WO2012081911A2 - Saliva purifying method - Google Patents

Abstract

The present invention relates to a method which denatures proteins contained in the saliva by suddenly varying a temperature and pH, to thereby purify the saliva. Particularly, the method of the present invention has higher accuracy compared to conventional saliva pre-treatment methods, and thus enables very small amount of hormone in the saliva to be detected, and is therefore very useful. That is, the method of the present invention may be valuably used in a pre-treatment process for a variety of analyses which use saliva as a sample.

Description

Saliva Purification Method

The present invention relates to a method for removing substances other than hormones for the analysis of hormones in saliva, and more particularly, to a method for effectively purifying saliva through specific pH control and temperature control.

Analytical steroid hormones are composed of male hormone (androgen), female hormone (estrogen), progestin, corticosteroid (corticoid), which are synthesized by the action of various enzymes using cholesterol (cholesterol) as a precursor Sterols and the like. Steroid hormones that control the endocrine system cause diseases when biological changes occur due to enzyme deficiency or excess of enzymes involved in their metabolism. Recently, changes in related steroid hormones in certain physiological and pathological conditions After analyzing the results and interpreting them in relation to physiological functions, studies are being actively conducted to investigate the causes of disease development.

Assays for evaluating various steroid hormones have been extensively developed [Endo. J., 2003, 50: 783-792], whereby 1) measuring relative changes between highly active steroids, 2) identifying changes in metabolic balance between precursors and products on metabolic pathways, and 3) appearing on metabolic pathways. By measuring the activity of enzymes related thereto from the imbalance, it is possible to comprehensively evaluate the metabolic processes of endogenous substances according to disease.

Since saliva contains a very small amount of steroid hormones, if you can accurately analyze them, you can easily identify the adrenal and gonad functions, and get information on stress levels and sexual dysfunctions.

Saliva sampling is non-invasive, so it is possible to collect saliva without the help of medical staff in accordance with the daily rhythm of the individual (wake up to bed), so that the circulating rhythm of hormone production and secretion of the individual can be grasped. Has high clinical utility because it is composed of active hormones that act on tissues (Wood, 2009)

However, because the concentration of steroids in saliva is as low as 1 / 100-1 / 1000 of the blood, it cannot be analyzed by commercially available hormone analysis kits.

Saliva contains digestive enzymes such as amylases, bacteria, mucosaccharides of slippery ingredients (Schenkels et al., 1995; Pogrel et al., 1997; Humphrey et al., 2001; Whembolua, et al., 2006 These substances can influence antigen-antibody responses during the quantitative analysis of steroid hormones in saliva by immunoassay (Tallon et al., 1985). In other words, various foreign substances, such as proteins, act as a factor of disturbance in the antigen-antibody reaction process, and thus show different results from the actual hormone levels, making accurate analysis very difficult.

Therefore, in order for saliva samples to be used for hormonal analysis for clinical purposes, a pretreatment process with accuracy and repoductivity is essential for immunoassay. Conventionally, after saliva was heat treated at 56 ° C. for 2 hours, steroid hormones contained in saliva were analyzed (Howard et al., 1989; Tamate et al., 1997), but it was difficult to have reliability in accuracy.

Accordingly, the present inventors, in order to accurately detect steroid hormones to be analyzed while effectively removing various substances contained in the collected saliva, increase the accuracy of steroid hormone detection through specific pH control and temperature control in addition to the conventional heat treatment method. It was confirmed that the present invention was completed.

An object of the present invention is to provide a method for purifying saliva for the analysis of hormones and the like in saliva.

In order to solve the above problems, the present invention provides a method for purifying saliva comprising the following steps:

(a) treating the collected saliva with a strong base of pH 12-14;

(b) cooling after heating;

(c) neutralization step; And

(d) mucus glycoprotein (mucosugar) removal step.

In particular, the method of the present invention is a step of pretreatment of saliva for the quantitative analysis of steroid hormones such as sex hormones or corticosteroids present in saliva, and can accurately detect steroid hormones using radioimmunoassay after pretreatment of the present invention. have.

