200538737 (1) 九、發明說明 【發明所屬之技術領域】 本發明爲有關內毒素之試驗方法及培養物之處理方法 ’特別關於爲確認於試料中有無內毒素之內毒素之試驗方 法及培養物之處理方法。 【先前技術】 近年來，生體親和性佳之膠原蛋白等天然來源之材料 ’多被利用於創傷披覆材料及組織培養製品之基礎材料等 醫療用具，但於另一方面看來，於此類天然來源之材料， 具有微生物污染之可能性，特別關注者爲內毒素引起之污 染。 內毒素爲革蘭氏陰性菌之內毒素，侵入人體時，具引 起惡寒伴隨著發燒及過敏性休克之可能性。爲防止這些倂 發症，對於使用天然來源之材料之醫療用具’要求內毒素 之管理，並且爲內毒素之檢出及定量，根據日本藥局方規 定內毒素試驗法（第十四改正日本藥局方’厚生勞動省， 2001年3月30日，第20頁〜第23頁），更進一步地其 改良方法等正被提案及實施。 例如，於「對於利用『天然來源物質』做爲創傷披覆 材料之使用」（厚生省藥物局醫療機器開發科事務聯絡 •審查事務聯絡（92-8)，平成4年4月14臼）’記載 以檢體1 〇倍之生理食鹽水於室溫下’進行7 2小時之萃取 ’測定其萃取液之內毒素之測定方法。 -4- 200538737 (2) 另外’於「天然來源醫用材料之內毒素回收法之開發 」（第24次日本生醫材料學會大會予稿集，20〇2年， P223 )指出’膠原蛋白等易吸附內毒素，以上述之藉由生 理食鹽水萃取之方法並不完備，另外，有報告指出，藉由 滅菌生理食鹽水精製膠原蛋白酶使膠原蛋白溶解，以此溶 解液爲基礎，進行內毒素之試驗，能夠使LPS (脂多糖， 亦即內毒素）之回收率飛躍地改善。 然而’一般易取得之膠原蛋白酶多來自微生物，因含 高量之內毒素’如上述方法於內毒素試驗之前處理使用膠 原蛋白酶時，須以內毒素去除管柱等，自膠原蛋白酶中去 除內毒素，相當費時。另外，因藉由酵素處理之溶解費時 ，爲進行正確的試驗，必須耗費長時間使試藥充分溶解。 另外爲利用於醫療用移植片之培養物，深切希望於使 用前對於各種成分進行迅速的試驗，以及必須盡可能地排 除費時之步驟。 本發明有鑒於上述先前技術之問題點，以提供即便使 用易吸附內毒素之材料，確認於試料中無內毒素，及能夠 簡便且迅速測量內毒素量之內毒素試驗方法爲目的。 另外本發明爲提供於培養物中不限於內毒素之各種成 分，簡便進行試驗之有效處理方法爲目的。 【發明內容】 本發明之內毒素試驗方法，其特徵爲對於試料進行內 毒素試驗之內毒素試驗方法，包含以酸性溶液溶解試料， -5- 200538737 (3) 獲得溶解試料液的步驟，及以具緩衝作用之溶液中和上述 溶解試料液，獲得中和試料液的步驟，及對上述中和試料 液進行內毒素之試驗的步驟。 B外本發明之培養物處理方法，其特徵包含將培養物 以酸性溶液溶解，獲得溶解試料液的步驟，及以具緩衝作 用之溶液中和上述溶解試料液，獲得中和試料液的步驟。 上述試料或培養物，最好是含有天然來源物質，特別 是最好含有膠原蛋白。另外，上述之試料最好是膠原蛋白 及以保持於此膠原蛋白之細胞所構成之培養物，且最好特 別是軟骨細胞包埋於膠原蛋白凝膠之培養軟骨。另外，上 述酸性溶液最好爲無機酸，且最好特別是鹽酸。另外，上 述具緩衝作用之溶液最好爲磷酸緩衝液。 若依據本發明，以酸性溶液溶解試料，接著因中和後 再進行內毒素試驗，無論是否爲易吸附內毒素之試料，對 於試料亦能夠簡便且迅速進行內毒素試驗。 另外若依據本發明，以酸性溶液溶解培養物，接著中 和’因其爲中和試料液，故不限於內毒素，可簡便且迅速 進行各種易吸附於培養物之成分之試驗。另外，若依據本 發明’可較藉由酵素等試藥溶解，更早期地溶解試料。 【實施方式】 〔實施發明之最佳型態〕 本發明之內毒素試驗方法，爲包含以酸性溶液溶解試 料’獲得溶解試料液的步驟，及以具緩衝作用之溶液中和 -6 - 200538737 (4) 上述溶解試料液，獲得中和試料液的步驟，及對上述中和 試料液進行內毒素試驗的步驟。 本試驗方法之對象試料爲存在含有內毒素之可能性， 若爲可以酸性溶液溶解之試料更佳，特別最好是其含有易 吸著或者混入內毒素之物質。試料中含有之物質爲天然來 源物質，可舉出例如膠原蛋白、明膠、殼多醣、植物樹膠 、果膠、藻酸、血纖蛋白、白蛋白、透明質酸、彈性蛋白等 ’這些物質無論爲單獨或者爲2種以上之組合均可。特別 最好是易吸附內毒素，以普通之萃取方法正確測定有困難 且含有膠原蛋白之試料。膠原蛋白爲水溶性膠原蛋白或非 水彳谷性膠原蛋白任何一種均可，另外自膠原蛋白去除端 後所得之端膠原（atelo-collagen)亦可。 尙且’試料中所含物質爲不僅只有一部分之物質存在 於試料全體中，亦包含構成試料全體之大部分時。 另外，試料爲含有細胞及細胞外間質（ECM : Extra Cellular Matrix)以及細胞激素等各種因子，或具含有之 可能性者亦可。此類細胞例如軟骨細胞、造骨細胞、肝細 胞、表皮細胞、纖維母細胞、上皮細胞（包含角膜上皮細 胞、黏膜上皮細胞、羊膜上皮細胞等）、內皮細胞、平滑 肌細胞、肌肉母細胞（筋芽細胞）、實質細胞、心肌細胞 、胰島細胞等。接著，可舉出試料爲以上述之天然來源物 質所構成之基礎材料上，進行細胞培養而得培養物，例如 膠原蛋白及以保持於此膠原蛋白之細胞所構成之培養物， 最好爲將軟骨細胞包埋於膠原蛋白凝膠之培養軟骨才符合 -7- 200538737 (5) 。特別希望培養軟骨等培養物適用於本發明時，能擔保對 此培養物於其後使用時具高信賴感。 於本試驗方法’ g式料以酸性溶液於溶解的步驟中溶解 ’由此可獲得藉酸性溶液所溶解之試料液，而認爲試料中 存在之成分分散於溶解試料液中。 此處所指之酸性溶液，爲若能夠溶解試料之酸性溶液 爲佳’且能對應試料之種類適宜地決定。考慮試料之溶解 時間等，ρΗ0·1〜5.9範圍之酸，最好特別爲 範圍之強酸。此類之強酸，於業界爲一般已知之強酸性物 質無機酸（例如鹽酸、硫酸、硝酸）及有機酸（例如三 氯乙酸、石碳酸），單獨或組合使用，能更容易製作。另 外，使用一般所知之弱酸性物質有機酸（例如醋酸、檸檬 酸、號拍酸、硝酸），可用於調整製作強酸於上述p Η 0.1 〜3 · 5之範圍’亦可使用弱酸性物質與強酸性物質之組合 〇 此類型之酸性溶液，做爲試料時因種類而異，例如使 用鹽酸時以ImM〜5 00mM之濃度，最好使用3〇mM〜 1 OOmM之濃度。少於ImM時，因無法溶化膠原蛋白故不 希望使用，若高於5 OOmM，則無法以緩衝溶液充分中和 ’亦不希望使用。 另外使用酸性溶液溶解試料時，可使用試料之1〜5 0 倍量之酸性溶液，最好是2〜1 0倍量之酸性溶液。若酸性 溶液量少於試料之1倍，其試驗精確度變低，多於5 0倍 ’則試驗敏感度下降。尙且’酸性溶液特別最好爲無內毒 -8 - 200538737 (6) 素者，若溶液中之內毒素濃度不會對於試驗結果有 影響程度低，例如於檢測質之下，即能夠利用。另 慮內毒素於酸性溶液之安定性，希望提供溶解後快 和步驟。 尙且，以酸性溶液溶解試料之前，是否除去試 能中和酸性溶液之成分，爲解決此問題，最好使用 (低pH値）之酸性溶液。 如此所得之溶解試料溶液，其酸性之pH ρΗ0·3〜3，特別最好以pHl〜3之範圍做爲試料之 圍，使用於後續中和步驟。p Η値低於p Η 0 · 3者， 液變爲不易中和，故不希望使用，pH値高於ρΗ3 爲不能充分溶解膠原蛋白亦不希望使用。 於溶解步驟所得之溶解試料液，於中和步驟以 作用之溶液中和。由此可得中和試料液。 此處所指具有緩衝作用之緩衝溶液，若爲具有 酸性溶液溶解試料後所得溶解試料溶液者爲佳，能 地對應酸性溶液及試料種類而決定。具體而言可舉 緩衝液、BSE(N，N—雙（2 —乙基羥）一 2 —牛磺 衝液、三羥甲基氨基甲烷緩衝液等。此類溶液其鹽 愈多，緩衝能不會過高，另外不會因中和溶解試料 之量變得過大，而使試驗敏感度下降，於本發明是 意的。由緩衝能強度及能維持高內毒素試驗檢出率 看來，最好是使用磷酸緩衝液。 中和溶解液時，藉由使溶解試料液之pH値變 影響， 外，考 速之中 料中可 高濃度 最好爲 pH範 緩衝溶 者，變 具緩衝 中和以 夠適宜 出磷酸 酸）緩 類析出 液所需 令人滿 此觀點 爲6.0 200538737 (7) 〜8 · 0，可獲得中和試料液。如此對於ρ Η値之調整，一般 而言係以具緩衝作用之溶液，將溶解試料液稀釋1 0〜1 00 倍而進行，可適宜的選擇因應具有上述緩衝作用之溶液。 於中和步驟中所得之中和試料液，提供於試驗步驟中 內毒素之試驗用。 此處所指之試驗係不只包含中和試料液中，內毒素量 之試驗方法，亦包含單純地檢出有無內毒素。若爲業者， 可以容易地自業界週知之方法中選擇一個最適宜之方法實 行。例如，可舉出使用內毒素檢測（Limulus )試藥（ Lysate試藥）所用之凝膠化法、比濁法及比色法等，曰本 藥局方所定之內毒素試驗法等爲符合者。尙且，採用內毒 素試驗法時’最好實行已知之反應干擾因子試驗等以確認 檢出能力。 尙且，於試驗進行之時，爲求適當之試料濃度，可以 蒸餾水等再稀釋中和試料液。 接著說明對本發明之培養物之處理方法。 本發明之培養物之處理方法，係包含以酸性溶液溶解 培養物，獲得溶解試料液之溶解步驟，及以具緩衝作用之 溶液’中和上述溶解試料液，獲得中和試料液的步驟。 此培養物之處理方法所指之培養物，係包含可能適用 於上述內毒素試驗方法。此類型之培養物適宜者，爲與做 爲培養對象之細胞所決定之培養基存在下，定期培養所得 者’特別最好爲與基礎材料共同培養。