1、1REAGENTS AND TEST SOLUTIONS 試劑和測(cè)試溶液試劑和測(cè)試溶液Amoebocyte Lysate A lyophilized product obtained from the lysate of amoebocytes (white blood cells) from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). This reagent refers only to a product manufactured in accordance with the regulations

2、 of the competent authority. NOTEAmoebocyte Lysate reacts to some -glucans in addition to endotoxins. Amoebocyte Lysate preparations that do not react to glucans are available: they are prepared by removing the G factor reacting to glucans from Amoebocyte Lysate or by inhibiting the G factor reactin

3、g system of Amoebocyte Lysate and may be used for endotoxin testing in the presence of glucans. 變形細(xì)胞溶解液 由鱟的（白血細(xì)胞）變形細(xì)胞溶解液制得的凍干產(chǎn)品。該試劑僅指那些按照相關(guān)的監(jiān)管機(jī)構(gòu)的法規(guī)要求制得的產(chǎn)品。注：變形細(xì)胞溶解液除了與內(nèi)毒素反應(yīng)外，還會(huì)與某些 -葡聚糖反應(yīng)。可以制成不與 -葡聚糖發(fā)生反應(yīng)的變形細(xì)胞溶解液制品：可通過從變形細(xì)胞溶解液中去除會(huì)與葡聚糖反應(yīng)的 G 因子或抑制變形細(xì)胞溶解液的 G 因子反應(yīng)系統(tǒng)來制得該制品，這些制品可用于在存在葡聚糖時(shí)進(jìn)行內(nèi)毒素檢測(cè)。Water for B

4、acterial Endotoxins Test (BET) Use Water for Injection or water produced by other procedures that shows no reaction with the lysate employed, at the detection limit of the reagent. 細(xì)菌內(nèi)毒素檢測(cè)（BET）用水 使用注射用水或用其他方式制得的不會(huì)與鱟試劑檢測(cè)限對(duì)應(yīng)濃度的溶菌液發(fā)生反應(yīng)的水。Lysate TS Dissolve Amoebocyte Lysate in Water for BET, or in a bu

5、ffer recommended by the lysate manufacturer, by gentle stirring. Store the reconstituted lysate, refrigerated or frozen, according to the specifications of the manufacturer.鱟試劑 在緩慢攪拌的同時(shí)，將變形細(xì)胞溶解液溶于細(xì)菌內(nèi)毒素檢測(cè)用水中或溶于溶菌液生產(chǎn)商推薦的緩沖液中。按照生產(chǎn)商的說明來冷藏或冷凍儲(chǔ)存該再生溶菌液。2GEL-CLOT TECHNIQUE 凝膠法凝膠法The Gel-Clot Technique is fo

6、r detecting or quantifying endotoxins based on clotting of the lysate reagent in the presence of endotoxin. The minimum concentration of endotoxin required to cause the lysate to clot under standard conditions is the labeled sensitivity of the lysate reagent. To ensure both the precision and validit

7、y of the test, perform the tests for confirming the labeled lysate sensitivity and for interfering factors as described under Preparatory Testing. 凝膠法通過鱟試劑與內(nèi)毒素產(chǎn)生凝集反應(yīng)的原理，來檢測(cè)或定量?jī)?nèi)毒素。在標(biāo)準(zhǔn)條件下使溶菌液凝結(jié)所需的最低內(nèi)毒素濃度即為鱟試劑的標(biāo)示靈敏度。為了確保化驗(yàn)的精密度和有效性，按照凝膠法預(yù)備試驗(yàn)項(xiàng)所述來確認(rèn)鱟試劑標(biāo)示靈敏度和干擾因子。Preparatory Testing 凝膠法預(yù)備試驗(yàn)?zāi)z法預(yù)備試驗(yàn)Test for

8、Confirmation of Labeled Lysate Sensitivity Confirm in four replicates the labeled sensitivity, , expressed in EU/mL of the lysate prior to use in the test. The test for confirmation of lysate sensitivity is to be carried out when a new batch of lysate is used or when there is any change in the test

9、conditions that may affect the outcome of the test. Prepare standard solutions having at least four concentrations equivalent to 2 , , 0.5 , and 0.25 by diluting the USP Endotoxin RS with Water for BET. 確認(rèn)鱟試劑標(biāo)示靈敏度 在進(jìn)行化驗(yàn)之前，要對(duì)鱟試劑標(biāo)示靈敏度（EU/ml）重復(fù)確認(rèn) 4 次。當(dāng)使用新批次的鱟試劑或當(dāng)實(shí)驗(yàn)條件出現(xiàn)任何可能影響實(shí)驗(yàn)結(jié)果的變更時(shí)，均應(yīng)進(jìn)行鱟試劑靈敏度的確認(rèn)。用細(xì)菌內(nèi)毒

