A quick quantitative detection card for abrin

Abstract

The application discloses a quick quantitative detection card for abrin, which comprises a PVC base plate, a water absorption pad, a nitrocellulose membrane, a marker pad, a sample pad and a diluent pad in a shell; the nitrocellulose membrane is fixed on the PVC base plate, the water absorption pad is connected to the left end of the nitrocellulose membrane, the diluent pad is connected to the right end of the nitrocellulose membrane, the marker pad is arranged on the right part of the middle part of the nitrocellulose membrane, the sample pad is arranged on the marker pad, the quality control line and the detection line are arranged between the water absorption pad and the marker pad on the nitrocellulose membrane; the window holes corresponding to the detection line and the quality control line are formed on the upper shell, the quantitative identification line is arranged on the side of the window hole corresponding to the detection line and the marker pad, the sample adding hole corresponding to the sample pad is formed, and the cleaning hole corresponding to the diluent pad is formed. The chromatographic immunization method of the application adds the sample through the sample adding hole, adds the diluent through the cleaning hole and controls the sample lateral flow liquid amount through the quantitative identification line, so that the sensitivity and the precision of the detected object are improved, and the quick and simple characteristics of the quick diagnosis detection method are retained.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a rapid quantitative detection card for abreast toxin. Background Technology

[0002] Abrin toxin (AT) is a toxic protein extracted from the seeds of the legume vine *Abrus precatorius*. Its molecular structure and mechanism of poisoning are extremely similar to ricin toxin (RT). With a relative molecular weight between 63 and 67 kDa, it exhibits an AB chain structure and is a type II ribosome-inactivating protein, making it highly toxic. The LD50 of AT in mice is 0.04 μg / kg, while the lethal dose in humans is 0.1–1 μg / kg, with an ingested lethal dose of 5.0–7.0 μg / kg, which is more than 70 times higher than that of ricin toxin (mouse LD50 3.0 μg / kg).

[0003] However, since abrinogen lacks specific chemical groups, it is difficult to identify using physical and biochemical methods, and can only rely on immunological detection methods. Among these, ELISA is the most classic international method, with sensitivity reaching ng / mL, but its application is relatively limited due to its complex operation, large equipment size, and long processing time. Colloidal gold immunochromatographic test strips utilize the visual localization properties of colloidal gold labeling technology, overcoming the limitations of dedicated instruments, but can only achieve semi-quantitative detection, and its sensitivity is also relatively low. Therefore, seeking new rapid and effective identification methods for abrinogen is of great practical value. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a dual-well quantitative detection card for abrin toxins.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] First, this invention provides a test strip for absinthecetium lappa toxin detection, comprising a PVC base plate, an absorbent pad, a nitrocellulose membrane, a marker pad, a sample pad, and a diluent pad; the nitrocellulose membrane is fixed on the PVC base plate, with one end of the nitrocellulose membrane overlapping the absorbent pad and the other end overlapping the diluent pad; the marker pad is placed on the nitrocellulose membrane and located in front of the diluent pad, the direction of the absorbent pad being defined as forward; the sample pad is disposed on the marker pad; a detection line (T line) and a control line (C line) are sequentially disposed on the nitrocellulose membrane from the marker pad to the absorbent pad; a quantitative identification line (M line) is disposed between the marker pad and the detection line (T line);

[0007] The marker pad is coated with marker-labeled chicken IgY antibody and marker-labeled abrin toxin polyclonal antibody; the detection line is coated with abrin toxin polyclonal antibody, and the control line is coated with goat anti-chicken IgY antibody IgG.

[0008] The labeling agents are applicable to colloidal gold, fluorescein, lanthanide fluorescence, colored latex, quantum dots, upconversion fluorescent substances, magnetic fluorescent microspheres, as well as enzyme labels and chemiluminescent substances; specifically, they can be lanthanide fluorescent microspheres.

[0009] Preferably, a corresponding filter pad, such as a blood filter pad, can be provided on the sample pad.

[0010] Preferably, the marker pad is also coated with pigment to indicate the side flow process of the sample solution to be tested, which is convenient for visual observation or automatic monitoring and control by the instrument.

[0011] Preferably, when the pigment is rinsed clean by the diluent and flows back into the detection window after the diluent has been exhausted, the result interpretation should be stopped.

[0012] Preferably, the overlap width between the nitrocellulose membrane and the absorbent pad is 1-2 mm; the overlap width between the nitrocellulose membrane and the diluent pad is 1-2 mm. The marker pad is placed in front of the diluent pad, leaving a 1-10 mm gap of exposed nitrocellulose membrane between the marker pad and the diluent pad.

