Toxoplasma gondii IgM antibody detection test strip

Abstract

The invention relates to the field of toxoplasma gondii detection, and discloses a toxoplasma gondii IgM antibody detection test strip, which comprises a substrate, a sample loading pad, a colloidal gold adsorption pad marked with a C line, a colloidal gold adsorption pad marked with a T line, and a water absorption pad, a sample loading pad, a colloidal gold adsorption pad marked with a C line, a colloidal gold adsorption pad marked with a T line, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the substrate along the liquid chromatography direction; the solid-phase colloidal gold T line is labeled with a TOX recombinant antigen, and the solid-phase colloidal gold C line is labeled with a mouse monoclonal antibody; the nitrocellulose membrane is sequentially coated with a detection line and a quality control line; the detection line is coated with an anti-human IgM chain monoclonal antibody, and the quality control line is coated with an anti-mouse IgG polyclonal antibody; the test strip has the technical advantages of high sensitivity and high specificity, few types of raw materials are used, the use amount is low, the production cost is effectively controlled, the quantity of samples required for detection is extremely small, the efficiency is high, special instruments are not needed, direct judgment can be carried out through naked eyes, and the operation is more convenient and efficient.

Description

Technical Field

[0001] This invention relates to the field of Toxoplasma gondii detection, specifically to a Toxoplasma gondii IgM antibody test strip. Background Technology

[0002] In Traditional Chinese Medicine (TCM), Toxoplasma gondii is known as the "Three Corpse Worm." It is an obligate intracellular parasite, taxonomically belonging to the subclass Coccidia, order Eucoccidales, family Isosporidiidae, and genus Toxoplasma. This parasite lives within the host's cells and can spread throughout the body via the bloodstream, damaging vital organs such as the brain, heart, and retina. It weakens the immune system, leading to various diseases; infection with this parasite results in toxoplasmosis. The main transmission routes are: felines often carry oocysts in their feces, and various animals (including humans) can become infected by ingesting food or water containing Toxoplasma gondii cysts, oocysts, or live parasites.

[0003] Acute toxoplasmosis can be classified into severe and mild forms. Severe acute infection is rare clinically but is extremely dangerous, manifesting as systemic symptoms: body temperature rises to 40°C or above with chills, the fever pattern is mostly remittent or continuous, lasting 2-3 weeks and the symptoms worsen with the rise in body temperature, specifically including severe headache, generalized muscle pain (especially in the eyes and back), facial flushing, conjunctival congestion, and neuropsychiatric symptoms such as lethargy, drowsiness, coma, or mania, accompanied by gastrointestinal reactions such as loss of appetite, diarrhea or constipation; some patients may develop maculopapular rash, measles-like rash, or erythematous nodular rash, which may leave colorless millet-like rashes or purplish-brown scars (which can last for decades) after about two weeks, and is often accompanied by lymphadenopathy, hepatosplenomegaly, and is prone to serious complications such as meningoencephalitis, hepatitis, pneumonia, myocarditis, pericarditis, etc., which can lead to death if not treated in time. Some patients may also develop eye damage such as retinitis and choroiditis. The systemic symptoms of mild acute infections are often atypical, resembling those of the common cold or acute gastroenteritis, and are easily overlooked clinically.

[0004] Currently, clinically used toxoplasmosis detection methods have low detection rates, and clinicians' insufficient understanding of toxoplasmosis leads to frequent misdiagnosis, delayed treatment, or inappropriate interventions such as the misuse of hormones, ultimately causing toxoplasmosis outbreaks. Even after diagnosis via cerebrospinal fluid testing in large hospitals and treatment with internationally accepted protocols (such as pyrimethamine, sulfadiazine combined with azithromycin), patients still suffer severe damage to their liver, kidneys, and hematopoietic system. Even with subsequent standardized treatment, irreversible sequelae may remain after recovery. Therefore, developing an efficient and accurate toxoplasmosis detection method is of significant clinical importance for improving the early diagnosis rate of this disease and improving patient prognosis, and is an urgent technical need. Summary of the Invention

[0005] This invention provides a Toxoplasma gondii antibody (IgM) test strip (colloidal gold method). The test strip includes a substrate, a sample pad, and an absorbent pad, with a nitrocellulose membrane positioned below the sample pad and the absorbent pad. The sample pad has a solidified colloidal gold T-line and a solidified colloidal gold C-line at its connecting end. The solidified colloidal gold T-line is a TOX recombinant antigen, and the solidified colloidal gold C-line is a mouse monoclonal antibody. The nitrocellulose membrane is sequentially coated with a detection line and a control line. The detection line is coated with anti-human IgM µ-chain monoclonal antibody, and the control line is coated with anti-mouse IgG polyclonal antibody. This test strip developed and produced by our company has the technical advantages of high sensitivity and high specificity. It uses fewer types and amounts of raw materials, effectively controlling production costs. It requires very little sample for detection, has a short result interpretation time, requires no special instruments, and can be directly interpreted visually. It is convenient and efficient to operate, and has significant value for the clinical diagnosis and prevention of toxoplasmosis.

[0006] On the one hand, a method for preparing a test strip for detecting Toxoplasma gondii IgM antibodies, the method comprising: The preparation steps of nitrocellulose membrane are as follows: the detection line and the quality control line are coated on the nitrocellulose membrane to obtain the coated nitrocellulose membrane; the coated nitrocellulose membrane is immersed in the sealing treatment soaking solution to obtain the sealed nitrocellulose membrane; the sealed nitrocellulose membrane is pasted onto the substrate to obtain the substrate with the nitrocellulose membrane pasted on. Preparation steps of colloidal gold adsorption pads labeled with C-lines or T-lines: Adjust the pH of the colloidal gold solution containing chloroauric acid and trisodium citrate to obtain the adjusted colloidal gold solution; add TOX antigen or mouse monoclonal antibody to the adjusted colloidal gold solution to obtain a mixture; add casein to the mixture, then stir and centrifuge to obtain a precipitate; reconstitute the precipitate with colloidal gold reconstitution solution and cast it onto the colloidal gold adsorption pad to obtain the colloidal gold adsorption pad labeled with C-lines or T-lines; Assembly steps: The substrate is sequentially arranged along the liquid chromatography direction with a sample pad, a colloidal gold adsorption pad marked with a C line, a colloidal gold adsorption pad marked with a T line, a nitrocellulose membrane, and an absorbent pad to obtain a test strip for detecting Toxoplasma gondii IgM antibodies.

