KR20180049846A - A method for diagnosis of Laryngopharyngeal Reflux by detection of pepsin content in saliva and a preparation method of a sample therefor - Google Patents

Abstract

The present invention relates to a method for providing information for diagnosing laryngopharyngeal reflux and a method for preparing sample therefor, comprising the steps of: i) preparing a sample by a pre-saliva collection method; and ii) measuring pepsin content in the sample. The method for diagnosing laryngopharyngeal reflux of the present invention maximizes a concentration of pepsin in the collected saliva by controlling time and method of collecting the saliva to enable sensitive and selective detection.

Description

TECHNICAL FIELD The present invention relates to a method for diagnosing reflux phosgitus by measuring the content of pepsin in saliva and a method for preparing the sample for preparing the same.

The present invention relates to a method for providing information for diagnosing refluxing rhinopathy and a sample preparation method for the same, wherein i) a sample is prepared by a total saliva collection method, and ii) a pepsin content in the sample is measured.

Reflux laryngitis refers to the chronic inflammation of the larynx and pharynx or damage to the mucous membrane of the larynx and pharynx that is caused by the contents of the stomach, including the stomach, through the esophagus and back to the larynx and pharynx. Although the number of patients complaining with refluxing pharyngitis has been increasing due to the change in modern habits of eating habits and industrialization and urbanization, and climate and atmosphere changes, there is no simple and accurate method for diagnosing reflux lyphitis. Most of the methods of treatment are restricted to the inhibition of gastric acid secretion. Therefore, it is difficult to treat effectively such as symptoms persist after treatment.

The 24 hour esophageal acid method, known as the gold standard for refluxing rhinopharyngeitis, is costly, time-consuming, inconvenient, difficult to use, and unpredictable in patients with atypical symptoms. Patients with refluxing rhinopharyngeitis often present with nonspecific symptoms such as foreign body sensation, coughing, and voice change rather than reflux symptoms, but it is difficult to diagnose it accurately. The need for the development of noninvasive and objective diagnostic methods.

On the other hand, pepsin is a proteolytic enzyme produced only in the stomach, so saliva pepsin detection technology can be useful in a sensitive but noninvasive way to counteract the stomach contents. Saritas et al. Reported that detection of pepsin in saliva was helpful in the diagnosis of reflux esophagitis using a rapid lateral flow device (Saritas Yuksel, E. et al. , Laryngoscope, 2012, 122 (6 ): 1312-1316), and Darija et al. Found that the amount of pepsin in saliva was significantly higher in patients with reflux lice than in healthy subjects (Darija, B. et al., Collegium Antropologicum, 2012, Supp. 86). Currently, the United States and other countries have developed a simpler, more reliable, and noninvasive diagnostic method, but currently there is no research or development in Korea. In addition, the method of collecting saliva and the timing of harvesting of pepsin in saliva for the diagnosis of reflux pharyngitis is not yet known. It is essential to secure preemptive technology.

One object of the present invention is to provide a method of preparing saliva, comprising: a first step of preparing a sample by collecting saliva from an individual suspected of refluxing afterglowth infection by saliva collecting method; And a second step of measuring the content of pepsin in the sample prepared from the previous step.

Another object of the present invention is to provide a method for collecting saliva, comprising: a first step of collecting saliva within 30 minutes immediately after a gas phase; And a second step of storing the collected saliva at 0? 25 占 폚. The present invention also provides a saliva sample preparation method for detecting pepsin.

According to one aspect of the present invention, there is provided a method for preparing a test sample, comprising the steps of: preparing a sample by collecting saliva from an individual suspected of having refluxing rhinitis; And a second step of measuring the content of pepsin in the sample prepared from the previous step, to provide information for diagnosing refluxing phosophobia.

