本试剂盒采用双抗体夹心ELISA法检测样品中人uPA的浓度。人uPA捕获抗体已经预包被于酶标板上，当加入样品或标准品时，其中的人uPA会与捕获抗体结合，而其它游离成分则会通过洗涤被除去。接着，再加入生物素标记的人uPA抗体后，抗人uPA抗体与人uPA接合，形成夹心的免疫复合物，其它游离成分则通过洗涤被除去。随后加入酶复合物，生物素与酶复合物特异性结合，这样酶复合物上的HRP就与夹心的免疫复合物连接起来，而其它游离成分则通过洗涤被除去。最后加入显色剂，若样品中存在人uPA，则会形成免疫复合物，其上连接的HRP会催化无色的显色剂氧化生成蓝色物质，而后加入终止液，最终产物呈黄色。通过酶标仪检测，读取450 nm处的OD值，人uPA浓度与OD₄₅₀值之间呈正比，通过检测标准品绘制标准曲线，对照未知样品中OD值，即可计算出样品中人uPA的浓度。
操作步骤
1. 样品制备
(1) 根据样品种类选择相应的处理方法：
A. 细胞上清：将细胞培养上清液100~500×g离心5 min，去除悬浮物后即可；
B. 血清样品：将全血在室温下静置0.5~2 h，待其自然凝固并析出血清后，离心取黄色上清即可 (4℃, 1000~2000×g, 10 min)，注意请勿吸取沉淀，制备好的血清需置于冰上待用，请勿在其中添加任何防腐剂或抗凝剂；
C. 血浆样品：使用EDTA对全血进行抗凝处理后，混合均匀置于冰上，离心取黄色上清即可 (4℃, 1000~2000×g, 10 min)，注意请勿吸取沉淀，制备好的血浆需置于冰上待用；
D. 组织匀浆/体液：离心去除沉淀即可。
注意：① 若待测样品无法及时检测，样品制备完成后，请分装冻存于-20℃，避免反复冻融；
② 请保证待测样品清澈透明，检测前如发现样品中有悬浮物，需通过离心去除；
③ 为了保证检测结果准确，请勿使用溶血、黄疸、高血脂或污染的样品。
(2) 稀释样品
查阅相关文献，预估样品中待测因子的含量，从而确定适当的稀释倍数，使稀释后样品中待测因子的浓度处于ELISA试剂盒的最佳检测范围。根据待测因子含量的不同，分别采取不同的稀释方案：
① 待测因子含量在10~100 ng/mL范围内，一般按1:100稀释，即向297 μL样品稀释液中加入3 μL样品；
② 待测因子含量在1~10 ng/mL范围内，一般按1:10稀释，即向225 μL样品稀释液中加入25 μL样品；
③ 待测因子含量在15.62~1000 pg/mL范围内，一般按1:2稀释，即向100 μL样品稀释液中加入100 μL样品；
④ 待测因子含量≤15.62 pg/mL，样品一般无需稀释。
以上方案仅供参考，实验中请详细记录样品的稀释方法。
2. 检测准备工作
(1) 试剂盒自 4℃冰箱取出后，请置于室温平衡20 min；如从-20℃取出，各组分需彻底融化后再平衡20 min；检测完成后，剩余试剂请及时置于4℃或-20℃保存；
(2) 将浓缩洗涤液 (25×)用双蒸水或去离子水稀释成1×洗涤液；
(3) 重组人uPA标准品的稀释和使用 (在使用前2 h内准备，室温操作，请严格控制在25~28℃)
① 配制10 ng/mL标准品：取1 mL样品稀释液加入标准品管内，盖好后静置15 min以上，然后反复颠倒/搓动以助溶解；
② 配制1000 pg/mL标准品：取100 µL 10 ng/mL的标准品加入有900 µL样品稀释液的EP管中，混匀，做上标记；
③ 按下表将1000 pg/mL标准品用样品稀释液进行倍比梯度稀释。(最高浓度为1000 pg/mL，将标准品稀释液作为浓度0 pg/mL。)
| 管号 | 稀释液用量 (μL) | 复溶后标准品用量 (μL) | 标准品的最终浓度 (pg/mL) | | A | 0 | 1,000 | 1000 | | B | 300 | 300 (从A管中取) | 500 | | C | 300 | 300 (从B管中取) | 250 | | D | 300 | 300 (从C管中取) | 125 | | E | 300 | 300 (从D管中取) | 62.5 | | F | 300 | 300 (从E管中取) | 31.25 | | G | 300 | 300 (从F管中取) | 15.62 | | H | 300 | 0 | 0 |
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注意：标准品复溶加样后，剩余部分请丢弃。
(4) 准备生物素标记人uPA抗体工作液
① 按每孔需添加100 μL抗体工作液，计算其总用量 (为弥补操作中的损耗，需多配制100~200 μL)；
② 按1 μL生物素标记人uPA抗体添加99 μL抗体稀释液的比例配制工作液，轻轻混匀。
(5) 准备酶复合物工作液 (需在使用前1 h内准备)
