Hepatitis a and e virus igm antibody combined detection kit and preparation method

Abstract

The application discloses a combined detection kit for IgM antibodies of hepatitis A and hepatitis E viruses and a preparation method, and belongs to the technical field of in-vitro detection. The gold mark pad has a double-layer structure composed of a bottom slow-release layer and an upper fast-release layer. The upper surface of the bottom slow-release layer is subjected to a solidification treatment with anhydrous ethanol to form a dense interface. The colloidal gold-labeled mouse anti-human IgM monoclonal antibody is obtained by modifying the surface of colloidal gold particles with alpha-mercapto-omega-hydroxyl polyethylene glycol, labeling the antibody and performing target stabilization. The sample pad is loaded with a zwitterionic nanohydrogel network. The coating buffer of the detection line T1 contains a macromolecular crowding agent and a viscosity regulator. The application is technically innovative in terms of molecular modification, formula optimization and structure design. Through the technical solutions of ion pre-programming, inert isolation, space-time programmed release and differential micro-area enhancement, the key technical problem that it is difficult to effectively balance the signals of two detection lines in a unified chromatographic channel is solved.

Description

Technical Field

[0001] This invention belongs to the field of in vitro detection technology and relates to a combined detection kit for hepatitis A and hepatitis E virus IgM antibodies and its preparation method. Background Technology

[0002] Hepatitis A virus (HAV) and hepatitis E virus (HEV) are both hepatotropic viruses transmitted via the fecal-oral route and are the main pathogens causing acute viral hepatitis in humans. The clinical symptoms of infections caused by the two viruses are highly similar, both presenting with fever, fatigue, jaundice, and elevated transaminase levels, making clinical differentiation difficult based solely on symptoms. However, their epidemiological characteristics, high-risk populations, and prognostic management differ significantly. For example, the mortality rate of hepatitis E in pregnant women can reach 20%–25%, while hepatitis A rarely leads to severe illness. Therefore, achieving rapid and simultaneous differential diagnosis of HAV and HEV infections is of great significance for guiding clinical treatment decisions and public health prevention and control.

[0003] In clinical testing, the diagnosis of acute HAV and HEV infections primarily relies on the detection of specific IgM antibodies in serum. Colloidal gold immunochromatography, with its advantages of ease of operation, rapid detection, and intuitive result interpretation, has become the preferred technology platform for primary healthcare institutions and on-site screening during outbreaks. Integrating HAV-IgM and HEV-IgM detection onto a single reagent strip to form a combined detection kit allows for simultaneous identification of both pathogens with a single sample addition, further improving detection efficiency.

[0004] However, currently available commercially available HAV / HEV IgM combined detection kits suffer from a significant technical problem in practical applications: the signal intensity of the two detection lines is difficult to balance. Specifically, due to inherent differences in immunoreactivity, isoelectric point, and affinity for antibodies between recombinant HAV antigen and recombinant HEV antigen, when they are coated on the same nitrocellulose membrane for tandem chromatography under the same reaction conditions, the color intensity of one detection line is often significantly weaker than the other, even leading to missed detections of weak positive samples. This signal imbalance severely interferes with the accuracy of clinical interpretation, easily resulting in false negatives for weak signal lines, and consequently affecting the selection of subsequent treatment plans.

[0005] Current solutions mainly follow two paths. One is to compensate for signal differences by adjusting the concentration of coating antigen or the amount of labeled antibody for each detection line. However, this approach has an inherent contradiction: increasing the coating concentration of weak signal lines increases the non-specific adsorption of labeled antibodies by the solid-phase antigen, thus raising the background; increasing the total amount of labeled antibody to balance the signal leads to preferential saturation of strong signal lines due to their higher capture efficiency, further amplifying the signal difference between the two lines. The second approach attempts to improve the physical structure, such as constructing two independent chromatography channels on a nitrocellulose membrane, each with its own binding pad and buffer conditions for different detection lines. However, this approach requires precisely dividing a clinical sample into two equal parts and adding them separately, increasing the complexity of the sample addition operation. Furthermore, the difficulty in ensuring uniformity in the micro-distribution of whole blood samples makes it practically impossible to parallel and control the reaction conditions of the two channels.

[0006] Therefore, how to overcome the limitations of single gold-labeled antibody supply within a unified chromatography channel and achieve effective signal balance for two detection lines with different activities has become a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0007] The purpose of this invention is to provide a combined detection kit and preparation method for hepatitis A and hepatitis E virus IgM antibodies. In order to achieve effective balance of signals from the two detection lines within a unified chromatography channel, this invention addresses the problems existing in the prior art, overcomes the technical bottleneck in existing combined detection kits that cannot meet the differentiated needs of the two detection lines due to the limited supply of a single gold-labeled antibody, and thereby improves the clinical interpretation accuracy and detection reliability of the kit.

[0008] The technical solution adopted in this invention is: The key feature of the combined detection kit for hepatitis A and E virus IgM antibodies is that it includes: The sample pad, gold label pad, reaction membrane coated with detection line T1, detection line T2 and quality control line C, and absorbent pad are fixed on the PVC base plate. The aforementioned gold-labeled pad has a two-layer structure consisting of a bottom slow-release layer and an upper fast-release layer. The upper surface of the bottom slow-release layer is cured with anhydrous ethanol to form a dense interface. The bottom slow-release layer contains hydroxypropyl methylcellulose, and the top fast-release layer contains polyvinylpyrrolidone. Both of them are loaded with colloidal gold-labeled mouse anti-human IgM monoclonal antibody. The above-mentioned colloidal gold-labeled mouse anti-human IgM monoclonal antibody was obtained by modifying the surface of colloidal gold particles with α-mercapto-ω-hydroxy polyethylene glycol, labeling the antibody, and then performing targeted stabilization treatment with boric acid-functionalized polyamide-amine dendritic macromolecules. The sample pads described above are loaded with zwitterionic nanohydrogel networks; The above-mentioned detection line T1 is a hepatitis A virus detection line coated with recombinant hepatitis A virus antigen, and its coating buffer contains macromolecular crowding agent and viscosity modifier; The above-mentioned detection line T2 is a hepatitis E virus detection line coated with recombinant hepatitis E virus antigen, and its coating buffer does not contain the above-mentioned macromolecular crowding agent and viscosity modifier. The kit also includes a sample diluent containing polyvinylpyrrolidone and sulfobetaine, with a pH of 6.1–6.3.

[0009] Furthermore, the molecular weight of the aforementioned α-mercapto-ω-hydroxy polyethylene glycol is 2kDa to 3kDa; the aforementioned boric acid-functionalized polyamide-amine dendritic macromolecule is obtained by coupling 3-aminophenylboronic acid with the polyamide-amine dendritic macromolecule under the action of a coupling agent.

[0010] Furthermore, the aforementioned macromolecular crowding agent is dextran, and its concentration in the coating buffer of detection line T1 is 30 g / L to 50 g / L; the aforementioned viscosity modifier is hydroxyethyl cellulose, and its concentration in the coating buffer of detection line T1 is 1.5 g / L to 2.5 g / L; the aforementioned coating buffer of detection line T2 contains trehalose at a concentration of 15 g / L to 25 g / L.

[0011] Furthermore, the aforementioned zwitterionic nanohydrogel is formed by the polymerization of nanoparticles through reverse microemulsion with [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide monomer, N,N'-methylenebisacrylamide crosslinking agent and sodium di(2-ethylhexyl)succinate sulfonate emulsifier, which are then impregnated and dried to form a hydrophilic network structure on the surface of the sample pad fiber.

[0012] The preparation method of the combined detection kit for hepatitis A and hepatitis E virus IgM antibodies, used to prepare the above-mentioned kit, mainly includes the following steps: S1. Preparation of sample diluent: Prepare a sample diluent containing polyvinylpyrrolidone and sulfobetaine, and adjust the pH to 6.1-6.3; S2. Preparation of Gold Labeled Pads: Colloidal gold was synthesized and concentrated to obtain a concentrated colloidal gold solution. The surface of the colloidal gold was modified with α-mercapto-ω-hydroxy polyethylene glycol, and then labeled with mouse anti-human IgM monoclonal antibody to obtain a colloidal gold-antibody complex. The complex was then targeted and stabilized using boric acid-functionalized polyamide-amine dendritic macromolecules, and after blocking, a stable colloidal gold-labeled antibody solution was obtained. The above colloidal gold-labeled antibody solution was divided into two equal parts and resuspended in fast-release and slow-release solutions, respectively, to obtain a fast-release component and a slow-release component. The slow-release component was first sprayed to form a bottom slow-release layer, which was then cured with anhydrous ethanol. The fast-release component was then sprayed to form an upper fast-release layer, and after drying, the finished gold-labeled pads were obtained. S3. Preparation of sample pad: zwitterionic nanohydrogel was prepared by reverse microemulsion polymerization. It was mixed with anhydrous ethanol to prepare a sample pad treatment solution. The glass fiber sample pad was immersed in the above sample pad treatment solution and dried to obtain the finished sample pad. S4. Coating of the reaction membrane: Prepare a working solution for coating the detection line T1 containing macromolecular crowding agent and viscosity modifier, a working solution for coating the detection line T2 without macromolecular crowding agent and viscosity modifier, and a working solution for coating the quality control line C. Spray these solutions onto the nitrocellulose membrane in sequence to form the detection line T1, the detection line T2, and the quality control line C. After drying, the finished reaction membrane is obtained. S5. Assembly of reagent strips: The finished sample pad, finished gold label pad, finished reaction membrane and absorbent pad are sequentially pasted onto the PVC base plate, and after cutting, shelling and packaging, the above reagent kit is obtained.

[0013] Specifically, in step S1 above: The above sample diluent contains 3.4 g / L to 3.8 g / L disodium hydrogen phosphate dodecahydrate, 1.0 g / L to 1.2 g / L citric acid, 18.0 g / L to 22.0 g / L polyvinylpyrrolidone, 4.0 g / L to 6.0 g / L sulfobetaine, 8.0 g / L to 9.0 g / L sodium chloride, 9.0 g / L to 11.0 g / L bovine serum albumin, 0.4 mL / L to 0.6 mL / L preservative, and water as the solvent.

[0014] Furthermore, in step S2 above: The above-mentioned synthetic colloidal gold is prepared by reducing tetrachloroauric acid with trisodium citrate. The specific process is as follows: a 10 g / L tetrachloroauric acid solution is mixed with ultrapure water at a volume ratio of (0.9-1.1):100, heated to boiling, and a 10 g / L trisodium citrate solution is added while stirring at 300-500 r / min. The mixture is kept boiling for 5-10 minutes, then heating is stopped. After cooling, membrane filtration, centrifugation, and concentration, a concentrated colloidal gold solution is obtained. The volume ratio of the above-mentioned trisodium citrate solution to the above-mentioned ultrapure water is (1.7-1.9):100; The reaction conditions for modifying colloidal gold surfaces with α-mercapto-ω-hydroxy polyethylene glycol are as follows: reaction at 20℃~30℃ in the dark for 3h~5h, adding α-mercapto-ω-hydroxy polyethylene glycol stock solution with a concentration of 4mmol / L~6mmol / L to the reaction system, and the volume ratio of α-mercapto-ω-hydroxy polyethylene glycol stock solution to concentrated colloidal gold solution is (0.75~1.25):100.