此處所指之基礎材 料適宜者’爲上述之天然來源物質，特別最好爲膠原蛋白 -10- 200538737200538737 (1) IX. Description of the invention [Technical field to which the invention belongs] The present invention relates to a test method for endotoxin and a method for processing a culture ', and particularly to a test method and culture for confirming the presence or absence of endotoxin in a sample The processing method. [Prior art] In recent years, materials of natural origin, such as collagen with good biocompatibility, have been used in medical devices such as wound covering materials and basic materials for tissue culture products. Materials of natural origin have the potential for microbial contamination, with special attention being given to endotoxin-induced contamination. Endotoxin is an endotoxin of gram-negative bacteria. When it invades the human body, it may cause chills with fever and anaphylactic shock. In order to prevent these outbreaks, the management of endotoxin is required for medical devices that use materials of natural origin, and for the detection and quantification of endotoxin, the Endotoxin Test Method (14th Correction of Japanese Medicine The bureau's Ministry of Health, Labour and Welfare, March 30, 2001, pages 20 to 23), and further improvements are being proposed and implemented. For example, "Use of" naturally-derived substances "as a wound covering material" (Information Liaison and Examination Affairs Liaison (92-8), Medical Device Development Division, Medical Bureau, Ministry of Health, and Welfare, April 14, 2004) A method for measuring endotoxin in the extract by 'extracting for 72 hours at room temperature' using physiological saline solution at 10 times the specimen. -4- 200538737 (2) In addition, "In the development of the endotoxin recovery method for medical materials of natural origin" (Proceedings of the 24th Japan Society of Biomedical Materials Congress, 2002, P223) pointed out that The method of adsorbing endotoxin by the above-mentioned extraction with physiological saline is not complete. In addition, there are reports that collagen is dissolved by refining collagenase by sterilizing physiological saline to dissolve collagen. The test can dramatically improve the recovery of LPS (lipopolysaccharide, also known as endotoxin). However, 'generally available collagenases mostly come from microorganisms because of high content of endotoxins'. As mentioned above, when using collagenase before the endotoxin test, the endotoxin removal column must be used to remove endotoxin from collagenase. Quite time consuming. In addition, since the dissolution by enzyme treatment is time-consuming, it takes a long time to fully dissolve the test reagent in order to perform a correct test. In addition, in order to use the culture of medical grafts, it is strongly desired that the various components be tested quickly before use and that time-consuming steps must be eliminated as much as possible. The present invention has been made in view of the problems of the above-mentioned prior art, and aims to provide an endotoxin test method for confirming the absence of endotoxin in a sample even if a material that easily adsorbs endotoxin is used, and the amount of endotoxin can be simply and quickly measured. In addition, the present invention aims to provide an effective treatment method that is not limited to endotoxin in the culture and can be easily tested. [Abstract] The endotoxin test method of the present invention is characterized in that the endotoxin test method for performing an endotoxin test on a sample includes dissolving the sample with an acidic solution, -5- 200538737 (3) a step of obtaining a dissolved sample liquid, and A solution having a buffering effect neutralizes the above-mentioned dissolved sample liquid to obtain a neutralized sample liquid, and a step of performing an endotoxin test on the above-mentioned neutralized sample liquid. In addition, the culture treatment method of the present invention includes a step of dissolving the culture in an acidic solution to obtain a dissolving sample solution, and a step of neutralizing the dissolving sample solution with a buffering solution to obtain a neutralization sample solution. The sample or culture preferably contains a substance of natural origin, and particularly preferably contains collagen. In addition, the above-mentioned sample is preferably a collagen and a culture composed of cells retained in the collagen, and particularly a cultured cartilage in which chondrocytes are embedded in a collagen gel. In addition, the above-mentioned acidic solution is preferably an inorganic acid, and particularly preferably hydrochloric acid. In addition, the buffering solution is preferably a phosphate buffer solution. If the sample is dissolved in an acidic solution according to the present invention, and then the endotoxin test is performed after neutralization, the endotoxin test can be easily and quickly performed on the sample regardless of whether it is a sample that easily adsorbs endotoxin. In addition, according to the present invention, if the culture is dissolved in an acidic solution and then neutralized, since it is a neutralization sample liquid, it is not limited