Kit for diagnosing inhalation pneumonia and preparation method thereof

Abstract

The invention discloses a kit for diagnosing inhalation pneumonia and a preparation method thereof, and belongs to the technical field of inhalation pneumonia detection. The kit for diagnosing the inhalation pneumonia comprises a PVC (polyvinyl chloride) bottom plate, a sample pad, a gold mark pad, a nanofiber membrane, a liquid absorption pad and a plastic card shell, a detection line 1, a detection line 2 and a quality control line are fixed on the nanofiber membrane; the detection line 1 and the detection line 2 consist of a colloidal gold labeled pepsinogen I antibody secondary antibody, a colloidal gold labeled pepsinogen II antibody secondary antibody; the preparation method comprises the following steps: under a nitrogen atmosphere, firstly, heating a chloroauric acid aqueous solution to be boiled, quickly adding a trisodium citrate aqueous solution, and boiling; cooling to room temperature, slowly adding an SH-PEG-COOH solution, reacting, standing overnight, centrifuging, washing, and adding a confining liquid to obtain a colloidal gold solution; the kit prepared by the invention has better accuracy and specificity and is convenient to operate.

Description

Technical Field

[0001] This invention relates to the field of aspiration pneumonia detection technology, specifically to a reagent kit for diagnosing aspiration pneumonia and its preparation method. Background Technology

[0002] Aspiration pneumonia refers to the infectious consequence of inhaling a relatively large amount of substance. However, healthy individuals often inhale small amounts of pharyngeal secretions while sleeping, and under normal circumstances, the body's mechanical and immune responses can prevent pneumonia from occurring. Pneumonia occurs when the host's defense mechanisms are unable to limit bacterial proliferation.

[0003] Aspiration pneumonia can be divided into bacterial aspiration pneumonia and chemical aspiration pneumonia. Bacterial aspiration pneumonia refers to an infectious disease caused by inhaling pathogenic bacteria that colonize the oropharynx. Under normal circumstances, the oropharynx contains a variety of bacteria. When the body's defense function is impaired, such as in elderly people with reduced swallowing function or long-term bedridden patients with weakened cough reflex, bacteria in the oropharynx are easily inhaled into the lower respiratory tract, multiply in large numbers in the lungs, and lead to lung infection.

[0004] Chemical aspiration pneumonia primarily results from the inhalation of irritants such as stomach acid and stomach contents, triggering a chemical inflammatory response in the lungs. When stomach contents reflux into the respiratory tract, the stomach acid and other components directly irritate and damage the respiratory mucosa and lung tissue, causing an inflammatory reaction. For example, during some surgical anesthesia procedures, improper positioning or gastrointestinal dysfunction can lead to the aspiration of stomach contents. The stomach acid irritates the airway, causing congestion and edema of the airway mucosa, and damaging alveolar surfactant, thus affecting gas exchange and leading to chemical aspiration pneumonia. The onset of chemical aspiration pneumonia is often rapid, with symptoms appearing shortly after inhaling stomach contents. Patients mainly present with an irritating cough, which is quite severe due to the irritation of the airway by stomach acid, and may be accompanied by shortness of breath and difficulty breathing; in severe cases, cyanosis may occur. Early systemic infection symptoms are relatively mild; for example, fever may be low or appear only briefly. As the inflammation progresses, lung tissue may suffer more severe damage, leading to a series of complications such as acute respiratory distress syndrome or pulmonary fibrosis. These complications not only increase the difficulty of treatment but may also lead to a poor prognosis for patients. People may unconsciously ignore these symptoms, leading to a worsening of the condition later on.

[0005] Currently, the proportion of pneumonia caused by the reflux of gastric contents into the respiratory tract is gradually increasing. However, the corresponding detection methods for aspiration pneumonia have significant limitations. 24-hour esophageal pH monitoring can only detect acid reflux events and is not sensitive enough to substances such as bile and pepsin in non-acidic refluxate. Although endoscopy can detect esophagitis, it cannot confirm whether the refluxate has reached the respiratory tract. Furthermore, the diagnostic treatment using proton pump inhibitors has an efficacy rate of less than 50% in patients with non-acidic reflux. This diagnostic blind spot leads to a large number of patients being misdiagnosed as having "idiopathic cough" or "allergic asthma," delaying targeted treatment.

[0006] To avoid confusion with common pneumonia or other respiratory diseases, the diagnosis of aspiration pneumonia needs to be accurate and rapid in order to promptly identify and treat potential sources of infection or irritants, thereby effectively controlling the progression of the disease. Summary of the Invention

[0007] To address the technical deficiencies in this area, the present invention aims to provide a reagent kit for diagnosing aspiration pneumonia and its preparation method, thereby solving the technical problem in the prior art that the diagnosis of pneumonia caused by gastric reflux requires accuracy and speed.

[0008] To achieve the above objectives, the present invention provides a kit for diagnosing aspiration pneumonia, comprising a PVC base plate, a sample pad, a gold-labeled pad, a nanofiber membrane, a liquid absorption pad, and a plastic cartridge, characterized in that: colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody are immobilized on the gold-labeled pad; the nanofiber membrane is immobilized with detection line 1 and detection line 2, which are composed of secondary antibodies recognizing colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody, as well as a quality control line;

[0009] The preparation of the nanofiber membrane includes the following steps:

[0010] PCL and PEG were added to a mixed solution and stirred thoroughly to form a spinning solution. Electrospinning was carried out at room temperature and relative humidity of 40-60% to prepare nanofiber membranes.

[0011] The colloidal gold labeling material is derived from a colloidal gold solution;

[0012] The preparation of the colloidal gold solution includes the following steps:

[0013] Under a nitrogen atmosphere, chloroauric acid aqueous solution was first heated to boiling, and trisodium citrate aqueous solution was quickly added and boiled. After cooling to room temperature, SH-PEG-COOH solution was slowly added and reacted at room temperature for 2.5~4.5h. After standing overnight at 4℃, the solution was centrifuged, washed, and blocked with a blocking solution. The solution was stored at 4℃ in the dark to obtain colloidal gold solution.

