CN116699138A

CN116699138A - Semi-quantitative pepsin detection product for distinguishing physiological and pathological reflux

Info

Publication number: CN116699138A
Application number: CN202310720187.1A
Authority: CN
Inventors: 杨轶轩
Original assignee: Hunan Youbosi Biotechnology Co ltd
Current assignee: Hunan Youbosi Biotechnology Co ltd
Priority date: 2020-12-03
Filing date: 2020-12-03
Publication date: 2023-09-05
Also published as: CN113049825A

Abstract

The invention provides a semi-quantitative pepsin detection product for distinguishing physiological and pathological reflux, which comprises a device for detecting pepsin and a pepsin standard concentration control, wherein the pepsin standard concentration control at least comprises a pepsin standard concentration control of less than 50ng/ml and a pepsin standard concentration control of not less than 50 ng/ml. The product provided by the invention is used for detecting pepsin and simultaneously measuring the concentration of the pepsin in a semi-quantitative manner so as to distinguish physiological reflux from pathological reflux. The semi-quantitative pepsin detection product has high detection rate and sensitivity, is simple to operate, has been verified by clinical practice, and is suitable for further clinical popularization.

Description

Semi-quantitative pepsin detection product for distinguishing physiological and pathological reflux

Technical Field

The invention belongs to the technical field of medical and health, in particular to in vitro detection of LPR, which is particularly suitable for in vitro differentiation and detection of physiological and pathological gastric reflux entering throat (namely throat).

Background

LPR (reflux pharyngolaryngitis) refers to a group of diseases caused by reflux of gastric contents to the throat, which is reported in literature to be associated with numerous upper respiratory diseases such as laryngitis, asthma, chronic cough, and to be associated with secretory otitis media and dysarthria. GERD (gastroesophageal reflux disease) refers to the general term for gastroesophageal reflux and esophageal mucosa injury diseases caused by gastric juice stimulation of the esophageal mucosa, in which the contents of the stomach and duodenum reflux to the esophagus.

LPR has a correlation with GERD, and LPR is often the extra-esophageal manifestation of GERD. LPR has been attributed to the category of chronic pharyngitis, but has its unique characteristics in etiology, symptoms and treatment, and the American ear-nose-throat-head-neck surgical society formally adopted the name LPR in 2002. LPR is very common in clinical work, where patients tend to first visit the otorhinolaryngology due to throat discomfort, but clinicians tend to focus more on the patient's laryngeal characterization and ignore it as might be associated with GERD. The common chronic pharyngolaryngitis is usually treated according to the scheme, but the common chronic pharyngolaryngitis is not treated according to the etiology, so that symptoms are common, but the symptoms of patients are often not obviously relieved after long-time treatment. Effective treatment of LPR depends on proper knowledge and accurate diagnosis. LPR has been lacking in an objective and simple diagnostic method. The literature reports that 24H esophageal pH monitoring can be used as a diagnostic standard for LPR, but is invasive and not widely accepted by patients due to its high cost and long time consumption.

The composition of saliva in the mouth is complex and the use of changes in certain components of saliva in the mouth to predict or diagnose the presence of certain diseases is a good method. Pepsin is only produced in the stomach, and the presence of pepsin detected in the throat suggests the presence of reflux. PCT International patent application publication No. WO/1998/012349 critically discloses the positive significance of pharyngeal detection of pepsin for in vitro rapid diagnosis of gastric reflux disease. Compared with questionnaire and 24H gastric acid detection, the in-vitro detection method has the clinical advantages of non-intervention, simplicity, rapidness, small influence by human subjective factors and the like.

Based on the prior art information disclosed in WO/1998/012349, the invention finds that pepsin can also be detected in saliva of people without clinical symptoms, resulting in diagnosis of false positives. Although it is disclosed in the prior art that reflux can be diagnosed by detecting pepsin in a sample such as saliva, the presence of physiological reflux can also cause pepsin to remain in the laryngopharynx, oral cavity and other parts, and the prior art cannot distinguish the physiological reflux from pathological reflux well.

Therefore, how to solve the problem of accurately distinguishing physiological reflux from pathological reflux and develop an early diagnosis and identification product of LPR for clinical practical application is an industrial problem to be solved. According to NMPA official website disclosures, no such clinically available medical device products currently have been approved for marketing.