Saliva collection is preferably performed within 1 hour after the gas phase. For example, it may be collected immediately after the weather, 30 minutes after the weather and 1 hour after the weather, and used to analyze the change.

In step (a), a strong base having a pH of 12 to 14 may be one selected from the group consisting of NaOH, LiOH, KOH, and Ca (OH) ₂ . In one embodiment of the present invention 1N NaOH was used.

In addition, the heating in step (b) is preferably made of a temperature of 70 ~ 90 ℃, more preferably about 80 ℃. In addition, the cooling may be performed by quenching using, for example, an ice bath.

And, after the step (b), it is preferable to further include the step of removing the denatured digestive enzymes and bacteria by pH and heat from saliva by centrifugation.

In addition, the (c) neutralization step is most typically carried out by treating a strong acid of pH 1-2, in particular, the strong acid of pH 1-2 is in the group consisting of HCl, H ₂ SO ₄ , HNO ₃ It may be one selected. In one embodiment of the present invention 1N HCl was used.

In addition, in step (d), the removal of mucus (mucosaccharides) is achieved by treating the mucus glycoprotein (mucosaccharide) degrading enzyme. Since the mucoglycoprotein (mucosaccharide) is not denatured by heat, a method of removing by using an enzyme is used.

At this time, the mucolytic enzyme can be any enzyme capable of degrading β-Nacetylhexosamine- [1 → 4] glycosidic bond, and in one embodiment of the present invention, hyaluronidase type 1 (H 3506, Sigmaaldrich). chemical co.) 500 IU was used.

Further, after removing the mucoglycoprotein (mucosugar), it is more preferable to perform the step of removing the mucoglycoprotein (mucosaccharide) degrading enzyme by centrifugation.

Finally, by further performing the step of mixing the buffer, it is possible to finish the saliva purification process. At this time, the buffer solution preferably contains the same amount of gelatin as saliva. This gelatin leads to a homogeneous distribution of trace estradiol in saliva and maintains optimum acidity even when saliva is exposed to outside air during the analysis.

The present invention is characterized by using a process of denaturing protein in saliva using rapid temperature changes and pH changes in order to accurately measure the concentration of steroid hormones in saliva, such as estradiol, testosterone, cortisol and the like. By accurate analysis of hormone concentrations, it is possible to collect information necessary for diagnosing physiological activity of women and diagnosing stress.

The present invention relates to a method for purifying saliva by denaturing proteins contained in saliva through rapid temperature changes and pH changes. In particular, the present invention is very useful because it can further increase the accuracy compared to the conventional saliva pretreatment method, it is possible to detect a small amount of hormones in saliva.

1 is a graph showing the difference in absorbance (A280 nm) after saliva sample pretreatment by the method of the present invention in combination with the conventional heat treatment method and acidity (pH) change / mucosaccharide decomposition.

Figure 2 is a graph showing the difference in steroid recovery after saliva sample pretreatment by the method of the present invention in combination with the conventional heat treatment method and acidity (pH) change / mucosaccharide decomposition.

Definitions of terms used in the present invention are as follows.

"Subject" or "subject" refers to an animal, in some embodiments a mammal, and in other embodiments a human being to be treated, observed or tested. In particular, in the present invention, a human female was targeted.

"Sample" or "sample" means a collection of similar cells obtained from a tissue of a subject or patient. Sources of tissue or cell samples may include solid tissue from fresh, frozen and / or preserved organ or tissue samples or biopsies or aspirates; Blood or any liquid tissue. In the present invention, saliva is used.

A steroid hormone is a hormone that has a steroid ring (cyclopentanohydrophenanthrene ring), a 4-membered ring hydrocarbon, as a basic structure. It is a male and female gonad hormone (sex hormone) found in higher animals and humans. ) And corticosteroids.

