## 4 Conclusions  
In summary, we developed an ultrasensitive, rapid, and stable fluorescent sandwich ELISA method for the trace detection of IGF-1 using ADHP as a fluorogenic substrate in the HRP enzymatic reaction, in which the LOD of IGF-1 reduces from 1.018 ng/mL to 77.29 pg/mL, the experimental time is shortened from 3 h to 1 h, and the resulting signal is maintained for 1 h in comparison with 15 min in tradition ELISA. The established method has the advantages of a low LOD, high sensitivity, a fast experimental process, a stable signal, and good specificity. Moreover, a multi-step pretreatment process for bovine colostrum powders including dissolution, centrifugation, and acid treatment is proposed, and thus, IGF-1 content can be detected in real dairy products by the fluorescent sandwich ELISA method with high accuracy and precision. The established fluorescent sandwich ELISA method combined with the multi-step pretreatment process has great potential for quantitatively detecting ultra-low contents of other specific targets in bovine colostrum powders, which are expected to become evidence of nutritional supplements for human bodies.  
T.H.: Writing--original draft, Visualization. B.L.: Investigation, Writing--original draft. S.H.: Investigation. Y.T.: Formal analysis. Z.P.: Conceptualization, Supervision, Writing--review and editing. All authors have read and agreed to the published version of the manuscript.  
This research was funded by the National Natural Science Foundation of China (52372158, 22404150), Zhejiang Provincial Natural Science Foundation of China (LZ18B050002, LQ24B020010), and Scientific Research Foundation for Scholars funded by Zhejiang University of Technology (2022181010002).  
Not applicable.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multicolumn{1}{c}{**Added IGF-1**} & \multicolumn{1}{c}{**Calculated IGF-1**} & \multicolumn{1}{c}{**Recovery Rate**} & \multicolumn{1}{c}{**CV a**} & \multicolumn{1}{c}{**Contention in**} & \multicolumn{1}{c}{**Contention in**} \\ \multicolumn{1}{c}{**Concentration**} & \multicolumn{1}{c}{**Concentration**} & \multicolumn{1}{c}{**(\%)**} & \multicolumn{1}{c}{**(n = 3, \%)**} & \multicolumn{1}{c}{**Bovine**} & \multicolumn{1}{c}{**Bovine**} \\ \multicolumn{1}{c}{**(ng/mL)**} & \multicolumn{1}{c}{**(ng/mL)**} & \multicolumn{1}{c}{**(\%)**} & \multicolumn{1}{c}{**(n = 3, \%)**} & \multicolumn{1}{c}{**Colostrum**} & \multicolumn{1}{c}{**Colostrum**} \\ \multicolumn{1}{c}{} & \multicolumn{1}{c}{} & \multicolumn{1}{c}{} & \multicolumn{1}{c}{**Powder (ng/g)**} & \multicolumn{1}{c}{**Powder (ng/g)**} \\ \hline \hline \multicolumn{1}{c}{Fluorescent sandwich ELISA} & & & & & \\ \hline
0 & 14.06 & / & 3.81 & 1125.00 & & \\
1 & 15.08 & 102.32 & 5.54 & 1126.85 & & \\
5 & 18.87 & 96.09 & 3.34 & 1109.36 & & \\
15 & 27.82 & 91.71 & 1.99 & 1025.50 & & \\ \hline \multicolumn{1}{c}{Traditional ELISA} & & & & & \\ \hline
0 & 32.08 & / & 3.12 & 1026.56 & & \\
25 & 57.25 & 100.69 & 5.89 & 1032.09 & & \\
60 & 97.93 & 109.75 & 3.89 & 1213.86 & & \\
80 & 113.70 & 102.02 & 2.50 & 1078.39 & & \\ \hline \hline \end{tabular}  
*: CV represents the coefficient of variation.  
\end{table}
Table 2: Measurements of IGF-1 in pretreated bovine colostrum powders.  
Data Availability Statement: The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.  
We are grateful to [PERSON] (Hangzhou Peptide Technology Co., Ltd., Hangzhou, 310063, China) for help with the methodology.  
The authors declare no conflicts of interest.