Kit for detecting PLA2R antibody in urine

Abstract

The invention relates to the technical field of detection, and discloses a kit for detecting a PLA2R antibody in urine, which comprises a non-invasive urine sampling device, a pore plate detection module and a time-resolved fluorescence detection module, the non-invasive urine sampling device comprises a urine collecting cup, a collecting cup and a U-shaped siphon which are matched up and down; a spiral flow guide groove for guiding the collection cup is formed in the inner wall of the urine collection cup; the collecting cup is located at the bottom of the urine collecting cup, and scale marks are arranged on the side wall of the collecting cup; the inlet end of the U-shaped siphon is communicated with the side wall of the collecting cup and is higher than the bottom of the collecting cup, and the outlet end of the U-shaped siphon extends into the urine collecting cup. According to the kit, urine is used for replacing serum to serve as a PLA2R-IgG antibody detection sample, the problems that traditional serological detection cannot achieve real-time monitoring and is high in cost are solved through the simplified process of non-invasive sampling-time resolution detection, and a convenient solution can be provided for rapid screening of membranous nephropathy.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, specifically to a kit for ultrasensitive detection of PLA2R-IgG antibodies in urine. In particular, the device utilizes a siphon-type liquid separation mechanism and a backflow prevention mechanism to automatically collect midstream urine and prevent contamination. The device is suitable for use in clinical or home settings, such as testing for membranous nephropathy and routine urinalysis, where sample contamination must be avoided. Background Art

[0002] Traditional PLA2R-IgG antibody detection relies on serological methods, which have the following inherent defects:

[0003] (1) Invasive sampling: venous blood drawing is required, patient compliance is low, and frequent sampling is not possible to achieve dynamic disease monitoring; (2) Limited detection scenarios: serum testing relies on laboratory equipment and professionals, and is difficult to adapt to the real-time monitoring needs at home or at the grassroots level; (3) Insufficient real-time performance: serum antibody levels reflect the intensity of the patient's immune response and ignore the basic health status of the kidneys; the changes in serological PLA2R-IgG antibody concentrations are slightly different from the disease progression and cannot reflect the individual disease status of patients with membranous nephropathy.

[0004] In TRFIA testing, the test kit is the carrier for key components such as reagents, reaction plates, and calibrators. Recent studies have shown that PLA2R-IgG antibodies can enter the urine through glomerular filtration or local production. Compared to serum testing, urine testing offers the advantages of non-invasiveness and real-time monitoring. Therefore, if urine is used as a sample for PLA2R-IgG antibody testing, real-time detection of PLA2R-IgG antibodies in the body can be achieved, enabling timely monitoring of membranous nephropathy progression, better medication guidance, and thus reducing the risk of renal failure. However, there are currently no test kits that can detect PLA2R-IgG antibodies in urine.

[0005] In summary, there is an urgent need in this field for a TRFIA ultrasensitive detection kit that can perform real-time sampling and safe and non-invasive detection of PLA2R-IgG in urine. Summary of the Invention

[0006] The present invention provides a kit for detecting PLA2R antibodies in urine. This kit uses urine instead of serum as the sample for PLA2R-IgG antibody detection. Through a simplified non-invasive sampling and time-resolved detection process, it addresses the limitations of traditional serological testing, including the lack of real-time monitoring and high costs. This kit can provide a convenient solution for rapid screening of membranous nephropathy.

[0007] The specific technical solution of the present invention is a kit for detecting PLA2R antibodies in urine, comprising a non-invasive urine sampling device, a 96-well plate, and a time-resolved fluorescence detection module. The non-invasive urine sampling device comprises a urine collection cup, a collection cup, and a U-shaped siphon tube, which fit together at the top and bottom. The top of the urine collection cup is a backflow-proof cup mouth shaped to fit the curve of the female vulva. The inner wall of the urine collection cup is provided with a spiral guide groove that guides the collection cup. The collection cup is located at the bottom of the urine collection cup, and the side walls of the collection cup are provided with scale lines. The inlet end of the U-shaped siphon tube is connected to the side wall of the collection cup and is higher than the bottom of the collection cup, and the outlet end of the U-shaped siphon tube extends into the urine collection cup. The microwells of the 96-well plate are pre-coated with PLA2R antigens to directly capture PLA2R-IgG antibodies in urine. The time-resolved fluorescence detection module comprises a fluorescence reader, a pipette, a buffer, an enhancement solution, a standard, and Eu3+-labeled anti-human PLA2R-IgG antibodies.