[0015] Specifically, in step S2 above: The above-mentioned rapid release treatment solution is a 40mmol / L to 60mmol / L PBS buffer containing bovine serum albumin 8g / L to 12g / L, trehalose 40g / L to 60g / L, and polyvinylpyrrolidone 2g / L to 4g / L, with a pH of 7.3 to 7.5. The slow-release treatment solution mentioned above is a 40mmol / L to 60mmol / L PBS buffer containing bovine serum albumin 8g / L to 12g / L, trehalose 40g / L to 60g / L, and hydroxypropyl methylcellulose 4g / L to 6g / L, with a pH of 7.3 to 7.5. The above-mentioned anhydrous ethanol surface curing treatment involves spraying the slow-release layer surface with a spraying amount of 0.1 mL / strip to 0.2 mL / strip and then blowing it dry. The mass ratio of the colloidal gold-labeled antibody in the fast-release component to that in the slow-release component is 1:1.

[0016] Furthermore, in step S3 above, the preparation process of the zwitterionic nanohydrogel is as follows: [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide was dissolved in deionized water, and N,N'-methylenebisacrylamide was added as a crosslinking agent to form the aqueous phase. The aqueous phase was then added dropwise to cyclohexane containing sodium di(2-ethylhexyl)succinate sulfonate and stirred to form a water-in-oil microemulsion. Under nitrogen protection, ammonium persulfate and tetramethylethylenediamine were added to initiate polymerization. After the reaction was completed, the microemulsion was broken, washed, and centrifuged to obtain nano-hydrogel particles, which were dispersed in PBS to prepare a nano-hydrogel working solution of 4 g / L to 6 g / L. The above-mentioned nano-hydrogel working solution was mixed with anhydrous ethanol at a volume ratio of (3.5-4.5):1 to obtain the sample pad treatment solution. The glass fiber sample pad is immersed in the above sample pad treatment solution and then dried to obtain the sample pad.

[0017] Furthermore, in step S4 above: The T1 coating working solution for the above-mentioned detection line is prepared by dissolving recombinant hepatitis A virus antigen in 8 mmol / L to 12 mmol / L PBS buffer containing 30 g / L to 50 g / L dextran, 1.5 g / L to 2.5 g / L hydroxyethyl cellulose, and 15 g / L to 25 g / L trehalose, with an antigen concentration of 0.9 g / L to 1.1 g / L. The T2 coating working solution for the above-mentioned detection line is prepared by dissolving recombinant hepatitis E virus antigen in 8 mmol / L to 12 mmol / L PBS buffer containing 15 g / L to 25 g / L trehalose and 0.08 g / L to 0.12 g / L Tween, with an antigen concentration of 1.4 g / L to 1.6 g / L. The above-mentioned quality control line C coating working solution is made by dissolving goat anti-mouse IgG antibody in 8mmol / L to 12mmol / L PBS buffer containing 15g / L to 25g / L trehalose, with an antibody concentration of 1.9g / L to 2.1g / L.

[0018] Compared with the prior art, the present invention has the following advantages: The hepatitis A and E virus IgM antibody combined detection kit provided by this invention addresses the technical challenge of signal balance between the two detection lines in a unified chromatography channel in existing combined detection technologies. Through a synergistic design of the sample diluent, sample pad, gold-labeled pad, and reaction membrane, it achieves effective signal balance while maintaining sensitivity comparable to single-detection kits, while significantly improving anti-interference capability and long-term stability. Specific beneficial effects include: First, this invention constructs a four-ring synergistic technical solution encompassing ion pre-programming, inert isolation, spatiotemporally programmed release, and differentiated micro-region enhancement, successfully solving the key challenge of signal balance in joint detection. This invention lays the initial ionic conditions for downstream differentiated signal modulation by precisely setting the pH of the sample diluent to the midpoint of the isoelectric points of the two antigens; the inert isolation of the zwitterionic nanohydrogel network in the sample pad ensures stable conduction within this ionic environment; the spatiotemporally programmed release of the gold-labeled antibody is achieved through the fast-and-slow-release bilayer structure of the gold-labeled pad and surface curing treatment with anhydrous ethanol, creating a preferential and sufficient reaction opportunity for weak signal lines; and the differentiated configuration of macromolecular crowding agents and viscosity modifiers in the detection line coating solution fully locks in and amplifies the aforementioned advantages. Therefore, the technical solution of this invention upgrades the passive compensation design concept of the prior art to active regulation, enabling two detection lines with different intrinsic activities to obtain uniform signal output within the same reaction system.

[0019] Secondly, this invention significantly enhances the anti-interference capability of the combined assay kit in complex sample environments through the synergistic innovation of the sample pad and sample diluent. The zwitterionic nanohydrogel network loaded in the sample pad utilizes its electrically neutral three-dimensional hydrophilic structure to effectively trap large molecular interferences such as fibrin filaments and lipoprotein aggregates in whole blood samples through physical sieving, while avoiding the introduction of additional ions or active groups that could disturb downstream reactions. The sulfobetaine introduced into the sample diluent, as a zwitterionic surfactant, gently lyses red blood cells and efficiently dissolves lipoproteins, while its electrically neutral properties do not disrupt the native conformation of IgM antibodies. The synergistic effect of these two components constructs a highly efficient sample purification system at the start of chromatography, enabling the combined assay kit to maintain a clear and stable positive signal even under conditions where high concentrations of interference are known to cause missed detections in single-analytical products.

[0020] Third, this invention effectively extends the shelf life of the diagnostic kit through PEGylation modification of colloidal gold particles and targeted stabilization treatment of antibodies. The colloidal gold surface is modified with HS-PEG-OH, and a dense hydrophilic steric hindrance layer is constructed using Au-S bonds and long PEG chains, fundamentally improving the long-term dispersion stability of the colloidal gold particles. Furthermore, the antibody is stabilized in an ideal conformation through the specific binding of boric acid-functionalized polyamide-amine dendritic macromolecules to the Fc glycosyl group of the antibody.

[0021] In summary, this invention has made systematic technological innovations at three levels: molecular modification, formulation optimization, and structural design. Through a four-ring synergistic scheme of ion pre-programming, inert isolation, spatiotemporal programming release, and differentiated micro-region enhancement, it has successfully overcome the key technical challenge of achieving effective signal balance between two detection lines in a unified chromatography channel in existing combined detection technologies. This provides an accurate and reliable technical means for the rapid and synchronous differential diagnosis of hepatitis A and hepatitis E. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Unless otherwise specified in the examples, the procedures can be performed under standard conditions. If the manufacturers of the reagents or instruments used are not specified, they are all commercially available products, including recombinant hepatitis A virus antigen, recombinant hepatitis E virus antigen, mouse anti-human IgM monoclonal antibody, and goat anti-mouse IgG antibody.

[0028] The polyamide-amine dendritic macromolecule used is a type of dendritic polyamide-amine-amino copolymer. The commercial name of this type of substance used in this invention is PAMAM G4-NH2 dendritic polyamide-amine fourth-generation copolymer, abbreviated as PAMAMG4.

[0029] The α-mercapto-ω-hydroxy polyethylene glycol used, abbreviated as HS-PEG-OH, is a commercially available product with a molecular weight specification of 2kDa or 3kDa. The molecular weight specification is the nominal number-average molecular weight (Mn) provided by the manufacturer.

[0030] The [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonylpropyl)ammonium hydroxide used is also known as sulfobetaine methacrylate, abbreviated as SBMA; the sodium di(2-ethylhexyl)succinate sulfonate used is abbreviated as AOT; the polyvinylpyrrolidone used is polyvinylpyrrolidone K30, abbreviated as PVP-K30; the hydroxypropyl methylcellulose used is abbreviated as HPMC; the hydroxyethyl cellulose used is abbreviated as HEC; bovine serum albumin is abbreviated as BSA; and the dextran 70 used is a commercially available product with a molecular weight of 70 kDa.

[0031] The coupling agent used in this invention is composed of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1:1, and is referred to as EDC / NHS coupling agent.

[0032] In this invention, the borate buffer solutions of different pH values ​​all refer to borate buffer systems with a concentration of 10 mmol / L. The target pH is obtained by adjusting the molar ratio of boric acid to sodium tetraborate. Example 1

[0033] The preparation process of the combined detection kit for hepatitis A and hepatitis E virus IgM antibodies in this embodiment is as follows: S1. Preparation of sample diluent: Prepare the sample diluent according to the following formula: Disodium hydrogen phosphate dodecahydrate: 3.6 g / L; Citric acid: 1.1 g / L; PVP-K30: 20.0 g / L; Sulfobetaine: 5.0 g / L; Sodium chloride: 8.5 g / L; BSA: 10.0 g / L; ProClin 300: 0.5 mL / L; Solvent: purified water; Adjust the pH to 6.2 using 0.1 mol / L NaOH solution; The sample dilution was obtained by filtration using a 0.22 μm aqueous filter membrane and stored at 4 °C.

[0034] S2, Preparation of the gold-labeled pad: S21. Synthesis and preliminary purification of colloidal gold particles: Take 100 mL of ultrapure water, add 1000 μL of 10 g / L tetrachloroauric acid solution, and heat to a vigorous boil; Add 1.8 mL of a 10 g / L trisodium citrate solution while stirring vigorously at 400 r / min. Keep boiling for 8 minutes until the solution turns a clear bright red color. Stop heating, stir and cool at room temperature, filter through a 0.22 μm aqueous phase membrane, and centrifuge at 4 °C and 9000 × g for 25 minutes using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa to concentrate to 1 / 5 of the original volume, thus obtaining a concentrated colloidal gold solution.

[0035] S22, PEGylation modification of colloidal gold particle surface: HS-PEG-OH (molecular weight specification 2.5kDa) was used as the modifying molecule. HS-PEG-OH was dissolved in ultrapure water to prepare a 5mmol / L HS-PEG-OH stock solution. Under continuous stirring at 400 r / min, 200 μL of HS-PEG-OH stock solution was added dropwise to 20 mL of concentrated colloidal gold solution, and the reaction was carried out continuously at 25 °C in the dark for 4 h. After the reaction was completed, the ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa was used to wash the sample with 10 mmol / L borate buffer at pH 8.5 and centrifuged at 4 °C and 9000 × g for 25 min. The supernatant was discarded and the washing was repeated twice. The final precipitate was resuspended in 10 mL of 10 mmol / L borate buffer (pH 8.5) to obtain a PEGylated colloidal gold solution.

[0036] S23. Antibody labeling and preparation of boric acid functionalized polymers: S231, Antibody labeling: Adding 170 μL of 1 g / L mouse anti-human IgM monoclonal antibody to 10 mL of PEGylated colloidal gold caused a slight decrease in the pH of the system. The pH was readjusted to 8.5 using a 0.1 mol / L K₂CO₃ solution. Mix at 300 rpm for 50 min to obtain colloidal gold-antibody complex.

[0037] Preparation of S232 and boric acid-functionalized PAMAM: Take 10 mg of PAMAM G4 and dissolve it in 1 mL of borate buffer at pH 8.5; Add 7 mg of 3-aminophenylboronic acid and 9 mg of EDC / NHS coupling agent, and react at 25°C for 3.5 h; Boronic acid-functionalized PAMAM was obtained by purification using a dialysis bag with a molecular weight cutoff of 3.5 kDa. The solution was brought to a final volume of 1 mL with 10 mmol / L borate buffer at pH 8.5 to obtain the BA-PAMAM solution.

[0038] S24. Target stabilization and post-processing: S241, Targeted Stabilization Response: Slowly add 1 mL of BA-PAMAM solution to the colloidal gold-antibody complex prepared in step S231; The target-stabilized colloidal gold-antibody complex was obtained by incubating at 25°C and 80 rpm for 2.5 h in the dark.

