TWI793238B - Method and kit of detecting gastrogenic protein in biological sample in vitro - Google Patents
Method and kit of detecting gastrogenic protein in biological sample in vitro Download PDFInfo
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本發明是有關於一種檢測方法及其套組,特別是有關於一種體外檢測生物樣品之胃源性蛋白的方法及套組。 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.
胃部疾病是現代人常見的文明病。大體上,胃部疾病的檢查方式有胃鏡檢查、酸鹼測定檢查、食道機能檢查等。 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.
胃鏡檢查是利用上消化道內視鏡直接觀察食道、胃、十二指腸。然而,胃鏡檢查並非百分百精確,檢查過程也會帶來一些不適,即使併用麻醉雖然無痛,但可能有潛在風險反應。 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.
酸鹼測定檢查是將酸鹼測定儀連接感應器的管線一端,經由鼻腔進入食道後,將端點置於胃食道括約肌上方的定位點,透過管線將資訊傳回另一端的主機,進行24小時的監測並記錄定位點的酸鹼值。酸鹼測定儀檢查不需麻 醉,不過由於胃部疾病患者(例如胃食道逆流)在治療時常須服用抑酸劑,可能會使檢測結果出現偽陰性。 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.
食道機能檢查是將食道機能檢測儀的細管,由鼻子放置到食道裡,可測試食道在吞嚥時將食物送到胃的運作是否正常,亦可測量下食道括約肌鬆弛的程度是否正常,以診斷食道功能是否失調或功能不良。食道機能檢查不需麻醉,不過食道機能檢查也無法百分百確認胃食道逆流。
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
另外,每年尚有數以千計的病人因胃部、食道、癌症等治療需求,須接受各種侵入式導管置入,以給藥或補充營養。前述侵入式導管例如鼻胃管(nasogastic tube,NGT)、鼻腸管(nasoduodenal tube,NDT;或稱nasojejunal tube,NJT)、口胃管(orogastric tube)、胃造廔口管(gastrostomy tube;亦稱胃管;或稱經皮內視鏡胃造口術,percutaneous endoscopic gastrostomy,PEG)及空腸造廔管(jejunostomy tube)等。一般侵入式導管之置入準確性的判斷方式之一是針對酸鹼值進行確認,惟如前述,目前胃腸疾病(例如胃食道逆流)患者在治療時常須服用抑酸劑,可能使檢測結果出現偽陰性。 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.
綜上所述,現行檢測方法多針對酸鹼值進行確認,但胃部疾病患者在治療時常須服用抑酸劑,可能會使檢測結果出現偽陰性。另外,食道與氣道的位置相近,若誤將胃管插入肺部,可能會導致吸入性肺炎或其他病症。 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.
有鑑於此,亟需發展一種體外檢測生物樣品之胃源性蛋白的方法及套組,以提供克服習知檢測方式的種種 缺點。 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.
因此,本發明之一態樣是在提供一種體外檢測生物樣品之胃源性蛋白的方法,其係根據複數種免疫檢測結果,快速且準確判斷生物樣品是否具有胃源性蛋白。 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.
本發明之另一態樣係在提供一種體外檢測生物樣品之胃源性蛋白的套組,其包含樣品導入單元、免疫檢測單元以及顯示單元,以利於快速且準確執行上述體外檢測方法。 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.
根據本發明之上述態樣,提出一種體外檢測生物樣品之胃源性蛋白的方法。在一實施例中,上述方法可包含進行第一免疫檢測步驟,以檢測生物樣品中是否存在胃蛋白酶,並獲得第一檢測結果。其次,進行第二免疫檢測步驟,以檢測該生物樣品中是否存在胃內因子,並獲得第二檢測結果。然後,根據第一免疫檢測結果及第二免疫檢測結果,判斷生物樣品是否具有胃源性蛋白。當生物樣品具有胃蛋白酶及胃內因子之至少一者時,判斷生物樣品具有胃源性蛋白。 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.