[0014] Optionally, the preparation method of the SH-PEG-COOH includes the following steps:

[0015] S1: Add PEG and succinic anhydride to dichloromethane, add catalyst, stir, heat to react, and after the reaction is completed, purify to obtain terminally carboxylated PEG;

[0016] S2: Dissolve terminally carboxylated PEG in DMF, add EDC and NHS sequentially under nitrogen protection, stir; add thiol reagent, heat to react, and after the reaction is complete, purify to obtain SH-PEG-COOH;

[0017] In S1, the mass ratio of PEG, succinic anhydride, dichloromethane and catalyst is (200~240):(7~9):(3000~5000):(0.7~0.9);

[0018] Specifically, in S1, the heating reaction is carried out by stirring at a temperature of 25~30℃ for 4~6 hours.

[0019] In S1, the molecular weight of PEG is 2000~5000;

[0020] In S1, the purification operation specifically involves: adding 10 times the volume of the reaction solution to ice-cold ether, allowing the mixture to stand at 4°C for 12 hours to precipitate, centrifuging at 3000 rpm for 10 minutes, and discarding the supernatant; washing the precipitate repeatedly with ice-cold ether 2-3 times; and vacuum drying at 30-35°C for 4-6 hours to obtain terminally carboxylated PEG.

[0021] In S2, the mass ratio of terminal carboxylated PEG, DMF, NHS, EDC and mercapto reagent is (400~500):(4000~6000):(18~26):(32~40):(13~15);

[0022] In S2, the thiol reagent is 2-mercaptoethylamine;

[0023] Specifically, in S2, the heating reaction is carried out at a temperature of 30~40℃ for 2~3 hours.

[0024] Specifically, in step S2, the stirring operation is performed by stirring at a speed of 200-300 r / min for 1-2 hours.

[0025] In S2, the purification operation specifically involves: transferring the reaction solution to a dialysis bag with a molecular weight cutoff of 3500~5000 Da, dialyzing with ultrapure water for 48~72 h, and changing the water every 6~8 h; filtering the dialyzed solution through a 0.22 μm filter membrane, and vacuum drying at 30~35℃ for 2~4 h to obtain SH-PEG-COOH.

[0026] Optionally, the blocking solution comprises 100-140 mmol / L of lysine and 2-5% BSA by mass.

[0027] Optionally, the catalyst is DMAP.

[0028] Optionally, the preparation of the SH-PEG-COOH solution includes the following steps:

[0029] SH-PEG-COOH and deionized water are mixed in a certain mass ratio and stirred evenly to obtain an SH-PEG-COOH solution.

[0030] The mass ratio of SH-PEG-COOH to deionized water is (10~20):(100~200).

[0031] Optionally, the mass ratio of the chloroauric acid aqueous solution, trisodium citrate aqueous solution, and SH-PEG-COOH solution is (10~18):(16~28):(1~3); wherein the mass concentration of the trisodium citrate aqueous solution is 1~3%, and the mass concentration of the chloroauric acid aqueous solution is 0.01%.

[0032] Optionally, the electrospinning process parameters are: voltage 18kV, spinning solution feed rate 1.5mL / h, and distance between the needle and the aluminum foil receiving plate controlled at 15~20cm.

[0033] Optionally, the mass ratio of PCL, PEG and the mixed solution is (30~90):(60~80):(350~650).

[0034] Optionally, the quality control line is goat anti-mouse IgG.

[0035] Optionally, the mixed solution is composed of tetrahydrofuran and anhydrous ethanol in a mass ratio of 5:1.

[0036] This invention also provides a method for preparing the reagent kit for diagnosing aspiration pneumonia, comprising the following steps:

[0037] Step (1) Soak the cut glass cellulose membrane in PBS containing 0.5% Tween-20 for 10 min, then remove it and dry it in a drying oven at 60°C for later use; coat the treated glass cellulose membrane with a mixture of colloidal gold-labeled pepsinogen I antibody / colloidal gold-labeled pepsinogen II antibody, and dry it to obtain a gold-labeled pad.

[0038] Step (2) The secondary antibodies that recognize colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody are coated on the detection line 1 and detection line 2 of the nanofiber membrane, respectively. Goat anti-mouse IgG is coated on the quality control line of the nanofiber membrane to obtain the coated nanofiber membrane.

[0039] Step (3) The coated nanofiber membrane was soaked and blocked in PBS containing 1% BSA and pH 7.2 for 1 hour. After washing twice with PBS, it was placed in a drying oven to dry and obtain the treated nanofiber membrane.

[0040] Step (4) Attach the treated nanofiber membrane to the PVC base plate to form a large plate; attach the gold label pad and liquid absorption pad to both ends of the nanofiber membrane in the large plate, attach the sample pad to the gold label pad, and then put the assembly into the plastic casing.

[0041] Optionally, in the preparation method of the kit for diagnosing aspiration pneumonia, the preparation method of the mixture of colloidal gold-labeled pepsinogen I antibody / colloidal gold-labeled pepsinogen II antibody in step (1) includes the following steps:

[0042] The colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody were mixed and stirred evenly to obtain a mixture of colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody.

[0043] Optionally, in the preparation method of the kit for diagnosing aspiration pneumonia, in step (1), the volume ratio of colloidal gold-labeled pepsinogen I antibody to colloidal gold-labeled pepsinogen II antibody in the mixture of colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody is 1:1.

[0044] Optionally, in the preparation method of the kit for diagnosing aspiration pneumonia, the preparation of colloidal gold-labeled pepsinogen I antibody in step (1) is as follows:

[0045] Add the carboxylic acid activator to the colloidal gold solution and stir at room temperature for 30 min to activate. Purify and remove excess carboxylic acid activator. Add pepsinogen I antibody and incubate at room temperature for 40 min. Add BSA at a volume equal to the pepsinogen I antibody (2 g / 100 mL), centrifuge at 14000 g for 40 min at 4°C, resuspend the precipitate with suspension buffer, and centrifuge at 14000 g for 40 min at 4°C. Resuspend the precipitate again with suspension buffer, centrifuge again at 14000 g for 40 min at 4°C, add suspension buffer, and store at 4°C for later use.

