Colloidal gold test paper card for rapidly determining rice yeast acid in whole blood

Abstract

The utility model discloses a colloidal gold test paper card for rapidly determining rice yeast acid in whole blood. The colloidal gold test paper card comprises a bottom plate as well as a glass cellulose membrane, a filtration binding area, a detection area and a water absorption pad which are sequentially arranged on the bottom plate from left to right, the filtration binding area adopts an antigen-antibody binding technology, red blood cells in whole blood can be retained on a filter membrane of a sample pad without moving, other substances in the whole blood can smoothly pass through, and the detection effect is good. According to the present invention, the kit can be combined with the rice yeast acid so as to eliminate the background interference, can be combined with the rice yeast acid, and can be detected through the detection region, such that the rice yeast acid in the whole blood can be accurately detected, the accuracy and the specificity of the rice yeast acid determination in the whole blood can be effectively improved, the application range and the efficiency of the trace whole blood sample in the detection can be expanded, and the redundant pretreatment operation is not required; the method is simple in operation and rapid in detection, and can obtain a result within 15 minutes only by taking 10 microliters of trace whole blood, and the lowest detection limit of 50 micrograms / L is reached.

Description

Technical Field

[0001] This utility model belongs to the technical field of medical testing and immunodiagnostic systems, specifically relating to a colloidal gold test strip for rapid determination of bongkrekic acid in whole blood. Background Technology

[0002] *Pseudomonas cocovenenans* subspecies *farinofermentans* (hereinafter referred to as "Pseudomonas cocovenenans") is the only pathogenic species of *Burkholderia gladioli* that can cause food poisoning in humans. This bacterium easily contaminates fermented cereal products, wet rice flour products, spoiled tremella, potato products, and wet starch products, often causing food poisoning during the summer and autumn seasons when temperature and humidity are suitable for its growth. Bongkrekic acid (BA) is its main toxic metabolite, readily soluble in organic solvents and alkaline aqueous solutions, extremely heat-resistant (not destroyed by boiling water at 100°C or high pressure), and highly toxic to humans and animals. Studies have shown that a BA concentration of 200-300 μg / L in human blood can be fatal. BA poisoning incidents caused by consuming food contaminated with *Pseudomonas cocovenenans* have been reported frequently in my country.

[0003] Whole blood is a complex matrix composed of plasma and blood cells. Its biomarkers can reflect real-time changes in the body's internal environment, effectively providing information for disease diagnosis, drug monitoring, and personal health. Qualitative determination of bongkrekic acid (BA) in whole blood can provide crucial support for rapid disease diagnosis, thereby improving patient prognosis. However, current analytical techniques for BA are mainly applied in the food industry, such as rice noodles and rice vermicelli, and primarily employ instrumental methods such as high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). These methods are limited by laboratory conditions, are complex and costly, require professional personnel, and cannot meet the needs of rapid on-site testing. To date, no research has been reported on the rapid detection of bongkrekic acid in whole blood using colloidal gold methods, indicating a significant research gap in this field. Furthermore, due to the complex matrix characteristics of whole blood, existing BA analytical techniques are difficult to adapt for rapid on-site qualitative detection of BA in whole blood. Therefore, a simple, on-site, and rapid screening technique for bongkrekic acid poisoning needs to be developed. Summary of the Invention

[0004] To address the shortcomings and deficiencies of existing technologies, the purpose of this invention is to provide a colloidal gold test strip for the rapid determination of bongkrekic acid in whole blood. This test strip contains a filtration binding zone and a detection zone. The filtration binding zone utilizes antigen-antibody binding technology, allowing red blood cells in the whole blood to remain immobilized on the filter membrane of the sample pad, while other substances in the whole blood pass through smoothly, thus eliminating background interference and binding with bongkrekic acid. The detection zone then detects the bongkrekic acid in whole blood, accurately improving the accuracy and specificity of bongkrekic acid determination. It also expands the application range and efficiency of micro-volume whole blood samples in detection, requiring no additional pretreatment and allowing for on-site testing. Biomarkers in whole blood can reflect dynamic changes in the internal environment in real time. Through the qualitative determination of bongkrekic acid in whole blood, this invention will provide crucial support for the rapid diagnosis of diseases, thereby improving patient prognosis.

