ELISA檢測試劑盒的開發(fā)及其方法學(xué)驗證

北京義翹神州科技股份有限公司ELISA檢測試劑盒的開發(fā)及其方法學(xué)驗證CONTENT義翹神州123EliSA試劑盒應(yīng)用及開發(fā)相關(guān)概述ELISA試劑盒的方法學(xué)驗證內(nèi)容及法規(guī)要求ELISA試劑盒開發(fā)案例分享4ELISA試劑盒開發(fā)服務(wù)介紹01義翹神州ELISA試劑盒應(yīng)用及開發(fā)相關(guān)概述義翹神州添加標(biāo)題添加標(biāo)題添加標(biāo)題中和抗體篩選ADAPK宿主蛋白殘留檢測免疫后血清滴度檢測抑制劑/中和抗體競爭篩選上清目標(biāo)分析物含量測定從研發(fā)到生產(chǎn)、質(zhì)控，貫穿生物試劑及醫(yī)藥研發(fā)的每一步ELISA檢測試劑盒應(yīng)用前景ELISA檢測類型B競爭ELISA底物抗體酶標(biāo)記二抗檢測信號抗原A間接ELISAC夾心ELISA原理概述：酶聯(lián)免疫吸附（ELISA）測定法的基礎(chǔ)是抗原與抗體之間的免疫學(xué)反應(yīng)，將抗原或抗體進(jìn)行固相化及酶標(biāo)記，加入酶反應(yīng)的底物后，底物被酶催化成為有色產(chǎn)物，產(chǎn)物的量與標(biāo)本中受檢物質(zhì)的量直接相關(guān)，由此進(jìn)行定性或定量分析。B酶標(biāo)二抗抗體藥靶點蛋白/biotin待測抗體包被抗體檢測抗體靶點蛋白義翹神州間接法ELISA檢測試劑盒間接法的優(yōu)點：方法建立相對容易，針對不同靶點適用性強(qiáng)，實驗條件易于調(diào)整成功建立方法的關(guān)鍵點：蛋白純度高，無雜質(zhì)排除非特異IgG的干擾待測抗體酶標(biāo)抗體顯色底物洗板洗板溫育讀數(shù)包被抗原洗板溫育義翹神州雙抗體夾心法ELISA檢測試劑盒夾心法的優(yōu)點：特異性強(qiáng)，可對蛋白準(zhǔn)確定量成功建立方法的關(guān)鍵點：排除內(nèi)源性物質(zhì)（類風(fēng)濕因子、自身抗體、異嗜性抗體等）及外源性物質(zhì)（標(biāo)本溶血、污染等）干擾酶標(biāo)抗體顯色底物洗板洗板溫育待測抗原讀數(shù)包被抗體溫育洗板義翹神州義翹神州競爭法ELISA檢測試劑盒B配體蛋白待測抗體受體蛋白BBB洗板溫育洗板顯色底物酶標(biāo)抗體BBB溫育洗板B讀數(shù)BBBB配體蛋白受體蛋白洗板溫育洗板顯色底物酶標(biāo)抗體BBBBBB溫育洗板讀數(shù)對照組：競爭組：02義翹神州ELISA試劑盒的方法學(xué)驗證內(nèi)容及法規(guī)要求義翹神州FDA法規(guī)驗證內(nèi)容標(biāo)準(zhǔn)曲線稀釋線性選擇性特異性準(zhǔn)確度&精密度靈敏度穩(wěn)定性義翹神州標(biāo)準(zhǔn)曲線反應(yīng)了分析物濃度與儀器響應(yīng)值之間的關(guān)系驗證要素可接受標(biāo)準(zhǔn)校正標(biāo)樣：至少6個有效校正標(biāo)樣，空白零濃度在相同基質(zhì)中進(jìn)行復(fù)孔檢測使用四參數(shù)、五參數(shù)擬合可使用錨定點進(jìn)行輔助擬合

定量下限/上限：回算濃度在標(biāo)示值的±25%，其它校正標(biāo)樣：回算濃度在標(biāo)示值的±20%

至少75%的校正標(biāo)樣滿足回算濃度在標(biāo)示值的±20%范圍內(nèi)錨定點可不遵循上述標(biāo)準(zhǔn)標(biāo)準(zhǔn)曲線1：2稀釋1：4

