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Article

Fluorescent Sandwich ELISA Method for Specific and Ultra-Sensitive Trace Detection of Insulin-like Growth Factor-1 in Bovine Colostrum Powders

by
Tianyu Hu
,
Bingying Liu
,
Siqian He
,
Yuanjie Teng
and
Zaifa Pan
*
College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(7), 3972; https://doi.org/10.3390/app15073972
Submission received: 21 February 2025 / Revised: 31 March 2025 / Accepted: 2 April 2025 / Published: 3 April 2025
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Abstract

:
Insulin-like growth factor-1 (IGF-1) is a regulatory factor closely associated with diabetes, obesity, and breast cancer, and it also acts as one of the most abundant growth factors in bovine colostrum. Current methods generally have the problem of low sensitivity, a time-consuming nature, and low stability, which makes it difficult to crack down on the false advertising of IGF-1 content in dairy products. In this work, an ultrasensitive fluorescent enzyme-linked immunosorbent assay (ELISA) is proposed, where the antibody and the target are combined in the form of a “sandwich” to ensure the accuracy and specificity of the assay. IGF-1 is quantified based on an effective hydrogen peroxide (H2O2) probe with 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) as the fluorogenic substrate. The proposed fluorescent sandwich ELISA has a low limit of detection (LOD) of 77.29 pg/mL, fast experimental process within 1 h, and stable signal of 1 h. Furthermore, multi-step pretreatment methods for bovine colostrum powders are established to remove interfering substances, including fat, casein, and binding proteins, achieving the accurate and specific detection of IGF-1. IGF-1 recovery studies on treated bovine colostrum powders exhibit good recovery rates ranging from 91.71% to 102.32%, which proves the feasibility of detecting IGF-1 in real bovine colostrum.