[0013] Preferably, the quantitative marking line (M line) is placed on a viewing window between the T line and the marker pad, which facilitates visual inspection and automatic detection instrument identification, so as to control the timing of adding the diluent.

[0014] The quantitative label line (M line) setting includes: meeting the set sample addition amount, meeting the set delay time for the first immune reaction after sample addition, the sample trace moving to the designated quantitative label line (M) position on the shell, or the sample trace appearing in the detection window.

[0015] The present invention also provides a two-well quantitative detection card for abrin toxin.

[0016] The present invention provides a dual-well quantitative detection card for abrinogen toxin, comprising an outer shell and a rapid test strip for abrinogen toxin disposed within the outer shell; the outer shell includes an upper cover and a lower cover, the upper cover and the lower cover being fastened together; a detection window is provided on the upper cover in the interval corresponding to the detection line "T" and the control line "C" in the test strip, a sample application hole is provided corresponding to the sample pad, and a cleaning hole is provided corresponding to the diluent pad; a "quantitative identification line" is provided between the edge of the detection window near the sample application hole and the "T" line, and a mark is provided on the corresponding outer shell.

[0017] Preferably, after adding the sample solution, diluent is added to the cleaning well after the liquid trace reaches the quantitative marker line (M line). The quantitative marker line (M line) setting includes: meeting the set sample addition volume, meeting the set delay time for the first immune reaction after sample addition, the sample trace moving to the designated quantitative marker line (M line) position on the shell, or the sample trace appearing in the detection window.

[0018] Preferably, if the liquid trace does not reach the quantitative marker line within the set time after adding the sample solution, more sample can be added until the trace line reaches the quantitative marker line. This ensures that the sample solutions participating in the immune reaction are consistent. This preference is especially important for samples with a viscous matrix.

[0019] Preferably, after the sample solution is added, it passes through the filter pad, sample pad, and marker pad, and then undergoes lateral flow chromatography along the nitrocellulose membrane in two directions: towards the absorbent pad and the diluent pad. This characteristic of two lateral flow chromatography directions is fundamentally different from existing technologies.

[0020] (1) As required, due to the increased flow towards the "dilution pad", the liquid traces after the sample reacts with the labeling pad will not reach the T line quickly. As the sample solution is depleted, the sample solution and labeling that have penetrated into the nitrocellulose membrane will gradually slow down until they stop. This stagnant buffering process, compared with the prior art, increases the reaction time of the antigen (or antibody) conjugated with the sample solution and labeling, thereby improving the sensitivity.

[0021] (2) Due to the bilateral flow, the test sample and the labeled antigen (or antibody) are evenly distributed symmetrically around the center of the labeling pad, which can improve the phenomenon of "dead zone" formed by unilateral flow.

[0022] (3) In addition to controlling the amount of sample added, the amount of diluent added, and the time of immune reaction, the volume of the first immune complex can be accurately controlled by using the "quantitative label line (M)".

[0023] Unlike existing technologies where both sample solution and markers tend to be completely released onto the nitrocellulose membrane, this invention involves the markers flowing into the nitrocellulose membrane after the sample solution is added. As the markers flow to the quantitative mark line (M) on the liquid side, a "quantitative" amount of sample solution and markers permeates into the nitrocellulose membrane. At this point, the marker pad is in a semi-dry, semi-wet state, and the absorbency of the marker pad, sample pad, and filter pad is much lower than that of the nitrocellulose membrane. Therefore, when diluent is added to the diluent pad, the humidity of the nitrocellulose membrane will be greater than that of the sample pad and marker pad. Only a very small amount of the remaining markers and sample solution that did not enter the nitrocellulose membrane can permeate into the chromatography membrane. The diluent can only precipitate the portion of sample solution and markers existing on the nitrocellulose membrane to the absorbent pad. Therefore, the rapid detection card of this invention can precisely control the amount of sample solution entering the nitrocellulose membrane. Existing technologies, although quantitatively adding samples, suffer from variations in the matrix between samples, leading to differences in the total amount of sample entering the nitrocellulose membrane, directly affecting the accuracy and stability of the detection results.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] The test strip and test card provided by this invention have a simple structure and reasonable design. Sample is added through the sample application hole, and diluent is added through the cleaning hole. Since the diluent does not pass through the sample pad, filter pad, and marker pad, secondary release of the marker-sample solution binding solution from the marker pad is reduced. Furthermore, the requirement that diluent be added through the cleaning hole only after the sample solution has reached the quantitative mark (M) allows for precise control of the amount of liquid involved in the immunobinding reaction between the sample and the marker pad. This not only improves the sensitivity and accuracy of the analyte but also retains the speed and convenience of rapid diagnostic methods.