在一些實施例中,上述第一免疫檢測步驟可例如使用專一性辨識胃蛋白酶的第一抗體進行,而第二免疫檢測步驟可例如使用專一性辨識胃內因子的第二抗體進行。在上述實施例中,第一抗體或第二抗體可例如為單株抗體或多株抗體。 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.
在一些實施例中,上述生物樣品之形式不拘, 可包括但不限於例如細胞、組織、分泌物、血液、淋巴液、組織液、體液及上述任意組合。 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.
在一些實施例中,上述生物樣品可例如源自於口腔、食道、胃、腸道、氣管或侵入式導管。在上述實施例中,侵入式導管可包括但不限於鼻胃管、鼻腸管、口胃管、胃造廔口管以及空腸造瘻管。 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.
在一些實施例中,上述生物樣品可源自於哺乳類動物,例如人類。 In some embodiments, the above-mentioned biological sample can be derived from a mammal, such as a human.
根據本發明之另一態樣,提供一種體外檢測生物樣品之胃源性蛋白的套組,其包含樣品導入單元、免疫檢測單元以及顯示單元,其中免疫檢測單元可與樣品導入單元接觸,而顯示單元可呈現免疫檢測單元的檢測結果。 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.
應用本發明之體外檢測生物樣品之胃源性蛋白的方法及套組,其係根據複數種免疫檢測結果,快速且準確判斷生物樣品是否具有胃源性蛋白,例如輔助判斷胃食道逆流或侵入式導管之置入胃部準確性。 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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為讓本發明之上述和其他目的、特徵、優點與實施例能更明顯易懂,所附圖式之詳細說明如下: 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:
〔圖1〕係顯示根據本發明一實施例之數株抗胃蛋白酶抗體對胃蛋白酶的西方墨點分析圖。 [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.
〔圖2〕係繪示本發明一實施例之數株抗胃蛋白酶抗體利用三明治ELISA法檢測胃蛋白酶的標準曲線圖。 [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.
〔圖3〕係繪示本發明另一實施例之數株抗胃蛋白酶抗體利用三明治ELISA法檢測胃食道逆流患者之唾液檢體的曲線圖。 [ 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.
〔圖4A〕與〔圖4B〕係繪示根據本發明一實施例之體外檢測生物樣品之胃源性蛋白的套組檢測生物樣品的直條圖。 [ 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.
〔圖5〕係顯示根據本發明一實施例之數株抗胃內因子抗體對胃內因子的西方墨點法的影像。 [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.
〔圖6〕係繪示本發明一實施例之數株抗胃內因子抗體利用三明治ELISA法檢測胃內因子的標準曲線圖。 [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.
〔圖7〕係繪示利用三明治ELISA法評估本發明一實施例之體外檢測生物樣品之胃源性蛋白的套組檢測生物樣品之胃內因子的曲線圖。 [ 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.
承前所述,本發明提供一種體外檢測生物樣品之胃源性蛋白的方法及套組,其係根據複數種免疫檢測結果,快速且準確判斷生物樣品是否具有胃源性蛋白。 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.
本發明前述所稱的胃源性蛋白,一般可包括胃部本身或由胃部細胞分泌之內生性蛋白的總稱,例如胃蛋白酶(pepsin)及/或胃內因子(gastric intrinsic factor,GIF),但不包括胃部以外的其他外生性蛋白,惟本發明不限於此處所載。在上述實施例中,胃蛋白酶及/或胃內因子的物種來源不拘,可源自於哺乳動物,例如人類。在其他實 施例中,亦可源自於人類以外的其他哺乳動物。 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.