Disclosure of Invention

In order to improve the accuracy and efficiency of early diagnosis of gastric reflux, the invention provides a novel semi-quantitative pepsin detection product, and the concentration of pepsin is measured in a semi-quantitative manner while pepsin is detected, so as to distinguish physiological reflux from pathological reflux. The semi-quantitative pepsin detection product has high detection rate and sensitivity, is simple to operate, has been verified by clinical practice, and is suitable for further clinical popularization.

The semi-quantitative pepsin detection product provided by the invention comprises a pepsin detection device and a pepsin standard concentration control, wherein the pepsin standard concentration control at least comprises a pepsin standard concentration control of less than 50ng/ml and a pepsin standard concentration control of not less than 50 ng/ml. According to the unexpected discovery of the invention, clinical practice proves that the sample tissues such as saliva, sputum, mucus and the like taken from the oral cavity/laryngopharynx are as follows: physiological reflux is usually performed for a short time due to the fact that the reflux frequency is low, the pepsin concentration is usually less than 50ng/ml, and about 0, 5, 10 and 20ng/ml are common; the concentration of protease in pathological reflux is usually 50ng/ml due to high frequency, long retention time, high concentration and the like, and even 100ng/ml or more, more usually about 50, 100, 200ng/ml or more depending on the degree of reflux severity exceeding 50 ng/ml. Based on the principle, the semi-quantitative pepsin detection product provided by the invention comprises pepsin standard concentration contrast, and the pepsin standard concentration contrast at least establishes key demarcation point contrast of 50ng/ml which is verified to be feasible by clinical practice of the invention so as to perform semi-quantitative measurement of the pepsin concentration of the detected sample and assist in performing high-accuracy and high-efficiency rapid diagnosis of the LPR in early stage. The pepsin analysis and detection are relatively mature prior art, and the corresponding pepsin detection devices can be applied to the invention, for example, a colloidal gold method, an electrophoresis method, an immunofluorescence method, a direct competition method, an indirect competition method, an ELISA method, a RIA method, a flow cytometry method or an immunochromatography method can be adopted, and the practice of the invention verifies that the colloidal gold method is adopted preferentially. The test sample of the pepsin testing device may be saliva, sputum, oral or laryngopharyngeal mucus, blood or urine, preferably saliva, sputum, oral or laryngopharyngeal mucus. The pepsin detection device detects pepsin from an extra-esophageal region including the laryngopharynx, oral cavity, sinus cavity, airway, tracheobronchial, lung, blood vessels and urinary tract, preferably the laryngopharynx, oral cavity, sinus cavity or airway. Matching with corresponding detection technology can match with the matched pepsin concentration standard substance contrast, which is also available in the prior art, and can be specifically and respectively selected according to actual conditions, and the invention is not listed in detail.

The pepsin standard concentration controls, as practiced according to the present invention, included pepsin standard concentration controls of 0ng/ml, 25ng/ml, 50ng/ml, and 100 ng/ml. Preferably, pepsin standard concentration controls of 5ng/ml, 10ng/ml and 200ng/ml may be additionally included.

According to the specific practice of the embodiment of the invention, the pepsin detection device is a colloidal gold pepsin detection test strip, and the pepsin standard substance concentration is controlled to be 0ng/ml, 5ng/ml, 10ng/ml, 25ng/ml, 50ng/ml, 100ng/ml and 200ng/ml pepsin standard substance concentration semi-quantitative standard color bands. The colloidal gold pepsin detection test strip comprises a sample pad, a binding pad, a detection pad and a water absorption pad which are sequentially connected, wherein a detection line and a quality control line are arranged on the detection pad, pepsin monoclonal antibodies are coated on the detection line, and DNP-BSA is coated on the quality control line.

On the basis of the semi-quantitative pepsin detection product provided by the invention, the invention also provides application of the product in preparing a medical device for diagnosing and distinguishing physiological reflux and pathological reflux, the medical device detects pepsin from an extra-esophageal region, the detected pepsin concentration is compared with a pepsin standard substance concentration control, the detected pepsin concentration is less than 50ng/ml, the physiological reflux is judged, and the detected pepsin concentration is not less than 50 ng/ml.

The colloidal gold pepsin detection test strip provided by the invention can be manufactured by adopting a prior art method, can also be directly obtained from common markets, and the pepsin monoclonal antibody, each component of the test strip and the connecting mode can be manufactured by adopting the prior art method, and can also be directly obtained from the common markets.