"Gonadotropin or sex hormone" is a hormone secreted from the gonads of females and males of vertebrates serves to promote the development of the genitals and maintain its function. The gonads that secrete hormones are hepatocytes in the testes in males and the females are mainly follicles or corpus luteum in the ovaries. Not only the gonads but also the adrenal cortex secrete substances with the action of sex hormones. During pregnancy, sex hormones are also released from the placenta, contributing to the maintenance of pregnancy. All sex hormones are steroidal substances

As used herein, the terms "about", "substantially", and the like, are used at, or in close proximity to, numerical values when manufacturing and material tolerances inherent in the meanings indicated are intended to aid the understanding of the invention. Accurate or absolute figures are used to assist in the prevention of unfair use by unscrupulous infringers.

All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. Also described herein are preferred methods or samples, but similar or equivalent ones are within the scope of the present invention. The contents of all publications described herein by reference are incorporated into the present invention.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention relates to a pretreatment process necessary for the analysis of specific proteins, hormones, etc. present in saliva, and most preferably, it is possible to efficiently remove other substances except steroid hormones for steroid hormone analysis. It relates to a method for purifying saliva.

In one embodiment of the present invention, the following steps may be included:

(a) treating the collected saliva with a strong base of pH 12-14;

(b) cooling after heating;

(c) neutralization step; And

(d) mucus glycoprotein (mucosugar) removal step.

At this time, after the step (b), it is preferable to further include the step of removing the digestive enzymes and bacteria modified by pH and heat from the saliva by centrifugation, and after the step (d), mixing the buffer solution It is more preferable to carry out further.

The following will focus on each of the above steps in more detail.

The present invention relates to a pretreatment process for the use of "saliva" as analytical sample .

In the meantime, hormone concentrations were measured mainly in blood. From an endocrinology point of view, blood is not a hormone's target organ, but a simple hormone carrier. Most blood protein hormones (growth hormones, insulin, etc.) are well soluble in water and present in an activated form in the blood, but steroids produced in the adrenal cortex and gonads are insoluble in water and therefore combined with other media (carrier proteins). Is present in the blood. When steroids are combined with the carrier, they do not act on the hormone receptors in the target organs, so they are called 'inactive' and cannot induce physiological mechanisms. Most of the steroids in the blood (95-99%) are present in this inactive state and only a fraction (1-4%) are known to be in an unbound state (active state). Therefore, the concentration of steroids in the blood can be said to be the concentration of most inactive rather than the concentration of the hormone in the active state.

On the contrary, unlike the blood, the steroid contained in the saliva is not in an active state in which all of the steroids are bound to the carrier medium, and the active steroid contained in the extracelluar fluid is the salivary gland by passive diffusion. Will be moved to. Therefore, the steroid concentration contained in saliva is recognized as an active steroid contained in the tissue.

In addition, saliva, unlike blood, can be easily collected by the general public in accordance with the individual's sleep cycle without the help of medical personnel, so it has the advantage of easily conducting research on the hormonal circadian rhythm of the individual. In other words, using saliva has the advantage of easy access to the concentration of active steroids and the hormonal circadian rhythms, which were difficult to access to blood.

The present invention relates to a method for purifying saliva that can be used for analysis using saliva as a sample, and the above advantages can be exhibited effectively.

Saliva collection can be done by spitting saliva into the collection tube and using salm that can penetrate the saliva.However, when collecting saliva using materials such as cotton, the steroid hormones contained in saliva The risk of quantitative concentrations differing from the actual amount of hormones may be due to combinations with the material or the effects of the material itself. Therefore, the preferred saliva collection method in the present invention is a method to spit saliva directly into the collection container.

In particular, for the analysis of sex hormones among steroid hormones, the saliva samples are spited directly into the provided sampling tube at the moment of waking up in the morning (within 1 minute immediately after waking up), 30 minutes after waking up and 60 minutes after waking up, respectively. Can be harvested. At this time, brushing your teeth, smoking, drinking caffeine-containing beverages for one hour after waking up, and do not eat snacks or meals.

In order to measure trace hormone concentrations contained in saliva, the present invention uses the principle of protein denaturation utilizing "pH control and abrupt temperature change", not the conventional heating method.

In a typical saliva purification process, heat treatment is performed at about 60 ° C. for 2 hours, followed by cooling, followed by centrifugation and direct analysis (Howard et al., 1989; Tamate et al., 1997). When the temperature is gradually lowered, there is a property to return to the original state and there is a problem that the separation is difficult by centrifugation.

