TWI793238B - Method and kit of detecting gastrogenic protein in biological sample in vitro - Google Patents

Abstract

The present invention relates to a method and a kit of detecting a gastrogenic protein in a biological sample in vitro, which can achieve a fast and accurately determination of the gastrogenic protein in the biological sample in vitro or a positional accuracy of an invasive catheter placed into the stomach according to a plurality of immunologically detecting results of the method.

Description

Method for in vitro detection of stomach-derived protein in biological samples and sets

The present invention relates to a detection method and its set, in particular to a method and set for in vitro detection of stomach-derived protein in biological samples.

Stomach disease is a common civilized disease in modern people. In general, the examination methods for stomach diseases include gastroscopy, acid-base measurement examination, and esophageal function examination.

Gastroscopy is the direct observation of the esophagus, stomach, and duodenum with an upper gastrointestinal endoscope. However, gastroscopy is not 100% accurate, and the examination process will also bring some discomfort. Even though anesthesia is used together, although it is painless, there may be potential risk reactions.

The acid-base measurement check is to connect the acid-base meter to the end of the pipeline of the sensor, enter the esophagus through the nasal cavity, place the end point on the positioning point above the gastroesophageal sphincter, and transmit the information back to the host computer at the other end through the pipeline for 24 hours Monitor and record the pH value of the positioning point. No anesthesia required for acid-base tester inspection However, because patients with gastric diseases (such as gastroesophageal reflux) often need to take acid suppressants during treatment, the test results may be false negative.

Esophageal function test is to place the thin tube of the esophageal function detector into the esophagus through the nose. It can test whether the operation of the esophagus to send food to the stomach during swallowing is normal. It can also measure the degree of relaxation of the lower esophageal sphincter to diagnose the esophagus. Whether it is dysfunctional or dysfunctional. Esophageal function test does not require anesthesia, but esophageal function test cannot confirm gastroesophageal reflux 100%.

In addition, thousands of patients have to undergo various invasive catheter placements every year for the treatment of stomach, esophagus, cancer, etc., in order to give medicine or supplement nutrition. The aforementioned invasive catheters such as nasogastric tube (nasogastic tube, NGT), nasoduodenal tube (nasoduodenal tube, NDT; or nasojejunal tube, NJT), orogastric tube (orogastric tube), gastrostomy tube (gastrostomy tube; also known as Gastric tube; or percutaneous endoscopic gastrostomy, percutaneous endoscopic gastrostomy, PEG) and jejunostomy tube (jejunostomy tube), etc. One of the ways to judge the accuracy of invasive catheter placement is to confirm the pH value. However, as mentioned above, patients with gastrointestinal diseases (such as gastroesophageal reflux) often need to take antacids during treatment, which may cause the test results to be inaccurate. false negative.

To sum up, the current detection methods mostly confirm the pH value, but patients with stomach diseases often need to take acid-suppressing drugs during treatment, which may make the test results false negative. In addition, the esophagus is close to the airway, and if the stomach tube is inserted into the lung by mistake, it may cause aspiration pneumonia or other diseases.

In view of this, there is an urgent need to develop a method and kit for in vitro detection of stomach-derived proteins in biological samples, so as to provide various methods to overcome conventional detection methods. shortcoming.

Therefore, one aspect of the present invention is to provide a method for in vitro detection of stomach-derived protein in a biological sample, which quickly and accurately judges whether the biological sample has stomach-derived protein according to a plurality of immunoassay results.

Another aspect of the present invention is to provide a set for in vitro detection of stomach-derived protein in biological samples, which includes a sample introduction unit, an immunological detection unit, and a display unit, so as to facilitate the rapid and accurate execution of the above in vitro detection method.

According to the above aspects of the present invention, a method for in vitro detection of stomach-derived proteins in biological samples is proposed. In one embodiment, the above method may include performing a first immunoassay step to detect whether pepsin exists in the biological sample, and obtain a first detection result. Secondly, a second immunoassay step is performed to detect whether intragastric factor exists in the biological sample, and a second detection result is obtained. Then, according to the first immunoassay result and the second immunoassay result, it is judged whether the biological sample has stomach-derived protein. When the biological sample has at least one of pepsin and gastric factor, it is judged that the biological sample has stomach-derived protein.