10、素檢測(cè)用水稀釋內(nèi)毒素 USP 標(biāo)準(zhǔn)品，配制一系列標(biāo)準(zhǔn)溶液，至少含有與 2 , , 0.5 , 和 0.25 相當(dāng)?shù)乃姆N濃度。Mix a volume of the Lysate TS with an equal volume (such as 0.1-mL aliquots) of one of the Standard Endotoxin Solutions in each test tube. When single test vials or ampuls containing lyophilized lysate are used, add solutions directly to

11、the vial or 3ampul. Incubate the reaction mixture for a constant period according to the directions of the lysate manufacturer (usually at 37 1 for 60 2 minutes), avoiding vibration. To test the integrity of the gel, take each tube in turn directly from the incubator and invert it through about 180

12、in one smooth motion. If a firm gel has formed that remains in place upon inversion, record the result as positive. A result is negative if an intact gel is not formed. The test is considered valid when the lowest concentration of the standard solutions shows a negative result in all replicate tests

13、.在每個(gè)試管中將一定體積的鱟試劑與等體積（例如 0.1mL 的等份）的其中一種標(biāo)準(zhǔn)內(nèi)毒素溶液進(jìn)行混合。當(dāng)使用含有凍干鱟試劑的一次性實(shí)驗(yàn)瓶或者安瓿時(shí)，向瓶或安瓿中直接加入溶液。按照鱟試劑生產(chǎn)商的規(guī)定，要在恒定時(shí)段內(nèi)將反應(yīng)混合物保持恒溫（通常是 37 1 條件下 60 2 分鐘），避免振動(dòng)。為檢驗(yàn)?zāi)z體的完整性，依次直接從恒溫箱中取出各個(gè)試管，并平滑地將其翻轉(zhuǎn) 180。如果已經(jīng)形成的牢固凝膠在倒置后保持原位，則將結(jié)果記錄為陽性。如果沒有形成完整的凝膠，則結(jié)果為陰性。只有當(dāng)最低濃度的標(biāo)準(zhǔn)溶液在所有重復(fù)試驗(yàn)中均呈陰性時(shí)，該試驗(yàn)方為有效。The endpoint is the smallest conc

14、entration in the series of decreasing concentrations of standard endotoxin that clots the lysate. Determine the geometric mean endpoint by calculating the mean of the logarithms of the endpoint concentrations of the four replicate series and then taking the antilogarithm of the mean value, as indica

15、ted in the following formula: 反應(yīng)終點(diǎn)就是引起溶菌液凝結(jié)的系列遞減的標(biāo)準(zhǔn)內(nèi)毒素濃度中的最低一個(gè)濃度。如下式所示，計(jì)算四次重復(fù)試驗(yàn)的系列終點(diǎn)濃度的對(duì)數(shù)平均值，然后計(jì)算該平均值的反對(duì)數(shù)，從而得到幾何平均終點(diǎn)。Geometric Mean Endpoint Concentration = antilog (Se/f)幾何平均終點(diǎn)濃度=（Se / f）的反對(duì)數(shù)where Se is the sum of the log endpoint concentrations of the dilution series used, and f is the number of r

16、eplicate test tubes. The geometric mean endpoint concentration is the measured sensitivity of the lysate (in EU/mL). If this is not less than 0.5 and not more than 2 , the labeled sensitivity is confirmed and is used in tests performed with 4this lysate. 其中，Se 指的是所使用的系列稀釋液的終點(diǎn)濃度的對(duì)數(shù)和，f 是重復(fù)試驗(yàn)的次數(shù)。幾何平均終點(diǎn)

17、濃度即為鱟試劑靈敏度的測(cè)定值（以 EU/mL 為單位）。如果該值不小于 0.5 且不大于 2 ，則標(biāo)示靈敏度得到了確認(rèn)，并可用于使用此溶菌液進(jìn)行的試驗(yàn)。Test for Interfering Factors Usually prepare solutions (AD) as shown in Table 1, and perform the inhibition/enhancement test on the Sample Solutions at a dilution less than the MVD, not containing any detectable endotoxins,

