【正文】 test for interfering factors using the preparation being examined to which the standard endotoxin has been added and which has then been submitted to the chosen treatment.當(dāng)內(nèi)毒素的回收率不在指定范圍內(nèi)，應(yīng)根據(jù)凝膠法章節(jié)的“（ii）干擾因素實(shí)驗(yàn)”項(xiàng)下的說(shuō)明排除干擾因素。(3)溶液D（陰性對(duì)照）的試驗(yàn)結(jié)果不得大于所用鱟試劑的說(shuō)明書(shū)中規(guī)定的空白值的限度或小于鱟試劑的內(nèi)毒素檢測(cè)限。4. TEST檢查法(i) ProcedureFollow the procedure described in 3. Preparatory testing,(ii) Test for interfering factors.按 “（3）預(yù)備試驗(yàn)下的（ii）干擾因素試驗(yàn)”項(xiàng)下操作步驟。– the result with solution D does not exceed the limit of the blank value required in the description of the lysate reagent employed, or it is less than the endotoxin detection limit of the lysate reagent employed.試驗(yàn)必須符合以下條件方有效： ； 溶液D（陰性對(duì)照）的試驗(yàn)結(jié)果不得大于所用鱟試劑的說(shuō)明書(shū)中規(guī)定的空白值的限度，或小于鱟試劑的內(nèi)毒素檢測(cè)限。在鱟試劑生產(chǎn)商規(guī)定的內(nèi)毒素濃度范圍內(nèi)，相關(guān)系數(shù)的絕對(duì)值︱r︱。根據(jù)鱟試劑生產(chǎn)商的建議，光度測(cè)定試驗(yàn)應(yīng)在孵育溫度下進(jìn)行（通常為37177。C). 根據(jù)鱟試劑生產(chǎn)商的建議，光度測(cè)定試驗(yàn)應(yīng)在孵育溫度下進(jìn)行（通常為37177。如供試品的所有結(jié)果均為陽(yáng)性，應(yīng)記為內(nèi)毒素濃度大于或等于最大的稀釋倍數(shù)乘以λ（如，初始稀釋倍數(shù)8λ）。從通過(guò)干擾試驗(yàn)的稀釋倍數(shù)開(kāi)始，用BET用水稀釋至1倍、2倍、4倍和8倍，每一濃度準(zhǔn)備兩份稀釋液，最后的稀釋倍數(shù)不得超過(guò)MVD，并且確定稀釋液通過(guò)了干擾試驗(yàn)。When a positie result is found for one replicate of solution A and a negative result is found for the others, repeat the test.當(dāng)溶液A的平行管其中一個(gè)檢查結(jié)果為陽(yáng)性，另一個(gè)為陰性時(shí)，重復(fù)試驗(yàn)。實(shí)驗(yàn)方法見(jiàn)（1）預(yù)備試驗(yàn)的（i）鱟試劑標(biāo)示靈敏度的復(fù)核試驗(yàn)項(xiàng)。Table Solution Endotoxin concentration/solution to which endotoxin is addedDiluent Dilution factorEndotoxin concentrationNumber of replicatesANone/Test solution4B2λ/Test solutionTest solution12482λ1λ4444C2λ/Water for BETWater for BET12482λ1λ2222DNone/Water for BET2Solution A = solution of the preparation being examined that is free of detectable endotoxins.Solution B = test for interference.Solution C = control of the labeled lysate sensitivity.Solution D = negative control (water for BET).溶液內(nèi)毒素濃度/配制內(nèi)毒素的溶液稀釋劑稀釋倍數(shù)稀釋后內(nèi)毒素的濃度平行管數(shù)A無(wú)/供試品溶液－－－4B2λ/供試品溶液供試品溶液12482λ1λ4444C2λ/BET用水BET用水12482λ1λ2222D0/BET用水－－－2A=經(jīng)檢查無(wú)內(nèi)毒素的溶液B=干擾實(shí)驗(yàn)用C=鱟試劑標(biāo)示靈敏度的對(duì)照品D=陰性對(duì)照品（BET檢查用水）The test for interfering factors must be repeated when any changes are made to the experimental conditions that are likely to influence the result of the test.如試驗(yàn)條件發(fā)生了任何可能影響到試驗(yàn)結(jié)果的變化，須重新進(jìn)行干擾因素試驗(yàn)。如未能形成堅(jiān)實(shí)且不變形的凝膠，該項(xiàng)檢查的結(jié)果即為陰性。1?C for 60177。7. GELCLOT TECHNIQUE (METHODS A AND B) 凝膠法（方法A和B）The gelclot techniques allow detection or quantification of endotoxins and is based on clotting of the lysate in the presence of endotoxins. The minimum concentration of endotoxins required to cause the lysate to clot under standard conditions is the labeled lysate sensitivity. To ensure both the precision and validity of the test, confirm the labeled lysate sensitivity and perform the test for interfering factors as described under 1. Preparatory testing.凝膠法系通過(guò)鱟試劑與內(nèi)毒素產(chǎn)生凝集反應(yīng)的原理來(lái)檢測(cè)或定量?jī)?nèi)毒素的方法。緩沖液必須經(jīng)過(guò)驗(yàn)證不含內(nèi)毒素和干擾因子。4. PREPARATION OF THE STANDARD ENDOTOXIN SOLUTIONS（ 內(nèi)毒素標(biāo)準(zhǔn)溶液的制備）After vigorously mixing the standard endotoxin stock solution, prepare appropriate serial dilutions of this solution using water for BET.