In some embodiments, the above-mentioned first immunodetection step can be performed, for example, using a first antibody that specifically recognizes pepsin, and the second immunodetection step can be performed, for example, using a second antibody that specifically recognizes gastric factors. In the above embodiments, the first antibody or the second antibody may be, for example, a monoclonal antibody or a polyclonal antibody.

In some embodiments, the above-mentioned biological sample is of any form, It may include, but is not limited to, cells, tissues, secretions, blood, lymph, interstitial fluid, body fluid, and any combination of the above.

In some embodiments, the above-mentioned biological sample may originate, for example, from the oral cavity, esophagus, stomach, intestinal tract, trachea, or invasive catheter. In the above embodiments, invasive catheters may include, but are not limited to, nasogastric tubes, nasoenteric tubes, orogastric tubes, gastrostomy tubes, and jejunostomy tubes.

In some embodiments, the above-mentioned biological sample can be derived from a mammal, such as a human.

According to another aspect of the present invention, a kit for in vitro detection of stomach-derived proteins in biological samples is provided, which includes a sample introduction unit, an immunodetection unit, and a display unit, wherein the immunodetection unit can be in contact with the sample introduction unit to display The unit may present the detection results of the immunodetection unit.

The method and kit for in vitro detection of gastric-derived proteins in biological samples of the present invention are used to quickly and accurately determine whether a biological sample has gastric-derived proteins based on multiple immune detection results, such as assisting in the determination of gastroesophageal reflux or invasive The accuracy of catheter placement in the stomach.

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In order to make the above and other objects, features, advantages and embodiments of the present invention more comprehensible, the detailed description of the accompanying drawings is as follows:

[FIG. 1] is a diagram showing the western blot analysis of several strains of anti-pepsin antibodies against pepsin according to an embodiment of the present invention.

[FIG. 2] is a standard curve diagram showing the detection of pepsin by several strains of anti-pepsin antibodies using the sandwich ELISA method according to an embodiment of the present invention.

[ FIG. 3 ] is a graph showing the detection of several strains of anti-pepsin antibodies in saliva specimens of patients with gastroesophageal reflux by sandwich ELISA according to another embodiment of the present invention.

[ FIG. 4A ] and [ FIG. 4B ] are bar graphs showing a kit for detecting stomach-derived proteins in a biological sample in vitro according to an embodiment of the present invention.

[FIG. 5] is an image showing western blot method of several strains of anti-intrinsic factor antibodies against intragastric factor according to an embodiment of the present invention.

[FIG. 6] is a standard curve diagram showing the detection of intragastric factor by several strains of anti-intrinsic factor antibodies using the sandwich ELISA method according to an embodiment of the present invention.

[ FIG. 7 ] is a graph showing a kit for detecting stomach-derived proteins in a biological sample in vitro using a sandwich ELISA method to evaluate gastric factors in a biological sample according to an embodiment of the present invention.

Based on the foregoing, the present invention provides a method and kit for in vitro detection of stomach-derived proteins in biological samples, which can quickly and accurately determine whether a biological sample has stomach-derived proteins based on a plurality of immunological detection results.

The stomach-derived protein referred to in the present invention can generally include the general term of endogenous proteins secreted by the stomach itself or by gastric cells, such as pepsin (pepsin) and/or gastric intrinsic factor (gastric intrinsic factor, GIF), However, other exogenous proteins other than the stomach are not included, but the present invention is not limited to those contained herein. In the above embodiments, the origin of pepsin and/or intragastric factor is not limited, and may be derived from mammals, such as humans. in other real In an embodiment, it may also be derived from mammals other than humans.

Pepsin is a digestive protease whose precursor is pepsinogen, which is secreted by the principal cells of the gastric mucosa. Its function is to decompose the protein in the food into small peptide fragments. Intrinsic factor is secreted by parietal cells (also known as acid oxyntic cells). Parietal cells can secrete hydrochloric acid and GIF. Hydrochloric acid converts pepsinogen to pepsin. GIF helps the small intestine absorb vitamin B12 to make red blood cells.

The above two stomach-derived proteins only appear in the gastrointestinal tract and do not appear in normal oral saliva or secretions. The present invention can quickly and accurately determine whether the biological sample is derived from the stomach by detecting whether the biological sample (such as saliva or oral secretion specimen) contains the above two stomach-derived proteins.