The term " laryngopharyngeal reflux (LPR) "of the present invention contaminates the throat (pharynx) which maintains a neutral pH at the steady state due to stomach contents, especially acidic substances with low pH, due to various causes. Symptoms can include, for example, diseases in which the throat is sick and sore, the chest is tight, the stomach is stiff, the voice changes, sputum comes out and cough is frequent. Such a refluxing pharyngitis may be suspected if the above symptoms are uncomfortable but no abnormal findings are found in the examination results. Although reflux phos- phuritis occurs in this way, there are many cases in which the diagnosis is usually normal, even if the gastrointestinal endoscopy is performed in an otolaryngology department or there is no special abnormality. At present, the most accurate method to diagnose this is the 24 hour esophageal acidity test, which has the disadvantage that it can cause inconvenience to the patient during the examination. Therefore, it is necessary to find a method that can accurately diagnose reflux phos- phatitis without non-invasive and uncomfortable patients.

The term "subject" of the present invention is intended to encompass any animal, including, but not limited to, a monkey, a cow, a horse, a sheep, a pig, a chicken, a turkey, a quail, a cat, a dog, a mouse, a rat, a rabbit or a guinea pig It can mean all animals, including.

The term "saliva " of the present invention is a secretion liquid secreted from the salivary glands into the mouth, often referred to as saliva. It contains more than 99% water (moisture), less than 1% of total components, various electrolytes, proteins, enzymes, immunoglobulins, antimicrobials, mucosal glycoproteins, several kinds of polypeptides and oligopeptides And very small amounts of albumin.

The term "diagnosis" of the present invention means confirming the presence or activity of a specific pathology or physiological condition. In the present invention, the diagnosis is made by examining whether the contents including gastric acid are passed through the esophagus to the larynx and pharynx Chronic inflammation or mucosal damage.

The term "pepsin" of the present invention is a digestive enzyme secreted from the above, and a pepsinogen secreted from the above chief cell is activated by hydrochloric acid. Pepsin breaks down proteins into polypeptides and is most active in acidic environments as low as pH 2. Pepsin in saliva can be used as a marker of reflux pharyngitis because pepsin is present in the acidic environment at steady state and is not present in the rhythm of the mouth, which is at neutral pH.

Thus, the present inventors have found that by measuring the concentration of pepsin in saliva, reflux lyophagitis can be diagnosed. Furthermore, optimal diagnostic collection and / or storage conditions were developed to maximize diagnostic accuracy and improve positive and negative predictive values.

The term "pre-saliva collection method" of the present invention is a method of collecting saliva by allowing an individual to spit directly on a container without using tools such as a cotton ball. Specifically, , The sputum is spit into the test tube by using a funnel once or twice a minute or the sputum is spilled out naturally and the saliva secreted during the stabilization can be collected in the test tube. Since the concentration of pepsin in the saliva collected using the saliva collecting method according to the present invention shows an average value higher than the concentration of pepsin in the saliva collected by the conventional Salivette method, it can be detected with higher sensitivity Based on this, a more accurate diagnosis can be made (FIG. 1 and Table 1).

In the first step, the saliva can be collected in a container containing 10 to 200 μmol of citric acid to stabilize pepsin, which is an analyte to be detected. The citric acid may be provided as a solution or a self-powder dissolved in a solvent. For example, it may be provided in the form of an aqueous solution of 0.05 M to 0.5 M, but is not limited thereto. When used in the form of a solution, if the concentration is too low, to less than 0.05 M, an increased volume of solution should be used to provide the same number of moles of citric acid, thus diluting the sample. Or a solution having a concentration exceeding 0.5 M, there is a large change in the number of moles of citric acid due to volume variation. To prevent this, a solution having a concentration in the above range may be used, but the present invention is not limited thereto. If the amount of citric acid used is less than 10 占 퐉 ol, it is difficult to achieve the above-mentioned purpose, which may cause decomposition of pepsin in the sample or cause infection and / or contamination of the sample. In case of more than 200 占 퐉 ol, unnecessary reagent may be wasted And may interfere with the detection signal in analyzing the sample later.