[0007] Furthermore, the detection line in the preparation step of the nitrocellulose membrane is coated with IgM µ-chain monoclonal antibody; Preferably, the concentration of the IgM µ-chain monoclonal antibody is 0.1-0.2 mg / ml.

[0008] Furthermore, the quality control line mentioned in the preparation step of the nitrocellulose membrane is coated with anti-mouse IgG polyclonal antibody; Preferably, the concentration of the anti-mouse IgG polyclonal antibody is 1-1.5 mg / ml.

[0009] Furthermore, in the preparation steps of the colloidal gold adsorbent pad labeled with C-line or T-line, the pH of the colloidal gold solution used for the colloidal gold adsorbent pad labeled with C-line is 6.5-7.0.

[0010] Furthermore, in the preparation steps of the colloidal gold adsorbent pad marked with C-line or T-line, the pH of the colloidal gold solution used for the colloidal gold adsorbent pad marked with T-line is 7.5-8.0.

[0011] Furthermore, in the preparation steps of the colloidal gold adsorption pad labeled with C-line or T-line, the antigen labeled with the colloidal gold adsorption pad labeled with C-line is a mouse monoclonal antibody. Preferably, the concentration of the mouse monoclonal antibody is 10-12 ug / ml.

[0012] Furthermore, in the preparation steps of the colloidal gold adsorption pad labeled with C-line or T-line, the antigen labeled with the colloidal gold adsorption pad labeled with T-line is TOX antigen. Preferably, the concentration of TOX antigen is 1-2 ug / ml.

[0013] Furthermore, the environmental conditions during the preparation process include: a temperature of 20-30℃ and a humidity of ≤40%.

[0014] On the other hand, the present invention provides a test strip for detecting Toxoplasma gondii IgM antibodies, the test strip being prepared using the preparation method described above.

[0015] Furthermore, the test strip is provided with a sample loading pad, a nitrocellulose membrane, an absorbent pad, a solidified colloidal gold T-line, a solidified colloidal gold C-line, a detection line, and a quality control line.

[0016] The technical solution of this invention has the following advantages: Our company's Toxoplasma gondii antibody (IgM) test strip (colloidal gold method) combines the technical advantages of high sensitivity and high specificity. Furthermore, the kit uses fewer types and smaller quantities of raw materials, effectively controlling production costs. In addition, the product requires very little sample volume, has a short result interpretation time, and requires no specialized instruments; results can be determined directly by visual inspection. It is convenient and efficient, possessing significant social and economic value for the clinical diagnosis and prevention of toxoplasmosis.

[0017] This test strip can be used as an auxiliary diagnostic tool for toxoplasmosis infection. It has the core characteristics of being fast, simple, accurate and sensitive. It has important application value in the auxiliary diagnosis of spontaneous abortion and fetal congenital defects and in clinical rapid detection scenarios. It can be widely used in clinical medical systems, health care institutions and scientific research fields. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the structure of the present invention; Figure 2This is a schematic diagram of the test strip structure of the present invention, including 1-test strip, 2-plastic card cover, 3-plastic card base, 1-1-substrate, 1-2-sample pad, 1-3-absorbent pad, 1-4-nitrocellulose membrane, 1-5-solidified colloidal gold T-line, 1-6-solidified colloidal gold C-line, T-detection line, C-control line; Figure 3 This is a schematic diagram of a random sampling test strip. Detailed Implementation

[0019] Example 1. Preparation method of Toxoplasma gondii antibody (IgM) test strip (colloidal gold method) This embodiment provides a Toxoplasma gondii antibody (IgM) test strip, which consists of a plastic casing cover, a plastic casing base, and a test strip. The plastic casing base has a slot in which... Figure 1 The toxoplasmosis IgM antibody test strip shown; The Figure 1 The toxoplasmosis IgM antibody test strip shown includes a substrate (1-1). On the upper surface of the substrate (1-1), along the chromatography direction, a sample loading pad (1-2), a nitrocellulose membrane (1-4), and an absorbent pad (1-3) are sequentially arranged, with the nitrocellulose membrane (1-4) located below the sample loading pad (1-2) and the absorbent pad (1-3). At the connection point between the sample loading pad (1-2) and the nitrocellulose membrane (1-4), along the chromatography direction, a solid-phase colloidal gold T-line (1-5) and a solid-phase colloidal gold C-line (1-6) are sequentially arranged. The active ingredient of the solid-phase colloidal gold T-line (1-5) is... Toxoplasmosis (TOX) recombinant antigen; the active ingredient of the solid-phase colloidal gold C line (1-6) is a murine monoclonal antibody; a detection line T and a control line C are sequentially arranged along the chromatography direction on the nitrocellulose membrane (1-4); the detection line (T line) is coated with anti-human IgM μ-chain monoclonal antibody to specifically capture the immune complex formed between the toxoplasmosis IgM antibody and the colloidal gold T line (1-5) in the sample; the control line (C line) is coated with anti-mouse IgG polyclonal antibody to specifically capture the free colloidal gold C line (1-6) to verify the effectiveness of the detection system.

