A time-resolved fluorescence immunochromatographic POCT test strip for simelglumide competition method and application

Abstract

The application provides a time-resolved fluorescence immunochromatography POCT test strip for semaglutide competition, which comprises a PVC bottom plate and a sample pad, a fluorescent microsphere combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially connected to the PVC bottom plate; the fluorescent microsphere combination pad is loaded with time-resolved fluorescent microsphere-labeled anti-semaglutide nanobody fusion protein; the nitrocellulose membrane is provided with a T line and a C line, the T line is coated with a semaglutide-carrier protein conjugate antigen, and the C line is coated with a quality control antibody capable of specifically combining with the labeled fusion protein. The application also provides a preparation method, a detection method and an application of the test strip. The application adopts specific VHH nanobody combined with time-resolved fluorescent microspheres to build a stable, low-interference and small-molecule polypeptide-adaptable competition immunochromatography detection platform, and the detection platform has no cross reaction with GLP-1 homologous polypeptides, so that rapid, stable and accurate quantitative detection of semaglutide biological samples is realized, and the on-site quantitative detection requirement is met.

Description

Technical Field

[0001] This invention belongs to the field of point-of-care testing (POCT) technology, and relates to immunofluorescence chromatography detection technology, particularly to a semaglutide competitive time-resolved fluorescence immunochromatographic POCT test strip and its application. Background Technology

[0002] Glucagon-like peptide-1 (GLP-1) receptor agonists have become important drugs for the treatment of type 2 diabetes and obesity due to their good hypoglycemic and weight-loss effects. Semaglutide, obtained by modifying the amino acids and fatty acid side chains of natural GLP-1, has a long in vivo half-life and high receptor affinity, allowing for once-weekly dosing. It has shown good clinical application value in diabetes treatment, obesity management, and cardiovascular disease risk control. With the expanding clinical application of semaglutide, the demand for monitoring drug concentrations in patients, pharmacokinetic studies, and product quality supervision is increasing.

[0003] However, semaglutide is a small polypeptide with a molecular weight of approximately 4 kDa, possessing only a limited number of antigenic epitopes. Furthermore, its amino acid sequence shows high homology with natural human GLP-1. Therefore, establishing a detection method that combines high sensitivity, high specificity, and rapid detection capabilities presents significant technical challenges. Currently, semaglutide detection primarily employs methods such as liquid chromatography-tandem mass spectrometry (LC-MS / MS), high-performance liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA). While LC-MS / MS offers advantages such as high sensitivity and accurate quantification, the equipment is expensive, sample pretreatment is complex, and the detection cycle is long, requiring a high level of expertise from testing personnel, making it difficult to meet the needs of primary healthcare institutions and rapid on-site testing. Although ELISA is relatively simple to operate, the detection cycle is long, typically requiring several hours, and it is not suitable for point-of-care testing (POCT) applications.

[0004] In recent years, immunochromatography has been widely used due to its fast detection speed, simple operation, low cost, and ease of field application. Among these, colloidal gold immunochromatography is relatively mature; however, because colloidal gold primarily relies on visible light for color development, its detection sensitivity is limited, its linear detection range is narrow, and the results are easily influenced by operator subjectivity, making it difficult to meet the needs of therapeutic drug monitoring and precise quantitative detection. In contrast, time-resolved fluorescent microsphere immunochromatography utilizes rare earth elements (such as Eu)... 3+It features long fluorescence lifetime, high quantum yield and low background interference. It can be used with a matching immunofluorescence analyzer to quantitatively acquire fluorescence signals of the detection line and control line, effectively improving detection sensitivity, repeatability and quantitative accuracy, and is gradually becoming an important development direction for POCT quantitative detection.

[0005] However, several key issues remain regarding competitive immunofluorescence chromatography detection of semaglutide. First, because semaglutide possesses only a single antigenic determinant, traditional double-antibody sandwich assays are unsuitable for detecting this type of small molecule peptide, typically requiring a competitive immunoassay. Second, there are few existing specific antibodies against semaglutide, and some antibodies exhibit varying degrees of cross-reactivity with structurally similar peptides such as endogenous GLP-1, liraglutide, and exenatide, affecting the accuracy of the detection results. Finally, the stability of the fluorescently labeled probe, the consistency of the fluorescence signal, and its resistance to interference in complex biological matrices also directly impact the performance of immunofluorescence chromatography.

[0006] Therefore, there is an urgent need to develop novel recognition antibodies with high affinity and high specificity, and to build a stable, low-interference, and adaptable competitive time-resolved fluorescence immunochromatographic point-of-care testing (POCT) platform for small molecule peptides to meet the demand for rapid and accurate on-site quantitative detection of smegglutinin. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a competitive time-resolved fluorescence immunochromatographic POCT test strip for semaglutide and its application. It uses a specific VHH nanobody obtained through self-screening as a recognition element, combined with time-resolved fluorescent microspheres, to build a stable, low-interference, and adaptable competitive immunochromatographic detection platform for small molecule peptides. There is no cross-reaction of GLP-1 homologous peptides, which enables rapid, stable, and accurate quantitative detection of semaglutide biological samples and meets the needs of on-site real-time quantitative detection.

