本品采用双抗夹心法检测样本中BsaI含量。将BsaI特异性抗体预包被在微孔板上，使用时将标准品与待测样本分别加入到已包被特异性抗体的反应孔内，样本中的BsaI会与微孔板中的抗体结合，孵育后洗涤以去除微孔板内未结合的物质，加入BsaI检测抗体 (HRP标记)，形成抗体-抗原-HRP标记抗体的复合物，加入TMB进行孵育显色，显色程度与BsaI浓度成比例关系，通过测定BsaI标准品的显色来判断样本中BsaI残留程度。
检测范围：0.188~12 ng/ml
定量限：0.188 ng/ml
检测限：0.094 ng/ml
器材
酶标仪、微孔板恒温振荡器、涡旋振荡器、移液器、封板膜等。
试剂配制
1. 平衡：将所需试剂转移到室温 (25±3℃)平衡至少30 min；确定测定运行的孔数，取出所需的酶标条，其它放回装有干燥剂包的铝箔袋中，然后重新密封并置于2~8℃保存。
2. 1×漂洗液配制：根据本次试验所需洗板液体积，量取漂洗液 (20×)适量，用去离子水按照1: 20体积比稀释，混匀后备用。
3. 标准品溶液配制 (示例)：
| 序号 | 标准液浓度 (ng/ml) | 标准液体积 (μl) | 稀释液体积 (μl) | 总体积 (μl) | 终浓度 (ng/ml) | 剩余体积 (μl) | | StA | 10000 | 20 | 1800 | 2000 | 100 | 1940 | | St8 | 100 | 60 | 440 | 500 | 12 | 250 | | St7 | 12 | 250 | 250 | 500 | 6 | 250 | | St6 | 6 | 250 | 250 | 500 | 3 | 250 | | St5 | 3 | 250 | 250 | 500 | 1.5 | 250 | | St4 | 1.5 | 250 | 250 | 500 | 0.75 | 250 | | St3 | 0.75 | 250 | 250 | 500 | 0.375 | 250 | | St2 | 0.375 | 250 | 250 | 500 | 0.188 | 250 | | St1 (Blank) | / | / | 300 | 300 | 0 | 300 |
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实验流程
1. 混匀：临用前将平衡至室温的试剂轻轻混匀。
2. 加样：分别加入100 μl标准品溶液 (St1~St8)或待测样品至相应孔中，加盖或封板膜封板后在室温 (25±3℃)条件下400~500 rpm孵育1 h。
3. 洗涤：甩掉孔内液体，每孔加入至少1×漂洗液350 μl (可用多通道移液器或者洗瓶)，再甩去孔内液体后于无尘纸上轻拍至拍干。重复3次。（注意：在加入BsaI检测抗体溶液前勿使板孔长时间过分干燥。洗涤不当可能使复孔间CV值偏大，信号偏高或偏低）
4. 标记：每孔加入BsaI检测抗体 (HRP标记)溶液100 μl，加盖或封板膜封板后在室温 (25±3℃)条件下 400~500 rpm孵育30 min。
5. 洗涤：甩掉孔内液体，每孔加入至少1×漂洗液350 μl (可用多通道移液器或者洗瓶)，再甩去孔内液体后于无尘纸上轻拍至拍干。重复3次。（注意：在加入TMB底物前勿使板孔长时间过分干燥）
6. 显色：每孔加入TMB底物100 μl，室温 (25±3℃)避光显色20 min。
7. 终止：每孔加入终止液100 μl，轻轻敲击孔板使混匀，终止反应。
8. 测定：加入终止液后10 min内，在450 nm波长处读取吸光值。
结果计算
本品建议采用浓度取10为底的对数后采用四参数Logistic模型拟合方式进行拟合计算。
表1 典型标准曲线数据表
| 标准品浓度 (ng/ml) | 吸光值测定1 | 吸光值测定2 | 吸光值均值 | | 12 | 2.342 | 2.29 | 2.316 | | 6 | 1.562 | 1.497 | 1.529 | | 3 | 0.875 | 0.866 | 0.871 | | 1.5 | 0.524 | 0.534 | 0.529 | | 0.75 | 0.346 | 0.34 | 0.343 | | 0.375 | 0.266 | 0.254 | 0.26 | | 0.188 | 0.205 | 0.204 | 0.205 | | 0 | 0.156 | 0.155 | 0.156 |
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典型标准曲线图谱 (四参数Logistic模型)
性能
1. 检测限：0.094 ng/ml。将 20个空白对照的平均吸光值加上2个标准偏差后代入标曲确定。
2. 定量限：0.188 ng/ml。确保变异系数 (n=6)在20%以内的最低浓度。
3. 线性：测定不同浓度的标准品溶液，并绘制曲线，R²≥0.98。
4. 重复性：三个已知浓度样品重复测试20次，评估重复性。
5. 中间精密度：三个已知浓度样品不同时间不同人测试3次，评估中间精密度。
| 名称 | 重复性 (n=20)
低浓度 | 重复性 (n=20)
中间浓度 | 重复性 (n=20)
高浓度 | 中间精密度 (n=3)
低浓度 | 中间精密度 (n=3)
中间浓度 | 中间精密度 (n=3)
高浓度 | | 均值 (ng/ml) | 0.72
| 2.90
| 9.74
| 0.75
| 2.95
| 9.97
| | SD | 0.063
| 0.166
| 0.305
| 0.023
| 0.093
| 0.606
| | CV (%) | 8.7
| 5.7
| 3.1
| 3.1
| 3.2
| 6.1
|
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6. 回收率：评估了在相关基质中在整个测定范围内加标至不同水平的BsaI的回收率。
| 平均回收率 (%) (n=3) | 回收率范围 (%) | | 100.5 | 90.2~107.3 |
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注意事项
1. 本产品仅用于科研，不能用于临床诊断或治疗。
2. 试剂应该按照标签标明的条件进行储存，试剂和待测样本使用前应平衡至室温。