[0039] S242, Closure and Stability: Add 1.1 mL of 100 g / L BSA solution to the targeted and stable colloidal gold-antibody complex and continue the reaction for 15 min to block non-specific sites. Then add 1.1 mL of trehalose solution with a concentration of 500 g / L as a protective agent to obtain a stable colloidal gold-labeled antibody solution; All stable colloidal gold-labeled antibody solutions were divided into two equal volumes, denoted as Solution I and Solution II.

[0040] S243. Preparation of fast-release colloidal gold-labeled antibody concentrate: Centrifuge solution I at 4℃ and 13000×g for 40 min; Discard the supernatant and resuspend the precipitate in 1 mL of rapid release buffer to obtain a rapid release colloidal gold-labeled antibody concentrate, denoted as component A. The rapid release buffer contains 10 g / L BSA, 50 g / L trehalose, 3 g / L PVP-K30 in 50 mmol / L PBS buffer, pH 7.4.

[0041] S244. Preparation of slow-release colloidal gold-labeled antibody concentrate: Centrifuge solution II at 4℃ and 13000×g for 40 min; Discard the supernatant and resuspend the precipitate in 1 mL of slow-release processing solution to obtain a slow-release colloidal gold-labeled antibody concentrate, denoted as component B; wherein the slow-release processing solution contains 10 g / L BSA, 50 g / L trehalose, 5 g / L HPMC in 50 mmol / L PBS buffer, pH 7.4.

[0042] S25, double-layer spraying and drying of gold standard pads: S251, Underlying slow-release layer spraying: Mix 1 mL of component B with 5 mL of slow-release treatment solution to obtain the slow-release working solution; Spray the coating at a uniform rate of 0.75 mL / strip onto a 30cm×1cm glass fiber membrane, distributing it in the bottom layer of the gold standard pad substrate. Dry at 37℃ for 18 minutes until there are no obvious liquid marks on the surface.

[0043] S252, Slow-release layer surface curing treatment: The dried slow-release layer surface was sprayed with anhydrous ethanol at a rate of 0.15 mL / strip, and then dried with a clean airflow at 37°C for 1.5 min.

[0044] S253, Top Fast-Release Layer Spraying: Mix 1 mL of component A with 5 mL of fast-release treatment solution to obtain the fast-release working solution; A second coating is applied to the slow-release layer after surface curing, using a uniform spraying amount of 0.75 mL / strip, so that it is distributed on the surface area of ​​the gold-labeled pad substrate. Dry at 37℃ for 3 hours, place the sprayed double-layer gold label pad in an aluminum foil bag, add desiccant, seal and store to obtain the gold label pad.

[0045] S3. Preparation of the sample pad: S31. Preparation of zwitterionic nanohydrogels: Dissolve 2.0 g SBMA in 10 mL of deionized water, add 20 mg N,N'-methylenebisacrylamide, stir to dissolve, and use as the aqueous phase; The aqueous phase was slowly added dropwise to 100 mL of cyclohexane containing 5.0 g AOT, and the mixture was stirred to form a water-in-oil microemulsion. Under nitrogen protection, 10 mg of ammonium persulfate and 5 μL of tetramethylethylenediamine were added, and the mixture was reacted at 25 °C for 4 h. After the reaction was completed, ethanol was added to break the emulsion, and the nano-hydrogel particles were collected by centrifugation at 13000×g. The precipitate was washed alternately with anhydrous ethanol and purified water and centrifuged, and the process was repeated 3 times to obtain the nano-hydrogel. The nanohydrogel was dispersed in PBS at pH 7.4 and 10 mmol / L to prepare a 5 g / L working solution of the nanohydrogel, which was stored at 4 °C.

[0046] S32. Preparation of sample pad treatment solution: The working solution of the nanohydrogel obtained by S31 was mixed with anhydrous ethanol at a volume ratio of 4:1 to obtain the sample pad treatment solution, in which no BSA, surfactant or blocking antibody was added.

[0047] S33. Impregnation and drying of the sample pad: Immerse the glass fiber sample pad completely in the sample pad treatment solution prepared in step S32 for 8 minutes; After being removed, the sample pads were rapidly air-dried in a clean airflow at 40°C for 3 hours, then placed in an aluminum foil bag with a desiccant, sealed, and stored to obtain the sample pads.

[0048] S4. Coating of the reaction membrane: S41. Preparation and coating of T1 (Hepatitis A) detection line coating solution: The recombinant hepatitis A virus antigen was dissolved in T1 line coating buffer to prepare a T1 line coating working solution with a concentration of 1.0 g / L; wherein, the T1 line coating buffer is a 10 mmol / L PBS buffer containing 40 g / L dextran-70, 2.0 g / L HEC, and 20 g / L trehalose, pH 7.4. Using a gold spraying apparatus, the working solution for coating the T1 line was sprayed onto the nitrocellulose membrane at a distance of 2.2 cm from the sample pad end at a spray rate of 0.10 μL / mm to form the detection line T1.

[0049] S42. Preparation and coating of T2 (Hepatitis E) detection line coating solution: Recombinant hepatitis E virus antigen was dissolved in T2 line coating buffer to prepare a T2 line coating working solution with a concentration of 1.5 g / L; wherein, the T2 line coating buffer is a 10 mmol / L PBS buffer containing 20 g / L trehalose and 0.10 g / L Tween-20, pH 7.4. Using a gold spraying apparatus, the coating solution for line T2 is precisely sprayed onto line T1 at a distance of 0.6 cm above line T1 with a spraying volume of 0.10 μL / mm, thus forming the detection line T2.

[0050] S43. Preparation and coating of coating solution for quality control line: Goat anti-mouse IgG antibody was dissolved in C-line coating buffer to prepare a C-line coating working solution with a goat anti-mouse IgG antibody concentration of 2.0 g / L; wherein, the C-line coating buffer is a 10 mmol / L PBS buffer containing 20 g / L trehalose, pH 7.4; Using a gold spraying apparatus, the C-line coating solution was precisely sprayed onto the same nitrocellulose membrane at a spraying rate of 0.10 μL / mm, 0.6 cm above and 0.6 cm from the T2 line.

[0051] S44. Drying and storage of the reaction membrane: The reaction membrane coated with T1, T2 and C lines was placed in a 37°C constant temperature oven and dried for 3 hours. After drying, the reaction membrane is placed in a high-strength, water- and oxygen-proof aluminum foil bag, and a desiccant is added. The bag is then sealed and stored to obtain the reaction membrane.

[0052] S5. Assembly of reagent strips: S51. Component cutting and preparation: The gold label pad, sample pad, reaction membrane, absorbent pad, and PVC base plate were cut into strips using a CNC cutting machine in a clean environment; the width of each component was 3.8 cm.

[0053] S52. Adhesion and assembly of reagent strips: On the PVC base plate, attach the following components in order from right to left, overlapping each other by 1.5mm: sample pad, gold label pad, reaction membrane, and absorbent pad. Press them together three times with a roller to ensure that each component is firmly bonded to the base plate without air bubbles.

[0054] S53. Final cutting and packaging of reagent strips: The assembled large plate is cut into individual test strips using a CNC cutting machine; the cut test strips are placed into plastic card holders, the caps are closed, and the holders are pressed tightly to obtain complete test cards; the complete test cards are placed into aluminum foil bags, each bag contains a desiccant, and the bags are sealed using a heat sealer; the sealed finished product is stored at room temperature or in a cool, dry, and light-proof place to obtain the finished product of the Hepatitis A and Hepatitis E Virus IgM Antibody Combined Detection Kit (Colloidal Gold Method), which is designated as Kit Sample 1. Example 2

[0055] The preparation process of the combined detection kit for hepatitis A and hepatitis E virus IgM antibodies in this embodiment is as follows: S1. Preparation of sample diluent: Prepare the sample diluent according to the following formula: Disodium hydrogen phosphate dodecahydrate: 3.8 g / L; Citric acid: 1.0 g / L; PVP-K30: 22.0 g / L; Sulfobetaine: 4.0 g / L; Sodium chloride: 9.0 g / L; BSA: 9.0 g / L; ProClin 300: 0.6 mL / L; Solvent: purified water; Adjust the pH to 6.3 using 0.1 mol / L NaOH solution; The sample dilution was obtained by filtration using a 0.22 μm aqueous filter membrane and stored at 2 °C.

[0056] S2, Preparation of the gold-labeled pad: S21. Synthesis and preliminary purification of colloidal gold particles: Take 100 mL of ultrapure water, add 1100 μL of 10 g / L tetrachloroauric acid solution, and heat to a vigorous boil; Add 1.7 mL of a 10 g / L trisodium citrate solution while stirring vigorously at 500 r / min. Keep boiling for 5 minutes until the solution turns a clear bright red color. Stop heating, stir and cool at room temperature, filter through a 0.22 μm aqueous phase membrane, and centrifuge at 2 °C and 10000 × g for 20 minutes using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa. Concentrate to 1 / 5 of the original volume to obtain a concentrated colloidal gold solution.

[0057] S22, PEGylation modification of colloidal gold particle surface: HS-PEG-OH (molecular weight specification 3kDa) was used as a modifying molecule. HS-PEG-OH was dissolved in ultrapure water to prepare a 6mmol / L HS-PEG-OH stock solution. Under continuous stirring at 500 r / min, 250 μL of HS-PEG-OH stock solution was added dropwise to 20 mL of concentrated colloidal gold solution, and the reaction was carried out continuously at 30 °C in the dark for 3 h. After the reaction was completed, the ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa was used to wash the sample with 10 mmol / L borate buffer at pH 8.7, and centrifuged at 2°C and 10000×g for 20 min. The supernatant was discarded and the washing was repeated twice. The final precipitate was resuspended in 10 mL of 10 mmol / L borate buffer (pH 8.6) to obtain a PEGylated colloidal gold solution.

[0058] S23. Antibody labeling and preparation of boric acid functionalized polymers: S231, Antibody labeling: Adding 180 μL of 1 g / L mouse anti-human IgM monoclonal antibody to 10 mL of PEGylated colloidal gold caused a slight decrease in the pH of the system. Adjust the pH to 8.6 using a 0.1 mol / L K₂CO₃ solution; Mix at 200 rpm for 55 min to obtain colloidal gold-antibody complex.

[0059] Preparation of S232 and boric acid-functionalized PAMAM: Take 10 mg of PAMAM G4 and dissolve it in 1 mL of borate buffer at pH 8.6; Add 8 mg of 3-aminophenylboronic acid and 10 mg of EDC / NHS coupling agent, and react at 20°C for 4 h; Boronic acid-functionalized PAMAM was obtained by purification using a dialysis bag with a molecular weight cutoff of 3.5 kDa. The solution was brought to a final volume of 1 mL with 10 mmol / L borate buffer at pH 8.6 to obtain the BA-PAMAM solution.

[0060] S24. Target stabilization and post-processing: S241, Targeted Stabilization Response: Slowly add 1 mL of BA-PAMAM solution to the colloidal gold-antibody complex prepared in step S231; The target-stabilized colloidal gold-antibody complex was obtained by incubating at 30℃ and 50r / min in the dark for 2 hours.

[0061] S242, Closure and Stability: Add 1.2 mL of 100 g / L BSA solution to the targeted and stable colloidal gold-antibody complex and continue the reaction for 12 min to block non-specific sites. Add 1.2 mL of trehalose solution with a concentration of 500 g / L as a protective agent to obtain a stable colloidal gold-labeled antibody solution; All stable colloidal gold-labeled antibody solutions were divided into two equal volumes, denoted as Solution I and Solution II.

