential analysis and hydrated particle size (DLS) analysis respectively.

[0066] Interpretation of results: Figure 3 As shown, the AIE microspheres labeled antigen-antibody complex had no effect on the fluorescence spectrum. Compared with AIE microspheres, the antigen-antibody AIE microspheres labeled had an increased Zeta potential and an increased average particle size, indicating that TOXO antigen-AIE fluorescent microspheres and chicken IgY antibody-AIE fluorescent microspheres were successfully labeled.

[0067] Example 3 Test strip detection method and result determination

[0068] 1. A method for detecting antibodies to Toxoplasma gondii using a test strip and a sample diluent, comprising the following steps:

[0069] 1) Sample processing: collect whole blood from animals at 4000×g, centrifuge for 5 min or incubate at 4°C overnight to precipitate naturally, and take serum as the test sample;

[0070] 2) Dilute the serum 50 times with sample diluent, mix well, and drip 80 μL of the diluted sample vertically into the sample well;

[0071] 3) Wait for 10 to 20 minutes, observe and judge the results.

[0072] 2. Interpretation of results

[0073] Irradiate the test strip after the reaction with a 365nm ultraviolet emitter. If both the C line and the T line of the test strip are colored, the result is judged as positive, and the darker the color of the T line, the higher the content of Toxoplasma gondii antibodies in the sample. When the C line of the test strip is colored and the T line is not colored, the result is judged as negative, and there is no Toxoplasma gondii antibody in the sample. If the C line of the test strip is not colored, the test result of the test strip is invalid.

[0074] Example 4 Test Strip Optimization

[0075] 1. Optimization of the optimal labeling amount of AIE microspheres coupled with TOXO antigen

[0076] 1) Optimization of coupling ratio with TOXO antigen: 50 μL of 10 mg / mL AIE microspheres were coupled with 10 μg, 25 μg, 50 μg, 75 μg, and 100 μg of TOXO antigen respectively. The coating concentration of the detection line and the quality control line on the nitrocellulose membrane was set to 1 mg / mL. The clinical positive serum was diluted 1:50 times as the calibrator A, and the clinical negative serum was diluted 1:50 times as the calibrator B for detection to screen the optimal coupling ratio of AIE microspheres and TOXO antigen.

[0077] 2) Interpretation of results: After adding the calibrant to the test strip, let it stand for 15 minutes, and use a dry fluorescence quantitative instrument to measure the fluorescence intensity of the test line and the quality control line. The TOXO result is as follows: Figure 4 As shown in A, 500 μg AIE microspheres labeled with 25 μg TOXO antigen is the optimal labeling amount.

[0078] 2. Optimization of the concentration of the test line and quality control line of nitrocellulose membrane coating

[0079] 1) Optimization of the concentration of TOXO antigen coated on the test line: TOXO antigens of different concentrations were coated on nitrocellulose membranes, which were 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, and 1 mg / mL, respectively. TOXO antigen-AIE microspheres (25 μg) and chicken IgY antibody-AIE microspheres (10 μg) were sprayed on the conjugate pad at a volume ratio of 2:1, and the calibration products A and B in step 1 of Example 3 were used for detection to screen out the optimal concentration of the antigen coated on the test line.

[0080] 2) Optimization of the concentration of goat anti-chicken IgY antibody for the quality control line coating: Same as step 21), except that the coating was changed from TOXO antigen to goat anti-chicken IgY, and the rest was the same, to screen out the optimal concentration of the antibody for the quality control line coating.

[0081] 3) Interpretation of results: Figure 4 As shown in B and 4C, the optimal coating concentration of TOXO antigen is 0.25 mg / mL, and the optimal coating concentration of goat anti-chicken IgY is 1 mg / mL.

[0082] 3. Determination of optimal reaction time

[0083] Take calibrants A and B as samples for detection, and repeat the measurement 5 times for each sample, starting from 5 minutes after adding the sample, and measuring every 5 minutes until 25 minutes, to select the optimal reaction time.