[0046] Optionally, in the preparation method of the kit for diagnosing aspiration pneumonia, the preparation of colloidal gold-labeled pepsinogen II antibody in step (1) is as follows:

[0047] Add the carboxylic acid activator to the colloidal gold solution and stir at room temperature for 30 min to activate. Purify and remove excess carboxylic acid activator. Add pepsinogen II antibody and incubate at room temperature for 40 min. Add BSA at a volume equal to the pepsinogen II antibody (2 g / 100 mL), centrifuge at 14000 g for 40 min at 4°C, resuspend the precipitate with suspension buffer, and centrifuge at 14000 g for 40 min at 4°C. Resuspend the precipitate again with suspension buffer, centrifuge again at 14000 g for 40 min at 4°C, add suspension buffer, and store at 4°C for later use.

[0048] Optionally, the gold particles in the colloidal gold solution have a particle size of 30 nm.

[0049] Optionally, the pH of the PBS is 7.2.

[0050] Optionally, the carboxylic acid activator contains EDC and NHS, wherein the mass ratio of EDC to NHS is (1.5~2):1.

[0051] Optionally, both the colloidal gold-labeled pepsinogen I antibody and the colloidal gold-labeled pepsinogen II antibody are monoclonal antibodies.

[0052] Optionally, the preparation of the coated nanofiber membrane includes the following steps:

[0053] Secondary antibodies containing pepsinogen I and pepsinogen II at concentrations of 0.3–1.2 mg / ml and goat anti-mouse IgG at concentrations of 0.3–1 mg / ml were sprayed onto the detection line 1, detection line 2, and control line of the nanofiber membrane at speeds of 1 μL / cm and 10 cm / s, respectively.

[0054] Optionally, the gold-labeled pad is prepared by spraying a mixture of colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody at a concentration of 0.1 mg / ml onto the gold-labeled pad at a concentration of 30 μL / cm.

[0055] Optionally, the detection method of the kit for diagnosing aspiration pneumonia includes the following steps:

[0056] Add the sample to be tested into the sample well, and read the result after 10-15 minutes. The sample loading volume is 50-100 μL.

[0057] Optionally, the judgment criteria in the detection method of the kit for diagnosing aspiration pneumonia are as follows:

[0058] When the control line (C line) shows color, and either test line 1 (T1) or test line 2 (T2) shows color, the result is considered positive.

[0059] When the control line (C line) shows color, but neither test line 1 (T1) nor test line 2 (T2) shows color, the result is considered negative.

[0060] If the control line (C line) does not develop color, it is considered invalid regardless of whether test line 1 (T1) or test line 2 (T2) develops color.

[0061] Optionally, the storage conditions for the kit for diagnosing aspiration pneumonia are: storage temperature 4℃~30℃, protected from light, not frozen, and shelf life of 18 months; applicable conditions are: use within 1 hour under environmental conditions of temperature 18℃~25℃ and humidity ≤70%.

[0062] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0063] 1. The nanofiber membrane prepared by electrospinning in this invention has a high specific surface area, increasing loading capacity and enhancing the capture ability of low-concentration antigens. Optimized pore structure ensures rapid migration of the gold-labeled antibody-antigen complex, improving detection efficiency. Compared to traditional nitrocellulose membranes, the nanofiber membrane not only increases protein loading but also improves sensitivity, thus enhancing accuracy. The nanofiber membrane in this invention contains hydrophilic PEG, and the hydrophilic surface reduces the physical adsorption of impurities. Furthermore, the SH-PEG-COOH modified colloidal gold encapsulates gold particles through PEG chains, exposing only antigen-binding sites and blocking non-specific binding to impurities. This further reduces the false positive problem caused by the non-specific binding of homologous proteins to unmodified colloidal gold surfaces, thereby further improving detection accuracy and reliability. Simultaneously, when the secondary antibody in the detection line binds to the gold-labeled antibody-antigen complex, the gold particles can still aggregate through antibody bridging. Since PEG is colorless, it does not affect the color presentation of the gold particles, and the optical properties after aggregation remain unchanged, which aligns with the visualization effect of the kit.

[0064] 2. This invention employs SH-PEG-COOH modification of colloidal gold. Due to steric hindrance, the PEG chain structure prevents gold particle aggregation, ensuring the detectability of low-concentration antigens. Furthermore, SH-PEG-COOH binds to gold particles through coordination bonds, enhancing the stability of the colloidal gold. The gold particles in the colloidal gold match the porous structure of the nanofiber membrane, reducing chromatographic resistance, accelerating the migration of the "gold-labeled antibody-antigen" complex, and shortening detection time. Moreover, the SH-PEG-COOH-modified colloidal gold exhibits better uniformity, facilitating secondary antibody binding and eliminating signal heterogeneity issues. The synergistic effect of colloidal gold and immunochromatography in this invention not only optimizes the stability and uniformity of the label but also enables a rapid and convenient detection process.

[0065] 3. The reagent kit for diagnosing aspiration pneumonia proposed in this invention not only improves sensitivity, specificity, and accuracy, but also provides an efficient and convenient solution for the early diagnosis of aspiration pneumonia. Clinical trials of the reagent kit for diagnosing aspiration pneumonia prepared according to this invention showed an average overall clinical concordance rate of 97.5%. Detailed Implementation

[0066] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0067] Example 1

[0068] This embodiment provides a method for preparing a reagent kit for diagnosing aspiration pneumonia, including the following steps:

[0069] Step (1) Preparation of gold label pad

[0070] A1: Preparation of pretreated glass cellulose membrane

[0071] The cut glass cellulose membrane was immersed in PBS containing 0.5% Tween-20 at pH 7.2 for 10 minutes, then removed and dried in a drying oven at 60°C to obtain the pretreated glass cellulose membrane.

[0072] A2: Preparation of colloidal gold-labeled pepsinogen I antibody

[0073] Add EDC and NHS at a mass ratio of 1.5:1 to the colloidal gold solution, stir and activate at room temperature for 30 min, centrifuge at 13000g for 20 min at 4℃ to remove excess carboxylic acid activator; add pepsinogen I antibody, incubate at room temperature for 40 min; add BSA at 2g / 100mL with an equal volume to pepsinogen I antibody, centrifuge at 14000g for 40 min at 4℃, resuspend the precipitate with suspension buffer, centrifuge at 14000g for 40 min at 4℃; resuspend the precipitate again with suspension buffer, centrifuge again at 14000g for 40 min at 4℃, add suspension buffer, and store at 4℃ for later use;

[0074] A3: Preparation of colloidal gold-labeled pepsinogen II antibody

[0075] The preparation process of colloidal gold-labeled pepsinogen II antibody is the same as that used in A2 for the preparation of colloidal gold-labeled pepsinogen I antibody.