[0008] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A semaglutide competitive time-resolved fluorescence immunochromatographic POCT test strip includes a PVC base plate, and a sample pad, a fluorescent microsphere binding pad, a nitrocellulose membrane (NC membrane), and an absorbent pad that are sequentially overlapped and fixed on the PVC base plate along the liquid chromatography direction. The fluorescent microsphere binding pad is loaded with a time-resolved fluorescent microsphere-labeled antisemaglutide nanobody fusion protein; the antisemaglutide nanobody in the fusion protein is a single-domain antibody lacking a light chain, and its amino acid sequence is shown in SEQ ID NO.1; The nitrocellulose membrane is provided with a detection line (T line) and a control line (C line); the T line is coated with semaglutide-carrier protein conjugate antigen, and the C line is coated with a control antibody that can specifically bind to the antisemaglutide nanobody fusion protein labeled with the microspheres.

[0009] As one of the preferred embodiments of the present invention, the time-resolved fluorescent microspheres are Eu 3+ Carboxylated time-resolved fluorescent microspheres with a particle size of 50~500nm, more preferably 100~300nm, and most preferably 200nm.

[0010] As one of the preferred embodiments of the present invention, the antisemaglutide nanobody fusion protein is formed by fusing an antisemaglutide single-domain nanobody (VHH) with a chicken IgY Fc fragment, wherein the fusion sequence is VHH-(G4S)3 flexible linker peptide-chicken IgY Fc.

[0011] As one of the preferred embodiments of the present invention, the semaglutide-carrier protein conjugate antigen is a conjugate of semaglutide and a carrier protein covalently bound together, wherein the carrier protein is selected from at least one of BSA, OVA, and KLH, and more preferably BSA.

[0012] As one of the preferred embodiments of the present invention, the quality control antibody is a goat anti-chicken IgY antibody.

[0013] As one of the preferred embodiments of the present invention, the test strip adopts the principle of competitive immunochromatography; when the sample to be tested migrates from the sample pad to the fluorescent microsphere binding pad, the free semaglutide in the sample competitively binds to the time-resolved fluorescent microsphere-labeled anti-semaglutide nanobody fusion protein on the detection line with the semaglutide-carrier protein conjugate antigen, and the T / C fluorescence signal is collected by an immunofluorescence analyzer to realize the quantitative detection of semaglutide.

[0014] A method for preparing the above-mentioned test strip includes the following steps: (1) Preparation of labeled probe: Time-resolved fluorescent microspheres were activated by EDC / NHS and then covalently coupled with antisemaglutide nanobody fusion protein. After blocking and centrifugation purification, the antisemaglutide nanobody fusion protein labeled with time-resolved fluorescent microspheres was obtained, which is the labeled probe. (2) Preparation of fluorescent microsphere binding pad: The labeled probe was diluted and uniformly sprayed onto a glass fiber pad, and then dried to obtain a fluorescent microsphere binding pad; (3) Coating treatment of nitrocellulose membrane: Spray smegglutinin-carrier protein conjugate antigen onto the detection line T of the nitrocellulose membrane and spray quality control antibody onto the control line C, and dry for later use; (4) Sample pad pretreatment: Immerse the sample pad in PBS pretreatment buffer containing 1% BSA, 0.05% Tween-20, 0.02% Proclin-300, pH 7.4, and dry thoroughly; (5) Test strip assembly: In accordance with the chromatographic flow direction, the pretreated sample pad, fluorescent microsphere binding pad, coated nitrocellulose membrane, and absorbent pad are sequentially overlapped on the PVC base plate. The overlap between adjacent layers is 2-3 mm. After cutting into single test strips, they are sealed and stored.

[0015] As one of the preferred embodiments of the present invention, the test strip has a detection limit of 0.18 ng / mL and an effective detection concentration range of 0.2~100 ng / mL; the intra-batch precision coefficient of variation is less than 8%, and the serum sample spike recovery rate is 98%~103%; and there is no cross-reactivity with endogenous GLP-1, glucagon, insulin, exenatide, liraglutide, and dulaglutide.

[0016] A non-diagnostic method for detecting semaglutide, using the above-mentioned test strip, comprises the following steps: (1) Sample loading chromatography: Add 80~100μL of the sample to be tested to the sample pad and allow it to chromatographically at room temperature for 10~15min; (2) Fluorescence signal acquisition: Place the chromatographically completed test strip into the immunofluorescence analyzer, acquire the T-line and C-line fluorescence signals respectively, and calculate the T / C ratio; (3) Result determination: The obtained T / C ratio is combined with the gradient semaglutide standard curve to obtain the precise concentration of semaglutide in the sample.