[0062] S243. Preparation of fast-release colloidal gold-labeled antibody concentrate: Centrifuge solution I at 8℃ and 14000×g for 35 min; Discard the supernatant and resuspend the precipitate in 1 mL of rapid release buffer to obtain a rapid release colloidal gold-labeled antibody concentrate, denoted as component A. The rapid release buffer contains 12 g / L BSA, 60 g / L trehalose, 4 g / L PVP-K30 in 60 mmol / L PBS buffer, pH 7.3.

[0063] S244. Preparation of slow-release colloidal gold-labeled antibody concentrate: Centrifuge solution II at 8℃ and 14000×g for 35 min; Discard the supernatant and resuspend the precipitate in 1 mL of slow-release processing solution to obtain a slow-release colloidal gold-labeled antibody concentrate, denoted as component B; wherein the slow-release processing solution contains 12 g / L BSA, 60 g / L trehalose, 6 g / L HPMC in 60 mmol / L PBS buffer, pH 7.3.

[0064] S25, double-layer spraying and drying of gold standard pads: S251, Underlying slow-release layer spraying: Mix 1 mL of component B with 6 mL of slow-release treatment solution to obtain the slow-release working solution; Spray the coating at a uniform rate of 0.9 mL / strip onto a 30cm×1cm glass fiber membrane, distributing it in the bottom layer area of ​​the gold standard pad substrate. Dry at 40℃ for 15 minutes until there are no obvious liquid marks on the surface.

[0065] S252, Slow-release layer surface curing treatment: The dried slow-release layer surface was sprayed with anhydrous ethanol at a rate of 0.2 mL / strip, and then dried with a clean airflow at 40°C for 1 min.

[0066] S253, Top Fast-Release Layer Spraying: Mix 1 mL of component A with 6 mL of fast-release treatment solution to obtain the fast-release working solution; A second coating is applied to the slow-release layer after surface curing, using a uniform spraying amount of 0.9 mL / strip, so that it is distributed on the surface area of ​​the gold-labeled pad substrate. Dry at 40℃ for 2.5 hours, place the sprayed double-layer gold label pad in an aluminum foil bag, add desiccant, seal and store to obtain the gold label pad.

[0067] S3. Preparation of the sample pad: S31. Preparation of zwitterionic nanohydrogels: Dissolve 2.2 g SBMA in 11 mL of deionized water, add 22 mg N,N'-methylenebisacrylamide, stir to dissolve, and use as the aqueous phase; The aqueous phase was slowly added dropwise to 110 mL of cyclohexane containing 5.5 g AOT, and the mixture was stirred to form a water-in-oil microemulsion. Under nitrogen protection, 11 mg of ammonium persulfate and 6 μL of tetramethylethylenediamine were added, and the mixture was reacted at 28 °C for 3.5 h. After the reaction was completed, ethanol was added to break the emulsion, and the nano-hydrogel particles were collected by centrifugation at 14000×g. The precipitate was washed alternately with anhydrous ethanol and purified water and centrifuged, and the process was repeated 3 times to obtain the nano-hydrogel. The nanohydrogel was dispersed in PBS at pH 7.5 and 12 mmol / L to prepare a 6 g / L working solution of the nanohydrogel, which was stored at 8 °C.

[0068] S32. Preparation of sample pad treatment solution: The working solution of the nano-hydrogel obtained by S31 was mixed with anhydrous ethanol at a volume ratio of 4.5:1 to obtain the sample pad treatment solution.

[0069] S33. Impregnation and drying of the sample pad: Immerse the glass fiber sample pad completely in the sample pad treatment solution prepared in step S32 and soak for 10 minutes. After being removed, the sample pads were rapidly air-dried in a clean airflow at 45°C for 2.5 hours, then placed in an aluminum foil bag with a desiccant, sealed, and stored to obtain the sample pads.

[0070] S4. Coating of the reaction membrane: S41. Preparation and coating of T1 (Hepatitis A) detection line coating solution: The recombinant hepatitis A virus antigen was dissolved in T1 line coating buffer to prepare a T1 line coating working solution with a concentration of 1.1 g / L; wherein, the T1 line coating buffer is a 12 mmol / L PBS buffer containing 50 g / L dextran-70, 1.5 g / L HEC, and 25 g / L trehalose, pH 7.3. Using a gold spraying apparatus, the working solution for coating the T1 line was sprayed onto the nitrocellulose membrane at a spray rate of 0.12 μL / mm, 2.5 cm from the sample pad end, to form the detection line T1.

[0071] S42. Preparation and coating of T2 (Hepatitis E) detection line coating solution: Recombinant hepatitis E virus antigen was dissolved in T2 line coating buffer to prepare a T2 line coating working solution with a concentration of 1.6 g / L; wherein, the T2 line coating buffer is a 12 mmol / L PBS buffer containing 25 g / L trehalose and 0.12 g / L Tween-20, pH 7.3; Using a gold spraying apparatus, the coating solution for line T2 is precisely sprayed onto line T1 at a distance of 0.5 cm above line T1 with a spraying volume of 0.12 μL / mm, thus forming the detection line T2.

[0072] S43. Preparation and coating of coating solution for quality control line: Goat anti-mouse IgG antibody was dissolved in C-line coating buffer to prepare a C-line coating working solution with a goat anti-mouse IgG antibody concentration of 2.1 g / L; wherein, the C-line coating buffer is a 12 mmol / L PBS buffer containing 25 g / L trehalose, pH 7.3; Using a gold spraying apparatus, the C-line coating solution was precisely sprayed onto the same nitrocellulose membrane at a spraying rate of 0.12 μL / mm, 0.5 cm above and 0.5 cm from the T2 line.

[0073] S44. Drying and storage of the reaction membrane: The reaction membrane coated with T1, T2 and C lines was placed in a 35℃ constant temperature oven and dried for 3.5 hours. After drying, the reaction membrane is placed in a high-strength, water- and oxygen-proof aluminum foil bag, and a desiccant is added. The bag is then sealed and stored to obtain the reaction membrane.

[0074] S5. Assembly of reagent strips: S51. Component cutting and preparation: The gold label pad, sample pad, reaction membrane, absorbent pad, and PVC base plate were cut into strips using a CNC cutting machine in a clean environment; the width of each component was 3.5cm.

[0075] S52. Adhesion and assembly of reagent strips: On the PVC base plate, attach the following components in order from right to left, overlapping each other by 1.0mm: sample pad, gold label pad, reaction membrane, and absorbent pad. Press them together three times with a roller to ensure that each component is firmly bonded to the base plate without air bubbles.

[0076] S53. Final cutting and packaging of reagent strips: The assembled large plate is cut into individual test strips using a CNC cutting machine; the cut test strips are placed into plastic card holders, the caps are closed, and the holders are pressed tightly to obtain complete test cards; the complete test cards are placed into aluminum foil bags, each bag contains a desiccant, and the bags are sealed using a heat sealer; the sealed finished product is stored at room temperature or in a cool, dry, and light-proof place to obtain the finished product of the Hepatitis A and Hepatitis E Virus IgM Antibody Combined Detection Kit (Colloidal Gold Method), which is designated as Kit Sample 2. Example 3

[0077] The preparation process of the combined detection kit for hepatitis A and hepatitis E virus IgM antibodies in this embodiment is as follows: S1. Preparation of sample diluent: Prepare the sample diluent according to the following formula: Disodium hydrogen phosphate dodecahydrate: 3.4 g / L; Citric acid: 1.2 g / L; PVP-K30: 18.0 g / L; Sulfobetaine: 6.0 g / L; Sodium chloride: 8.0 g / L; BSA: 11.0 g / L; ProClin 300: 0.4 mL / L; Solvent: purified water; Adjust the pH to 6.1 using 0.1 mol / L NaOH solution; The sample dilution was obtained by filtration using a 0.22 μm aqueous filter membrane and stored at 8 °C.

[0078] S2, Preparation of the gold-labeled pad: S21. Synthesis and preliminary purification of colloidal gold particles: Take 100 mL of ultrapure water, add 900 μL of 10 g / L tetrachloroauric acid solution, and heat until it boils vigorously; Add 1.9 mL of a 10 g / L trisodium citrate solution while stirring vigorously at 300 r / min. Keep boiling for 10 minutes until the solution turns a clear bright red color. Stop heating, stir and cool at room temperature, filter through a 0.22 μm aqueous phase membrane, and centrifuge at 8°C and 8000×g for 30 minutes using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa to concentrate to 1 / 5 of the original volume, thus obtaining a concentrated colloidal gold solution.

[0079] S22, PEGylation modification of colloidal gold particle surface: Using HS-PEG-OH (molecular weight specification 2kDa) as the modifying molecule, HS-PEG-OH was dissolved in ultrapure water to prepare a 4mmol / L HS-PEG-OH stock solution; Under continuous stirring at 300 r / min, 150 μL of HS-PEG-OH stock solution was added dropwise to 20 mL of concentrated colloidal gold solution, and the reaction was carried out continuously at 20 °C in the dark for 5 h. After the reaction was completed, the ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa was used to wash the tube with 10 mmol / L borate buffer at pH 8.3 and centrifuged at 8°C and 8000×g for 30 min. The supernatant was discarded and the washing was repeated 3 times. The final precipitate was resuspended in 10 mL of 10 mmol / L borate buffer (pH 8.4) to obtain a PEGylated colloidal gold solution.

[0080] S23. Antibody labeling and preparation of boric acid functionalized polymers: S231, Antibody labeling: Adding 160 μL of 1 g / L mouse anti-human IgM monoclonal antibody to 10 mL of PEGylated colloidal gold caused a slight decrease in the pH of the system. Adjust the pH to 8.4 using a 0.1 mol / L K₂CO₃ solution; Mix at 400 rpm for 45 min to obtain colloidal gold-antibody complex.

[0081] Preparation of S232 and boric acid-functionalized PAMAM: Take 10 mg of PAMAM G4 and dissolve it in 1 mL of borate buffer at pH 8.4; Add 6 mg of 3-aminophenylboronic acid and 8 mg of EDC / NHS coupling agent, and react at 30°C for 3 h; Boronic acid-functionalized PAMAM was obtained by purification using a dialysis bag with a molecular weight cutoff of 3.5 kDa. The solution was brought to a final volume of 1 mL with 10 mmol / L borate buffer at pH 8.4 to obtain the BA-PAMAM solution.

[0082] S24. Target stabilization and post-processing: S241, Targeted Stabilization Response: Slowly add 1 mL of BA-PAMAM solution to the colloidal gold-antibody complex prepared in step S231; The target-stable colloidal gold-antibody complex was obtained by incubating at 20℃ and 100r / min in the dark for 3 hours.

[0083] S242, Closure and Stability: Add 1.0 mL of 100 g / L BSA solution to the targeted and stable colloidal gold-antibody complex and continue the reaction for 18 min to block non-specific sites. Add 1.0 mL of trehalose solution with a concentration of 500 g / L as a protectant to obtain a stable colloidal gold-labeled antibody solution; All stable colloidal gold-labeled antibody solutions were divided into two equal volumes, denoted as Solution I and Solution II.

[0084] S243. Preparation of fast-release colloidal gold-labeled antibody concentrate: Centrifuge solution I at 2℃ and 12000×g for 45 min; Discard the supernatant and resuspend the precipitate in 1 mL of rapid release buffer to obtain a rapid release colloidal gold-labeled antibody concentrate, denoted as component A. The rapid release buffer contains 8 g / L BSA, 40 g / L trehalose, 2 g / L PVP-K30 in 40 mmol / L PBS buffer, pH 7.5.

