Respiratory pathogen antigen combined detection kit based on triplex card and preparation method thereof

Abstract

The application discloses a triple-card respiratory tract pathogen antigen combined detection kit and a preparation method, and belongs to the technical field of in-vitro detection. The sample pad is a glass fiber membrane on which cross-linked jacalin is fixed on the surface after being immersed in a basic treatment liquid for pre-passivation. The jacalin is cross-linked with bovine serum albumin to form a covalent cross-linking network on the surface after being pre-protected by galactose, and the activity of the sugar chain is recovered after elution and activation treatment. The kit further comprises a sample treatment liquid. The application realizes systematic technical innovation from three aspects of conformation compensation, interference capture and microenvironment regulation. Through the organic cooperation of the synergistic system of arginine, histidine-and calcium ions and the complete fixation process of lectin, the two technical problems of limited sensitivity of Victoria subtypes and serious interference of complex samples in the existing combined detection technology are successfully solved.

Description

Technical Field

[0001] This invention belongs to the field of in vitro detection technology, and relates to a respiratory pathogen antigen detection kit based on a triple card and its preparation method. Background Technology

[0002] Respiratory infections are among the leading causes of disease-related morbidity and mortality worldwide. Influenza A virus, influenza B virus, and Mycoplasma pneumoniae are the three main pathogens causing acute respiratory infections. Influenza A virus can be classified into several subtypes based on differences in the structure of its surface hemagglutinin and neuraminidase; currently, the H1N1 and H3N2 subtypes are the most prevalent infecting humans. Influenza B virus is divided into the Victoria and Yamagata lineages, which alternate in circulation. Mycoplasma pneumoniae is one of the most common atypical pathogens, with a relatively mild onset, characterized by a prominent irritating dry cough, which is clearly distinct from the severe symptoms of influenza virus, such as high fever and body aches. However, the clinical symptoms caused by these three pathogens highly overlap, all presenting with fever, cough, and sore throat, and co-infections can occur, making clinical differentiation difficult based solely on symptoms.

[0003] Colloidal gold immunochromatography has become the preferred technology platform for rapid screening in primary healthcare institutions and emergency departments due to its ease of operation, rapid detection, and intuitive result interpretation. In recent years, triplet test kits integrating antigen detection for influenza A virus, influenza B virus, and mycoplasma pneumoniae onto the same test card have been launched on the market. These kits enable simultaneous detection of three pathogens with a single sample addition, significantly improving detection efficiency.

[0004] However, in practical applications, existing triple antigen test kits show significant differences in detection sensitivity between the Victoria and Yamagata lineages of influenza B virus. The root cause of this problem lies in the ongoing antigenic drift of the hemagglutinin protein in the Victoria lineage virus, with the emergence of variants carrying amino acid deletions in recent years. This results in a naturally lower affinity for the detection antibody compared to the Yamagata lineage. This difference in sensitivity between subtypes is particularly pronounced in triple antigen tests. When the detection signal for the Victoria lineage is significantly weaker than that for the Yamagata lineage, interpreters struggle to determine whether the weak signal is a true positive or background interference, leading to difficulties in clinical interpretation and false negative results. Furthermore, the abundant presence of large glycoproteins such as mucin in oropharyngeal swab samples severely interferes with the detection process. Mucin readily adheres to the nitrocellulose membrane during chromatography, clogging the pores and increasing the T-line background, further complicating signal interpretation.

[0005] Therefore, how to eliminate the difference in detection sensitivity between the Victoria and Yamagata subtypes of influenza B virus and effectively reduce the interference of mucin in oropharyngeal swab samples has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a respiratory pathogen antigen detection kit and preparation method based on a triple card, in order to solve the risk of false negatives caused by structural defects of the Victoria subtype antigen and background interference caused by mucin in existing triple detection kits, thereby improving the detection sensitivity, result interpretation accuracy and clinical reliability of the kit.

[0007] The technical solution adopted in this invention is: The key features of the respiratory pathogen antigen detection kit based on the triple card are: Three independent detection channels are fixed on the PVC base plate. Each detection channel includes a sample pad, a gold label pad, a reaction membrane coated with detection lines and quality control lines, and an absorbent pad. The sample dispensing holes of the three detection channels are independent of each other. The corresponding gold-labeled pads of the three detection channels are respectively loaded with colloidal gold-labeled influenza A virus antibody 1, colloidal gold-labeled influenza B virus antibody 1, or colloidal gold-labeled mycoplasma pneumoniae antibody 1; The sample pad described above is a glass fiber membrane that has been pre-passivated by impregnation with a basic treatment solution, and then has cross-linked jackfruit lectin fixed on its surface. The jackfruit lectin is pre-treated with galactose for sugar preprotection, then cross-linked with bovine serum albumin to form a covalent cross-linked network fixed on the surface of the sample pad. After elution and activation treatment, its sugar chain binding activity is restored. The basic treatment solution contains bovine serum albumin 10.0 g / L–25.0 g / L, trehalose 30.0 g / L–80.0 g / L, polyvinylpyrrolidone 3.0 g / L–10.0 g / L, Tween 20 1.0 mL / L–5.0 mL / L, preservative 0.2 mL / L–1.0 mL / L, purified water as the solvent, and a pH of 7.3–7.5. The above kit also includes a sample processing solution having the following components: Tris(hydroxymethyl)aminomethane 3.0 g / L–4.2 g / L, sodium chloride 5.0 g / L–6.5 g / L, 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate 1.8 g / L–2.3 g / L, Tween 80 0.8 mL / L~1.2 mL / L, bovine serum albumin 8.0 g / L~12.0 g / L, trehalose 25.0 g / L~35.0 g / L, polyethylene glycol 0.8 g / L~1.2 g / L, arginine 3.6 g / L~5.0 g / L, histidine 2.8 g / L~4.5 g / L, calcium chloride 0.16 g / L~0.25 g / L, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride 0.1 g / L~0.15 g / L, preservative 0.4 mL / L~0.6 mL / L, pH 6.4~6.6.

[0008] Specifically, the mass ratio of arginine, histidine, and calcium chloride in the above sample processing solution is (20–22.5):(17.5–19):1.

[0009] Furthermore, the corresponding reaction membranes of the three detection channels are respectively equipped with influenza A virus detection lines, influenza B virus detection lines, mycoplasma pneumoniae detection lines, and quality control lines; the influenza A virus detection lines are coated with influenza A virus antibody 2, the influenza B virus detection lines are coated with influenza B virus antibody 2, and the mycoplasma pneumoniae detection lines are coated with mycoplasma pneumoniae antibody 2; the coating concentrations of influenza A virus antibody 2 and influenza B virus antibody 2 are both 0.2 g / L to 0.4 g / L, and the coating concentration of mycoplasma pneumoniae antibody 2 is 0.3 g / L to 0.5 g / L; the influenza A virus antibody 2, influenza B virus antibody 2, and mycoplasma pneumoniae antibody 2 are respectively paired antibodies of influenza A virus antibody 1, influenza B virus antibody 1, and mycoplasma pneumoniae antibody 1.

[0010] The preparation method of the respiratory pathogen antigen detection kit based on the triple card, used to prepare the above-mentioned kit, mainly includes the following steps: S1. Preparation of sample processing solution: Prepare a sample processing solution containing arginine and histidine, and adjust the pH to 6.4-6.6; S2. Preparation of gold-labeled pads: Colloidal gold was synthesized and concentrated. Influenza A virus antibody 1, influenza B virus antibody 1 and mycoplasma pneumoniae antibody 1 were labeled on colloidal gold particles respectively. After blocking, centrifugation, and reconstitution with colloidal gold conjugate diluent, the respective labeled solutions were obtained. They were then sprayed onto glass fiber membranes and dried to obtain gold-labeled pads with three independent detection channels. S3. Preparation of sample pads: The glass fiber membrane is impregnated with the basic treatment solution for pre-passivation; after sugar preprotection treatment of jackfruit lectin and galactose, bovine serum albumin and glutaraldehyde are added to make a cross-linking mixture, which is sprayed onto the pre-passivated glass fiber membrane. After standing for cross-linking, it is then rinsed with elution buffer to activate it. After drying, sample pads with three independent detection channels are obtained. S4. Coating of the reaction membrane: Prepare coating working solutions for influenza A virus antibody 2, influenza B virus antibody 2, mycoplasma pneumoniae antibody 2 and goat anti-mouse IgG polyclonal antibody respectively, and spray them onto the nitrocellulose membrane in sequence to form detection lines and control lines. After drying, a reaction membrane with three independent detection channels is obtained. S5. Assembly of reagent strips: After assembling the sample pads, gold label pads, reaction membranes and absorbent pads of the three independent detection channels, fix them on the same PVC base plate, cut, shell and package them to obtain the above reagent kit. The influenza A virus antibody 2, influenza B virus antibody 2, and mycoplasma pneumoniae antibody 2 mentioned above are paired antibodies of influenza A virus antibody 1, influenza B virus antibody 1, and mycoplasma pneumoniae antibody 1 mentioned above, respectively.

[0011] Furthermore, in step S2 above, the colloidal gold conjugate diluent contains bovine serum albumin 8.0 g / L to 12.0 g / L, trehalose 45.0 g / L to 55.0 g / L, and Tween 20 4.0 mL / L to 6.0 mL / L, and the solvent is PBS buffer with a pH of 7.2 to 7.6.

[0012] Furthermore, in step S2 above, the specific method for labeling the colloidal gold antibody is as follows: Adjust the pH of the concentrated colloidal gold solution to 6.8–7.2 with potassium carbonate solution and let it stand for 5–15 minutes. Add the above-mentioned influenza A virus antibody 1, influenza B virus antibody 1 and mycoplasma pneumoniae antibody 1 to each of them, so that their final concentrations are 0.010 g / L to 0.014 g / L, and let stand for 35 min to 45 min; Add bovine serum albumin to a final concentration of 1.5 g / L to 2.5 g / L, and let stand for 12 to 18 minutes. Centrifuge at 9000 to 11000 r / min and 2 to 8℃ for 35 to 45 minutes, discard the supernatant, and mix the precipitate with the colloidal gold conjugate diluent at a volume ratio of 1:4.5 to 1:5.5.