[0020] The specific preparation method is as follows: 1. Preparation of nitrocellulose membranes: (1) Coating of quality control lines and testing lines: Select YN140 nitrocellulose membrane and cut it into sizes with a width of 2.0cm or more and a length of 30.5cm as needed, for later use; Using 0.01M phosphate buffer as solvent, prepare 0.2 mg / ml of anti-human IgM µ-chain monoclonal antibody solution for detection line coating and 1.5 mg / ml of anti-mouse IgG polyclonal antibody solution for quality control line coating. The antibody-coated surface of the nitrocellulose membrane was labeled using a quantitative spraying method. Anti-human IgM µ-chain monoclonal antibody solution and anti-mouse IgG polyclonal antibody solution were uniformly coated on the membrane surface in parallel directions. The distance between the detection line and the control line was controlled to be 0.5 cm. The coated nitrocellulose membrane was then stored at a constant temperature of 2℃ to 8℃ for later use. (2) Enclosed nitrocellulose membrane: Prepare a blocking treatment soaking solution, which consists of 0.1 mol / L phosphate buffer, 0.5% trehalose, 1% casein, and 0.05% NaN3. Stir the solution evenly during preparation for at least 10 minutes to obtain the blocking treatment soaking solution for later use. The coated nitrocellulose membranes were placed in a treatment tank, and a sealing treatment soaking solution was added. Each coated nitrocellulose membrane was ensured to be completely submerged in the solution for 30 minutes, and the membranes were guaranteed not to move or overlap. The soaked nitrocellulose membranes were obtained after 30 minutes. The soaked nitrocellulose membranes were then removed from the treatment tank and allowed to air dry slightly on gauze to obtain sealed nitrocellulose membranes. (3) Drying the plate: Using tweezers, attach the sealed nitrocellulose membrane to the blank space in the center of the adhesive substrate, avoiding air bubbles. Control the indoor temperature to 18-28℃ and the relative humidity to ≤40%, ensuring air circulation in the drying room and preventing the dehumidifier's air from blowing directly onto the sealed nitrocellulose membrane. The drying time is ≥4 hours to obtain the substrate with the nitrocellulose membrane attached, for later use. 2. Preparation of solid-phase colloidal gold T-wires and colloidal gold C-wires (1) Preparation of colloidal gold complex solution: Prepare a solution containing 5% trehalose, 2% bovine serum albumin, 0.5% trisodium citrate, 0.05% polyethylene glycol, and 0.05% NaN3 by mass. The solution should be stirred thoroughly for more than 30 minutes during preparation. (2) Preparation of colloidal gold: Add 8 ml of chloroauric acid (mass fraction of 2%) to 792 ml of purified water, heat to boiling, and then quickly add 14 ml of trisodium citrate (mass fraction of 1%). The color of the reaction solution first turns black, then turns red. Boil for 5 minutes until the color no longer changes. Then cool at room temperature or in ice water. Finally, dilute to 800 ml with process water, seal with plastic wrap, and obtain colloidal gold solution. (3) Marking the T-line: Measure the required amount of colloidal gold solution using a graduated cylinder, adjust the pH to 7.5-8.0 to obtain the adjusted colloidal gold solution A; the pH adjustment is achieved by adding 0.6% by volume 0.2mol / L potassium carbonate solution to the colloidal gold solution; after stirring the adjusted colloidal gold solution A on a magnetic stirrer for 15 minutes, take TOX recombinant antigen and dilute it with 5% by volume double-distilled water, and add the diluted TOX recombinant antigen to the colloidal gold solution A at a final concentration of 1ug / ml and mix evenly, then stir on a magnetic stirrer for 30 minutes to obtain mixture A; add 0.5‰ casein to mixture A, stir for 30 minutes and centrifuge to collect the precipitate; redissolve the precipitate with colloidal gold redissolved solution at a volume percentage of 3%, and stir on a magnetic stirrer until evenly mixed to obtain the colloidal gold solution for marking the T-line, for later use; Take the colloidal gold solution marked with the T line, redissolve it with colloidal gold reconstitution solution at a volume percentage of 40%, and mix thoroughly on a magnetic stirrer to obtain mixture B; mix mixture B at a concentration of 50 μl / cm 2 The colloidal gold absorbent pads with T-lines are poured onto them to obtain the colloidal gold absorbent pads with T-lines. The colloidal gold absorbent pads with T-lines are placed in a drying room to dry for ≥4 hours. The temperature of the drying room is controlled at 18-28℃ and the relative humidity is ≤40%. Ensure that the air is unobstructed and that the airflow does not blow directly onto the colloidal gold absorbent pads with T-lines. After drying, the colloidal gold absorbent pads with T-lines are placed in an aluminum foil bag containing desiccant, sealed and stored for later use. Note: Colloidal gold is cast because cast colloidal gold can be freely cut to different widths, thus making it easy to adjust the depth and lightness of the product's color.

[0021] (3) Preparation of colloidal gold adsorption pads marked with C-lines: Measure the required amount of colloidal gold solution using a graduated cylinder, and adjust the pH to 6.5-7.0 to obtain adjusted colloidal gold solution B. The pH adjustment involves adding 0.3% (v / v) of 0.2 mol / L potassium carbonate solution to the colloidal gold solution. Stir the adjusted colloidal gold solution B on a magnetic stirrer for 15 minutes. Dilute the mouse monoclonal antibody with 5% (v / v) double-distilled water, and add the diluted mouse monoclonal antibody to the colloidal gold solution B at a final concentration of 11 μg / ml. Mix thoroughly, and then stir on a magnetic stirrer for 30 minutes to obtain mixture C. Add 0.5‰ (w / v) casein to mixture C, stir for 30 minutes, centrifuge, and collect the precipitate. Reconstitute the precipitate with a colloidal gold reconstitution solution at 3% (v / v), and stir on a magnetic stirrer until homogeneous to obtain the colloidal gold solution marked with line C, for later use. Take the colloidal gold solution marked with line C above, redissolve it with colloidal gold reconstitution solution at 40% by volume, mix thoroughly on a magnetic stirrer to obtain mixture D; mix mixture D at 50 μl / cm 2 The colloidal gold absorbent pad is poured onto the colloidal gold absorbent pad to obtain the C-line marking. The C-line marking colloidal gold absorbent pad is placed in a drying room to dry for ≥4 hours. The temperature of the drying room is controlled at 18-28℃ and the relative humidity is ≤40%. Ensure that the air is unobstructed and that the airflow does not blow directly onto the C-line marking colloidal gold absorbent pad. After drying, the C-line marking colloidal gold absorbent pad is placed in an aluminum foil bag containing desiccant, sealed and stored for later use. Note: Colloidal gold is cast because cast colloidal gold can be freely cut to different widths, thus making it easy to adjust the depth and lightness of the product's color.

[0022] 3. Assembly and cutting: Take the substrate on which the nitrocellulose membrane is attached. Cut the colloidal gold adsorption pad marked with the C line into strips of 0.5cm x 30cm, 0.5cm wide, and attach them to the substrate, ensuring an overlap of approximately 1mm with the nitrocellulose membrane. Cut the colloidal gold adsorption pad marked with the T line into strips of 0.5cm x 30cm, 0.5cm wide, and attach them behind the C line of the solidified colloidal gold along the chromatography direction, ensuring an overlap of approximately 1mm with the C line. Composite the absorbent pad to the upper end of the substrate membrane of the nitrocellulose membrane, overlapping it with the nitrocellulose membrane by approximately 1mm. Composite the sample loading pad to the lower end of the T line of the solidified colloidal gold, overlapping it by approximately 1mm. Mark the pads and set them aside. According to the corresponding reaction device, the assembled and prepared substrate is cut into strip test strips, and the test strips are marked for later use.

