BioQuick-plus-抗生素(β-內(nèi)酰胺類)檢測試劑盒方案實施總結(jié)報告

PAGEword文檔可自由復(fù)制編輯BioQuickplus抗生素（β-內(nèi)酰胺類）檢測試劑盒方案實施總結(jié)報告目錄項目簡介·································································2技術(shù)路線·································································2研發(fā)內(nèi)容·································································4PBPs蛋白的選擇·························································4Amp/SA乳膠的包被·······················································4反應(yīng)緩沖體系（凍干緩沖體系）的驗證······································9常用緩沖體系··························································9緩沖系統(tǒng)初步篩選······················································10不同體系的膜反應(yīng)實驗··················································11膜反應(yīng)的進(jìn)一步檢測····················································11確認(rèn)緩沖系統(tǒng)··························································12緩沖體系使用量的確認(rèn)··················································13凍干反應(yīng)管的穩(wěn)定性檢測················································13膜處理試驗·····························································15層析膜處理方法初步試驗················································15層析膜處理后的牛奶檢測················································15層析膜處理后的老化實驗················································17不同氯化鈉濃度溶液處理膜··············································17Amp乳膠點膜后干燥時間的確認(rèn)以及加速老化試驗····························18Amp乳膠點膜后干燥時間的確認(rèn)··········································18加速老化試驗結(jié)果······················································19試劑盒性能的確認(rèn)·······················································22青霉素G靈敏度、重復(fù)性及檢測范圍的確認(rèn)·································22頭孢噻呋靈敏度、重復(fù)性及檢測范圍的確認(rèn)·································22其他抗生素靈敏度確認(rèn)···················································23研發(fā)驗證β-內(nèi)酰胺類抗生素靈敏度總結(jié)···································25特異性檢測·····························································26試劑盒加速老化試驗·····················································26質(zhì)控品·································································27PencillinG質(zhì)控品····················································27其他質(zhì)控品···························································29其他···································································29PBP2x原料功能性檢測··················································29PBP2x-HRP中間品檢測方法的建立········································30青霉素鉀鹽、鈉鹽比較；水解測定·······································30輸出文件·······························································31研發(fā)總結(jié)································································31項目簡介目前我們的抗生素檢測試劑盒由于抗體受其特異性的影響，不能同時檢測青霉素類抗生素和頭孢類抗生素，市場上主要的B-內(nèi)酰胺類抗生素檢測試劑盒有IDEXX公司的Snap試劑盒、Delvo公司的DelvoSP，Charm公司的CharmROSA，根據(jù)技術(shù)調(diào)研這幾家產(chǎn)品都能是使用青霉素受體蛋白（Penicillin-BindingProteins，簡稱PBPs）作為固定相，同時檢測青霉素類抗生素和頭孢類抗生素。它的原理是受體配體之間的特異性識別反應(yīng)。PBPs是一類存在于細(xì)菌細(xì)胞膜或細(xì)胞質(zhì)中的酶，種類很多（我們主要用到的是PBP2x和PBP5）。β-內(nèi)酰胺類抗生素中的β內(nèi)酰胺環(huán)結(jié)構(gòu)能夠與PBPs共價結(jié)合形成復(fù)合物，并且該結(jié)合過程不可逆轉(zhuǎn)，由于PBPs與β內(nèi)酰胺類抗生素親合能力，所以PBPs吸附分析法是繼免疫吸附法之后檢測抗生素殘留的有力工具，在檢測該類抗生素中具備十分廣闊的應(yīng)用前景。技術(shù)路線我們在研發(fā)初期設(shè)置3條技術(shù)路線，如下：Latex-PBPsLatex-PBPsAmp-HRPSamplePGLatex-MIgGR-A-MIgG-HRPA路線Latex-SALatex-SAAmp-HRPSamplePGLatex-MIgGR-A-MIgG-HRPBiotin-PBPsB路線Latex-AmpPBPs-HRPSamplePGLatex-AmpPBPs-HRPSamplePGLatex-MIgGR-A-MIgG-HRPC路線2.2技術(shù)路線B：通過固相包被技術(shù)將SA（鏈霉親和素）固定到微納米乳膠顆粒上通過固相包被技術(shù)將小鼠IgG固定到微納米乳膠顆粒上通過辣根過氧化酶(HRP)標(biāo)記技術(shù)，將Amp和兔抗小鼠抗體標(biāo)記HRP將SA包被的微納米乳膠顆粒和小鼠IgG包被微納米乳膠顆粒稀釋到一定濃度點樣到層析膜上將生物素標(biāo)記PBPs、HRP標(biāo)記的Amp和兔抗小鼠抗體標(biāo)記HRP稀釋到一定濃度，凍干。