to endotoxin, and various components that are easily adsorbed on the culture can be easily and quickly tested. In addition, according to the present invention, it is possible to dissolve the sample earlier than to dissolve it by a reagent such as an enzyme. [Embodiment] [The best form of implementing the invention] The endotoxin test method of the present invention includes a step of dissolving a sample with an acidic solution to obtain a dissolving sample liquid, and neutralizing the solution with a buffering effect-6-200538737 ( 4) The steps of dissolving the sample liquid, obtaining a neutralized sample liquid, and performing the endotoxin test on the neutralized sample liquid. The target sample of this test method is likely to contain endotoxin. If it is an acid solution that can be dissolved, it is more preferable, and it is particularly preferable that it contains a substance that is easily absorbed or mixed with endotoxin. The substance contained in the sample is a substance of natural origin, and examples thereof include collagen, gelatin, chitin, plant gum, pectin, alginic acid, fibrin, albumin, hyaluronic acid, and elastin. They can be used alone or in combination of two or more. Particularly, it is preferable to easily adsorb endotoxin, and it is difficult to accurately determine the common extraction method and the sample contains collagen. The collagen may be either water-soluble collagen or non-water gluten collagen, and atelo-collagen may be obtained by removing the ends of the collagen. In addition, the substance contained in the 'sample is not only a part of the substance present in the entire sample, but also includes a large part of the entire sample. In addition, the sample may contain various factors such as cells and extracellular matrix (ECM: Extra Cellular Matrix) and cytokines, or may contain them. Such cells are, for example, chondrocytes, osteoblasts, hepatocytes, epidermal cells, fibroblasts, epithelial cells (including corneal epithelial cells, mucosal epithelial cells, amnion epithelial cells, etc.), endothelial cells, smooth muscle cells, muscle mother cells (tendons) Bud cells), parenchymal cells, cardiomyocytes, islet cells, etc. Next, the sample may be a cell culture obtained by culturing the sample on the basic material composed of the above-mentioned natural source material, such as collagen and a culture composed of cells retained in the collagen. Chondrocytes are embedded in collagen gel for cultured cartilage to comply with -7-200538737 (5). In particular, when a culture such as cultured cartilage is suitable for use in the present invention, it can be guaranteed that the culture will have a high degree of trust in subsequent use. In this test method, 'g formula is dissolved in the dissolving step with an acidic solution', thereby obtaining a sample liquid dissolved by the acidic solution, and it is considered that the components present in the sample are dispersed in the dissolved sample liquid. The acidic solution referred to here is preferably an acidic solution capable of dissolving the sample, and is appropriately determined depending on the type of the sample. Considering the dissolution time of the sample, etc., an acid in the range of ρΗ0.1 to 5.9 is particularly preferably a strong acid in the range. Such strong acids are generally known in the industry as strong acidic inorganic acids (such as hydrochloric acid, sulfuric acid, nitric acid) and organic acids (such as trichloroacetic acid, phenolic acid), which can be used alone or in combination to make it easier. In addition, the use of generally known weakly acidic organic acids (such as acetic acid, citric acid, beating acid, and nitric acid) can be used to adjust the production of strong acids in the above range of p Η 0.1 to 3 · 5 '. Weakly acidic substances and Combination of strongly acidic materials. This type of acidic solution varies depending on the type of sample. For example, when using hydrochloric acid at a concentration of ImM ~ 500mM, it is best to use a concentration of 30mM ~ 100mM. If it is less than ImM, it is not desirable to use collagen because it cannot dissolve collagen. If it is more than 500 mM, it cannot be sufficiently neutralized with a buffer solution. In addition, when using an acidic solution to dissolve the sample, an acidic solution of 1 to 50 times the amount of the sample may be used, and an acidic solution of 2 to 10 times the amount is preferable. If the amount of acidic solution is less than 1 time of the sample, the test accuracy becomes lower, and if it is more than 50 times, the test