[0023] Place the prepared, qualified test strips into the qualified plastic card, with the handle paper on top, cover the card, and pinch it tightly.

[0024] Example 2. Screening of nitrocellulose membranes This embodiment tests the chromatography speed of nitrocellulose membranes from different manufacturers and with different pore sizes to obtain a nitrocellulose membrane that is more suitable for detecting Toxoplasma gondii antibodies.

[0025] 1. Experimental Materials and Methods (1) Test materials: NC membranes include: Milipore-135 (Milipore - imported), YN100 (Shantou Yineng), YN120 (Shantou Yineng), YN140 (Shantou Yineng), and YN180 (Shantou Yineng); Reagents: physiological saline; gold solution obtained by adding 0.5‰ casein to colloidal gold solution.

[0026] (2) Experimental design: Prepare 30 o A slanted water bath was used to immerse a 4 cm long nitrocellulose membrane (hereinafter referred to as NC membrane) in 500 μl of physiological saline or gold solution (the membrane length in the immersion solution is about 4 mm). A stopwatch was used to record the capillary chromatography time under various conditions (three sets of experiments were performed for each type of membrane).

[0027] 2. Test Results: The results are shown in Table 1.

[0028] Table 1: Test results of nitrocellulose membranes (NC membranes) from different manufacturers

[0029] According to the quality control standards, the required chromatography speed is 150 seconds on a 4cm NC membrane. The chromatography times of four nitrocellulose membranes—YN100, YN120, YN140, and MILLIPORE-135—all meet the requirements. MILLIPORE-135 is an imported membrane, which is relatively expensive and less readily available than domestically produced membranes. In contrast, the four NC membranes from Shantou Yineng are relatively cheaper than imported membranes, and their procurement is more convenient and faster. Therefore, Shantou Yineng's NC membranes were chosen for preparing the test strips.

[0030] To prevent non-specific binding during chromatography, the NC membrane is blocked. However, since the blocked proteins fill some of the pores in the NC membrane, it will affect the chromatographic performance of the membrane. Therefore, it is necessary to measure the chromatographic performance of the blocked NC membrane. Each type of NC membrane (YN100, YN120, YN140) was divided into three groups, and each group was blocked with membrane blocking buffer (30 min), dried, and then tested. Using a 30° inclined water bath, a 4 cm long piece of membrane was immersed in 500 μl of solution, with a 4 mm immersion length (three groups of tests were performed for each type of membrane). The chromatography time was measured and is shown in Table 2.

[0031] Table 2: Detection results of different nitrocellulose membranes (NC membranes) after sealing

[0032] By comparing several membranes (YN100, YN120, YN140), it was found that the Shantou nitrocellulose membrane, after being sealed, still had a chromatography time of ≤150s, which met the quality control standards of NC membranes. It also had a clean, smooth, and flat appearance, a moderate pore size, and a reasonable price. However, only YN140 had a clean background, so YN140 was selected to prepare the test strips.

[0033] Example 3. Screening for the size of colloidal gold particles in colloidal gold In colloidal gold, the size of the gold particles has a significant impact on the sensitivity and specificity of the reagent. Larger gold particles result in higher detection sensitivity but lower specificity; conversely, smaller gold particles result in lower detection sensitivity but higher specificity. Therefore, the appropriate selection of gold particle size greatly affects reagent quality. This experimental example uses the trisodium citrate reduction method to prepare colloidal gold solutions. By varying the amount of reducing agent and preparation conditions, colloidal gold solutions containing different sizes of colloidal gold particles were obtained.

[0034] Specific operating steps: Take four 100ml Erlenmeyer flasks, add 1ml of 2% chloroauric acid to each flask and add it to 99ml of process water. Heat to boiling, and then quickly add 4.00ml, 3.00ml, 2.00ml, and 1.50ml of 1% trisodium citrate aqueous solution respectively. The color will first turn black, then red. Boil for another 5 minutes until the color no longer changes. Then cool at room temperature or in ice water. Finally, bring the volume to 100ml with process water, seal with plastic wrap, and label.

[0035] Table 3: Preparation and properties of four colloidal gold compounds

[0036] Since the size of colloidal gold particles determines the color and casein content of the solution, larger particles result in a deeper color, gradually changing from red to purple; smaller particles produce a brighter red solution. To improve reagent sensitivity, colloidal gold solutions with larger particles that have not aggregated or precipitated are generally selected. Therefore, optimization experiments were conducted using colloidal gold with a color between red and purplish-red, i.e., a particle size between 30nm and 50nm. The colloidal gold was prepared as follows: Table 4: Preparation and properties of five types of colloidal gold

[0037] All five colloidal gold solutions were red, clear, and free of turbidity. After cooling to room temperature, 3 ml of colloidal gold was taken and placed in a cuvette. The maximum absorption peak A was measured at 520 nm to 560 nm using a UV spectrophotometer.

[0038] The above-mentioned colloidal gold was labeled and assembled into TOX test strips. Performance indicators were then tested, and the results are shown in Table 5. Table 5: Performance Indicators of TOX Test Strips Prepared from Five Colloidal Golds

[0039] Note: -: indicates the test line is not colored; +: indicates the test line is colored and the color is clear. Ten parallel tests were performed on the precision reference standard R.

[0040] The results above show that: When 1% trisodium citrate was added in 16 ml of colloidal gold 1: the particles were small, the positive color was light, and the sensitivity was low.

[0041] When 1% trisodium citrate was added in 15ml of colloidal gold 2: the particles were relatively large, the sensitivity was acceptable, and there were no non-specific reactions.

[0042] When 1% trisodium citrate was added in 14 ml of colloidal gold 3: the particles were larger, the positive samples showed deeper color, the sensitivity was better, and there were no non-specific reactions.

[0043] When 13 ml of trisodium citrate (1%) was added (colloidal gold 4): due to the large particle size and deep color development, false positives occurred in sensitivity L3.

[0044] When 1% trisodium citrate was added in 12ml (colloidal gold 5): due to the large particle size and deep color development, false positives occurred at sensitivity L3.

[0045] Therefore, the preparation methods for colloidal gold 1-colloidal gold 3 are more suitable.