18、operating as described for Test for Confirmation of Labeled Lysate Sensitivity. The geometric mean endpoint concentrations of Solutions B and C are determined using the formula described in the Test for Confirmation of Labeled Lysate Sensitivity. 干擾因子檢測(cè)：按照表 1 所示配制溶液 A、溶液 B、溶液 C、溶液 D，并且按照上述的確認(rèn)鱟試劑標(biāo)示靈敏

19、度項(xiàng)所述，對(duì)稀釋倍數(shù)不超過最大有效稀釋倍數(shù)（MVD）、不含有任何內(nèi)毒素的樣品溶液進(jìn)行抑制/增強(qiáng)試驗(yàn)。用確認(rèn)鱟試劑標(biāo)示靈敏度項(xiàng)中提及的公式來測(cè)定溶液B 和溶液C 的幾何平均終點(diǎn)濃度。Table 1. Preparation of Solutions for the Inhibition/Enhancement Test for Gel-Clot Techniques 表 1：用于凝膠法抑制/增強(qiáng)試驗(yàn)的溶液制備Solution 溶液Endotoxin Concentration/Solution to which Endotoxin is Added 內(nèi)毒素濃度內(nèi)毒素濃度/加入內(nèi)加入內(nèi)毒素的溶液毒

20、素的溶液Diluent稀釋液DilutionFactor稀釋因子Endotoxin Concentration內(nèi)毒素濃度Number ofReplicates重復(fù)次數(shù)重復(fù)次數(shù)AaNone/Sample Solution無/樣品溶液4Bb2 /Sample Solution 2 /樣品溶液Sample Solution樣品溶液12482 10.50.254444Cc2 /Water for BET 2 /細(xì)菌內(nèi)毒素檢測(cè)（BET）用水Water for BET細(xì)菌內(nèi)毒素檢測(cè)用水122 1225Solution 溶液Endotoxin Concentration/Solution to which

21、Endotoxin is Added 內(nèi)毒素濃度內(nèi)毒素濃度/加入內(nèi)加入內(nèi)毒素的溶液毒素的溶液Diluent稀釋液DilutionFactor稀釋因子Endotoxin Concentration內(nèi)毒素濃度Number ofReplicates重復(fù)次數(shù)重復(fù)次數(shù)480.50.2522DdNone/Water for BET無/細(xì)菌內(nèi)毒素檢測(cè)用水2a Solution A: A Sample Solution of the preparation under test that is free of detectable endotoxins. 溶液A：用于化驗(yàn)的不含可檢測(cè)到的內(nèi)毒素的樣品溶液。b

22、Solution B: Test for interference. 溶液B：干擾因子檢測(cè)。c Solution C: Control for labeled lysate sensitivity溶液C：對(duì)鱟試劑標(biāo)示靈敏度的控制d Solution D: Negative control of Water for BET溶液D：細(xì)菌內(nèi)毒素檢測(cè)用水陰性對(duì)照The test is considered valid when all replicates of Solutions A and D show no reaction and the result of Solution C confir

23、ms the labeled sensitivity.僅當(dāng)溶液A 和溶液D 的所有重復(fù)試驗(yàn)均顯示無反應(yīng)，且溶液C 的結(jié)果確認(rèn)標(biāo)示靈敏度符合要求時(shí)，試驗(yàn)方為有效。If the sensitivity of the lysate determined in the presence of Solution B is not less than 0.5 and not greater than 2 , the Sample Solution does not contain factors that interfere under the experimental conditions used.

24、Otherwise, the Sample Solution to be examined interferes with the test. 如果用溶液B 的供試品溶液測(cè)得的該溶菌液的靈敏度不小于 0.5 且不大于 2 ，則該樣品溶液中在實(shí)驗(yàn)條件下不含有干擾因子。否則，該樣品溶液會(huì)干擾此試驗(yàn)。If the sample under test does not comply with the test at a dilution less than the MVD, repeat the test using a greater dilution, not exceeding the MVD.