將牛奶樣品加入到試管中，45℃將反應(yīng)后的樣品加入到點有SA-乳膠顆粒和小鼠IgG-乳膠顆粒的層析膜進(jìn)行雙向免疫層析反應(yīng)。檢測讀數(shù)，根據(jù)SA-乳膠顆粒和小鼠IgG-乳膠顆粒反應(yīng)信號進(jìn)行分析和判定。輸出檢測結(jié)果2.3技術(shù)路線C：通過固相包被技術(shù)將BSA偶聯(lián)的氨芐青霉素（Amp）固定到微納米乳膠顆粒上；或?qū)㈡溍褂H和素固定到微納米乳膠顆粒上，通過生物素標(biāo)記Amp，將Amp包被到微納米乳膠顆粒表面。通過固相包被技術(shù)將小鼠IgG固定到微納米乳膠顆粒上通過辣根過氧化酶(HRP)標(biāo)記技術(shù)，將受體蛋白（PBPs）和兔抗小鼠抗體標(biāo)記HRP。將Amp包被到微納米乳膠顆粒和小鼠IgG包被微納米乳膠顆粒稀釋到一定濃度點樣到層析膜上。將HRP標(biāo)記的受體蛋白（PBPs）和兔抗小鼠抗體標(biāo)記HRP稀釋到一定濃度，凍干。將牛奶樣品加入到凍存有受體蛋白（PBPs）和兔抗小鼠抗體標(biāo)記HRP的試管中，45℃將反應(yīng)后的樣品倒到點有Amp-乳膠顆粒和小鼠IgG-乳膠顆粒的層析膜進(jìn)行雙向免疫層析反應(yīng)。檢測讀數(shù)，根據(jù)Amp-乳膠顆粒和小鼠IgG-乳膠顆粒反應(yīng)信號進(jìn)行分析和判定。輸出檢測結(jié)果研發(fā)內(nèi)容PBPs蛋白選擇PBPs來源十分廣泛，大多數(shù)細(xì)菌中都含有對β內(nèi)酰胺類抗生素敏感的一種或多種PBPs。根據(jù)各種PBPs對β內(nèi)酰胺類抗生素的敏感程度，我們選擇了serine-typeD-Ala-D-Alacarboxypeptidase（絲氨酸型D-丙氨酰-D-丙氨酸羧肽酶;也被稱為PBP5）作為提取目標(biāo)。該酶是一種最為典型的β內(nèi)酰胺類抗生素結(jié)合蛋白，對這類抗生素靈敏度高，且目前對其研究較多，技術(shù)資料相對比較全面，便于開展開發(fā)工作。我們計劃從嗜熱脂肪芽苞桿菌（ATCC15952、12980）中提取該酶。另外的途徑是通過外購PBPs蛋白（英國PBP2x和國內(nèi)PBPs蛋白），保證項目的順利進(jìn)行。詳細(xì)實驗結(jié)果見附件1：附件1：青霉素結(jié)合蛋白實驗報告.ppt我們選擇英國PBP2x和吉林PBPs蛋白進(jìn)行進(jìn)一步實驗，由于吉林PBPs蛋白不能穩(wěn)定供應(yīng)，最后我們在英國PBP2x基礎(chǔ)上進(jìn)行開發(fā)實驗。Amp/SA乳膠的包被SA包被乳膠由于SA包被乳膠成本比較高，且在反應(yīng)管中仍然需要存在2個點，一個點是Biotin-PBPs，另一個點是Amp-HRP和R-A-MIgG-HRP，對工藝要求比較高，不適用于大批量生產(chǎn)，所以不考慮該技術(shù)路線。BSA-Amp包被乳膠Amp（或其他抗生素）直接包被乳膠不同方法Amp和Ceftiofur包被乳膠，經(jīng)檢測包被效率不高，在MAbuffer中0和4ppb的信號差小于5，基本確認(rèn)該方法不可行。BSA-Amp（或其他抗生素偶聯(lián)BSA后）包被乳膠Latex-BSA/OVA-Amp，PBP2X-HRP反應(yīng)BSA-Amp（或其他抗生素偶聯(lián)BSA后）包被乳膠，檢測信號差（0和4ppb的信號差）基本小于10，包被效率比較低或其他原因，確認(rèn)該方法不可行。Latex-BSA/OVA-Cef,PBP2X-HRP反應(yīng)Cef包被乳膠后檢測，Cef包被上了乳膠，在MABuffer顯色明顯（絕對信號可以達(dá)到130），但由于PBP2x和牛奶的非特異反應(yīng)，在牛奶中的檢測信號很低。所以，Cef包被方法包被效率比較低，該方法不可行。Latex-BSA/OVA-Amp,PBP2X-Biotin，SA-HRP反應(yīng)BSA-Amp包被在96孔板，PBP2X-Biotin，SA-HRP反應(yīng)，如下圖，信號差異明顯。SA-HRP用于現(xiàn)有平臺的檢測，用MAbuffer替代牛奶，見下：信號平均信號差0ppb125131128134ppb137144141SA-HRP用于現(xiàn)有平臺的檢測，用牛奶樣本稀釋Amp標(biāo)準(zhǔn)品信號0ppb1431504ppb//調(diào)整試劑用量后的檢測情況如下：從實驗數(shù)據(jù)看，新的技術(shù)路線（方法）下，0ppb和4ppb的氨芐青霉素或0ppb和4ppb的青霉素G可以區(qū)分開，明顯比原技術(shù)路線有優(yōu)勢?，F(xiàn)在銷售的試劑盒氨芐青霉素靈敏度可以達(dá)到4ppb，但青霉素G的靈敏度只能達(dá)到8ppb左右。新的技術(shù)方法RandoxPBP2x蛋白青霉素G和氨芐青霉素的靈敏度都可以達(dá)到4ppb。分析原因，主要可能原因：1.牛奶樣本存在非特異性的反應(yīng)；2.包被效率不夠高，BSA分子量比較大，雖然BSA表面可以連接多個Amp，但BSA連接latex的概率會下降，導(dǎo)致總的Amp連接到latex上的量偏少；3.BSA-Amp連接到latex上后，由于BSA和latex相對于Amp來說都是大分子物質(zhì)，在Amp和PBPs蛋白反應(yīng)的時候可能存在空間位阻左右，抑制Amp和PBPs蛋白的反應(yīng)。總的來說，由于在牛奶樣本存在非特異性的反應(yīng)，信號降低明顯。根據(jù)繼續(xù)加大酶或者PBPs蛋白的用量，信號略微有增加，牛奶中的樣本信號差能夠達(dá)到15左右，但會導(dǎo)致本底增加或者成本大量上升，主要解決方法還是可能需要提高Amp包被效率。BSA包被乳膠，偶聯(lián)Amp本方法通過EDC和Sulfo-NHS將BSA偶聯(lián)到latex，然后通過BS3將Amp偶聯(lián)到BSA。本方法由于第一步先將BSA偶聯(lián)到latex，BSA氨基可以和latex上的羧基一對一連接，可以將盡量多的將BSA偶聯(lián)到latex，同時由于BSA上的剩余氨基將通過BS3將Amp上的氨基和BSA上的氨基偶聯(lián)，提高偶聯(lián)效率同時又增加Amp和BSA以及l(fā)atex之間的距離，降低空間位阻作用。由于考慮到可以增加包被效率，下面實驗采用HRP直接標(biāo)記PBPs蛋白，沒有采用SA-bition系統(tǒng)，降低反應(yīng)系統(tǒng)的復(fù)雜程度，降低成本。在MAbuffer中的反應(yīng)信號情況編號信號值A(chǔ)-19899B-165注：左側(cè)數(shù)據(jù)不同編號為不同的BSA，Amp，BS3的濃度76注：左側(cè)數(shù)據(jù)不同編號為不同的BSA，Amp，BS3的濃度A-29397B-27481A-38793B-35864A-48887B-47174信號增加明顯，陰性樣本信號基本可以達(dá)到100以內(nèi)，可以滿足檢測要求。在牛奶中的檢測情況（初步測試0ppb的牛奶和4ppbPenicillin以及Ceftiofur）檢測點信號值檢測時間0ppb4ppb5min130/131149/15210min115/117127/131結(jié)果認(rèn)為初步可以達(dá)到檢測要求根據(jù)上述結(jié)果，再進(jìn)行進(jìn)一步的包被條件確認(rèn)：根據(jù)下面不同條件，進(jìn)行包被乳膠，然后進(jìn)行點樣測試（稀釋倍數(shù)比較大手工點樣）A1：乳膠先與BS3反應(yīng)30min，再加入氨芐。（BS340μl+Amp42μl）A2：乳膠先與BS3反應(yīng)30min，再加入氨芐。（BS340μl+Amp14μl）A3：乳膠先與BS3反應(yīng)30min，再加入氨芐。（BS340μl+Amp28μl）B1：乳膠與BS3、氨芐同時反應(yīng)。