[0013] Furthermore, in step S3 above, during the cross-linking fixation process, bovine serum albumin is added to a final concentration of 2.0 g / L to 5.0 g / L and glutaraldehyde to a final concentration of 0.5 g / L to 1.0 g / L, and the mixture is allowed to stand at 4℃ to 10℃ for 1 h to 2 h for cross-linking.

[0014] Furthermore, in step S3 above, the conditions for the sugar preprotection treatment are as follows: 1.0 g / L to 1.5 g / L of jackfruit lectin and 9.0 g / L to 18.0 g / L of galactose are incubated in phosphate buffer at 2°C to 8°C for 30 min to 60 min.

[0015] Furthermore, in step S3 above, the elution buffer contains the following components: galactose 9.0 g / L to 18.0 g / L, bovine serum albumin 5.0 g / L, Tween 20 0.5 mL / L, and the solvent is phosphate buffer with a pH of 7.3 to 7.5; the elution activation conditions are: rinsing with the above elution buffer, standing for 5 min to 10 min, and then drying.

[0016] Furthermore, in step S4 above, the coating buffer is PBS buffer of 20 mmol / L to 50 mmol / L, pH 7.2 to 7.6; the coating concentrations of the influenza A virus antibody 2 and influenza B virus antibody 2 are both 0.2 g / L to 0.4 g / L, the coating concentration of the mycoplasma pneumoniae antibody 2 is 0.3 g / L to 0.5 g / L, and the coating concentration of the goat anti-mouse IgG polyclonal antibody is 0.8 g / L to 1.2 g / L.

[0017] Compared with the prior art, the present invention has the following advantages: The respiratory pathogen antigen detection kit based on a triplet card provided by this invention addresses the dual technical challenges of insufficient sensitivity of influenza B virus Victoria subtype due to HA protein structural defects and severe interference from mucins in oropharyngeal swab samples in existing detection technologies. Through a systematic design from sample processing solution and sample pad to reaction membrane, it achieves efficient detection of three pathogens while significantly improving the detection sensitivity, resistance to mucin interference, and long-term stability of weaker targets. Specific beneficial effects include: First, this invention constructs a conformational compensation scheme based on the synergistic effect of arginine, histidine, and calcium ions, successfully overcoming the key challenge of insufficient detection sensitivity for the Victoria subtype of influenza B virus. This invention integrates the directional hydrogen bonding of the guanidinium group of arginine, the pH-stabilizing function of the imidazole ring of histidine, and the structural rigidity-enhancing effect of calcium ions into a unified whole. This synergistic effect from three dimensions—structural rigidity enhancement, directional conformational compensation, and microenvironmental stabilization—compensates for the local conformational looseness defect caused by amino acid deletion in the Victoria subtype HA protein, restoring its immunoreactivity to a level comparable to the structurally intact Yamagata subtype. The technical solution of this invention fundamentally compensates for the conformational defects of the target antigen, upgrading passive adaptation to active repair, resulting in an order-of-magnitude improvement in the detection sensitivity of the Victoria subtype.

[0018] Secondly, this invention effectively eliminates chromatographic interference from mucin in oropharyngeal swab samples through a complete three-step process: sugar pre-protection, cross-linking fixation, and elution activation of bromelain on the sample pad. The sugar pre-protection step utilizes galactose to occupy the glycan binding sites of the lectin, protecting its essential primary amino groups from glutaraldehyde cross-linking damage. The subsequent elution activation step removes the protective galactose, allowing the lectin's glycan binding activity to be fully restored. This process ensures that the bromelain immobilized on the sample pad can efficiently and specifically capture large mucin molecules in the sample, physically intercepting interference at the start of chromatography, avoiding chromatographic obstruction and signal attenuation caused by lectin inactivation or lack of activation in existing technologies. These three steps are interconnected; the absence of any step will severely reduce anti-interference capability. Their completeness plays an irreplaceable role in ensuring the signal stability of each target under complex sample conditions. For weak targets with already limited detection sensitivity, this anti-interference guarantee is particularly crucial.

[0019] Third, this invention, through the synergistic effect of the sample processing solution and the sample pad, simultaneously achieves targeted enhancement of weak targets and broad-spectrum interception of common interfering substances in the joint detection system, significantly extending the effective usage period of the kit. The combination of arginine and histidine in the sample processing solution not only provides conformational compensation but also exhibits a synergistic protective effect on bioactive components in accelerated stability testing, effectively delaying the degradation of reagent performance. Meanwhile, the functional stability of the sample pad ensured by the lectin immobilization process allows the kit to maintain efficient mucin capture capabilities even after long-term storage, avoiding performance degradation caused by the accumulation of interfering substances.

[0020] In summary, this invention has made systematic technological innovations in three aspects: conformation compensation, interfering substance capture, and microenvironment regulation. Through the synergistic system of arginine, histidine-, and calcium ions, and the organic combination of lectin fixation process, it has successfully solved the two major technical problems of limited sensitivity of Victoria subtype and severe interference from complex samples in existing joint detection technologies. This provides an accurate, reliable, and highly stable technical means for the rapid and simultaneous differential diagnosis of influenza A virus, influenza B virus, and mycoplasma pneumoniae. Detailed Implementation

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] Unless otherwise specified in the examples, the standard conditions can be followed; unless the manufacturer of the reagents or instruments used is specified, they are all commercially available products.

[0027] Among them, influenza A virus antibody 1 and influenza A virus antibody 2 are monoclonal antibodies produced in mice after immunization with influenza A virus antigen, purchased from Weixian Jubang New Materials Technology Co., Ltd. They are paired antibodies, used for colloidal gold labeling and detection line coating, respectively. Influenza B virus antibody 1 and influenza B virus antibody 2 are monoclonal antibodies produced in mice after immunization with influenza B virus antigen, purchased from Weixian Jubang New Materials Technology Co., Ltd. They are paired antibodies, used for colloidal gold labeling and detection line coating, respectively. Mycoplasma pneumoniae antibody 1 and Mycoplasma pneumoniae antibody 2 are monoclonal antibodies produced in mice after immunization with Mycoplasma pneumoniae antigen, purchased from Xiamen Wankelong Biotechnology Co., Ltd. They are paired antibodies, used for colloidal gold labeling and detection line coating, respectively. Goat anti-mouse IgG polyclonal antibody was purchased from Beijing Wanyu Meilan Technology Co., Ltd.

[0028] The bovine serum albumin used is abbreviated as BSA; the polyvinylpyrrolidone K30 used is abbreviated as PVP-K30; the polysorbate 20 used is abbreviated as Tween-20; the Tween 80 used is abbreviated as Tween-80; and the polyethylene glycol 20000 used is abbreviated as PEG20000. Example 1

[0029] The respiratory pathogen antigen detection kit and its preparation method for the triple card in this embodiment are as follows: S1. Preparation of sample processing solution: Prepare the sample treatment solution according to the following formula: Tris(hydroxymethyl)aminomethane: 3.6 g / L; Sodium chloride: 5.8 g / L; 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate: 2.0 g / L; Tween-80: 1.0 mL / L; BSA: 10.0 g / L; Trehalose: 30.0 g / L; PEG20000: 1.0 g / L; Arginine: 4.2 g / L; Histidine: 3.8 g / L; Calcium chloride: 0.2 g / L; 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride: 0.12 g / L; ProClin 300: 0.5 mL / L; Solvent: purified water; pH adjusted to 6.5 with 0.5 mol / L HCl solution; wherein the mass ratio of arginine, histidine and calcium chloride is 21:19:1; The sample was filtered through a 0.22 μm aqueous filter membrane to obtain the sample treatment solution, which was then stored at 4 °C in the dark.

[0030] S2, Preparation of the gold-labeled pad: S21. Synthesis and preliminary purification of colloidal gold particles: Take 100 mL of purified water, add 4.0 mL of 10 g / L tetrachloroauric acid solution, and heat until it boils vigorously; Add 6.5 mL of 10 g / L trisodium citrate solution at 400 r / min; Keep boiling for 8 minutes until the solution turns a clear bright red color. Stop heating, cool, and filter through a 0.22 μm aqueous phase filter membrane. Use an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa to centrifuge at 4 °C and 9000 × g for 25 minutes to concentrate to 1 / 5 of the original volume to obtain a concentrated colloidal gold solution.

[0031] S22. Preparation of colloidal gold conjugate diluent: Prepare a colloidal gold conjugate diluent based on the following formulation: BSA: 10.0 g / L; Trehalose: 50.0 g / L; Tween-20: 5.0 mL / L; Solvent: PBS buffer (50 mmol / L, pH 7.4); Stir until completely dissolved, filter through a 0.22 μm filter membrane to obtain a diluted colloidal gold conjugate solution.

[0032] S23, Colloidal gold-labeled antibody: Preparation of S231, Influenza A virus antibody 1 labeled solution: Take the concentrated colloidal gold solution, add 0.1 mol / L potassium carbonate solution to adjust the pH to 7.0, and let it stand for 10 min; Add influenza A virus antibody 1 to a final concentration of 0.012 g / L, mix well, and let stand at 25°C for 40 min; Add BSA to a final concentration of 2.0 g / L and let stand for another 15 minutes; Centrifuge at 10000 r / min and 4℃ for 40 min, discard the supernatant, and mix the precipitate with the colloidal gold conjugate dilution solution prepared by S22 at a volume ratio of 1:5.0 to obtain the influenza A virus antibody 1 labeled solution.

[0033] S232. Preparation of Influenza B Virus Antibody 1 Label Solution: The preparation method is the same as step S231, except that influenza B virus antibody 1 is replaced with an equal mass of influenza A virus antibody 1 to prepare an influenza B virus antibody 1 labeled solution.

[0034] Preparation of S233, Mycoplasma pneumoniae antibody 1 labeled solution: The preparation method is the same as step S231, except that the same mass of Mycoplasma pneumoniae antibody 1 is used instead of influenza A virus antibody 1 to prepare Mycoplasma pneumoniae antibody 1 labeled solution.

[0035] S24, Spraying and drying of gold standard pads: Cut the fiberglass film into 30cm×1cm pieces and lay them flat on the worktable of the gold spraying machine. The influenza A virus antibody 1-labeled solution, influenza B virus antibody 1-labeled solution, and mycoplasma pneumoniae antibody 1-labeled solution were sprayed sequentially onto their respective gold-labeled pad areas at a spraying volume of 1.2 mL / strip, and then dried at 38°C for 3 hours.