胃蛋白酶是一種消化性蛋白酶,其前驅物為胃蛋白酶原(pepsinogen),係由胃黏膜主細胞所分泌,功能是將食物中的蛋白質分解為小的胜肽片段。胃內因子係由壁細胞(又名泌酸細胞)所分泌。壁細胞可分泌鹽酸及GIF。鹽酸使胃蛋白酶原轉變為胃蛋白酶。GIF幫助小腸吸收維生素B12,以製造紅血球。 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.
上述二種胃源性蛋白僅出現於腸胃道,不會出現於正常口腔唾液或分泌物中。本發明藉由檢測生物樣品(例如唾液或口腔分泌物檢體)是否含有上述二種胃源性蛋白,可快速且準確判斷生物樣品是否源自於胃。 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.
本發明前述所稱的複數種免疫檢測結果係指利用複數種抗體偵測生物樣品之複數種胃源性蛋白所得的結果,藉此確認生物樣品是否為胃源性,以輔佐臨床相關應用。在上述實施例中,複數種免疫檢測結果可例如包含檢測生物樣品之胃蛋白酶及胃內因子的免疫檢測結果。 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.
在一實施例中,上述體外檢測生物樣品之胃源性蛋白的方法可至少包含以下步驟。首先,進行第一免疫檢測步驟,以檢測生物樣品中是否存在胃蛋白酶,並獲得第一檢測結果。其次,進行第二免疫檢測步驟,以檢測上述生物樣品中是否存在胃內因子,並獲得第二檢測結果。 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.
在一實施例中,上述生物樣品(或稱為檢體)之形式不拘,可包括但不限於例如細胞、組織、分泌物、血液、淋巴液、組織液、體液及上述任意組合。在一些實施例中, 上述生物樣品可例如源自於口腔、食道、胃、腸道、氣管或侵入式導管。在一些具體例中,前述侵入式導管可包括但不限於鼻胃管、鼻腸管、口胃管、胃造廔口管以及空腸造瘻管等。 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.
在一些實施例中,第一免疫檢測步驟與第二免疫檢測步驟可使用習知方式進行,其方式可包含但不限於例如直接酵素連結免疫吸附分析(enzyme-linked immunosorbent assay;ELISA)法、間接ELISA法、三明治ELISA法、西方墨點分析法、競爭性ELISA、免疫組織化學染色、側層流分析法、多重免疫分析法、放射免疫分析法、免疫放射量測定分析法、螢光免疫分析法、化學發光免疫分析法及免疫濁度測定法之至少一者,惟本發明不限於上述所舉。 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.
在一些實施例中,上述第一抗體或第二抗體並無特別限制,可例如為單株抗體或多株抗體。在間接ELISA法檢測的例子中,上述第一抗體或第二抗體可為初級抗體。在三明治ELISA法檢測的例子中,上述第一抗體或第二抗體可為捕捉及檢測抗體(capture and detection antibody)。補充說明的是,本發明所屬技術領域中具有通常知識者應可理解,抗胃蛋白酶及胃內因子的抗體已為熟知且普遍的技術,上述第一抗體或第二抗體可自行篩選獲得或使用市售商品,故不另贅述。 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.
在一些實施例中,上述第一免疫檢測步驟與第二免疫檢測步驟可為同步或不同步進行。在上述實施例中, 進行第一免疫檢測步驟之前,亦可先進行第二免疫檢測步驟。在另一些實施例中,第一免疫檢測步驟及/或第二免疫檢測步驟之分析靈敏度(analytical sensitivity),亦可稱為檢測下限值(lower limit of detection;LOD),一般而言不低於62.5ng/mL。在其他例示中,第一免疫檢測步驟及/或第二免疫檢測步驟之分析靈敏度可不低於125ng/mL。 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.
然後,根據第一免疫檢測結果及第二免疫檢測結果,判斷生物樣品是否具有胃源性蛋白。當生物樣品(例如胃食道逆流患者的口腔分泌物)具有胃蛋白酶及胃內因子之至少一者時,判斷生物樣品具有胃源性蛋白。在其他實施例中,由侵入式導管之生物樣品(黏附於臨床鼻胃插管患者的導管上的分泌物)檢測結果,可快速且準確判斷生物樣品是否具有胃源性蛋白,以輔助判斷例如胃食道逆流或侵入式導管之置入胃部準確性。 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.