[0017] As one of the preferred embodiments of the present invention, the sample to be tested in step (1) is selected from any one of serum, plasma, and saliva; wherein, serum and plasma are directly loaded, and saliva is centrifuged to obtain the supernatant and then dripped onto the sample pad.

[0018] An application of the above-mentioned test strip in the preparation of a semagrastim assay kit, wherein the assay kit is used for semagrastim blood concentration detection, pharmacokinetic studies, administration effect monitoring, screening for illegal addition of semagrastim to samples, and other semagrastim-related in vitro detections.

[0019] The advantages of this invention compared to the prior art are: (1) In this invention, the anti-semaglutide single-domain nanobody VHH obtained through independent screening is used as a specific recognition element. This antibody has high binding activity (EC). 50 With a concentration of 40.26 ng / mL, it can specifically recognize smegglutinin and has no cross-reactivity with structurally similar homologous peptides such as endogenous GLP-1, glucagon, insulin, and exenatide. It also exhibits excellent resistance to matrix interference. VHH has a small molecular weight and stable structure, making it easy to recombinantly express, engineer, and couple with time-resolved fluorescent microspheres. The resulting fluorescently labeled probes have good stability.

[0020] (2) In view of the fact that smegglutinin is a small molecule polypeptide with only a single antigenic epitope, and cannot be detected by the double antibody sandwich method, this invention constructs a competitive method Eu 3+ A time-resolved fluorescence immunochromatography system; utilizing the advantages of long fluorescence lifetime and low background interference of rare earth fluorescent microspheres, and using an immunofluorescence analyzer to collect T / C fluorescence signals and combine them with a standard curve to achieve accurate quantification, overcoming the limitation of colloidal gold immunochromatography which can only perform qualitative analysis and has insufficient quantitative capabilities, and achieving highly sensitive quantitative detection.

[0021] (3) The present invention engineered the antisemaglutide single-domain nanobody VHH by fusing a chicken IgY Fc fragment with a flexible peptide at its C-terminus as an independent quality control tag, which can specifically pair with the C-line coated goat anti-chicken IgY antibody to build a stable and reliable quality control system. This fusion modification method does not interfere with the antigen recognition center structure of VHH and does not affect the specific binding of the antibody to semaglutide, effectively ensuring good repeatability of the detection results.

[0022] (4) The whole detection operation is simple, without the need for complex sample pretreatment or large precision instruments such as LC-MS / MS. The detection can be completed with just a portable immunofluorescence analyzer. The chromatographic reaction can produce results in 10-15 minutes. The detection speed is fast, which meets the needs of rapid quantitative detection in POCT and solves the problems of expensive equipment, complicated pretreatment, long detection cycle and difficulty in field application of traditional mass spectrometry methods.

[0023] (5) The detection system of the present invention has excellent comprehensive performance, with a detection limit of 0.18 ng / mL, an effective linear range of 0.2~100 ng / mL, an intra-batch precision coefficient of variation of less than 8%, and a sample spike recovery rate of 98%~103%. It is compatible with various test samples such as serum, plasma, and saliva, and can be applied to various in vitro detection scenarios such as semaglutide blood concentration detection, pharmacokinetic studies, drug administration effect monitoring, and screening for illegal addition of semaglutide in samples. It has broad prospects for industrial application. Attached Figure Description

[0024] Figure 1 This is an SDS-PAGE electrophoresis image of the purified antisemaglutide VHH from Example 2 (in the image, "M" represents the protein molecular weight marker, and "1" represents the purified antisemaglutide VHH protein sample). Figure 2 This is the ELISA fitting curve of the binding activity between antisemaglutide VHH and semaglutide in Example 3; Figure 3 This is the Logistic calibration curve of the four parameters of the competitive immunochromatography of smegglutinin in Example 7; Figure 4This is a schematic diagram of the competitive immunofluorescence chromatography detection principle of smegglutinin in Example 7; Figure 5 This is a linear correlation curve between the measured concentration and the theoretical spiking concentration of smegglutinin recovered by spiked treatment in Example 8. Detailed Implementation

[0025] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents, peptides, carrier proteins, antigens, antibodies, and other materials used in the following embodiments are all commercially available products conventional in the art; the instruments and equipment used are all commercially available equipment generally used in the art; and the experimental methods used are all conventional methods in the art and will not be described further.

[0026] Example 1: Screening of anti-semaglutide single-domain nanobody VHH: 1. Antigen coating 200 pmol of BSA-semaglutide-biotin (prepared by biotin-NHS ester labeling after conjugation of semaglutide with BSA) was incubated with 20 μg of M280-streptavidin magnetic beads at room temperature for 1 h to allow the antigen to bind to the surface of the magnetic beads. After washing three times with PBS buffer, 5% skim milk powder-PBS buffer (PBST) was added for blocking for 1 h to reduce non-specific binding.