3. 预包被酶标板使用前，应平衡至室温后再打开包装袋取用，剩余的板条应立即放回包装中重新密封， 置于2~8℃保存。剩余试剂也应重新密封后按标签标明的条件进行储存。
4. BsaI标准品、BsaI检测抗体 (HRP标记)溶液应混匀后再使用。
5. 洗涤过程中板孔残留的液体应在洁净的无尘纸上拍干，拍干后的板孔不宜长时间过分干燥，否则不恰当的洗涤过程会造成复孔的CV值偏高或者空白 (0 ng/ml)吸光值偏高，影响结果准确性。
6. TMB底物应该是无色透明液体，如果显示淡蓝色，证明该溶液已经被污染，不适合进行后续分析使用。
7. 样本处在极端pH值 (<5 .0或>8 .5)，或者含高盐、高聚糖、高尿素、高有机溶剂和高的去污剂的溶液中，均有可能导致较低回收率。
8. 由于生物样本基质的复杂性，建议每次试验加阳性质控试验样品，确保试验的准确性，并采用复孔进行试验。
安全须知
1. 终止液为酸性溶液，使用时应注意安全。
2. 操作者应穿戴个人防护装备， 如实验服，手套，口罩和护目镜等。
This kit adopts the double-antibody sandwich ELISA method to detect BsaI content in samples. BsaI-specific antibodies are pre-coated on microplate wells. During detection, standards and samples are added separately into antibody-coated wells. BsaI in samples binds to the immobilized antibodies. After incubation, unbound substances are washed away. HRP-labeled BsaI detection antibody is added to form antibody-antigen-HRP antibody complexes. TMB substrate is added for color development. The color intensity is proportional to BsaI concentration. The residual BsaI in samples is determined by detecting color development of BsaI standards.
Detection Range: 0.188~12 ng/ml
Quantitation Limit: 0.188 ng/ml
Limit of Detection: 0.094 ng/ml
Required Instruments
Microplate reader, microplate constant temperature shaker, vortex mixer, pipettes, plate sealing film, etc.
Reagent Preparation
1. Equilibration: Allow all required reagents to equilibrate at room temperature (25±3℃) for at least 30 minutes. Determine the number of wells needed, take out required microplate strips, and return unused strips to aluminum foil bags with desiccant. Reseal and store at 2–8℃.
2. Prepare 1× Washing Buffer: Dilute 20× Washing Buffer with deionized water at a volume ratio of 1:20 according to required volume, mix well for standby use.
3. Standard Solution Dilution (Example):
| No. | Standard Concentration (ng/ml) | Standard Volume (μl) | Diluent Volume (μl) | Total Volume (μl) | Final Concentration (ng/ml) | Remaining Volume (μl) | | StA | 10000 | 20 | 1800 | 2000 | 100 | 1940 | | St8 | 100 | 60 | 440 | 500 | 12 | 250 | | St7 | 12 | 250 | 250 | 500 | 6 | 250 | | St6 | 6 | 250 | 250 | 500 | 3 | 250 | | St5 | 3 | 250 | 250 | 500 | 1.5 | 250 | | St4 | 1.5 | 250 | 250 | 500 | 0.75 | 250 | | St3 | 0.75 | 250 | 250 | 500 | 0.375 | 250 | | St2 | 0.375 | 250 | 250 | 500 | 0.188 | 250 | | St1 (Blank) | / | / | 300 | 300 | 0 | 300 |
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Assay Procedure
1. Mix all reagents equilibrated to room temperature gently before use.
2. Pipette 100 μL of standard solutions (St1~St8) or test samples into corresponding wells. Seal the plate with a plate sealer, then incubate at 25±3 ℃ and 400–500 rpm for 1 hour.