sensitivity decreases. ’And the acidic solution is particularly preferably non-endotoxin-8-200538737 (6). If the concentration of endotoxin in the solution does not have a low degree of influence on the test result, for example, it can be used if it is below the detection quality. In addition to the stability of endotoxin in acidic solutions, it is desirable to provide a rapid post-dissolution procedure. In addition, whether to remove the test solution to neutralize the components of the acidic solution before dissolving the sample with the acidic solution. To solve this problem, it is best to use (low pH) acidic solution. The solubilized sample solution thus obtained has an acidic pH ρΗ0 ~ 3 ~ 3, and a pH range of 1 ~ 3 is particularly preferred as the sample range for subsequent neutralization steps. If p Η 値 is lower than p Η 0 · 3, the liquid becomes difficult to neutralize, so it is not desirable to use. If pH 値 is higher than ρ Η 3, it is not sufficient to dissolve collagen and it is not desirable. The dissolving sample liquid obtained in the dissolving step is neutralized with the solution acting in the neutralizing step. Thus, a neutralization sample liquid can be obtained. The buffer solution having a buffering effect here is preferably a dissolving sample solution obtained by dissolving the sample with an acidic solution, and can be determined according to the type of the acidic solution and the sample. Specific examples include buffer solutions, BSE (N, N-bis (2-ethylhydroxy) -2taurine flushing solution, trimethylolaminomethane buffer solution, etc. The more salts there are in such solutions, the greater the buffer energy. It will be too high, and the sensitivity of the test will not decrease due to the amount of neutralized dissolved sample being too large, which is intended by the present invention. From the perspective of buffering energy strength and maintaining high detection rate of endotoxin, it is best Phosphate buffer is used. When neutralizing the dissolving solution, the pH of the dissolving sample solution will be affected. In addition, the high concentration in the test material is preferably the pH range buffering solution. Enough for the production of phosphoric acid) Slow-type precipitation solution is satisfactory. This view is 6.0 200538737 (7) ~ 8 · 0, and a neutralization sample liquid can be obtained. In this way, the adjustment of ρ Η 値 is generally performed by diluting the dissolved sample solution 10 to 100 times with a solution having a buffering effect, and a solution having the above buffering effect can be appropriately selected. The neutralization sample liquid obtained in the neutralization step is provided for the test of endotoxin in the test step. The test referred to here includes not only the test method for neutralizing the amount of endotoxin in the sample liquid, but also the simple detection of the presence or absence of endotoxin. If you are a professional, you can easily choose the most suitable method from the methods known in the industry. For example, the gelation method, turbidimetric method, and colorimetric method used for the endotoxin detection (Limate) test reagent can be cited, and the endotoxin test method prescribed by the Pharmacy Bureau is acceptable. . In addition, when using the endotoxin test method, it is best to perform a known reaction interference factor test and the like to confirm the detection ability. In addition, at the time of the test, in order to obtain an appropriate sample concentration, the sample liquid may be neutralized by diluting it with distilled water or the like. Next, a method for treating the culture of the present invention will be described. The treatment method of the culture of the present invention includes a dissolving step of dissolving the culture with an acidic solution to obtain a dissolving sample solution, and a step of neutralizing the dissolving sample solution with a buffering solution 'to obtain a neutralizing sample solution. This culture refers to a culture that includes methods that may be applicable to the endotoxin test described above. The culture of this type is suitable, and in the presence of a culture medium determined by the cells to be cultured, it is particularly preferable to co-culture with the base material. The suitable base material here is the above-mentioned natural source material, and particularly preferably collagen -10- 200538737