[0026] Preferably, dye is added to the label pad to trace the label release process. When the colored liquid from the sample-labeled binding solution flows laterally to the quantitative label line (M), it indicates the need for diluent. After the diluent chromatography is complete, as the humidity of the nitrocellulose membrane decreases, the remaining label and sample binding solution on the label pad may begin to permeate back onto the membrane. When the colored liquid reappears, the interpretation of the results should be stopped. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the structure of the abrin toxin detection test strip of the present invention;

[0028] Figure 2 This is a top view of the outer shell of the abrin toxin detection card of the present invention. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Unless otherwise specified, the methods described are conventional methods. Unless otherwise specified, the raw materials are all available from publicly available commercial sources.

[0030] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0031] like Figure 1 As shown, a rapid test strip for abrinogen toxin includes a PVC base plate 1, an absorbent pad 2, a nitrocellulose membrane 3, a marker pad 5, a sample pad 8, a filter pad 9, and a diluent pad 4. The nitrocellulose membrane 3 is fixed on the PVC base plate 1, with its left end overlapping the absorbent pad 2 and its right end overlapping the diluent pad 4. The marker pad 5 is placed on the nitrocellulose membrane 3 and located in front of the diluent pad 4, with the direction of the absorbent pad 2 defined as forward. A 1-10 mm space of exposed nitrocellulose membrane is maintained between the marker pad 5 and the diluent pad 4. The sample pad 8 is placed on the marker pad 5. A detection line (T line) 7 and a control line (C line) 6 are sequentially arranged along the direction from the marker pad 5 to the absorbent pad 2 on the nitrocellulose membrane 3 between the absorbent pad 2 and the marker pad 5. A quantitative identification line (M line) 13 is arranged between the marker pad 5 and the detection line (T line) 7.

[0032] The labeling pad 5 is impregnated with polyclonal antibody against abrinogen toxin and chicken IgY antibody against lanthanide fluorescent microspheres; polyclonal antibody against abrinogen toxin is coated on the detection line 7, and IgG anti-chicken IgY antibody from goats is coated on the control line 6.

[0033] As a preferred embodiment, the marker pad 5 contains a pigmented marker.

[0034] like Figure 2 As shown, a two-well rapid quantitative test strip for abrinogen toxin includes a housing 14 and the aforementioned two-well rapid quantitative test strip disposed within the housing 14; a detection window 10 is provided on the upper shell of the housing corresponding to the interval between the detection line 7 and the control line 6; a quantitative marking line (M line) 13 is provided on the edge of the detection window (the edge of the detection window can also be used as the quantitative marking line); a sample application hole 11 is provided corresponding to the sample pad, and a cleaning hole 12 is provided corresponding to the diluent pad 4.

[0035] This invention also provides a method for detecting samples using the above-mentioned abrinogen toxin dual-well rapid quantitative detection card.

[0036] The method for testing samples provided by the present invention includes the following steps: adding sample liquid through the sample application hole of the rapid test card, then waiting for the watermark of the sample liquid to move to the quantitative identification line in the test strip of the test card, adding diluent through the cleaning hole, and finally observing through the detection window or interpreting the result through a corresponding instrument.

[0037] The instruments mentioned include gold standard reading instruments, fluorescence analyzers, and upconversion analyzers.

[0038] In practical use, the sample solution (generally 5-50 μL) is added through the sample application well 11. After passing through the filter pad 9, sample pad 8, and marker pad 5, the sample solution enters the nitrocellulose membrane 3. Since the diluent pad 4 and absorbent pad 2 are both dry, and the combined liquid capacity of the sample pad 8 and marker pad 5 is much smaller than that of the absorbent pad 2 and diluent pad 4 on both sides, the sample solution carries the marker and permeates bidirectionally to both sides of the chromatography membrane until it is exhausted. This causes the sample solution containing the immunoreaction complex marker to remain near the quantitative label line on the nitrocellulose membrane 3. Compared to existing technologies, this invention allows the biological material coupled to the sample solution in the marker pad more reaction time and prevents it from reaching the detection line 7. After a certain reaction time (generally 0.5-5 minutes) between the sample solution and the label, when the trace of the flowing sample solution appears at the quantitative marker line (M) in the detection window, diluent (which can be purified water or PBS buffer) is added to the cleaning well 12. The diluent first passes through the diluent pad 4, which may contain reagent components (such as blocking agents) to eliminate non-specific reactions. After mixing with the diluent, the sample immunoassay solution containing the labeled tracer is diluted. Under the action of the absorbent pad 2, as the diluent flows towards the absorbent pad 2, it immunobindes with the antigen (or antibody) coated on the T and C lines and is captured and remains on the T and C lines. The remaining substance continues to flow on the nitrocellulose membrane through lateral chromatography and is absorbed by the absorbent pad until the immunochromatography is completed. The result is observed through the detection window 10 or detected and interpreted by the instrument.