（BS340μl+Amp42μl）B2：乳膠與BS3、氨芐同時反應(yīng)。（BS340μl+Amp14μl）B3：乳膠與BS3、氨芐同時反應(yīng)。（BS340μl+Amp28μl）B4：乳膠與BS3、氨芐同時反應(yīng)。（BS320μl+Amp14μl）B5：乳膠與BS3、氨芐同時反應(yīng)。（BS380μl+Amp28μl）0ppb4ppb青霉素GA1141.490147.838平均//平均信號差139.728144.790143.462144.624149.066146.8453.384A2143.252149.726145.002145.836140.872145.524144.844144.070147.678145.6470.803A3139.134143.150147.568149.892139.848143.066141.300150.108145.908148.3697.070B1134.790129.242132.364134.218125.724131.174130.233137.608136.906135.2745.042B2127.950127.846135.800138.524122.326121.220124.836129.474132.870134.1679.332B3127.532126.724131.386143.266118.210123.780124.062132.924140.010136.89712.835B4131.124129.700131.474133.052129.306129.794129.981146.498136.308136.8336.852B5122.098124.938130.234131.108129.018129.768126.456130.420133.610131.3434.887從實驗數(shù)據(jù)看，B3反應(yīng)條件包被的乳膠，檢測效果更好。BSA包被乳膠，偶聯(lián)Amp后保存的穩(wěn)定性檢測Amp通過BS3和BSA偶聯(lián)乳膠后保存于現(xiàn)有配方的乳膠稀釋液中，由于生產(chǎn)工藝需要，驗證其保存穩(wěn)定性。0ppb6ppb青霉素G3d97.26499.514平均136.072139.610平均信號差99.186103.92699.973135.804140.152137.91037.9376d96.308107.708133.886141.802106.058107.548104.406133.032138.822136.88632.48010d117.688115.112129.294140.480105.720100.936109.864137.384133.960135.28025.41616d102.866105.520135.046138.144111.158106.014106.390133.990138.736136.47930.09028d122.530125.722145.560148.568114.588120.320120.790142.120132.470142.18021.390從上述結(jié)果看，乳膠包被氨芐后，可在4℃總的來說，新的包被方法信號明顯提高，信號差也有提高，0ppb和4ppb的Penicillin以及和50ppb的Ceftiofur的信號差基本可以達(dá)到15以上，可以滿足檢測初步要求。從乳膠的穩(wěn)定性保存的結(jié)果看，后期工藝改進(jìn)的需要對乳膠稀釋液進(jìn)行進(jìn)一步的改進(jìn)，主要方向可能：乳膠稀釋液PH值，適當(dāng)調(diào)整乳膠稀釋液的PH值有助有改善保存穩(wěn)定。適當(dāng)加入一些穩(wěn)定劑緩沖系統(tǒng)反應(yīng)緩沖體系（凍干緩沖體系）的驗證常用緩沖系統(tǒng)：主要以以下系統(tǒng)為主：PBS，PH在7.0～8.0之間檸檬酸系統(tǒng)，PH在5.0～7.0之間Tris系統(tǒng)，PH在8.0～9.0之間CBS系統(tǒng)，PH在9.0以上硼酸系統(tǒng)，PH在7.0～8.0之間HEPES系統(tǒng)，PH在7.0～8.0之間通過不同緩沖系統(tǒng)的PH值，鹽離子濃度，加入不同濃度的抑制劑以及緩沖體系的體積，如：BSA，硝基硫酸亞鐵，葡聚糖等，來調(diào)節(jié)反應(yīng)。對BSA濃度進(jìn)行梯度實驗，發(fā)現(xiàn)當(dāng)BSA濃度超過2%以后，會出現(xiàn)不同程度的邊緣效應(yīng)，濃度越高，邊緣效應(yīng)約明顯。對硝基硫酸亞鐵和葡聚糖加入進(jìn)行初步檢測，具有一定的降低非特異性反應(yīng)的能力，預(yù)計可以增加信號差。緩沖系統(tǒng)初步篩選由于PBPs蛋白和牛奶中某些成份存在非特異反應(yīng)，對不同緩沖系統(tǒng)對非特異反應(yīng)的影響進(jìn)行初步檢測（96孔板反應(yīng)檢測）。實驗一、BSA-Amp包被，PBP2x-HRP檢測，樣本分別為水和牛奶，不加任何抗生素。實驗結(jié)果顯示，效果不明顯MAA-1A-2A-3A-4A-5H2O0.9361.0832.551.2090.8791.2390.8920.8622.5060.920.9041.003Milk0.2710.2730.2640.2630.2290.2620.2420.2530.2510.227B-1B-2C-1D-1D-2D-3H2O0.1570.1190.1250.340.3480.3180.1410.0360.150.2950.3320.273Milk0.1190.0350.0560.1260.1620.1340.1080.0760.0480.1280.1440.125A/B/C/D為不同反應(yīng)緩沖體系，主要為PBS，Tris，CBS，Citrate系統(tǒng)A-10.1MCitrateBufferPH6.42%BSAA-20.1MCitrateBufferPH6.42%BSA+0.1%SDSA-30.1MCitrateBufferPH6.42%BSA+0.5%PEG2000A-40.1MCitrateBufferPH6.42%BSA+0.5%PEG8000A-50.1MCitrateBufferPH6.42%BSA+0.5%PVP40B-110mMTris-EDTA(EDTA1mM)PH8.02%BSAB-210mMTris-EDTA(EDTA1mM)PH8.02%BSA+2.5ul羧肽酶C-150mMCBSPH9.62%BSAD-115mMPBSPH7.22%BSA+2.5ul羧肽酶D-215mMPBSPH7.22%BSA+0.1%SDS+2.5ul羧肽酶D-315mMPBSPH7.22%BSA+0.5%PVP40+2.5ul羧肽酶實驗二、對照MA為MAbuffer，檢測系統(tǒng)主要是不同濃度BSA和Tris緩沖體系，加入一定量的SOS以及SDS等表面活性劑。實驗結(jié)果顯示：E,G緩沖體系抑制牛奶和PBP2X蛋白非特異反應(yīng)明顯，進(jìn)一步在膜系統(tǒng)實驗。MAbuffer0.5410.612J0.0570.016milk0.0530.035K0.1030.143A0.3780.38L0.0640.059B0.2930.265M0.2060.178C0.4080.441N0.1570.15D0.4420.449O0.020.04E0.5080.537P0.1370.14F0.5380.44Q0.2580.205G0.5710.517R0.0910.067H0.4610.419S0.220.223I0.0320.041T0.3220.322初步篩選后，認(rèn)為E,G系統(tǒng)有進(jìn)一步篩選的可能，但在下一步膜反應(yīng)驗證中發(fā)現(xiàn)，SOS在5min的反應(yīng)后牛奶樣本凝固現(xiàn)象明顯，基本可以在膜反應(yīng)中進(jìn)行檢測，所以排除此類系統(tǒng)。不同體系的膜反應(yīng)實驗：Tris系統(tǒng)，CBS系統(tǒng)，PBS系統(tǒng)和HEPES+硼酸系統(tǒng)（加入一定量的BSA，硝基硫酸亞鐵和葡聚糖）膜反應(yīng)實驗。