In the specific practice of the embodiment of the invention, the monoclonal antibody used by the colloidal gold pepsin detection test strip can also be prepared by the following method: (1) constructing a pepsin eukaryotic expression vector; (2) eukaryotic expression of pepsin; (3) Pepsin is used as antigen for immunoreaction, and pepsin monoclonal antibody is prepared by culture treatment.

In the specific practice of the embodiment of the invention, the invention further discovers that the production process of the colloidal gold pepsin detection test strip has critical influence on the quality of products. The production process of the colloidal gold pepsin detection test strip comprises the steps of heating and stirring water to boil, adding sodium ascorbate and trisodium citrate, continuously heating to boil, adding tetrachloroauric acid, continuously heating and reacting, and then treating to obtain the colloidal gold particles. Wherein the addition amount of the sodium ascorbate is 0.1mol/L, the addition amount of the trisodium citrate can be selected to be 0.5wt%, and the addition amount of the tetrachloroauric acid can be selected to be 1wt%; the time for continuing the heating after the addition of the tetrachloroauric acid is preferably 15 minutes.

Drawings

FIGS. 1 to 3 are spectral scans of three batches of colloidal gold solutions prepared in example 1 of the present invention.

Detailed Description

Example 1:

a semi-quantitative pepsin detection product comprises a pepsin detection device, namely a colloidal gold pepsin detection test strip and a pepsin standard substance concentration control are pepsin standard substance concentration semi-quantitative standard color bands of 0ng/ml, 5ng/ml, 10ng/ml, 25ng/ml, 50ng/ml, 100ng/ml and 200 ng/ml.

The preparation method comprises the following steps:

pepsin eukaryotic expression pPICZ alpha A vector construction

The primer is designed by taking a nucleotide sequence corresponding to pepsin as a template. Through PCR amplification, double enzyme digestion of PCR product and carrier, cloning the nucleotide sequence corresponding to pepsin into pPICZ alpha A carrier, converting the obtained carrier into Pichia pastoris, and inducing culture.

Eukaryotic expression of pepsin

Recombinant engineering saccharomycete construction (pepsin) expression and screening,

pepsin fermentation process

Collecting glycerol bacteria in working seed library at-80deg.C, inoculating 1:100 into YPD medium, shake culturing at 200rpm and 30deg.C for 16hr, growing OD600 to 2-3, and transferring to seed solution.

Seed liquid culture

The above activated culture solution is transferred to YPD medium at 1:100, shake-cultured at 200rpm and 30deg.C for 18hr, and OD600 grows to 2-3, and transferred to a tank.

Inoculation and culture

A BSM culture medium is placed on a tank, the pH value after sterilization is about 6.8, the seed solution is placed on the tank at a ratio of 1:10 (v/v), and the ventilation is 0.5VVM; the dissolved oxygen speed is linked, the pH detection is not controlled, and the dissolved oxygen is controlled to be not lower than 20%.

About 12hr after the tank is filled, the dissolved oxygen rises, 15% YT feed is fed in, the initial flow acceleration is selected to be 1rpm/10L tank volume, the rotating speed is regulated to maintain the dissolved oxygen between 20% and 30%, and the YT feed feeding rate is gradually increased. Loading into tank for 15 hr.

Induction

Culturing in a jar for 18hr, measuring OD600 to grow to about 15, adding 0.5-0.8% methanol, and inducing for 72 hr; during induction, the supernatant was sampled every 12hr and the protein expression level was measured by HPLC.

Centrifuging in lower tank

After the induction is finished, 1M (NH 4) 2SO4 is added into the fermentation liquor according to the volume, and the fermentation liquor is placed at the temperature of 4 ℃ from supernatant fluid and transferred into a purification process.

Purification process flow

The purified water washes the pipeline until the bubbles are exhausted, and is connected with the chromatographic column;

column cleaning: the chromatographic column is washed with 2CV of purified water, 2CV of 0.5mol/L sodium hydroxide solution, 3CV of purified water, 2CV of 1mol/L sodium chloride solution and 3CV of purified water in sequence;

column balance: washing the chromatographic column with equilibrium buffer to reach electric conductivity, stable ultraviolet absorption and zero ultraviolet detection;

loading balance: loading according to the target protein load of 20+/-5 mg/ml; 2CV to UV280nm baseline stabilization by equilibration wash of chromatographic column

Elution 1: washing the chromatographic column with eluent I for 3-5CV; equilibration buffer washing chromatographic column 2-4CV to UV280nm baseline stabilization;

elution 2: washing the chromatographic column by eluent II, starting to collect effluent after UV280nm absorption is higher than 100mV, and stopping collecting when UV280nm absorption is lower than 100 mV;

column cleaning and preservation

Eluting the 2 samples at-20 ℃ for later use.