Protein denaturation means that proteins are affected by physical factors (heating, drying, stirring, pressure, X-rays, ultrasound, vibration, freezing), chemical factors (acids, bases, urea, organic solvents, heavy metals, surfactants) and enzymatic reactions. It refers to a phenomenon in which the unique higher-order structure (secondary, third-order, fourth-order) changes without changing the peptide structure. Among them, the temperature of the thermal denaturation is usually 60 ~ 70 ℃ in the protein and the higher the temperature, the faster the denaturation rate.

In addition, since the concentration of steroids in saliva is very low, such as 1 / 100-1 / 1000 of the blood, accurate analysis was not easy due to the uncertainty of protein heat denaturation and trace amounts of steroid hormones.

However, in the present invention, by using a method of denaturing proteins more effectively by exposing and quenching to a high heat for a short time of 20-30 minutes after the strong base treatment, the accuracy during centrifugation is increased.

More specifically, first, the saliva is treated with a strong base of pH 12-14. The strong base of pH 12-14 is not limited, but may be one strong base solution selected from the group consisting of NaOH, LiOH, KOH, Ca (OH) ₂ , preferably about 1N concentration.

Next, the saliva treated with the strong base is heated to a temperature of 70 ° C. to 90 ° C. for about 20 to 30 minutes, and then the temperature is rapidly lowered. In one embodiment of the present invention was heated for about 20 minutes at 80 ℃ and then quenched using an ice bath.

As such, the present invention purifies saliva using the principle of protein denaturation through pH and rapid temperature changes, thereby making it possible to detect trace steroid hormones in saliva with high accuracy. That is, compared with the conventional heating and cooling process, the present invention is exposed to a high temperature of 20-30 minutes after the strong base treatment and quenched using an ice bath, so that the denatured protein does not have a problem of returning to its original structure. This has the effect of increasing the accuracy of hormone detection and reducing the analysis time.

In the present invention, after the heating and quenching process as described above, centrifugation is performed to remove digestive enzymes, bacteria, and the like contained in saliva. At this time, it is preferably performed for about 15000 ~ 17000rpm, 3 ~ 5 ℃, 5 ~ 10 minutes. For example, it is performed for 5 minutes at 4 ℃ at 16000 rpm.

Next, in the present invention, a neutralization step of adjusting the pH to the neutral pH is performed.

The most representative method can be used to treat strong acid solutions of pH 1-2. The strong acid having a pH of 1 to 2 may be used, for example, one selected from the group consisting of HCl, H ₂ SO ₄ , HNO ₃ , and the like, and may be used at a concentration of about 1N.

Preferably, the supernatant obtained after centrifugation is treated with a strong acid solution having a pH of 1 to 2 at a 1N concentration to adjust the neutral acidity (pH).

The obtained neutral sample is treated with a mucin glycoproteinase to remove mucus glycoprotein (mucosaccharide) in saliva. In particular, since the mucin glycoprotein is not denatured by heat, it is highly likely that the above-described denaturation process alone will be an obstacle in obtaining accurate results in the subsequent hormonal analysis.

Therefore, in the present invention, the mucus glycoprotein (mucosaccharide) is included with the step of decomposing using an enzyme.

Mucus is a biological liquid that can form a gel, which is a mixture of components that contain water and secretions of various cells. Mucins are also called mucus glycoproteins or epithelial glycoproteins, and a number of disaccharide side chains are linked by N- and O-links to the peptide core. It is a major component of the mucus and glycoconjugates that are characterized

Since the mucoglycoprotein (mucosaccharide) is not denatured by heat, various mucolytic agents or mucoglycoproteinases that can directly degrade it are used.

The mucin glycoprotein (mucosaccharide) degrading enzyme of the present invention may be any enzyme capable of degrading β-Nacetylhexosamine- [1 → 4] glycosidic bond, and in one embodiment of the present invention, hyaluronidase type 1 (H 3506, Sigmaaldrich chemical co.) 500 IU was used.