[0027] 2. Phage library binding The pre-constructed synthetic nanobody phage display library was diluted to 10. 12 Add pfu / mL to the above magnetic beads and incubate with gentle shaking at room temperature for 1 hour to ensure that the phage and antigen are fully bound.

[0028] 3. Washing and elution Discard unbound phages and wash 10 times with PBST containing 0.05% Tween-20 to remove non-specifically bound phages. Add 0.1M glycine-HCl elution buffer (pH 2.2), incubate at room temperature for 10 min, and immediately neutralize the elution buffer with 1M Tris-HCl (pH 9.0) to collect antigen-bound phages.

[0029] 4. Amplification and Enrichment The eluted phages were used to infect logarithmically growing E. coli TG1 competent cells and cultured at 37°C in a shaker for 30 min. Amplification was then performed using 2×YT medium containing ampicillin and glucose. The phage supernatant was concentrated using PEG / NaCl precipitation. This coating, binding, elution, and amplification process was repeated 2-3 times, increasing the number of washes and the Tween-20 concentration in each round to enhance the selection pressure and thus enrich a population of high-affinity smegglutinin-binding phages.

[0030] 5. Monoclonal screening and identification Enriched bacteriophages were used to infect *E. coli*, and single-clone colonies were selected to induce the expression of nanobody fragments. Identification was performed using a soluble supernatant ELISA method: BSA-coated wells were used as detection wells, and PBS-coated wells were used as control wells. The binding activity and specificity of different clones to semaglutide were detected. Finally, OD... 450 Positive clones with significantly elevated levels and no obvious signal in the control wells.

[0031] 6. Sequencing analysis The nanobody genes of positive clones were sequenced to analyze the diversity of CDR region sequences, and representative clones were selected for subsequent expression and purification.

[0032] This embodiment ultimately yielded an antisemaglutide single-domain nanobody (VHH) with the amino acid sequence shown in SEQ ID NO.1.

[0033] Example 2: Gene synthesis, vector construction, expression and purification of anti-semaglutide nanobody VHH (containing His-tag).

[0034] 1. Based on the amino acid sequence shown in SEQ ID NO.1, and according to the codon preference of E. coli, the DNA sequence encoding the nanobody was chemically synthesized (SEQ ID NO.2).

[0035] 2. Using restriction endonucleases Nco I and Xho I. The synthesized DNA fragment and expression vector pET-22b (+) were double-digested. After recovering the digestion products, they were ligated with T4 DNA ligase and transformed into E. coli DH5α competent cells to construct the recombinant plasmid pET-22b-VHH.

[0036] 3. The recombinant plasmid was sequenced and verified. The plasmid with the correct sequence was transformed into Escherichia coli BL21 (DE3) competent cells, plated on LB agar plates containing 100 μg / mL ampicillin, and incubated overnight at 37°C to obtain positive expression strains.

[0037] 4. Pick a single colony of the positive expression strain obtained in the previous steps, inoculate it into LB liquid medium containing 100 μg / mL ampicillin, and incubate at 37°C with shaking at 220 rpm until OD reaches 100%. 600 The value reaches 0.6~0.8.

[0038] 5. Add isopropyl-β-D-thiogalactoside (IPTG) to the culture medium to a final concentration of 0.5 mM and induce expression at 28°C for 16 h.

[0039] 6. After the induction expression is completed, the bacterial cells are collected by centrifugation, and after sonication, the supernatant is collected by centrifugation.

[0040] 7. The histidine-tag fusion nanobody in the supernatant was purified using a Ni-NTA affinity chromatography column and eluted with an elution buffer containing 20 mM Tris, 150 mM NaCl, 5% glycerol and 500 mM imidazole.

[0041] 8. The purity of the eluted fraction was determined by SDS-PAGE electrophoresis, and the results are as follows: Figure 1 As shown: There are no obvious impurities, indicating that the high-purity anti-smegglutinin single-domain nanobody VHH was successfully obtained (due to errors in the His-tag label and electrophoresis system, there is a slight reasonable shift in the band).

[0042] Example 3: Affinity verification of anti-semaglutide nanobody VHH with semaglutide (ELISA method): 1. Coating: Dilute smegglutinin to 2 μg / mL with PBS buffer, and coat 100 μL per well in a 96-well microplate. Incubate overnight at 4°C.

[0043] 2. Blocking: After washing the plate 3 times, add PBS solution containing 3% BSA and block at 37°C for 2 hours.