① 按每孔需添加100 μL酶复合物工作液，计算其总用量 (为弥补操作中的损耗，需多配制100~200 μL）；
② 按1 μL酶复合物添加99 μL酶复合物稀释液的比例配制工作液，轻轻混匀。
3. 检测流程
(1) 通过计算确定一次实验所需的板条数，取出所需板条放置于框架内，多余的板条请放回铝箔袋密封，保存于4℃或-20℃;
注意：① 标准品和样品建议做双复孔检测；
② 每次实验均需绘制标准曲线。
(2) 将用样品稀释液稀释后的样品和不同浓度标准品 (100 μL/孔)分别加入相应孔中，用封板胶纸封住反应孔，37℃孵育90 min；
注意：① 请查阅相关文献确定样品中待检测蛋白的大致浓度，若其大于本试剂盒标准曲线的最大标准品浓度，请将样品适当稀释后再进行检测；
② 整个加样过程不宜超过10 min，否则可能会影响检测结果。
(3) 甩去酶标板内液体，无需洗板，将板倒扣在吸水纸上拍干；
(4) 加入稀释后的生物素标记人uPA抗体工作液 (100 μL/孔)，用封板胶纸封住反应孔，37℃孵育60 min；
(5) 洗板5次，每孔1×洗涤液用量为300 μL，注入与吸出间隔15~30 s，洗完后将板倒扣在吸水纸上拍干；
注意：洗涤过程至关重要，洗涤不充分会导致结果产生较大误差。
(6) 加入稀释后的酶复合物 (100 μL/孔)，用封板胶纸封住反应孔，37℃避光孵育30 min；
(7) 洗板5次，方法同步骤 (5) ；
(8) 加入显色剂TMB (100 μL/孔 )，用封板胶纸封住反应孔，避光37℃反应10~25 min；
注意：① 在保存和使用时，请勿将TMB接触氧化剂和金属；
② 因实验室条件差异，最佳显色时间会有所不同，反应充分时肉眼可见标准品的前3~4孔有明显的梯度蓝色。
(9) 加入终止液 (100 μL/孔 )，混匀后即刻使用酶标仪测量OD₄₅₀，同时设定540 nm或570 nm作为校正波长，即可计算得到校正吸光度值 (OD₄₅₀-OD₅₄₀或OD₄₅₀-OD₅₇₀)；
注意：读取OD值建议在10 min内完成。
4. 数据分析
(1) 绘制标准曲线。以标准品浓度作横坐标，OD值作纵坐标，利用计算机软件作四参数逻辑 (4-PL) 曲线拟合创建标准曲线，通过样品的OD值即可在标准曲线上计算出其相应浓度。
注意：① 复孔OD值在20%的差异范围内结果才有效，复孔OD值取平均后可作为测量值；
② 若样品OD值高于标准曲线上限，应适当稀释后重测，计算浓度时应乘以稀释倍数。
(2) 典型数据
该标准曲线仅供演示使用。每批检测样本均需单独绘制标准曲线。
| 标准品浓度 (pg/ml) | O.D. | | 0 | 0.152 | | 15.63 | 0.201 | | 31.25 | 0.236 | | 62.5 | 0.335 | | 125 | 0.498 | | 250 | 0.837 | | 500 | 1.535 | | 1000 | 2.262 |
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(3) 精密度
① 板内精密度 (同批次检测精密度)：取3份已知浓度样本，在同一块酶标板上重复检测20次，用以评估板内精密度。
② 板间精密度 (批次间检测精密度)：取3份已知浓度样本，进行独立批次检测，用以评估板间精密度。实验由至少三名实验人员、使用两个不同批号的试剂盒组分完成。
| 板内精密度 | 板内精密度 | 板内精密度 | 板间精密度 | 板间精密度 | 板间精密度 | | 样本编号 | 1 | 2 | 3 | 1 | 2 | 3 | | 检测数量 (n) | 20 | 20 | 20 | 20 | 20 | 20 | | 平均值 (pg/ml) | 61 | 163 | 59 | 59 | 62 | 91 | | 标准差 | 1.5 | 5.5 | 2.4 | 2.4 | 3.2 | 5.1 | | 变异系数 (%) | 2.4 | 3.3 | 4 | 4 | 5.1 | 5.6 |
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(4) 回收率
在不同基质样本中，于检测量程范围内加入高、中、低三个浓度梯度的人尿激酶uPA，对样本加标回收率进行评估。
| 样本类型 | 平均回收率 (%) | 回收率范围 | | 细胞培养上清液 (n=10) | 101 | 94%~119% | | 血清 (n=9) | 94 | 89%~112% | | 肝素抗凝血浆 (n=9) | 99 | 90%~114% |
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(5) 线性关系