[0076] A4: Preparation of a mixture of colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody

[0077] Colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody were mixed at a volume ratio of 1:1 and stirred evenly to obtain a mixture of colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody.

[0078] Among them, the colloidal gold-labeled pepsinogen I antibody and the colloidal gold-labeled pepsinogen II antibody are both monoclonal antibodies;

[0079] A5: Preparation of gold-labeled pads

[0080] The mixture of 0.1 mg / ml colloidal gold-labeled pepsinogen I antibody / colloidal gold-labeled pepsinogen II antibody prepared in A4 was sprayed at 30 μL / cm onto the pretreated glass cellulose membrane prepared in A1 and dried to obtain the gold-labeled pad.

[0081] Step (2) Preparation of the coated nanofiber membrane

[0082] Secondary antibodies containing pepsinogen I and pepsinogen II at a concentration of 0.3 mg / ml and goat anti-mouse IgG at a concentration of 0.3 mg / ml were respectively sprayed onto the detection line 1, detection line 2 and control line of the nanofiber membrane at a speed of 1 μL / cm and 10 cm / s to obtain the coated nanofiber membrane.

[0083] Step (3) Preparation of the treated nanofiber membrane

[0084] The coated nanofiber membrane was immersed and blocked in PBS containing 1% BSA and pH 7.2 for 1 hour. After washing twice with PBS, it was dried in a drying oven to obtain the treated nanofiber membrane.

[0085] Step (4) Assembly

[0086] The treated nanofiber membrane is pasted onto a PVC base plate to form a large plate; the gold label pad and liquid absorption pad obtained in step (1) are pasted onto both ends of the nanofiber membrane in the large plate, the sample pad is pasted onto the gold label pad, and the sample is assembled and then inserted into a plastic casing.

[0087] The preparation of the nanofiber membrane includes the following steps:

[0088] PCL, PEG, and a mixed solution were mixed at a mass ratio of 30:60:350 and stirred thoroughly to form a spinning solution. Electrospinning was then performed at room temperature and 40% relative humidity to prepare nanofiber membranes.

[0089] The mixed solution is composed of tetrahydrofuran and anhydrous ethanol in a mass ratio of 5:1.

[0090] The electrospinning process parameters are as follows: voltage 18kV, spinning solution feed rate 1.5mL / h, and distance between the needle and the aluminum foil receiving plate controlled at 15cm.

[0091] The colloidal gold labeling material is derived from a colloidal gold solution;

[0092] The preparation of the colloidal gold solution includes the following steps:

[0093] Under a nitrogen atmosphere, a 0.01 wt% chloroauric acid aqueous solution was first heated to boiling, and a 1 wt% trisodium citrate aqueous solution was quickly added and boiled. After cooling to room temperature, SH-PEG-COOH solution was slowly added, and the reaction was carried out at room temperature for 2.5 h. The mixture was then allowed to stand overnight at 4 °C, centrifuged, washed, and blocked with 100 mmol / L lysine and 2% BSA for 1 h. The mixture was then stored at 4 °C in the dark to obtain a colloidal gold solution.

[0094] The mass ratio of chloroauric acid aqueous solution, trisodium citrate aqueous solution and SH-PEG-COOH solution is 10:16:1;

[0095] In the SH-PEG-COOH solution, the mass ratio of SH-PEG-COOH to deionized water is 10:100.

[0096] The preparation method of SH-PEG-COOH includes the following steps:

[0097] S1: PEG, succinic anhydride, dichloromethane, and catalyst DMAP were mixed in a mass ratio of 200:7:3000:0.7 and stirred at 25°C for 6 hours. After the reaction was completed, 10 times the volume of the reaction solution was added to the reaction solution, and the mixture was allowed to stand at 4°C for 12 hours to precipitate. The mixture was then centrifuged at 3000 rpm for 10 minutes, and the supernatant was discarded. The precipitate was washed three times with ice-cold ether and dried under vacuum at 30°C for 6 hours to obtain terminally carboxylated PEG.

[0098] S2: Dissolve terminally carboxylated PEG in DMF, add EDC and NHS sequentially under nitrogen protection, and stir at 200 r / min for 2 h; add 2-mercaptoethylamine, heat to 30 °C and react for 3 h. After the reaction is complete, transfer to a dialysis bag with a molecular weight cutoff of 3500 Da, dialyze with ultrapure water for 48 h, changing the water every 6 h; filter the dialyzed solution through a 0.22 μm filter membrane and vacuum dry at 30 °C for 4 h to obtain SH-PEG-COOH;

[0099] The mass ratio of terminally carboxylated PEG, DMF, NHS, EDC and 2-mercaptoethylamine is 400:4000:18:32:13.

[0100] The molecular weight of PEG is 2000.

[0101] Example 2

[0102] This embodiment provides a method for preparing a reagent kit for diagnosing aspiration pneumonia, including the following steps:

[0103] Step (1) Preparation of gold label pad

[0104] B1: Preparation of pretreated glass cellulose membrane

[0105] The cut glass cellulose membrane was immersed in PBS containing 0.5% Tween-20 at pH 7.2 for 10 minutes, then removed and dried in a drying oven at 60°C to obtain the pretreated glass cellulose membrane.

[0106] B2: Preparation of colloidal gold-labeled pepsinogen I antibody

[0107] Add EDC and NHS at a mass ratio of 1.8:1 to the colloidal gold solution, stir and activate at room temperature for 30 min, centrifuge at 13000g for 20 min at 4℃ to remove excess carboxylic acid activator; add pepsinogen I antibody, incubate at room temperature for 40 min; add BSA at 2g / 100mL with an equal volume to pepsinogen I antibody, centrifuge at 14000g for 40 min at 4℃, resuspend the precipitate with suspension buffer, centrifuge at 14000g for 40 min at 4℃; resuspend the precipitate again with suspension buffer, centrifuge again at 14000g for 40 min at 4℃, add suspension buffer, and store at 4℃ for later use;

[0108] B3: Preparation of colloidal gold-labeled pepsinogen II antibody

[0109] The preparation process of colloidal gold-labeled pepsinogen II antibody is the same as that used in B2 for the preparation of colloidal gold-labeled pepsinogen I antibody.