[0039] Initially, the nitrocellulose membrane 3 is dry, and the sample solution and label are rapidly released onto it, largely unaffected by sample differences. Subsequently, the label and sample solution remaining in the label pad 5 slowly enter the nitrocellulose membrane 3. The release rate at this point is significantly affected by sample differences, impacting the stability of the immunochromatography. Therefore, this invention positions the sample well in the middle of the nitrocellulose membrane, slightly towards the dilution well. After the sample solution and label permeate the membrane, they precipitate to both sides until the sample solution is exhausted and remains near the quantitative mark line. At this point, diluent is added. Because the humidity of the nitrocellulose membrane 3 is higher than that of the label pad 5, the remaining label in the label pad 5 continues to be released into the nitrocellulose membrane 3. The amount released is minimal compared to the initial amount that remained on the membrane 3 after the initial split.

[0040] This invention employs a dual-pore structure design that terminates the release of the marker after the diluent is injected. This design prevents the release of sample solution and marker into the nitrocellulose membrane 3 as required, ensuring a stable quantity of sample solution and marker entering the nitrocellulose membrane 3 and improving the stability and precision of immunochromatography. Furthermore, by adding the sample solution and diluent separately, this invention improves the cleaning efficiency of self-cleaning methods relying on the sample solution, maximizing the background cleanliness of the nitrocellulose membrane 3 in the detection window 10.

[0041] This invention achieves, in addition to controlling the amount of the first sample added and the quantitative control of the immune reaction time, an additional control over the amount of reaction between the sample and the labeled substance, thereby improving the sensitivity and precision of the lateral flow chromatography detection method.

[0042] The following is a detailed explanation of the manufacturing process of this test card.

[0043] In the following embodiments:

[0044] Chicken IgY was purchased from Hangzhou Qitai Biotechnology Co., Ltd.

[0045] The sheep anti-chicken IgY IgG was purchased from Hangzhou Qitai Biotechnology Co., Ltd.

[0046] Abrus precatorius toxin polyclonal antibodies were prepared in our laboratory: Abrus precatorius toxin (GI: 543748, sequence: qdrpikfstegatsqsykqf iealrerlrg glihdipvlp dpttlqernr yitvelsnsd tesievgidv tnayvvayragtqsyflrda pssasdylft gtdqhslpfy gtygdlerwa hqsrqqiplg lqalthgisf frsggndneekartliviiq mvaeaarfry isnrvrvsiq tgtafqpdaa mislennwdn lsrgvqesvq dtfpnqvtltnirnepvivd slshptvavl almlfvcnpp nanqspllir sivekskics sryeptvrig grdgmcvdvydngyhngnri imwkckdrle enqlwtlksd ktirsngkcl ttygyapgsy vmiydctsav aeatyweiwdngtiinpksa lvlsaesssm ggtltvqtne ylmrqgwrtg nntspfvtsi sgysdlcmqa qgsnvwmadcdsnkkeqqwa lytdgsirsv qntnncltsk dhkqgstill mgcsngwasq rwvfkndgsi yslyddmvmdvkgsdpslkq iilwpytgkp nqiwltlf) was used as an antigen to immunize rabbits, and serum was collected and purified to obtain absinthecetine polyclonal antibodies;

[0047] Lanthanide fluorescent microspheres were purchased from Chengdu Microray Biotechnology Co., Ltd.