稀釋基質(zhì)中無干擾因素影響，線性稀釋檢測目標(biāo)分析物結(jié)果準(zhǔn)確未稀釋檢測值=81:2稀釋校正值=81:4稀釋校正值=8未稀釋檢測值=41:2稀釋校正值=6基質(zhì)中有干擾因素影響，可干擾目標(biāo)分析物檢測，可通過線性稀釋實驗進(jìn)行驗證分析物干擾因子義翹神州稀釋線性利用質(zhì)控樣品進(jìn)行稀釋線性的考察考察是否存在“前帶”或者“鉤狀效應(yīng)”每個稀釋因子做5個重復(fù)準(zhǔn)確度：回算濃度在標(biāo)示值±20%精密度：回算濃度的精密度±20%CV驗證要素可接受標(biāo)準(zhǔn)1：2稀釋1：4

稀釋1:4稀釋校正值=8特異性選擇性義翹神州驗證要素對含內(nèi)源性物質(zhì)的基質(zhì)進(jìn)行并行考察分析≥10個不同個體空白基質(zhì)基質(zhì)中存在非相關(guān)物質(zhì)的情況下，準(zhǔn)確測定分析物的能力樣本中存在干擾物質(zhì)的情況下，分析方法準(zhǔn)確測定分析物的能力≥80%的樣品準(zhǔn)確度在±20%定量下限水平，則準(zhǔn)確度在±25%

未摻入分析物的基質(zhì)的測量值低于定量下限可接受標(biāo)準(zhǔn)驗證要素可接受標(biāo)準(zhǔn)評估交叉反應(yīng)分子、合用藥物等考察可能的干擾物質(zhì)，同時測量不加分析物的基質(zhì)質(zhì)控樣品準(zhǔn)確度在±20%定量下限及定量上限準(zhǔn)確度在±25%義翹神州準(zhǔn)確度&精密度驗證要素可接受標(biāo)準(zhǔn)運行至少6個獨立的分析批包含5個質(zhì)控品濃度每個質(zhì)控濃度水平≥3個重復(fù)定量下限和定量上限批內(nèi)和批間準(zhǔn)確度應(yīng)在標(biāo)示值的±25%其他濃度水平質(zhì)控的批內(nèi)和批間精密度均不應(yīng)超過標(biāo)示值的20%每個濃度水平質(zhì)控品總誤差不應(yīng)超過30％定量下限和定量上限不應(yīng)超過40％驗證要素靈敏度校準(zhǔn)曲線最低非0標(biāo)準(zhǔn)品濃度定義為靈敏度，即定量下限至少6個分析批，≥3次重復(fù)123總誤差≤40%不夠準(zhǔn)確度應(yīng)為標(biāo)示濃度的±25%精密度±25%CV可接受標(biāo)準(zhǔn)穩(wěn)定性使用高、低濃度質(zhì)控品（至少3個重復(fù)）考察室溫條件、凍融次數(shù)、低溫放置等條件下的穩(wěn)定性驗證要素

每個濃度水平的質(zhì)控樣品的回算濃度在標(biāo)示值的±20%可接受標(biāo)準(zhǔn)03義翹神州ELISA試劑盒開發(fā)案例分享義翹神州范例01：游離抗體藥濃度檢測試劑盒開發(fā)及驗證01分析方法設(shè)計02分析方法建立03分析方法驗證類型：單抗與單抗配對目標(biāo)：檢測血清樣品中游離抗體藥抗體配對篩選配對抗體最佳濃度（棋盤法）試驗條件、試劑的準(zhǔn)備MRD、基質(zhì)干擾、鉤狀效應(yīng)標(biāo)準(zhǔn)曲線定量限ULOQ,LLOQ靈敏度精密度、準(zhǔn)確度選擇性特異性稀釋線性穩(wěn)定性“““義翹神州captureXXXMM05Detectionantibodydrug/ng/mlOD450-1OD450-2averageOD-BXXXMM14/HRP-1:800-Blank0.0720.0530.063