[0008] The kit of the present invention uses urine instead of serum as the sample for PLA2R-IgG antibody detection, and achieves non-invasive, real-time dynamic monitoring based on high-throughput detection using a well plate. Traditional PLA2R antibody detection kits typically use serum as the detection target, requiring invasive wounds to the human body. Current studies have shown that the levels of PLA2R antibodies in urine are significantly positively correlated with the levels of PLA2R antibodies in serum, so the levels of PLA2R antibodies in urine can be used as a basis for detecting membranous nephropathy. The present invention innovatively uses urine instead of serum as the detection target, and non-invasive PLA2R antibody detection can be achieved using a non-invasive urine sampling device.

[0009] The anti-backflow cup mouth of the urine collection cup of the present invention fits the human body and can prevent contamination by menstrual blood or urethral secretions; the spiral guide groove can guide urine to flow into the collection cup in a one-way manner.

[0010] Preferably, the anti-backflow cup mouth is an annular edge extending toward the outer side of the cup mouth.

[0011] Preferably, the spiral guide groove has a groove width of 5-8mm and an inclination angle of 15-25°. This design achieves a balance between efficient urine collection, anti-backflow and user experience by scientifically matching the groove width and inclination angle, and is a precise application of fluid mechanics and ergonomics. The groove width of the spiral guide groove is 5-8mm: (1) Flow rate and flow stability. If the groove width is too narrow (<5mm), the flow resistance will increase, resulting in a decrease in urine flow rate, which may cause splashing or poor urination for the user; if the groove width is too wide (>8mm), turbulence may be generated due to excessive urine flow rate, destroying the unidirectional flow stability and increasing the risk of backflow. The width of 5-8mm can balance the laminar flow and turbulent flow state, ensuring that urine flows along the guide groove in a stable laminar flow form; (2) Adapting to the human body's urine flow rate. The normal adult urine flow rate is about 10-20mL / s. The 5-8mm groove width combined with the spiral structure can accommodate the typical flow range and avoid overflow or backflow; (3) Anti-splash and anti-backflow. This width can suppress the splashing of droplets when urine flows, and at the same time form a physical barrier through the end baffle to further prevent backflow. The inclination angle of the spiral guide groove is 15-25°: (1) Gravity guidance and flow efficiency. When the inclination angle is <15°, the gravity component is insufficient, and urine may be retained in the groove due to viscous resistance, resulting in slow flow or even backflow; when the inclination angle is >25°, the flow rate is too fast and turbulence is easily generated, and urine may rebound and splash after hitting the baffle due to inertia. The inclination angle of 15-25° can make full use of the gravity component to achieve uniform unidirectional flow; (2) Adapt to the human body's urination angle. When the human body urinates naturally, the angle between the urine flow and the horizontal plane is usually between 10-30°. The inclination angle of the guide groove of 15-25° can seamlessly connect the physiological angle and reduce the user's operating burden. (3) Anti-backflow enhancement. The inclination angle and the spiral structure work together to guide urine to the outside of the groove through centrifugal force, blocking the backflow path.

[0012] Preferably, the number of the spiral guide grooves is 1-4.

[0013] Preferably, the U-shaped siphon has a diameter of 4-6 mm, its inlet end is 5-6 cm higher than the bottom of the collection cup, and its outlet end is 3-4 cm lower than the inlet end. The parameter design of the U-shaped siphon achieves efficient and stable directional transfer of urine by accurately matching the laws of fluid mechanics and ergonomic requirements, while also having anti-backflow, anti-clogging and easy-to-clean characteristics. Its scientificity and practicality have been verified by theoretical calculations and experiments. The diameter is 4-6 mm. (1) Balance between flow rate and viscous resistance. The viscosity of urine is about 1-1.5 mPa·s (close to that of water). If the diameter is too small (<4 mm), the flow resistance will increase significantly, which may cause difficulty in starting the siphon or interruption of flow; if the diameter is too large (>6 mm), the urine flow rate in the siphon will be reduced, the ability to maintain negative pressure will be weakened, and the siphon may fail due to air mixing. The tube diameter of 4-6mm can ensure that the urine flow rate is adapted to the human urination rate (10-20mL / s) while maintaining a stable laminar flow (Reynolds number Re <2000; (2) anti-clogging and cleanliness. The tube diameter needs to accommodate tiny particles that may exist in urine (such as crystals, epithelial cells) to avoid the risk of clogging (clinical data show that the average particle size of urine particles is <2mm). At the same time, this size is convenient for flushing or disinfection and meets the hygiene requirements of medical equipment. The design basis of the inlet end being 5-6cm high is: (1) the liquid level difference drives the siphon effect; (2) to prevent residue and contamination. The design basis of the outlet end being 3-4cm lower than the inlet end is: (1) maintaining a continuous siphon pressure gradient; (2) anti-backflow and liquid seal protection.

[0014] Preferably, the siphon triggering liquid volume of the U-shaped siphon is 18-22 mL, and the urine drainage flow rate is 15-20 mL / s.