The multiple immunoassay results mentioned above in the present invention refer to the results obtained by using multiple antibodies to detect multiple stomach-derived proteins in biological samples, so as to confirm whether the biological samples are stomach-derived, and to assist clinical related applications. In the above embodiment, the plurality of immunoassay results may include, for example, the immunoassay results of detecting pepsin and intragastric factor in the biological sample.

In one embodiment, the above-mentioned method for in vitro detection of stomach-derived protein in a biological sample may at least include the following steps. Firstly, a first immunoassay step is performed to detect whether pepsin exists in the biological sample, and a first detection result is obtained. Secondly, a second immunoassay step is carried out to detect whether there is intragastric factor in the above-mentioned biological sample, and obtain a second detection result.

In one embodiment, the above-mentioned biological sample (or referred to as specimen) is not limited in form, and may include but not limited to, for example, cells, tissues, secretions, blood, lymph, interstitial fluid, body fluid, and any combination of the above. In some embodiments, The aforementioned biological sample may eg originate from the oral cavity, esophagus, stomach, intestinal tract, trachea or invasive catheter. In some specific examples, the aforementioned invasive catheters may include, but are not limited to, nasogastric tubes, nasoenteric tubes, orogastric tubes, gastrostomy tubes, and jejunostomy tubes.

In some embodiments, the first immunoassay step and the second immunoassay step can be performed using conventional methods, which may include but not limited to, for example, direct enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay; ELISA) method, indirect ELISA, sandwich ELISA, western blot assay, competitive ELISA, immunohistochemical staining, lateral laminar flow assay, multiplex immunoassay, radioimmunoassay, immunodosimetry assay, fluorescent immunoassay , at least one of chemiluminescent immunoassay and immunoturbidimetric assay, but the present invention is not limited to the above.

In some embodiments, the above-mentioned first antibody or second antibody is not particularly limited, and may be, for example, a monoclonal antibody or a polyclonal antibody. In the example of indirect ELISA detection, the above-mentioned primary antibody or secondary antibody can be a primary antibody. In the example of sandwich ELISA detection, the above-mentioned primary antibody or secondary antibody can be a capture and detection antibody. It should be added that those with ordinary knowledge in the technical field of the present invention should understand that antibodies against pepsin and gastric factor are well-known and common techniques, and the above-mentioned first antibody or second antibody can be obtained by self-screening or used Commercially available products, so no further details.

In some embodiments, the first immunodetection step and the second immunodetection step can be performed synchronously or asynchronously. In the above example, Before performing the first immunoassay step, the second immunoassay step may also be performed first. In other embodiments, the analytical sensitivity (analytical sensitivity) of the first immunoassay step and/or the second immunoassay step, also referred to as the lower limit of detection (LOD), is generally not low At 62.5ng/mL. In other examples, the analytical sensitivity of the first immunoassay step and/or the second immunoassay step may not be lower than 125 ng/mL.

Then, according to the first immunoassay result and the second immunoassay result, it is judged whether the biological sample has stomach-derived protein. When the biological sample (such as the oral secretion of a gastroesophageal reflux patient) has at least one of pepsin and gastric factor, it is judged that the biological sample has stomach-derived protein. In other embodiments, from the test results of biological samples of invasive catheters (secretion adhering to catheters of patients with clinical nasogastric intubation), it is possible to quickly and accurately determine whether the biological samples contain stomach-derived proteins, so as to assist in the determination of, for example, Gastroesophageal reflux or invasive catheter placement accuracy in the stomach.

In application, the present invention further provides a kit for in vitro detection of stomach-derived proteins in biological samples, so as to facilitate the rapid and accurate execution of the above in vitro detection method. In one embodiment, the kit for in vitro detection of stomach-derived proteins in biological samples may include a sample introduction unit, an immunodetection unit in contact with the sample introduction unit, and a display unit. The sample introduction unit can introduce biological samples in vitro. The immunodetection unit may include, but not limited to, a first antibody that specifically recognizes pepsin and a second antibody that specifically recognizes gastric factors, and the marking methods and/or fixed positions of the first antibody and the second antibody may be different. Facilitate the detection of biological samples. The display unit can present the detection results of the immunodetection unit.