为评估检测方法的线性，选取高浓度人尿激酶uPA天然样本及加标样本，采用校准品稀释液进行梯度倍比稀释，使样本浓度落在试剂盒有效检测线性范围内。
| 稀释比例 | 指标 | 细胞培养上清液 (n=10) | 血清 (n=10) | 肝素抗凝血浆 (n=10) | | 1:4 | 预期值均值 (%) | 105 | 90 | 101 | | 1:4 | 范围 (%) | 83~120 | 83~97 | 93~119 | | 1:8 | 预期值均值 (%) | 91 | 100 | 95 | | 1:8 | 范围 (%) | 86~106 | 96~102 | 89~118 | | 1:16 | 预期值均值 (%) | 102 | 90 | 104 | | 1:16 | 范围 (%) | 82~108 | 88~94 | 92~111 | | 1:32 | 预期值均值 (%) | 100 | 91 | 102 | | 1:32 | 范围 (%) | 90~104 | 86~99 | 96~115 |
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This kit adopts the double-antibody sandwich ELISA method to detect human uPA concentration in samples. Human uPA capture antibodies are pre-coated on microplates. When samples or standards are added, human uPA binds specifically to capture antibodies, while unbound components are removed by washing. Subsequently, biotin-labeled human uPA antibodies are added to form sandwich immune complexes with uPA, and excess free reagents are washed away. Enzyme conjugates are then added, where biotin binds specifically to the enzyme conjugate, linking HRP on the conjugate to the immune complex. Unbound substances are washed again. Finally, TMB substrate is added. If human uPA is present in the sample, HRP catalyzes colorless substrate to produce blue color. After stop solution is added, the reaction turns yellow. OD values at 450 nm are measured by a microplate reader. Human uPA concentration is positively correlated with OD450. The sample concentration can be calculated by comparing sample OD values with the standard curve established from standards.
Assay Procedures
Sample Preparation
1. Select appropriate pretreatment according to sample type:
A. Cell supernatant: Centrifuge cell culture supernatant at 100–500×g for 5 min to remove suspended particles.
B. Serum sample: Allow whole blood to stand at room temperature for 0.5–2 h for natural coagulation. Centrifuge at 1000–2000×g for 10 min at 4 ℃ and collect yellow supernatant. Do not aspirate precipitates. Keep serum on ice. Do not add preservatives or anticoagulants.