[0044] 3. Gradient antibody incubation: Wash the plate, add a series of concentration gradients (0.1 nM~100 nM) of the purified anti-semaglutide nanobody VHH from Example 2, and incubate at 37°C for 1 h. Wash the plate again, add mouse anti-His tag primary antibody, and incubate at 37°C for 1 h.

[0045] 4. Secondary antibody incubation: Wash the plate, add HRP-labeled goat anti-mouse secondary antibody, and incubate at 37℃ for 1 hour.

[0046] 5. OD signal detection: After washing the plate, add TMB substrate solution, develop the color in the dark, terminate the reaction with 2M H2SO4, and measure the absorbance at a wavelength of 450 nm. Results are shown below. Figure 2 The nanobody can bind specifically to smegglutinin in a dose-dependent manner, and its EC50 can be calculated. 50The value was 40.26 ng / ml, confirming that the nanobody and smegglutinin have good binding activity.

[0047] Example 4: Construction and expression of anti-semaglutide nanobody fusion protein (VHH-IgY Fc): 1. The DNA sequence (SEQ ID NO.2) encoding the anti-smegglutinin nanobody VHH shown in SEQ ID NO.1 was linked to the DNA sequence of the chicken IgY Fc segment (a known sequence) via a flexible linker peptide (G4S)3. The fusion gene was chemically synthesized in the order of "VHH-(G4S)3-Fc". At the same time, restriction enzyme sites (Hind III and Xba I) matching the commercially available mammalian expression vector pcDNA3.4 were introduced at both ends of the gene.

[0048] 2. The synthesized fusion gene and the commercially available mammalian expression vector pcDNA3.4 were double-digested with Hind III and Xba I enzymes; the target fragment and vector backbone were recovered by electrophoresis and ligated overnight at 16°C using T4 DNA ligase; the cells were transformed into E. coli DH5α competent cells, plated on LB agar plates containing ampicillin, and incubated overnight at 37°C; single colonies were picked, recombinant plasmids were extracted and sequenced to verify the recombinant expression plasmid pcDNA3.4-VHH-IgY Fc.

[0049] 3. Using polyethyleneimine (PEI) transfection reagent, the recombinant plasmid pcDNA3.4-VHH-IgY Fc was transfected into Expi293F mammalian suspension cells. The cells were cultured at 37°C and 8% CO2 for 7 days, and the cell culture supernatant was collected.

[0050] 4. The VHH-IgY Fc fusion protein in the supernatant was captured and purified using an anti-chicken IgY affinity chromatography column. The anti-chicken IgY affinity chromatography column was pre-equilibrated with Binding Buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4). After filtering the supernatant through a 0.45 μm filter membrane, the sample was loaded and washed with 5 column volumes of Binding Buffer to remove impurities. The target protein was then eluted with Elution Buffer (0.1 mol / L citric acid, pH 3.0). The eluent was collected and immediately neutralized to pH 7.0 with 1 mol / L Tris-HCl (pH 9.0).

[0051] 5. After identifying the purified product by SDS-PAGE electrophoresis, the dimeric form of antisemaglutide-IgY Fc fusion antibody (VHH-IgY Fc fusion protein) was obtained, which is the target antisemaglutide nanobody fusion protein.

[0052] Example 5: Preparation of time-resolved fluorescent microsphere-labeled VHH probes: This embodiment uses Eu 3+ Carboxylated time-resolved fluorescent microspheres were used as fluorescent signal carriers. The VHH-IgY Fc fusion protein (prepared in Example 4) was covalently coupled to the surface of the fluorescent microspheres via an EDC / NHS activation coupling method to prepare the Eu-VHH fluorescent probe required for competing immunofluorescence chromatography detection reagents.

[0053] 1. Take Eu 3+ Take an appropriate amount of carboxylated time-resolved fluorescent microsphere suspension (microsphere diameter 200 nm), shake thoroughly to mix, and centrifuge at 11000×g for 11 min, discarding the supernatant. Resuspend the microspheres in MES buffer (50 mmol / L, pH 5.8) and sonicate for 30–60 s to ensure uniform dispersion of the fluorescent microspheres and prevent particle aggregation.

[0054] 2. Add freshly prepared EDC and NHS solutions sequentially to the above microsphere suspension to activate the carboxyl groups on the microsphere surface and form NHS ester intermediates. Gently shake the mixture at room temperature for 20 minutes.

[0055] 3. After activation, centrifuge at 11000×g for 10 min and discard the supernatant. Resuspend the activated fluorescent microspheres in PBS buffer (pH 7.3). Add the purified VHH-IgY Fc fusion protein and mix slowly. Incubate at room temperature with gentle shaking for 1.5 h to allow the amino groups in the VHH molecule to undergo amide bond coupling with the activated carboxyl groups on the microsphere surface, forming a stable Eu-VHH fluorescent probe. The mass ratio of microspheres to VHH is 1:10.