Specifically, in the first step, the saliva can be collected within 30 minutes immediately after the vapor phase. For example, the saliva can be collected without rinsing the mouth after the gas phase, without taking food or water, or without brushing the mouth.

The sample to be used in the second step may be stored at 0 to 10 DEG C for more than 0 hours and less than 24 hours after collection. Specifically, it may be stored for not less than 0 hours but not more than 12 hours, more specifically not less than 0 hours but not longer than 6 hours, but is not limited thereto. Alternatively, the sample used in the second step may be stored at 10 to 25 DEG C for not less than 0 hours but not longer than 6 hours after collection. Specifically, it may be stored for not less than 0 hours but not longer than 4 hours, but is not limited thereto. A sample stored at 0 to 10 ° C for more than 24 hours or stored at 10 to 25 ° C for more than 6 hours may have a markedly decreased amount of pepsin contained therein, Therefore, in the case of judging the onset of reflux pharyngitis based on the measured values, a case may occur in which the actual pepsin concentration is significantly decreased and judged to be negative even in a patient who is actually positive. On the other hand, when stored at 0 ° C or below under freezing conditions, the amount of pepsin rapidly decreases and falls to less than 50% within 6 hours, so that the accuracy of diagnosis may not be reliable and detection itself may not be possible (FIG.

And determining that the reflux disease occurs if the value measured from the second step is 4 ng / mL or more.

According to another aspect, the present invention provides a method for purifying saliva comprising: a first step of collecting saliva within 30 minutes immediately after the vapor phase; And a second step of storing the collected saliva at 0 to 25 DEG C, wherein the saliva sample preparation method is for detecting pepsin.

In the first step, the saliva can be collected in a container containing 10 to 200 μmol of citric acid to stabilize pepsin, which is an analyte to be detected. The citric acid may be provided as a solution or a self-powder dissolved in a solvent. For example, it may be provided in the form of an aqueous solution of 0.05 M to 0.5 M, but is not limited thereto. When used in the form of a solution, if the concentration is too low, to less than 0.05 M, an increased volume of solution should be used to provide the same number of moles of citric acid, thus diluting the sample. Or a solution having a concentration exceeding 0.5 M, there is a large change in the number of moles of citric acid due to volume variation. To prevent this, a solution having a concentration in the above range may be used, but the present invention is not limited thereto.

The second step may be carried out at 0 to 10 < 0 > C for more than 0 hours but less than 24 hours. Specifically, it may be performed at a time of more than 0 hours but not more than 12 hours, more specifically, more than 0 hours and less than 6 hours, but is not limited thereto. Alternatively, the second step may be carried out at 10 to 25 占 폚 for more than 0 hours but not longer than 6 hours. Specifically, it may be performed in a range from 0 hours to 4 hours, but the present invention is not limited thereto. Preferably at 0 to 10 DEG C for 6 hours or less, but is not limited thereto.

According to a specific embodiment of the present invention, a sample stored at room temperature, for example, or up to 6 hours when stored at 10 to 25 캜, maintains the pepsin content at a reduced level of less than about 10% relative to the initial pepsin content, / Or pepsin can be detected with selectivity, thus diagnosing refluxing pharyngitis may be possible. Further, when stored at 0 to 10 ° C under refrigerated conditions, pepsin can be detected with high sensitivity and / or selectivity because the pepsin concentration is maintained at the same level as the initial level even after storage for 6 hours. Thus, diagnosis of reflux phothyitis May be possible.

The method of diagnosing reflux liceitis according to the present invention is non-invasive, and it is possible to detect sensitively and selectively by maximizing the concentration of pepsin in collected saliva by controlling the time and method of collecting saliva and the storage conditions and time of collected saliva, High positive and negative predictive values can be used to diagnose reflux phosophobia in place of the 24-hour esophageal acidity test which is uncomfortable to the patient in the past.