The product form of the above-mentioned kit for in vitro detection of stomach-derived protein in a biological sample is not limited, and it can be a conventional product form. For example, when the set is an immunochromatographic test strip, the sample introduction unit can be a sample pad, and the display unit can be a display observation window, directly displaying whether the test result is positive or negative. In addition, the above-mentioned kit for in vitro detection of stomach-derived protein in biological samples can also be combined with other conventional detection components/equipment, such as flow cytometer, biochip, etc., to facilitate other detections.

Several examples are used below to illustrate the application of the present invention, but it is not intended to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various modifications and changes without departing from the spirit and scope of the present invention. retouch.

Example 1. Establishment and evaluation of a kit for in vitro detection of stomach-derived proteins in biological samples (I)—detection of pepsin

1.1 Evaluation of the affinity of anti-pepsin monoclonal antibody

This example evaluates the affinity of anti-pepsin monoclonal antibodies by indirect ELISA. First, spread 100 μL of gastroesophageal reflux patient’s saliva (dissolved in phosphate buffered saline (PBS)) in each well of a 96-well cell culture plate, and react at 4°C overnight. Next, a blocking solution [PBS containing 1% bovine serum albumin (BSA)] was added into the wells, and the blocking reaction was carried out at 37° C. for 1 hour. After removing the blocking solution, rinse each well with PBS, then add 100 μL of primary antibody to each well and react at 37°C for 1 hour, wherein the primary antibody is serially diluted anti-pepsin antibody strain 85-5 and antibody strain 90 -1 and antibody strain HP-1. Then, use PBS to wash away the unbound primary antibody in each well, and add 100 μL of horseradish catalase (horse radish peroxidase; HRP)-conjugated anti-mouse IgG (IgG-HRP) to each well at a dilution of 1:5000. Proportions were dissolved in PBST (Vicrion Biomedical, Taiwan) as the secondary antibody and reacted at 37°C for 1 hour. After that, 100 μL of tetramethylbenzidine (TMB) was added to each well to react for 10 minutes at 37°C, and then 50 μL of 0.1N sulfuric acid (H ₂ SO ₄ ) was added to each well for 10 minutes to terminate the reaction. reaction. Next, use a commercially available enzyme immunoassay analyzer (ELISA reader) to read the absorbance at 450 nm, and the results are shown in Table 1, thereby confirming whether the above antibody can recognize pepsin in the saliva sample. Each value is in triplicate. The operation method of the above-mentioned secondary antibody is carried out with reference to the manufacturer's operation manual, which should be well known to anyone with ordinary knowledge in the technical field of the present invention, so it will not be described in detail.

It can be seen from the results in Table 1 that compared with the negative control group, the absorbance at 450nm of the anti-pepsin antibody strain 85-5, antibody strain 90-1 and antibody strain HP-1 at a serial dilution concentration of 62.5ng/mL was still higher than that of the negative control group. In the control group, it was confirmed that all three anti-pepsin antibody strains could recognize pepsin in saliva samples.

1.2 Western blot analysis to evaluate the effect of anti-pepsin monoclonal antibody detection in biological samples

In this example, Western blot analysis was used to evaluate the effect of anti-pepsin monoclonal antibody in the detection of biological samples.

Western blot analysis can be performed using conventional means. Firstly, the protein in the saliva of the gastroesophageal reflux patients was extracted using the modified radioimmunoprecipitation assay buffer solution (RIPA buffer).

The cell lysates obtained above (take 10 μL of cell lysates for each lane) were electrophoresed on SDS-PAGE, and the cell proteins were transferred from SDS-PAGE colloid to polyvinylidene difluoride membrane (polyvinylidene difluoride membrane, PVDF) , using primary antibodies to act overnight at 4°C, wherein the primary antibodies include anti-pepsinol antibody strain 85-5, antibody strain 90-1 and antibody strain HP-1. Then, the secondary antibody conjugated with peroxidase was used to sense at room temperature for 1.5 hours. Afterwards, the expression level of the protein was detected using a commercially available color development kit (such as ECL kit, PerkinElmer, Inc.), and the results are shown in FIG. 1 . Protein extraction and western blot analysis are well known to those skilled in the art of the present invention, and details will not be repeated here.

Please refer to FIG. 1 , which shows a western blot analysis diagram of several strains of anti-pepsin antibodies against pepsin according to an embodiment of the present invention, wherein the color band of pepsin is indicated by arrow 101 .

As can be seen from the results in FIG. 1 , the anti-pepsin antibody strain 85-5, antibody strain 90-1 and antibody strain HP-1 can all detect pepsin in saliva samples.