[0110] B4: Preparation of a mixture of colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody.

[0111] Colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody were mixed at a volume ratio of 1:1 and stirred evenly to obtain a mixture of colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody.

[0112] Among them, the colloidal gold-labeled pepsinogen I antibody and the colloidal gold-labeled pepsinogen II antibody are both monoclonal antibodies;

[0113] B5: Preparation of gold-labeled pads

[0114] The mixture of 0.1 mg / ml colloidal gold-labeled pepsinogen I antibody / colloidal gold-labeled pepsinogen II antibody prepared in B4 was sprayed at 30 μL / cm onto the pretreated glass cellulose membrane prepared in B1 and dried to obtain the gold-labeled pad.

[0115] Step (2) Preparation of the coated nanofiber membrane

[0116] Secondary antibodies containing pepsinogen I and pepsinogen II at a concentration of 1 mg / ml and goat anti-mouse IgG at a concentration of 0.8 mg / ml were respectively sprayed onto the detection line 1, detection line 2 and control line of the nanofiber membrane at a speed of 1 μL / cm and 10 cm / s to obtain the coated nanofiber membrane.

[0117] Step (3) Preparation of the treated nanofiber membrane

[0118] The coated nanofiber membrane was immersed and blocked in PBS containing 1% BSA and pH 7.2 for 1 hour. After washing twice with PBS, it was dried in a drying oven to obtain the treated nanofiber membrane.

[0119] Step (4) Assembly

[0120] The treated nanofiber membrane is pasted onto a PVC base plate to form a large plate; the gold label pad and liquid absorption pad obtained in step (1) are pasted onto both ends of the nanofiber membrane in the large plate, the sample pad is pasted onto the gold label pad, and the sample is assembled and then inserted into a plastic casing.

[0121] The preparation of the nanofiber membrane includes the following steps:

[0122] PCL, PEG, and a mixed solution were mixed at a mass ratio of 60:70:500 and stirred thoroughly to form a spinning solution. Electrospinning was then performed at room temperature and 50% relative humidity to prepare nanofiber membranes.

[0123] The mixed solution is composed of tetrahydrofuran and anhydrous ethanol in a mass ratio of 5:1.

[0124] The electrospinning process parameters are as follows: voltage 18kV, spinning solution feed rate 1.5mL / h, and distance between the needle and the aluminum foil receiving plate controlled at 18cm.

[0125] The colloidal gold labeling material is derived from a colloidal gold solution;

[0126] The preparation of the colloidal gold solution includes the following steps:

[0127] Under a nitrogen atmosphere, a 0.01 wt% chloroauric acid aqueous solution was first heated to boiling, and a 2 wt% trisodium citrate aqueous solution was quickly added and boiled. After cooling to room temperature, SH-PEG-COOH solution was slowly added, and the reaction was carried out at room temperature for 3.5 h. The mixture was then allowed to stand overnight at 4 °C, centrifuged, washed, and blocked with 120 mmol / L lysine and 3% BSA for 1 h. The mixture was then stored at 4 °C in the dark to obtain a colloidal gold solution.

[0128] The mass ratio of chloroauric acid aqueous solution, trisodium citrate aqueous solution and SH-PEG-COOH solution is 14:22:2;

[0129] In the SH-PEG-COOH solution, the mass ratio of SH-PEG-COOH to deionized water is 15:150.

[0130] The preparation method of SH-PEG-COOH includes the following steps:

[0131] S1: PEG, succinic anhydride, dichloromethane, and catalyst DMAP were mixed in a mass ratio of 220:8:4500:0.8 and stirred at 27°C for 5 hours. After the reaction was completed, 10 times the volume of the reaction solution was added to the reaction solution, and the mixture was allowed to stand at 4°C for 12 hours to precipitate. The mixture was then centrifuged at 3000 rpm for 10 minutes, and the supernatant was discarded. The precipitate was washed three times with ice-cold ether and dried under vacuum at 33°C for 5 hours to obtain terminally carboxylated PEG.

[0132] S2: Dissolve terminally carboxylated PEG in DMF, add EDC and NHS sequentially under nitrogen protection, and stir at 250 r / min for 1.5 h; add 2-mercaptoethylamine, heat to 35 °C and react for 2.5 h. After the reaction is complete, transfer to a dialysis bag with a molecular weight cutoff of 4250 Da, dialyze with ultrapure water for 60 h, changing the water every 7 h; filter the dialyzed solution through a 0.22 μm filter membrane and vacuum dry at 33 °C for 3 h to obtain SH-PEG-COOH;

[0133] The mass ratio of terminally carboxylated PEG, DMF, NHS, EDC and 2-mercaptoethylamine is 450:5000:22:36:14.

[0134] The molecular weight of PEG is 3500.

[0135] Example 3

[0136] This embodiment provides a method for preparing a reagent kit for diagnosing aspiration pneumonia, including the following steps:

[0137] Step (1) Preparation of gold label pad

[0138] C1: Preparation of pretreated glass cellulose membranes

[0139] The cut glass cellulose membrane was immersed in PBS containing 0.5% Tween-20 at pH 7.2 for 10 minutes, then removed and dried in a drying oven at 60°C to obtain the pretreated glass cellulose membrane.

[0140] C2: Preparation of colloidal gold-labeled pepsinogen I antibody

[0141] Add EDC and NHS at a mass ratio of 2:1 to the colloidal gold solution, stir and activate at room temperature for 30 min, centrifuge at 13000g for 20 min at 4℃ to remove excess carboxylic acid activator; add pepsinogen I antibody and incubate at room temperature for 40 min; add BSA at 2g / 100mL with an equal volume to pepsinogen I antibody, centrifuge at 14000g for 40 min at 4℃, resuspend the precipitate with suspension buffer, centrifuge at 14000g for 40 min at 4℃; resuspend the precipitate again with suspension buffer, centrifuge again at 14000g for 40 min at 4℃, add suspension buffer, and store at 4℃ for later use;

[0142] C3: Preparation of colloidal gold-labeled pepsinogen II antibody

[0143] The preparation process of colloidal gold-labeled pepsinogen II antibody is the same as that used in C2 for the preparation of colloidal gold-labeled pepsinogen I antibody.