[0015] Preferably, the scale lines are fluorescent scale lines. Further preferably, the scale lines include three capacity levels: 50 mL, 100 mL, and 150 mL.

[0016] Preferably, the antigen coating concentration of the 96-well plate is 8-12 μg / mL, the coating process is microarray printing, the spot diameter is 0.5-1 mm, and the density uniformity CV is less than 5%.

[0017] Preferably, the Eu3+ labeled anti-human PLA2R-IgG antibody is coupled via a BHHCT chelating agent, with an excitation wavelength of 337 nm, an emission wavelength of 615 nm, and a fluorescence lifetime greater than 1 ms.

[0018] Preferably, the fluorescence reader integrates a 96-channel fiber optic sensor, supports delayed detection (delay time 400 μs), the whole plate detection time is ≤30 seconds, and the linear range is 0.5-200 ng / mL.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) Non-invasive dynamic monitoring: Urine sampling is painless and non-invasive, and can achieve continuous monitoring for multiple days, accurately tracking the dynamic changes of antibodies, and achieving early diagnosis and treatment.

[0021] (2) High throughput and precision: 96 samples can be tested at a time using a 96-well plate, which is suitable for the batch requirements of clinical laboratories and improves efficiency by 80%. The detection limit of TRFIA is 0.5 ng / mL, which is 5 times more sensitive than ELISA (enzyme-linked immunosorbent assay).

[0022] (3) Full-scenario coverage: In home scenarios, patients take samples independently and the data is remotely transmitted to the doctor's end; in laboratory scenarios, it is adapted to automated workstations to support large-scale screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is an integrated appearance diagram of the kit of the present invention.

[0024] Figure 2 It is a schematic diagram of the internal distribution of the kit of the present invention.

[0025] Figure 3 It is a structural schematic diagram of the urine sampling device of the present invention.

[0026] The accompanying drawings are marked as follows: non-invasive urine sampling device 1, 96-well plate 2, fluorescence reader 3, urine collection cup 4, collection cup 5, spiral guide groove 6, U-shaped siphon 7, fluorescence scale line 8, pipette 9, buffer 10, enhancement solution 11, standard 12, Eu3+-labeled anti-human PLA2R-IgG antibody 13. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to the embodiments.

[0028] Example 1

[0029] A kit for detecting PLA2R antibodies in urine, such as Figure 1 and Figure 2 As shown, it includes a non-invasive urine sampling device 1, a 96-well plate 2, and a time-resolved fluorescence detection module 3. Specifically:

[0030] Non-invasive urine sampling device1: Figure 3 As shown, the non-invasive urine sampling device includes a urine collecting cup 4, a collecting cup 5 and a U-shaped siphon 7 that fit together at the top and bottom.

[0031] The inner wall of the urine collection cup is provided with three spiral guide grooves 6 (6mm wide and 20° inclination angle) that guide the urine collection cup. Functional verification: In a simulated menstrual female urination test (with 10mL of artificial menstrual blood mixture added), the menstrual blood backflow rate was <1% (compared to >30% for traditional urine cups).

[0032] The collection cup is located at the bottom of the urine collection cup. The side wall of the collection cup is provided with fluorescent scale lines 8 (including three capacity levels of 50mL, 100mL, and 150mL; ultraviolet excitation fluorescent paint, excitation wavelength 365nm), and the reading can be clearly read in a dim environment by illuminating with a mobile phone flashlight;

[0033] The U-shaped siphon is made of transparent PP plastic. Its inlet is connected to the side wall of the collection cup, and its outlet extends into the urine collection cup. The diameter of the U-shaped siphon is 5mm, and the inlet is 5cm above the bottom of the collection cup, while the outlet is 3cm below the inlet. The siphon trigger liquid volume of the U-shaped siphon is 20mL, and the urine extraction flow rate is 15-20mL / s. Performance test: 50mL of simulated urine is injected, the midstream urine collection volume is 30mL±1.5mL (error ≤5%), and the terminal urine residual volume is 10mL±2mL.

[0034] The microwells of the 96-well plate are pre-coated with PLA2R antigen, which can directly capture PLA2R-IgG antibodies in urine. The antigen coating concentration is 10 μg / mL, and the coating process is microarray printing. The spot diameter is 0.8 mm, and the density uniformity CV is less than 5%.

[0035] The time-resolved fluorescence detection module includes a fluorescence reader 3, a pipette 9, a buffer 10, an enhancement solution 11, a standard 12, and an Eu3+-labeled anti-human PLA2R-IgG antibody 13. The fluorescence reader integrates a 96-channel fiber optic sensor, supports delayed detection (delay time 400 μs), a full-plate detection time ≤ 30 seconds, and a linear range of 0.5-200 ng / mL. The Eu3+-labeled anti-human PLA2R-IgG antibody is coupled via a BHHCT chelator, with an excitation wavelength of 337 nm, an emission wavelength of 615 nm, and a fluorescence lifetime >1 ms. The pipette is a disposable tip (volume 100 μL ± 2 μL) adapted to the microwell diameter (6 mm) of a 96-well plate to prevent cross-contamination.