FIG. 1 is a graph showing an average value and an error range of the amount of pepsin in the saliva measured from the saliva collected from the reflux phosophobia group using the saliva collection method and the salivette.
FIG. 2 is a graph comparing amounts of pepsin in the saliva collected from the saliva collected from the reflux phosophagitis patient group and the normal control group at the time of the weather, breakfast, lunch, and dinner.
3 is a graph showing changes in pepsin amount with storage temperature and / or time.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1: Reflux due to detection of pepsin in saliva by saliva collection method Pharyngitis  Diagnostic power

The saliva was collected from the patients diagnosed with reflux laryngitis by a 24-hour multi-channel intraluminal impedance test and the normal controls without reflux by the following two methods.

1) Pre-saliva collection method : Before taking saliva, take a rest from sitting on a chair, then close your lips and spit the saliva in your mouth once or twice a minute with a funnel to the test tube or spontaneously The total saliva secreted at rest was collected in a test tube.

2) Salivette Usage : Salivette (Sarstedt, Numbrecht, Germany) was gently chewed for 1 minute, then placed in a tube and centrifuged at 1,000 g for 2 minutes to collect the extracted saliva.

Pepsin in the collected saliva was analyzed using an enzyme-linked immunosorbent assay (ELISA) kit (Cloud-Clone, Houston, TX, USA). First, the saliva was put into a 96-well plate, and detection reagent A was immediately added thereto, followed by incubation at 37 ° C for 1 hour. After washing, the detection reagent B was added and incubation was further performed at 37 DEG C for 30 minutes. After washing again, TMB substrate solution was added and incubated at 37 ° C for 15 minutes. After stopping the reaction by adding stop solution, the absorbance was measured at 450 nm using a microplate reader. 3 times repeatedly, and one person who does not know the information of the subject was allowed to read.

The mean value and the error range of the measured amounts of pepsin in saliva are shown in Fig. As shown in Fig. 1, the mean value of pepsin detected in the saliva collected by the saliva collection method was 20.92 ± 20.74 ng / mL and ranged from 2.66 to 80.22 ng / mL. On the other hand, the mean value of pepsin detected in Salivette-collected saliva was 10.30 ± 6.56 ng / mL and ranged from 2.46 to 25.86 ng / mL. The sensitivity and specificity for the detection of pepsin in saliva collected by the above two methods and the positive and negative predictive values for the diagnosis of refluxing rhinitis using the same were calculated as shown in Table 1 below.

As shown in Table 1, both positive and negative predictive values for the diagnosis of refluxing rhinitis, as well as the sensitivity and specificity of saliva pepsin detection, were significantly better than those obtained using Salivette method . Especially, the two methods showed a remarkable difference in the negative predictive value. The saliva collection method, which showed excellent results in detecting pepsin and refluxing rhinitis in the two saliva extraction methods, was used in the subsequent experiments.

Saliva collection method Salivette Method responsiveness 92.0% 68.7% Specificity 83.3% 50.5% Positive predictive value 95.8% 95.6% Negative prediction 71.4% 9.1%

In Table 1, the sensitivity is defined as the probability that pepsin is detected in saliva collected from patients with reflux phosophagitis, the probability that pepsin will not be detected in saliva collected from a normal individual with no specific reflux phosophobia, Among the patients who had detected pepsin in the saliva, the percentage of patients who had actually diagnosed reflux phthophagitis and the negative predictive value showed the fraction of individuals who did not have pepsin in the saliva, Are derived from the following equations, respectively.