[0046] Example 4. Optimal pH for colloidal gold plating during the preparation of colloidal gold adsorbent pads marked with T lines: Take 20 mL of colloidal gold solution and distribute it evenly into 10 glass test tubes, ensuring that the volume of colloidal gold solution in each test tube is 2.0 mL. Adjust the pH to 5.5–9.5 with 0.2 M K₂CO₃, stir well, and then add a uniform concentration of qualified TOX antigen to each test tube. The labeling results are shown in Table 6. Table 6: Colloidal gold labeling effect of TOX antigen at different pH values

[0047] -: The test line does not develop color; +: The test line develops color and the color is clear; Ten parallel tests are performed on the precision reference material R.

[0048] The results showed that colloidal gold solutions labeled with a pH < 7.5 exhibited fine particulate precipitates after centrifugation, while those labeled with a pH > 8.0 showed poor protein binding, low titer, and weak color development, resulting in undetectable P3-P5 levels in positive control serum. In contrast, colloidal gold solutions labeled with a pH between 7.5 and 8.0 performed well, showing no precipitation and meeting the required sensitivity. Therefore, the optimal pH for TOX labeling is 7.5 ≤ pH ≤ 8.0.

[0049] Example 5. Screening of antigen and antibody concentrations This embodiment examines the application effects of the provided TOX-Ag and anti-human IgM μ-chain monoclonal antibody in a colloidal gold system.

[0050] In this embodiment, the test antigen and antibody were sourced from: TOX-Ag (TOX antigen) and anti-human IgM μ chain monoclonal antibody, which were purchased from Shenzhen Feipeng Biotechnology Co., Ltd.

[0051] 1. Results of activity assay and purity identification: The purity results of the SDS-PAGE electrophoresis method are shown in Table 7. Table 7: Results of Antigen Activity and Purity Identification

[0052] 2. Sample small-scale test method Screening of IgM μ-chain monoclonal antibody concentration: The IgM μ-chain monoclonal antibody coated with NC membrane in the coating steps of the control line and the test line was set to 0.1 mg / ml to 1.5 mg / ml, and the other steps remained unchanged.

[0053] Screening of TOX antigen concentration: The final concentration of TOX antigen in the preparation of solid-phase colloidal gold T-wires and colloidal gold C-wires was set to 0.5 ug / ml-2.0 ug / ml, respectively, with the remaining steps unchanged. The TOX enterprise internal control panel was tested. The test results are shown in Table 8: Table 8: Pairing results of antigen-antibody concentration optimization test

[0054] -: Negative reaction (only the control line is clearly visible); +: Positive reaction, the test line is relatively clear. Ten parallel tests were performed on the precision reference standard R.

[0055] 4. Test conclusions: Based on the above results, the ideal concentration of colloidal gold-labeled TOX-Ag is about 1 μg / ml, and the coating concentration of anti-human IgM μ chain monoclonal antibody is at least 0.10 mg / ml.

[0056] Further concentration optimization was then performed, and the results are shown in Table 9.

[0057] Table 9: Paired Results of Antigen-Antibody Concentration Optimization Test

[0058] -: Negative reaction (only the control line is clearly visible); +: Positive reaction, the test line is relatively clear. Ten parallel tests were performed on the precision reference standard R.

[0059] 5. Test conclusion: Based on the above results, the ideal concentration of colloidal gold-labeled TOX antigen is 1.0 ug / ml, and the coating concentration of anti-human IgM μ chain monoclonal antibody is 0.2 mg / ml.

[0060] Example 6. Selection of drying time for a carrier adsorbed with colloidal gold The drying times for the colloidal gold adsorption pads used to mark the T-lines in the preparation of solid-phase colloidal gold T-lines and colloidal gold C-lines were set to 3 hours, 4 hours, and 5 hours, respectively, while other steps remained unchanged. The TOX-IgM internal control sample was tested, and the results are shown in Table 10. Table 10: Effect of different drying times on TOX results

[0061] -: The test line is not colored; +: The test line is clearly colored. Ten parallel tests were performed on the precision reference standard R.

[0062] The results showed that the optimal drying time for the colloidal gold adsorption pads marked with T lines was more than 4 hours in the drying room. If the drying time was too short, the stability would be poor and the detection sensitivity would decrease after 3 days. If the drying time was too long, the efficiency would be low. Therefore, the optimal drying time for the colloidal gold adsorption pads marked with T lines was 4 hours in the drying room.

[0063] Example 7. Optimization of temperature and humidity conditions during production. During the production process, temperature is a key factor affecting the performance and shelf life of the colloidal gold-labeled T and C lines. The test strips are exposed to the air for an average of 30-60 minutes from the time they are pasted on to the time they are packaged into aluminum foil bags containing desiccant. Therefore, temperature and humidity must be strictly controlled during the production process.

[0064] The following tests were conducted to determine the minimum temperature and humidity requirements for the drying chamber and the laminating, cutting, and inner packaging chambers. In the temperature- and humidity-controlled drying chamber, after the temperature and relative humidity stabilized within the required experimental range (20%–50%), 400 TOX-IgM test strips (qualified and ready for packaging) were quickly placed inside. After 1 hour, they were removed and placed in an aluminum foil bag containing desiccant. After 3 days at (2–30)℃, the TOX-IgM control samples were tested, and the results are shown in Table 11. Table 11: Effects of drying chamber temperature and relative humidity on TOX-IgM test strips

[0065] -: The test line is not colored; +: The test line is clearly colored. Ten parallel tests were performed on the precision reference standard R.

[0066] The results showed that excessively high temperatures (above 30℃) and humidity (above 40%) affected the test strips, decreasing not only sensitivity but also the color development of positive and precision reference samples. The experiment also observed that the flow rate of colloidal gold marking the T-line on the membrane was affected, resulting in an uneven mobile phase. During the experiment, the test strips were stored overnight at 25℃ and (20-40)% humidity; after three days, the sensitivity and specificity remained unchanged. In practice, controlling humidity at 20% is difficult; therefore, humidity ≤ 40% and temperature 25±5℃ are the optimal conditions for the drying chamber, lamination, cutting, and inner packaging chamber.

[0067] Example 8. Selection of the time for observing the results after the reaction of the reagent To determine the optimal time range for diagnostic efficacy, 60 μL of TOX control serum was added to the test strips prepared in Example 1, and the results were observed at 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 minutes after sample addition. The results are shown in Table 12. Table 12: Effect of different observation times on the diagnostic efficacy of TOX

[0068] -: The test line is not colored; +: The test line is clearly colored. Ten parallel tests were performed on the precision reference standard R.