1. Introduction

Insulin-like growth factor-1 (IGF-1) is a regulatory factor with a key role in cell proliferation and differentiation, as well as in the growth and development of the human skeleton [1,2]. The pathogenesis of diseases such as diabetes, acne, obesity, and breast cancer is also thought to be closely related to IGF-1 levels [3,4,5,6]. Because of the identical amino acid sequence, bovine IGF-1 can be recognized by the human IGF-1 receptor (IGF-1R), making it an ideal complement to IGF-1 for humans [7,8,9]. Studies have shown that IGF-1 is one of the most abundant growth factors in bovine colostrum [10,11], and milk products have a better effect on increasing IGF-1 levels than other dietary proteins [5]. Therefore, in addition to relying on human’s liver and peripheral tissues to produce IGF-1, the intake of IGF-1-rich foods such as colostrum is also an important way to supplement the content of IGF-1 in the body [12,13].
Colostrum milk powder contains large amounts of lipids, proteins, and pigments that are highly likely to have a matrix effect on IGF-1 detection. Proteins in colostrum are mainly water-insoluble casein, which accounts for about 75% of the total protein content in colostrum [14]. Water-soluble whey proteins are the second most abundant protein in colostrum besides casein, mainly including α-lactalbumin, β-lactoglobulin, lactoferrin, immunoglobulins, and growth factors [15]. Centrifugation for skimming is a common step in almost all milk pretreatment processes when detecting a component of whey protein [16,17]. Generally, the skimmed milk obtained by centrifugation still contains a large amount of casein, which needs to be further removed by acid precipitation or rennet treatment [15,18,19]. The resulting colostrum whey is suitable for the detection of immune globulins, growth factors, and other trace amounts of proteins.
Current methods for the detection of IGF-1 in milk and its derivatives include liquid chromatography–tandem mass spectrometry (LC-MS) [20,21], radioimmunoassay (RIA) [22,23], immunoradiometric analysis (IRMA) [24], and enzyme-linked immunosorbent assay (ELISA) [25]. Remaggi et al. developed a liquid chromatography–triple quadrupole mass spectrometry (LC-MS/MS) method for the detection of IGF-1 in a wide range of commercially available milk [20], which had a limit of detection (LOD) of 1–5 ng/mL. However, the method has high requirements for sample pretreatment, and the complicated sample preparation process probably causes a large amount of target loss. RIA methods usually have higher specificity and sensitivity than other methods; for example, Daxenberger et al. showed a LOD as low as 0.1 ng/mL IGF-1 [26]. However, the waste disposal of radioactive materials is a major challenge for RIA methods. Radioactive materials are potentially hazardous to human health, and their half-life and irradiation problems affect the stability of radionuclide-labeled antibodies or antigens [27]. The same issues also exist in IRMA. In contrast, the operation process of ELISA is safe and non-harmful for human beings and the environment. Currently, the emergence of commercial ELISA kits makes the detection of IGF-1 more convenient. These commercial ELISA kits are generally based on the traditional ELISA method, in which a chromogenic reaction happens due to the specific binding of antigens and antibodies inducing the catalysis of a substrate with the help of an enzyme. Tetramethylbenzidine (TMB) is the most common substrate, because it exhibits an obvious blue color during the process of reducing hydrogen peroxide (H2O2) to water via horseradish peroxidase (HRP) [28]. IGF-1 content is determined on the basis of the degree of chromogenic absorption. However, traditional ELISA methods do not have a low enough sensitivity due to the limit of ultraviolet–visible spectrophotometry. For example, JonlnbioTM (Shanghai Jianglai Biotechnology Co., Ltd., Shanghai, China) and Thermo InvitrogenTM ELISA kits (Thermo Fisher Scientific, Waltham, MA, USA) have detection ranges of 1.56–100 ng/mL and 1.23–300 ng/mL, respectively, in which both LOD values are higher than some reported retail milk with <0.5 ng/mL IGF-1 content [21].
With the growing prevalence of bovine colostrum products in the market, the quality of these products has become a matter of great concern [10,29]. In particular, the false advertising of IGF-1 content has become a problem in the dairy market; the actual content is usually lower than the broadcast one. Therefore, ultrasensitive IGF-1 detection is necessary for the supervision of the quality of bovine colostrum products. The fluorescent sandwich ELISA method was developed as a good substitute for the traditional ELISA method because of its excellent sensitivity and stable signal [30]. 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) is the most widely used fluorogenic substrate for horseradish peroxidase (HRP) [31]. ADHP is non-fluorescent, but when it reacts with H2O2 under enzyme catalysis, a new product of resorufin with red fluorescence is produced; therefore, the fluorescence intensity is used to indicate the content of HRP or other HRP-linked substances. Research on the use of ADHP for the detection of other targets has been documented. Meng et al. compared the effects of different substrates on the sensitivity and accuracy of ELISA [32], showing that ADHP enhanced the detection sensitivity by approximately five folds in comparison to TMB.
Herein, a new kind of ultrasensitive, rapid, and stable fluorescent sandwich ELISA method was developed in comparison with traditional ELISA, to detect trace amounts of IGF-1 in bovine colostrum powders, and the scheme of the fluorescent sandwich ELISA using a fluorogenic peroxidase substrate of ADHP is shown in Figure 1. The immobilized capture antibody and detection antibody are matched antibody pairs, which specifically bind with IGF-1, forming a “sandwich”. The biotinylated detection antibody is further connected with streptavidin–HRP based on the streptavidin–biotin interaction. In the presence of HRP, transparent and non-fluorescent ADHP reacts with H2O2 to produce resorufin with stable fluorescent signals, and thus, the amount of IGF-1 is related to the fluorescence intensity. The fluorescence working solution is optimized according to the contents of the enzyme and substrate and environmental factors. The standard curve, linear range, and LOD of IGF-1 are obtained using the established fluorescent sandwich ELISA method. To effectively remove the interfering impurities and retain as much IGF-1 in bovine colostrum powders, three different multi-step pretreatments are compared, and furthermore, IGF-1 recovery assays are performed on treated colostrum samples to validate the accuracy of the fluorescent sandwich ELISA and quantify the IGF-1 content in colostrum powders. Finally, the specificity of the fluorescent sandwich ELISA is verified by low cross-reactivity rates.