[0144] C4: Preparation of a mixture of colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody.

[0145] Colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody were mixed at a volume ratio of 1:1 and stirred evenly to obtain a mixture of colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody.

[0146] Among them, the colloidal gold-labeled pepsinogen I antibody and the colloidal gold-labeled pepsinogen II antibody are both monoclonal antibodies;

[0147] C5: Preparation of gold-labeled pads

[0148] The mixture of 0.1 mg / ml colloidal gold-labeled pepsinogen I antibody / colloidal gold-labeled pepsinogen II antibody prepared in C4 was sprayed at 30 μL / cm onto the pretreated glass cellulose membrane prepared in C1 and dried to obtain the gold-labeled pad.

[0149] Step (2) Preparation of the coated nanofiber membrane

[0150] Secondary antibodies containing pepsinogen I and pepsinogen II at a concentration of 1.2 mg / ml and goat anti-mouse IgG at a concentration of 1 mg / ml were respectively sprayed onto the detection line 1, detection line 2 and control line of the nanofiber membrane at a speed of 1 μL / cm and 10 cm / s to obtain the coated nanofiber membrane.

[0151] Step (3) Preparation of the treated nanofiber membrane

[0152] The coated nanofiber membrane was immersed and blocked in PBS containing 1% BSA and pH 7.2 for 1 hour. After washing twice with PBS, it was dried in a drying oven to obtain the treated nanofiber membrane.

[0153] Step (4) Assembly

[0154] The treated nanofiber membrane is pasted onto a PVC base plate to form a large plate; the gold label pad and liquid absorption pad obtained in step (1) are pasted onto both ends of the nanofiber membrane in the large plate, the sample pad is pasted onto the gold label pad, and the sample is assembled and then inserted into a plastic casing.

[0155] The preparation of the nanofiber membrane includes the following steps:

[0156] PCL, PEG, and a mixed solution were mixed at a mass ratio of 90:80:650 and stirred thoroughly to form a spinning solution. Electrospinning was then performed at room temperature and 60% relative humidity to prepare nanofiber membranes.

[0157] The mixed solution is composed of tetrahydrofuran and anhydrous ethanol in a mass ratio of 5:1.

[0158] The electrospinning process parameters are as follows: voltage 18kV, spinning solution feed rate 1.5mL / h, and distance between the needle and the aluminum foil receiving plate controlled at 20cm.

[0159] The colloidal gold labeling material is derived from a colloidal gold solution;

[0160] The preparation of the colloidal gold solution includes the following steps:

[0161] Under a nitrogen atmosphere, a 0.01 wt% chloroauric acid aqueous solution was first heated to boiling, and a 3 wt% trisodium citrate aqueous solution was quickly added and boiled. After cooling to room temperature, SH-PEG-COOH solution was slowly added, and the reaction was carried out at room temperature for 4.5 h. The mixture was then allowed to stand overnight at 4 °C, centrifuged, washed, and blocked with 140 mmol / L lysine and 5% BSA for 1 h. The mixture was then stored at 4 °C in the dark to obtain a colloidal gold solution.

[0162] The mass ratio of chloroauric acid aqueous solution, trisodium citrate aqueous solution and SH-PEG-COOH solution is 18:28:3.

[0163] In the SH-PEG-COOH solution, the mass ratio of SH-PEG-COOH to deionized water is 20:200.

[0164] The preparation method of SH-PEG-COOH includes the following steps:

[0165] S1: PEG, succinic anhydride, dichloromethane, and catalyst DMAP were mixed in a mass ratio of 240:9:5000:0.9 and stirred at 30°C for 4 hours. After the reaction was completed, 10 times the volume of the reaction solution was added to the reaction solution, and the mixture was allowed to stand at 4°C for 12 hours to precipitate. The mixture was then centrifuged at 3000 rpm for 10 minutes, and the supernatant was discarded. The precipitate was washed three times with ice-cold ether and dried under vacuum at 35°C for 4 hours to obtain terminally carboxylated PEG.

[0166] S2: Dissolve terminally carboxylated PEG in DMF, add EDC and NHS sequentially under nitrogen protection, and stir at 300 r / min for 1 h; add 2-mercaptoethylamine, heat to 40 °C and react for 2 h. After the reaction is complete, transfer to a dialysis bag with a molecular weight cutoff of 5000 Da, and dialyze with ultrapure water for 72 h, changing the water every 8 h; filter the dialyzed solution through a 0.22 μm filter membrane and vacuum dry at 35 °C for 2 h to obtain SH-PEG-COOH;

[0167] The mass ratio of terminally carboxylated PEG, DMF, NHS, EDC and 2-mercaptoethylamine is 500:6000:26:40:15.

[0168] The molecular weight of PEG is 5000.

[0169] Comparative Example 1

[0170] The difference between this comparative example and Example 1 is that the preparation method of the nanofiber membrane is omitted, and an equal amount of nitrocellulose membrane is used instead of the nanofiber membrane.

[0171] Comparative Example 2

[0172] The difference between this comparative example and Example 1 is that the preparation process of the nanofiber membrane is omitted, and an equal amount of traditional nitrocellulose membrane is used instead of the nanofiber membrane; an equal amount of traditional colloidal gold solution is used instead of the colloidal gold solution.

[0173] The traditional method for preparing colloidal gold solutions is as follows:

[0174] Under a nitrogen atmosphere, first heat 100 ml of 0.01 wt% chloroauric acid aqueous solution to boiling, then quickly add 1 wt% trisodium citrate aqueous solution, boil again, and a transparent wine-red color appears. Finally, add distilled water to 100 ml to obtain a traditional colloidal gold solution.