[0069] When performing TOX internal control tests, some positive and sensitivity reference samples show negative reactions within 3-5 minutes, which does not meet the testing requirements; after 25 minutes, some negative reference samples show false positive reactions, which also does not meet the testing requirements. Therefore, it is best to observe and judge the results within 15-20 minutes after sample addition.

[0070] Example 9. Optimization of Sample Loading Amount Take the assembled test strip card, place it horizontally, and add 60 μL, 70 μL, 80 μL, 90 μL, and 100 μL of TOX sample to the sample inlet end respectively. Observe the results. The sensitivity and specificity of the test strip card are shown in Table 13. Table 13: Selection Criteria for Different TOX Doses

[0071] -: The test line is not colored; +: The test line is clearly colored. Ten parallel tests were performed on the precision reference standard R.

[0072] When preparing TOX internal control samples, if the sample loading volume is 100 μL, sample overflow occurs; if the sample loading volume is less than 70 μL, the mobile phase cannot reach the quality control zone. Therefore, a sample loading volume of 70-90 μL is selected.

[0073] Experimental Example 1. Specific Detection Steps (1) Bring the test card (without opening the foil bag), the sample to be tested, and the diluent to room temperature (20-30℃); (2) Dilute the serum or plasma sample to be tested with the diluent at a ratio of 1:50 (e.g., add 20 μl of serum or plasma directly to 980 μl of diluent and mix well); (3) Remove the test card from the foil bag and label it with the patient or sample number, then place the test card flat on the table; (4) Use a disposable plastic pipette to draw up the diluted serum or plasma and add 2 drops into the sample well; (5) Positive samples can be detected within 1-15 minutes. It is recommended to observe and record the experimental results after 15 minutes. Results displayed after 20 minutes have no clinical significance. Note: Test strips removed from the aluminum foil packaging should be used as soon as possible within 1 hour if the room temperature is below 30°C and the relative humidity is less than 65%; if the room temperature is above 30°C or the relative humidity is greater than 65%, they should be used immediately.

[0074] The test results are as follows: (1) Invalid: No purple-red band appeared at the control line, indicating incorrect operation or kit failure; (2) Negative (-): Only one purple-red band appeared at the control line on the test strip. This indicates that Toxoplasma gondii specific antibody IgM was not detected in the sample; (3) Positive (+): Two purple-red bands appeared at the test line and the control line on the test strip. This indicates that Toxoplasma gondii specific antibody IgM was present in the sample.

[0075] This invention applies the principles of capture and immunochromatography to qualitatively detect Toxoplasma gondii (TOX) IgM antibodies in human serum (or plasma) in vitro, and is suitable for the clinical auxiliary diagnosis of Toxoplasma gondii infection.

[0076] Example 2: Performance testing of the test strip 1. Detection range (analytical sensitivity): This kit qualitatively detects Toxoplasma gondii IgM antibodies in human serum (or plasma) for clinical auxiliary diagnosis. The sensitivity requirements for this kit are: minimum detection limit: L1 should detect positive, L2 may detect positive or negative, and L3 should detect negative. Specific experimental results are shown in Table 14. Table 14: Serum Detection Results of TOX-IgM Antibody Sensitivity

[0077] -: No color development on the test line, indicating a negative result; +: Color development on the test line, indicating a positive result.

[0078] 2. Positive reference sample compliance rate: Three batches of Toxoplasma gondii antibody (IgM) detection kits (colloidal gold method) were used to detect positive reference samples in the TOX-IgM enterprise internal control reference materials. The positive reference sample concordance rate was 100%, and the results are shown in Table 15: Table 15: Detection Results of Positive Reference Samples

[0079] 3. Negative reference sample compliance rate: Three batches of Toxoplasma gondii antibody (IgM) detection kits (colloidal gold method) were used to test the negative reference samples in the TOX-IgM enterprise internal control reference materials. The negative reference sample compliance rate was 100%, and the results are shown in Table 16. Table 16: Test Results of Negative Reference Samples

[0080] 4. Detection results of tests interfering with endogenous substances (hemolysis, jaundice samples, rheumatoid factor (RF factor), hyperlipidemia samples, lupus erythematosus positive, HBsAg positive, HCV positive, HEV positive, hepatitis G positive, TP positive, HIV positive, etc.). (1) Results of the detection of 6 rheumatoid factor (Rf) positive sera: The Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1 and the Toxoplasma gondii IgM antibody diagnostic kit (enzyme-linked immunosorbent assay) produced by Zhuhai Haitai Co., Ltd. were used to detect six Rf-positive sera in parallel. The results are shown in Table 17. Table 17: Results of parallel detection of 6 Rf factor-positive sera using the Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) from Nantong Yishi Company and the TOX-IgM kit from Zhuhai Haitai Company.

[0081] As can be seen from the results in the table above, the detection results of the two methods are consistent, and no false positives were found, indicating that the Rf factor does not interfere with the detection of Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1.

[0082] (2) Detection results of two serum samples that were positive for lupus erythematosus: Two lupus-positive serum samples were tested in parallel using the Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1 and the Toxoplasma gondii IgM antibody diagnostic kit (enzyme-linked immunosorbent assay) produced by Zhuhai Haitai Co., Ltd. The results are shown in Table 18: Table 18: Results of parallel detection of two lupus-positive serum samples using the Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) from Nantong Yishi Company and the TOX-IgM kit from Zhuhai Haitai Company.

[0083] As can be seen from the results in the table, the detection results of the two methods are consistent, and no false positives were found, indicating that the positive result of lupus erythematosus does not interfere with the detection of Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1.

[0084] (3) Detection results of 7 HBsAg-positive serum samples: The Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1 and the Toxoplasma gondii IgM antibody diagnostic kit (enzyme-linked immunosorbent assay) produced by Zhuhai Haitai Company were used to test seven HBsAg positive serum samples in parallel. The specific test results are shown in Table 19.

[0085] Table 19: Results of parallel testing of 7 HBsAg-positive serum samples using the Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) from Nantong Yishi Company and the TOX-IgM kit from Zhuhai Haitai Company.

[0086] The results in the table show that the detection results of the two methods are consistent, and no false positives were found. This indicates that HBsAg positivity does not interfere with the detection of Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1.