[0036] S3. Preparation of the sample pad: S31. Preparation of basic treatment solution: Prepare the basic treatment solution according to the following formula: BSA: 18.0 g / L; Trehalose: 55.0 g / L; PVP-K30: 6.5 g / L; Tween-20: 3.0 mL / L; ProClin300: 0.6 mL / L; Solvent: purified water; Adjust pH to 7.4 with 0.5 mol / L NaOH, stir until completely dissolved, and filter through a 0.45 μm filter membrane to obtain the basic treatment solution.

[0037] S32, Pre-passivation of the sample pad: Immerse the 30cm×1cm glass fiber membrane completely in the base treatment solution prepared by S31, soak at 25℃ for 25 minutes, and gently turn it twice during the process. Remove the sample pad, drain off the liquid, and dry at 38℃ for 3.5h to obtain the pre-passivated glass fiber membrane.

[0038] S33. Preparation of pre-protection and cross-linking mixture of lectins: S331, Sugar Preprotection Treatment: Prepare the sugar preprotection solution according to the following formula: Jackfruit lectin: 1.2 g / L; galactose: 13.5 g / L; solvent: phosphate buffer (10 mmol / L, pH 7.4); stir until completely dissolved, incubate at 5°C for 45 min to obtain sugar preprotection solution.

[0039] S332, Preparation of cross-linking mixture: Add BSA to the sugar preprotection solution to a final concentration of 3.5 g / L and glutaraldehyde to a final concentration of 0.8 g / L, mix well, and allow to stand at 7°C for cross-linking for 1.5 h to obtain a lectin cross-linking mixture. Filter the mixture through a 0.22 μm filter membrane for sterilization.

[0040] S34. Spraying and fixing of crosslinking mixture: The pre-passivated glass fiber membrane after drying S32 was laid flat on the worktable of the gold spraying machine. The gold spraying machine was used to spray the lectin crosslinking mixture prepared with S332 onto the surface, with a spraying amount of 0.8 mL / strip. After spraying, allow it to stand at 7°C for 1.5 hours for crosslinking, and then dry it at 38°C for 1.2 hours after the reaction is complete.

[0041] S35. Elution, Activation, and Post-Processing: S351. Preparation of elution buffer: Prepare the elution buffer according to the following formula: Galactose: 13.5 g / L; BSA: 5.0 g / L; Tween-20: 0.5 mL / L; Solvent: Phosphate buffer (10 mmol / L, pH 7.4).

[0042] S352, Elution Activation: The dried glass fiber membrane of S34 was rinsed with elution buffer prepared with S351 at a volume of 1.5 mL / strip and allowed to stand at 25°C for 8 min. The rinsed sample pads were then laid flat in a single layer on a stainless steel mesh frame and dried at 38°C for 0.8 h to obtain the sample pads. The dried sample pads were then placed in aluminum foil bags with desiccant in each bag, sealed, and kept for later use.

[0043] S4. Coating of the reaction membrane: S41. Preparation and coating of coating solution for the T-line (influenza A virus): Influenza A virus antibody 2 was dissolved in coating buffer to prepare a T-line coating working solution with a concentration of 0.3 g / L. The coating buffer was 30 mmol / L PBS buffer, pH 7.4. Using a gold spraying apparatus, the T-line coating working solution was sprayed onto the detection line position of the nitrocellulose membrane at a spraying rate of 1.0 μL / cm.

[0044] S42. Preparation and coating of coating solution for the T-line (influenza B virus): Influenza B virus antibody 2 was dissolved in coating buffer to prepare a T-line coating working solution with a concentration of 0.3 g / L. The coating buffer was the same as in step S41. Using a gold spraying apparatus, the T-line coating working solution was sprayed onto the detection line position of the nitrocellulose membrane at a spraying rate of 1.0 μL / cm.

[0045] S43. Preparation and coating of coating solution for T-line (Mycoplasma pneumoniae) detection lines: Dissolve Mycoplasma pneumoniae antibody 2 in coating buffer to prepare a T-line coating working solution with a concentration of 0.4 g / L. The coating buffer is the same as in step S41. Using a gold spraying apparatus, the T-line coating working solution was sprayed onto the detection line position of the nitrocellulose membrane at a spraying rate of 1.0 μL / cm.

[0046] S44, Coating of the quality control line C: Goat anti-mouse IgG polyclonal antibody was dissolved in coating buffer to prepare a C-line coating working solution with a concentration of 1.0 g / L. The coating buffer was the same as in step S41. Using a gold sprayer, spray the C-line coating working solution onto the control line of the same nitrocellulose membrane at a spray rate of 1.0 μL / cm, above the test line (away from the sample application end).

[0047] S45. Drying and storage of the reaction membrane: The reaction membrane coated with T and C lines was dried at 38°C for 3 hours. After drying, the reaction membrane was placed in an aluminum foil bag with desiccant, sealed, and stored for later use.

[0048] S5. Assembly of reagent strips: The gold label pad, sample pad, reaction membrane, absorbent pad, and PVC base plate were cut into strips in a clean environment, with each component having a width of 3.8cm. On the PVC base plate, attach the following components in sequence, overlapping each other, according to the sample flow direction (from the sample application end to the water absorption end): The sample pad, gold label pad, reaction membrane, and absorbent pad are overlapped by 1.5mm and pressed together twice with rollers to ensure that each component is firmly bonded to the base plate without air bubbles. Among them, influenza A virus, influenza B virus, and mycoplasma pneumoniae are assembled into independent reagent strips, and each reagent strip has its own sample pad, gold label pad, reaction membrane and absorbent pad. Insert three independent reagent strips into the three independent channels of the same plastic cartridge, with each channel corresponding to one sample dispensing well; The assembled large board is cut into individual test strips using a CNC cutting machine, each strip being 3.0mm wide; Insert the cut test strips into the corresponding channels of the plastic card, close the cap and press it tightly to obtain a complete test card. Each test card contains three independent test channels, and each channel corresponds to a sample dispensing hole. The test cards were placed in aluminum foil bags, each bag was filled with desiccant, and sealed to obtain a respiratory pathogen antigen detection kit based on triple cards (colloidal gold immunochromatography), which was designated as kit sample 1. Example 2

[0049] The respiratory pathogen antigen detection kit and its preparation method for the triple card in this embodiment are as follows: S1. Preparation of sample processing solution: Prepare the sample treatment solution according to the following formula: Tris(hydroxymethyl)aminomethane: 4.2 g / L; Sodium chloride: 6.5 g / L; 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate: 2.3 g / L; Tween-80: 1.2 mL / L; BSA: 12.0 g / L; Trehalose: 35.0 g / L; PEG20000: 1.2 g / L; Arginine: 5.0 g / L; Histidine: 4.5 g / L; Calcium chloride: 0.25 g / L; 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride: 0.1 g / L; ProClin 300: 0.4 mL / L; Solvent: purified water; pH adjusted to 6.6 with 0.5 mol / L HCl solution; wherein the mass ratio of arginine, histidine and calcium chloride is 20:18:1; The sample was filtered through a 0.22 μm aqueous filter membrane to obtain the sample processing solution, which was then stored at 2°C in the dark.

[0050] S2, Preparation of the gold-labeled pad: S21. Synthesis and preliminary purification of colloidal gold particles: Take 100 mL of purified water, add 4.5 mL of 10 g / L tetrachloroauric acid solution, and heat until it boils vigorously; Add 7.0 mL of 10 g / L trisodium citrate solution at 500 r / min; Keep boiling for 5 minutes until the solution turns a clear bright red color. Stop heating, cool, and filter through a 0.22 μm aqueous phase filter membrane. Use an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa to centrifuge at 2 °C and 10,000 × g for 20 minutes to concentrate to 1 / 5 of the original volume to obtain a concentrated colloidal gold solution.

[0051] S22. Preparation of colloidal gold conjugate diluent: Prepare a colloidal gold conjugate diluent based on the following formulation: BSA: 12.0 g / L; Trehalose: 55.0 g / L; Tween-20: 6.0 mL / L; Solvent: PBS buffer (55 mmol / L, pH 7.6); Stir until completely dissolved, filter through a 0.22 μm filter membrane to obtain a diluted colloidal gold conjugate solution.

[0052] S23, Colloidal gold-labeled antibody: Preparation of S231, Influenza A virus antibody 1 labeled solution: Take the concentrated colloidal gold solution, add 0.1 mol / L potassium carbonate solution to adjust the pH to 7.2, and let it stand for 5 minutes; Add influenza A virus antibody 1 to a final concentration of 0.014 g / L, mix well, and let stand at 28°C for 35 min; Add BSA to a final concentration of 2.5 g / L and let stand for another 12 minutes. Centrifuge at 11000 r / min and 2℃ for 35 min, discard the supernatant, and mix the precipitate with the colloidal gold conjugate dilution solution prepared by S22 at a volume ratio of 1:5.5 to obtain the influenza A virus antibody 1 labeled solution.

[0053] S232. Preparation of Influenza B Virus Antibody 1 Label Solution: The preparation method is the same as step S231, except that influenza B virus antibody 1 is replaced with an equal mass of influenza A virus antibody 1 to prepare an influenza B virus antibody 1 labeled solution.

[0054] Preparation of S233, Mycoplasma pneumoniae antibody 1 labeled solution: The preparation method is the same as step S231, except that the same mass of Mycoplasma pneumoniae antibody 1 is used instead of influenza A virus antibody 1 to prepare Mycoplasma pneumoniae antibody 1 labeled solution.

[0055] S24, Spraying and drying of gold standard pads: Cut the fiberglass film into 30cm×1cm pieces and lay them flat on the worktable of the gold spraying machine. The influenza A virus antibody 1-labeled solution, influenza B virus antibody 1-labeled solution, and mycoplasma pneumoniae antibody 1-labeled solution were sprayed sequentially onto their respective gold pad areas at a spraying volume of 1.4 mL / strip, and then dried at 40℃ for 2.5 h.

[0056] S3. Preparation of the sample pad: S31. Preparation of basic treatment solution: Prepare the basic treatment solution according to the following formula: BSA: 25.0 g / L; Trehalose: 80.0 g / L; PVP-K30: 10.0 g / L; Tween-20: 5.0 mL / L; ProClin300: 0.2 mL / L; Solvent: purified water; Adjust pH to 7.5 with 0.5 mol / L NaOH, stir until completely dissolved, and filter through a 0.45 μm filter membrane to obtain the basic treatment solution.