[0085] S244. Preparation of slow-release colloidal gold-labeled antibody concentrate: Centrifuge solution II at 2℃ and 12000×g for 45 min; Discard the supernatant and resuspend the precipitate in 1 mL of slow-release processing solution to obtain a slow-release colloidal gold-labeled antibody concentrate, denoted as component B; wherein the slow-release processing solution contains 8 g / L BSA, 40 g / L trehalose, 4 g / L HPMC in 40 mmol / L PBS buffer, pH 7.5.

[0086] S25, double-layer spraying and drying of gold standard pads: S251, Underlying slow-release layer spraying: Mix 1 mL of component B with 4 mL of slow-release treatment solution to obtain the slow-release working solution; Spray the coating at a uniform rate of 0.6 mL / strip onto a 30cm×1cm glass fiber membrane, distributing it in the bottom layer of the gold standard pad substrate. Dry at 35℃ for 20 minutes until there are no obvious liquid marks on the surface.

[0087] S252, Slow-release layer surface curing treatment: The dried slow-release layer surface was sprayed with anhydrous ethanol at a rate of 0.1 mL / strip, and then dried with a clean airflow at 35°C for 2 minutes.

[0088] S253, Top Fast-Release Layer Spraying: Mix 1 mL of component A with 4 mL of fast-release treatment solution to obtain the fast-release working solution; A second coating is applied to the slow-release layer after surface curing, using a uniform spraying amount of 0.6 mL / strip, so that it is distributed on the surface area of ​​the gold-labeled pad substrate. Dry at 35℃ for 3.5 hours, place the sprayed double-layer gold label pad in an aluminum foil bag, add desiccant, seal and store to obtain the gold label pad.

[0089] S3. Preparation of the sample pad: S31. Preparation of zwitterionic nanohydrogels: Dissolve 1.8 g SBMA in 9 mL of deionized water, add 18 mg N,N'-methylenebisacrylamide, stir to dissolve, and use as the aqueous phase; The aqueous phase was slowly added dropwise to 90 mL of cyclohexane containing 4.5 g AOT, and the mixture was stirred to form a water-in-oil microemulsion. Under nitrogen protection, 9 mg of ammonium persulfate and 4 μL of tetramethylethylenediamine were added, and the mixture was reacted at 22 °C for 4.5 h. After the reaction was completed, ethanol was added to break the emulsion, and the nano-hydrogel particles were collected by centrifugation at 12000×g. The precipitate was washed alternately with anhydrous ethanol and purified water and centrifuged, and the process was repeated 3 times to obtain the nano-hydrogel. The nanohydrogel was dispersed in PBS at pH 7.3 and 8 mmol / L to prepare a 4 g / L working solution of the nanohydrogel, which was stored at 6 °C.

[0090] S32. Preparation of sample pad treatment solution: The working solution of the nano-hydrogel obtained by S31 was mixed with anhydrous ethanol at a volume ratio of 3.5:1 to obtain the sample pad treatment solution.

[0091] S33. Impregnation and drying of the sample pad: Immerse the glass fiber sample pad completely in the sample pad treatment solution prepared in step S32 for 5 minutes; After being removed, the sample pads were rapidly air-dried in a clean airflow at 35°C for 3.5 hours, then placed in an aluminum foil bag with a desiccant, sealed, and stored to obtain the sample pads.

[0092] S4. Coating of the reaction membrane: S41. Preparation and coating of T1 (Hepatitis A) detection line coating solution: The recombinant hepatitis A virus antigen was dissolved in T1 line coating buffer to prepare a T1 line coating working solution with a concentration of 0.9 g / L; wherein, the T1 line coating buffer is an 8 mmol / L PBS buffer containing 30 g / L dextran-70, 2.5 g / L HEC, and 15 g / L trehalose, pH 7.5. Using a gold spraying apparatus, the working solution for coating the T1 line was sprayed onto the nitrocellulose membrane at a spray rate of 0.08 μL / mm, 2.0 cm from the sample pad end, to form the detection line T1.

[0093] S42. Preparation and coating of T2 (Hepatitis E) detection line coating solution: Recombinant hepatitis E virus antigen was dissolved in T2 line coating buffer to prepare a T2 line coating working solution with a concentration of 1.4 g / L; wherein, the T2 line coating buffer is an 8 mmol / L PBS buffer containing 15 g / L trehalose and 0.08 g / L Tween-20, pH 7.5; Using a gold spraying apparatus, the coating solution for line T2 is precisely sprayed onto line T1 at a distance of 0.8 cm above line T1 with a spraying volume of 0.08 μL / mm, thus forming the detection line T2.

[0094] S43. Preparation and coating of coating solution for quality control line: Goat anti-mouse IgG antibody was dissolved in C-line coating buffer to prepare a C-line coating working solution with a goat anti-mouse IgG antibody concentration of 1.9 g / L; wherein, the C-line coating buffer is an 8 mmol / L PBS buffer containing 15 g / L trehalose, pH 7.5; Using a gold spraying apparatus, the C-line coating solution was precisely sprayed onto the same nitrocellulose membrane at a spraying rate of 0.08 μL / mm, 0.8 cm above and 0.8 cm from the T2 line.

[0095] S44. Drying and storage of the reaction membrane: The reaction membrane coated with T1, T2 and C lines was placed in a constant temperature oven at 40℃ and dried for 2.5 hours. After drying, the reaction membrane was placed in a high-strength water- and oxygen-proof aluminum foil bag, and desiccant was added. The bag was then sealed and stored to obtain the reaction membrane.

[0096] S5. Assembly of reagent strips: S51. Component cutting and preparation: In a clean environment, the gold label pad, sample pad, reaction membrane, absorbent pad, and PVC base plate were cut into strips using a CNC cutting machine. The width of each component—sample pad, gold label pad, reaction membrane, and absorbent pad—was 4.0 cm.

[0097] S52. Adhesion and assembly of reagent strips: On the PVC base plate, attach the following components in order from right to left, overlapping each other by 2.0mm: sample pad, gold label pad, reaction membrane, and absorbent pad. Press them together twice with a roller to ensure that each component is firmly bonded to the base plate without air bubbles.

[0098] S53. Final cutting and packaging of reagent strips: The assembled large plate is cut into individual test strips using a CNC cutting machine; the cut test strips are placed into plastic card holders, the caps are closed, and the cards are pressed tightly to obtain complete test cards; the complete test cards are placed into aluminum foil bags, each bag contains a desiccant, and the bags are sealed using a heat sealer; the sealed finished products are stored at room temperature or in a cool, dry, and light-proof place to obtain the finished product of the Hepatitis A and Hepatitis E Virus IgM Antibody Combined Detection Kit (Colloidal Gold Method), which is designated as Kit Sample 3. Comparative Example 1

[0099] This comparative example provides a combined detection kit for hepatitis A and E virus IgM antibodies. The specific implementation method is the same as in Example 1, except that: in the sample pad preparation process, no zwitterionic nanogel is loaded; instead, a conventional glass fiber sample pad treatment method is used. The specific preparation process is as follows: S1. Preparation of sample diluent: Same as S1 in Example 1.

[0100] S2, Preparation of the gold-labeled pad: Same as S2 in Example 1.

[0101] S3. Preparation of the sample pad: Immerse the glass fiber sample pad completely in 50 mmol / L PBS buffer (pH 7.4) containing 10 g / L BSA, 50 g / L trehalose, and 5 g / L Tween-20 for 8 min. After being removed, the sample pads were rapidly air-dried in a clean airflow at 40°C for 3 hours, then placed in an aluminum foil bag with a desiccant, sealed, and stored to obtain the sample pad reference standard.

[0102] S4. Coating of the reaction membrane: Same as S4 in Example 1.

[0103] S5. Assembly of reagent strips: Same as S5 in Example 1, but replace the sample pad prepared in the example with the sample pad control prepared in step S3 to obtain the combined detection kit for hepatitis A and hepatitis E virus IgM antibodies, denoted as kit control 1. Comparative Example 2

[0104] This comparative example provides a combined detection kit for hepatitis A and E virus IgM antibodies. The specific implementation method is the same as in Example 1, except that the same conventional coating buffer is used for both the T1 and T2 lines of the reaction membrane. Specifically, no macromolecular crowding agents or viscosity modifiers are added to the T1 line coating buffer. The specific preparation process is as follows: S1. Preparation of sample diluent: Same as S1 in Example 1.

[0105] S2, Preparation of the gold-labeled pad: Same as S2 in Example 1.

[0106] S3. Preparation of sample pad: Same as S3 in Example 1.

[0107] S4. Coating of the reaction membrane: S41. Preparation and coating of T1 (Hepatitis A) detection line coating solution: The recombinant hepatitis A virus antigen was dissolved in T1 line coating buffer control to prepare a T1 line coating working solution control with a concentration of 1.0 g / L; wherein, the T1 line coating buffer control is a 10 mmol / L PBS buffer containing 20 g / L trehalose and 10 g / L Tween-200, pH 7.4. Using a gold spraying apparatus, the working solution for coating the T1 line was sprayed onto the nitrocellulose membrane at a distance of 2.2 cm from the sample pad end at a spray rate of 0.10 μL / mm to form the detection line T1.

[0108] S42. Preparation and coating of the T2 (Hepatitis E) detection line coating solution: Same as S42 in Example 1.

[0109] S43. Preparation and coating of the coating solution for the quality control line: Same as S43 in Example 1.

[0110] S44. Drying and preservation of the reaction membrane: Same as S44 in Example 1, to prepare the reaction membrane control.

[0111] S5. Assembly of reagent strips: Same as S5 in Example 1, but replace the reaction membrane prepared in the example with the reaction membrane control prepared in step S4 to obtain the hepatitis A and hepatitis E virus IgM antibody combined detection kit, which is denoted as kit control 2. Comparative Example 3

[0112] This comparative example provides a combined detection kit for hepatitis A and hepatitis E virus IgM antibodies. The specific implementation method is the same as in Example 1, except that the pH of the sample dilution solution is adjusted to the conventional 7.4. The specific preparation process is as follows: S1. Preparation of sample diluent: Prepare the sample dilution control standard according to the following formula: Disodium hydrogen phosphate dodecahydrate: 3.6 g / L; Citric acid: 1.1 g / L; PVP-K30: 20.0 g / L; Sulfobetaine: 5.0 g / L; Sodium chloride: 8.5 g / L; BSA: 10.0 g / L; ProClin 300: 0.5 mL / L; Solvent: purified water; The pH was adjusted to 7.4 using 0.1 mol / L NaOH solution; the sample dilution was filtered through a 0.22 μm aqueous filter membrane to obtain the reference standard, which was stored at 4 °C.

[0113] S2, Preparation of the gold-labeled pad: Same as S2 in Example 1.

[0114] S3. Preparation of sample pad: Same as S3 in Example 1.

[0115] S4. Coating of the reaction membrane: Same as S4 in Example 1.

[0116] S5. Assembly of reagent strips: Same as S5 in Example 1, to obtain the combined detection kit for hepatitis A and hepatitis E virus IgM antibodies, denoted as kit control 3. Comparative Example 4

[0117] This comparative example provides a combined detection kit for hepatitis A and E virus IgM antibodies. The specific implementation method is the same as in Example 1, except that the anhydrous ethanol surface curing step is omitted in the double-layer spraying of the gold-labeled pad. The specific preparation process is as follows: S1. Preparation of sample diluent: Same as S1 in Example 1.

[0118] S2, Preparation of the gold-labeled pad: S21. Synthesis and preliminary purification of colloidal gold particles: Same as S21 in Example 1.

[0119] S22, PEGylation modification of colloidal gold particles: Same as S22 in Example 1.

[0120] S23, Antibody labeling and preparation of boric acid functionalized polymer: Same as S23 in Example 1.