將latex-BSA-Amp點樣(非BS3偶聯(lián))，兩點同時點latex-BSA-Amp，加入PBPs-HRP，樣本為0ppb的牛奶和含4ppbPenicillin的牛奶，對照樣本為MAbuffer。（100ul緩沖液+300ul牛奶，對照400ulMAbuffer）緩沖系統(tǒng)0ppb4ppbTris.HClPH8.0152/151無法識別Tris.HClPH8.5無法識別無法識別Tris.HClPH9.0147/148無法識別CBSPH9.6155/159無法識別HEPES+硼酸鹽,PH8.0152/152149/152PBS+HEPES，PH8.0無法識別無法識別PBS，PH7.8137/140135/142MAbuffer（對照）123/129128/129初步在膜上的檢測結(jié)果顯示，PBS系統(tǒng)信號比較高。膜反應(yīng)的進(jìn)一步檢測：BSA包被乳膠，偶聯(lián)Amp后，不同緩沖系統(tǒng)在膜上的檢測實驗在上面3.2.3的實驗基礎(chǔ)上，調(diào)整緩沖系統(tǒng)，進(jìn)行進(jìn)一步的檢測實驗。將latex-BSA-Amp點樣(BS3偶聯(lián))，同時點latex-MouseIgG，加入PBPs-HRP和R-A-M-HRP，樣本為0ppb的牛奶和含4ppbPenicillin的牛奶緩沖系統(tǒng)0ppb4ppb比值信號備注比值信號備注A500.731104/127顯色略慢0.924117/121A1000.64288/122流速非常慢//流不動A200//流不動//流不動B500.744107/1280.879121/128B1000.66399/134流速慢0.769113/132流速慢B2000.64995/130漏奶，流速慢0.739108/131流速慢C500.815116/1280.987127/128C1000.770102/1190.767101/118D500.845115/1250.870115/119D1000.73496/1170.902115/121漏奶，快D2000.68889/1190.929123/127漏奶，快，異常E501.178115/1061.236124/112E1001.247116/1041.264129/114E2001.053116/1141.206118/108確認(rèn)緩沖系統(tǒng)在上面3.3.5的實驗基礎(chǔ)上對A50，C50，D100，D200等緩沖體系進(jìn)行進(jìn)一步的測試。將latex-BSA-Amp點樣(BS3偶聯(lián))，同時點latex-MouseIgG，加入PBPs-HRP和R-A-M-HRP，樣本為0ppb的牛奶和含4ppbPenicillin的牛奶。（在C50體系中，PBPs-HRP用量大于A50緩沖體系）0ppb4ppb0ppb4ppb0ppb4ppb比值信號比值信號比值信號比值信號比值信號比值信號A500.67879/1041.064107/1040.665811/1090.985102/1030.71895/1191.109119/113C500.86193/990.982107/1080.77293/1081.078108/1050.807106/1171.114124/118D1000.739106/1290.929128/1330.79897/1091.168113/105D2000.699122/1430.799123/138在后續(xù)的試驗中還有對A50和C50的測試，結(jié)果表明在陰性牛奶中C50的檢測值明顯要高于A50，相對來說0ppb和4ppb的牛奶信號差要小于A50，且PBPs-HRP用量大于A50緩沖體系。根據(jù)上述表格中的結(jié)果，確認(rèn)使用A50體系。緩沖體系使用量的確認(rèn)在后續(xù)試驗中，驗證得到A50緩沖體系使用量在40～80ul/test，如果A50緩沖體系使用量少會出現(xiàn)陰性牛奶檢測比值偏大，如果A50緩沖體系使用量過量，會導(dǎo)致檢測出現(xiàn)邊緣效應(yīng)，或者出現(xiàn)牛奶流速嚴(yán)重變慢，甚至于停止流動。所以，需要控制A50緩沖體系使用量在40～80ul/test。緩沖液體積0ppb4ppb青霉素G信號差對照點總平均40μl0.908107.170112.1961.006119.488119.2260.820100.180109.8101.045123.384120.8940.781106.936122.6101.019118.508117.4880.766102.442119.7181.019122.758121.7200.879105.090111.6661.132117.620110.9680.916114.944120.4041.053116.974114.230平均0.845106.127116.0671.046119.789117.42113.662116.744SD0.075.095.450.052.694.144.67CV7.69%4.80%4.69%4.38%2.24%3.52%4.00%50μl0.879122.322119.2141.201122.284112.2040.792109.776124.1061.260124.050109.9420.861106.226113.8880.908122.210127.8720.881105.088111.5021.049129.918127.0020.804103.622114.7381.034126.720124.7720.889103.946109.7941.114128.660122.390平均0.851108.497115.5401.094125.640120.69717.144118.119SD0.047.135.280.133.297.736.86CV4.96%6.57%4.57%11.53%2.62%6.40%5.81%80μl0.861110.828118.8800.964114.486116.5140.870102.110108.9001.062121.664118.2460.875102.608109.2381.008117.620117.2320.818106.008116.2561.049127.236124.4440.799102.860118.2160.995125.774126.1060.888106.514112.7081.021125.228124.044平均0.852105.155114.0331.017122.001121.09816.847117.565SD0.043.344.410.045.064.225.53CV4.13%3.18%3.86%3.53%4.14%3.48%4.70%根據(jù)上述表格中的結(jié)果，50μl緩沖液體積的試劑盒檢測后得到的信號差更大。凍干反應(yīng)管的穩(wěn)定性檢測初步調(diào)試一批試劑，將調(diào)試后的酶液和凍干緩沖液進(jìn)行凍干，凍干程序如下：①-25℃②-15℃③-5℃④5℃⑤25℃將凍干后的反應(yīng)管放置于37℃，進(jìn)行加速老化（10天），同時放置2～8℃0ppb比值檢測點下降值下降比值對照點下降值下降比值對照0.918104.497106.9681天0.921109.6905.1935.0%111.7244.7574.4%3天0.939114.62110.1249.7%116.1699.2028.6%5天0.904123.02718.53117.7%126.67519.70718.4%7天0.942135.50131.00429.7%136.82929.86227.9%10天0.906150.17645.68043.7%154.89747.93044.8%4ppb比值檢測點下降值下降比值對照點下降值下降比值對照1.311124.702108.5051天1.333127.1542.4532.0%109.0940.5890.5%3天1.269135.03710.3358.3%120.74512.24111.3%5天1.224142.91418.21314.6%130.51822.01320.3%7天1.157150.06925.36720.3%140.28431.78029.3%10天1.078157.74833.04726.5%150.81242.30839.0%從實驗結(jié)果看，凍干反應(yīng)管放置37℃最后，從實際的實驗結(jié)果來看，緩沖體系的改進(jìn)是下一步工作重點，主要考慮：PBS溶液體系中PB的濃度，降低牛奶流速過慢的風(fēng)險海藻糖的使用量（5%的海藻糖使用量偏少，造成凍干后試劑溶液分散成散塊狀或粉狀）膜處理試驗層析膜處理方法初步試驗由于膜供應(yīng)商對膜批間控制不夠，各批次之間的膜差異，特別是牛奶的流速方面，經(jīng)過試驗后認(rèn)為主要是膜的空隙控制不夠嚴(yán)格。