In addition, based on the mechanisms of action of mucin glycoproteinases, mucolytic agents or mucin glycoproteinases can often be classified into the following groups: proteases that break down the protein core of mucin glycoproteins (Eg, pronase, papain); Sulfhydryl compounds that cleave mucoprotein disulfide linkages; And detergents that destroy non-covalent bonds in mucus (eg, Triton X-100, Tween 20). Additional compounds may be used in this context, but not limited to, bile salts and surfactants such as sodium deoxycholate, sodium taurodeoxycholate, sodium glycolate, and Lysophosphatidylcholine.

In the present invention, the mucus glycoproteinase is treated and reacted at about 37 ° C. for 10 to 15 minutes to break down the mucus glycoprotein in saliva.

And, similarly to the previous step, the sample is heated again to a temperature of 70 ° C ~ 90 ° C, and then rapidly lowered to induce denaturation of the mucoglycoproteinase. In one embodiment of the present invention was heated for about 10 minutes at 80 ℃ and then quenched using an ice bath. Thereafter, the denatured mucin glycoproteinase is removed by centrifugation.

Finally, it is preferable to finish the saliva purification process by mixing the buffer with the obtained sample.

In particular, the buffer preferably contains the same amount of gelatin as saliva. The gelatin induces a homogeneous distribution of trace steroids, estradiol and maintains optimal acidity even when saliva is exposed to outside air during the analysis.

The saliva purification process of the present invention takes about 25 to 30 minutes for the strong base treatment, heating and quenching steps, and takes about 20 to 25 minutes for the removal of mucus glycoprotein (mucosugar) and the enzyme used therein, It takes 50 to 60 minutes. This has the effect of being much shorter in time compared to the conventional saliva purification process, which required heating at about 56 ° C. for at least 2 hours.

In addition, the greatest effect of the saliva purification process of the present invention is to bring high accuracy in the subsequent analysis of hormones and the like. That is, the present invention effectively removes impurities and other components other than steroid hormones present in trace amounts in saliva. This can be confirmed through the embodiment of the present invention.

Since the method of the present invention relates to a saliva pretreatment process for accurate analysis of a target protein contained in saliva, most preferably a steroid hormone such as sex hormone, corticosteroid, etc. Detect steroid hormones in an appropriate way. Examples include the following hormones.

"Sex hormones" express the development and function of male or female reproductive organs. In testes, testosterone is produced, and the name androgen is used according to its action. In the ovary, two steroid hormones are made. Mature follicle epithelium

胞 上皮) follicle hormone estradiol, the corpus luteum hormone progesterone is made. When they pay attention to the action, the names estrogen and gestagen are used respectively.

The "androgen" is, for example, dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), testosterone (testosterone), 5α-androstan-3α, 17β-diol (5α-androstane- 3α, 17β-diol, androstenedione, epipitosterone, 5α-androstan-3β, 17β-diol, 5α-androstane-3β, 17β-diol, androstenediol, andros Theronsterone, etiocholanolone, 11-keto-androsterone (11-keto-A), 11-keto-etiocholanolone: 11- keto-E), 11-hydroxy-androsterone (11-OH-A), 11-hydroxy-etiocholanolone (11-OH-E) and 5α Androstanedione (5α-androstanedione) and the like.

The "estrogens" include estrone, 17β-estradiol, estriol, 2-hydroxy-estrone (2-OH-E1), 2-hydrate. 2-hydroxy-estradiol (2-OH-E2), 17-epiestriol, 4-hydroxy-estrone (4-OH-E1), 4-hydroxy-estradiol (4-OH-E2), 2-methoxy-estrone (2-MeO-E1), 2-methoxy-estradiol (2 -methoxy-estradiol: 2-MeO-E2), 16α-hydroxy-estrone (16α-hydroxy-estrone: 16α-OH-E1), and the like.

In addition, in the adrenal cortex, steroid hormones (adrenal cortex hormones), collectively called cortinoids, are produced. These are all indispensable for life support, and among them, aldosterone (inorganic corticoid), corticosterone, cortisol and cortisone (sugar corticosteroid) are important.