Preparation of pepsin monoclonal antibody

Antigen pretreatment, mixing a proper amount of antigen with an equal volume of Freund's complete adjuvant, and sucking and emulsifying the mixture into a water-in-oil state by using a syringe to immunize a mouse.

Immunization, carrying out secondary immunization one week after the primary immunization, replacing a complete adjuvant with an incomplete adjuvant, and carrying out the rest steps as above; third immunization: three-way is carried out two weeks after two-way, incomplete adjuvant is used for replacing complete adjuvant, and the rest are the same as above; enhancing immunity.

And (3) detecting serum titers of mice, wherein one week after three-phase mice are immunized, the mice are subjected to tail-breaking blood sampling to separate serum, the serum titers are measured by adopting an indirect ELISA method, and the mice with the serum titers of more than 10000 are selected for cell fusion.

Cell fusion and myeloma cell preparation. The SP2/0 cell cryopreservation tube was removed from the liquid nitrogen tank and thawed rapidly. Centrifugation at 1200r,2min, and supernatant discarded. SP2/0 cells were resuspended in 10ml of complete medium and added to cell culture dishes and incubated in a 5% CO2 incubator at 37 ℃.

Preparing immune spleen cells, fusing, taking a mouse with enhanced immunity, removing eyeballs, taking blood, centrifuging, and taking supernatant as a positive control; the cervical dislocation method is killed and then soaked in 75% alcohol for 5min; transferring to an ultra-clean workbench, taking out the spleen aseptically, grinding on a 200-mesh copper wire mesh, preparing spleen cell suspension and collecting in a 50ml centrifuge tube; centrifugation at 1200rpm for 5min, discarding supernatant, re-suspending in DMEM, and placing in an incubator at 37deg.C for use.

Cell fusion spleen cells and myeloma cells were mixed in a 50ml centrifuge tube in proportion, and the cell suspension was added to a 96-well cell culture plate with feeder cells spread thereon, 100ul per well, and incubated in a 5% CO2 incubator at 37 ℃.

Positive hybridoma cells clone, and positive wells obtained in the screening may contain a plurality of hybridoma clones, so that wells contain no monoclonal antibodies. In order to obtain monoclonal antibodies secreted by the same hybridoma cells, the hybridoma cells must be subcloned.

And (3) preparing ascites and collecting the ascites. The abdomen of the mouse is obviously enlarged, when the mouse is touched by hands, the skin is tense, namely, the ascites can be collected by the 12-gauge needle, the ascites is sprayed out after the needle is penetrated into the abdominal cavity, and if no ascites flows out, the needle can be lightly screwed, and the operation is performed with tolerance. Typically, the collection will be 3-4 times in succession, typically 3-5ml ascites per mouse. The ascites was centrifuged (5 min at 2000 rpm), the uppermost adipose tissue was aspirated, cellular components and other sediments were removed, and the supernatant was collected and frozen in a-20℃refrigerator.

Fourth, colloidal gold pepsin detection test strip and pepsin standard substance concentration contrast preparation

And (3) screening the antibodies, respectively coating and marking the prepared antibodies according to the process requirements of pepsin, and then pairing the coated and marked antibodies to detect antigen solution with a certain concentration and blank. And detecting the antigen solution to generate a positive signal and a blank solution signal with the maximum ratio, namely the optimal pairing antibody. Semi-quantitative standard color bands corresponding to 0ng/ml, 5ng/ml, 10ng/ml, 25ng/ml, 50ng/ml, 100ng/ml and 200ng/ml respectively are prepared according to the color development intensity of the detection line and the pepsin standard concentration.