由于膜存在批間差，我們對膜進(jìn)行初步預(yù)處理。初步的處理結(jié)果如下：半條膜吸水前吸水后1h2h3h4h6h吸水率殘留生理鹽水-0.5%吐溫60℃10.6218.8910.6777.87%0.0510.6319.3810.7482.32%0.1137℃10.8719.8911.0082.94%0.1310.9919.5511.0677.88%0.06干燥房10.8719.0913.4775.56%2.6010.8719.4310.9478.74%0.0710.8219.1710.8977.14%0.07干燥房10.9919.0512.1973.37%1.20去離子水-0.5%吐溫11.1319.3611.1173.91%-0.0211.0919.6311.0777.00%-0.02層析膜處理后的牛奶檢測針對新到的層析膜流速慢、顯色后檢測點顏色不均勻現(xiàn)象，對點樣緩沖液進(jìn)行調(diào)整以及膜的預(yù)處理處理和干燥。干燥后的膜進(jìn)行點樣和檢測，結(jié)果如下：流速/S顯色/S陰性讀數(shù)3.5ppb青霉素G差值a1乳膠加吐溫，生理鹽水-吐溫處理膜60℃42280110.942114.85250248109.064110.03443262105.452105.3750250105.488108.094a2生理鹽水-吐溫處理膜60℃5225598.272107.40652240108.048107.57640250108.294107.61460245116.4109.818d1乳膠加吐溫，生理鹽水-吐溫處理膜37℃70247108.376104.26412.36770250108.066115.55273241121.452124.61475250115.294124.366d2生理鹽水-吐溫處理膜37℃80243106.262104.54218.9288824094.71499.5690255122.87118.67880260119.26119.98f1乳膠加吐溫，生理鹽水-吐溫處理膜室溫4h54252101.714109.06416.25253255105.386105.7355255110.05119.70652270121.552123.918f2生理鹽水-吐溫處理膜室溫4h60285103.332106.1069.865325699.83104.83861275113.93121.3862272111.522114.708g1乳膠加吐溫，生理鹽水-吐溫處理膜室溫6h55220105.132108.2317.83170280104.83105.05257258122.654125.66270250121.972124.28g2生理鹽水-吐溫處理膜室溫6h70270105.398106.97217.04768270102.372106.90262245121.158125.6660277122.22120.794I1乳膠加吐溫，去離子水-吐溫處理膜室溫4h55295100.84107.6416.92250277102.774107.30850290118.72127626120.714I2去離子水-吐溫處理膜室溫4h50283104.638109.20617.44145268105.762104.1345292120.59124.51250290123.164125.232J1乳膠加吐溫，去離子水-吐溫處理膜室溫6h44300112.554113.23215.29445315111.47122932129.08853301127.98129.852J2去離子水-吐溫處理膜室溫6h45295116.396111.4146.87850290106.224115.4245306118.444123.88645305121.546121.276根據(jù)處理后的膜檢測陽性和陰性牛奶的差值，D2和G1差值最大。選取這兩種處理方法再處理一批層析膜，用于加速穩(wěn)定性試驗。層析膜處理后的老化實驗加速時間ABC0d2018191d1528193d1717205d1620207d1817129d152413注：A膜為層析膜在0.5%吐溫20-0.85%氯化鈉溶液浸泡后，在干燥房烘干，進(jìn)行點樣后置于37℃加速的檢測信號差B膜為層析膜在0.5%吐溫20-0.85%氯化鈉溶液浸泡后，在37℃條件下烘干，進(jìn)行點樣后置于37℃加速的檢測信號C膜為層析膜在0.5%吐溫20-0.85%氯化鈉溶液浸泡后，在干燥房烘干，然后將膜置于45℃加速老化后，進(jìn)行點樣，然后再檢測的信號差（0ppb和4ppb），加速時間是指45結(jié)果顯示：0.5%吐溫20-0.85%氯化鈉溶液浸泡、室溫干燥房干燥的膜流速及顯色時間偏差較小。37℃干燥的膜流速偏差大，不適合生產(chǎn)使用。所以，下一步試生產(chǎn)采用處理后的膜預(yù)計可以在室溫的條件下保存9個月。不同氯化鈉濃度溶液處理膜流速顯色陰性檢測值陽性檢測值差值備注去離子水-0.5%吐溫2038290121124頂部有藍(lán)線流向整齊均勻30292128128452961141163829312242290144140432951381404630513713944297140180.45%氯化鈉-0.5%吐溫2045292118119流向分層44278112118頂部有藍(lán)線442881161244428611848310129135頂部有藍(lán)線453051361375527713913849297136180.85%氯化鈉-0.5%吐溫2053265118116流向部分鋸齒，分層472551151205025811811950259118/28013413843300133136462781321344528613517由于在檢測中發(fā)現(xiàn)，用0.85%氯化鈉-0.5%吐溫20溶液處理的膜，在牛奶進(jìn)行層析擴(kuò)散時存在分層現(xiàn)象，所以，對處理溶液再一次進(jìn)行確認(rèn)。分別用去離子水-0.5%吐溫20，0.45%氯化鈉-0.5%吐溫20，0.85%氯化鈉-0.5%吐溫20溶液進(jìn)行處理，然后干燥房烘干后點膜測試。從數(shù)據(jù)結(jié)果看，重復(fù)試驗不同的浸泡溶液對操作過程及結(jié)果的影響，三種溶液對檢測的絕對值、0ppb和4ppb的信號差沒有太大區(qū)別，但去離子水溶液對流向現(xiàn)象改善較明顯，牛奶層析輪廓明顯，沒有出現(xiàn)分層現(xiàn)象。對本試驗進(jìn)行重復(fù)一次后，結(jié)果相同，顯示使用去離子水吐溫溶液浸泡后液體流速均勻性好，流向一致，且三種溶液檢測結(jié)果沒有明顯差異。最終選定去離子水-0.5%吐溫20溶液作為膜處理液，浸泡45min后干燥房室溫晾干6小時。由于實驗時間限制，對去離子水-0.5%吐溫20溶液作為膜處理液，浸泡45min后干燥房室溫晾干6小時的膜及試劑盒穩(wěn)定性的影響將在試生產(chǎn)好的試劑盒上進(jìn)行檢測。