[0005] The colloidal gold test strip for determining bongkrekic acid in whole blood described in this invention is simple to operate and fast to detect. Only 10 µL of whole blood is needed, and results can be obtained within 15 minutes, reaching a minimum detection limit of 50 µg / L.

[0006] The objective of this utility model is achieved through the following technical solution:

[0007] A colloidal gold test strip for rapid determination of bongkrekic acid in whole blood includes a base plate and a glass cellulose membrane, a filter binding zone, a detection zone, and an absorbent pad arranged from left to right on the base plate.

[0008] Preferably, the filtration binding region includes a red blood cell filtration membrane and a colloidal gold binding pad.

[0009] Preferably, the red blood cell filter membrane is a nitrocellulose membrane containing mouse anti-human red blood cell antibodies.

[0010] The pore size of the red blood cell filtration membrane is 7-12 µm.

[0011] When whole blood passes through the erythrocyte filtration membrane, an antigen-antibody reaction occurs to form a erythrocyte-mouse anti-human erythrocyte antibody complex. Finally, the erythrocytes are retained on the erythrocyte filtration membrane, thereby achieving the purpose of removing erythrocytes.

[0012] Preferably, the colloidal gold conjugate pad is a nitrocellulose membrane labeled with bongkrekic acid monoclonal antibody-colloidal gold.

[0013] Preferably, the detection area is a nitrocellulose membrane, which is provided with a detection line coated with bongkrekic acid antigen carrier protein conjugate and a quality control line coated with goat anti-mouse secondary antibody.

[0014] Preferably, the left side of the filter binding zone is located below the right side of the glass cellulose membrane.

[0015] Preferably, the left side of the detection area is located below the right side of the filter binding area.

[0016] Preferably, the right side of the detection area is located below the left side of the absorbent pad.

[0017] Preferably, the base plate is a PVC base plate.

[0018] Preferably, the colloidal gold test strip for rapid determination of bongkrekic acid in whole blood includes a base plate and glass cellulose membrane, red blood cell filter membrane, colloidal gold binding pad, detection area and absorbent pad arranged from left to right on the base plate.

[0019] The left side of the red blood cell filtration membrane is located below the right side of the glass cellulose membrane, and the left side of the colloidal gold conjugate pad is located below the right side of the red blood cell filtration membrane; the left side of the detection area is located below the right side of the colloidal gold conjugate pad.

[0020] Preferably, the colloidal gold test strip for rapid determination of bongkrekic acid in whole blood includes a base plate and glass cellulose membrane, colloidal gold conjugation pad, red blood cell filter membrane, detection area and absorbent pad arranged from left to right on the base plate.

[0021] The left side of the colloidal gold conjugate pad is located below the right side of the glass fiber membrane, and the left side of the erythrocyte filtration membrane is located below the right side of the colloidal gold conjugate pad; the left side of the detection area is located below the right side of the erythrocyte filtration membrane. Preferably, the colloidal gold test strip for rapid determination of bongkrekic acid in whole blood is 3–5 cm wide.

[0022] This colloidal gold test strip uses a competitive design. During testing, the blood sample flows into the colloidal gold conjugate pad via capillary action. If the sample contains bongkrekic acid, it will bind to the specific antibody labeled with colloidal gold, forming an antigen-antibody colloidal gold complex. This complex then migrates forward to the detection area, including the test line (T line) and the control line (C line), under capillary action. Because the T line is pre-coated with a bongkrekic acid antigen-carrier protein conjugate, when the antigen-antibody colloidal gold complex flows through the T line, the antibody already bound to the target antigen cannot bind to the antigen fixed on the T line, causing the T line to fade or even disappear. Meanwhile, the goat anti-mouse secondary antibody coated on the C line binds to the free colloidal gold-labeled antibody, ensuring the C line develops color to verify the test's validity. Therefore, if the concentration of bongkrekic acid in the sample is high, the T line will fade significantly or even disappear, while the C line will remain visible, indicating a positive result. If the sample contains no or only trace amounts of bongkrekic acid, the colloidal gold-labeled antibody is not saturated with the antigen and can bind to the fixed antigen at the T line, causing the T line to darken while the C line remains visible, which indicates a negative result.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] This invention discloses a colloidal gold test strip for the rapid determination of bongkrekic acid in whole blood. This colloidal gold test strip utilizes whole blood filtration membrane technology and colloidal gold-labeled antibodies to achieve rapid and accurate determination of bongkrekic acid in whole blood samples. Compared with other colloidal gold methods for detecting bongkrekic acid, this test strip has the following main advantages:

[0025] (1) High filtration efficiency: The red blood cell filter membrane is set up, which has a higher filtration efficiency and effectively removes the interference of colored components in whole blood; at the same time, the amount of anti-human red blood cell antibody used on the red blood cell filter membrane is low, and the cost is low.

[0026] (2) Making rapid detection of bongkrekic acid toxin in whole blood a reality: Due to the interference of red blood cells, the color development of colloidal gold test strips is difficult to distinguish; at the same time, their presence can easily block the pores of the test strip, affecting the flow of reagents on the test strip card. In response to this problem, this utility model adopts an innovative red blood cell removal technology, making it possible to detect bongkrekic acid toxin in whole blood samples, and the color development results are clear, avoiding common interferences and improving the reliability and sensitivity of the detection.

[0027] (3) Support for rapid on-site diagnosis: This test strip supports rapid on-site testing, providing immediate biomarker data for early detection and timely treatment of diseases, which helps improve the treatment prognosis of patients, especially in acute diseases and public health emergencies, and can provide valuable decision support.

[0028] In summary, the colloidal gold test strip of this invention is superior to existing technologies in terms of ease of operation, detection efficiency, and result accuracy. It is particularly suitable for clinical and public health settings that require rapid response, and contributes an important technology to disease management and precision medicine. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the structure of the colloidal gold test strip for rapid determination of bongkrekic acid in whole blood as described in Embodiment 1 of this utility model, wherein 1 is a PVC base plate, 2 is a glass cellulose membrane, 3 is a red blood cell filter membrane, 4 is a colloidal gold conjugate pad, 5 is a nitrocellulose membrane, 5.1 is a detection line, 5.2 is a quality control line, and 6 is an absorbent pad. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0031] Unless otherwise specified in the embodiments of this utility model, the conditions shall be performed according to conventional conditions or conditions recommended by the manufacturer. All raw materials and reagents used, unless otherwise specified, are commercially available products.

[0032] Example 1: Colloidal gold test strip for rapid determination of bongkrekic acid in whole blood

[0033] like Figure 1 As shown, the colloidal gold test strip consists of a PVC base plate (1) and a glass cellulose membrane (2), an 8 µm erythrocyte filter membrane (3), a colloidal gold conjugate pad (4), a nitrocellulose membrane (5), and an absorbent pad (6) on the base plate (1). The left side of the erythrocyte filter membrane (3) is located below the right side of the glass cellulose membrane (2), while the right side of the erythrocyte filter membrane (3) is located above the left side of the colloidal gold conjugate pad (4). The left side of the nitrocellulose membrane (5) is located below the right side of the colloidal gold conjugate area (4), while its right side is located below the left side of the absorbent paper (6). The erythrocyte filter membrane (3) contains mouse anti-human erythrocyte antibody. The colloidal gold conjugate pad (4) contains bongkrekic acid monoclonal antibody-colloidal gold marker. The nitrocellulose membrane (5) is coated with a detection line T (5.1) and a control line C (5.2), wherein the detection line T is coated with bongkrekic acid antigen carrier protein conjugate; and the control line C is coated with goat anti-mouse secondary antibody. The test strips are cut into 4 mm wide strips by machine, placed in specially made plastic cards, and stored at 25°C for 12 months.