[0056] 4. After the coupling reaction is complete, blocking buffer is added to block unreacted active carboxyl sites to reduce non-specific adsorption in subsequent immunochromatography. The blocking buffer is 1% bovine serum albumin (BSA), and the mixture is incubated at room temperature for 25 minutes.

[0057] 5. After blocking, centrifuge at 10000~15000×g for 10 min. Discard the supernatant. Wash 2~3 times with PBS buffer to thoroughly remove uncoupled VHH, EDC, NHS and other small molecule impurities. If necessary, ultrafiltration or gel filtration can be used to further purify the fluorescent probe.

[0058] 6. The purified Eu-VHH probe was resuspended in a preservation solution. The preservation solution included PBS buffer, trehalose, Tween-20, and ProClin 300 preservative. The obtained Eu-VHH probe was stored at 4°C protected from light for later use.

[0059] 7. The changes in protein concentration before and after conjugation were determined using ultraviolet absorption, BCA protein quantification, or other protein quantification methods, and the antibody conjugation efficiency was calculated. Simultaneously, dynamic light scattering (DLS) was used to detect changes in the size of the fluorescent microspheres before and after conjugation, a Zeta potentiometer was used to measure changes in the surface potential of the microspheres, and a time-resolved fluorescence analyzer was used to detect the fluorescence signal intensity and stability of the Eu-VHH probe to evaluate the quality of the fluorescent probe preparation.

[0060] Example 6: Preparation of competitive immunofluorescence chromatography test strips based on Eu-VHH probes: A PVC base plate is used as the support substrate for the test strip. The sample pad, fluorescent microsphere binding pad, NC membrane, and absorbent pad are sequentially overlapped along the liquid chromatography direction, with each component overlapping by approximately 2.5 mm to ensure continuous liquid migration. The fluorescent microsphere binding pad is loaded with a time-resolved fluorescent microsphere-labeled VHH probe (prepared in Example 5). The NC membrane has T-lines and C-lines. The T-line is coated with semaglutide-BSA conjugate antigen (semaglutide polypeptide and bovine serum albumin prepared by chemical cross-linking and covalent bonding), and the C-line is coated with a goat anti-chicken IgY antibody (quality control antibody) that specifically binds to the time-resolved fluorescent microsphere-labeled VHH probe.

[0061] The specific preparation method is as follows: (1) Preparation of fluorescent microsphere binding pad: The fluorescent microspheres labeled with VHH probe prepared in Example 5 were diluted and uniformly sprayed onto a glass fiber pad. After drying at 37°C, the fluorescent microsphere binding pad was obtained.

[0062] (2) NC membrane coating treatment: The smegglutinin-BSA conjugate antigen was sprayed on the T line position of the NC membrane using a biological spotting instrument, and the goat anti-chicken IgY antibody was sprayed on the C line position. The membrane was then dried for later use.

[0063] (3) Sample pad pretreatment: Immerse the sample pad in PBS pretreatment buffer containing 1% BSA, 0.05% Tween-20, 0.02% Proclin-300, pH 7.4, and dry thoroughly.

[0064] (4) Test strip assembly: In accordance with the chromatographic flow direction, the pretreated sample pad, fluorescent microsphere binding pad, coated NC membrane, and absorbent pad are sequentially overlapped on the PVC base plate. The overlap between adjacent layers is 2.5 mm. Then, the strips are cut into individual test strips with a width of about 4 mm and sealed in an aluminum foil bag containing desiccant for later use.

[0065] Example 7: Establishment of a competitive immunofluorescence chromatography detection method: This embodiment is used to establish a rapid quantitative detection method for smegglutinin based on time-resolved fluorescent microsphere competitive immunochromatography.

[0066] 1. Sample detection: Take 90 μL of the sample to be tested and add it to the sample pad of the test strip. The sample enters the fluorescent microsphere binding pad through the sample pad, and after being fully mixed with the Eu-VHH probe, a competitive binding reaction occurs, and the chromatography continues along the NC membrane.

[0067] 2. Fluorescence signal acquisition: After the chromatography reaction is completed (reaction time 12 min), insert the test strip into the immunofluorescence analyzer. The instrument acquires the fluorescence signals of the T line and C line respectively, and automatically calculates the fluorescence intensity ratio of the test line to the control line (T / C value).

[0068] 3. Result Interpretation: Based on the pre-established standard curve, the T / C value obtained from the detection is converted into smegglutinin concentration to achieve rapid quantitative detection.

[0069] The method for establishing the standard curve is as follows: Using smegglutide standard, negative serum or sample diluent was used as the dilution matrix to prepare standards with different concentration gradients. The above method was used to detect the standards with different concentration gradients. Each concentration standard was detected repeatedly for no less than 3 times. The fluorescence intensity of the corresponding T line and C line was recorded, and the T / C value was calculated.