[0121] S24. Target stabilization and post-processing: S241, Targeted Stabilization Response: Same as S241 in Example 1.

[0122] S242, Sealing and Stability: Same as S242 in Example 1.

[0123] S243, Preparation of fast-release colloidal gold-labeled antibody concentrate: Same as S243 in Example 1.

[0124] S244, Preparation of slow-release colloidal gold-labeled antibody concentrate: Same as S244 in Example 1.

[0125] S25, double-layer spraying and drying of gold standard pads: S251, Underlying slow-release layer spraying: Same as S251 in Example 1.

[0126] S252, Top Fast-Release Layer Spraying: Mix 1 mL of component A with 5 mL of fast-release treatment solution to obtain the fast-release working solution; A second coating is applied directly on top of the slow-release layer at a uniform spraying rate of 0.75 mL / strip, so that it is distributed on the surface area of ​​the gold-labeled pad substrate. After drying at 37℃ for 3 hours, the coated double-layer gold label pad was placed in an aluminum foil bag with desiccant, sealed and stored to obtain gold label pad control 1.

[0127] S3. Preparation of sample pad: Same as S3 in Example 1.

[0128] S4. Coating of the reaction membrane: Same as S4 in Example 1.

[0129] S5. Assembly of reagent strips: Same as S5 in Example 1, but replace the gold-labeled pad prepared in Example 1 with the gold-labeled pad control 1 prepared in step S2 to obtain the combined detection kit for hepatitis A and hepatitis E virus IgM antibodies, which is denoted as kit control 4. Comparative Example 5

[0130] This comparative example provides a combined detection kit for hepatitis A and E virus IgM antibodies. The specific implementation method is the same as in Example 1, except that cysteine ​​is used instead of HS-PEG-OH for surface modification of colloidal gold particles during the preparation of the gold pad. The specific preparation process is as follows: S1. Preparation of sample diluent: Same as S1 in Example 1.

[0131] S2, Preparation of the gold-labeled pad: S21. Synthesis and preliminary purification of colloidal gold particles: Same as S21 in Example 1.

[0132] S22, Cysteine ​​modification on the surface of colloidal gold particles: Cysteine ​​was used as a linker molecule, and cysteine ​​was dissolved in ultrapure water to prepare a 10 mmol / L cysteine ​​stock solution. Under continuous stirring at 400 r / min, 200 μL of cysteamine stock solution was added dropwise to 20 mL of concentrated colloidal gold solution (to make the final concentration 0.1 mmol / L), and the reaction was carried out continuously at 25 °C in the dark for 5 h. After the reaction was completed, the ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa was used to wash the sample with 10 mmol / L borate buffer at pH 8.5 and centrifuged at 4 °C and 9000 × g for 25 min. The supernatant was discarded and the washing was repeated twice. The final precipitate was resuspended in 10 mL of 10 mmol / L borate buffer (pH 8.5) to obtain a colloidal gold solution with surface amino groups modified.

[0133] S23, Antibody labeling and preparation of boric acid functionalized polymer: Same as S23 in Example 1.

[0134] S24, Targeted stabilization and post-processing: Same as S24 in Example 1.

[0135] S25, Double-layer spraying and drying of the gold label pad: Same as S25 in Example 1, to obtain gold label pad control sample 2.

[0136] S3. Preparation of sample pad: Same as S3 in Example 1.

[0137] S4. Coating of the reaction membrane: Same as S4 in Example 1.

[0138] S5. Assembly of reagent strips: Same as S5 in Example 1, but replace the gold-labeled pad prepared in Example 1 with the gold-labeled pad control 2 prepared in step S2 to obtain the combined detection kit for hepatitis A and hepatitis E virus IgM antibodies, which is denoted as kit control 5.

[0139] Analysis and Testing

[0140] In this invention, " " indicates negative; "±" indicates borderline / vague; "+" indicates clear weak positive; "++" indicates positive; "+++" indicates strong positive.

[0141] Commercially available reference standard 1: Hepatitis A virus IgM antibody detection kit (colloidal gold method), a single-test product of our company; Commercially available reference standard 2: Hepatitis E virus IgM antibody detection kit (colloidal gold method), a single-test product of our company; Commercially available reference standard 3: Hepatitis A virus IgM antibody diagnostic kit (enzyme-linked immunosorbent assay), Beijing Hyundai Gaoda Biotechnology Co., Ltd.

[0142] Commercially available reference standard 4: Hepatitis E virus IgM antibody diagnostic kit (enzyme-linked immunosorbent assay), Beijing Hyundai Gaoda Biotechnology Co., Ltd.

[0143] I. Detection Sensitivity Test To evaluate the core detection capabilities of the kit of this invention, the detection limits of T1 line (hepatitis A) and T2 line (hepatitis E) were determined respectively.

[0144] 1. Determination of the detection limit of T1 line (hepatitis A): Clinically confirmed hepatitis A virus IgM antibody-positive mixed serum samples were used as the starting samples; serial dilutions were performed using negative human serum (1:2, 1:4, 1:8, 1:16, 1:32); commercially available control standard 3 (enzyme-linked immunosorbent assay) was used to test all dilution samples, and the OD of each dilution gradient was recorded. 450 Values ​​and positive / negative interpretation.

[0145] Samples at each concentration gradient were tested using kit samples 1-3, kit controls 1-5, and commercially available control 1, and the results are shown in Table 1.

[0146] Table 1: Results of T1 line (Hepatitis A) detection limit determination

[0147] 2. Determination of the detection limit for T2 line (hepatitis E): Serum samples from individuals clinically diagnosed as positive for hepatitis E virus IgM antibody and negative for hepatitis A virus IgM antibody were used as the starting samples. These negative human serum samples were serially diluted (1:2, 1:4, 1:8, 1:16, 1:32). All dilutions were tested using commercially available control 4 (enzyme-linked immunosorbent assay), and the OD value of each dilution was recorded. 450 Values ​​and positive / negative interpretation.

[0148] Samples at each concentration gradient were tested using kit samples 1-3, kit controls 1-5, and commercially available control 2. The results are shown in Table 2.

[0149] Table 2: Results of T2 line (Hepatitis E) detection limit determination

[0150] As shown in Tables 1 and 2, the detection limits of the kit samples 1-3 prepared in the embodiments of the present invention all reached 1:8 dilution at both the T1 and T2 lines, consistent with the sensitivity levels of commercially available single-sample kits (commercially available reference standards 1 and 2). This indicates that integrating the HAV-IgM and HEV-IgM detection systems onto the same test strip did not result in a loss of detection sensitivity. At a dilution of 1:16, both the example samples and the commercially available single-sample kits showed "±" (critical value), and at 1:32, both were negative, further verifying the equivalence of the combined detection kit of the present invention with mature single-sample products in terms of basic sensitivity.

[0151] At a 1:8 dilution, the results for each comparative example were mostly "±", indicating a slightly lower sensitivity than the examples. Specifically, in serum-only conditions, the physical sieving function of the nanohydrogel was unnecessary due to the lack of macromolecular interfering substances in whole blood, thus its sensitivity was the same as the examples. This confirms that the core value of the nanohydrogel lies in handling complex samples rather than improving basic sensitivity.

[0152] The kit's control standard 2 exhibited a unique sensitivity differentiation characteristic: the T1 line decayed to "±" at a ratio of 1:8, while the T2 line remained "+". This is because the molecular crowding effect of dextran-70 and the microviscosity-modulating function of HEC only act on local microregions of the T1 line. Their absence significantly reduced the efficiency of the T1 line in capturing the gold-labeled antibody, while the T2 line, due to its intact coating conditions and independent formulation without crowding agents or viscosity modifiers, remained unaffected in sensitivity. This inherent difference between weak T1 and strong T2 already foreshadows the risk of signal imbalance in the combined detection at the baseline sensitivity level, which will be further exposed in subsequent signal balance tests.

[0153] The sensitivities of the T1 and T2 lines of both reagent kit control 3 and reagent kit control 4 decreased to ±. The former's degradation stemmed from the sample dilution solution's pH of 7.4 deviating from the midpoint between HAV-Ag (isoelectric point approximately 6.5) and HEV-Ag (isoelectric point approximately 5.8). Both antigens carry a negative charge, resulting in the loss of the differential charge regulation effect provided by the ion pre-programming strategy, leading to a comprehensive reduction in immunoreactivity. The latter was due to the absence of the anhydrous ethanol solidification step, causing component mixing of the fast and slow release layers during preparation. This disrupted the release sequence of the gold-labeled antibody, failing to provide sufficient and timely probe supply to the target detection line.

[0154] It is worth noting that the T1 line of reagent kit control 5 decayed to "±", and the T2 line also decreased to "±". This differs from the unidirectional decay pattern of the T1 line of reagent kit control 2, reflecting the dual negative effects caused by cysteine ​​modification: On the one hand, the short-arm amine group of cysteine ​​gives the colloidal gold surface a positive charge, which forms a protein crown by electrostatically adsorbing negatively charged proteins such as albumin in serum, partially obscuring the antigen binding site of the antibody Fab segment and reducing the effective labeled antibody concentration. This effect is more pronounced in the T1 line (upstream); on the other hand, the cysteine-modified surface lacks the compatibility of the PEG chain with the HPMC polyether backbone in the slow-release layer, impairing the uniformity of the hybrid gel network and reducing the controllability of sustained release. This global effect also affects the T2 line, resulting in a decrease in the basic sensitivity of both detection lines.

[0155] The above results clearly show that, under ideal sample conditions, the embodiments of the present invention have demonstrated sensitivity comparable to commercially available single-inspection products. However, each comparative example has shown varying degrees of performance degradation due to the lack of key innovative modules, and the degradation pattern is highly consistent with the functional positioning of the missing modules, laying the foundation for further amplification of these differences in subsequent tests.

[0156] II. Joint Detection Specificity and Signal Equalization Test To evaluate the identification ability and signal balancing effect of the combined detection kit of the present invention in mixed infection scenarios, single positive and double positive samples were used for verification.

[0157] All samples were pre-confirmed as positive or negative for the corresponding IgM antibodies using commercially available control 3 (Hepatitis A ELISA kit) and commercially available control 4 (Hepatitis E ELISA kit) to ensure the accuracy of the sample positive and negative results.

[0158] Test method: Ten clinical serum samples were selected from each of the following groups: those confirmed by enzyme-linked immunosorbent assay (ELISA) to be positive for hepatitis A alone (HAV-IgM+ / HEV-IgM-), hepatitis E alone (HAV-IgM- / HEV-IgM+), and both hepatitis A and hepatitis E (HAV-IgM+ / HEV-IgM+). Samples 1-3 and controls 1-5 from the kit were used for detection. The results were read 15-20 minutes after sample addition, and the T1 and T2 line color development results were recorded.

[0159] If a single positive sample shows color on only the detection line and the other line does not show color, it is considered to have no cross-interference; if both lines of a double positive sample show color and the intensity difference does not exceed one color level, it is considered to have balanced signal. The results are shown in Table 3.

[0160] Table 3: Results of Joint Detection Specificity and Signal Uniformity Measurement

[0161] As shown in Table 3, the reagent kit samples 1-3 prepared in the embodiments of the present invention all exhibit excellent performance in terms of joint detection specificity and signal balance, fully demonstrating the key role of each innovative module of the present invention in achieving joint detection signal balance.