[0036] The operating procedures of the kit of this embodiment are as follows:

[0037] (1) 96-well plate: Antigen pre-coating process PLA2R antigen: recombinant human PLA2R antigen (purity > 95%), coating concentration 10 μg / mL, dissolved in PBS buffer, pH = 7.4; microarray printing process (spot diameter 0.8 mm, spacing 2 mm), coating uniformity CV = 3.2% (n = 96). Blocking and storage: Block nonspecific sites with 1% BSA, freeze-dry, seal and store in a dark place (shelf life 12 months).

[0038] (2) Detection operation steps: First add 100 μL of the sample to be tested to the 96-well plate coated with PLA2R, shake for 1 hour, and then wash with Tris-HCl solution to wash away the unbound sample. Then add Eu3+ labeled anti-human PLA2R-IgG antibody to the 96-well plate for incubation reaction. The incubation condition is shaken at room temperature for 1 hour to obtain the coated antigen-PLA2R-IgG antibody-Eu 3+ Label the anti-human PLA2R-IgG antibody complex. Wash again with Tris-HCl. Add enhancement solution and shake for 5 minutes. Measure fluorescence intensity on a microscope to determine the PLA2R-IgG antibody content.

[0039] Unless otherwise specified, the raw materials and equipment used in the present invention are commonly used in the art; the methods used in the present invention are conventional methods in the art unless otherwise specified.

[0040] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A kit for detecting PLA2R antibodies in urine, characterized in that: Includes non-invasive urine sampling device, 96-well plate, and time-resolved fluorescence detection module; The non-invasive urine sampling device includes a urine collection cup, a collection cup, and a U-shaped siphon tube that fit together. The top of the urine collection cup is a backflow prevention cup mouth that is shaped to fit the curve of the female vulva. The inner wall of the urine collection cup is provided with a spiral guide groove that guides the collection cup. The collection cup is located at the bottom of the urine collection cup, and the side wall of the collection cup is provided with scale lines. The inlet end of the U-shaped siphon tube is connected to the side wall of the collection cup and is higher than the bottom of the collection cup. The outlet end of the U-shaped siphon tube extends into the urine collection cup. The microwells of the 96-well plate are pre-coated with PLA2R antigen; The time-resolved fluorescence detection module includes a fluorescence reader, a pipette, a buffer solution, an enhancement solution, a standard substance and an Eu3+-labeled anti-human PLA2R-IgG antibody.

2. The kit for detecting PLA2R antibodies in urine according to claim 1, wherein The anti-backflow cup mouth is an annular edge extending toward the outer side of the cup mouth.

3. The kit for detecting PLA2R antibodies in urine according to claim 1, wherein The spiral guide groove has a groove width of 5-8 mm and an inclination angle of 15-25 degrees.

4. The kit for detecting PLA2R antibodies in urine according to claim 1 or 3, wherein: The number of the spiral guide grooves is 1-4.

5. The kit for detecting PLA2R antibodies in urine according to claim 1, wherein The diameter of the U-shaped siphon tube is 4-6 mm, the inlet end thereof is 5-6 cm higher than the bottom of the collection cup, and the outlet end thereof is 3-4 cm lower than the inlet end.

6. The kit for detecting PLA2R antibodies in urine according to claim 4, wherein: The siphon triggering liquid volume of the U-shaped siphon is 18-22 mL, and the urine drainage flow rate is 15-20 mL / s.

7. The kit for detecting PLA2R antibodies in urine according to claim 1, wherein The scale lines are fluorescent scale lines.

8. The kit for detecting PLA2R antibodies in urine according to claim 1, wherein The antigen coating concentration of the 96-well plate is 8-12 μg / mL, the coating process is microarray printing, the spot diameter is 0.5-1 mm, and the density uniformity CV is less than 5%.

9. The kit for detecting PLA2R antibodies in urine according to claim 1, wherein The Eu3+-labeled anti-human PLA2R-IgG antibody is coupled via a BHHCT chelating agent, has an excitation wavelength of 337 nm, an emission wavelength of 615 nm, and a fluorescence lifetime greater than 1 ms.

10. The kit for detecting PLA2R antibodies in urine according to claim 1, wherein The fluorescence reader integrates a 96-channel fiber optic sensor, supports delayed detection, has a whole-plate detection time of ≤30 seconds, and a linear range of 0.5-200 ng / mL.

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