[0070] Plotting semaglutide concentration on the x-axis and T / C fluorescence ratio on the y-axis, a four-parameter Logistic (4PL) model was used to fit the calibration curve. The fitting equation is as follows: y =(0.962-0.038) / [1+ ( x / 2.8) -1.15 +0.038; where y represents the T / C value and x represents the concentration of semaglutide (ng / mL); the asymptotic upper limit of the curve is 0.962, the asymptotic lower limit is 0.038, the slope factor is -1.15, and the half-maximal inhibitory concentration (IC50) is... 50 =2.8 ng / mL, fitting correlation coefficient R 2 =0.9987. A calibration curve was plotted based on the test data. Figure 3 The standard curve parameters are imported into the immunofluorescence analyzer to automatically calculate the concentration of semaglutide in the sample.

[0071] A schematic diagram illustrating the principle of the competitive immunofluorescence chromatography-integrity detection of this invention is shown below. Figure 4 .

[0072] Example 8: Evaluation of Detection Methodology The detection method established in Example 7 was used to evaluate the methodology of the competitive immunofluorescence chromatography test strip of the present invention.

[0073] 1. Preparation of standards: Dilute semaglutide-negative serum with 0.2~100 ng / mL gradient standards.

[0074] 2. Sample addition and detection: Add 90 μL of each concentration to the test paper and compare with the standard curve after 12 min; the data are shown in Table 1.

[0075] Table 1

[0076] 3. Sensitivity: The detection limit was calculated to be 0.18 ng / mL based on 20 blank serum samples.

[0077] 4. Linearity: Spiked in the range of 0.2~100 ng / mL, the recovery linearity was normal, R0 2 >0.99, linear correlation results are shown in [link to results]. Figure 5 The data is shown in Table 2.

[0078] Table 2

[0079] 5. Intra-batch precision: For the three concentrations of 1.5, 15, and 80 ng / mL, the CVs were 6.34%, 4.84%, and 4.10% respectively in 24 replicates on the same plate, all <8%; data are shown in Table 3.

[0080] Table 3

[0081] 6. Spike recovery rate: The recovery rate of serum spiked samples at 1.0, 10, and 50 ng / mL was 98%–103%. Data are shown in Table 4.

[0082] Table 4

[0083] Example 9: Evaluation of specificity and anti-interference ability: 1. Preparation of interfering substances: GLP-1, glucagon, insulin, exenatide, liraglutide, and dulaglutide were each prepared into a high-concentration solution of 1000 ng / mL; negative buffer and 20 ng / mL smegglutide were prepared for quality control. 2. Test strip detection: Samples from each group were loaded separately, and fluorescence detection results were read after 12 minutes; 3. Result Interpretation: All homologous interfering reagent test strips showed clear fluorescence at the T-line, with equivalent detection concentrations below the detection limit of 0.18 ng / mL, showing no difference from the negative control; the positive control T-line was significantly lighter, with no cross-reaction. Data are shown in Table 5.

[0084] Table 5

[0085] Example 10, Suitability Testing of Different Biological Samples (Serum / Plasma): 1. Sample grouping: 30 serum samples, 30 EDTA anticoagulated plasma samples, and 30 saliva samples from healthy individuals; each group was supplemented with 1 ng / mL, 20 ng / mL, and 80 ng / mL smegglutide, respectively; serum and plasma were directly loaded onto the samples.

[0086] 2. Test strip testing: Each sample is tested in parallel 3 times, and the concentration range is determined by comparing with the standard curve.

[0087] 3. Results: The recovery rates for all three types of samples were between 98% and 103%, with intra-assay CV < 8%. There was no significant interference from the saliva matrix, and the test strips are compatible with three commonly used clinical biological samples. Data are shown in Table 6.

[0088] Table 6

[0089] Example 11, Accelerated stability test of test strips: 1. Acceleration conditions: The sealed test strips were placed in a 37℃ constant temperature incubator for 30 days; sealed test strips at 25℃ room temperature were set up as a control.

[0090] 2. Testing indicators: Low, medium and high concentration quality control samples were tested at 0, 10, 20 and 30 days respectively.

[0091] 3. Evaluation criteria: Compare the intensity of T-line fluorescence detection, recovery rate, and CV value.

[0092] 4. Test Results: After 30 days of accelerated testing at 37℃, the sensitivity, specificity, and precision of the test strips showed no significant attenuation. The C-line exhibited normal fluorescence detection, and the product's shelf life at room temperature can reach over 12 months. Data are shown in Table 7.