C. Plasma sample: Anticoagulate whole blood with EDTA, mix well and keep on ice. Centrifuge at 1000–2000×g for 10 min at 4 ℃ and collect supernatant. Avoid precipitates and keep plasma on ice.
D. Tissue homogenate & body fluid: Centrifuge to remove sediment.
Notes:① If samples cannot be tested immediately, aliquot and store at -20 ℃. Avoid repeated freeze-thaw cycles.
② Samples must be clear. Centrifuge to remove turbidity before detection.
③ Hemolyzed, lipemic, icteric or contaminated samples are not recommended.
(2) Sample Dilution
Estimate target concentration according to literature and choose proper dilution ratio to keep samples within optimal detection range.
① 10–100 ng/mL: Dilute 1:100 (add 3 μL sample into 297 μL sample diluent)
② 1–10 ng/mL: Dilute 1:10 (add 25 μL sample into 225 μL sample diluent)
③ 15.62–1000 pg/mL: Dilute 1:2 (add 100 μL sample into 100 μL sample diluent)
④ ≤15.62 pg/mL: No dilution required.
Above protocols are for reference only. Record detailed dilution procedures.
Pre-assay Preparation
3. Equilibrate kit to room temperature for 20 min after taking out from 4 ℃. For reagents from -20 ℃, fully thaw then equilibrate for 20 min. Store remaining reagents at 4 ℃ or -20 ℃ after assay.
4. Dilute 25× concentrated washing buffer to 1× working buffer with distilled/deionized water.
(3) Dilution and Use of Recombinant Human uPA Standard (Prepare within 2 hours before use; operate at room temperature and strictly control the temperature at 25–28℃)
① Prepare 10 ng/mL standard: Add 1 mL sample diluent into standard vial, stand for ≥15 min, mix thoroughly.
② Prepare 1000 pg/mL standard: Add 100 μL 10 ng/mL standard into 900 μL diluent, mix and label.
③ Serial two-fold dilution of 1000 pg/mL standard as below (0 pg/mL=diluent blank).
| Tube No. | Diluent Volume (μL) | Reconstituted Standard Volume (μL) | Final Standard Concentration (pg/mL) | | A | 0 | 1000 | 1000 | | B | 300 | 300 (from Tube A) | 500 | | C | 300 | 300 (from Tube B) | 250 | | D | 300 | 300 (from Tube C) | 125 | | E | 300 | 300 (from Tube D) | 62.5 | | F | 300 | 300 (from Tube E) | 31.25 | | G | 300 | 300 (from Tube F) | 15.62 | | H | 300 | 0 | 0 |
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Note: Discard any remaining reconstituted standard after pipetting.
6. Prepare biotin-antibody working solution
① Calculate total volume (add extra 100–200 μL for loss). 100 μL per well.
② Dilute antibody at 1:99 (1 μL antibody + 99 μL diluent), mix gently.
7. Prepare enzyme conjugate working solution (prepare within 1 h before use)
① Calculate total volume (add extra 100–200 μL). 100 μL per well.
② Dilute conjugate at 1:99 (1 μL enzyme conjugate + 99 μL diluent), mix gently.
Detection Steps
8. Take required strips, assemble into frame. Seal unused strips in aluminum pouch and store at 4 ℃ / -20 ℃.
Note:
Note: ① Duplicate wells are recommended for both standards and samples.
② A standard curve must be established for each independent assay.
9. Add 100 μL diluted samples and standards into corresponding wells. Seal plate and incubate at 37 ℃ for 90 min.
Note:
Note: ① Please refer to relevant literature to estimate the approximate concentration of the target protein in samples. If the concentration exceeds the maximum value of the kit standard curve, dilute the sample appropriately before detection.
② The entire sample adding procedure shall be completed within 10 minutes, otherwise the test results may be affected.
10. Discard liquid, blot dry on absorbent paper without washing.
11. Add 100 μL biotin-antibody working solution per well, seal and incubate at 37 ℃ for 60 min.
12. Wash plate 5 times with 300 μL 1× buffer per well, interval 15–30 s, then blot dry. Insufficient washing causes large errors.