The colloidal gold labeling process optimizing method optimizes the colloidal gold reaction condition, and the produced gold particles are stable, uniform and small in batch-to-batch difference. The order of addition of the reactants is important. If a small amount of trisodium citrate is added to a large amount of chloroauric acid, it takes a long time for trisodium citrate to disperse uniformly, and small regions with significantly different concentrations appear in the reactor therebetween, the reaction rate will be different, and thus the nucleation rate will be different, and thus the quality of colloidal gold will be poor. In contrast, if chloroauric acid is added rapidly to a trisodium citrate solution, there is little chance of forming a region of non-uniform concentration. The reactions were performed in the optimization process according to the loading sequence described below: stirring ultrapure water to boil, adding 0.1mol/L sodium ascorbate and 0.5wt% of trisodium citrate, continuously heating to boil, adding 1wt% of tetrachloroauric acid, and continuously heating for 15 minutes. In addition, the concentration of the reactant is low, and the yield is low. Thus, to achieve high yields, the reactant concentration is increased. However, when the concentration of the reactant is too high, the yield of the target product is often insufficient and a large amount of byproducts are produced. The concentration of the reactant is insufficient to ensure that the size of gold particles is not opposite or irregular in shape, the concentration of the reactant in the gold particle preparation method is optimized, the prepared gold particles are stable and uniform, and once colloid is formed, the particle size of gold is not changed no matter how long the reaction time is prolonged.

In practical operation, the quality of the colloidal gold solution is generally evaluated by three indices, that is, an absorption peak (λ), a peak width (Δλ), and an absorbance value (λod) at the absorption peak. Absorption peak (λ): according to the size of the colloidal gold particles prepared by the purpose, an absorption peak corresponding to the particle size of the purpose can be established (for example, full-wavelength scanning between 400 and 600 is used), and the index is used for evaluating whether the prepared colloidal gold solution meets the expected requirement. Even if all conditions can be uniform and stable enough in practical work, the maximum absorption peak obtained when the colloidal gold particles prepared are scanned each time is slightly deviated, and the deviation represents the difference from the target particle size, so that the established index is a range, and the smaller the range is (for example, within +/-3 nm) the better. Peak width (Δλ): the desired colloidal gold solution, ideally the particle size is uniform, but in practice it is not possible to do, and must be within a certain acceptable range, so an acceptable range of particle size distribution can be established, although the smaller the range the better, indicating that the colloidal gold solution prepared is of more uniform particle size. Absorbance value (OD lambda peak): the absorbance value of the prepared colloidal gold solution at the maximum absorption peak represents the concentration of the target particle diameter, and the larger the absorbance value is, the higher the concentration of the target particle diameter is, and the lower the concentration is otherwise. This concentration also affects the stability of the colloidal gold itself and the efficiency of subsequent labelling of the protein. The optimum value is 1+ -0.1.

FIGS. 1 to 3 are spectral scans of three batches of colloidal gold solutions prepared after optimizing the reaction conditions of the colloidal gold according to the present invention. The combination of the spectral scans of FIGS. 1-3 shows that the colloidal gold solution obtained by the optimized colloidal gold process of the invention has stable and uniform particles, small batch-to-batch difference and good quality.

And coating, namely sequentially connecting a sample pad, a binding pad, a detection pad and a water absorption pad, and further assembling the colloidal gold pepsin detection test strip.

Three batches were produced continuously according to the method, batch number: 202003001, 202003002, 202003003.

Example 2:

semi-quantitative pepsin detection product clinical evaluation

And selecting saliva of 20 clinical asymptomatic physiological reflux patients and saliva of 25 pathological reflux patients, which are collected from a second hospital infection department affiliated to Chongqing medical university, according to a hospital biosafety management system. Three batches produced continuously from the quality management system: and 202003001, 202003002 and 202003003, randomly extracting colloidal gold pepsin detection test strips, detecting, comparing the detection result with pepsin standard substance concentration semi-quantitative standard color bands, determining the pepsin semi-quantitative concentration of the detection sample, determining that the patient of the detection sample is in physiological reflux or pathological reflux according to the detected pepsin semi-quantitative concentration, determining that the pepsin semi-quantitative concentration of the detection sample is 0ng/ml, less than 5ng/ml, 10ng/ml or 25ng/ml, determining that the patient is in physiological reflux, and determining that the pepsin semi-quantitative concentration of the detection sample is 50ng/ml, 100ng/ml, 200ng/ml or more than 200 ng/ml. And comparing the detection diagnosis result with the clinical result by adopting the semi-quantitative pepsin detection product, and checking the detection sensitivity and the specificity of the semi-quantitative pepsin detection product. The test results are shown in Table 1 below:

TABLE 1

Compared with the prior art, the invention has the following advantages and beneficial effects:

the invention relates to a semi-quantitative pepsin detection product, in particular to a novel semi-quantitative gastric protein colloidal gold rapid diagnosis kit which has the advantage of distinguishing physiological or pathological reflux when pepsin is detected. From clinical verification, the diagnosis result of the semi-quantitative pepsin detection product is consistent with the clinical result, and the semi-quantitative pepsin detection product has high sensitivity and specificity. Has great clinical significance and application prospect in the aspect of early differential auxiliary diagnosis of physiological or pathological reflux.