回收率SDCV(%)4.68750.1360.1160.1260.063104%0.01412%9.3750.1660.1740.1700.10795%0.0053%18.750.2880.2890.2880.225101%0.0000%37.50.5280.5100.5190.456100%0.0133%751.0070.9840.9950.932100%0.0172%1501.9231.9351.9291.866100%0.0090%3003.5133.5873.5503.487100%0.0521%四參數(shù)Logistic曲線擬合================================================方程:y=(A-D)/[1+(x/C)^B]+D------------------------------------------------------------------A=13.93049B=-1.11735C=804.00201D=0.01643r^2=1.00000================================================Anti-ID抗體MM05（非抑制型）Anti-ID抗體MM14（抑制型）抗體藥PK方法建立——雙抗體夾心抗體分型——競爭方法B酶標(biāo)二抗抗體藥靶點蛋白/biotin待測抗體義翹神州理論濃度（ng/mL）實測濃度(ng/mL)批間精密度分析批1分析批2分析批3分析批4分析批5分析批6MeanSDCV(%)240274.66261.84257.75264.57273.32254.51262.3810.684.07277.82248.28255.40271.17277.92252.20273.61255.21263.00263.00258.10240.56Mean275.36255.11258.72266.25269.78249.09SD2.196.783.894.3310.377.47CV(%)0.792.661.501.633.843.006057.5259.8956.7463.4662.8364.2858.823.295.6055.8657.1457.4861.9661.0360.5354.0556.7154.1354.1359.4361.54Mean55.8157.9156.1259.8561.1062.12SD1.741.721.765.011.701.94CV(%)3.112.983.138.372.793.122019.2821.1819.8721.9220.7722.6820.791.014.8519.4221.7821.1422.1920.7021.2119.9520.9120.1520.1521.5419.40Mean19.5521.2920.3921.4221.0021.10SD0.360.440.671.110.461.64CV(%)1.822.093.285.162.217.79ELISA法定量XXX抗體藥的方法學(xué)驗證——精密度注：CV（%）=SD/mean×100；CV（%）≤20%。批次LLOQ理論濃度（ng/ml）

實測濃度（ng/mL）準(zhǔn)確度（%）分析批19.389.54101.74分析批210.25109.26分析批310.34110.27分析批49.98106.34分析批59.57101.98分析批69.3899.95

Mean9.84

SD0.40

CV(%)4.11

準(zhǔn)確度(%)104.93

批次ULOQ理論濃度（ng/ml）實測濃度（ng/mL）準(zhǔn)確度（%）分析批1300347.87115.96分析批2303.13101.04分析批3314.55104.85分析批4320.13106.71分析批5323.82107.94分析批6313.70104.57

Mean320.53

SD15.12

CV(%)4.72

準(zhǔn)確度(%)106.84

注：CV（%）=SD/mean×100；CV（%）≤25%；準(zhǔn)確度（實測值的平均值/理論值×100%）介于75-125%義翹神州理論濃度（ng/mL）準(zhǔn)確度（%）分析批1分析批2分析批3分析批4分析批5分析批6240114.44109.10107.39110.24113.88106.04115.76103.45106.42112.99115.80105.08114.00106.34109.58109.58107.54100.24準(zhǔn)確度(%)114.73106.30107.80110.94112.41103.796096.4199.8194.56105.77104.72107.1393.0995.2495.8103.27101.71100.8890.0990.0994.5190.2299.05102.57準(zhǔn)確度(%)93.2096.5293.5399.76101.83103.532095.87108.5599.35109.58103.85113.4297.08105.89105.72110.96103.50106.0699.77108.88100.77100.77107.6997.01準(zhǔn)確度(%)97.57107.77101.95107.10105.01105.20ELISA法定量XXX抗體藥的方法學(xué)驗證——準(zhǔn)確度注：準(zhǔn)確度介于80%-120%義翹神州ELISA法定量XXX抗體藥的方法學(xué)驗證——選擇性*未能符合標(biāo)準(zhǔn)的樣品。理論濃度（ng/mL）準(zhǔn)確度(%)樣品1樣品2樣品3樣品4樣品5樣品6樣品7樣品8樣品9樣品10樣品11樣品12240386.46235.42250.43241.75248.90242.05264.65262.76227.16245.77240.26243.79準(zhǔn)確度(%)161.03*98.09104.35100.73103.71100.86110.27109.4894.65102.40100.11101.589.3817.887.437.777.237.507.438.057.577.097.307.238.58準(zhǔn)確度(%)190.62*79.2582.8877.0679.9779.2580.7075.6077.7977.0691.4391.43義翹神州實測濃度(ng/mL)樣品/ng/mL即刻室溫放置4h-70℃以下1周-70℃以下3周-70℃以下5周-70℃以下8周反復(fù)凍融3次1000000010821684.6010066813.7011148994.307888785.108953095.708764507.1010193309.0010346744.7010471936.109630740.808650029.809089166.808764507.109684242.9013131387.9010214407.808867099.709827165.409277315.908268558.109582747.50Mean10433272.4010251052.539882278.278788660.109106526.138599190.779820099.80回收率（%）104.33102.5198.8287.8991.0785.9998.20SD353189.28205032.111161556.74976597.83162805.70286336.29327168.40CV(%)3.39%2.00%11.75%11.11%1.79%3.33%3.33%100000102002.68100745.7093731.7890164.2887200.8596999.8496104.18104071.02102221.9093131.0988235.6397722.01102433.0995550.85106660.68104328.1989700.0193540.8592059.1293045.6297488.84Mean104244.79102431.9392187.6290646.9292327.3397492.8596381.29回收率（%）104.24102.4392.1990.6592.3397.4993.38SD2333.861800.462175.172685.345265.704713.11998.27CV(%)2.24%1.76%2.36%2.96%5.70%4.83%1.04%10000937.57948.88973.38919.01855.19919.30965.66920.44914.54917.58959.54798.931034.71935.24946.99965.76931.31932.51888.191018.28923.29Mean935.00943.06940.76937.02847.44990.76941.40回收率（%）93.5094.3194.0893.7084.7499.0894.14SD13.4626.1029.0720.6445.1362.4321.81CV(%)1.44%2.77%3.09%2.20%5.33%6.30%2.32%ELISA法定量XXX抗體藥的方法學(xué)驗證——高濃度穩(wěn)定性義翹神州范例02：針對新冠病毒的抗體滴度檢測試劑盒SARS-CoV-2