[0057] S32, Pre-passivation of the sample pad: Immerse the 30cm×1cm glass fiber membrane completely in the base treatment solution prepared by S31, soak at 28℃ for 20 minutes, and gently turn it once during the process. Remove the sample pad, drain off the liquid, and dry at 40℃ for 3 hours to obtain the pre-passivated glass fiber membrane.

[0058] S33. Preparation of pre-protection and cross-linking mixture of lectins: S331, Sugar Preprotection Treatment: Prepare the sugar preprotection solution according to the following formula: Jackfruit lectin: 1.5 g / L; galactose: 18.0 g / L; solvent: phosphate buffer (12 mmol / L, pH 7.5); stir until completely dissolved, incubate at 2°C for 60 min to obtain sugar preprotection solution.

[0059] S332, Preparation of cross-linking mixture: Add BSA to the sugar preprotection solution to a final concentration of 5.0 g / L and glutaraldehyde to a final concentration of 1.0 g / L, mix well, and allow to stand at 4°C for 2 hours to crosslink, to obtain a lectin crosslinking mixture, which is then filtered through a 0.22 μm filter membrane for sterilization.

[0060] S34. Spraying and fixing of crosslinking mixture: The pre-passivated glass fiber membrane after drying S32 was laid flat on the worktable of the gold spraying machine. The gold spraying machine was used to spray the lectin crosslinking mixture prepared with S332 onto the surface, with a spraying amount of 0.9 mL / strip. After spraying, allow it to stand at 4℃ for 2 hours for crosslinking, and then dry it at 40℃ for 1 hour after the reaction is complete.

[0061] S35. Elution, Activation, and Post-Processing: S351. Preparation of elution buffer: Prepare the elution buffer according to the following formula: Galactose: 18.0 g / L; BSA: 5.0 g / L; Tween-20: 0.5 mL / L; Solvent: Phosphate buffer (12 mmol / L, pH 7.5).

[0062] S352, Elution Activation: The dried glass fiber membrane of S34 was rinsed with elution buffer prepared with S351 at a volume of 2.0 mL / strip and allowed to stand at 22℃ for 10 min. The rinsed sample pads were then laid flat in a single layer on a stainless steel mesh frame and dried at 35℃ for 1 h to obtain the sample pads. The dried sample pads were then placed in aluminum foil bags with desiccant in each bag, sealed, and kept for later use.

[0063] S4. Coating of the reaction membrane: S41. Preparation and coating of coating solution for the T-line (influenza A virus): Influenza A virus antibody 2 was dissolved in coating buffer to prepare a T-line coating working solution with a concentration of 0.4 g / L. The coating buffer was 50 mmol / L PBS buffer, pH 7.6. Using a gold spraying apparatus, the T-line coating working solution was sprayed onto the detection line position of the nitrocellulose membrane at a spraying rate of 1.2 μL / cm.

[0064] S42. Preparation and coating of coating solution for the T-line (influenza B virus): Influenza B virus antibody 2 was dissolved in coating buffer to prepare a T-line coating working solution with a concentration of 0.4 g / L. The coating buffer was the same as in step S41. Using a gold spraying apparatus, the T-line coating working solution was sprayed onto the detection line position of the nitrocellulose membrane at a spraying rate of 1.2 μL / cm.

[0065] S43. Preparation and coating of coating solution for T-line (Mycoplasma pneumoniae) detection lines: Dissolve Mycoplasma pneumoniae antibody 2 in coating buffer to prepare a T-line coating working solution with a concentration of 0.5 g / L. The coating buffer is the same as in step S41. Using a gold spraying apparatus, the T-line coating working solution was sprayed onto the detection line position of the nitrocellulose membrane at a spraying rate of 1.2 μL / cm.

[0066] S44, Coating of the quality control line C: Goat anti-mouse IgG polyclonal antibody was dissolved in coating buffer to prepare a C-line coating working solution with a concentration of 1.2 g / L. The coating buffer was the same as in step S41. Using a dip-coating gold sprayer, spray the C-line coating working solution onto the control line of the same nitrocellulose membrane at a spray rate of 1.2 μL / cm, above the test line (away from the sample application end).

[0067] S45. Drying and storage of the reaction membrane: The reaction membrane coated with T and C lines was dried at 40°C for 2.5 hours. After drying, the reaction membrane was placed in an aluminum foil bag with desiccant, sealed, and stored for later use.

[0068] S5. Assembly of reagent strips: The gold label pad, sample pad, reaction membrane, absorbent pad, and PVC base plate were cut into strips in a clean environment, with each component having a width of 4.0 cm. On the PVC base plate, attach the following components in sequence, overlapping each other, according to the sample flow direction (from the sample application end to the water absorption end): The sample pad, gold label pad, reaction membrane, and absorbent pad are overlapped by 2.0 mm and pressed together three times with rollers to ensure that each component is firmly bonded to the base plate without air bubbles. Among them, influenza A virus, influenza B virus, and mycoplasma pneumoniae are assembled into independent reagent strips, and each reagent strip has its own sample pad, gold label pad, reaction membrane and absorbent pad. Insert three independent reagent strips into the three independent channels of the same plastic cartridge, with each channel corresponding to one sample dispensing well; The assembled large board is cut into individual test strips using a CNC cutting machine, each strip being 3.2mm wide; Insert the cut test strips into the corresponding channels of the plastic card, close the cap and press it tightly to obtain a complete test card. Each test card contains three independent test channels, and each channel corresponds to a sample dispensing hole. The test cards were placed in aluminum foil bags, each bag was filled with desiccant, and sealed to obtain a respiratory pathogen antigen detection kit based on triple cards (colloidal gold immunochromatography), which was designated as kit sample 2. Example 3

[0069] The respiratory pathogen antigen detection kit and its preparation method for the triple card in this embodiment are as follows: S1. Preparation of sample processing solution: Prepare the sample treatment solution according to the following formula: Tris(hydroxymethyl)aminomethane: 3.0 g / L; Sodium chloride: 5.0 g / L; 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate: 1.8 g / L; Tween-80: 0.8 mL / L; BSA: 8.0 g / L; Trehalose: 25.0 g / L; PEG20000: 0.8 g / L; Arginine: 3.6 g / L; Histidine: 2.8 g / L; Calcium chloride: 0.16 g / L; 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride: 0.15 g / L; ProClin 300: 0.6 mL / L; Solvent: purified water; pH adjusted to 6.4 with 0.5 mol / L HCl solution; wherein the mass ratio of arginine, histidine and calcium chloride is 22.5:17.5:1; The sample was filtered through a 0.22 μm aqueous filter membrane to obtain the sample processing solution, which was then stored at 8°C in the dark.

[0070] S2, Preparation of the gold-labeled pad: S21. Synthesis and preliminary purification of colloidal gold particles: Take 100 mL of purified water, add 3.5 mL of 10 g / L tetrachloroauric acid solution, and heat until it boils vigorously; Add 6.0 mL of 10 g / L trisodium citrate solution at 300 r / min; Keep boiling for 10 minutes until the solution turns a clear bright red color. Stop heating, cool, and filter through a 0.22 μm aqueous phase filter membrane. Use an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa to centrifuge at 8℃ and 8000×g for 30 minutes to concentrate to 1 / 5 of the original volume, thus obtaining a concentrated colloidal gold solution.

[0071] S22. Preparation of colloidal gold conjugate diluent: Prepare a colloidal gold conjugate diluent based on the following formulation: BSA: 8.0 g / L; Trehalose: 45.0 g / L; Tween-20: 4.0 mL / L; Solvent: PBS buffer (45 mmol / L, pH 7.2); Stir until completely dissolved, filter using a 0.22 μm filter membrane to obtain a diluted colloidal gold conjugate solution.

[0072] S23, Colloidal gold-labeled antibody: Preparation of S231, Influenza A virus antibody 1 labeled solution: Take the concentrated colloidal gold solution, add 0.1 mol / L potassium carbonate solution to adjust the pH to 6.8, and let it stand for 15 min; Add influenza A virus antibody 1 to a final concentration of 0.010 g / L, mix well, and let stand at 22°C for 45 min; Add BSA to a final concentration of 1.5 g / L and let stand for another 18 minutes. Centrifuge at 9000 r / min and 8℃ for 45 min, discard the supernatant, and mix the precipitate with the colloidal gold conjugate dilution solution prepared by S22 at a volume ratio of 1:4.5 to obtain the influenza A virus antibody 1 labeled solution.

[0073] S232. Preparation of Influenza B Virus Antibody 1 Label Solution: The preparation method is the same as step S231, except that influenza B virus antibody 1 is replaced with an equal mass of influenza A virus antibody 1 to prepare an influenza B virus antibody 1 labeled solution.

[0074] Preparation of S233, Mycoplasma pneumoniae antibody 1 labeled solution: The preparation method is the same as step S231, except that the same mass of Mycoplasma pneumoniae antibody 1 is used instead of influenza A virus antibody 1 to prepare Mycoplasma pneumoniae antibody 1 labeled solution.

[0075] S24, Spraying and drying of gold standard pads: Cut the fiberglass film into 30cm×1cm pieces and lay them flat on the worktable of the gold spraying machine. The influenza A virus antibody 1-labeled solution, influenza B virus antibody 1-labeled solution, and mycoplasma pneumoniae antibody 1-labeled solution were sprayed sequentially onto their respective gold pad areas at a spraying volume of 1.0 mL / strip, and then dried at 35°C for 3.5 h.

[0076] S3. Preparation of the sample pad: S31. Preparation of basic treatment solution: Prepare the basic treatment solution according to the following formula: BSA: 10.0 g / L; Trehalose: 30.0 g / L; PVP-K30: 3.0 g / L; Tween-20: 1.0 mL / L; ProClin300: 1.0 mL / L; Solvent: purified water; Adjust pH to 7.3 with 0.5 mol / L NaOH, stir until completely dissolved, and filter through a 0.45 μm filter membrane to obtain the basic treatment solution.

[0077] S32, Pre-passivation of the sample pad: Immerse the 30cm×1cm glass fiber membrane completely in the base treatment solution prepared by S31, soak at 22℃ for 30 minutes, and gently turn it twice during the process. Remove the sample pad, drain off the liquid, and dry at 35°C for 4 hours to obtain the pre-passivated glass fiber membrane.

