ELISA检测试剂盒的开发及其方法学验证

北京义翘神州科技股份有限公司ELISA检测试剂盒的开发及其方法学验证CONTENT义翘神州123EliSA试剂盒应用及开发相关概述ELISA试剂盒的方法学验证内容及法规要求ELISA试剂盒开发案例分享4ELISA试剂盒开发服务介绍01义翘神州ELISA试剂盒应用及开发相关概述义翘神州添加标题添加标题添加标题中和抗体筛选ADAPK宿主蛋白残留检测免疫后血清滴度检测抑制剂/中和抗体竞争筛选上清目标分析物含量测定从研发到生产、质控，贯穿生物试剂及医药研发的每一步ELISA检测试剂盒应用前景ELISA检测类型B竞争ELISA底物抗体酶标记二抗检测信号抗原A间接ELISAC夹心ELISA原理概述：酶联免疫吸附（ELISA）测定法的基础是抗原与抗体之间的免疫学反应，将抗原或抗体进行固相化及酶标记，加入酶反应的底物后，底物被酶催化成为有色产物，产物的量与标本中受检物质的量直接相关，由此进行定性或定量分析。B酶标二抗抗体药靶点蛋白/biotin待测抗体包被抗体检测抗体靶点蛋白义翘神州间接法ELISA检测试剂盒间接法的优点：方法建立相对容易，针对不同靶点适用性强，实验条件易于调整成功建立方法的关键点：蛋白纯度高，无杂质排除非特异IgG的干扰待测抗体酶标抗体显色底物洗板洗板温育读数包被抗原洗板温育义翘神州双抗体夹心法ELISA检测试剂盒夹心法的优点：特异性强，可对蛋白准确定量成功建立方法的关键点：排除内源性物质（类风湿因子、自身抗体、异嗜性抗体等）及外源性物质（标本溶血、污染等）干扰酶标抗体显色底物洗板洗板温育待测抗原读数包被抗体温育洗板义翘神州义翘神州竞争法ELISA检测试剂盒B配体蛋白待测抗体受体蛋白BBB洗板温育洗板显色底物酶标抗体BBB温育洗板B读数BBBB配体蛋白受体蛋白洗板温育洗板显色底物酶标抗体BBBBBB温育洗板读数对照组：竞争组：02义翘神州ELISA试剂盒的方法学验证内容及法规要求义翘神州FDA法规验证内容标准曲线稀释线性选择性特异性准确度&精密度灵敏度稳定性义翘神州标准曲线反应了分析物浓度与仪器响应值之间的关系验证要素可接受标准校正标样：至少6个有效校正标样，空白零浓度在相同基质中进行复孔检测使用四参数、五参数拟合可使用锚定点进行辅助拟合

定量下限/上限：回算浓度在标示值的±25%，其它校正标样：回算浓度在标示值的±20%

至少75%的校正标样满足回算浓度在标示值的±20%范围内锚定点可不遵循上述标准标准曲线1：2稀释1：4

稀释基质中无干扰因素影响，线性稀释检测目标分析物结果准确未稀释检测值=81:2稀释校正值=81:4稀释校正值=8未稀释检测值=41:2稀释校正值=6基质中有干扰因素影响，可干扰目标分析物检测，可通过线性稀释实验进行验证分析物干扰因子义翘神州稀释线性利用质控样品进行稀释线性的考察考察是否存在“前带”或者“钩状效应”每个稀释因子做5个重复准确度：回算浓度在标示值±20%精密度：回算浓度的精密度±20%CV验证要素可接受标准1：2稀释1：4

稀释1:4稀释校正值=8特异性选择性义翘神州验证要素对含内源性物质的基质进行并行考察分析≥10个不同个体空白基质基质中存在非相关物质的情况下，准确测定分析物的能力样本中存在干扰物质的情况下，分析方法准确测定分析物的能力≥80%的样品准确度在±20%定量下限水平，则准确度在±25%

未掺入分析物的基质的测量值低于定量下限可接受标准验证要素可接受标准评估交叉反应分子、合用药物等考察可能的干扰物质，同时测量不加分析物的基质质控样品准确度在±20%定量下限及定量上限准确度在±25%义翘神州准确度&精密度验证要素可接受标准运行至少6个独立的分析批包含5个质控品浓度每个质控浓度水平≥3个重复定量下限和定量上限批内和批间准确度应在标示值的±25%其他浓度水平质控的批内和批间精密度均不应超过标示值的20%每个浓度水平质控品总误差不应超过30％定量下限和定量上限不应超过40％验证要素灵敏度校准曲线最低非0标准品浓度定义为灵敏度，即定量下限至少6个分析批，≥3次重复123总误差≤40%不够准确度应为标示浓度的±25%精密度±25%CV可接受标准稳定性使用高、低浓度质控品（至少3个重复）考察室温条件、冻融次数、低温放置等条件下的稳定性验证要素