本處理方法係與內毒素試驗方法相同，若可自培養物 獲得溶解試料液及中和試料液，對於其條件可原封不動地 適用內毒素試驗方法中之條件。 若以此方法所獲得之中和試料液中、因培養物中存在 內毒素等成分以可被檢出之型態存在，本方法能繼續容易 地進行該成分之試驗。 如本方法之試驗，其培養物中可能含有之成分，藉酸 性溶液溶解，若爲不會變質之成分，業者能容易地判斷因 應所使用酸性溶液及具緩衝作用之衝溶液之種類。 另外，提供處理方法需要之藥劑整合於一個試劑套組 亦可，屆時將可含有加入酸性溶液之容器，以及加入具緩 衝作用溶液之容器，及記載此類藥劑使用方法之使用說明 書。 本發明對於內毒素等成分進行試驗時，因可不需要藉 由酵素等來自生物之試藥進行前處理，能夠簡便且迅速地 進行試驗。因此’在需要以更高精密度及迅速檢測內毒素 等成分之試驗時，例如常使用易吸附內毒素材料時，如用 於醫療用移植片，及用於無菌操作試料，或於確認培養物 有無該成分時’能夠有效地利用。另外，本方法能夠較以 酵素等試藥來溶解試料，更早期地溶解試料。 實施例 以下舉一實施例說明本發明。於試料則舉包埋於膠原 -11 - 200538737 (9) 蛋白凝膠中之軟骨細胞培養後之培養軟骨爲例。 〔實施例1〕 (1 )檢量線之作成 爲保證於各實施例中使用之試藥之精準度及其有效性 ，檢量線之信賴性之確認實驗，會較培養軟骨之內毒素試 驗先實行。This treatment method is the same as the endotoxin test method. If a dissolved sample solution and a neutralized sample solution can be obtained from the culture, the conditions in the endotoxin test method can be applied as they are. If the components such as endotoxin in the neutralized sample solution obtained in this method exist in a detectable form, this method can continue to easily test the components. For the test of this method, the components that may be contained in the culture are dissolved by the acidic solution. If it is a component that does not deteriorate, the industry can easily determine the type of acidic solution and buffer solution with buffering effect. In addition, the reagents required to provide the processing method can be integrated into a reagent kit. At that time, the container can contain an acidic solution, a container with a buffering solution, and an instruction manual describing the method of using such a reagent. In the present invention, when tests are performed on components such as endotoxin, it is possible to perform the test simply and quickly because pretreatment by a biologically-derived reagent such as an enzyme is not required. Therefore, 'when it is necessary to test with higher precision and rapid detection of endotoxin and other components, for example, when easy-to-adsorb endotoxin materials are often used, such as for medical grafts, and for aseptic manipulation of samples, or to confirm culture The presence or absence of this component can be effectively used. In addition, this method can dissolve the sample earlier than using a reagent such as an enzyme to dissolve the sample. Examples The following examples illustrate the invention. In the sample, chondrocytes cultured in collagen -11-200538737 (9) protein gel were used as an example. [Example 1] (1) The calibration curve is used to ensure the accuracy and effectiveness of the test reagents used in the examples, and the confirmation test of the reliability of the calibration curve will be compared with the endotoxin test of cultured cartilage. Do it first.
來自E.coli UKT-B之標準內毒素（CSE: 1000 EU/ ml，和光純藥工業社製）以杜比克氏磷酸緩衝液（DPBS )稀釋，調製〇.〇13、0.025、0.050 EU/ml之內毒素標準 液。 這些內毒素標準液以各50//1分裝於多孔盤，與50 # 1之內毒素檢測試藥（內毒素試劑組（Endospesy )〔登 記商標〕生化學工業社製）混合後，於3 7 °C下反應3 0分 鐘。反應終了後，依序加入重氮偶聯試藥（毒素呈色試劑 組（Toxicolar )〔登記商標〕生化學工業社製），測定其 吸光度，測定5 4 5 n m與6 3 0 n m吸光度之差値。表1爲測 定結果。檢量線以全對數作圖法作成，進行直線回歸分析 ，確認相關係數爲〇 · 9 8以上。 -12- 200538737 (10) 表1 內毒素濃度 (EU/ mL) 吸光度 f 545 -63 0nm) 平均土 S D 0.013 0.25 1 0.250 0·25 1±0·001 0.025 0.633 0.528 0·58 1 土 0·074 0.050 1.164 1.261 1.213+0.069 log〔吸光度〕=1.617 + 1.17xlog〔內毒素濃度（EU/ml)〕 (2 )鹽酸處理之影響 其次，確認使用鹽酸對於內毒素試驗之溶解處理及中 和處理之影響。Standard endotoxin (CSE: 1000 EU / ml, manufactured by Wako Pure Chemical Industries, Ltd.) from E.coli UKT-B was diluted with Dubbock's phosphate buffer solution (DPBS) to prepare 0.013, 0.025, 0.050 EU / ml endotoxin standard solution. These endotoxin standard solutions are divided into 50 // 1 portions on multiwell plates, mixed with 50 # 1 endotoxin detection reagent (Endospesy [Registered Trademark] Biochemical Industry Co., Ltd.), and mixed in 3 Reaction at 7 ° C for 30 minutes. After the reaction is completed, a diazo coupling reagent (Toxicolar [registered trademark] Biochemical Industry Co., Ltd.) is sequentially added, the absorbance is measured, and the difference between the absorbance at 5 4 5 nm and 6 30 nm is measured. value. Table 1 shows the measurement results. The calibration curve was created using the full logarithmic plotting method, and linear regression analysis was performed to confirm that the correlation coefficient was above 0.98. -12- 200538737 (10) Table 1 Endotoxin concentration (EU / mL) Absorbance f 545 -63 0nm) Average soil SD 0.013 0.25 1 0.250 0 · 25 1 ± 0 · 001 0.025 0.633 0.528 0 · 58 1 soil 0 · 074 0.050 1.164 1.261 1.213 + 0.069 log [Absorbance] = 1.617 + 1.17xlog [Endotoxin concentration (EU / ml)] (2) Effect of hydrochloric acid treatment Next, confirm the effect of using hydrochloric acid on the dissolution treatment and neutralization treatment of the endotoxin test .