[0087] (4) Detection results of 6 HCV-positive serum samples: The Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1 and the Toxoplasma gondii IgM antibody diagnostic kit (enzyme-linked immunosorbent assay) produced by Zhuhai Haitai Company were used to test six HCV-positive serum samples in parallel. The specific test results are shown in Table 20.

[0088] Table 20: Results of parallel testing of 6 HCV-positive serum samples using the Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) from Nantong Yishi Company and the TOX-IgM kit from Zhuhai Haitai Company.

[0089] As can be seen from the results in the table, the detection results of the two methods are consistent, and no false positives were found, indicating that HCV positivity does not interfere with the detection of Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1.

[0090] (5) Detection results of 6 serum samples that were positive for HEV-IgM antibodies Six HEV-IgM positive serum samples were tested in parallel using the Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1 and the Toxoplasma gondii IgM antibody diagnostic kit (enzyme-linked immunosorbent assay) produced by Zhuhai Haitai Company. The specific test results are shown in Table 21.

[0091] Table 21: Results of parallel testing of 6 HEV-IgM positive serum samples using the Nantong Yishi Company's Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) and the Zhuhai Haitai Company's TOX-IgM kit.

[0092] The results in the table show that the detection results of the two methods are consistent, and no false positives were found. This indicates that HEV-IgM positivity does not interfere with the detection of Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1.

[0093] (6) Detection results of 6 serum samples that were positive for hepatitis G virus: The Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1 and the Toxoplasma gondii IgM antibody diagnostic kit 2-18 (enzyme-linked immunosorbent assay) produced by Zhuhai Haitai Company were used to detect six HGV-IgM positive serum samples in parallel. The specific test results are shown in Table 22.

[0094] Table 22: Results of parallel testing of 6 serum samples positive for hepatitis G using the Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) from Nantong Yishi Company and the TOX-IgM kit from Zhuhai Haitai Company.

[0095] The results in the table show that the detection results of the two methods are consistent, and no false positives were found, indicating that hepatitis G positivity does not interfere with the detection of Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1.

[0096] (7) Detection results of 6 TP-positive serum samples: The Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1 and the Toxoplasma gondii IgM antibody diagnostic kit (enzyme-linked immunosorbent assay) produced by Zhuhai Haitai Company were used to test six TP-positive serum samples in parallel. The specific test results are shown in Table 23.

[0097] Table 23: Results of parallel testing of 6 TP-positive serum samples using the Nantong Yishi Company's Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) and the Zhuhai Haitai Company's TOX-IgM kit.

[0098] The results in the table show that the detection results of the two methods are consistent, and no false positives were found, indicating that TP positivity does not interfere with the detection of Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1.

[0099] (8) Test results of two HIV-positive serum samples: Two HIV-positive serum samples were tested in parallel using the Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1 and the Toxoplasma gondii IgM antibody diagnostic kit (enzyme-linked immunosorbent assay) produced by Zhuhai Haitai Company. The specific test results are shown in Table 24.

[0100] Table 24: Results of parallel testing of two HIV-positive serum samples using the Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) from Nantong Yishi Company and the TOX-IgM kit from Zhuhai Haitai Company.

[0101] The results in the table show that the two methods yielded consistent results with no false positives, indicating that HIV positivity did not interfere with the detection of Toxoplasma gondii antibody (IgM) using the colloidal gold method prepared in Example 1.

[0102] (9) Test results of 6 samples with high blood lipids: The Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1 and the Toxoplasma gondii IgM antibody diagnostic kit (enzyme-linked immunosorbent assay) produced by Zhuhai Haitai Company were used to test six serum samples with high blood lipids in parallel. The specific test results are shown in Table 25.

[0103] Table 25: Results of parallel testing of 6 hyperlipidemia samples using the Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) produced by Nantong Yishi Company and the Toxoplasma gondii IgM antibody diagnostic kit (enzyme-linked immunosorbent assay) produced by Zhuhai Haitai Company.

[0104] The results in the table show that the two methods yielded consistent results with no false positives, indicating that high blood lipids did not interfere with the detection of Toxoplasma gondii antibody (IgM) using the colloidal gold method prepared in Example 1.

[0105] (10) Detection results of 6 jaundice samples (bilirubin ≤213.27μmol / L): Six jaundice serum samples were tested in parallel using the Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1 and the Toxoplasma gondii IgM antibody diagnostic kit (enzyme-linked immunosorbent assay) produced by Zhuhai Haitai Company. The specific test results are shown in Table 26. Table 26: Results of parallel testing of 6 jaundice serum samples using the Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) from Nantong Yishi Company and the TOX-IgM kit from Zhuhai Haitai Company.

[0106] The results in the table show that the two methods yielded consistent results with no false positives, indicating that jaundice did not interfere with the detection of Toxoplasma gondii antibody (IgM) using the colloidal gold method prepared in Example 1.

[0107] (11) Detection results of 6 hemolyzed samples (hemoglobin concentration ≤890mg / dl): Six hemolyzed samples were tested in parallel using the Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1 and the Toxoplasma gondii IgM antibody diagnostic kit (enzyme-linked immunosorbent assay) produced by Zhuhai Haitai Company. The specific test results are shown in Table 27.

[0108] Table 27: Results of parallel testing of 6 hemolyzed samples using the Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) from Nantong Yishi Company and the TOX-IgM kit from Zhuhai Haitai Company.

[0109] As can be seen from the results in the table, the detection results of the two methods are consistent, and no false positives were found, indicating that hemolysis of the specimens did not interfere with the detection of Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method) prepared in Example 1.

[0110] (12) Effects on different anticoagulants Twenty whole blood samples were collected from a clinical hospital (10 confirmed positive and 10 confirmed negative). All samples were divided into four portions. One portion was centrifuged to obtain a serum sample. Another portion was mixed thoroughly with heparin sodium to obtain a plasma sample (containing 0.2 mg / ml of heparin sodium). The remaining two portions were mixed thoroughly with EDTA-2Na (containing 150 mmol / L of EDTA-2Na) and sodium citrate, respectively, to obtain plasma samples (containing 0.25% sodium citrate). The Toxoplasma gondii antibody (IgM) detection kit prepared in Example 1 (colloidal gold method) was used for detection. The results are summarized in Table 28. Table 28: Results of parallel testing of 20 serum samples using the Yishi Biosciences Toxoplasma gondii antibody (IgM) detection kit (colloidal gold method)

[0111] The results in Table 28 indicate that the detection results of Toxoplasma gondii antibody (IgM) test kit (colloidal gold method) produced by Yishi Biotechnology Co., Ltd. are not affected by plasma samples anticoagulated with conventional anticoagulants compared with serum samples. Example 2: Sampling and testing of test strips Random sampling shall be conducted from the same batch, with a minimum sample size of no less than three times the amount to be tested.