[0048] Example 1: Preparation of Abrus precatorius toxin detection card

[0049] The detection principle of the abrinosa toxin test card (double-antibody sandwich method): For example... Figure 1The test card shown has goat anti-chicken IgY IgG coated at control line 6 of the nitrocellulose membrane and abrinogen toxin polyclonal antibody coated at test line 7. When a sample containing abrinogen is added, as it passes through the labeled pad, the abrinogen binds to the lanthanide fluorescent microspheres labeled with abrinogen toxin polyclonal antibody to form a complex. When it flows to the quantitative label line (M), diluent is added to allow the complex to flow on the nitrocellulose membrane. The AT of the lanthanide fluorescent microspheres is captured by the AT polyclonal antibody coated at line T, forming a double antibody sandwich complex fixed on line T. The amount of abrinogen toxin conjugate captured at test line (T) is proportional to the amount of the labeled lanthanide fluorescent antibody. When the sample contains abrinogen toxin, the detection line shows fluorescence. When the sample contains no abrinogen or only a very small amount, the polyclonal antibody against AT on the detection line cannot capture the AT conjugated to the labeled lanthanide fluorescent microspheres, thus the detection line does not show fluorescence. Lanthanide fluorescent microspheres labeled with chicken IgY are captured by goat anti-chicken IgY IgG on the control line, causing the control line to fluoresce. The fluorescence ratio between the detection line and the reference line is obtained by a lanthanide fluorescence immunoassay analyzer. By converting this ratio using a preset standard curve, the content of abrinogen toxin in the sample can be determined. This enables rapid and accurate quantitative detection of abrinogen toxin.

[0050] Preparation of abrin toxin detection card:

[0051] Step 1: Adhere the nitrocellulose membrane (Sartorius CN140) to the PVC base plate. Using a special dot-coating gold spraying machine, spray the nitrocellulose membrane with the control line (C line) formed by goat anti-chicken IgY IgG diluted to 0.15 mg / ml and the detection line (T line) formed by AT polyclonal antibody diluted to 3 mg / ml. The spraying volume is 1 μl / cm. Then bake at 37°C for 16 hours.

[0052] Step 2: Prepare lanthanide fluorescent microspheres labeled with chicken IgY and lanthanide fluorescent microspheres labeled with AT polyclonal antibodies. Add 1 mL of lanthanide fluorescent microspheres to 5 mL of MES (2-(N-morpholino)ethanesulfonic acid) buffer (0.05 M, pH 7.2), then add 10 mg of carbodiimide (EDC) and 10 mg of N-hydroxysuccinimide sodium salt and stir to dissolve. After reacting at room temperature for 30 minutes, centrifuge. Redissolve the precipitate with 50 mM borate buffer (pH 8.2), add 2 mg of dialyzed chicken IgY, and stir to react at room temperature for 24 hours. Then centrifuge, block, and store in dilution buffer (storage temperature 2–8 °C) to obtain lanthanide fluorescent microspheres labeled with chicken IgY. Use the same method to label lanthanide fluorescent microspheres labeled with AT polyclonal antibodies.

[0053] Step 3: Lanthanide fluorescent microspheres labeled with chicken IgY and AT polyclonal antibodies were diluted to concentrations of 0.1 μg / ml and 8 μg / ml, respectively, and mixed together. The mixture was then sprayed onto a polyester film using a gold spraying machine to form a labeled pad, with a spraying volume of 2.5 μl / cm. The pad was then baked at 37°C for 8 hours.

[0054] Step 4: Cut the marker pad into 8mm lengths and the sample pad into 8mm lengths as well. Then, stick them together with double-sided tape. Then, use a continuous cutter to cut them into 5mm wide sample strips and assemble them under the sample application hole on the top cover of the test card.

[0055] Step 5: Attach the 305mm long and 12mm wide dilution pad and the 305mm long and 17mm wide absorbent pad to the two ends of the nitrocellulose membrane on the PVC base plate to assemble a large card. Then, use a continuous cutter to cut it into 4.15mm wide test strips and assemble them inside the lower cover of the test card shell. Snap the upper and lower covers of the test card together to obtain the abrinosa toxin test card.

[0056] During the test, add 40 μL of sample to the sample well. After 3 minutes, when the sample liquid trace moves to the quantitative marker line (M), add 80 μL of cleaning solution to the cleaning well and perform chromatography for 17 minutes. Finally, interpret the results using a fluorescence analyzer.

[0057] Preparation of a standard curve for the detection of abrin toxins:

[0058] Six standard solutions containing abrinogen toxin were prepared at concentrations of 0, 100, 200, 500, 1000, and 2000 ng / mL. 40 μL of each standard solution was added to the middle well of a pre-assembled two-well test card, followed by 80 μL of diluent in the subsequent wells. The total chromatographic immunoassay time was 20 minutes. Detection was performed using a lanthanide fluorescence immunoassay analyzer. Linear regression analysis was performed on the six detection data to generate a standard curve for abrinogen toxin.