在應用時,本發明另提供一種體外檢測生物樣品之胃源性蛋白的套組,以利於快速且準確執行上述體外檢測方法。在一實施例中,體外檢測生物樣品之胃源性蛋白的套組可包括樣品導入單元、與樣品導入單元接觸之免疫檢測單元以及顯示單元。樣品導入單元可於體外導入生物樣品。免疫檢測單元可包含但不限於專一性辨識胃蛋白酶的第一抗體以及專一性辨識胃內因子的第二抗體,第一抗體與第二抗體的標定方式及/或固定的位置可為不同,以利於檢測生物樣品。顯示單元可呈現免疫檢測單元的檢測結果。 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.
上述體外檢測生物樣品之胃源性蛋白的套組,其產品形式不拘,可為習知的產品形式。舉例而言,當套組為免疫層析試片時,樣品導入單元可為樣品墊,顯示單元可為顯示觀測窗,直接呈現檢測結果為陽性或陰性。另外,上述體外檢測生物樣品之胃源性蛋白的套組亦可結合其他習知檢測用的元件/設備,例如流式細胞儀、生物晶片等,以利於進行其他檢測。 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.
以下利用數個實施例以說明本發明之應用,然其並非用以限定本發明,本發明技術領域中具有通常知識者,在不脫離本發明之精神和範圍內,當可作各種之更動與潤飾。 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.
1.1評估抗胃蛋白酶單株抗體的親和力1.1 Evaluation of the affinity of anti-pepsin monoclonal antibody
此實施例利用間接ELISA法評估抗胃蛋白酶單株抗體的親和力。首先,將100μL之胃食道逆流患者之唾液〔溶於磷酸鹽緩衝溶液(PBS)中〕塗佈在96孔細胞培養盤之各孔內,4℃反應至隔夜。接著,將阻隔溶液〔含1%牛血清白蛋白(BSA)之PBS〕加入孔內,於37℃進行阻隔反應達1小時。在去除阻隔溶液後,利用PBS潤洗各孔,再於每孔加入100μL之初級抗體於37℃反應達1小時,其中初級抗體為經序列稀釋之抗胃蛋白酶抗體株85-5、抗體株90-1以及抗體株HP-1。然後,利用PBS洗去各孔未結合的 初級抗體,於每孔加入100μL之結合辣根過氧化氫酶(horse radish peroxidase;HRP)之抗小鼠IgG(IgG-HRP,以1:5000的稀釋比例溶於PBST中,偉喬生醫,台灣)作為二級抗體,於37℃反應達1小時。之後,各孔加入100μL之四甲基聯苯胺(tetramethyl benzidine,TMB)於37℃反應達10分鐘後,再於每孔加入50μL之0.1N硫酸(H2SO4)反應達10分鐘,以終止反應。接下來,利用市售酵素免疫分析儀(ELISA reader)讀取450nm的吸光值,其結果如表1所示,藉此確認上述抗體是否可辨識唾液檢體中的胃蛋白酶。每個數值為三重複。上述二級抗體的操作方式係參照製造商之操作手冊進行,此應為本發明所屬技術領域中任何具有通常知識者所熟知,故不另贅述。 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.
由表1結果可知,相較於陰性控制組,抗胃蛋白酶抗體株85-5、抗體株90-1及抗體株HP-1於62.5ng/mL之序列稀釋濃度的450nm吸光值仍高於陰性控制組,確認三株抗胃蛋白酶抗體株皆可辨識唾液檢體中的胃蛋白酶。 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西方墨點分析法評估抗胃蛋白酶單株抗體檢測生物樣品的效果1.2 Western blot analysis to evaluate the effect of anti-pepsin monoclonal antibody detection in biological samples
此實施例利用西方墨點分析法評估抗胃蛋白酶單株抗體檢測生物樣品的效果。 In this example, Western blot analysis was used to evaluate the effect of anti-pepsin monoclonal antibody in the detection of biological samples.