[0078] S33. Preparation of pre-protection and cross-linking mixture of lectins: S331, Sugar Preprotection Treatment: Prepare the sugar preprotection solution according to the following formula: Jackfruit lectin: 1.0 g / L; galactose: 9.0 g / L; solvent: phosphate buffer (8 mmol / L, pH 7.3); stir until completely dissolved, incubate at 8°C for 30 min to obtain sugar preprotection solution.

[0079] S332, Preparation of cross-linking mixture: Add BSA to the sugar preprotection solution to a final concentration of 2.0 g / L and glutaraldehyde to a final concentration of 0.5 g / L, mix well, and let stand at 10°C for 1 hour to crosslink, to obtain a lectin crosslinking mixture, and filter it with a 0.22 μm filter membrane for sterilization.

[0080] S34. Spraying and fixing of crosslinking mixture: The pre-passivated glass fiber membrane after drying S32 was laid flat on the worktable of the gold spraying machine. The gold spraying machine was used to spray the lectin crosslinking mixture prepared with S332 onto the surface, with a spraying amount of 0.6 mL / strip. After spraying, allow it to stand at 10℃ for 1 hour for crosslinking, and then dry it at 35℃ for 1.5 hours after the reaction is complete.

[0081] S35. Elution, Activation, and Post-Processing: S351. Preparation of elution buffer: Prepare the elution buffer according to the following formula: Galactose: 9.0 g / L; BSA: 5.0 g / L; Tween-20: 0.5 mL / L; Solvent: Phosphate buffer (8 mmol / L, pH 7.3).

[0082] S352, Elution Activation: The dried glass fiber membrane of S34 was rinsed with elution buffer prepared with S351 at a volume of 1.0 mL / strip and allowed to stand at 28°C for 5 min. The rinsed sample pads were then laid flat in a single layer on a stainless steel mesh frame and dried at 40°C for 0.5 h to obtain the sample pads. The dried sample pads were then placed in aluminum foil bags with desiccant in each bag, sealed, and kept for later use.

[0083] S4. Coating of the reaction membrane: S41. Preparation and coating of coating solution for the T-line (influenza A virus): Influenza A virus antibody 2 was dissolved in coating buffer to prepare a T-line coating working solution with a concentration of 0.2 g / L. The coating buffer was 20 mmol / L PBS buffer, pH 7.2. Using a gold spraying apparatus, the T-line coating working solution was sprayed onto the detection line position of the nitrocellulose membrane at a spraying rate of 0.8 μL / cm.

[0084] S42. Preparation and coating of coating solution for the T-line (influenza B virus): Influenza B virus antibody 2 was dissolved in coating buffer to prepare a T-line coating working solution with a concentration of 0.2 g / L. The coating buffer was the same as in step S41. Using a gold spraying apparatus, the T-line coating working solution was sprayed onto the detection line position of the nitrocellulose membrane at a spraying rate of 0.8 μL / cm.

[0085] S43. Preparation and coating of coating solution for T-line (Mycoplasma pneumoniae) detection lines: Dissolve Mycoplasma pneumoniae antibody 2 in coating buffer to prepare a T-line coating working solution with a concentration of 0.3 g / L. The coating buffer is the same as in step S41. Using a gold spraying apparatus, the T-line coating working solution was sprayed onto the detection line position of the nitrocellulose membrane at a spraying rate of 0.8 μL / cm.

[0086] S44, Coating of the quality control line C: Goat anti-mouse IgG polyclonal antibody was dissolved in coating buffer to prepare a C-line coating working solution with a concentration of 0.8 g / L. The coating buffer was the same as in step S41. Using a dip-coating gold sprayer, spray the C-line coating working solution onto the control line of the same nitrocellulose membrane at a spray rate of 0.8 μL / cm, above the test line (away from the sample application end).

[0087] S45. Drying and storage of the reaction membrane: The reaction membrane coated with T and C lines was dried at 35°C for 3.5 hours. After drying, the reaction membrane was placed in an aluminum foil bag with desiccant, sealed, and stored for later use.

[0088] S5. Assembly of reagent strips: The gold label pad, sample pad, reaction membrane, absorbent pad, and PVC base plate were cut into strips in a clean environment, with each component being 3.5cm wide. On the PVC base plate, attach the following components in sequence, overlapping each other, according to the sample flow direction (from the sample application end to the water absorption end): The sample pad, gold label pad, reaction membrane, and absorbent pad are overlapped by 1.0 mm and pressed together twice with rollers to ensure that each component is firmly bonded to the base plate without air bubbles. Among them, influenza A virus, influenza B virus, and mycoplasma pneumoniae are assembled into independent reagent strips, and each reagent strip has its own sample pad, gold label pad, reaction membrane and absorbent pad. Insert three independent reagent strips into the three independent channels of the same plastic cartridge, with each channel corresponding to one sample dispensing well; The assembled large board is cut into individual test strips using a CNC cutting machine, each strip being 2.8mm wide; Insert the cut test strips into the corresponding channels of the plastic card, close the cap and press it tightly to obtain a complete test card. Each test card contains three independent test channels, and each channel corresponds to a sample dispensing hole. The test cards were placed in aluminum foil bags, each bag was filled with desiccant, and sealed to obtain a respiratory pathogen antigen detection kit based on triple cards (colloidal gold immunochromatography), which was designated as kit sample 3. Comparative Example 1

[0089] This comparative example provides a respiratory pathogen antigen detection kit based on a triplet card. The specific implementation method is the same as in Example 1, except that arginine and histidine are not added to the sample processing solution. The specific preparation process is as follows: S1. Preparation of sample processing solution: Prepare sample processing solution reference standard 1 according to the following formula: Tris(hydroxymethyl)aminomethane: 3.6 g / L; Sodium chloride: 5.8 g / L; 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate: 2.0 g / L; Tween-80: 1.0 mL / L; BSA: 10.0 g / L; Trehalose: 30.0 g / L; PEG20000: 1.0 g / L; Calcium chloride: 0.22 g / L; 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride: 0.12 g / L; ProClin 300: 0.5 mL / L; Solvent: purified water; pH adjusted to 6.5 with 0.5 mol / L HCl solution; The sample processing solution reference standard 1 was obtained by filtration using a 0.22 μm aqueous filter membrane and stored at 4 °C protected from light.

[0090] S2. Preparation of the gold-labeled pad: Same as S2 in Example 1.

[0091] S3. Preparation of sample pad: Same as S3 in Example 1.

[0092] S4. Coating of the reaction membrane: Same as S4 in Example 1.

[0093] S5. Assembly of reagent strips: Same as S5 in Example 1, but replace the sample processing solution in Example 1 with the sample processing solution control 1 prepared in step S1 to obtain reagent kit control 1. Comparative Example 2

[0094] This comparative example provides a respiratory pathogen antigen detection kit based on a triplet card. The specific implementation method is the same as in Example 1, except that histidine is not added to the sample processing solution, while the amount of arginine is increased. The specific preparation process is as follows: S1. Preparation of sample processing solution: Prepare sample processing solution reference standard 2 according to the following formula: Tris(hydroxymethyl)aminomethane: 3.6 g / L; Sodium chloride: 5.8 g / L; 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate: 2.0 g / L; Tween-80: 1.0 mL / L; BSA: 10.0 g / L; Trehalose: 30.0 g / L; PEG20000: 1.0 g / L; Arginine: 8.0 g / L; Calcium chloride: 0.22 g / L; 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride: 0.12 g / L; ProClin 300: 0.5 mL / L; Solvent: purified water; pH adjusted to 6.5 with 0.5 mol / L HCl solution; The sample processing solution was filtered through a 0.22 μm aqueous filter membrane to obtain standard 2, which was stored at 4 °C protected from light.

[0095] S2. Preparation of the gold-labeled pad: Same as S2 in Example 1.

[0096] S3. Preparation of sample pad: Same as S3 in Example 1.

[0097] S4. Coating of the reaction membrane: Same as S4 in Example 1.

[0098] S5. Assembly of reagent strips: Same as S5 in Example 1, except that the sample processing solution 2 prepared in step S1 is used instead of the sample processing solution in Example 1 to obtain reagent kit 2. Comparative Example 3

[0099] This comparative example provides a respiratory pathogen antigen detection kit based on a triplet card. The specific implementation method is the same as in Example 1, except that the sample processing solution uses PBS buffer instead of the tris(hydroxymethyl)aminomethane-HCl buffer system. The specific preparation process is as follows: S1. Preparation of sample processing solution: Replace the tris(hydroxymethyl)aminomethane and sodium chloride in Example 1S1 with the following formulation to prepare sample processing solution control 3: Dissolve the following components in 50 mmol / L PBS buffer (pH 6.5): 3-[(3-cholanamidopropyl)dimethylammonium]-1-propanesulfonate: 2.0 g / L; Tween-80: 1.0 mL / L; BSA: 10.0 g / L; Trehalose: 30.0 g / L; PEG20000: 1.0 g / L; Arginine: 4.2 g / L; Histidine: 3.8 g / L; Calcium chloride: 0.22 g / L; 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride: 0.12 g / L; ProClin 300: 0.5 mL / L; do not add additional sodium chloride; adjust pH to 6.5. The sample processing solution was filtered through a 0.22 μm aqueous filter membrane and stored at 3-4 °C in the dark to obtain the reference standard.

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

[0101] S3. Preparation of sample pad: Same as S3 in Example 1.

[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, except that the sample processing solution 3 prepared in step S1 is used instead of the sample processing solution in Example 1 to obtain reagent kit 3. Comparative Example 4

[0104] This comparative example provides a respiratory pathogen antigen detection kit based on a triple card. The specific implementation method is the same as in Example 1, except that: jackfruit lectin is directly cross-linked and fixed with BSA and glutaraldehyde, omitting the sugar preprotection treatment step. The specific preparation process is as follows: S1. Preparation of sample processing solution: Same as S1 in Example 1.

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

[0106] S3. Preparation of the sample pad: S31. Preparation of basic treatment solution: Same as S31 in Example 1.

[0107] S32, Pre-passivation of sample pad: Same as S32 in Example 1.