13. Add 100 μL enzyme conjugate per well, seal and incubate at 37 ℃ for 30 min in dark.
14. Wash plate 5 times as step 12.
15. Add TMB substrate solution (100 μL per well), seal the wells with plate sealing film, and incubate at 37 ℃ for 10–25 minutes away from light.
Note:
Note: ① Avoid contact between TMB and oxidants or metals during storage and operation.
② The optimal color development time varies with laboratory conditions. Distinct gradient blue color can be visually observed in the first 3–4 wells of standards when the reaction is complete.
(9) Add stop solution (100 μL per well). Mix well immediately and measure OD₄₅₀ with a microplate reader. Set 540 nm or 570 nm as reference wavelength to calculate corrected absorbance (OD₄₅₀-OD₅₄₀或OD₄₅₀-OD₅₇₀).
Note: OD reading should be completed within 10 minutes.
4. Data Analysis
17. Establish the standard curve. Take standard concentration as the X-axis and OD value as the Y-axis. Fit the curve with four-parameter logistic (4-PL) regression using software. The sample concentration can be calculated according to its OD value on the standard curve.
Note: ① Results are valid only when the OD difference between duplicate wells is within 20%. The average OD value of duplicates is adopted for calculation.
② If sample OD exceeds the upper limit of the standard curve, dilute the sample and retest. Multiply the final concentration by the dilution factor.
(2) Typical Data
This standard curve is provided for demonstration only. A standard curve should be generated for each set of samples assayed.
| Standard concentration (pg/ml) | O.D. | | 0 | 0.152 | | 15.63 | 0.201 | | 31.25 | 0.236 | | 62.5 | 0.335 | | 125 | 0.498 | | 250 | 0.837 | | 500 | 1.535 | | 1000 | 2.262 |
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(3) Precision
① Intra-Assay Precision (Precision within an assay): Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.
② Inter-Assay Precision (Precision between assays): Three samples of known concentration were tested in separate assays to assess inter-assay precision. Assays were performed by at least three technicians using two lots of components.
| Intra-Assay Precision | Intra-Assay Precision | Intra-Assay Precision | Inter-Assay Precision | Inter-Assay Precision | Inter-Assay Precision | | Sample | 1 | 2 | 3 | 1 | 2 | 3 | | n | 20 | 20 | 20 | 20 | 20 | 20 | | Mean (pg/ml) | 61 | 163 | 59 | 59 | 62 | 91 | | Standard Deviation | 1.5 | 5.5 | 2.4 | 2.4 | 3.2 | 5.1 | | CV% | 2.4 | 3.3 | 4 | 4 | 5.1 | 5.6 |
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(4) Recovery
The recovery of human uPA/Urokinase spiked to three levels throughout the range of the assay in various matrices was evaluated.
| Sample Type | Average Recovery (%) | Range (%) | | Cell culture supernates (n=10) | 101 | 94%-119% | | Serum (n=9) | 94 | 89%-112% | | Herparin plasma (n=9) | 99 | 90%-114% |
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(5) Linearity
To assess the linearity of the assay, samples containing and/or spiked with high concentrations of human uPA/Urokinase were serially diluted with calibrator diluent to produce samples with values within the dynamic range of the assay.
| Dilution Ratio | Index | Cell Culture Supernatants (n=10) | Serum (n=10) | Heparin Plasma (n=10) | | 1:4 | Average of Expected (%) | 105 | 90 | 101 | | 1:4 | Range (%) | 83–120 | 83–97 | 93–119 | | 1:8 | Average of Expected (%) | 91 | 100 | 95 | | 1:8 | Range (%) | 86–106 | 96–102 | 89–118 | | 1:16 | Average of Expected (%) | 102 | 90 | 104 | | 1:16 | Range (%) | 82–108 | 88–94 | 92–111 | | 1:32 | Average of Expected (%) | 100 | 91 | 102 | | 1:32 | Range (%) | 90–104 | 86–99 | 96–115 |
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ZJ26F0535520
危险品化学品经营许可证（带存储）