西方墨點分析法可使用習知方式進行。首先,利用修改配方的放射免疫沉澱測定緩衝溶液(RIPA buffer)抽取上述胃食道逆流患者唾液的蛋白質。 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).
上述所得之細胞溶解產物〔每道(lane)取10μL之細胞溶解產物〕於SDS-PAGE進行電泳後,將細胞蛋白質由SDS-PAGE膠體轉漬至聚偏氟乙烯膜(polyvinylidene difluoride membrane,PVDF),利用一級抗體於4℃作用至隔夜,其中一級抗體包括抗胃蛋白醇抗體株85-5、抗體株90-1以及抗體株HP-1。然後,利用結合過氧化酶(peroxidase)之二級抗體於室溫感作1.5小時。之後,利用市售呈色套組(例如ECL kit,PerkinElmer,Inc.)檢測蛋白質的表現量,其結果如圖1所示。關於抽取蛋白質及西方墨點分析法誠屬本發明所屬技術領域具有通常知識者所熟知,在此不另贅述細節。 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.
請參閱圖1,其顯示根據本發明一實施例之數株抗胃蛋白酶抗體對胃蛋白酶的西方墨點分析圖,其中箭頭101所指處為胃蛋白酶的色帶。
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
由圖1的結果可知,抗胃蛋白酶抗體株85-5、抗體株90-1以及抗體株HP-1皆可檢測到唾液檢體中的胃蛋白酶。 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建立抗胃蛋白酶單株抗體檢測胃蛋白酶的標準曲線1.3 Establish a standard curve for the detection of pepsin by anti-pepsin monoclonal antibody
此實施例利用三明治ELISA法評估抗胃蛋白酶單株抗體檢測胃蛋白酶的效果。 In this example, a sandwich ELISA method was used to evaluate the effect of anti-pepsin monoclonal antibody in detecting pepsin.
前述之三明治ELISA法可使用習知方式進行。首先,將捕獲抗體(capture antibody)加入96孔細胞培養盤之各孔內,於4℃作用至隔夜,以固著於孔內,其中捕獲抗體包括抗胃蛋白酶抗體株85-5以及抗體株90-1(2μg/mL,溶於PBST,每孔50μL)。接著,將300μL之阻隔溶液〔含1% BSA之PBST〕加入孔內,於37℃進行阻隔反應達1小時。然後,將50μL經序列稀釋之胃蛋白酶〔溶於磷酸鹽緩衝溶液(PBS)中〕加入各孔內,於37℃進行反應達1小時。之後,將偵測抗體(detection antibody)加入各孔內,於37℃進行反應達1小時,其中偵測抗體包括結合生物素(biotin)的抗體株HP-1(2μg/mL,溶於1×PBST,每孔50μL)。 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).
接著,加入鏈黴親和素(streptavidin)-HRP(溶於PBST中)作為二級抗體,於室溫(4℃至40℃)反應達30分鐘。然後,各孔加入50μL之TMB於37℃反應達10分鐘後,每孔加入50μL之1N硫酸(H2SO4)反應達10分鐘,以終止反應。之後,利用市售酵素免疫分析儀讀取450nm的吸光值,其結果如圖2及圖3所示,藉此驗證上述抗體辨識唾液檢體中的胃蛋白酶之標準曲線。在圖2及圖3中,每個數值為三重複。上述二級抗體的操作方式係參照製造商之操作手冊進行,此應為本發明所屬技術領域中任何具有通常知 識者所熟知,故不另贅述。 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.