[0162] Specifically, in the detection of single-positive samples, samples 1-3 of the kit showed no cross-reaction between the T1 and T2 lines. For hepatitis A single-positive samples, only the T1 line showed color, and for hepatitis E single-positive samples, only the T2 line showed color, with a false positive rate of 0%. In the detection of double-positive samples, both the T1 and T2 lines showed 100% color development, and the signal intensity difference between the two lines was consistently controlled within one color level, demonstrating a highly balanced joint detection effect. This directly proves that the present invention can effectively coordinate two detection lines with different intrinsic activities to obtain uniform signal output within the same reaction system.

[0163] In each comparative example, although the positive rates for both T1 and T2 were 100% and there were no false positives in kit control 1, the signal difference between the two lines in double-positive samples was one color level. Compared to the sensitivity performance comparable to the examples in pure serum, this difference stems from the presence of trace amounts of matrix components such as lipoproteins and fibrin fragments in real clinical serum samples. Sample pads treated with conventional chemical blocking agents cannot provide the thorough inert isolation that the zwitterionic nanohydrogel network offers. These trace interfering substances undergo slight non-specific adsorption with the nitrocellulose membrane and detection lines during chromatography, resulting in local background heterogeneity between the two lines and a decrease in signal uniformity. This demonstrates the unique value of the nanohydrogel network of this invention in real sample environments.

[0164] In hepatitis A single-positive samples, reagent kit control 2 showed a 10% false negative rate on the T1 line, and in double-positive samples, only 90% of the T1 line showed color development, while the T2 line consistently maintained a 100% positive rate. The signal intensity difference between the two lines exceeded three color levels, exhibiting a clear imbalance with one line being darker and the other lighter. This further validated the weak T1 / strong T2 differentiation trend already exposed in the first part of the detection limit test, indicating that the differential micro-enhancement of the T1 line is a key element in achieving signal balance in joint detection. Even if the spatiotemporal programming release of the gold-labeled pad still functions normally after the T1 line is missing, it cannot compensate for the insufficient capture efficiency of the T1 line, making signal imbalance an inevitable result.

[0165] Both reagent kit control 3 and reagent kit control 5 showed a signal intensity difference of approximately two color levels between the two lines in double-positive samples. Reagent kit control 3, due to its sample dilution solution pH being set to the standard 7.4, deviated from the median isoelectric point of HAV-Ag and HEV-Ag, thus losing the initial ionic conditions for differential charge regulation of the two antigens. Consequently, the immunoreactivity of both detection lines was affected to varying degrees, and the intrinsic activity difference between the T1 and T2 lines could not be effectively compensated, resulting in deteriorated signal uniformity. Reagent kit control 5, due to the positive charge on the colloidal gold surface after cysteine ​​modification, showed significantly reduced compatibility with HPMC in the slow-release layer compared to the PEG chain, weakening the controllability of sustained release. Simultaneously, the positively charged surface electrostatically adsorbed negatively charged proteins such as albumin in serum, forming a protein crown, reducing the effective labeled antibody concentration. This loss of activity was more pronounced in the T1 line (located upstream), resulting in a significantly weaker signal intensity in the T1 line compared to the T2 line.

[0166] Of particular concern is reagent kit control 4, which uniquely exhibited both false positives and false negatives, with performance declining in all three sample groups. In single-positive samples, both the T1 and T2 lines showed a 10% false positive rate, with the positive rate dropping to 90%. In double-positive samples, the T1 and T2 positive rates were also only 90%. This dual negative effect is directly attributed to the absence of the anhydrous ethanol curing step. Specifically, water from the fast-release working solution permeated into the slow-release layer, causing partial dissolution of HPMC and mixing with PVP-K30. This blurred the release sequence boundary between the two layers, disrupting the release sequence of the gold-labeled antibody's leading and trailing phases. This disordered release sequence resulted in non-specific retention of some gold-labeled antibody in non-target detection line areas, generating false positive signals; furthermore, it led to insufficient effective gold-labeled antibody concentration in the target detection line area, resulting in decreased sensitivity and false negatives. This result confirms the irreplaceable role of anhydrous ethanol surface curing in maintaining the structural integrity of the bilayer gold-labeled pad and the accuracy of the release sequence.

[0167] In summary, this invention integrates a four-ring synergistic technology scheme of ion pre-programming, inert isolation, spatiotemporal programming release, and differentiated micro-region enhancement into a single synergistic technology scheme. This successfully achieves signal balance between two detection lines while ensuring the specificity of joint detection, thus solving the signal imbalance problem that is common in existing joint detection technologies.

[0168] III. Analytical Specificity and Anti-interference Ability Test To evaluate the reliability of the combined detection kit of the present invention in complex real sample environments, its resistance to common endogenous interfering substances was examined.

[0169] Serum samples confirmed to be negative for both hepatitis A and hepatitis E IgM antibodies were collected and prepared into the following interfering samples using artificial interfering substances: rheumatoid factor positive serum (rheumatoid factor concentration of 480 IU / mL), high bilirubin serum (bilirubin concentrations of 40 mM and 80 mM, respectively), hemolyzed serum (hemoglobin concentrations of 5 g / L and 10 g / L, respectively), and high lipid serum (lipidemia concentrations of 6 mM and 9 mM, respectively). The above interfering samples were tested using kit samples 1–3, kit controls 1–5, and commercially available controls 1–2, respectively. Each sample was tested 10 times, and the color development intensity was recorded visually. If the 10 results were completely consistent, the color level symbol was directly recorded in the table; if the 10 results were inconsistent, the most frequent color level was recorded and specifically described in the analysis text. The results are shown in Tables 4 and 5.

[0170] Table 4: Colorimetric results of interfering agents on the T1 line (hepatitis A) of negative samples

[0171] Table 5: Colorimetric results of interfering agents on the T2 line (hepatitis E) of negative samples

[0172] Simultaneously, mixed serum samples from clinically confirmed hepatitis A / E double-positive cases were collected and diluted to a 1:4 ratio (weak positive level) using the aforementioned interfering sera. These were then used to test various samples to investigate the effect of the interfering agents on the weak positive signal intensity of the T1 and T2 lines. No significant signal attenuation was observed in any of the test samples under moderate concentrations of interfering agents; this test focused on high concentrations of interfering agents for comparison. If the color intensity of the detection line remained unchanged compared to normal serum conditions (still "+") under the presence of interfering agents, it was considered that there was no signal attenuation; if the color intensity weakened to "±" or turned negative, it was considered that there was signal attenuation. The results are shown in Tables 6 and 7.

[0173] Table 6: Results of the influence of interfering substances on the signal intensity of weakly positive samples (T1 line, hepatitis A)

[0174] Table 7: Results of the influence of interfering substances on the signal intensity of weakly positive samples with T2 line (hepatitis E).

[0175] As shown in Tables 4 to 7, samples 1-3 of the kit prepared in this embodiment of the invention did not exhibit non-specific color development on the T1 and T2 lines in all interfering substance tests (Tables 4 and 5). Furthermore, under conditions of 80 mM high bilirubin, 10 g / L hemolysis, and 9 mM high lipids, the weak positive signal intensity of the T1 and T2 lines remained consistently "+" without any attenuation (Tables 6 and 7), demonstrating excellent anti-interference ability. This result is attributed to the synergistic effect of the physical sieving of the zwitterionic nanohydrogel network in the sample pad and the mild cleavage and lipid dissolution function of sulfobetaine in the sample diluent, effectively removing large molecular interfering substances from whole blood samples and ensuring the reliability of the combined detection system in complex sample environments.

[0176] The anti-interference performance of each comparative example highly corresponds to the missing innovative module, thus verifying the independent value of each module of this invention from the opposite perspective. In 10 g / L hemolyzed and 9 mM hyperlipidemic samples, the T1 and T2 lines of the reagent kit control 1 showed "+", indicating a clear false positive (see Tables 4 and 5). Simultaneously, the weak positive signal completely decayed to "-" or "±" (see Tables 6 and 7). This demonstrates the irreplaceable role of the nano-hydrogel network as the inert isolation layer of the sample pad. Sample pads treated with conventional chemical blocking agents cannot effectively retain high concentrations of hemoglobin fragments and lipoprotein aggregates. These large molecular interferences clog membrane pores and undergo non-specific adsorption with the detection lines during chromatography, leading to false positives and severely interfering with the specific immune response.

[0177] Under conditions of 10 g / L hemolysis and 9 mM hyperlipidemia, the weak positive signals of both T1 and T2 lines in kit control 2 were attenuated to "±" (see Tables 6 and 7). Although no false positives were observed, the anti-interference ability was significantly weaker than that of the example. This is because the defect of kit control 2 lies in the absence of dextran-70 and HEC in the T1 line coating solution. However, signal attenuation also occurred in the T2 line at this point. In double-positive or weakly positive samples, the decreased capture efficiency of the T1 line led to more unbound gold-labeled antibodies continuing to move forward with the liquid flow, resulting in an abnormally high concentration of gold-labeled antibodies in the T2 line region. When interfering substances coexisted, this exacerbated the risk of non-specific adsorption, thus affecting the T2 line signal as well. This indirect effect of the T1 line defect on the T2 line further highlights the crucial role of the differential micro-region enhancement of the T1 line in the entire combined detection system.

[0178] In the kit, control 4 showed non-specific color development of "±" on both the T1 and T2 lines under conditions of 10 g / L hemolysis and 9 mM hyperlipidemia (see Tables 4 and 5), and the weak positive signal decayed to "±" (see Tables 6 and 7). This indicates that the mixing effect of fast and slow release layers is more severe in the presence of interfering substances. This is because high concentrations of hemoglobin and lipid components alter the viscosity and surface tension of the sample solution, further disrupting the already fragile release sequence and leading to increased retention of labeled antibodies in non-target detection line areas.

[0179] Neither reagent kit control 3 nor reagent kit control 5 showed attenuation of the weak positive signals in the T1 and T2 lines in the presence of interfering substances (see Tables 6 and 7), consistent with the performance in the examples. Combined with the results of decreased signal balance in the second part, it can be seen that the lack of ion pre-programming in reagent kit control 3 and the surface charge problem in reagent kit control 5 mainly affect the signal balance of the two lines, rather than absolute sensitivity or anti-interference ability. This distinction further verifies that the functional positioning of each module in the four-ring synergistic system of this invention has its own focus and does not overlap.

[0180] It is noteworthy that all test samples showed negative T1 and T2 line color development results in rheumatoid factor-positive serum (see Tables 4 and 5), indicating that the IgM capture method used in this invention and each comparative example can effectively avoid interference from rheumatoid factor. Under conditions of 40 mM bilirubin and 5 g / L hemoglobin, no nonspecific color development was observed in any of the samples (see Tables 4 and 5), indicating that these interfering substances did not exceed the tolerance threshold of each protocol at moderate concentrations.

[0181] IV. Accelerated Stability Test To evaluate the shelf stability of the combined detection kit of the present invention, accelerated stability testing was performed.

[0182] Test Method: Samples 1-3 of the kit, controls 1-5 of the kit, and commercially available controls 1-2 were placed in a 37°C incubator and removed on days 7, 14, 21, and 28. At each time point, samples diluted to a 1:4 ratio (weak positive level) using clinically confirmed hepatitis A and hepatitis E double-positive mixed serum were tested. The color intensity of the T1 and T2 lines was compared before and after acceleration. If the detection lines remained clearly visible after acceleration, the sensitivity was considered to have been maintained. The results are shown in Table 8.

[0183] Table 8: Results of Accelerated Stability Measurement

[0184] As shown in Table 8, after accelerated aging at 37°C for 28 days, samples 1 and 2 of the kit prepared in this embodiment of the invention still showed clear positive results for weakly positive samples in terms of T1 and T2 lines, demonstrating excellent long-term stability. Among them, sample 3 showed a decrease in T1 and T2 line color to "±" at 28 days, but maintained stable positive results in tests up to 21 days later, indicating that its stability was still significantly better than all comparative samples.