25、 The use of a more sensitive lysate permits a greater dilution of the sample to be examined and this may contribute to the elimination of interference.如果供試品在稀釋倍數(shù)小于最大有效稀釋倍數(shù)（MVD）的情況下不符合該試驗(yàn)的要求，則用一個(gè)更大的稀釋倍數(shù)（但不超過 MVD）來重復(fù)該試驗(yàn)。使用靈敏度更大的溶菌液允許對(duì)樣品進(jìn)行更大倍數(shù)的稀釋，這樣可以起到排除干擾的作用。Interference may be overcome by suitable t

26、reatment, such as filtration, neutralization, dialysis, or heating. To establish that the chosen treatment effectively eliminates 6interference without loss of endotoxins, perform the assay described above using the preparation to be examined to which USP Endotoxin RS has been added and which has th

27、en been submitted to the chosen treatment.可以通過適當(dāng)?shù)奶幚韥砜朔蓴_，例如過濾、中和、透析、或加熱。為了確定所選用的處理方法能有效地排除干擾且不會(huì)造成內(nèi)毒素的減少，要使用加入了內(nèi)毒素 USP 標(biāo)準(zhǔn)品并用所選方法進(jìn)行了處理的供試品溶液，進(jìn)行上述化驗(yàn)。Limit Test 限度檢測(cè)限度檢測(cè)Procedure Prepare Solutions A, B, C, and D as shown in Table 2, and perform the test on these solutions following the procedure for Tes

28、t for Confirmation of Labeled Lysate Sensitivity under Preparatory Testing. 程序 根據(jù)表 2 配制溶液A、溶液B、溶液C、溶液D，并按照凝膠法預(yù)備試驗(yàn)中鱟試劑標(biāo)示靈敏度的確認(rèn)項(xiàng)所述對(duì)這些溶液進(jìn)行檢測(cè)。Table 2. Preparation of Solutions for the Gel-Clot Limit Test 表 2：凝膠限度檢測(cè)，溶液的制備Solution* 溶液*Endotoxin Concentration/Solution to which Endotoxin is Added 內(nèi)毒素濃度/加入內(nèi)毒

29、素的溶液Number of Replicates 重復(fù)次數(shù)ANone/Diluted Sample Solution 無/稀釋的樣品溶液2B2 /Diluted Sample Solution 2 /稀釋的樣品溶液2C2 /Water for BET 2 /細(xì)菌內(nèi)毒素檢測(cè)用水2DNone/Water for BET 無/細(xì)菌內(nèi)毒素檢測(cè)用水2* Prepare Solution A and the positive product control Solution B using a dilution not greater than the MVD and treatments as for

30、the Test for Interfering Factors under Preparatory Testing. The positive control Solutions B and C contain the Standard Endotoxin Solution at a concentration corresponding to twice the labeled lysate sensitivity. The negative control Solution D consists of Water for BET.使用不超過最大有效稀釋倍數(shù)（MVD）的稀釋度并按照凝膠法預(yù)

31、備試驗(yàn)中干擾因子的檢測(cè)項(xiàng)規(guī)定的處理方法來配制溶液A 和陽性產(chǎn)品對(duì)照溶液B。陽性對(duì)照溶液B 和 C 含有濃度為標(biāo)示稱鱟試劑靈敏度兩倍的標(biāo)準(zhǔn)內(nèi)毒素溶液。陰性對(duì)照溶液D由細(xì)菌內(nèi)毒素檢測(cè)用水組成。Interpretation The test is considered valid when both replicates of Solution B and C are positive and those of Solution D are negative. When a negative result is found for 7both replicates of Solution A, th

32、e preparation under test complies with the test. When a positive result is found for both replicates of Solution A, the preparation under test does not comply with the test. 說明：只有當(dāng)陽性對(duì)照溶液B和C 的兩次重復(fù)試驗(yàn)均呈陽性，且陰性對(duì)照溶液D 均呈陰性，此試驗(yàn)方為有效。若溶液A 的兩次重復(fù)檢測(cè)結(jié)果呈陰性，則溶液配制符合試驗(yàn)要求。當(dāng)溶液 A 的兩次重復(fù)檢測(cè)均呈陽性結(jié)果，則供試品不符合此檢測(cè)的要求。When a posit

33、ive result is found for one replicate of Solution A and a negative result is found for the other, repeat the test. In the repeat test, the preparation under test complies with the test if a negative result is found for both replicates of Solution A. The preparation does not comply with the test if a

34、 positive result is found for one or both replicates of Solution A. However, if the preparation does not comply with the test at a dilution less than the MVD, the test may be repeated using a greater dilution, not exceeding the MVD.當(dāng)溶液 A 的兩次重復(fù)測(cè)試中，1 個(gè)呈陽性結(jié)果，1 個(gè)呈陰性結(jié)果時(shí)，須重復(fù)該實(shí)驗(yàn)。在重復(fù)該實(shí)驗(yàn)時(shí)，如果溶液A 均呈陰性結(jié)果，則溶液配制符