1.3 Establish a standard curve for the detection of pepsin by anti-pepsin monoclonal antibody

In this example, a sandwich ELISA method was used to evaluate the effect of anti-pepsin monoclonal antibody in detecting pepsin.

The aforementioned sandwich ELISA method can be carried out using a known method. First, add the capture antibody to each well of the 96-well cell culture plate, and let it act at 4°C overnight to fix in the well. The capture antibody includes anti-pepsin antibody strain 85-5 and antibody strain 90 -1 (2 μg/mL, dissolved in PBST, 50 μL per well). Then, 300 μL of blocking solution [PBST containing 1% BSA] was added into the wells, and the blocking reaction was carried out at 37° C. for 1 hour. Then, 50 μL of serially diluted pepsin [dissolved in phosphate buffered saline (PBS)] was added to each well, and the reaction was carried out at 37° C. for 1 hour. Afterwards, detection antibody (detection antibody) was added to each well, and the reaction was carried out at 37°C for 1 hour, wherein the detection antibody included antibody strain HP-1 (2 μg/mL, dissolved in 1× PBST, 50 μL per well).

Next, streptavidin-HRP (dissolved in PBST) was added as a secondary antibody and reacted at room temperature (4° C. to 40° C.) for 30 minutes. Then, 50 μL of TMB was added to each well and reacted at 37° C. for 10 minutes, and then 50 μL of 1N sulfuric acid (H ₂ SO ₄ ) was added to each well for 10 minutes to terminate the reaction. Afterwards, the absorbance at 450nm was read using a commercially available enzyme immunoassay analyzer, and the results are shown in Figures 2 and 3, thereby verifying the standard curve of the antibody recognizing pepsin in the saliva specimen. In Figures 2 and 3, each value is in triplicate. The operation method of the above-mentioned secondary antibody is carried out with reference to the manufacturer's operation manual, which should be well known to anyone with ordinary knowledge in the technical field of the present invention, so it will not be described in detail.

Please refer to FIG. 2 , which is a standard curve diagram for the detection of pepsin by several strains of anti-pepsin antibodies using the sandwich ELISA method according to an embodiment of the present invention, wherein curve 201 represents the standard of antibody strain 85-5 and different concentrations of pepsin Curve, curve 203 represents the standard curve of antibody strain 90-1 and different concentrations of pepsin.

From the results in Figure 2, it can be seen that the anti-pepsin antibody strain 85-5 and the antibody strain 90-1 were paired with the antibody strain HP-1, and both of them could detect pepsin, and their analytical sensitivity (or LOD) was not lower than 62.5ng /mL.

1.4 Evaluate the effect of anti-pepsin monoclonal antibody detection biological samples (I)

This example uses several anti-pepsin antibody strains to detect the effect of the saliva samples of patients with gastroesophageal reflux, which can be carried out in the same way as point 1.3 or the conventional method, the difference is that the antigen is tested with the saliva samples of patients with gastroesophageal reflux body, and the results are shown in Figure 3.

Please refer to FIG. 3 , which is a graph showing several strains of anti-pepsin antibodies of another embodiment of the present invention using the sandwich ELISA method to detect saliva samples from patients with gastroesophageal reflux. Curve 301 represents the difference between antibody strain 85-5 and different The standard curve of pepsin at serial dilution ratios, curve 303 represents the standard curve of antibody strain 90-1 and pepsin at different serial dilution ratios.

It can be seen from the results in Figure 3 that the anti-pepsin antibody strain 85-5 and antibody strain 90-1 can indeed detect pepsin in saliva samples, and can still be detected when the serial dilution ratio is as high as 400 times.

1.5 Evaluation of the effect of anti-pepsin monoclonal antibody detection biological samples (II)

This example uses several anti-pepsin antibody strains to detect the effect of saliva samples from patients with gastroesophageal reflux or normal people, which can be carried out in the same way as point 1.3 or conventional methods, except that the antigen is used in patients with gastroesophageal reflux Or normal human saliva (dissolved in phosphate buffered saline solution (PBS)), the capture antibody includes anti-pepsin antibody strain 85-5, and the detection antibody includes biotin-binding antibody strain HP-1 (2μg/mL dissolved in 1% BSA in PBST), and the pepsin concentration in the specimen was back-estimated according to the standard curve in Figure 3, and the results are shown in Figure 4A and Figure 4B.