[0175] In the embodiments and comparative examples of this invention, the gold particles in the colloidal gold solution all had a particle size of 30 nm. The suspension buffer was specifically a 20 mmol / L Tris / HCl buffer with a pH of 7.5, containing 1% (w / v) BSA, 3% (w / v) sucrose, and 0.02% sodium azide. The pepsinogen I antibody was the monoclonal antibody PGI-8012 from Medix. The pepsinogen II antibody was the monoclonal antibody PGII-8105 from Medix. The goat anti-mouse IgG was from Beijing Tiangen Pharmaceuticals. The relative molecular mass of PCL was 45,000. All other products were commercially available.

[0176] The kits prepared in Examples 1-3 and Comparative Examples 1-2 were subjected to corresponding tests.

[0177] (1) Sensitivity test: PGI-8012 was prepared into solutions with concentrations of 0, 5, 10, 12, 14, 16, and 18 ng / mL using PBS buffer (pH 7.2, 1% BSA), and PGII-8105 was prepared into solutions with concentrations of 0, 1, 2, 3, 4, and 5 ng / mL. Five parallel samples were prepared for each concentration. 60 μL of each sample was added to the sample well of the test strip, incubated for 30 min, and the results were interpreted after 10 min. The interpretation criteria were as follows: The results are as follows: a positive result is determined when the control line (C line) shows color and either test line 1 (T1) or test line 2 (T2) shows color; a negative result is determined when the control line (C line) shows color but neither test line 1 (T1) nor test line 2 (T2) shows color; and an invalid result is determined regardless of whether test line 1 (T1) or test line 2 (T2) shows color if the control line (C line) does not show color. The sensitivity test results for PGI-8012 are shown in Table 1; and the sensitivity test results for PGII-8105 are shown in Table 2.

[0178] Table 1

[0179]

[0180] Table 2

[0181]

[0182] As can be seen from Tables 1 and 2, the kit prepared by the present invention has high sensitivity, which is higher than that of the kits prepared in Comparative Examples 1-2.

[0183] (2) Accuracy test: 20 negative samples and 20 positive samples were tested using the kits of Examples 1-3. 60 μL of each sample was dropped into the sample slot of the test strip and incubated for 30 min. The results were read after 10 min. 60 μL of the sample was dropped into the sample slot of the test strip according to the same procedure as the sensitivity test and read after 10 min. The specific test results are shown in Table 3 below.

[0184] Table 3

[0185]

[0186] As can be seen from the data in Table 3, the accuracy of the reagent kits prepared by this invention is higher than that of the comparative example, indicating higher accuracy.

[0187] The above steps were repeated 10 times for sensitivity and accuracy testing. The results showed that the intra-batch and inter-batch repeatability of the kit was 100%.

[0188] (3) Specificity test: Pepsinogen I, pepsinogen II, salivary amylase, trypsin and lipase were prepared into standard solutions with concentrations of 0, 5 mg / L, 10 mg / L and 20 mg / L, respectively. The kits prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention were used for detection. Each concentration was tested three times. The specific test results are shown in Table 4 below.

[0189] Table 4

[0190]

[0191] As shown in Table 4, the kits in Examples 1-3 exhibit good accuracy. Specifically, the kits in Examples 1-3 were negative for interfering enzymes such as salivary amylase, trypsin, and lipase at concentrations ranging from 0 to 20 mg / L, indicating that the nanofiber membrane synergistic with colloidal gold prepared in this invention effectively avoids cross-reactions with non-target analytes and demonstrates good specificity. In Comparative Example 1, a traditional nitrocellulose membrane was used instead of the nanofiber membrane. Due to the uneven antibody loading on the nitrocellulose membrane, cross-reactions with salivary amylase and trypsin occurred at high concentrations, leading to increased non-specific adsorption. Comparative Example 2 used a nitrocellulose membrane and traditional colloidal gold: at lower concentrations, the detection effect was not significant, and at higher concentrations, the traditional colloidal gold exhibited poor stability and was prone to non-specific binding with other proteins, resulting in poor specificity. Analysis of the above data shows that the kit prepared in this invention solves the false positive problem caused by cross-reactions in traditional kits.

[0192] (4) Clinical performance validation test of the reagent kit

[0193] Sample Collection: The sample collection tube should be pre-filled with 0.5 ml of 0.01 M deionized citrate solution. After coughing and clearing their throat, the test subject should add at least 1 ml of saliva sample to the collection tube. Samples should be collected one hour after a meal. Record the sampling time and any other special symptoms. Clinical samples that cannot be tested within 8 hours at room temperature should be refrigerated and tested within 3 days. Precautions for sample collection include: avoiding sample collection within 1 hour of consuming caffeinated beverages, carbonated drinks, or smoking; avoiding sample collection within 48 hours of consuming alkaline water or beverages, antacids, or alginate antacids; and avoiding sample collection immediately after strenuous exercise. Collected samples should be tested within 2 hours at room temperature. Samples stored at 2-8°C should not be kept for more than 3 days, and frozen samples at -20°C should not be stored for more than 12 months. Samples must be fully thawed to room temperature before testing. Frozen samples must be fully thawed to room temperature and thoroughly mixed before use; the number of freeze-thaw cycles should not exceed 3.

[0194] Detection method of the kit: Before use, allow the kit to reach room temperature. Remove the test card and place it on a horizontal work surface. Add 60 μL of the collected sample to the sample well. Interpret the results after 10 minutes. The specific interpretation criteria are as follows:

[0195] When the control line (C line) shows color, and either test line 1 (T1) or test line 2 (T2) shows color, the result is considered positive.

[0196] When the control line (C line) shows color, but neither test line 1 (T1) nor test line 2 (T2) shows color, the result is considered negative.

[0197] If the control line (C line) does not develop color, it is considered invalid regardless of whether test line 1 (T1) or test line 2 (T2) develops color.

[0198] Saliva samples were collected from 50 patients diagnosed with pneumonia caused by gastric reflux and 50 healthy individuals. The samples were tested using the kits prepared in Examples 1-3 and Comparative Examples 1-2. The specific test results are shown in Table 5.

[0199] Table 5

[0200]

[0201] As can be seen from the test results in Table 5, the average total compliance rate of the kits prepared in Examples 1-3 of this invention is 97.5%, which is higher than the total compliance rate of the kits in Comparative Examples 1-2, indicating that they have high accuracy.