將來自E. coli UKT-B之標準內毒素（CSE: 1000 EU / ml、和光純藥工業社製）以注射用蒸餾水稀釋成之內毒 素標準液（25 EU/ ml )，加入此標準液9倍之50mM之 鹽酸（HC1)，放置於室溫下5分鐘。50mM之鹽酸爲使 用1 N之鹽酸（和光純藥工業社製）以注射用蒸餾水稀釋 ，並以0.45 // m過濾器過濾滅菌。放置後，以DPBS稀釋 1 00倍後，做爲試料溶液，進行如同上述檢量線之製作步 驟，調查鹽酸處理對測定値造成之影響。 其結果爲以5 OmM之鹽酸處理之內毒素標準液之測定 値爲理論値之1 3 0% (未做圖示），因於標準偏差之內’ 確認藉由鹽酸處理不會有內毒素被分解等影響。 〔實施例2〕 根據比色法之內毒素試驗 -13- 200538737 (11) (1 )培養軟骨組織之製作 採取日本白色家兔之膝蓋、大腿、肩關節軟骨，以胰 蛋白酶EDTA溶液及膠原蛋白酶溶液，進行酵素處理，分 離·回收軟骨細胞。將所獲得之軟骨細胞洗淨後，加入含 有10%牛胎兒血淸（？88)之〇£]\4£(〇11156(^〇’3改良的 Eagle’s培養基），調製細胞密度爲1 xlO7個/ml之細胞 懸浮液。將細胞懸浮液與端膠原植入物（高硏社製），以 1 : 4比例混合（包埋），將1 00 // 1此混和液，以略成半 圓球狀，設置於培養皿上。因藉由此步驟細胞密度被稀釋 ，以1 X 1 07個/ ml濃度調製細胞懸浮液時，包埋於膠原 蛋白時之濃度變爲2xl06個/cm3(2xl05個/100//1) 〇 將設置好的混合液，於5%C02、37T：條件下，S爭置 0.5〜1小時，待使其凝膠化後，加入培養基並開始培養。 培養基爲包含有50// g/ ml之抗環血酸（L 一抗環血酸磷 酸酯化鎂鹽η水合物：C6H609P· 3/2Mg· nH20;日光 化學有限公司製）、及使用含有10 %牛胎兒血淸之DEME ，調製時含有50//g / ml之正大黴素及250ng/ml之兩性 黴素B，在37°C、5%C02條件下，進行3或4周之培養。 培養後可得直徑約l〇mm、厚度約2mm之培養軟骨。 (2 )反應干擾因子試驗 其次，爲保證內毒素試驗之精確度及有效性，進行反 應干擾因子試驗。另外實驗時所使用之檢量線之可信度， 因使用實施例1所採用之檢量線，已被確認。 -14- 200538737 (12) 試驗之前處理爲測定以上述製作方法製作之培養軟骨 量，加入9倍量之5 OmM之鹽酸，於室溫下攪拌並溶解培 養軟骨。確認溶解培養軟骨後’使用DPBS進行1〇〇倍稀 釋做爲試料溶液（稀釋倍率：1 〇〇〇倍）。另外準備不含 軟骨細胞之溶解的膠原蛋白凝膠試料液。此膠原蛋白凝膠 試料液準備有100倍稀釋（稀釋倍率：1 000倍）、200倍 稀釋（稀釋倍率：2000倍）、3 00倍稀釋（稀釋倍率： • 3 000 倍）。 反應干擾因子試驗爲遵循日本藥局方所定之方法進行 。具體而言，首先將內毒素標準液（2.5 EU/ml )，以上 述之試料液稀釋1 00倍，調製成添加內毒素之試驗檢體（ 0.02 5 EU/ml)。接著，與製作檢量線相同之步驟，測定 添加內毒素群與未添加內毒素群之各試驗檢體其內毒素濃 度。由添加內毒素群減去未添加內毒素群之內毒素量，算 出回收量。結果表示於表2 表2 試料 稀釋倍率X 內毒素濃度（EU/ mL: 未添加（n = 2) 添加（η = 2) 回收量 培養軟骨 1000 0·000±0·000 0.034土0.000 0.034 膠原蛋白 1000 0·002±0·001 0·031±〇.〇〇〇 0.029 凝膠 2000 0.002* 0.029±0.001 0.027 3000 0.005* 0.035±0.001 0.030 ※：來自培養軟骨或膠原蛋白凝膠之換算 * : 之測定値 -15- 200538737 (13) 如表2所示，於各個試驗檢體回收量與添加內毒素濃 度（0.02 5 EU/ ml )之差，大部分不認定其具差異性，不 被認定爲反應干擾因子。 (3 )內毒素試驗 首先，由上述內毒素比色法試驗，於未添加內毒素群 軟骨培養之試驗溶液之測定値所算出之內毒素濃度，未達 檢量線之最小濃度（0.013 EU/ ml )。因試料溶液爲自培 養軟骨稀釋1〇〇〇倍換算，確認培養軟骨中之內毒素濃度 未達 1 3 EU/ cm3。 接著，將做爲培養軟骨之構成成分之膠原蛋白凝膠及 膠原蛋白，以鹽酸溶解，用DPBS中和後之中和試料溶液 ，未損及檢量線之可信度，亦確認未顯示有反應干擾作用 存在。基於此結果，以比色法進行內毒素試驗，能夠簡便 地測定培養軟骨中之內毒素濃度。 〔實施例3〕 根據凝膠化法之內毒素試驗 首先，將與實施例1中使用之相同之標準內毒素（和 光純藥工業社製），以注射用蒸餾水3倍稀釋之DPBS ( 以下稱作3倍稀釋DPBS )溶解，調製1〇〇〇 EU / ml之內 毒素標準原液。此原液以3倍稀釋DPBS稀釋，製作出 6.25、0.062 5、0.0313、0.0156、0.0078 EU/ml 之內毒素 標準液（C )。 其次，測定培養軟骨之重量，加入9倍量之5 OmM鹽 •16- 200538737 (14) 酸，於室溫下攪拌並溶解。5 OmM之鹽酸調製爲與上述相 同。確認培養軟骨溶解後，使用DPBS進行100倍稀釋， 再將此溶液以注射用蒸餾水稀釋3倍後成爲試料溶液（A )° 6.2 5 EU/ ml之濃度之內毒素標準液，以試料溶液稀 釋100倍，調製成添加內毒素之試驗溶液（0.0625 EU/ ml)，接下來以試料溶液反覆進行2倍稀釋，調製0.0313 EU/ml、0.0156 EU/ml、0.0078 EU/ml 之添力 D 內毒素群 之各試驗溶液（B )。 表3所示爲各試驗溶液2 0 0 // 1，添加至內毒素檢測試 藥（鱟試驗法HS-J單一試驗組：和光純藥工業社製）’ 經3 7°C水浴加熱60分鐘後，倒置含有各試驗溶液之內毒 素檢測試藥，觀察凝膠化之有無。此時，內容物未凝固者 判定爲陽性。結果表示於表4。An endotoxin standard solution (25 EU / ml) diluted with distilled water for injection was added to the standard endotoxin (CSE: 1000 EU / ml, manufactured by Wako Pure Chemical Industries, Ltd.) from E. coli UKT-B. 9 50 mM hydrochloric acid (HC1), and left at room temperature for 5 minutes. 50 mM hydrochloric acid was diluted with distilled water for injection using 1 N hydrochloric acid (manufactured by Wako Pure Chemical Industries, Ltd.), and sterilized by filtration through a 0.45 // m filter. After being left to stand, it was diluted 100 times with DPBS. As a sample solution, the same steps as in the above calibration line were performed to investigate the effect of hydrochloric acid treatment on the measurement of radon. As a result, the measurement of endotoxin standard solution treated with 5 OmM hydrochloric acid was 130% of the theoretical value (not shown), because the standard deviation was within the standard deviation. 'It was confirmed that no endotoxin was treated by hydrochloric acid. Decomposition and other effects. [Example 2] Endotoxin test according to colorimetric method-13- 200538737 (11) (1) Production of cultured cartilage tissue The knee, thigh, and shoulder cartilage of Japanese white rabbits were used, and trypsin EDTA solution and collagenase were used. The solution is treated with an enzyme to separate and recover chondrocytes. After the obtained chondrocytes were washed, 10% bovine fetal blood 淸 (? 