Amp乳膠點膜后干燥時間的確認(rèn)以及加速老化試驗Amp乳膠點膜后干燥時間的確認(rèn)包被好的乳膠進(jìn)行手工點樣，37℃0ppb4ppb青霉素G信號差比值檢測點對照點比值檢測點對照點1h0.684101.790132.9720.873122.364130.3880.70497.720124.1480.904121.324127.2621.051124.950122.0940.951123.010126.022平均99.755128.560122.912126.44223.1572h0.719105.792131.7400.899122.228128.5080.71299.144124.4780.875117.210124.742平均102.468128.109119.719126.62517.251結(jié)果認(rèn)為點膜后37℃加速老化試驗結(jié)果包被好的乳膠點樣后37℃干燥40min，1h，1.5h，2h，2.5h，3h，3.5h，4h，然后將干燥后的膜放入37℃進(jìn)行加速老化實驗，同時放置2～8℃的膜作為對照，進(jìn)行檢測，確認(rèn)實驗一、40min，1h，1.5h的檢測結(jié)果0ppb檢測點40對照點40檢測點1對照點1檢測點1.5對照點1.5489.204114.969102.658124.89792.248116.4651d83.877126.55796.04122.3193.34123.7623d97.324119.94596.698128.03789.095120.5075d96.505121.47d99.859114.97990.7121.1289d100.789123.27293.941126.15998.251127.22510d104.326126.86190.447125.97395.165123.48平均95.90121.1096.05124.8093.13122.094ppb檢測點40對照點40檢測點1對照點1檢測點1.5對照點1.54119.26103.4118.725111.075113.105106.8551d121.294110.377119.883110.442112.749105.2283d122.436115.287121.291114.66116.522109.9065d122.023114.9177d120.705113.056115.838104.5279d121.325114.456117.054/116.287113.10710d121.509111.412118.509112.669113.284108.557平均121.09111.33119.58112.75114.63108.03實驗二、1h，1.5h，2h,2.5h,3h,3.5h,4h,4.5h的實驗結(jié)果數(shù)據(jù)1:0ppb結(jié)果1hour41.275115.032103.5261.103108.892103.6161d0.817107.85118.4860.867106.316113.663d0.908104.91109.811.101105.496100.8925d0.829102.546111.8980.869100.736107.6047d0.78491.936104.9760.83296.674105.29810d0.881108.766115.390.792102.306115.6720.868102.7536110.36861.5hours40.865101.776108.9260.8799.574106.1681d0.78998.47111.6560.78998.098111.3165d0.836115.514125.510.745101.432121.887d0.8108.268120.2420.799107.358120.45410d0.805105.816117.7260.817106.17116.5980.81104.25116.052hours40.906103.91108.8641.062106.988104.0061d0.797100.26112.7420.942106.61109.5825d0.72597.35120.040.73793.998114.0987d0.73995.708115.8260.75296.126114.34610d0.73296.194117.5640.72791.574112.7520.7797.23114.622.5hours40.909102.63107.3260.886103.56109.6741d0.806101.828113.2060.92101.902106.0185d0.936104.206107.5380.936104.49107.8327d0.78996.84109.9440.873100.012106.54210d0.929104.752108.5060.822104.746114.680.88102.50109.133hours40.80896.97107.0620.92697.958101.5241d0.80299.636111.282///5d0.82299.256108.6380.77699.028114.2567d0.798108.394121.6920.899106.17111.57410d0.881107.3113.7920.818105.236115.4440.84102.22111.703.5hours40.899101.146106.294///1d0.87104.914111.850.886103.448109.5565d0.875103.714110.4420.825105.49115.3827d0.838103.528112.3420.813102.098112.42210d0.815109.046119.9620.881106.696113.1880.86104.45112.384hours40.78795.41108.6180.77293.516108.3681d0.818107.962118.430.79699.452111.9525d0.893105.574111.2760.73997.644118.2487d0.838109.38118.7580.792105.038118.69210d0.867106.786114.1620.827106.472116.3620.81102.72114.494.5hours40.972108.248109.7560.891103.478109.161d0.831106.54115.320.962110.738112.7785d0.829106.356116.070.822102.776112.5687d0.805102.068113.5380.843102.26110.61210d0.817106.946117.450.845106.49115.0720.86105.59113.23數(shù)據(jù)2:4.5ppb結(jié)果1hour41.058122.23119.0681d0.94107.376110.583d1.043113.874111.6945d1.158105.916100.5567d0.916102.1106.45410d1.222116.118105.71.056111.269109.0091.5hours40.895108.83114.5761d1.01106.36105.8645d0.935116.566120.3027d1.034118.326116.5110d0.851114.662123.4880.882112.086122.7240.935112.805117.2442hours41.066119.664116.1341.222116.934106.441d0.924113.846118.1520.911103.266107.8585d0.873116.98124.581.103117.73112.5187d0.924110.732114.9640.97120.332122.16210d0.926117.282121.5660.958117.076119.470.988115.384116.3843hours41.23126.494114.7441.049117.00114.431d0.997115.268115.481.082119.00114.7245d0.964115.084117.1160.964113.886115.867d1.14114.828107.9361.058112.44109.56210d1.074116.358112.5861.116118.448112.5761.067116.881113.501分析：從陰性牛奶的數(shù)據(jù)看，基本都符合陰性標(biāo)準(zhǔn)，但從陽性數(shù)據(jù)看，干燥時間長后，檢測點和對照點的信號逐漸變?nèi)酰瑢φ丈厦?0min，1h，1.5h數(shù)據(jù)，我們選擇1h的干燥時間。