35、合此測(cè)試的要求。如果溶液A 的一次或者兩次的檢測(cè)結(jié)果都是陽性，則溶液配制不符合試驗(yàn)要求。但是，如果其稀釋度小于最大有效稀釋倍數(shù)（MVD）且溶液配制不符合實(shí)驗(yàn)要求，可以使用更大的稀釋度來重復(fù)該實(shí)驗(yàn)，但該稀釋度不得超過最大有效稀釋倍數(shù)（MVD）。Quantitative Test 定量檢測(cè)Procedure The test quantifies bacterial endotoxins in Sample Solutions by titration to an endpoint. Prepare Solutions A, B, C, and D as shown in Table 3, and

36、 test these solutions by following the procedure in the Test for Confirmation of Labeled Lysate Sensitivity under Preparatory Testing. 程序 通過滴定至終點(diǎn)來定量檢測(cè)樣品溶液中的細(xì)菌內(nèi)毒素。按照表 3 所示配制溶液A、溶液B、溶液C、溶液D，并且按照凝膠法預(yù)備試驗(yàn)中鱟試劑標(biāo)示靈敏度的確認(rèn)項(xiàng)所述來進(jìn)行檢測(cè)。Table 3. Preparation of Solutions for the Gel-Clot Assay 表 3：凝膠法定量檢測(cè)，溶液的制備8Solut

37、ion溶液Endotoxin Concentration/Solution to which Endotoxinis Added 內(nèi)毒素的含量/加入內(nèi)毒素的溶液Diluent 稀釋液Dilution Factor 稀釋因子Endotoxin Concentration內(nèi)毒素濃度Number of Replicates重復(fù)次數(shù)Aa無/樣品溶液None/Sample SolutionWater for BET 細(xì)菌內(nèi)毒素檢測(cè)用水1248 2222Bb2 /Sample Solution 2 /樣品溶液122Cc2 /Water for BET 2 /細(xì)菌內(nèi)毒素檢測(cè)用水Water for BET細(xì)菌

38、內(nèi)毒素檢測(cè)用水12482 10.50.252222DdNone/Water for BET無/細(xì)菌內(nèi)毒素檢測(cè)用水2a Solution A: Sample Solution under test at the dilution, not to exceed the MVD, with which the Test for Interfering Factors was completed. Subsequent dilution of the Sample Solution must not exceed the MVD. Use Water for BET to make a dilutio

39、n series of four tubes containing the Sample Solution under test at concentrations of 1, , , and 1/8 relative to the concentration used in the Test for Interfering Factors. Other dilutions up to the MVD may be used as appropriate. 溶液A：稀釋度不超過最大有效稀釋倍數(shù)（MVD）的供試品溶液，用于進(jìn)行干擾因子檢測(cè)。樣品溶液的后續(xù)稀釋一定不得超過最大有效稀釋倍數(shù)（MVD）

40、。使細(xì)菌內(nèi)毒素檢測(cè)用水來配制 4 管稀釋液，其中樣品溶液濃度分別為 干擾因子檢測(cè)中用到的濃度的 1、1/2、1/4、1/8 倍。必要時(shí)也可以使用其他稀釋度（不超過 MVD）的稀釋液。b Solution B: Solution A containing standard endotoxin at a concentration of 2 (positive product control).溶液B：含有濃度為 2 的內(nèi)毒素標(biāo)準(zhǔn)品的溶液A（陽性控制）。c Solution C: Two replicates of four tubes of Water for BET containing th

41、e standard endotoxin at a concentration of 2 , , 0.5 , and 0.25 , respectively.溶液 C：2 組裝有細(xì)菌內(nèi)毒素檢測(cè)用水的4 個(gè)試管，每個(gè)試管分別含有 2 、 、0.5 、0.25 的內(nèi)毒素標(biāo)準(zhǔn)品。d Solution D: Water for BET (negative control)溶液D：細(xì)菌內(nèi)毒素檢測(cè)用水（陰性對(duì)照）9Calculation and Interpretation The test is considered valid when the following three conditions are met: (1) Both replicates of negative control Solution D are negative; (2) Both replicates of positive product control Solution B are positive; and (3) The geometric mean endpoint concentration of Solution C is in the range of 0.5 to 2 .計(jì)算和說明：只有滿足下列三個(gè)條件時(shí)，化驗(yàn)才有效：(1)陰性對(duì)照溶液D 的兩次檢測(cè)結(jié)果均為陰性；(2)陽性產(chǎn)品