88) was added to ££] \ 4 £ (〇11156 (^ 〇'3 modified Eagle's medium), and the cell density was adjusted to 1 x 10 7 / ml of cell suspension. The cell suspension and collagen-terminated implant (manufactured by Takahata Co., Ltd.) were mixed (embedded) at a ratio of 1: 4, and the mixture was mixed with 1 00 // 1 to form a hemisphere. The cell density is diluted by this step. When the cell suspension is prepared at a concentration of 1 X 107 cells / ml, the concentration when embedded in collagen becomes 2xl06 cells / cm3 (2xl05 cells). / 100 // 1) 〇 S set the mixed solution under the conditions of 5% CO2, 37T: S for 0.5 to 1 hour, after it is gelled, add the culture medium and start the culture. The culture medium contains 50 // g / ml of ascorbic acid (L mono-ascorbic acid phosphate esterified magnesium salt η hydrate: C6H609P · 3 / 2Mg · nH20; manufactured by Nikko Chemical Co., Ltd.), and containing 10% bovine fetal blood DEDEME, containing 50 // g / ml of orthomycin and 250ng / ml of amphotericin B during the preparation, cultured at 37 ° C, 5% C02 for 3 or 4 weeks After culture, cultured cartilage with a diameter of about 10mm and a thickness of about 2mm can be obtained. (2) Response interference factor test Secondly, in order to ensure the accuracy and effectiveness of the endotoxin test, a response interference factor test is performed. In addition, it is used in experiments The credibility of the calibration curve has been confirmed by using the calibration curve used in Example 1. -14- 200538737 (12) Processing before the test is to measure the amount of cultured cartilage produced by the above production method, adding 9 times An amount of 5 OmM hydrochloric acid was stirred at room temperature to dissolve the cultured cartilage. After confirming that the cultured cartilage was dissolved, a 1000-fold dilution with DPBS was used as a sample solution (dilution ratio: 1,000-fold). Chondrocyte lysed collagen gel sample solution. This collagen gel sample solution is prepared with 100-fold dilution (dilution magnification: 1,000-fold), 200-fold dilution (dilution magnification: 2000-fold), and 300-fold dilution (diluted Magnification: • 3 000 times). The response interference factor test was performed in accordance with the method prescribed by the Japanese Pharmacy. Specifically, first, an endotoxin standard solution (2.5 EU / ml) was used for the above sample. Diluted 100 times to prepare an endotoxin-added test specimen (0.02 5 EU / ml). Next, the same procedure as in the preparation of the calibration curve was used to determine the test specimens with and without the endotoxin group. Endotoxin concentration. Calculate the recovered amount by adding the endotoxin group and subtracting the endotoxin amount without the endotoxin group. The results are shown in Table 2. Table 2 Sample dilution ratio X Endotoxin concentration (EU / mL: not added (n = 2 ) Add (η = 2) Recovered amount of cultured cartilage 1000 0 · 000 ± 0 · 000 0.034 ± 0.000 0.034 Collagen 1000 0 · 002 ± 0 · 001 0 · 031 ± 〇.〇〇〇 0.029 Gel 2000 0.002 * 0.029 ± 0.001 0.027 3000 0.005 * 0.035 ± 0.001 0.030 ※: Conversion from cultured cartilage or collagen gel *: Measurement 値 -15- 200538737 (13) As shown in Table 2, the recovered amount of each test specimen and the addition of endotoxin Most of the differences in concentration (0.02 5 EU / ml) were not considered to be different, and were not considered to be response interference factors. (3) Endotoxin test First, the concentration of endotoxin calculated from the above-mentioned endotoxin colorimetric test in the test solution of endotoxin group-free cartilage culture test, the minimum concentration that does not reach the calibration line (0.013 EU / ml). Since the sample solution was diluted 1,000 times from the cultured cartilage, it was confirmed that the endotoxin concentration in the cultured cartilage did not reach 13 EU / cm3. Next, the collagen gel and collagen, which are the constituents of the cartilage, were dissolved in hydrochloric acid and neutralized with DPBS to neutralize the sample solution. The credibility of the test line was not damaged, and it was confirmed that no Reaction interference exists. Based on this result, the endotoxin test by colorimetric method can easily measure the endotoxin concentration in cultured cartilage. [Example 3] Endotoxin test by gelation method First, the same standard endotoxin (manufactured by Wako Pure Chemical Industries, Ltd.) as that used in Example 1 was diluted with DPBS (hereinafter referred to as DPBS 3 times) Make a 3x dilution of DPBS) to prepare a 1000 EU / ml endotoxin standard stock solution. This stock solution was diluted with 3 times diluted DPBS to prepare 6.25, 0.062 5, 0.0313, 0.0156, 0.0078 EU / ml endotoxin standard solution (C). Next, measure the weight of the cultured cartilage, add 9 times the amount of 5 OmM salt • 16- 200538737 (14) acid, stir and dissolve at room temperature. 