試劑盒性能的確認(rèn)青霉素G靈敏度、重復(fù)性及檢測范圍的確認(rèn)比值檢測點對照點0ppb0.806110.236121.8800.703102.590120.3744ppb1.076124.714120.4981.062116.070112.8401.114120.584114.6521.062120.388117.0581.194123.454113.6681.097118.004113.0001.076119.266115.2721.103120.234114.938平均值1.098120.339115.241SD0.043CV0.03910ppb1.134122.330115.34020ppb1.405136.514111.34650ppb1.767158.822110.626100ppb1.771166.538115.786從數(shù)據(jù)看，青霉素G靈敏度可以達(dá)到4ppb，在4ppb濃度是的CV是3.9%，檢測范圍4-50pb有相當(dāng)?shù)木€性，儀器和試劑在濃度50ppb以上，信號基本達(dá)到飽和。頭孢噻呋靈敏度、重復(fù)性及檢測范圍的確認(rèn)比值檢測點對照點0ppb0.813110.014121.1180.867107.676115.1241ppb1.082120.872116.5021.084122.804118.2021.123117.57111.350.897114.206120.1261.053119.258116.3681.028108.14105.7881.16118.304110.3741.015117.444116.6641.078120.4116.2382ppb1.318132.644113.8481.166121.078112.6421.134124.86117.7561.227127.104115.5421.196131.912121.3481.234125.618113.5261.178130.452120.8081.178129.4119.8981.218127.252116.024平均1.205127.813116.821SD0.053CV0.0444ppb1.595152.638115.4110ppb1.769158.54110.312從數(shù)據(jù)看，頭孢噻呋靈敏度可以達(dá)到2ppb，在2ppb時的CV是4.4%，檢測范圍4-10pb有相當(dāng)?shù)木€性，儀器和試劑在濃度10ppb以上，信號基本達(dá)到飽和。其他抗生素靈敏度確認(rèn)頭孢匹林比值檢測點對照點0ppb0.789101.956115.8560.936108.5912.0584ppb0.811110.168121.348ppb1.076122.47118.3461.051117.198114.4881.13119.786113.1721.103117.726112.4781.154115.72108.231.064105.942102.8681.051113.82111.1881.091114.904110.3561.097112.488107.7061.184124.37114.8821.146117.976110.6941.06113.926110.8321.116115.86110.1161.053110.574107.941.109115.376109.9081.116115.72109.9641.093117.194112.4平均1.088115.701111.269SD0.0524.1223.397CV0.0480.0360.03110ppb1.146120.664113.2550ppb1.719159.182113.534頭孢匹林靈敏度可以達(dá)到8ppb氨芐青霉素（三水）濃度比值檢測點對照點4ppb1.230122.320110.9781.130119.280112.7281.093117.418112.6541.074119.358115.4441.269124.054109.1761.127116.824110.4201.101112.386107.4881.074113.084109.420平均1.137118.091111.039氨芐青霉素靈敏度達(dá)到4ppb阿莫西林比值檢測點對照點4ppb1.107116.72111.3621.047117.842115.3244.5ppb1.058117.628114.5881.036116.676114.81.043118.624116.3380.997115.522115.7240.972110.982112.4620.922113.534117.9181.043117.454115.2181.028118.214116.766ppb1.062127.436123.8641.266129.47114.2321.123121.628115.2761.136119.658112.7221.154118.292110.6141.14119.904112.761.204123.202113.0061.072115.776112.1441.148121.144113.631.148121.144113.61.259122.722108.868平均1.156121.852113.701SD0.0653.8863.791CV0.0570.0320.03310ppb1.299132.57611520ppb1.578147.96112.55250ppb1.904176.342115.312100ppb2.039171.242105.618阿莫西林靈敏度達(dá)到6ppb苯唑西林檢測值濃度比值檢測點對照點15ppb1.049107.212104.83630ppb1.236127.858115.364苯唑西林靈敏度達(dá)到15ppb頭孢氨芐比值檢測點對照點0ppb0.938100.486103.584200ppb0.883102.314108.3960.913105.266109.77300ppb1.01111.182110.7081.004109.414109.282350ppb1.182117.672108.7821.087114.834110.5221.118116.856110.9621.123116.058109.9981.076111.654107.9561.109110.992105.7841.13113.184106.9141.244110.91499.5060.955107.848110.271.03109.682108.17平均1.105112.969107.886SD0.0793.2863.383CV0.0710.0290.031400ppb1.18117.03108.3061.107107.97102.928500ppb1.116118.208110.0581.39124.028101.9881000ppb1.341131.304111.151.657135.1499.9685000ppb1.925164.736106.74從數(shù)據(jù)看頭孢氨芐的靈敏度只能達(dá)到350ppb。研發(fā)驗證β-內(nèi)酰胺類抗生素靈敏度總結(jié)：檢測項目MRL（EU）.ppbBioer.ppbSanp.ppb氨芐青霉素445青霉素G443頭孢噻呋10023頭孢匹林6083阿莫西林467頭孢氨芐10035025苯唑西林301540頭孢唑林506015上表主要是研發(fā)內(nèi)部驗證的數(shù)據(jù)，實際數(shù)據(jù)要在試生產(chǎn)后確認(rèn)。特異性檢測主要針對非β-內(nèi)酰胺類藥物干擾試驗，適當(dāng)高濃度的非β-內(nèi)酰胺類藥物進(jìn)行檢測。