[0059] The test card is used as follows: Prepare a 100 ng / mL standard solution using abrinogen toxin standard. Add 40 μL of sample according to the sample addition method in the example. When the liquid flows to the quantitative mark line, add 80 μL of diluent (PBS) and perform immunochromatography for 17 min. Then, use a WR-1608 fluorescence immunoassay analyzer manufactured by Chengdu Weirui Biotechnology Co., Ltd. to detect the test card and obtain the fluorescence signals of the test line and the control line. The fluorescence analyzer transmits the detection data to the client via Bluetooth. The client calculates the fluorescence signal intensity values ​​of the test line and the control line based on the detection data. The client obtains the abrinogen toxin standard curve from the cloud platform by scanning the barcode of this batch of test cards. Then, calculate the content of abrinogen toxin in the sample based on the comparison relationship between the standard curve and the fluorescence signal intensity values ​​of the control line and the test line.

[0060] The initial test result was 97.2 ng / mL. Following the same procedure, when 45 μL of sample and 80 μL of PBS were added, the result was 106.77 ng / mL. The results show that variations in sample volume had little impact on the final result.

[0061] Repeatability test data: The above sample was added in a dose of 40 μL, followed by 80 μL of diluent. The results of 10 repetitions were: 97.2 ng / mL, 92.58 ng / mL, 116.97 ng / mL, 110 ng / mL, 89.54 ng / mL, 96.8 ng / mL, 93 ng / mL, 108.2 ng / mL, 105.49 ng / mL, and 93.52 ng / mL, with a deviation of less than 9.2%.

[0062] Sensitivity experiment:

[0063] A standard solution of 800 ng / mL was prepared using abrinogen toxin standard, and then diluted 2, 4, 8, 16, and 32 times. Samples were added and tested respectively, and the results were as follows: 390.11 ng / mL, 209.57 ng / mL, 106.2 ng / mL, 0 ng / mL, and 0 ng / mL.

[0064] The results showed that the sensitivity could reach 100 ng / mL.

[0065] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A test strip for absinthecetine toxin detection, comprising a PVC base plate, an absorbent pad, a nitrocellulose membrane, a marker pad, a sample pad, and a diluent pad; wherein the nitrocellulose membrane is fixed on the PVC base plate, with one end of the nitrocellulose membrane overlapping the absorbent pad and the other end overlapping the diluent pad; The marker pad is placed on the nitrocellulose membrane and in front of the diluent pad, and the direction of the absorbent pad is defined as forward; the sample pad is placed on the marker pad; a detection line and a control line are sequentially arranged on the nitrocellulose membrane from the marker pad to the absorbent pad; A quantitative marking line is provided between the marker pad and the detection line; The marker pad is coated with marker-labeled chicken IgY antibody and marker-labeled abrin toxin polyclonal antibody; the detection line is coated with abrin toxin polyclonal antibody, and the control line is coated with goat anti-chicken IgY antibody IgG; The marker is a lanthanide fluorescent microsphere; A corresponding filter pad is provided on the sample pad; Pigment is added to the marker pad; The overlap width between the nitrocellulose membrane and the absorbent pad is 1-2 mm; the overlap width between the nitrocellulose membrane and the diluent pad is 1-2 mm; a 1-10 mm nitrocellulose membrane exposed space is maintained between the marker pad and the diluent pad; the sum of the liquid carrying capacity of the sample pad and the marker pad is much smaller than that of the absorbent pad and the diluent pad on both sides.

2. A two-well quantitative detection card for abrinogen toxin, comprising a housing and a brusinogen toxin test strip as described in claim 1 disposed within the housing; a detection window is provided on the housing corresponding to the interval between the detection line and the control line in the test strip; a sample application hole is provided corresponding to the sample pad, and a cleaning hole is provided corresponding to the diluent pad; a quantitative identification line is provided between the edge of the detection window and the detection line near the sample application hole, and a mark is provided on the corresponding housing.

3. The abrinogen toxin dual-well quantitative detection card according to claim 2, characterized in that: The outer shell includes an upper cover and a lower cover, which are fastened together. The detection window, sample application hole, and cleaning hole are located on the upper cover.

4. A method for detecting a sample using the abrinogen toxin dual-well quantitative detection card according to claim 2 or 3, comprising the following steps: adding sample liquid through the sample application well of the detection card, then waiting for the sample liquid trace to move to the quantitative marking line in the test strip of the detection card, then adding diluent through the cleaning well, and finally observing through the detection window or interpreting the result through an instrument.

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