NGL-Impact® A ELISA Kit Performance Summary
INTRODUCTION
The Repligen NGL-Impact® A ELISA kit (Catalog number 9222-1) is intended for the detection and quantitation of residual NGL-Impact® Protein A. It was been developed for users who require a highly sensitive assay to measure small amounts of contaminating NGL-Impact® A in antibody products. Testing for NGL-Impact® A contamination occurs in several different phases of development and commercial manufacturing which may include:
- 在特定条件下，针对填料配基脱落特性的工艺开发
- 生产，通常需对纯化工艺中多个不同点采集的洗脱样品进行分析
- 终产品放行时文件记录工艺污染物水平和批次间的一致性
The following summary report contains performance data collected from the evaluation of the NGL-Impact® A ELISA Kit in the presence of human Immunoglobulin G (hIgG). The data presented here demonstrates the following:
- Ability to detect NGL-Impact® A in the presence of up to 0.5 mg/ml hIgG in a PBS-T buffer using Dilute & Go method, or sample up to 10 mg/ml hIgG by Boil & Boost method
- 回收率（准确性）、分析间和分析内精确性、定量限和检测限
RESULTS SUMMARY
NGL-Impact® A in the presence of hIgG
The performance of the Repligen NGL-Impact® A ELISA kit was evaluated when detecting NGL-Impact® A in the presence of hIgG compared to a standard containing no hIgG. NGL-Impact® A spiked samples had a starting hIgG concentration of 0.5 mg/ml when diluted into the assay plate or had a 10 mg/ml hIgG initial concentration when using boiling method. Each spike sample was prepared in replicates of 3 and ELISA was performed according to the Kit’s standard protocol.
数据处理
Standard curve data points were fitted to linear regression line. This equation allowed back-calculation of sample NGL-Impact® A concentrations and calculation of LoQ values. Percent recovery was calculated as follows:
% Recovery = Calculated Conc X100
Theoretical Conc
分析内精确性
表1.标准曲线样品的分析内精确性（无hlgG）
| 浓度 (ng/ml) | 平均 % CV |
|---|---|
| 1.6 | 0.8 |
| 0.8 | 5.3 |
| 0.4 | 2.0 |
| 0.2 | 1.3 |
| 0.1 | 5.7 |
| 0.05 | 7.1 |
表2. 使用Dilute & Go时，含hlgG样品的分析内精确性
| 浓度 (ng/ml) | 计算的 浓度 |
平均 % CV |
|---|---|---|
| 1.0 | 1.09 | 1.0 |
| 0.5 | 0.58 | 2.6 |
| 0.25 | 0.26 | 2.4 |
表3.使用Boil & Boost时，含hlgG样品的分析内精确性
| 浓度 (ng/ml) | 计算的 浓度 |
平均 % CV |
|---|---|---|
| 1.0 | 1.10 | 3.4 |
| 0.5 | 0.50 | 3.5 |
| 0.25 | 0.24 | 1.6 |
Inter-Assay Percision
表4.标准曲线样品的分析间精确性（无hlgG）
| 浓度 (ng/ml) | 平均 % CV |
|---|---|
| 1.6 | 1.0 |
| 0.8 | 3.1 |
| 0.4 | 3.2 |
| 0.2 | 2.6 |
| 0.1 | 7.2 |
| 0.05 | 21.1* |
*浓度低于LLOQ
表5. 使用Dilute & Go时，含hlgG样品的分析间精确性
| 浓度 (ng/ml) | 计算的 浓度 |
平均 % CV |
|---|---|---|
| 1.0 | 1.09 | 6.2 |
| 0.5 | 0.58 | 2.4 |
| 0.25 | 0.26 | 5.5 |
表6. 使用Boil & Boost时，含hlgG样品的分析间精确性
| 浓度 (ng/ml) | 计算的 浓度 |
平均 % CV |
|---|---|---|
| 1.0 | 1.10 | 3.7 |
| 0.5 | 0.50 | 7.5 |
| 0.25 | 0.24 | 4.7 |
准确性
表7.标准曲线样本的准确性
| 浓度 (ng/ml) | 平均 % 误差 | 平均%回收率 |
|---|---|---|
| 1.6 | -2.5 | 97.5 |
| 0.8 | 6.3 | 106.3 |
| 0.4 | 14.2 | 114.2 |
| 0.2 | 8.6 | 108.6 |
| 0.1 | -10.6 | 89.4 |
| 0.05 | -51.6* | 48.4* |
*浓度低于LLOQ
表9. Boil & Boost对于含hlgG样品的准确度
| 浓度 (ng/ml) | 平均 % 误差 | 平均%回收率 |
|---|---|---|
| 1.0 | 9.8 | 109.8 |
| 0.5 | 0.1 | 100.1 |
| 0.25 | -5.6 | 94.4 |
表8. Dilute & Go对于含hlgG样品的准确度
| 浓度 (ng/ml) | 平均 % 误差 | 平均%回收率 |
|---|---|---|
| 1.0 | 8.6 | 108.6 |
| 0.5 | 15.1 | 115.1 |
| 0.25 | 3.7 | 103.7 |
定量限（LoQ）
The LoQ determined from the standard curve with no IgG present was calculated to be 0.07 ng/ml. The LoQ for the NGL-Impact® A spiked samples in the presence of hIgG was 0.87 ppm when using Dilute & Go method and 0.048 ppm for Boil & Boost method.