[0084] Result interpretation: The result is as follows: Figure 4 As shown in D, the optimal detection time is 10 min.

[0085] Example 5 Test Strip Performance Determination

[0086] 1. Establishment of standard curve

[0087] 1) Establishment method: Take calibrator A and dilute it 2-fold with sample diluent. The dilution multiples are set to 50, 100, 200, 400, 800, 1600, 3200, 6400, and 12800 times, respectively. Take calibrator B as the negative control. Take 80 μL of the diluted samples at different dilution multiples and add them to the sample pad of the test strip. Repeat 3 times for each dilution multiple and let it stand for 10 minutes.

[0088] 2) Result determination: The fluorescence intensity of the test strip test line and the quality control line is detected by a dry fluorescence quantitative instrument, and a standard curve is drawn. The serum has the ability to be quantitatively detected in the dilution range of 1:200 to 1:6400. The results are as follows: Figure 5 As shown in A, the standard curve is y = 220.79x + 0.042642, R 2 =0.9942.

[0089] 2. Repeatability determination

[0090] 1) Detection method: The calibrants A and B were detected using the test strips prepared under the optimal conditions of Example 4, and the C and T values ​​were read using a dry fluorescence quantitative instrument. The detection was repeated 13 times.

[0091] 2) Test results: Figure 5 As shown in B, the test results of calibrator A were all positive, with uniform fluorescence color development, close fluorescence intensity values, and a coefficient of variation of 5.20%; the test results of calibrator B were all negative, with uniform fluorescence color development, close fluorescence intensity values, and a coefficient of variation of 13.54%.

[0092] 3. Specificity Determination

[0093] 1) The sample diluent of the present invention was combined with the test strip prepared according to the optimal conditions of Example 4 and the method of Example 2 was used to detect 7 clinical sera of Trichinella spiralis, 3 clinical sera of Taenia asiatica, 8 clinical sera of Cysticercus cellulosa, 5 clinical sera of Cystoisospora cellulosa, 5 clinical sera of Sarcocystis suum, and 10 clinical sera of Ascaris suum.

[0094] 2) Test results: Test results such as Figure 6 As shown, the results were all negative, indicating that the test strip of the present invention had no cross reaction with other parasitic diseases of pigs and had good specificity.

[0095] 4. Methodological comparison

[0096] 1) The test strip prepared according to the optimal conditions of Example 4 was tested in parallel with the colloidal gold detection kit for porcine Toxoplasma gondii antibody (Cat. No. L8402) of Sinorica, that is, the serum dilution method of step 1 in Example 4 was adopted, 60 μL of the diluted sample was added to the colloidal gold sample well, and after standing for 15 minutes, the colloidal gold test strip results were visually observed. The specific results are shown in Tables 1 and Figure 5 C.

[0097] Table 1 Comparison of sensitivity between colloidal gold method and this method

[0098]

[0099]

[0100] 2) Test results: The clinical serum sample diluted 12,800 times by the test strip of the present invention was still positive, while the clinical serum sample diluted 800 times by the commercially available colloidal gold test strip was negative, indicating that the sensitivity of the test strip of the present invention is much higher than that of the commercially available colloidal gold test strip, which is increased by at least 32 times.

[0101] Example 6 Clinical Sample Detection of Pig Blood Samples

[0102] 20 samples of Toxoplasma gondii positive sera and 20 samples of Toxoplasma gondii negative sera retained in the laboratory were tested using the Toxoplasma gondii ELISA antibody detection kit (TOXOS-MS-2P) of French ID-VET and the test strip of the present invention, and each serum sample was tested 3 times, and the average value was calculated. The test results are shown in Figure 2. Figure 7 As shown, the consistency with the ELISA result is 100%, indicating that the test strip of the present invention has high reliability in clinical diagnosis.