[0202] The above description is merely an example and illustration of the structure of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the structure of the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

[0203] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0204] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to specific implementations. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A reagent kit for diagnosing aspiration pneumonia, comprising a PVC base plate, a sample pad, a gold label pad, a nanofiber membrane, a liquid absorption pad, and a plastic cartridge, characterized in that: The gold-labeled pad is immobilized with colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody. The nanofiber membrane is immobilized with detection line 1 and detection line 2, which consist of secondary antibodies that recognize colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody, as well as a quality control line. The preparation of the nanofiber membrane includes the following steps: PCL and PEG were added to a mixed solution and stirred thoroughly to form a spinning solution. Electrospinning was carried out at room temperature and relative humidity of 40-60% to prepare nanofiber membranes. The colloidal gold labeling material is derived from a colloidal gold solution; The preparation of the colloidal gold solution includes the following steps: Under a nitrogen atmosphere, chloroauric acid aqueous solution was first heated to boiling, and trisodium citrate aqueous solution was quickly added and boiled. After cooling to room temperature, SH-PEG-COOH solution was slowly added and reacted at room temperature for 2.5~4.5h. After standing overnight at 4℃, the solution was centrifuged, washed, and blocked with a blocking solution. The solution was stored at 4℃ in the dark to obtain colloidal gold solution.

2. The kit for diagnosing aspiration pneumonia according to claim 1, characterized in that, The preparation method of the SH-PEG-COOH includes the following steps: S1: Add PEG and succinic anhydride to dichloromethane, add catalyst, stir, heat to react, and after the reaction is completed, purify to obtain terminally carboxylated PEG; S2: Dissolve terminally carboxylated PEG in DMF, add EDC and NHS sequentially under nitrogen protection, stir; add thiol reagent, heat to react, and after the reaction is complete, purify to obtain SH-PEG-COOH.

3. The kit for diagnosing aspiration pneumonia according to claim 2, characterized in that, The catalyst is DMAP.

4. The kit for diagnosing aspiration pneumonia according to claim 1, characterized in that, The preparation of the SH-PEG-COOH solution includes the following steps: SH-PEG-COOH and deionized water are mixed in a certain mass ratio and stirred evenly to obtain an SH-PEG-COOH solution. The mass ratio of SH-PEG-COOH to deionized water is (10~20):(100~200).

5. The kit for diagnosing aspiration pneumonia according to claim 1, characterized in that, The mass ratio of the chloroauric acid aqueous solution, trisodium citrate aqueous solution, and SH-PEG-COOH solution is (10~18):(16~28):(1~3); The mass concentration of the trisodium citrate aqueous solution is 1-3%, and the mass concentration of the chloroauric acid aqueous solution is 0.01%.

6. A method for preparing a reagent kit for diagnosing aspiration pneumonia as described in claim 1, characterized in that, Includes the following steps: Step (1) Soak the cut glass cellulose membrane in PBS containing 0.5% Tween-20 for 10 min, then remove it and dry it in a drying oven at 60°C for later use; coat the treated glass cellulose membrane with a mixture of colloidal gold-labeled pepsinogen I antibody / colloidal gold-labeled pepsinogen II antibody, and dry it to obtain a gold-labeled pad. Step (2) The secondary antibodies that recognize colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody are coated on the detection line 1 and detection line 2 of the nanofiber membrane, respectively. Goat anti-mouse IgG is coated on the quality control line of the nanofiber membrane to obtain the coated nanofiber membrane. Step (3) The coated nanofiber membrane was soaked and blocked in PBS containing 1% BSA and pH 7.2 for 1 hour. After washing twice with PBS, it was placed in a drying oven to dry and obtain the treated nanofiber membrane. Step (4) Attach the treated nanofiber membrane to the PVC base plate to form a large plate; attach the gold label pad and liquid absorption pad to both ends of the nanofiber membrane in the large plate, attach the sample pad to the gold label pad, and then put the assembly into the plastic casing.

7. The method for preparing the reagent kit for diagnosing aspiration pneumonia according to claim 6, characterized in that, In step (1), the preparation method of the mixture of colloidal gold-labeled pepsinogen I antibody / colloidal gold-labeled pepsinogen II antibody includes the following steps: The colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody were mixed and stirred evenly to obtain a mixture of colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody.

8. The method for preparing the reagent kit for diagnosing aspiration pneumonia according to claim 7, characterized in that, In step (1), the volume ratio of colloidal gold-labeled pepsinogen I antibody to colloidal gold-labeled pepsinogen II antibody in the mixture of colloidal gold-labeled pepsinogen I antibody and colloidal gold-labeled pepsinogen II antibody is 1:

1.

9. The method for preparing the reagent kit for diagnosing aspiration pneumonia according to claim 6, characterized in that, In step (1), the preparation of colloidal gold-labeled pepsinogen I antibody is as follows: Add the carboxylic acid activator to the colloidal gold solution prepared according to claim 1, stir and activate at room temperature for 30 min, and purify to remove excess carboxylic acid activator; add pepsinogen I antibody, incubate at room temperature for 40 min; add BSA of equal volume (2 g / 100 mL) to pepsinogen I antibody, centrifuge at 14000 g for 40 min at 4°C, resuspend the precipitate with suspension buffer, centrifuge at 14000 g for 40 min at 4°C; resuspend the precipitate again with suspension buffer, centrifuge again at 14000 g for 40 min at 4°C, add suspension buffer, and store at 4°C for later use.

10. The method for preparing the reagent kit for diagnosing aspiration pneumonia according to claim 6, characterized in that, In step (1), the preparation of colloidal gold-labeled pepsinogen II antibody is as follows: Add the carboxylic acid activator to the colloidal gold solution prepared according to claim 1, stir and activate at room temperature for 30 min, and purify to remove excess carboxylic acid activator; add pepsinogen II antibody, incubate at room temperature for 40 min; add BSA of equal volume (2 g / 100 mL) to pepsinogen II antibody, centrifuge at 14000 g for 40 min at 4°C, resuspend the precipitate with suspension buffer, centrifuge at 14000 g for 40 min at 4°C; resuspend the precipitate again with suspension buffer, centrifuge again at 14000 g for 40 min at 4°C, add suspension buffer, and store at 4°C for later use.

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