The "adrenal cortex hormone" is triamcinolone, prednisolone, prednisone, prednisone, fluorochlorocortisone, 6α-methylprednisolone, betamethasone, dexamethasone, dexamethasone, dexamethasone, dexamethasone, dexamethasone, and dexamethasone. Flumethasone, beclomethasone, triamcinolone acetonide, desonide, flunisolide, flurandrenolide, fluorinolone acetonide ( fluocinolone acetonide, desoximethasone, budesonide, flucinonide, amcinonide, cortisol and cortisone, and the like.

On the other hand, there is no limitation as a method for detecting such steroid hormones in the purified saliva sample, it is preferable to use radioimmunoassay.

For example, enzyme immunoassay (EIA) using enzymes as markers, chemiluminescence immunoassay (CIA) with increased sensitivity with luminol or derivatives thereof or aminobutyl-ethylisoluminol (ABEI), or fluorescent label (fluorescent label) Fluorescence immunoassay (FIA) and the like can be used, and in particular, it is preferable to use radioimmunoassay.

Radioimmunoassays have many advantages, such as high sensitivity, precision, and accuracy, and allow for clear and clear detection after the reaction. Most preferably, radioimmunoassays can be used. For this purpose, a liquid-phased-double antibody method can be used. More detailed measurement procedures are available in the literature on hormone measurement using the liquid-dual antibody method (Salivary cortisol and DHEA levels in the Korean population; age-related differences, diurnal, and correlations with serum levels.Yonsei Med J. 2007; 48: 379 -88).

Most hormonal analyzes mainly use immunoassay using antigen-antibody reactions. Since various foreign substances act as disturbances in antigen-antibody reactions, they often show different results from actual hormone levels. By using the saliva sample purified by the various foreign substances such as this can be effectively removed, more accurate concentration measurement is possible.

Steroid hormone in saliva is very low level, but the saliva purified by the method of the present invention significantly increases the detection efficiency of the hormone, it can be accurately measured using radioimmunoassay.

 EXAMPLE

Hereinafter, the present invention will be described in more detail with reference to Examples.

These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Saliva Tablets

The saliva sample was checked for the purpose of treatment, menstrual cycle, menopause and timing, and weight, height and body mass index were measured. We included women with normal menstrual cycles for the last 3 months and menopausal women without normal menstrual cycles a year ago.

However, women taking hormone replacement therapy, oral contraceptives, drugs containing tamoxifen or estrogen, and progesterone cortisol; Women who have been diagnosed with abnormalities in pregnancy, lactation, infertility, sexually transmitted diseases or ovarian function; Women with severe disease such as cancer, diabetes, high / low blood pressure or abnormal body mass index (BMI ≤18 or ≥25) were excluded.

Women included in the screening were asked to spit 1 ml of saliva samples directly into the provided collection tubes each time they woke up in the morning (less than 1 minute after waking up), 30 minutes after waking up and 60 minutes after waking up.

Here, women included in the screening were not allowed to brush their teeth, smoke, drink intake of caffeine and eat snacks or meals for 1 hour after waking up, and the saliva collection time was marked on the surface of the collecting tube using an oil pen. .

Cortisol produced and secreted by the adrenal gland has a very pronounced circadian rhythm, especially at 30 minutes immediately after waking up. However, if saliva is not collected immediately after the weather, there is no difference from the cortisol concentration measured in the saliva 30 minutes after the weather. These results should be at least 2.8 nmol / L, which means that saliva was collected immediately after the weather. Therefore, after cortisol was quantitatively analyzed in the saliva of women included in the screening, only samples with a difference of more than 2.8 nmol / L were selected from samples taken immediately after the weather and 30 minutes after the weather. In addition, since sexual function is affected by internal and external stress factors, samples that are considered to have a shift in adrenal function due to extremely low or high cortisol concentration were excluded from screening.

Then, the collected saliva sample was purified.

First, saliva each treated with 1N NaOH was heated at 80 ° C. for about 20 minutes and then quenched using an ice bath. Then, centrifugation was performed to remove digestive enzymes, bacteria, and the like contained in saliva. The supernatant was then collected and treated with 1N HCl to adjust the pH to neutral acidity.