[0103] Example 7 Clinical Sample Detection of Cat Blood Samples

[0104] 13 cat Toxoplasma-positive sera and 17 cat Toxoplasma-negative sera retained in the laboratory were tested using the Toxoplasma ELISA antibody detection kit (TOXOS-MS-2P) of French ID-VET, the colloidal gold test strip (article number L8402) of Sinolikang Company and the test strip of the present invention, and each serum sample was tested 3 times, and the average value was calculated. The test results are shown in the figure. Figure 8 As shown, the consistency between the test strip of the present invention and the ELISA result is 100%, and no positive results of the cat colloidal gold test strip of Synergic Company were detected (possibly due to low sensitivity), indicating that the fluorescent test strip involved in the present invention has a very high credibility in clinical diagnosis.

[0105] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing an AIE fluorescent immunochromatographic test strip for detecting Toxoplasma gondii antibodies, characterized in that: The AIE fluorescent immunochromatographic test strip comprises a bottom plate, a sample pad, a conjugate pad, a nitrocellulose membrane and a water-absorbing pad. The preparation method of the AIE fluorescent immunochromatographic test strip comprises the following steps: 1) The glass fiber is soaked in a Tris buffer containing BSA, sucrose and trehalose and dried, and then sprayed with a dilution solution containing AIE microsphere-labeled TOXO antigen and AIE microsphere-labeled chicken IgY antibody, and then dried to obtain a conjugate pad; 2) TOXO antigen was coated on nitrocellulose to form the detection line - T line, and goat anti-chicken IgY antibody was coated on nitrocellulose to form the quality control line - C line; 3) The sample pad, conjugate pad, nitrocellulose membrane, absorbent paper and bottom plate are assembled to obtain the AIE fluorescent immunochromatography test strip for detecting Toxoplasma gondii antibodies; The amount of TOXO antigen labeled by the 50-250 μg AIE microspheres is 10-100 μg, the amount of chicken IgY labeled by the 50-250 μg AIE microspheres is 5-100 μg, the TOXO antigen coating concentration on the nitrocellulose membrane is 0.1-2 mg / mL, and the quality control line, i.e., the sheep anti-chicken coating concentration, is 0.1-2 mg / mL.

2. The preparation method according to claim 1, characterized in that: The amount of TOXO antigen labeled by the 50-250 μg AIE microspheres is 25-75 μg, and the amount of chicken IgY antibody labeled by the 50-250 μg AIE microspheres is 5-20 μg.

3. The preparation method according to claim 2, characterized in that: The amount of TOXO antigen labeled by the 50-250 μg AIE microspheres is 25 to 50 μg, and the amount of chicken IgY labeled by the 50-250 μg AIE microspheres is 5 to 10 μg.

4. The preparation method according to claim 1, characterized in that: The coating concentration of TOXO antigen on nitrocellulose membrane is 0.25-0.5 mg / mL, and the coating concentration of the quality control line, i.e., sheep anti-chicken, is 0.5-1 mg / mL.

5. The preparation method according to claim 1, characterized in that: In the step 1), the TOXO antigen labeled with AIE microspheres and the chicken IgY labeled with AIE microspheres are mixed at a volume ratio of 1-3:0.5-1.5 to prepare a dilution solution, and the original concentrations of the TOXO antigen labeled with AIE microspheres and the chicken IgY labeled with AIE microspheres are both 0.5-1.5 mg / mL.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The sample pad material is any one of glass fiber, cotton fiber, filter paper fiber or polyester fiber.

7. The preparation method according to claim 6, characterized in that: The bonding pad material is polyester fiber or glass fiber.

8. An AIE fluorescent immunochromatographic test strip prepared by the preparation method described in any one of claims 1 to 5 and 7.

9. The AIE fluorescent immunochromatographic test strip prepared by the preparation method according to claim 6.

10. Use of the AIE fluorescent immunochromatographic test strip described in claim 8 in the preparation of a Toxoplasma gondii antibody detection product.

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