## 3 Results and Discussion
### Multi-Step Pretreatment of Bovine Colostrum Powders  
In order to evaluate the feasibility of the proposed fluorescent sandwich ELISA method for IGF-1 detection in real samples, bovine colostrum powders were pretreated using different steps. A milky white suspension was obtained when the colostrum powders were dissolved and dispersed in water without any further treatment (Method I), in which large numbers of insoluble substances, lipids, and interfering proteins were contained. After centrifugation was conducted once, the solution was divided into three layers, the fat layer, the middle layer, and the precipitation layer, from top to bottom. After removing the fat and precipitation layers, slightly opalescent skimmed milk was obtained (Method II). However, some interfering substances, such as casein and binding proteins, which are difficult to precipitate by centrifugation, were still dispersed in it. At the isoelectric point of casein (pH 4.6), the maximal precipitation of the protein occurs due to charge neutralization [38], while IGF-1 release from its binding proteins is concurrently promoted under acidic conditions [39]. Therefore, acid treatment and centrifugation followed to obtain a transparent and light-yellow solution (Method III), which showed that the interfering matrix was effectively removed in the liquid bovine colostrum. Finally, the pH of the solution was readjusted to its original physiological level (pH 6.4) to ensure the optimal preservation of bioactive IGF-1. The solutions obtained by the three methods were diluted with PBS and added to a 96-well plate as samples for ELISA determination. As shown in Figure 6, the OD value gradually decreased with an increasing number of pretreatment steps. The observed turbidity in samples treated with either Method I or II was likely caused by the persistence of residual components (e.g., lipid and casein), which may have concurrently interfered with chromogenic detection and induced nonspecific binding interactions, ultimately increasing matrix effects. Therefore, the experimental results were false positive and a higher OD value was obtained in Method I or Method II. To verify this hypothesis, 25 ng/mL IGF-1 standard solutions were added into bovine colostrum powders with the multi-step pretreatment process of Methods I, II, and III, respectively, and the total concentrations of IGF-1 in the actual samples were detected based on the traditional ELISA method. A higher OD value was obtained for Method III, proving that many interfering  
Figure 5: The specificity of interfering substances in the fluorescent sandwich ELISA method for IGF-1 detection.  
substances in the samples obtained by Methods I and II occupied the specific recognition sites and prevented the effective binding of IGF-1 to the antibody, resulting in more accurate detection for Method III. Therefore, Method III is the optimal pretreatment method for colostrum powders and suitable for all types of ELISA methods. The established multi-step pretreatment of bovine colostrum powders is much simpler compared with the complex enzymolysis strategy for the LC-MS method [20], which further promotes the application and popularization of this method in real samples.