Then, the mucosaccharide degrading enzyme hyaluronidase type 1 (H 3506, Sigmaaldrich chemical co.) 500 IU was treated and reacted at about 37 ° C. for 10 minutes to decompose the mucus glycoprotein (mucosaccharide) in saliva. The sample was heated at 80 ° C. for about 10 minutes and then quenched using an ice bath. Thereafter, the denatured mucin glycoproteinase was removed by centrifugation.

Finally, the saliva purification process was completed by mixing saliva with a buffer containing 0.2% gelatin in the same amount.

The process took about 1 hour and 30 minutes in total.

Comparative Example 1 Comparison of Protein Change in Pretreatment by Existing Heat Treatment and Pretreatment by the Present Invention

For comparison with conventional methods of saliva, centrifugation of sputum and foreign substances in saliva was carried out in accordance with the existing method of saliva pretreatment (Howard et al., 1989; Tamate et al., 1997). 10 minutes) After removal, the saliva sample was heated at 56 ° C. for 2 hours, and then allowed to stand at room temperature (25 ° C.) for 10 minutes, followed by centrifugation (3000 g, 20 minutes) to obtain a supernatant. (About 3 hours). Absorbance at 280 nm (A280) was measured using a spectrophotometer (Beckman DU 800 UV / VIS).

Saliva samples treated with the method of the present invention of Example 1 were also measured for absorbance at 280 nm (A280) using a spectrophotometer (Beckman DU 800 UV / VIS).

A pretreated saliva sample was used as a control.

The results are shown in FIG.

As shown in FIG. 1, the absorbance of the untreated saliva sample was 0.05664 0.0002 and the absorbance of the previously treated saliva sample was 0.3794 0.0006. The absorbance of the saliva sample pretreated with the method of the present invention was 0.0361 00003.

As a result of Dunn's Multiple Comparison Test, it was confirmed that the samples pretreated by the conventional method and the method of the present invention had a significantly lower absorbance than the non-pretreated samples (p <0.05). There was no significant difference (p> 0.05).

This means that both the conventional method and the method of the present invention have the effect of removing protein-based material contained in saliva, and the difference in protein-based material removal effect between the two methods is not significant.

Comparative Example 2: Comparison of Steroid Hormone Loss Rate in Pretreatment by Conventional Heat Treatment and Pretreatment According to the Present Invention

When the saliva sample is pretreated by heating at 56 ° C (2 hours) and centrifuged (conventional method) and when the saliva sample is pretreated using a combination of acidity (pH) / mucosaccharide decomposition (method of the invention) Pretreatment of the saliva sample To determine whether there was a loss of steroid hormones during the course of the procedure, saliva samples were added with 5000 cpm of steroid hormones (cortisol, testosterone, estradiol) labeled with radioisotopes, and then the recovery rate of steroid hormones was examined.

The results are shown in FIG.

As a result of the Kruskal-Wallis test, there was no difference in the recovery rate of steroid hormones (cortisol, testosterone, estradiol) during the pretreatment when the saliva sample was pretreated with the conventional method and the method of the present invention (p> 0.05). There was no significant difference between the recovery of steroid hormones and the recovery of saliva samples without pretreatment (p> 0.05).

This means that steroid loss does not occur significantly in both processes.

Comparative Example 3 Comparison of Accuracy of Steroid Hormone Detection with Pretreatment by Conventional Heat Treatment and Pretreatment According to the Present Invention

As described above, steroid hormones were detected by radioimmunoassay to find out whether the conventional method and the method of the present invention have no significant difference in the amount of protein change or steroid loss rate. .

For this purpose, the saliva sample was first centrifuged (3000 g, 10 minutes) to remove the sputum component first, and the supernatant was taken. Dextrane-coated activated carbon (dextran coated 0.1% activated charcoal (w / v)) in the supernatant was stirred for 12 hours in a low temperature chamber maintained at 4 ° C to remove small molecular weight substances such as steroid hormones contained in saliva samples. (charcoal stripped saliva, CSS).