充分混合內(nèi)毒素儲(chǔ)備標(biāo)準(zhǔn)溶液后，用細(xì)菌內(nèi)毒素試驗(yàn)檢查用水（BET用水）稀釋?zhuān)瞥蛇m當(dāng)?shù)南盗邢♂屢?，即得BET用內(nèi)毒素標(biāo)準(zhǔn)溶液。(2) Lysate solution鱟試液Dissolve amoebocyte lysate in water for BET or in a buffer, as remended by the lysate manufacturer, by gentle stirring. Store the reconstituted lysate, refrigerated or frozen, as indicated by the manufacturer.通過(guò)緩慢攪拌，將鱟試劑溶于水或溶于廠家推薦的緩沖液中來(lái)進(jìn)行細(xì)菌內(nèi)毒素試驗(yàn)（BET）。若使用塑料器械，如微孔板和微量進(jìn)樣器配套的吸頭等，它們必須標(biāo)明無(wú)內(nèi)毒素并確對(duì)試驗(yàn)無(wú)干擾。 the turbidimetric technique, based on the development of turbidity after cleavage of an endogenouse substrate。The test is carried out in a manner that avoids endotoxin contamination.試驗(yàn)操作過(guò)程應(yīng)防止內(nèi)毒素的污染。 they are prepared by removing from amoebocyte lysate the G factor, which reacts with glucans, or by inhibiting the G factor reactoing system of amoebocyte lysate. These preparations may be used for endotoxin testing in the presence of glucans.注：除了內(nèi)毒素外，鱟試劑和β葡聚糖反應(yīng)。IU的換算見(jiàn)國(guó)際衛(wèi)生組織（WHO）公布的國(guó)際標(biāo)準(zhǔn)。——。— Units/mL if the endotoxin limit is specified by unit of biological activity (IU/Unit),— 當(dāng)內(nèi)毒素限度以（IU/ml）表示時(shí)，用ml/ml表示濃度。當(dāng)使用新批號(hào)的鱟試劑或試驗(yàn)條件發(fā)生了任何可能影響檢驗(yàn)結(jié)果的改變時(shí)，應(yīng)進(jìn)行鱟試劑靈敏度復(fù)核試驗(yàn)。2min)，在此過(guò)程中，不能振動(dòng)試管。(ii) Test for interfering factors干擾因素試驗(yàn)Prepare solutions A, B, C and D as shown in Table , and use the test solutions at a dilution less than the MVD, not containing any detectable endotoxins, operating as described under 1. preparatory testing, (i) Confirmation of the labeled lysate sensitivity.、B、C、D。Interference may be overe by suitable validated treatment, such as filtration, neutralization, dialysis or heat treatment. To establish that the treatment chose effectively eliminates interference without loss of endotoxins, repeat the test for interfering factors using the preparation being examined to which the standard endotoxin has been added and which has then been submitted to the chosen treatment.可通過(guò)其他適宜的方法（如過(guò)濾、中和、透析或加熱處理等）排除干擾。(ii) Interpretation結(jié)果判斷The test is considered valid when both replicates of solution B and C are positive and those of solution D are negative.只有溶液B和C的平行管的試驗(yàn)結(jié)果均為陽(yáng)性、溶液D的平行管的試驗(yàn)結(jié)果均為陰性時(shí)，試驗(yàn)方有效。、B、C、D。供試品的內(nèi)毒素濃度指這些平行管的反應(yīng)終點(diǎn)濃度的幾何平均值。The kineticturbidimetric test (Method C) is a method to measure either the time (onset time) needed for the reaction mixture to reach a predetermined absorbance or transmission, or the rate of turbidity development.動(dòng)態(tài)濁度法（方法C）是檢測(cè)反應(yīng)混合物的濁度達(dá)到某一預(yù)先設(shè)定的吸光度或透光率需要的反應(yīng)時(shí)間（起效時(shí)間），或是檢測(cè)濁度變化率。The kineticchromogenic test (Method D) measures either the time (onset time) needed for the reaction mixture to reach a predetermined absorbance, or the rate of colour development.The test is carried out at the incubation temperature remended by the lysate manufacturer (usually 37 177。用內(nèi)毒素標(biāo)準(zhǔn)溶液制成至少三個(gè)濃度的稀釋液，以獲取標(biāo)準(zhǔn)曲線。 溶液B=加入了標(biāo)準(zhǔn)曲線終點(diǎn)或靠近中點(diǎn)的一個(gè)已知濃度內(nèi)毒素的、且與溶液A有相同稀釋倍數(shù)的供試品溶液。重復(fù)干擾因素實(shí)驗(yàn)以驗(yàn)證處理的有效性。(iii) InterpretationThe preparation being examined plies with the test if the mean