[0034] The red blood cell filter membrane, colloidal gold conjugate pad, and detection area (nitrocellulose membrane) in the colloidal gold test strip card described in this embodiment are obtained by the following process:

[0035] Preparation of red blood cell filtration membrane

[0036] (1) Pretreatment of nitrocellulose membrane: First, pre-wet the nitrocellulose membrane with a pore size of 8 µm in PBS containing 0.1% Tween-20 at pH 7.5 for 10 minutes; (2) Antibody dilution and adsorption: Use PBS as a diluent to dilute mouse anti-human erythrocyte antibody to 0.2 mg / mL, and then uniformly drop it onto the pretreated nitrocellulose membrane and incubate overnight at 4 °C; (3) Washing and drying: Use PBS containing 0.1% Tween-20 to wash the membrane to remove unfixed antibodies and other possible impurities, and wash 3 times. (4) After washing, use 5% bovine serum albumin as a blocking agent and incubate the membrane in the blocking solution for 1 hour. Then dry the filter membrane in a 37 °C drying oven to obtain a nitrocellulose membrane with mouse anti-human erythrocyte antibody as an erythrocyte filtration membrane. When using, cut it into strips 0.5 cm long and 4 mm wide, carefully remove them and assemble them on the test strip card.

[0037] Colloidal gold bonding pad

[0038] Colloidal gold particles were prepared using the trisodium citrate reduction method. The pH of the colloidal gold particles was adjusted to 7–7.5. The bongkrekic acid monoclonal antibody was added to the colloidal gold solution at a rate of 35 μg of antibody per milliliter of colloidal gold solution. The solution was then centrifuged, washed, and resuspended. The reconstituted bongkrekic acid monoclonal antibody-colloidal gold label was then uniformly added dropwise to a nitrocellulose membrane, at a density of 1 mm. 2 10 µl of the reconstitution product was added to the nitrocellulose membrane, and the membrane was dried at 37 °C for 1 h. The membrane was then removed to obtain a nitrocellulose membrane labeled with bongkrekic acid monoclonal antibody-colloidal gold, i.e., a colloidal gold conjugate pad. The membrane was stored in a dry environment for later use.

[0039] nitrocellulose membrane

[0040] The bongkrekic acid antigen carrier protein conjugate and goat anti-mouse secondary antibody were diluted to 1 mg / mL and 0.2 mg / mL, respectively, with 0.2 mol / L phosphate buffer (pH=7.5). These were then sprayed onto nitrocellulose membranes using a coating apparatus to serve as the detection T line and control C line, respectively, with a coating amount of 1.0 μL / cm for both. The membranes were then incubated overnight at 45 ℃. This yielded a nitrocellulose membrane with a detection line coated with the bongkrekic acid antigen carrier protein conjugate and a control line coated with the goat anti-mouse secondary antibody.

[0041] Example 2: Detection limit of test strip

[0042] Take 10 µL of whole blood and add different concentrations of bongkrekic acid standards. Then, dilute it with 90 µL of PBS containing 0.5% Tween 20 and pH 7.5 to prepare standard solutions with final bongkrekic acid concentrations of 0 µg / L, 2 µg / L, 5 µg / L, 10 µg / L, 15 µg / L, 20 µg / L, 50 µg / L, and 100 µg / L. Next, add these standard solutions dropwise onto the glass fiber filter membrane of the test strip card obtained in Example 1, and repeat the experiment three times. The detection results are interpreted as follows: a negative result is indicated by a T line that is darker than or equal to the C line, while a positive result is indicated by a T line that is lighter than the C line or even not showing any color. The experimental results show that in the detection of bongkrekic acid standard solutions of different concentrations, when the final concentration of bongkrekic acid is 0 µg / L, the T line is clearly visible and similar in color to the C line, indicating a negative result. As the concentration of bongkrekic acid increases, the T line gradually fades. When the final concentration of bongkrekic acid reaches 5 µg / L, the T line color weakens significantly; this lowest detectable concentration is defined as the detection limit of the test strip. Further increasing the bongkrekic acid concentration (≥10 µg / L) causes the T line color to fade further or even disappear completely, while the C line remains visible, indicating a positive result. Ultimately, a final standard solution concentration of 5 µg / L is used as the detection limit for bongkrekic acid, corresponding to a detection limit of 50 µg / L for bongkrekic acid in whole blood.