[0108] S33. Preparation of cross-linking mixture (sugar preprotection treatment omitted): The cross-linking mixture was prepared directly according to the following formula: 1.2 g / L jackfruit lectin; 3.5 g / L BSA; 0.8 g / L glutaraldehyde; solvent: phosphate buffer (10 mmol / L, pH 7.4); mixed thoroughly, and allowed to stand at 7°C for 1.5 h for cross-linking to obtain the lectin cross-linking mixture control, which was then sterilized by filtration through a 0.22 μm filter membrane.

[0109] S34. Spraying and fixing the crosslinking mixture: Same as S34 in Example 1, except that the lectin crosslinking mixture control prepared in step S33 is used instead of the lectin crosslinking mixture in Example 1.

[0110] S35, Elution, Activation and Post-processing: Same as S35 in Example 1.

[0111] S4. Coating of the reaction membrane: Same as S4 in Example 1.

[0112] S5. Assembly of reagent strips: Same as S5 in Example 1, to obtain reagent kit control 4. Comparative Example 5

[0113] This comparative example provides a respiratory pathogen antigen detection kit based on a triplet card. The specific implementation method is the same as in Example 1, except that the elution and activation step is omitted, and the cross-linking and fixation are performed directly by drying without rinsing with elution buffer. The specific preparation process is as follows: S1. Preparation of sample processing solution: Same as S1 in Example 1.

[0114] S2. Preparation of the gold-labeled pad: Same as S2 in Example 1.

[0115] S3. Preparation of the sample pad: S31. Preparation of basic treatment solution: Same as S31 in Example 1.

[0116] S32, Pre-passivation of sample pad: Same as S32 in Example 1.

[0117] S33, Preparation of pre-protection and cross-linking mixture of lectin: Same as S33 in Example 1.

[0118] S34, Spraying and fixing of crosslinked mixture: Same as S34 in Example 1, but the subsequent elution and activation steps are omitted, and drying is performed directly.

[0119] S35, Drying: The sample pad after S34 crosslinking fixation was dried at 38℃ for 0.8h to obtain the sample pad reference.

[0120] S4. Coating of the reaction membrane: Same as S4 in Example 1.

[0121] S5. Assembly of reagent strips: Same as S5 in Example 1, except that the sample pad prepared in Example 1 is replaced with the sample pad control prepared in step S35 to obtain reagent kit control 5. Analysis and Testing

[0122] In this invention, " " indicates negative; "±" indicates borderline / vague; "+" indicates clear weak positive; "++" indicates positive; "+++" indicates strong positive.

[0123] Commercially available reference standard 1: Influenza A virus antigen detection kit (colloidal gold method), a single-test product of our company.

[0124] Commercially available reference standard 2: Influenza B virus antigen detection kit (colloidal gold method), a single-test product of our company.

[0125] Commercially available reference standard 3: Mycoplasma pneumoniae antigen detection kit (colloidal gold method), a single-test product of our company.

[0126] I. Detection Sensitivity Test To evaluate the detection sensitivity of the kit of this invention, particularly the difference in detection between the Victoria and Yamagata subtypes of influenza B virus, the limits of detection for influenza A virus (H1N1 and H3N2), influenza B virus (Victoria and Yamagata lineages), and Mycoplasma pneumoniae were determined. The limit of detection was defined as the lowest antigen concentration with a 95% positive detection rate.

[0127] 1. Limit of detection for H1N1 influenza virus antigen: Samples positive for H1N1 influenza A virus (known titer) confirmed by nucleic acid testing were serially diluted using a negative oropharyngeal swab matrix. Samples 1-3 from the kit, commercially available control 1, and kit controls 1-5 were tested separately. Results are shown in Table 1.

[0128] Table 1: Results of Detection Limit of Influenza A Virus H1N1 Antigen

[0129] As shown in Table 1, the detection limits for influenza A virus H1N1 in samples 1-3 of the kits in this embodiment are all 1.2 × 10⁻⁶. 1 The TCID50 / mL level was completely consistent with that of commercially available reference standard 1. The detection results of reference standards 1-3 in the kit were comparable to those in the examples, indicating that changes in the combination of arginine and histidine and the buffer system did not affect the detection of H1N1.

[0130] The reagent kit controls 4 and 5 were negative at a dilution of 1:625000, and the limit of detection decreased to 2.4 × 10⁻⁶. 2 The TCID50 / mL concentration represents a sensitivity loss of approximately 20 times compared to the kit sample, indicating that incomplete lectin fixation in the sample pad leads to decreased chromatography efficiency, which in turn affects the sensitivity of influenza A detection.

[0131] 2. Detection limit for influenza A virus H3N2 antigen: Samples positive for influenza A virus H3N2 (known titer) confirmed by nucleic acid testing were serially diluted using a negative oropharyngeal swab matrix. Samples 1-3 from the kit, commercially available control 1, and kit controls 1-5 were tested separately. Results are shown in Table 2.

[0132] Table 2: Results of Detection Limit of Influenza A Virus H3N2 Antigen

[0133] As shown in Table 2, the detection limits of the reagent kit samples 1-3 for influenza A virus H3N2 in this embodiment are consistent with those of the commercially available control 1, which is 1.0 × 10⁻⁶. 1The TCID concentration was 50 / mL, and the colorimetric intensity was no different from that of commercially available reference standard 1. The detection results of reference standards 1-3 in the kit were comparable to those in the examples, indicating that changes in the combination of arginine and histidine, as well as the buffer system, did not affect the detection of H3N2. Reference standards 4 and 5 remained positive ("+") at 250,000-fold dilution, but turned negative at 500,000-fold dilution, with a detection limit of approximately 2.0 × 10⁻⁶. 1 The TCID50 / mL concentration represents approximately two times the sensitivity loss compared to the previous example, further confirming the crucial role of the integrity of the sample pad lectin fixation process in ensuring chromatographic efficiency and detection sensitivity.

[0134] 3. Detection limit of influenza B virus Victoria lineage antigen Positive samples of influenza B virus (Victoria lineage) confirmed by nucleic acid testing (known titer) were serially diluted using a negative oropharyngeal swab matrix. Samples 1-3 from the kit, commercially available control 2, and kit controls 1-5 were used for testing. Results are shown in Table 3.

[0135] Table 3: Results of Detection Limit of Influenza B Virus Victoria Lineage Antigen

[0136] As shown in Table 3, the detection limit for commercially available reference standard 2 against the Victoria lineage of influenza B virus was 5.2 × 10⁻⁶. 2 TCID50 / mL. The detection limits for samples 1 and 2 in the kit of this invention are 5.2 × 10⁻⁶. 1 The TCID50 / mL concentration is an order of magnitude lower than commercially available kits, while increasing sensitivity tenfold; the detection limit for sample 3 in the kit is approximately 6.5 × 10⁻⁶. 1 The TCID50 / mL is also significantly superior to commercially available solutions. This is because the sample processing solution of this invention achieves conformational compensation through the synergistic effect of arginine, histidine, and calcium ions. Specifically, calcium ions can specifically coordinate with glutamic acid and aspartic acid residues on the surface of HA proteins, enhancing protein structural rigidity and compensating for local conformational looseness caused by amino acid deletions. The guanidino group of arginine interacts with the HA protein deletion region through hydrogen bonds, providing directional conformational compensation. The imidazole ring of histidine synergistically stabilizes the protein structure while maintaining the pH microenvironment. These three components work together from three dimensions—enhanced structural rigidity, directional conformational compensation, and microenvironmental stability—significantly improving the immunoreactivity compensation for the Victoria subtype, thus increasing the detection sensitivity for the Victoria subtype.

[0137] The detection limits of reagent kits 1 and 3 were essentially equivalent to those of commercially available reagent kit 2, indicating that the conformational compensation effect disappeared after the absence of arginine and histidine or the change of the buffer system, and the detection sensitivity of the Victoria subtype returned to the level of conventional products. Reagent kit 2 also showed similar detection results to reagent kit 1, indicating that simply increasing the concentration of arginine cannot replace the synergistic function of histidine.

[0138] Reference standard 4 in the kit was negative at all dilutions, while reference standard 5 was only very weakly positive at a 2500-fold dilution, indicating that the integrity of the lectin fixation process in the sample pad is crucial for the detection of the Victoria subtype. The glycan binding sites of jackfruit lectin contain primary amino groups essential for sugar binding. When covalently fixed with glutaraldehyde, the aldehyde group of glutaraldehyde can irreversibly react with the aforementioned primary amino groups, destroying the sugar-binding activity of the lectin. Galactose protects these essential groups by occupying the sugar-binding sites, thereby preventing cross-linking damage; after cross-linking, the protective galactose must be removed by elution to restore the glycan binding activity of the lectin. This demonstrates the irreplaceable nature of the three-step process of sugar pre-protection, glutaraldehyde cross-linking, and elution activation in the sample pad preparation of this invention.

[0139] 4. Detection limit of influenza B virus Yamagata lineage antigen Positive samples of influenza B virus Yamagata lineage (known titer) confirmed by nucleic acid testing were serially diluted using a negative oropharyngeal swab matrix. Samples 1–3 from the kit, commercially available control 2, and kit controls 1–5 were tested separately. Results are shown in Table 4.

[0140] Table 4: Results of Detection Limit of Influenza B Virus Yamagata Lineage Antigen

[0141] As shown in Table 4, the detection limit of commercially available reference standard 2 for influenza B virus Yamagata strain is 1.0 × 10⁻⁶. 1 TCID50 / mL. The detection limits for samples 1 and 2 in the kit of this embodiment are 5.0 × 10⁻⁶. 0 The TCID50 / mL concentration is approximately twice as sensitive as commercially available kits; the detection limit for sample 3 of the kit is comparable to that of commercially available kits.

[0142] The HA protein structure of the Yamagata subtype is intact, lacking the amino acid deletions characteristic of the Victoria subtype. Therefore, it has lower requirements for conformational compensation of arginine, histidine, and calcium ions. The limits of detection (LODs) of kit controls 1–3 are essentially equivalent to those of commercially available control 2, indicating that changes in the arginine and histidine combination and buffer system have minimal impact on the sensitivity of Yamagata subtype detection. Kit controls 4 and 5 become negative at 5000-fold dilution, with a significant decrease in the LOD, demonstrating that the integrity of the sample pad lectin fixation process is equally crucial for Yamagata subtype detection.