The RIA method confirmed that the CSS contained residual steroid hormones. The CSS did not contain cortisol and sex hormones (below detection range).

Known concentration in CSS (

Pretreatment of saliva samples by the conventional method and the method of the present invention is carried out by adding cortisol (500 pg / 100 ul), testosterone (50 pg / 100 ul) and estradiol (50 pg / 100 ul), respectively. Each of these steroid hormones was then quantified in saliva samples.

The results are shown in Table 1 below.

Table 1

	No pretreatment (pg / 100 ul)	Conventional Method (pg / 100 ul)	Method of the Invention (pg / 100 ul)
Cortisol addition (500 pg / 100 ul)	595.5 ± 27.5	495.1 ± 43.9	537.2 ± 1.9
Testosterone addition (50 pg / 100ul)	65.2 ± 3.7	42.1 ± 2.5	48.1 ± 1.9
Add estradiol (50 pg / 100ul)	56.2 ± 3.2	44.6 ± 2.3	47.9 ± 2.0

As shown in Table 1, steroid hormones were detected slightly higher than expected in saliva samples that were not pretreated. This is interpreted as the effect that the high molecular weight substances (proteins, mucosaccharides, etc.) that could not be removed by activated carbon contained in the saliva sample interfered with the antigen-antibody reaction in hormonal quantitative analysis.

In the saliva sample pretreated by the conventional method, sex hormones except cortisol were detected slightly lower than expected concentration, and the standard error value was also slightly larger, indicating that reliability was not high due to low accuracy or repeatability.

On the contrary, in the case of the method of the present invention, steroid hormones are detected without significant difference from the expected concentration, it can be seen that the method of the present invention improves the accuracy of the detection concentration.

If the detection is not accurate, there is no value as a sample of the quantitative analysis, the present invention is a technique that improves the accuracy of hormone extraction without the difference between the change in protein amount and steroid hormone loss rate.

Claims (16)

1.  Saliva purification method comprising the following steps:

   (a) treating the collected saliva with a strong base of pH 12-14;

   (b) cooling after heating;

   (c) neutralization step; And

   (d) mucus glycoprotein (mucosugar) removal step.
2. According to claim 1, The method is a saliva purification method, characterized in that the step of pretreatment saliva for quantitative analysis of steroid hormones.
3. The method of claim 2, wherein the steroid hormone is sex hormone or corticosteroid.
4. The method of claim 1, wherein the strong base having a pH of 12 to 14 in step (a) is one selected from the group consisting of NaOH, LiOH, KOH, Ca (OH) ₂ .
5. The method of claim 1, wherein the heating in step (b) is characterized in that at a temperature of 70 ~ 90 ℃.
6. The method of claim 1, wherein the cooling in step (b) is performed by quenching using an ice bath.
7. The method of claim 1, further comprising, after step (b), removing digestive enzymes and bacteria from saliva by centrifugation.
8. The method of claim 1, wherein the step (c) neutralization is carried out by treating a strong acid of pH 1-2.
9. The method of claim 8, wherein the strong acid having a pH of 1 to 2 is one selected from the group consisting of HCl, H ₂ SO ₄ , and HNO ₃ .
10. The method of claim 1, wherein in step (d), the mucus glycoprotein (mucosugar) removal is performed by treating the mucus glycoprotein (mucosaccharide) degrading enzyme.
11. The method of claim 10, wherein the mucoglycoprotein (mucosaccharide) degrading enzyme is characterized in that to decompose Nacetylhexosamine- [1 → 4] glycosidic bond.
12. The method of claim 1, further comprising removing mucus glycoprotein (mucosaccharide) degrading enzyme by centrifugation after removing the mucus glycoprotein (mucosaccharide) in the step (d).
13. The method of claim 12, further comprising the step of mixing the buffer after removing the mucoglycoprotein (mucosugar) degrading enzyme.
14. The method of claim 13, wherein the buffer comprises the same amount of gelatin as saliva.
15. The method of claim 1 wherein saliva is collected within 1 hour after the weather.
16. The method of claim 2, wherein the quantitative analysis of steroid hormones uses radioimmunoassay.

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