5 OmM hydrochloric acid was prepared in the same manner as described above. After confirming that the cultured cartilage was dissolved, the solution was diluted 100 times with DPBS, and then the solution was diluted 3 times with distilled water for injection to become the sample solution (A) ° 6.2 5 EU / ml concentration of endotoxin standard solution, diluted 100 times with the sample solution. Times to prepare a test solution with added endotoxin (0.0625 EU / ml), and then double-dilute the sample solution repeatedly to prepare 0.0313 EU / ml, 0.0156 EU / ml, and 0.0078 EU / ml. Test solution (B). Table 3 shows that each test solution 2 0 0 // 1 was added to the endotoxin detection reagents (鲎 test method HS-J single test group: manufactured by Wako Pure Chemical Industries, Ltd.) 'heated in a 37 ° C water bath for 60 minutes Then, the endotoxin detection reagent containing each test solution was inverted, and the presence or absence of gelation was observed. At this time, those whose contents were not solidified were judged to be positive. The results are shown in Table 4.
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表3 試驗溶液 添加內毒素 濃度（EU/ mL) A液 試料溶液 0 0.0625 B液 標準內毒素/添加試料溶液 0.0313 0.0156 0.0078 0.0625 C液 標準內毒素溶液/ 3倍稀釋DPBS 0.0313 0.0156 0.0078 D液 3倍稀釋D P B S 0Table 3 Endotoxin concentration in test solution (EU / mL) A sample solution 0 0.0625 B standard endotoxin / add sample solution 0.0313 0.0156 0.0078 0.0625 C solution standard endotoxin solution / 3 times diluted DPBS 0.0313 0.0156 0.0078 D solution 3 times DPBS 0
-18- 200538737 (16) 表4 添加內毒素 凝膠化 幾何平均終點 濃度（EU/ mL) 濃度（E u / m T A液 0 XXXX 0.0625 〇〇〇〇 0.0313 〇〇〇〇 B液 0.0156 XXXX 0.3 1 3 0.0078 XXXX 0.0625 〇〇 0.0313 〇〇 C液 0.0156 XX 0.313 0.0078 XX D液 0 XX ----—-- 〇： 陽性 X :陰性 Φ 如表4所示，自C液所求得之幾何平均終點濃度，爲 試驗用內毒素檢測試藥之表示敏感度（λ : 0.0 3 E U / m 1 ^ )之0 · 5〜2 · 0 λ之範圍，確認內毒素檢測試藥之表示敏感 . 度。另外，Α液及D液（3倍稀釋DPBS )，均確認爲陰 性。由B液所算出之幾何平均終點濃度亦在〇·5〜2〇λ之 範圍內，試料溶液中不被認爲具反應干擾因子。 力外由Α液（培養軟骨之中和試料液）均爲陰性 ，及B液及C液之結果看來，可判定試料溶液a中之內 參素濃度未達0.03 EU/ml。另外，田 / 另外，因試料溶液A爲自培 -19- 200538737 (17) 養軟骨稀釋3 000倍換算，確認培養軟骨中之內毒素濃度 未達 90 EU/ cm3。 如此以鹽酸溶解培養軟骨所得之溶解試料液，以 DPBS稀釋1〇〇倍並中和，其後，在不超過最大有效稀釋 倍率之範圍下，以蒸餾水稀釋之溶液，未顯示有反應干擾 作用，顯示其具高信賴性。另外’基於此結果，根據凝膠 化法之內毒素試驗，能夠簡便地測定培養軟骨中之內毒素 濃度。 如上述之說明，若藉由本發明以酸性溶液溶解含有內 毒素之試料，對於藉由具緩衝作用之溶液中和，所得中和 試料液，能夠以比色法及凝膠化法進行內毒素試驗。特別 爲無論是否爲易吸附內毒素之材料均可進行正確之測定。 另外’包含於試料中檢出對象不限於內毒素時，能夠 簡便地S周製出各種爲貫施g式驗之試料。進一步地，能夠較 藉由酵素等溶解試藥，更早期將試料溶g。 -20.-18- 200538737 (16) Table 4 Geometric mean endpoint concentration of endotoxin gelation (EU / mL) Concentration (Eu / m TA solution 0 XXXX 0.0625 〇〇〇〇0.0313 〇〇〇〇〇 B solution 0.0156 XXXX 0.3 1 3 0.0078 XXXX 0.0625 〇〇0.0313 〇〇C solution 0.0156 XX 0.313 0.0078 XX D solution 0 XX ------------ ○: positive X: negative Φ As shown in Table 4, the geometric mean endpoint obtained from the C solution The concentration is a range of 0 · 5 ~ 2 · 0 λ that indicates the sensitivity of the endotoxin detection reagent used in the test (λ: 0.0 3 EU / m 1 ^), and confirms the sensitivity of the endotoxin detection reagent. Solution A and Solution D (three-fold dilution of DPBS) were confirmed to be negative. The geometric mean endpoint concentration calculated from Solution B was also in the range of 0.5 to 2λ, and the sample solution was not considered to be reactive. Interfering factors. The force A was negative from the A solution (the cultured cartilage and the sample solution), and the results of the B and C solutions seemed to determine that the concentration of the endogenin in the sample solution a had not reached 0.03 EU / ml. ， 田 / In addition, because the sample solution A is self-cultivation-19- 200538737 (17) cartilage dilution 3 00 Zero-fold conversion confirmed that the endotoxin concentration in the cultured cartilage did not reach 90 EU / cm3. The solubilized sample solution obtained by dissolving the cultured cartilage in hydrochloric acid was diluted 100 times with DPBS and neutralized, and thereafter, it was effective at not exceeding the maximum. In the range of the dilution ratio, the solution diluted with distilled water does not show a reaction interference effect and shows high reliability. In addition, based on this result, the endotoxin test in the gelation method can be used to easily determine the Endotoxin concentration. As described above, if the sample containing endotoxin is dissolved in an acidic solution according to the present invention, the neutralized sample solution obtained by neutralizing the solution with a buffering effect can be colorimetric and gelatinized. Endotoxin test. Especially for materials that can easily absorb endotoxins, accurate measurements can be made. In addition, when the detection object included in the sample is not limited to endotoxins, it is possible to easily produce a variety of peristaltic g in S weeks. The test sample of the formula test. Further, the sample can be dissolved g earlier than the sample is dissolved by enzymes. -20.