# Development of a Calibration Strip for Immunohromatographic Assay Detection Systems
## 4 Experimental Results and Discussion
### Development of the Calibration Strip  
HCG solution, which has been used in preliminary selection of Down's syndrome and the diagnosis of early pregnancy, or ecycesis, is chosen as the reagent for extracting characteristic quantities. In addition, test strips are selected from diagnostic kits (Xiamen Boson Biotech Co., Ltd., Xiamen, China) for rapid quantitative determination of human choriogonadotropin (\(\beta\)-HCG) in the same patch and specification. The detection sensitivity of the strip is 10 mIU/mL. Additionally, the concentration of diluents involves 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 mIU/mL. Generally, the best detection time is 10 min-18 min after the sample solution is dropped into the sample pad. Therefore, the image acquisition device captures the strip image at 10 min, 12 min, 14 min, 16 min, and 18 min. Then, the OD value of the acquired strip image is immediately measured by a quantitative detection system for ICG assay (SWP-SC-2). To reduce errors, every concentration of diluent is repeatedly measured three times. The test strips are shown in Figure 7.  
As the light source of image acquisition device is not exactly stable, which results in the instability of \(V\) in the HSV color space, four characteristic quantities of the strip image are selected from \(H\) and \(S\), which are one- or two-dimensional mean values of \(H\), \(360\times H\), \(10\times H\times S\), and \(\sqrt{H^{2}+(360\times S)^{2}}\). The linear coefficient between these characteristic quantities and concentrations of HCG, and OD values, are shown in Tables 3 and 4, respectively. As shown, an average value of \(H\) at 14 min has the  
Figure 6: Process of image segmentation. (**a**) Original image; (**b**) the segmented test line; (**c**) HSV image with adjusted brightness; and (**d**) the result of test line segmentation. Figure 7: Test strips for colloidal gold-based ICG assay. maximum linear coefficient (95.24%) with the concentration of HCG. Further, the linear coefficient between an average value of \(H\) at 14 min and OD value is over 98%. Thus, an average value of \(H\) at 14 min is chosen as the characteristic quantity for the calibration test strip. Now, the empirical formula between \(H\) and OD value is obtained by:  
\[H=10.153\times OD+262.427 \tag{16}\]  
From Equation (16), the mean value of \(H\) can be calculated from the OD value. Selecting a number of OD values (1.5, 3, 4, 5, 6, 7, 8, 9, 9.5), the predicted mean values of \(H\) are shown in Table 5.  
In order to determine \(S\) and \(V\), the pixels of \(pix_{ij}\) that satisfied the inequality (\(H-0.25<H_{pix_{ij}}\leqslant H+0.25\)) are regarded as valid points. For example, if \(H=303.039\), the valid points of the segmented strip image is shown in Figure 8. Hence, \(S\) and \(V\) is calculated by Equation (17). \[S=\sum_{i}S_{pix_{ij}}/N;\ V=\sum_{i}V_{pix_{ij}}/N \tag{17}\] \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**CQ** & **Time (min)** & **10** & **12** & **14** & **16** & **18** \\ \hline \(H\) & 98.02 & 98.31 & 98.12 & 98.11 & 97.93 \\ \(360\times H\) & 81.33 & 87.87 & 90.91 & 92.18 & 93.11 \\ \(10\times H\times S\) & 86.77 & 90.92 & 93.02 & 93.90 & 94.85 \\ \(\sqrt{H^{2}+\left(360\times S\right)^{2}}\) & 81.64 & 88.02 & 91.03 & 92.27 & 93.56 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Linear coefficient between characteristic quantities and OD value (%). \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**CQ** & **Time (min)** & **10** & **12** & **14** & **16** & **18** \\ \hline \(H\) & 95.15 & 95.04 & 95.24 & 94.61 & 94.22 \\ \(360\times H\) & 79.58 & 84.70 & 88.34 & 89.45 & 91.35 \\ \(10\times H\times S\) & 84.57 & 87.54 & 90.21 & 90.01 & 91.88 \\ \(\sqrt{H^{2}+\left(360\times S\right)^{2}}\) & 79.84 & 84.81 & 88.41 & 89.50 & 90.82 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Linear coefficient between characteristic quantities and concentration of HCG (%).