Please refer to FIG. 4A and FIG. 4B , which are histograms showing the detection of pepsin in biological samples by a kit for detecting stomach-derived proteins in biological samples in vitro according to an embodiment of the present invention, wherein FIG. 4A is the use of anti-pepsin antibodies The detection result of the pairing of the antibody strain 85-5 and the antibody strain HP-1, and Fig. 4B is the detection result of the pairing of the antibody strain 90-1 and the antibody strain HP-1.

From the results in Figure 4A and Figure 4B, it can be known that the anti-pepsin antibody strain 85-5 and the antibody strain 90-1 were paired with the antibody strain HP-1, both of which could detect pepsin in the saliva samples of patients with gastroesophageal reflux, And its content is higher than 4000ng/mL. In contrast, pepsin was not detected in the saliva samples of normal people, and it is indeed possible to quickly and accurately determine whether a biological sample has stomach-derived protein.

Example 2. Establishment and evaluation of a kit for in vitro detection of gastric-derived proteins in biological samples (II) - detection of gastric intrinsic factor (GIF)

2.1 Evaluation of anti-GIF monoclonal antibody detection in biological samples by western blot analysis Effect

In this example, the western blot analysis method of the above point 1.2 is used to evaluate the effect of anti-GIF monoclonal antibody detection of biological samples. The difference is that the primary antibodies include anti-GIF antibody strain 50-1, antibody strain 16-1, and antibody strain 15. -3 and antibody strain 6-2, the results are shown in FIG. 5 .

Please refer to FIG. 5 , which shows the western blot images of several strains of anti-intrinsic factor antibodies against intragastric factor according to an embodiment of the present invention, where the arrow 501 is the color band of GIF.

As can be seen from the results in FIG. 5 , anti-GIF antibody strain 50-1, antibody strain 16-1, antibody strain 15-3, and antibody strain 6-2 can all detect GIF in saliva samples.

2.2 Establish a standard curve for the detection of GIF by anti-GIF monoclonal antibody

This example uses the sandwich ELISA method in point 1.3 above to evaluate the effect of anti-GIF monoclonal antibody detection of biological samples. The difference is that 100 μL of serially diluted GIF (dissolved in PBS) is added to each well, and the capture antibody includes anti-GIF For antibody strain 50-1, the detection antibody includes antibody strain 16-1 (1 μg/mL dissolved in PBST containing 1% BSA) that binds to HRP, and the results are shown in FIG. 6 .

Please refer to FIG. 6 , which is a standard curve diagram of several strains of anti-intrinsic factor antibodies of an embodiment of the present invention using a sandwich ELISA method to detect intragastric factor.

As can be seen from the results in Figure 6, the anti-GIF antibody strain 50-1 paired with the antibody strain 16-1 can detect GIF, and its analytical sensitivity (or LOD) is not lower than 125ng/mL.

2.3 Evaluation of the effect of anti-GIF monoclonal antibody detection of biological samples

This embodiment uses several anti-GIF monoclonal antibodies to detect the effect of biological samples, which can be carried out in the same way as point 1.3 or the conventional method, the difference is that the antigen is used in the saliva of patients with gastroesophageal reflux or normal people [soluble in phosphoric acid saline buffer solution (PBS)], the capture antibody includes anti-GIF antibody strain 50-1, and the detection antibody includes antibody strain 16-1 that binds to HRP (1 μg/mL dissolved in PBST containing 1% BSA), and according to the figure The standard curve of 6 back-calculates the concentration of GIF in the sample, and the results are shown in Figure 7.

Please refer to FIG. 7 , which is a histogram showing a kit for detecting gastric-derived proteins in biological samples in vitro and detecting gastric factors in biological samples according to an embodiment of the present invention, wherein "N.D." represents not detected.

From the results in Figure 7, it can be known that GIF was not detected in the saliva samples of normal people, but the anti-GIF antibody strain 50-1 paired with the antibody strain 16-1 can detect GIF in the saliva samples of gastroesophageal reflux patients, And its content is higher than 20000ng/mL, it can indeed quickly and accurately determine whether a biological sample has stomach-derived protein.