[0043] Example 3: Accuracy, Repeatability and Stability Experiments

[0044] The test strips from Example 1 were tested three times repeatedly with sample mixtures containing 0 µg / L and 50 µg / L concentrations of bongkrekic acid standard. No false positives or false negatives were observed. In the sample containing 0 µg / L, the T line was the same color as the C line, indicating a negative result; while in the sample containing 50 µg / L, the T line was lighter than the C line, indicating a positive result. Simultaneously, the test strips prepared in Example 1 were sealed and stored at 37 °C. Test strips with different storage periods were collected on days 0, 5, 10, 20, and 30 and tested with 50 µg / L concentrations of bongkrekic acid standard. The results showed that the test strips had good stability and no false negatives were observed. Example

[0045] The preparation of the colloidal gold test strip for bongkrekic acid in whole blood is the same as in Example 1, except that the positions of the erythrocyte filtration membrane and the colloidal gold conjugate pad are interchanged. Specifically, it consists of a PVC base plate and a glass fiber membrane, a colloidal gold conjugate pad, an erythrocyte filtration membrane, a nitrocellulose membrane, and an absorbent pad mounted on the base plate. The left side of the colloidal gold conjugate pad is located below the right side of the glass fiber membrane, while the right side of the colloidal gold conjugate pad is located above the left side of the erythrocyte filtration membrane. The left side of the nitrocellulose membrane is located below the right side of the erythrocyte filtration membrane, while its right side is located below the left side of the absorbent paper. The nitrocellulose membrane is coated with a detection line (T line) and a control line (C line).

[0046] The detection performance of the colloidal gold test strip for bongkrekic acid in whole blood obtained in this embodiment is the same as that in Example 1.

[0047] The above embodiments are preferred embodiments of the present utility model, but the embodiments of the present utility model are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present utility model shall be considered equivalent substitutions and shall be included within the protection scope of the present utility model.

Claims

1. A colloidal gold test paper card for rapidly determining mycophenolic acid in whole blood, characterized in that, The bottom plate and the glass fiber membrane, the filter combination area, the detection area and the water absorption pad arranged in sequence from left to right on the bottom plate; The filter combination area comprises a red blood cell filter membrane and a colloidal gold combination pad.

2. The colloidal gold test paper card according to claim 1, wherein, The red blood cell filter membrane is a nitrocellulose membrane provided with a mouse anti-human red blood cell antibody. The pore size of the red blood cell filter membrane is 7-12 μm.

3. The colloidal gold test paper card according to claim 1 or 2, characterized in that, The colloidal gold combination pad is a nitrocellulose membrane provided with a monoclone antibody of mycosterol acid-gold label.

4. The colloidal gold test paper card according to claim 1 or 2, characterized in that, The detection area is a nitrocellulose membrane provided with a detection line coated with a mycosterol acid hapten carrier protein conjugate and a quality control line coated with a goat anti-mouse secondary antibody.

5. The colloidal gold test paper card according to claim 1 or 2, characterized in that, The left side of the filter combination area is located below the right side of the glass fiber membrane.

6. The colloidal gold test paper card according to claim 1 or 2, characterized in that, The left side of the detection area is located below the right side of the filter combination area.

7. The colloidal gold test paper card according to claim 1 or 2, characterized in that, The right side of the detection area is located below the left side of the water absorption pad.

8. The colloidal gold test paper card according to claim 1 or 2, characterized in that, The bottom plate and the glass fiber membrane, the red blood cell filter membrane, the colloidal gold combination pad, the detection area and the water absorption pad arranged in sequence from left to right on the bottom plate; The left side of the red blood cell filter membrane is located below the right side of the glass fiber membrane, the left side of the colloidal gold combination pad is located below the right side of the red blood cell filter membrane, and the left side of the detection area is located below the right side of the colloidal gold combination pad.

9. The colloidal gold test paper card according to claim 1 or 2, characterized in that, The bottom plate and the glass fiber membrane, the colloidal gold combination pad, the red blood cell filter membrane, the detection area and the water absorption pad arranged in sequence from left to right on the bottom plate; The left side of the colloidal gold combination pad is located below the right side of the glass fiber membrane, the left side of the red blood cell filter membrane is located below the right side of the colloidal gold combination pad, and the left side of the detection area is located below the right side of the red blood cell filter membrane.

10. The colloidal gold test paper card according to claim 1 or 2, characterized in that, The colloidal gold test paper card for rapidly determining mycosterol acid in whole blood is 3-5 cm wide.

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