[0093] Table 7

[0094] In summary, this invention screened and obtained the anti-semagraft VHH nanobody, constructed the VHH-IgY Fc fusion protein, prepared a time-resolved fluorescent probe, and established a competitive immunofluorescence chromatography quantitative detection test strip and a corresponding detection method. Verification showed that the detection system has high sensitivity, excellent specificity, good precision and accuracy, is adaptable to various clinical biological samples, exhibits good stability, and can rapidly quantify the concentration of semagraft in samples. This test strip can be used to prepare a semagraft detection kit for real-time monitoring of semagraft blood concentrations in clinical settings, pharmacokinetic studies, efficacy evaluation, screening for illegal addition of semagraft to samples, and other semagraft-related in vitro detections.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A semaglutide competitive time-resolved fluorescence immunochromatographic POCT test strip, characterized in that, It includes a PVC base plate, and a sample pad, a fluorescent microsphere binding pad, a nitrocellulose membrane and an absorbent pad that are sequentially overlapped and fixed on the PVC base plate along the liquid chromatography direction; The fluorescent microsphere binding pad is loaded with a time-resolved fluorescent microsphere-labeled antisemaglutide nanobody fusion protein; the amino acid sequence of the antisemaglutide nanobody in the fusion protein is shown in SEQ ID NO.1; The nitrocellulose membrane is provided with a detection line (T line) and a control line (C line); the T line is coated with semaglutide-carrier protein conjugate antigen, and the C line is coated with a control antibody that can specifically bind to the anti-semaglutide nanobody fusion protein labeled with the microspheres.

2. The test strip according to claim 1, characterized in that, The time-resolved fluorescent microspheres are Eu 3+ Carboxylated time-resolved fluorescent microspheres with a particle size of 50-500 nm.

3. The test strip according to claim 1, characterized in that, The antisemaglutide nanobody fusion protein is formed by fusing the antisemaglutide single-domain nanobody VHH with a chicken IgY Fc fragment, with the fusion sequence being VHH-(G4S)3 flexible linker peptide-chicken IgY Fc.

4. The test strip according to claim 1, characterized in that, The semaglutide-carrier protein conjugate antigen is a conjugate of semaglutide and a carrier protein covalently bound together, wherein the carrier protein is selected from at least one of BSA, OVA, and KLH.

5. The test strip according to claim 1, characterized in that, The quality control antibody is a goat anti-chicken IgY antibody.

6. The test strip according to any one of claims 1 to 5, characterized in that, When the sample to be tested migrates from the sample pad to the fluorescent microsphere binding pad, the free semaglutide in the sample competitively binds to the time-resolved fluorescent microsphere-labeled anti-semaglutide nanobody fusion protein on the detection line. The T / C fluorescence signal is collected by an immunofluorescence analyzer to achieve quantitative detection of semaglutide.

7. A method for preparing a test strip as described in any one of claims 1 to 6, characterized in that, Includes the following steps: (1) Preparation of labeled probe: Time-resolved fluorescent microspheres were activated by EDC / NHS and then covalently coupled with antisemaglutide nanobody fusion protein. After blocking and centrifugation purification, the antisemaglutide nanobody fusion protein labeled with time-resolved fluorescent microspheres was obtained, which is the labeled probe. (2) Preparation of fluorescent microsphere binding pad: The labeled probe was diluted and uniformly sprayed onto a glass fiber pad, and then dried to obtain a fluorescent microsphere binding pad; (3) Coating treatment of nitrocellulose membrane: Spray smegglutinin-carrier protein conjugate antigen onto the detection line T of the nitrocellulose membrane and spray quality control antibody onto the control line C, and dry for later use; (4) Sample pad pretreatment: Immerse the sample pad in PBS pretreatment buffer containing 1% BSA, 0.05% Tween-20, 0.02% Proclin-300, pH 7.4, and dry thoroughly; (5) Test strip assembly: In accordance with the chromatographic flow direction, the pretreated sample pad, fluorescent microsphere binding pad, coated nitrocellulose membrane, and absorbent pad are sequentially overlapped on the PVC base plate. The overlap between adjacent layers is 2-3 mm. After cutting into single test strips, they are sealed and stored.

8. A method for detecting smegglutinin for non-diagnostic purposes, characterized in that, Using the test strip according to any one of claims 1 to 6, the steps are as follows: (1) Sample loading chromatography: Add 80~100μL of the sample to be tested to the sample pad and allow it to chromatographically at room temperature for 10~15min; (2) Fluorescence signal acquisition: Place the chromatographically completed test strip into the immunofluorescence analyzer, acquire the T-line and C-line fluorescence signals respectively, and calculate the T / C ratio; (3) Result determination: The obtained T / C ratio is combined with the gradient semaglutide standard curve to obtain the precise concentration of semaglutide in the sample.

9. The detection method according to claim 8, characterized in that, The sample to be tested in step (1) is selected from any one of serum, plasma, and saliva; wherein, serum and plasma are directly loaded, and saliva is centrifuged to obtain the supernatant and then dripped onto the sample pad.

10. The use of a test strip as described in any one of claims 1 to 6 in the preparation of a smegglutinin detection kit.

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