Claims

1. A semi-quantitative pepsin detection product for distinguishing between physiological and pathological reflux comprising a device for detecting pepsin and a pepsin standard concentration control comprising at least a pepsin standard concentration control of less than 50ng/ml and a pepsin standard concentration control of not less than 50 ng/ml.

2. The semi-quantitative pepsin detection product of claim 1 wherein the pepsin standard concentration control comprises a pepsin standard concentration control of 0ng/ml, 25ng/ml, 50ng/ml, and 100 ng/ml.

3. The semi-quantitative pepsin detection product of claim 1 wherein the pepsin standard concentration control comprises a pepsin standard concentration control of 0ng/ml, 5ng/ml, 10ng/ml, 25ng/ml, 50ng/ml, 100ng/ml, and 200 ng/ml.

4. The semi-quantitative pepsin detection product according to claim 1, wherein the pepsin concentration detection device is configured to detect pepsin concentration by colloidal gold method, electrophoresis method, immunofluorescence method, direct competition method, indirect competition method, ELISA method, RIA method, flow cytometry method or immunochromatography method, preferably colloidal gold method;

the test sample of the pepsin detection device can be saliva, sputum, oral or laryngopharyngeal mucus, blood or urine, preferably saliva, sputum, oral or laryngopharyngeal mucus;

the pepsin detection device detects pepsin from an extra-esophageal region including the laryngopharynx, oral cavity, sinus cavity, airway, tracheobronchial, lung, blood vessels and urinary tract, preferably the laryngopharynx, oral cavity, sinus cavity or airway.

5. The semi-quantitative pepsin detection product according to claim 1, wherein the pepsin detection device is a colloidal gold pepsin detection test strip, and the pepsin standard concentration control is a pepsin standard concentration semi-quantitative standard color band of 0ng/ml, 5ng/ml, 10ng/ml, 25ng/ml, 50ng/ml, 100ng/ml and 200 ng/ml.

6. The semi-quantitative pepsin detection product according to claim 5, wherein the colloidal gold pepsin detection test strip comprises a sample pad, a combination pad, a detection pad and a water absorption pad which are sequentially connected, a detection line and a quality control line are arranged on the detection pad, pepsin monoclonal antibodies are coated on the detection line, and DNP-BSA is coated on the quality control line.

7. Use of a semi-quantitative pepsin detection product according to any one of claims 1-6 for the manufacture of a medical device for diagnosing and distinguishing between physiological and pathological reflux, said medical device detecting pepsin from an extra-esophageal region, the measured pepsin concentration being compared to a pepsin standard concentration control, the measured pepsin concentration being less than 50ng/ml for determining physiological reflux, the measured pepsin concentration being not less than 50ng/ml for determining pathological reflux.

8. The method for preparing a semi-quantitative pepsin detection product according to claim 5 or 6, wherein the colloidal gold particles used in the colloidal gold pepsin detection test strip are prepared by the following method: heating water, stirring to boil, adding sodium ascorbate and trisodium citrate, heating to boil, adding tetrachloro-gold acid, and heating to react to obtain colloidal gold particles.

9. The preparation method of claim 8, wherein the addition amount of sodium ascorbate is 0.1mol/L, the addition amount of trisodium citrate is 0.5wt% or 1wt% of tetrachloroauric acid;

the time for continuing the heating after the addition of the tetrachloroauric acid is preferably 15 minutes.

10. The preparation method of claim 8, wherein the pepsin monoclonal antibody used by the colloidal gold pepsin detection test strip is prepared by the following method:

constructing a pepsin eukaryotic expression vector;

eukaryotic expression of pepsin;

pepsin is used as antigen for immunoreaction, and pepsin monoclonal antibody is prepared by culture treatment.