检测限 (LoD)
The LoD determined from the standard curve was calculated to be 0.06 ng/ml. The LoQ for the NGL-Impact® A spiked samples in the presence of hIgG was 0.64 ppm using Dilute & Go and 0.04 ppm for Boil & Boost.
稀释线性
图1. 在含有IgG的整个检测浓度范围内，使用Dilute & Go和Boil & Boost方法，稀释线性均显示出准确性的高相关性。
CONCLUSIONS
分析内数据显示，标准曲线样品的精确性在可接受参数范围内，%CV值范围为0.8-7.1%。分析内数据显示，样品分析相对准确且精确。
分析间数据显示数据具有可重复性。浓度等于或高于LoQ时，分析间变异系数低于或等于7.2%。样品分析间精确性范围为2.4-7.5%。
高于LoQ的浓度的准确性，以%相对误差计量，标准品为-10.6 - 14.2 ，使用Dilute & Go方法和Boil & Boost方法分析的样品为-5.6 - 15.1。
The LoQ determined from the standard curve with no IgG present was calculated to be 0.07 ng/ml. The LoQ for the NGL-Impact® A spiked samples in the presence of hIgG was 0.87 ppm when using Dilute & Go method and 0.048 ppm for Boil & Boost method.
The LoD determined from the standard curve was calculated to be 0.06 ng/ml. The LoQ for the NGL-Impact® A spiked samples in the presence of hIgG was 0.64 ppm using Dilute & Go and 0.04 ppm for Boil & Boost.
所有等于或高于LoQ的样品浓度的%CV值为1.0-3.5%，低于15%的限值。
EXPLANATION OF CALCULATIONS
精确性 (%CV)
Precision was calculated by determining the standard deviation between NGL-Impact® A spiked sample data points and dividing by the mean value. According to the ‘Guidance for Industry: Bio-analytical Method Validation’ text, precision should be within 15%.
分析内精确性
The intra-assay precision was calculated for each NGL-Impact® A spiked sample concentration by averaging the %CV values across all assays.
分析间精确性
通过确定三次分析中每次计算结果之间的标准偏差，然后除以平均值，可以计算出每个浓度点的分析间精确性。
定量限（LoQ）
The limit of quantitation (LoQ) was defined as 10 times the standard deviation of 0 ng/ml sample. The standard deviation of the 0 ng/ml OD value was multiplied by 10 then added to base 0 ng/ml OD value. The LoQ was then generated by entering the summed value into the linear equation. For each kit the LoQ was reported as ng NGL-Impact® A per ml (ng/ml) buffer, and ng NGL-Impact® A per mg hIgG (ppm) for NGL-Impact® A spiked samples run in presence of hIgG.
检测限 (LoD)
The limit of detection (LoD) was defined as 3 times the standard deviation of 0 ng/ml Protein A sample. The standard deviation of the 0 ng/ml OD value was multiplied by 3 then added to base 0 ng/ml OD value. The LoD was then generated by entering the summed value into the standard curve equation. For each kit a LoD was reported as ng NGL-Impact® A per ml (ng/ml) buffer and in parts per million (ppm) against IgG concentration.
准确性
准确性描述为与理论加标浓度相比，分析确定的回收率%
Example standard curve