[0112] After sampling, the samples to be inspected are placed in the yellow inspection area. Samples that can be inspected on the same day are inspected on the same day; otherwise, they must be inspected by the next day. The remaining paper strips that pass the inspection can be used as samples and temporarily placed in the qualified product area. Then, based on the production volume and sample retention requirements, a certain number of samples are randomly selected from the production line to be used as samples of the finished products.

[0113] 1. The sample dilution solution is 0.01M phosphate buffer (pH=7.4). 2. Physical Properties: Appearance Inspection: Remove the test strip and visually inspect it. The strip should be clean, intact, free of burrs, damage, and contamination; the material should be firmly adhered. Test Strip Width: Take 3 test strips and measure the width of the film on the strip using a ruler. Liquid Migration Speed: Take 3 test strips and operate according to the instructions. Start timing with a stopwatch (accuracy 0.01s) from the moment the test strip is immersed in the sample liquid until the liquid reaches the boundary between areas E and F as shown in the diagram below. Record the time taken as (t). Measure the length (A area + B area + E area) with a vernier caliper (precision 0.02mm) and record it as (L). Calculate L / t, which is the migration speed. Repeat the measurement for 3 test strips and take the average value. The result should meet the requirement that the liquid migration speed is not less than 10mm / min. Figure 3 . 3. Test the minimum detection limit: Use 3 of the company's internal control sensitivity reference samples L1~L3 for testing, and observe the results within 15 minutes. The minimum detection limit is: L1 should be positive, L2 can detect positive or negative, and L3 should be negative.

[0114] 4. Test the compliance rate of negative reference materials: Use 10 internal control negative reference materials N1~N10 for testing, and determine the results within 15 minutes. The compliance rate of 10 negative reference materials (- / -) is 10 / 10.

[0115] 5. Verification of positive reference sample compliance rate: Use 5 positive reference samples P1~P5 from the company's internal control for testing, and determine the results within 15 minutes. The compliance rate of the 5 positive reference samples is (+ / +) 5 / 5.

[0116] 6. Test repeatability: Use the company's internal control repeatability reference material for testing, perform 10 parallel tests, and determine the results within 15 minutes. All results must be positive and the anti-TOX strength must be consistent.

[0117] 7. Inter-batch variation test: Take three batches of test strips and test repeatability separately. The test results of the three batches should be consistent.

[0118] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a test strip for detecting Toxoplasma IgM antibody, characterized in that, The preparation method comprises: a preparation step of a nitrocellulose membrane: coating a detection line and a quality control line on a nitrocellulose membrane to obtain a coated nitrocellulose membrane; soaking the coated nitrocellulose membrane with a blocking treatment soaking solution to obtain a blocked nitrocellulose membrane; and pasting the blocked nitrocellulose membrane on a substrate; a preparation step of a colloidal gold adsorption pad for marking the C line or the T line: adjusting the pH of a colloidal gold solution containing chloroauric acid and trisodium citrate to obtain an adjusted colloidal gold solution; adding the adjusted colloidal gold solution into TOX antigen or mouse monoclonal antibody respectively to obtain a mixture; adding casein into the mixture, and then stirring and centrifuging to obtain a precipitate; resuspending the precipitate with a colloidal gold resuspension solution, and pouring it on a colloidal gold adsorption pad to obtain a colloidal gold adsorption pad for marking the C line or the T line; an assembly step: sequentially arranging a sample pad, a colloidal gold adsorption pad for marking the C line, a colloidal gold adsorption pad for marking the T line, a nitrocellulose membrane and a water absorption pad on the substrate in the direction of liquid chromatography to obtain a test strip for detecting Toxoplasma IgM antibody.

2. The preparation method according to claim 1, characterized in that, In the preparation step of the nitrocellulose membrane, the detection line is coated by IgM mu chain monoclonal antibody. Preferably, the concentration of the IgM mu chain monoclonal antibody is 0.1-0.2 mg / ml.

3. The preparation method according to claim 1, characterized in that, In the preparation step of the nitrocellulose membrane, the quality control line is coated by anti-mouse IgG polyclonal antibody. Preferably, the concentration of the anti-mouse IgG polyclonal antibody is 1-1.5 mg / ml.

4. The preparation method according to claim 1, characterized in that, In the preparation step of the colloidal gold adsorption pad for marking the C line or the T line, the pH of the colloidal gold solution used for the colloidal gold adsorption pad for marking the C line is 6.5-7.

0.

5. The method of claim 1, wherein the step of forming the first and second layers is performed by a process selected from the group consisting of: sputtering, evaporation, and chemical vapor deposition. In the preparation step of the colloidal gold adsorption pad for marking the C line or the T line, the pH of the colloidal gold solution used for the colloidal gold adsorption pad for marking the T line is 7.5-8.

0.

6. The method of claim 1, wherein the step of forming the first and second layers is performed by a process selected from the group consisting of: sputtering, evaporation, and chemical vapor deposition. In the preparation step of the colloidal gold adsorption pad for marking the C line or the T line, the antigen marked by the colloidal gold adsorption pad for marking the C line is mouse monoclonal antibody. Preferably, the concentration of the mouse monoclonal antibody is 10-12 ug / ml.

7. The method of claim 1, wherein the step of forming the first and second layers is performed by a process selected from the group consisting of: sputtering, evaporation, and chemical vapor deposition. In the preparation step of the colloidal gold adsorption pad for marking the C line or the T line, the antigen marked by the colloidal gold adsorption pad for marking the T line is TOX antigen. Preferably, the concentration of the TOX antigen is 1-2 ug / ml.

8. The process according to any one of claims 1 to 7, characterized in that, The environmental conditions during the preparation process include: temperature of 20-30℃, and humidity of ≤ 40%.

9. A test strip for detecting Toxoplasma IgM antibody, characterized in that, The test strip is prepared by using the preparation method according to any one of claims 1-8.

10. The test strip of claim 9, wherein, The test strip is provided with a sample pad, a nitrocellulose membrane, a water absorption pad, a solid-phase colloidal gold T line, a solid-phase colloidal gold C line, a detection line and a quality control line.

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