每个浓度水平的质控样品的回算浓度在标示值的±20%可接受标准03义翘神州ELISA试剂盒开发案例分享义翘神州范例01：游离抗体药浓度检测试剂盒开发及验证01分析方法设计02分析方法建立03分析方法验证类型：单抗与单抗配对目标：检测血清样品中游离抗体药抗体配对筛选配对抗体最佳浓度（棋盘法）试验条件、试剂的准备MRD、基质干扰、钩状效应标准曲线定量限ULOQ,LLOQ灵敏度精密度、准确度选择性特异性稀释线性稳定性“““义翘神州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%四参数Logistic曲线拟合================================================方程:y=(A-D)/[1+(x/C)^B]+D------------------------------------------------------------------A=13.93049B=-1.11735C=804.00201D=0.01643r^2=1.00000================================================Anti-ID抗体MM05（非抑制型）Anti-ID抗体MM14（抑制型）抗体药PK方法建立——双抗体夹心抗体分型——竞争方法B酶标二抗抗体药靶点蛋白/biotin待测抗体义翘神州理论浓度（ng/mL）实测浓度(ng/mL)批间精密度分析批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抗体药的方法学验证——精密度注：CV（%）=SD/mean×100；CV（%）≤20%。批次LLOQ理论浓度（ng/ml）

实测浓度（ng/mL）准确度（%）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

准确度(%)104.93

批次ULOQ理论浓度（ng/ml）实测浓度（ng/mL）准确度（%）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

准确度(%)106.84

注：CV（%）=SD/mean×100；CV（%）≤25%；准确度（实测值的平均值/理论值×100%）介于75-125%义翘神州理论浓度（ng/mL）准确度（%）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准确度(%)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准确度(%)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准确度(%)97.57107.77101.95107.10105.01105.20ELISA法定量XXX抗体药的方法学验证——准确度注：准确度介于80%-120%义翘神州ELISA法定量XXX抗体药的方法学验证——选择性*未能符合标准的样品。理论浓度（ng/mL）准确度(%)样品1样品2样品3样品4样品5样品6样品7样品8样品9样品10样品11样品12240386.46235.42250.43241.75248.90242.05264.65262.76227.16245.77240.26243.79准确度(%)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准确度(%)190.62*79.2582.8877.0679.9779.2580.7075.6077.7977.0691.4391.43义翘神州实测浓度(ng/mL)样品/ng/mL即刻室温放置4h-70℃以下1周-70℃以下3周-70℃以下5周-70℃以下8周反复冻融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抗体药的方法学验证——高浓度稳定性义翘神州范例02：针对新冠病毒的抗体滴度检测试剂盒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

1.56250.1000.0980.0990.0353.1250.1410.1420.1410.0776.250.2320.2300.2310.16712.50.4170.4300.4230.359250.7990.8020.8000.736501.5291.5731.5511.4871002.8022.8662.8342.770四参数Logistic曲线拟合===================================方程:y=(A-D)/[1+(x/C)^B]+D----------------------------------------------A=10.71988B=-1.11551C=257.27186D=-0.00017r^2=0.99999===================================底物抗体酶标记二抗检测信号抗原义翘神州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

0.3910.1200.1030.1110.0450.7810.1860.1610.1730.1071.5630.2640.2640.2640.1983.1250.4670.4710.4690.4036.2500.8510.8920.8710.80512.51.5441.5421.5431.477252.6152.6982.6572.591四参数Logistic曲线拟合===================================方程:y=(A-D)/[1+(x/C)^B]+D----------------------------------------------A=9.96607B=-1.00482C=70.63124D=-0.00740r^2=0.99995===================================04义翘神州ELISA试剂盒开发服务介绍义翘神州抗体亲和力筛选抗体标记抗体配对标曲建立样本检测、准确性验证检测方法建立及样本检测介入抗体亲和力筛选抗体配对标曲建立样本检测、准确性验证试剂盒生产及性能评估、验证稳定性准确性精密度灵敏度特异性试剂盒研发试剂盒生产、质控高品质的试剂盒ELISA试