3. Washing: Discard the liquid in wells. Add at least 350 μL of 1× Washing Buffer to each well (using a multichannel pipette or wash bottle). Discard residual liquid and pat the plate dry on lint-free absorbent paper. Repeat washing for 3 cycles.
(Note: Do not allow wells to dry excessively for a long time before adding BsaI detection antibody. Improper washing may cause high CV between duplicate wells and abnormal absorbance signals.)
4. Add 100 μL of HRP-labeled BsaI detection antibody to each well. Seal the plate and incubate at 25±3 ℃, 400–500 rpm for 30 minutes.
5. Washing: Discard the liquid in wells. Add at least 350 μL of 1× Washing Buffer to each well. Discard liquid and pat the plate dry. Repeat washing 3 times.
(Note: Avoid prolonged excessive drying of wells prior to TMB substrate addition.)
6. Color development: Add 100 μL TMB substrate to each well, and incubate for 20 minutes at room temperature in the dark.
7. Termination: Add 100 μL Stop Solution to each well, tap the plate gently to mix thoroughly and terminate the reaction.
8. Detection: Measure the absorbance at 450 nm within 10 minutes after adding Stop Solution.
Data Calculation
It is recommended to perform logarithmic transformation of concentration and fit standard curve with 4-Parameter Logistic regression model.
Table 1 Typical Standard Curve Data
| Standard Concentration (ng/ml) | OD Reading 1 | OD Reading 2 | Average OD Value | | 12 | 2.342 | 2.29 | 2.316 | | 6 | 1.562 | 1.497 | 1.529 | | 3 | 0.875 | 0.866 | 0.871 | | 1.5 | 0.524 | 0.534 | 0.529 | | 0.75 | 0.346 | 0.34 | 0.343 | | 0.375 | 0.266 | 0.254 | 0.26 | | 0.188 | 0.205 | 0.204 | 0.205 | | 0 | 0.156 | 0.155 | 0.156 |
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Typical Standard Curve (4PL Logistic Model)
Performance
1. Limit of Detection (LOD): 0.094 ng/ml. Calculated by substituting the average absorbance of 20 blank controls plus 2 standard deviations into the standard curve.
2. Limit of Quantitation (LOQ): 0.188 ng/ml. The lowest concentration with coefficient of variation (n=6) ≤ 20%.
3. Linearity: Standard curves are plotted with serially diluted standard solutions, with R² ≥ 0.98.
4. Repeatability: Evaluated by detecting three samples of known concentrations for 20 replicates.
5. Intermediate Precision: Evaluated by detecting three samples of known concentrations 3 times by different operators on different days.
| Parameter | Repeatability (n=20)
Low Concentration | Repeatability (n=20)
Mid Concentration | Repeatability (n=20)
High Concentration | Intermediate Precision (n=3)
Low Concentration | Intermediate Precision (n=3)
Mid Concentration | Intermediate Precision (n=)
High Concentration | | Mean (ng/mL) | 0.72
| 2.9
| 9.74
| 0.75
| 2.95
| 9.97
| | SD | 0.063
| 0.166
| 0.305
| 0.023
| 0.093
| 0.606
| | CV (%) | 8.7 | 5.7
| 3.1
| 3.1 | 3.2 | 6.1
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6. Recovery: The recovery rates of spiked BsaI at different levels within the full detection range in biological matrix were verified.
| Average Recovery (%) (n=3) | Recovery Range (%) | 100.5
| 90.2 ~ 107.3
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Precautions
1. For research use only, not for clinical diagnosis or treatment.
2. Store reagents as labeled. Equilibrate reagents and samples to room temperature before detection.
3. Equilibrate pre-coated microplates to room temperature before opening packages. Return unused strips to sealed bags and store at 2–8℃. Seal all leftover reagents properly.
4. Mix Bsal standard and HRP detection antibody thoroughly before use.
5. Pat washed wells dry on lint-free paper. Do not over-dry wells, which will increase CV and blank OD deviation.
6. TMB substrate should be colorless and transparent. Pale blue TMB indicates contamination and cannot be used.
7. Samples with extreme pH (<5.0 or >8.5), high salt, polysaccharide, urea, organic solvent or detergent may result in low recovery.
8. Due to complex biological matrix, use positive controls and duplicate wells to ensure accurate results.
Safety Instructions
1. Stop Solution is acidic. Operate with caution.
2. Wear proper personal protective equipment: lab coat, gloves, mask, goggles, etc.
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