[0143] 5. Detection limit of Mycoplasma pneumoniae antigen Samples of Mycoplasma pneumoniae positive (known concentration) confirmed by nucleic acid testing were serially diluted using a negative oropharyngeal swab matrix. Samples 1-3 from the kit, commercially available control 3, and kit controls 1-5 were tested separately. Results are shown in Table 5.

[0144] Table 5: Results of the detection limit determination of Mycoplasma pneumoniae antigen

[0145] As shown in Table 5, the detection limit for commercially available reference standard 3 against Mycoplasma pneumoniae was 1.0 × 10⁻⁶. 4 CFU / mL. Samples 1 and 2 of the kit in this embodiment of the invention were diluted 75,000 times (5.0 × 10⁻⁶). 3 The kit remained positive at CFU / mL, with a detection limit half that of commercially available kits and a sensitivity approximately two times higher. Sample 3 of the kit was borderline at 50,000-fold dilution, with a detection limit of approximately 7.5 × 10⁻⁶. 3 The CFU / mL concentration is also superior to commercially available products.

[0146] The limits of detection (LODs) of reagent kit controls 1 and 2 were essentially equivalent to those of commercially available control 3, indicating that the combination of arginine and histidine does not affect the detection of Mycoplasma pneumoniae. Reagent kit control 3 became negative at a dilution of 37,500 times, with a LOD of approximately 1.5 × 10⁻⁶. 4 The sensitivity decreased at CFU / mL, indicating that changes in the buffer system affect the stability of Mycoplasma pneumoniae antigen release. Kit controls 4 and 5 became negative at 12500-fold dilution, with a significant decrease in the detection limit. The reason is consistent with Tables 3 and 4: lectin inactivation prevents the capture of mucin, hindering chromatography and thus affecting Mycoplasma pneumoniae detection. Therefore, the detection sensitivity of Mycoplasma pneumoniae antigen mainly depends on the effective lysis of mycoplasma cell membranes by surfactants in the sample processing solution and the capture of mucin by lectin in the sample pad, ensuring chromatographic efficiency.

[0147] II. Verification of the specificity of the joint detection and the effect of eliminating mucin interference To evaluate the specificity of the triple detection kit of the present invention in the simultaneous detection of multiple targets, and the elimination effect of immobilized jackfruit lectin on mucin interference, cross-reactivity studies and mucin spiked interference experiments were conducted.

[0148] 1. Cross-reaction verification Ten samples were collected from each of the following groups: single positive for influenza A virus (H1N1 and H3N2), influenza B virus (Victoria lineage and Yamagata lineage), and Mycoplasma pneumoniae, as well as clinical oropharyngeal swab samples that were confirmed by nucleic acid testing to be positive for influenza A (H1N1 + Yamagata B), influenza A (H3N2 + Victoria B), influenza A (H1N1 + Mycoplasma pneumoniae), influenza A (H3N2 + Mycoplasma pneumoniae), influenza B (Victoria + Mycoplasma pneumoniae), and influenza B (Yamagata + Mycoplasma pneumoniae).

[0149] Samples 1-3 and controls 1-5 from the kit were used for testing. Readings were performed 15 minutes after sample addition, and the color development results of the three detection lines were recorded. The results showed that in all example samples and comparative samples, only the corresponding detection line showed color in each single-positive sample, while other detection lines did not show color. In double-positive samples, all corresponding detection lines showed color, with no cross-reaction. This indicates that there is no cross-interference between the three detection channels in the triple detection kit of this invention.

[0150] 2. Mucin spiked interference experiment Large glycoprotein molecules such as mucin, which are abundant in oropharyngeal swab samples, easily adhere to nitrocellulose membranes during chromatography, clogging membrane pores and increasing the T-line background. To verify the specific capture effect of immobilized jackfruit lectin on the sample pad on mucin, a mucin spiked interference experiment was designed.

[0151] Oropharyngeal swab samples that were weakly positive for influenza A (H1N1) virus, influenza B (Victoria and Yamagata lineages) virus, and Mycoplasma pneumoniae, as confirmed by nucleic acid testing, were collected. Different concentrations of porcine gastric mucin (0 mg / mL, 5 mg / mL, 10 mg / mL, and 20 mg / mL) were added as interfering agents. Samples 1–3 and controls 1–5 from the kit were used for detection. Readings were performed 15 minutes after sample addition, and the T-line intensity was recorded. Results are shown in Tables 6 and 7.

[0152] Table 6: Effects of mucin spiking on the detection signals of influenza A (H1N1) virus and mycoplasma pneumoniae.

[0153] Table 7: Effect of mucin spiking on the detection signal of influenza B virus

[0154] As shown in Tables 6 and 7, under different concentrations of mucin spiked in the kits of this invention, the T-line intensities of influenza A H1N1 virus, influenza B virus Victoria lineage, influenza B virus Yamagata lineage, and Mycoplasma pneumoniae gradually decreased with increasing mucin concentration. Even at a high concentration of 20 mg / mL, they were still detectable, demonstrating good anti-interference ability. The signal intensity of the Yamagata lineage was slightly higher than that of the Victoria lineage at all spiked concentrations, consistent with its inherently high detection sensitivity.

[0155] At a 10 mg / mL mucin spike, reference standard 1 in the kit showed that the signals of influenza A virus and mycoplasma pneumoniae decreased to "±", the Victoria lineage signal decreased to "-", and the Yamagata lineage signal decreased to "±". At a 20 mg / mL spike, all targets except the Yamagata lineage remained "±", and all others became negative. Because reference standard 1's sample pad lectin fixation process was complete and able to capture mucin, the lack of arginine and histidine in its sample processing solution led to the loss of conformational compensation for the Victoria subtype. This subtype itself has low sensitivity, and its signal disappeared first under high concentrations of mucin interference. Other targets, due to their relatively higher sensitivity, showed a slightly slower decrease in signal, but were still affected by changes in the ionic environment.

[0156] In kit control 2, at a 10 mg / mL mucin spike, the signals for influenza A virus, Mycoplasma pneumoniae, and the Yamagata lineage were all "+", while the signal for the Victoria lineage decreased to "±". At a 20 mg / mL spike, the signals for all three targets decreased to "±", and the Victoria lineage became negative. Its anti-interference ability was slightly better than control 1, but weaker than in the examples, indicating that simply increasing the arginine concentration cannot completely replace the synergistic function of histidine.

[0157] At a 10 mg / mL mucin spike, reagent kit control 3 showed positive signals for influenza A virus, mycoplasma pneumoniae, and the Yamagata lineage, while the Victoria lineage signal weakened to ±. At a 20 mg / mL spike, all three target signals weakened to ±, and the Victoria lineage became negative. This performance is similar to that of reagent kit control 2, indicating that changes in the buffer system alter the ionic strength and pH stability of the sample, thus affecting the chromatographic efficiency of all targets, with a particularly pronounced impact on the Victoria subtype.

[0158] In kit control 4, with a 5 mg / mL mucin spike, the signals for influenza A virus and mycoplasma pneumoniae decreased to "±", the Victoria lineage signal turned negative, and the Yamagata lineage signal turned negative with a 10 mg / mL spike. This showed the worst anti-interference ability among all controls, indicating that the lectin lost its activity due to cross-linking damage and was unable to capture mucin. The inactivated protein itself further hindered chromatography, leading to severe failure in the detection of each target.

[0159] At a 5 mg / mL mucin spike, the Victoria lineage signal of reagent kit control 5 was reduced to "±"; at a 10 mg / mL spike, the Victoria and Mycoplasma pneumoniae signals turned negative, while the influenza A virus and Yamagata lineage signals were reduced to "±". Its anti-interference ability was slightly better than reagent kit control 4, but still far worse than the examples, indicating that the failure to wash out galactose resulted in the inability to restore lectin activity and the failure of mucin capture function.

[0160] The above results demonstrate that the complete immobilization process of jackfruit lectin on the sample pad of this invention is crucial for achieving efficient mucin capture and ensuring stable detection signals for various targets (especially the Victoria subtype of influenza B virus). Simultaneously, the complete configuration of arginine, histidine, and the buffer system in the sample processing solution is equally indispensable for maintaining the detection sensitivity of the Victoria subtype under high mucin interference.

[0161] III. High-dose hook effect test To evaluate the reliability of the kit in detecting high-concentration samples, a hook effect study was conducted. Serial dilutions of influenza A virus H1N1, influenza A virus H3N2, influenza B virus Victoria, influenza B virus Yamagata, and Mycoplasma pneumoniae cultures were performed using a negative matrix. Kit sample 1 and kit controls 1–5 were used for detection, with each dilution repeated three times. Changes in the T-line intensity were observed. Results, with influenza A virus H1N1 as a representative example, are listed in Table 8; the results for other pathogens showed consistent trends.

[0162] Table 8: Results of High-Dose Hook Effect Test

[0163] As shown in Table 8, sample 1 of the reagent kit of the present invention was effective against influenza A virus at a titer of 1.5 × 10⁻⁶. 5 The strongest color development was achieved at TCID50 / mL, with a stock solution of 3.0 × 10⁻⁶. 6 The signal decreased slightly at TCID50 / mL, indicating that the hook effect threshold was higher than 1.5 × 10⁻⁶. 5 TCID50 / mL, not exceeding 1.5 × 10⁻⁶ 5The detection signal within the TCID50 / mL range either increases or remains stable with increasing concentration, and no signal decrease is observed, demonstrating that the safe detection range of the kit of this invention can meet the detection needs of routine clinical samples.

[0164] The hook effect thresholds of reagent kit controls 1 and 2 were consistent with those of reagent kit sample 1, indicating that the combination of arginine and histidine does not affect the antigen-antibody ratio in the reaction system. Reagent kit control 3 was at 1.5 × 10⁻⁶. 5 At TCID50 / mL, the signal was only "++", indicating a significant decrease in the threshold, suggesting that changes in the buffer system affect the immune response rate, leading to a relative decrease in the effective antibody concentration. The kit control 4 and the kit control at 1.5 × 10⁻⁶... 3 The signal weakened to "++" at TCID50 / mL, and at 1.5×10 2 At TCID50 / mL, the signal was only "+", with the lowest hook effect threshold. The overall signal intensity was lower than that of sample 1 in the kit, indicating that lectin inactivation led to chromatography obstruction and disordered release sequence of gold-labeled antibody, exacerbating the imbalance of antigen-antibody ratio.