請參閱圖2,其係繪示本發明一實施例之數株抗胃蛋白酶抗體利用三明治ELISA法檢測胃蛋白酶的標準曲線圖,其中曲線201代表抗體株85-5與不同濃度之胃蛋白酶的標準曲線,曲線203代表抗體株90-1與不同濃度之胃蛋白酶的標準曲線。
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
由圖2的結果可知,抗胃蛋白酶抗體株85-5以及抗體株90-1分別與抗體株HP-1配對,皆可檢測到胃蛋白酶,且其分析靈敏度(或LOD)不低於62.5ng/mL。 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評估抗胃蛋白酶單株抗體檢測生物樣品的效果(I)1.4 Evaluate the effect of anti-pepsin monoclonal antibody detection biological samples (I)
此實施例利用數個抗胃蛋白酶抗體株檢測胃食道逆流患者之唾液檢體的效果,其可使用與第1.3點或習知方式進行,不同之處在於抗原是用胃食道逆流患者之唾液檢體,其結果如圖3所示。 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.
請參閱圖3,其係繪示本發明另一實施例之數株抗胃蛋白酶抗體利用三明治ELISA法檢測胃食道逆流患者之唾液檢體的曲線圖,其中曲線301代表抗體株85-5與不同序列稀釋倍率之胃蛋白酶的標準曲線,曲線303代表抗體株90-1與不同序列稀釋倍率之胃蛋白酶的標準曲線。
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.
由圖3的結果可知,抗胃蛋白酶抗體株85-5以及抗體株90-1確實可檢測到唾液檢體中的胃蛋白酶,且於序列稀釋倍率高達400倍時,仍能檢測得到。 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評估抗胃蛋白酶單株抗體檢測生物樣品的效果(II)1.5 Evaluation of the effect of anti-pepsin monoclonal antibody detection biological samples (II)
此實施例利用數個抗胃蛋白酶抗體株檢測胃食道逆流患者或正常人之唾液檢體的效果,其可使用與第1.3點或習知方式進行,不同之處在於抗原是用胃食道逆流患者或正常人之唾液〔溶於磷酸鹽緩衝溶液(PBS)中〕,捕獲抗體包括抗胃蛋白酶抗體株85-5,偵測抗體包括結合生物素的抗體株HP-1(2μg/mL溶於含有1%BSA的PBST中),並根據圖3之標準曲線回推檢體中的胃蛋白酶濃度,其結果如圖4A及圖4B所示。 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.
請參閱圖4A及圖4B,其係繪示本發明一實施例之體外檢測生物樣品之胃源性蛋白的套組檢測生物樣品之胃蛋白酶的直條圖,其中圖4A是利用抗胃蛋白酶抗體株85-5與抗體株HP-1配對的檢測結果,而圖4B是利用抗體株90-1與抗體株HP-1配對的檢測結果。 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.
由圖4A及圖4B的結果可知,抗胃蛋白酶抗體株85-5以及抗體株90-1分別與抗體株HP-1配對,皆可檢測到胃食道逆流患者之唾液檢體中的胃蛋白酶,且其含量皆高於4000ng/mL。相較之下,正常人的唾液檢體未檢出胃蛋白酶,確實可以快速且準確判斷生物樣品是否具有胃源性蛋白。 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.
2.1西方墨點分析法評估抗GIF單株抗體檢測生物樣品的2.1 Evaluation of anti-GIF monoclonal antibody detection in biological samples by western blot analysis 效果Effect
此實施例利用上述第1.2點之西方墨點分析法評估抗GIF單株抗體檢測生物樣品的效果,不同之處在於一級抗體包括抗GIF抗體株50-1、抗體株16-1、抗體株15-3以及抗體株6-2,其結果如圖5所示。 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 .