Example  2: Amount of pepsin detected in saliva according to saliva collection time

To all test takers; One hour after breakfast, lunch and dinner; And to collect saliva in the event of symptoms such as coughing or foreign body reflux symptoms. Subjects harvested saliva without eating or drinking water during the weather. To stabilize pepsin in saliva, 0.5 mL of 0.1 M citric acid was added in advance to collect the saliva in the prepared tube. The saliva was collected in the same manner as the saliva collection method of Example 1. The collected samples were collected in a freezer at -80 ° C and used for analysis within 2 months. In the analysis, saliva was centrifuged at 1,000 g at 4 ° C, and only the upper layer was recovered and used for analysis. Pepsin analysis and reading using an ELISA kit were performed in the same manner as in Example 1. The measured results are shown in Fig. 2 and Table 2 below.

As shown in FIG. 2 and Table 2, the amount of pepsin in the saliva was significantly higher in patients diagnosed with reflux than in the normal control at all time points, and when measured at each time point, the measured value was 17.2 ng / mL, which was 4.4 to 6.6 times higher than that measured at other time points. The amount of pepsin in the saliva collected at the time of metastasis was significantly higher than that of other biomarkers in the saliva, depending on the hormonal cycle. This suggests that the accumulation of countercurrent and pepsin in the gastric juice continuously occurs in the supine position during sleep, and there is no swallowing during sleep, so dilution of pepsin by saliva swallowing can be minimized.

Time of collection Weather After breakfast after lunch After dinner When the symptom occurs Saliva
Pepsin concentration
(ng / mL) 17.2 3.0 3.0 2.6 3.9

Example  3: Stability of pepsin with storage temperature and / or time

0.5 mL of 0.1 M citric acid was added to 1 mL of a PBS solution containing 100 ng pepsin and incubated for 6 hours and 24 hours at room temperature (20 DEG C), cold (4 DEG C) and freezing (-20 DEG C) After storage, the concentration of pepsin was measured and the results are shown in FIG.

As shown in FIG. 3, the concentration of pepsin was decreased with the lapse of time during storage at the same temperature, and the rate of decrease was remarkably high especially in refrigerated storage. On the other hand, when stored at room temperature, the reduction rate was within 10% even after 6 hours, but decreased to about 65% after 24 hours. In addition, when stored in the refrigerator, the initial concentration remained unchanged even after storage for 6 hours. However, after 24 hours, the initial concentration decreased to 80%, which may adversely affect the sensitivity of the detection and the accuracy of the diagnosis .

Claims (12)

A first step of preparing a sample by collecting saliva from a subject suspected of refluxing pharyngitis by a saliva collecting method; And
The second step of measuring the pepsin content in the sample prepared from the previous step
And a method for providing information for the diagnosis of refluxing rhinitis. The method according to claim 1,
Wherein the saliva is collected in a container containing 10 to 200 占 퐉 ol of citric acid in the first step. The method according to claim 1,
Wherein the saliva is collected within 30 minutes immediately after the vapor phase in the first step. The method of claim 3,
Wherein the saliva is collected without rinsing the mouth, not consuming food or water, and without gargling. The method according to claim 1,
Wherein the sample used in the second step is stored at 0 to 10 DEG C for more than 0 hours and less than 24 hours after harvesting. The method according to claim 1,
Wherein the sample used in the second step is stored at 10 to 25 DEG C for not less than 0 hours but not more than 6 hours after collection. The method according to claim 1,
And determining that the reflux disease occurs if the value measured from the second step is 4 ng / mL or more. A first step of collecting saliva within 30 minutes immediately after the vapor phase; And
The second step of storing the collected saliva at 0 to 25 캜
≪ / RTI > wherein the method comprises the steps of: 9. The method of claim 8,
In the first step, the saliva is collected without rinsing the mouth, taking no food or water, and without brushing. 9. The method of claim 8,
Wherein the saliva is collected in a container containing 10 to 200 占 퐉 ol of citric acid in the first step. 9. The method of claim 8,
Wherein said second step is carried out at 0 to < RTI ID = 0.0 > 10 C < / RTI > 9. The method of claim 8,
Wherein the second step is carried out at 10 to 25 DEG C for longer than 0 hours but not longer than 6 hours.

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