It is supplemented that the anti-pepsin antibody strains and anti-GIF antibody strains of several embodiments of the present invention have good affinity and sensitivity, and can be applied to the kits and methods for detecting gastric-derived proteins for rapid and accurate detection in vitro Gastric-derived protein content in biological samples. For example, multiple antibody strains of the same kind or different anti-pepsin antibody strains and/or anti-GIF antibody strains can be used simultaneously to detect biological samples in vitro, thereby obtaining multiple immunoassays. test results. The applicable biological samples, methods, kits, components/equipment, etc. applicable to the detection of stomach-derived proteins have been described above and will not be repeated here.

In summary, although the present invention uses a specific monoclonal antibody, a specific analysis mode or a specific evaluation method as an example to illustrate the method and kit for in vitro detection of gastric-derived proteins in biological samples, the technology to which the present invention belongs Anyone with ordinary knowledge in the field can know that the present invention is not limited thereto. Without departing from the spirit and scope of the present invention, the method and kit for in vitro detection of gastric-derived proteins in biological samples of the present invention can also use other analysis mode or other evaluation methods.

It can be seen from the above examples that the method and kit for in vitro detection of stomach-derived proteins in biological samples of the present invention have the advantage of quickly and accurately judging whether a biological sample contains stomach-derived proteins based on multiple immunoassay results, and can be applied to To judge the accuracy of gastric insertion of biological samples or invasive catheters.

Although the present invention has been disclosed as above with several embodiments, it is not intended to limit the present invention. Anyone with ordinary knowledge in the technical field of the present invention can make various embodiments without departing from the spirit and scope of the present invention. Changes and modifications, so the scope of protection of the present invention should be defined by the scope of the appended patent application.

Claims (10)

A method for detecting gastric-derived protein in a biological sample in vitro, comprising: performing a first immunoassay step to detect whether pepsin exists in a biological sample, and obtaining a first detection result; performing a second immunoassay step , to detect whether there is a gastric factor in the biological sample, and obtain a second detection result; and according to the first immunological detection result and the second immunological detection result, determine whether the biological sample has the stomach-derived protein, Wherein, when the biological sample has at least one of the pepsin and the gastric factor, it is determined that the biological sample has the stomach-derived protein. According to the method for in vitro detection of stomach-derived proteins in biological samples described in item 1 of the scope of the patent application, wherein the first immunoassay step is performed using a first antibody that specifically recognizes the pepsin, and the first antibody is A monoclonal antibody or a polyclonal antibody. According to the method for in vitro detection of stomach-derived proteins in biological samples described in item 1 of the scope of the patent application, wherein the second immunoassay step is performed using a second antibody that specifically recognizes the gastric factor, and the second antibody It is a monoclonal antibody or a polyclonal antibody. According to the method for in vitro detection of gastric-derived protein in a biological sample according to claim 1 of the patent application, wherein the biological sample includes a secretion, a biological tissue, a blood and any combination of the above. According to the method for detecting gastric-derived protein in a biological sample in vitro according to claim 4, wherein the biological sample is derived from the oral cavity, esophagus, stomach, intestinal tract, trachea or an invasive catheter. According to the method for in vitro detection of stomach-derived protein in biological samples described in item 5 of the scope of the patent application, wherein the invasive catheter includes a nasogastric tube, a nasoenteric tube, an orogastric tube, a gastrostomy tube or a jejunum Fistula. According to the method for in vitro detection of stomach-derived protein in a biological sample according to claim 1, wherein the biological sample is derived from a mammal. According to the method for in vitro detection of stomach-derived protein in a biological sample according to claim 7, wherein the mammal is a human being. According to the method for in vitro detection of stomach-derived proteins in biological samples described in item 1 of the scope of the patent application, wherein the first immunoassay step and the second immunoassay step include direct enzyme-linked immunosorbent assay (ELISA) method, indirect ELISA, sandwich ELISA, western blot assay, competitive ELISA, immunohistochemical staining, lateral laminar flow assay, multiplex immunoassay, radioimmunoassay, immunodosimetry assay, fluorescent immunoassay , at least one of chemiluminescent immunoassay and immunoturbidimetric assay. A set for in vitro detection of gastric-derived protein in biological samples, comprising: a sample introduction unit for introducing a biological sample in vitro; an immunological detection unit in contact with the sample introduction unit, wherein the immunological detection unit includes specific recognition gastric A first antibody for protease and a second antibody for specifically recognizing gastric factors; and a display unit to present a detection result of the immunodetection unit.

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