[0165] Furthermore, the test results for influenza A virus H3N2, influenza B virus Victoria, influenza B virus Yamagata, and Mycoplasma pneumoniae showed trends consistent with those for influenza A virus H1N1, with each target showing a titer ≤1.5×10⁻⁶. 5 TCID50 / mL (Mycoplasma pneumoniae concentration ≤3.0×10⁻⁶) 8 No signal decrease was observed as the concentration increased (CFU / mL) at any of the concentration levels.

[0166] The above results demonstrate that the complete sample pad process of the present invention is an important guarantee for ensuring the effective concentration of antibodies and solid-phase capture efficiency in the reaction system, and for improving the hook effect threshold.

[0167] IV. Accelerated Stability Test Samples 1-3 of the reagent kit and controls 1-5 of the reagent kit were placed in a 37°C incubator and removed on days 7, 14, 21, and 28, respectively. Weakly positive samples (influenza A virus H1N1, H3N2, influenza B virus Victoria and Yamagata lineages, Mycoplasma pneumoniae, Ct value 30-32) were used for detection, and the color intensity of the T line before and after acceleration was compared. Influenza B virus Victoria lineage is the key detection target of this invention, and its results are representative, as shown in Table 9.

[0168] Table 9: Accelerated Stability Test Results

[0169] As shown in Table 9, after 28 days of accelerated testing at 37°C, samples 1 and 2 of the reagent kit of the present invention still showed interpretable (±) detection lines for the Victoria lineage of influenza B virus. Sample 3 attenuated to ± at 21 days and turned negative at 28 days, indicating that the reagent kit of the present invention has good thermal stability. Controls 1-3 of the reagent kit were essentially ineffective at 21 days, while controls 4 and 5 showed signal attenuation or disappearance at 7 days, indicating significantly inferior stability compared to the samples in the reagent kit of the present invention. It is evident that the synergistic protective effect of arginine and histidine in the sample processing solution and the complete immobilization process of lectins on the sample pad not only improved the detection sensitivity but also enhanced the long-term stability of the reagent kit under accelerated conditions. Among them, controls 4 and 5 showed the worst stability, further demonstrating the irreplaceable role of sugar preprotection and elution activation steps in maintaining lectin activity and ensuring the functional stability of the sample pad. The accelerated stability results for influenza A virus H1N1, H3N2, influenza B virus Yamagata lineage, and Mycoplasma pneumoniae showed the same trend as those for influenza B virus Victoria lineage; data omitted.

[0170] 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 respiratory pathogen antigen detection kit based on a triplet card, characterized in that, include: Three independent detection channels are fixed on the PVC base plate. Each detection channel includes a sample pad, a gold label pad, a reaction membrane coated with detection lines and quality control lines, and an absorbent pad. The sample dispensing holes of the three detection channels are independent of each other. The corresponding gold-labeled pads of the three detection channels are respectively loaded with colloidal gold-labeled influenza A virus antibody 1, colloidal gold-labeled influenza B virus antibody 1, or colloidal gold-labeled mycoplasma pneumoniae antibody 1; The sample pad is a glass fiber membrane that has been pre-passivated by impregnation with a basic treatment solution, and then has cross-linked bromelain fixed on its surface. The bromelain is pre-protected with galactose and then cross-linked with bovine serum albumin to form a covalent cross-linked network fixed on the sample pad surface. After elution and activation treatment, its glycan binding activity is restored. The basic treatment solution contains bovine serum albumin 10.0 g / L–25.0 g / L, trehalose 30.0 g / L–80.0 g / L, polyvinylpyrrolidone 3.0 g / L–10.0 g / L, Tween 20 1.0 mL / L–5.0 mL / L, preservative 0.2 mL / L–1.0 mL / L, purified water as solvent, and a pH of 7.3–7.

5. The kit also includes a sample processing solution having the following components: Tris(hydroxymethyl)aminomethane 3.0 g / L–4.2 g / L, sodium chloride 5.0 g / L–6.5 g / L, 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate 1.8 g / L–2.3 g / L, Tween 80 0.8 mL / L~1.2 mL / L, bovine serum albumin 8.0 g / L~12.0 g / L, trehalose 25.0 g / L~35.0 g / L, polyethylene glycol 0.8 g / L~1.2 g / L, arginine 3.6 g / L~5.0 g / L, histidine 2.8 g / L~4.5 g / L, calcium chloride 0.16 g / L~0.25 g / L, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride 0.1 g / L~0.15 g / L, preservative 0.4 mL / L~0.6 mL / L, pH 6.4~6.

6.

2. The reagent kit according to claim 1, characterized in that, The mass ratio of arginine, histidine, and calcium chloride in the sample processing solution was (20–22.5):(17.5–19):

1.

3. The reagent kit according to claim 1, characterized in that, The three detection channels are respectively equipped with an influenza A virus detection line, an influenza B virus detection line, a mycoplasma pneumoniae detection line, and a quality control line on their corresponding reaction membranes. The influenza A virus detection line is coated with influenza A virus antibody 2, the influenza B virus detection line is coated with influenza B virus antibody 2, and the mycoplasma pneumoniae detection line is coated with mycoplasma pneumoniae antibody 2. The coating concentrations of influenza A virus antibody 2 and influenza B virus antibody 2 are both 0.2 g / L to 0.4 g / L, and the coating concentration of mycoplasma pneumoniae antibody 2 is 0.3 g / L to 0.5 g / L. The influenza A virus antibody 2, influenza B virus antibody 2, and mycoplasma pneumoniae antibody 2 are paired antibodies of influenza A virus antibody 1, influenza B virus antibody 1, and mycoplasma pneumoniae antibody 1, respectively.

4. A method for preparing a respiratory pathogen antigen detection kit based on a triplet card, used to prepare the kit as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. Preparation of sample processing solution: Prepare a sample processing solution containing arginine and histidine, and adjust the pH to 6.4-6.6; S2. Preparation of gold-labeled pads: Colloidal gold was synthesized and concentrated. Influenza A virus antibody 1, influenza B virus antibody 1 and mycoplasma pneumoniae antibody 1 were labeled on colloidal gold particles respectively. After blocking, centrifugation, and reconstitution with colloidal gold conjugate diluent, the respective labeled solutions were obtained. They were then sprayed onto glass fiber membranes and dried to obtain gold-labeled pads with three independent detection channels. S3. Preparation of sample pad: Impregnate the glass fiber membrane with the basic treatment solution for pre-passivation; After pre-protecting the bromelain with galactose, bovine serum albumin and glutaraldehyde were added to prepare a cross-linking mixture, which was then sprayed onto a pre-passivated glass fiber membrane. After standing for cross-linking, the membrane was rinsed with elution buffer to activate it. After drying, a sample pad with three independent detection channels was obtained. S4. Coating of the reaction membrane: Prepare coating working solutions for influenza A virus antibody 2, influenza B virus antibody 2, mycoplasma pneumoniae antibody 2 and goat anti-mouse IgG polyclonal antibody respectively, and spray them onto the nitrocellulose membrane in sequence to form detection lines and control lines. After drying, a reaction membrane with three independent detection channels is obtained. S5. Assembly of reagent strips: After assembling the sample pads, gold label pads, reaction membranes and absorbent pads of the three independent detection channels, fix them on the same PVC base plate, cut, shell and package them to obtain the reagent kit. The influenza A virus antibody 2, influenza B virus antibody 2, and mycoplasma pneumoniae antibody 2 are respectively the paired antibodies of influenza A virus antibody 1, influenza B virus antibody 1, and mycoplasma pneumoniae antibody 1.

5. The preparation method according to claim 4, characterized in that, In step S2, the colloidal gold conjugate diluent contains bovine serum albumin 8.0 g / L to 12.0 g / L, trehalose 45.0 g / L to 55.0 g / L, and Tween 20 4.0 mL / L to 6.0 mL / L, and the solvent is PBS buffer with a pH of 7.2 to 7.

6.

6. The preparation method according to claim 4, characterized in that, In step S2, the specific method for labeling antibodies with colloidal gold is as follows: Adjust the pH of the concentrated colloidal gold solution to 6.8–7.2 with potassium carbonate solution and let it stand for 5–15 minutes. Add the influenza A virus antibody 1, influenza B virus antibody 1 and mycoplasma pneumoniae antibody 1 to each of them, so that their final concentrations are 0.010 g / L to 0.014 g / L, and let stand for 35 min to 45 min; Add bovine serum albumin to a final concentration of 1.5 g / L to 2.5 g / L, and let stand for 12 to 18 minutes. Centrifuge at 9000 to 11000 r / min and 2 to 8℃ for 35 to 45 minutes, discard the supernatant, and mix the precipitate with the colloidal gold conjugate diluent at a volume ratio of 1:4.5 to 1:5.

5.

7. The preparation method according to claim 4, characterized in that, In step S3, during the cross-linking fixation process, bovine serum albumin is added to a final concentration of 2.0 g / L to 5.0 g / L, and glutaraldehyde is added to a final concentration of 0.5 g / L to 1.0 g / L. The mixture is then allowed to stand at 4°C to 10°C for 1 to 2 hours for cross-linking.

8. The preparation method according to claim 4, characterized in that, In step S3, the sugar preprotection treatment conditions are as follows: 1.0 g / L to 1.5 g / L of jackfruit lectin and 9.0 g / L to 18.0 g / L of galactose are incubated in phosphate buffer at 2°C to 8°C for 30 min to 60 min.

9. The preparation method according to claim 4, characterized in that, In step S3, the elution buffer contains the following components: galactose 9.0 g / L to 18.0 g / L, bovine serum albumin 5.0 g / L, and Tween 20 0.5 mL / L, with phosphate buffer as the solvent and pH 7.3 to 7.

5. The elution activation conditions are: rinsing with the elution buffer, standing for 5 min to 10 min, and then drying.

10. The preparation method according to claim 4, characterized in that, In step S4, the coating buffer is PBS buffer with a concentration of 20 mmol / L to 50 mmol / L and a pH of 7.2 to 7.6; the coating concentrations of influenza A virus antibody 2 and influenza B virus antibody 2 are both 0.2 g / L to 0.4 g / L, the coating concentration of mycoplasma pneumoniae antibody 2 is 0.3 g / L to 0.5 g / L, and the coating concentration of goat anti-mouse IgG polyclonal antibody is 0.8 g / L to 1.2 g / L.

Images