結(jié)果如下：濃度名稱500ng/ml1000ng/ml比值檢測點對照點比值檢測點對照點磺胺嘧啶0.71296.460121.1060.72190.310111.970磺胺二甲嘧啶0.73095.766117.2780.67788.856117.130氯霉素0.66388.200118.6260.69289.950116.214土霉素0.67093.004123.6840.73798.612119.750金霉素0.71488.584110.9160.68992.994120.658慶大霉素0.66989.776119.6620.788102.558116.562鹽酸四環(huán)素0.73996.100116.2940.780101.838116.868從檢測結(jié)果看，1000ng/ml的上述各種抗生素對β-內(nèi)酰胺類抗生素的檢測沒有影響。所以，我們認(rèn)為這些抗生素對β-內(nèi)酰胺類抗生素的檢測不存在干擾現(xiàn)象。試劑盒加速老化試驗我們根據(jù)阿倫尼烏斯公式，正常保存溫度為2～8℃的試劑盒，加速老化溫度設(shè)定為37℃，我們選取調(diào)試好的試劑盒進(jìn)行37℃370ppb4ppb青霉素G0d比值檢測點對照點比值檢測點對照點0.84594.964102.6251.233101.84894.5940.80695.9106.081.082109.858105.8920.76893.33108.8941.17101.84894.8940.76190.368106.2661.21101.6692.942平均0.79593.641105.9661.174103.80497.0811d0.829112.664122.9881.053124.608121.6060.76100.764118.551.218123.262112.4480.947105.518108.2541.062122.494119.1020.72489.466110.5481.087121.08116.466平均0.815102.103115.0851.105122.861117.413d0.936127.026131.0581.06130.92127.4180.881120.902128.31.206137.682126.940.861126.664135.8141.093134.53129.090.974136.382138.0441.144134.132126.054平均0.913127.744133.3041.126134.316127.3765d0.972129.048130.7981.038137.552135.150.802115.686128.1221.198130.97120.3140.947120.754123.9661.152129.312121.050.853121.65130.851.206137.696126.1360.815129.96142.8181.109125.44119.578平均0.878123.420131.3111.141132.194124.4467d0.886131.396139.1581.157141.806132.5040.827130.048142.0181.184136.226125.8840.869124.436134.9781.235137.374124.030.877143.724152.7461.134130.036122.596平均0.865132.401142.2251.178136.361126.2549d0.847134.038144.7041.144131.444125.5180.995132.128132.431.134134.334126.6480.908131.872138.0241.144134.68126.520.893132.086139.1921.245139.464124.972平均0.911132.531138.5881.167134.981125.915從數(shù)據(jù)看，我們的試劑盒在37℃存放9天，檢測結(jié)果符合試劑盒的質(zhì)量標(biāo)準(zhǔn)，但檢測點和對照點絕對值都升高，從數(shù)據(jù)看7天的檢測信號升高30%以下。所以，從上面的數(shù)據(jù)看，根據(jù)阿倫尼烏斯公式的推算，我們認(rèn)為我們的試劑盒在2～8℃正常溫度（2～8℃）保存的穩(wěn)定性實驗需要等到試生產(chǎn)完成后開始。質(zhì)控品PenicillinG質(zhì)控品由于本試劑盒和原先的抗生素檢測試劑盒不同，且主要針對PenicillinG和頭孢的檢測，所以，本試劑盒的主要質(zhì)控品采用PenicillinG，而不是原先的Ampicillin。所以，本實驗對質(zhì)控品的制備進(jìn)行驗證，以建立PenicillinG質(zhì)控標(biāo)準(zhǔn)品的配制標(biāo)準(zhǔn)操作規(guī)程。由于PenicillinG有現(xiàn)成的標(biāo)準(zhǔn)品，要求采用德國Dr.青霉素G鉀鹽標(biāo)準(zhǔn)品，稱取適量的青霉素G鉀鹽標(biāo)準(zhǔn)品，用生理鹽水和去離子水分別進(jìn)行復(fù)溶，配制成100ng/ml的水溶液，取72ul加入2ml離心管，加入1728ul牛奶，分別用三個Snap試劑盒進(jìn)行檢測，計算平均值。根據(jù)結(jié)果，分別將100ng/ml的溶液進(jìn)行72ul/2ml分裝，然后凍干。凍干程序如下：程序節(jié)溫度速度時間Seg1:-25℃0.5Seg2:-10℃0.5Seg3:-0℃0.5Seg4:10℃1Seg5:25℃1.5分別取凍干后的兩種凍干品3個，用1800ul牛奶進(jìn)行復(fù)溶，分別用Snap試劑盒對每個樣本進(jìn)行檢測，計算平均值。數(shù)據(jù)如下：凍干溶液凍干前凍干后比值檢測點對照點比值檢測點對照點比值升降%去離子水1.5991641241.7181531091.8311641111.8441691131.3931411151.76164114平均1.6081561171.77416211210.35%標(biāo)準(zhǔn)偏差0.2213.286.660.068.192.65生理鹽水1.4571481171.4571351071.551421091.3141401201.4681451141.374140116平均1.4921451131.382138114-7.37%標(biāo)準(zhǔn)偏差0.0534.040.072.896.66根據(jù)檢測數(shù)據(jù)（Snap試劑盒03729-AG261），發(fā)現(xiàn)：去離子水溶解PenicillinG，凍干前檢測數(shù)據(jù)不穩(wěn)定，波動比較大去離子水溶解PenicillinG，凍干后檢測比值比凍干前的大，說明PenicillinG的濃度在凍干后提高了，可能存在比較大的誤差。生理鹽水溶解PenicillinG，檢測數(shù)據(jù)比較穩(wěn)定，凍干后比凍干前略有損耗。用去離子水溶解PenicillinG凍干后基本看不到凍干點，而生理鹽水溶解PenicillinG凍干后凍干點明顯。最后確認(rèn)用方法用生理鹽水溶解PenicillinG凍干，凍干后進(jìn)行檢測，根據(jù)凍干前后的數(shù)據(jù)，在復(fù)溶的時候進(jìn)行調(diào)整加入牛奶的體積。凍干一周后檢測結(jié)果發(fā)現(xiàn)：PenicillinG凍干質(zhì)控品不穩(wěn)定。實驗設(shè)計：用1.666ml牛奶復(fù)溶（1.8ml×92.6%）生理鹽水PenicillinG凍干質(zhì)控品，用Snap試劑盒03729-AG261檢測，檢測結(jié)果如下：時間檢測值檢測點對照點2011-11-231.1661281192011-11-241.06111108其他質(zhì)控品由于PenicillinG質(zhì)控品不穩(wěn)定，進(jìn)一步確認(rèn)其他質(zhì)控品：Ampicillin，Ccftiofur，Cefapirin。方法同PenicillinG質(zhì)控品，也是用生理鹽水作為凍干緩沖體系，凍干程序相同。其中Ampicillin凍干濃度理論6ppb（加入1.8ml牛奶，下同），Ccftiof