(B.1.1.529sublineageBA.4/BA.5/BA.5.2)

SpikeRBDAntibodyTiterAssayKit(Human)Cat:Kit040AcaptureSARS-CoV-2(BA.4/BA.5/BA.5.2)SpikeRBDProtein(HisTag)

Detection40150-D005

ng/mlOD450-1OD450-2averageOD-BMouseanti-humanIgG/HRPBlank0.0660.0630.064

1.56250.1000.0980.0990.0353.1250.1410.1420.1410.0776.250.2320.2300.2310.16712.50.4170.4300.4230.359250.7990.8020.8000.736501.5291.5731.5511.4871002.8022.8662.8342.770四參數(shù)Logistic曲線擬合===================================方程:y=(A-D)/[1+(x/C)^B]+D----------------------------------------------A=10.71988B=-1.11551C=257.27186D=-0.00017r^2=0.99999===================================底物抗體酶標(biāo)記二抗檢測信號抗原義翹神州SARS-CoV-2SpikeRBD(B.1.1.529sublineageBA.4/BA.5/BA.5.2)AntibodyTiterAssayKit(Mouse)Cat:Kit041captureSARS-CoV-2(BA.4/BA.5/BA.5.2)SpikeRBDProtein(HisTag)

Detection40592-MM117ng/mlOD450-1OD450-2averageOD-BRabbitanti-mouseIgG/HRPBlank0.0730.0600.066

0.3910.1200.1030.1110.0450.7810.1860.1610.1730.1071.5630.2640.2640.2640.1983.1250.4670.4710.4690.4036.2500.8510.8920.8710.80512.51.5441.5421.5431.477252.6152.6982.6572.591四參數(shù)Logistic曲線擬合===================================方程:y=(A-D)/[1+(x/C)^B]+D----------------------------------------------A=9.96607B=-1.00482C=70.63124D=-0.00740r^2=0.99995===================================04義翹神州ELISA試劑盒開發(fā)服務(wù)介紹義翹神州抗體親和力篩選抗體標(biāo)記抗體配對標(biāo)曲建立樣本檢測、準(zhǔn)確性驗證檢測方法建立及樣本檢測介入抗體親和力篩選抗體配對標(biāo)曲建立樣本檢測、準(zhǔn)確性驗證試劑盒生產(chǎn)及性能評估、驗證穩(wěn)定性準(zhǔn)確性精密度靈敏度特異性試劑盒研發(fā)試劑盒生產(chǎn)、質(zhì)控高品質(zhì)的試劑盒ELISA試