請參閱圖5,其係顯示根據本發明一實施例之數株抗胃內因子抗體對胃內因子的西方墨點法的影像,其中箭頭501所指處為GIF的色帶。
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
由圖5的結果可知,抗GIF抗體株50-1、抗體株16-1、抗體株15-3以及抗體株6-2皆可檢測到唾液檢體中的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建立抗GIF單株抗體檢測GIF的標準曲線2.2 Establish a standard curve for the detection of GIF by anti-GIF monoclonal antibody
此實施例利用上述第1.3點之三明治ELISA法評估抗GIF單株抗體檢測生物樣品的效果,不同之處在於100μL經序列稀釋之GIF(溶於PBS中)加入各孔內,捕獲抗體包括抗GIF抗體株50-1,偵測抗體包括結合HRP的抗體株16-1(1μg/mL溶於含有1%BSA的PBST中),其結果如圖6所示。 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 .
請參閱圖6,其係繪示本發明一實施例之數株抗胃內因子抗體利用三明治ELISA法檢測胃內因子的標準曲線圖。 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.
由圖6的結果可知,抗GIF抗體株50-1與抗體株16-1配對,可檢測到GIF,且其分析靈敏度(或LOD)不低於 125ng/mL。 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評估抗GIF單株抗體檢測生物樣品的效果2.3 Evaluation of the effect of anti-GIF monoclonal antibody detection of biological samples
此實施例利用數個抗GIF單株抗體檢測生物樣品的效果,其可使用與第1.3點或習知方式進行,不同之處在於抗原是用胃食道逆流患者或正常人之唾液〔溶於磷酸鹽緩衝溶液(PBS)中〕,捕獲抗體包括抗GIF抗體株50-1,偵測抗體包括結合HRP的抗體株16-1(1μg/mL溶於含有1%BSA的PBST中),並根據圖6之標準曲線回推檢體中的GIF濃度,其結果如圖7所示。 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.
請參閱圖7,其係繪示本發明一實施例之體外檢測生物樣品之胃源性蛋白的套組檢測生物樣品之胃內因子的直條圖,其中「N.D.」代表未檢出。 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.
由圖7的結果可知,正常人的唾液檢體未檢出有GIF,而抗GIF抗體株50-1與抗體株16-1配對,可檢測到胃食道逆流患者之唾液檢體中的GIF,且其含量高於20000ng/mL,確實可以快速且準確判斷生物樣品是否具有胃源性蛋白。 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.
補充說明的是,本發明數個實施例之抗胃蛋白酶抗體株及抗GIF抗體株具有良好的親和力及靈敏度,可應用於檢測胃源性蛋白的套組及方法,以於體外快速且準確檢測生物樣本中的胃源性蛋白含量。舉例而言,可同時使用上述同一種多個抗體株或不同種的抗胃蛋白酶抗體株及/或抗GIF抗體株,於體外檢測生物樣品,藉此獲得複數種免疫檢 測結果。關於適用的生物樣本、適用於檢測胃源性蛋白的方法、套組、元件/設備等已悉如前述,不再贅述。 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.
綜言之,本發明雖以特定的單株抗體、特定的分析模式或特定的評估方式作為例示,說明本發明之體外檢測生物樣品之胃源性蛋白的方法及套組,惟本發明所屬技術領域中任何具有通常知識者可知,本發明並不限於此,在不脫離本發明之精神和範圍內,本發明之體外檢測生物樣品之胃源性蛋白的方法及套組,亦可使用其他的分析模式或其他的評估方式進行。 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.
由上述實施例可知,本發明之體外檢測生物樣品之胃源性蛋白的方法及套組,其優點在於根據複數種免疫檢測結果,快速且準確判斷生物樣品是否具有胃源性蛋白,可應用於判斷生物樣品或侵入式導管之置入胃部準確性。 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.
雖然本發明已以數個實施例揭露如上,然其並非用以限定本發明,在本發明所屬技術領域中任何具有通常知識者,在不脫離本發明之精神和範圍內,當可作各種之更動與潤飾,因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。 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.
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