[0185] Reference standards 1, 3, and 5 all decayed to "±" after 21 days of accelerated degradation and became completely ineffective after 28 days. Specifically, reference standard 1, lacking the physical isolation and protection of a nano-hydrogel during preparation, was more susceptible to residual moisture and trace impurities during long-term storage. Reference standard 3 experienced accelerated antibody activity decay during long-term storage because the sample dilution pH deviated from the optimal stability range for antibody conformation under ion pre-programmed conditions. Reference standard 5, due to the significantly smaller steric hindrance of the short arms of cysteine ​​compared to the long chains of PEG, exhibited insufficient anti-aggregation ability of colloidal gold particles during long-term storage, leading to gradual particle aggregation and a decrease in the effective labeled antibody concentration. These three comparative examples collectively verify the necessity and effectiveness of this invention in ensuring the long-term effectiveness of the combined detection kit.

[0186] Among them, the reagent kit control 4 showed the worst stability, with signal attenuation appearing after 7 days of accelerated release and complete failure after 14 days. This is directly attributed to the intermingling of components in the fast and slow release layers during long-term storage. Specifically, the lack of a dense interfacial protective layer formed by ethanol curing allowed PVP-K30 in the fast release layer and HPMC in the slow release layer to gradually interpenetrate during storage. The accuracy of the release timing deteriorated over time, ultimately resulting in insufficient concentration of the gold-labeled antibody fronter to effectively saturate the T1 line.

[0187] The stability of the T1 line of reagent kit control 2 was significantly worse than that of the T2 line, decaying to "±" after 14 days and completely failing after 21 days, while the T2 line remained "+" at 28 days. This significant divergence in the stability of the two lines within the same sample reveals that the glassy protective layer constructed by trehalose and dextran-70 in the T1 line coating solution effectively maintained the native conformation of the coated antigen during long-term storage, while conventional formulations containing only trehalose could not provide the same protective effect. The T2 line stability of reagent kit control 2 was the best among all comparative examples, even comparable to that of the examples, which is completely consistent with its design of complete T2 line coating conditions and defects only in the T1 line, thus demonstrating the precision and effectiveness of the differentiated micro-region enhancement strategy of this invention.

[0188] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A combined detection kit for hepatitis A and hepatitis E virus IgM antibodies, characterized in that, include: The sample pad, gold label pad, reaction membrane coated with detection line T1, detection line T2 and quality control line C, and absorbent pad are fixed on the PVC base plate. The gold-labeled pad has a double-layer structure consisting of a bottom slow-release layer and an upper fast-release layer. The upper surface of the bottom slow-release layer is cured with anhydrous ethanol to form a dense interface. The bottom slow-release layer contains hydroxypropyl methylcellulose, and the top fast-release layer contains polyvinylpyrrolidone, both of which are loaded with colloidal gold-labeled mouse anti-human IgM monoclonal antibody. The colloidal gold-labeled mouse anti-human IgM monoclonal antibody was obtained by modifying the surface of colloidal gold particles with α-mercapto-ω-hydroxy polyethylene glycol, labeling the antibody, and then targeting and stabilizing it with boric acid-functionalized polyamide-amine dendritic macromolecules. The sample pad is loaded with a zwitterionic nanohydrogel network; The detection line T1 is a hepatitis A virus detection line coated with recombinant hepatitis A virus antigen, and its coating buffer contains macromolecular crowding agent and viscosity modifier. The detection line T2 is a hepatitis E virus detection line coated with recombinant hepatitis E virus antigen, and its coating buffer does not contain the macromolecular crowding agent and viscosity modifier. The kit also includes a sample diluent containing polyvinylpyrrolidone and sulfobetaine, with a pH of 6.1–6.

3.

2. The reagent kit according to claim 1, characterized in that, The molecular weight of the α-mercapto-ω-hydroxy polyethylene glycol is 2kDa to 3kDa; the boric acid-functionalized polyamide-amine dendritic macromolecule is obtained by coupling 3-aminophenylboronic acid with the polyamide-amine dendritic macromolecule under the action of a coupling agent.

3. The reagent kit according to claim 1, characterized in that, The macromolecular crowding agent is dextran, and its concentration in the coating buffer of detection line T1 is 30 g / L to 50 g / L; the viscosity modifier is hydroxyethyl cellulose, and its concentration in the coating buffer of detection line T1 is 1.5 g / L to 2.5 g / L; the coating buffer of detection line T2 contains trehalose at a concentration of 15 g / L to 25 g / L.

4. The reagent kit according to claim 1, characterized in that, The zwitterionic nanohydrogel is formed by the polymerization of nanoparticles through reverse microemulsion with [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide monomer, N,N'-methylenebisacrylamide crosslinking agent and sodium di(2-ethylhexyl)succinate sulfonate emulsifier, which are then impregnated and dried to form a hydrophilic network structure on the surface of the sample pad fiber.

5. A method for preparing a combined detection kit for hepatitis A and hepatitis E virus IgM antibodies, used to prepare the kit as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. Preparation of sample diluent: Prepare a sample diluent containing polyvinylpyrrolidone and sulfobetaine, and adjust the pH to 6.1-6.3; S2. Preparation of the gold-labeled pad: Colloidal gold was synthesized and concentrated to obtain a concentrated colloidal gold solution. The surface of the colloidal gold was modified with α-mercapto-ω-hydroxy polyethylene glycol, and then labeled with mouse anti-human IgM monoclonal antibody to obtain a colloidal gold-antibody complex. The complex was then targeted and stabilized using boric acid-functionalized polyamide-amine dendritic macromolecules, and after blocking, a stable colloidal gold-labeled antibody solution was obtained. The colloidal gold-labeled antibody solution was divided into two equal parts and resuspended in fast-release and slow-release solutions, respectively, to obtain a fast-release component and a slow-release component. The slow-release component was first sprayed to form a bottom slow-release layer, which was then cured with anhydrous ethanol. The fast-release component was then sprayed to form an upper fast-release layer, and after drying, the finished gold-labeled pad was obtained. S3. Preparation of sample pad: zwitterionic nanohydrogel is prepared by reverse microemulsion polymerization, and it is mixed with anhydrous ethanol to prepare a sample pad treatment solution. The glass fiber sample pad is immersed in the sample pad treatment solution and dried to obtain the finished sample pad. S4. Coating of the reaction membrane: Prepare a working solution for coating the detection line T1 containing macromolecular crowding agent and viscosity modifier, a working solution for coating the detection line T2 without macromolecular crowding agent and viscosity modifier, and a working solution for coating the quality control line C. Spray these solutions onto the nitrocellulose membrane in sequence to form the detection line T1, the detection line T2, and the quality control line C. After drying, the finished reaction membrane is obtained. S5. Assembly of reagent strips: The finished sample pad, finished gold label pad, finished reaction membrane and absorbent pad are sequentially pasted onto the PVC base plate, and after cutting, shelling and packaging, the reagent kit is obtained.

6. The preparation method according to claim 5, characterized in that, In step S1: The sample diluent contains 3.4 g / L to 3.8 g / L disodium hydrogen phosphate dodecahydrate, 1.0 g / L to 1.2 g / L citric acid, 18.0 g / L to 22.0 g / L polyvinylpyrrolidone, 4.0 g / L to 6.0 g / L sulfobetaine, 8.0 g / L to 9.0 g / L sodium chloride, 9.0 g / L to 11.0 g / L bovine serum albumin, 0.4 mL / L to 0.6 mL / L preservative, and water as the solvent.

7. The preparation method according to claim 5, characterized in that, In step S2: The synthesized colloidal gold is prepared by reducing tetrachloroauric acid with trisodium citrate. The specific process is as follows: a 10 g / L tetrachloroauric acid solution is mixed with ultrapure water at a volume ratio of (0.9-1.1):100, heated to boiling, and a 10 g / L trisodium citrate solution is added while stirring at 300-500 r / min. The mixture is kept boiling for 5-10 minutes, then heating is stopped. After cooling, membrane filtration, centrifugation, and concentration, a concentrated colloidal gold solution is obtained. The volume ratio of the trisodium citrate solution to the ultrapure water is (1.7-1.9):

100. The reaction conditions for modifying colloidal gold surfaces with α-mercapto-ω-hydroxy polyethylene glycol are as follows: reaction at 20℃~30℃ in the dark for 3h~5h, adding α-mercapto-ω-hydroxy polyethylene glycol stock solution with a concentration of 4mmol / L~6mmol / L to the reaction system, and the volume ratio of α-mercapto-ω-hydroxy polyethylene glycol stock solution to concentrated colloidal gold solution is (0.75~1.25):

100.

8. The preparation method according to claim 5, characterized in that, In step S2: The rapid release solution is a 40mmol / L to 60mmol / L PBS buffer containing 8g / L to 12g / L bovine serum albumin, 40g / L to 60g / L trehalose, and 2g / L to 4g / L polyvinylpyrrolidone, with a pH of 7.3 to 7.

5. The slow-release treatment solution is a 40mmol / L to 60mmol / L PBS buffer containing bovine serum albumin (8g / L to 12g / L), trehalose (40g / L to 60g / L), and hydroxypropyl methylcellulose (4g / L to 6g / L), with a pH of 7.3 to 7.

5. The anhydrous ethanol surface curing treatment involves atomizing and spraying the slow-release layer surface with a spraying amount of 0.1 mL / strip to 0.2 mL / strip and then drying it. The mass ratio of the colloidal gold-labeled antibody in the fast-release component to that in the slow-release component is 1:

1.

9. The preparation method according to claim 5, characterized in that, In step S3, the preparation process of the zwitterionic nanohydrogel is as follows: [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide was dissolved in deionized water, and N,N'-methylenebisacrylamide was added as a crosslinking agent to form the aqueous phase. The aqueous phase was then added dropwise to cyclohexane containing sodium di(2-ethylhexyl)succinate sulfonate and stirred to form a water-in-oil microemulsion. Under nitrogen protection, ammonium persulfate and tetramethylethylenediamine were added to initiate polymerization. After the reaction was completed, the microemulsion was broken, washed, and centrifuged to obtain nano-hydrogel particles, which were dispersed in PBS to prepare a nano-hydrogel working solution of 4 g / L to 6 g / L. The nano-hydrogel working solution was mixed with anhydrous ethanol at a volume ratio of (3.5-4.5):1 to obtain the sample pad treatment solution. The glass fiber sample pad is immersed in the sample pad treatment solution and then dried to obtain the sample pad.

10. The preparation method according to claim 5, characterized in that, In step S4: The T1 coating working solution for the detection line is prepared by dissolving recombinant hepatitis A virus antigen in 8 mmol / L to 12 mmol / L PBS buffer containing 30 g / L to 50 g / L dextran, 1.5 g / L to 2.5 g / L hydroxyethyl cellulose, and 15 g / L to 25 g / L trehalose, with an antigen concentration of 0.9 g / L to 1.1 g / L. The T2 coating working solution for the detection line is prepared by dissolving recombinant hepatitis E virus antigen in 8 mmol / L to 12 mmol / L PBS buffer containing 15 g / L to 25 g / L trehalose and 0.08 g / L to 0.12 g / L Tween, with an antigen concentration of 1.4 g / L to 1.6 g / L. The quality control line C coating working solution is prepared by dissolving goat anti-mouse IgG antibody in 8mmol / L to 12mmol / L PBS buffer containing 15g / L to 25g / L trehalose, resulting in an antibody concentration of 1.9g / L to 2.1g / L.

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