2. Materials and Methods

2.1. Reagents

The SignalUp™ super-sensitive ELISA kit (including ADHP, H2O2, and termination solution) was purchased from Beyotime Co., Ltd. (Shanghai, China). The Bovine IGF-1 ELISA kit (including coated 96-well plate, IGF-1 standard solution, biotinylated antibody, enzyme labeling reagent, dilution buffer, TMB, H2O2, and termination solution) was purchased from Bioswamp Co., Ltd. (Wuhan, China). Colostrum powder was provided by Hangzhou Pupai Technology Co., Ltd. (Hangzhou, China). Lactose was purchased from Titan Technology Co., Ltd. (Shanghai, China). Whey protein (80%), NaH2PO4, and Na2HPO4 were purchased from Maklin Reagent Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) and NaCl were obtained from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). NaOH and HCl were provided by Sinopharm Chemical Reagent Co. (Shanghai, China). Pure water (milliq) was obtained from Wahaha Co., Ltd. (Hangzhou, China).

2.2. Apparatus

The optical density (OD) value of the traditional ELISA was detected by the Spark multimode microplate reader (Tecan, Männedorf, Switzerland). The fluorescence intensity of the fluorescent sandwich ELISA was detected by the FluoroMax Plus fluorescence spectrometer (HORIBA, Kyoto, Japan). Centrifugation and mixing processes for all reagents were performed with a mySPIN mini centrifuge (Thermo, Waltham, MA, USA) and a MX-S vortexer (DLAB, Beijing, China), respectively. All warming processes were implemented in a 96RH microplate thermostatic oscillator (Bionoon, Shanghai, China). The pH of the buffer was adjusted using a PHS-3E pH meter (Leici, Shanghai, China).

2.3. Methods

2.3.1. Preparation of Traditional ELISA Working Solution

TMB substrate was mixed with H2O2 at a stoichiometric ratio of 1:1 to formulate a traditional ELISA working solution.

2.3.2. Optimization of Fluorescent Sandwich ELISA Working Solution

The fluorescence working solution was made of ADHP, H2O2, and PBS buffer. The optimization included 1. the volume ratio of ADHP to H2O2; 2. the total volume of ADHP and H2O2; 3. the acidity and alkalinity of the working solution; 4. the reaction temperature. Fluorescence intensities at an emission wavelength of 585 nm for different fluorescence working solutions were compared to evaluate these reaction conditions.
  • The volume ratio of ADHP to H2O2: 20 μL of H2O2 was mixed with 10, 20, 40, and 80 μL of ADHP, respectively. Then, PBS buffer was added to dilute the fluorescence working solution to a total volume of 1 mL, making the final concentrations of ADHP 62.5 μM, 125 μM, 250 μM, and 500 μM, respectively.
  • The total volume of ADHP and H2O2: The total volume of ADHP and H2O2 with an optimal ratio was changed from 20 to 40, 60, and 80 μL, and PBS buffer was added to dilute the fluorescence working solution to a total volume of 1 mL.
  • The acidity and alkalinity of the working solution: 50 mL of 50 mM PBS buffer (10.4 mM NaH2PO4, 39.6 mM Na2HPO4, 150 mM NaCl) was prepared, and the pH value of the buffer was adjusted to 5, 7, and 9, respectively. The fluorescence working solutions were prepared with the above buffers.
  • The reaction temperature: The reaction temperature of the fluorescent working solution was varied by changing the incubation temperature of the microplate thermostatic oscillator to 25 °C, 37 °C, and 50 °C, respectively.

2.3.3. Detection Process

  • Traditional ELISA Method:
IGF-1 standard solution was diluted sequentially with dilution buffer to obtain five IGF-1 standards at concentrations of 0, 6.25, 12.5, 25, 50, and 100 ng/mL, respectively. A total of 50 μL of IGF-1 standards (biotinylated antibodies included) and 50 μL of enzyme-labeled reagents were added to the coated wells. As the blank control, 100 μL of dilution buffer was added to the wells instead. After incubation at 37 °C for 30 min, the plates were washed four times with 300 μL of wash buffer. A total of 100 μL of the working solution prepared in Section 2.3.1 was then added to each well and the reaction was carried out at 37 °C for 15 min in a dark environment. Finally, 50 μL of termination solution was added to stop the reaction. The OD value of each well was measured at 450 nm by the multimode microplate reader. Error bars were obtained according to the standard deviation and mean value obtained from three experiments. The LOD of the method was calculated from the standard deviation of the OD value of the blank well (σa) and the slope of the standard curve (s) according to Equation (1).
L O D = 3 σ a s
  • Fluorescent sandwich ELISA method:
Similarly to the procedure above, 0, 0.1, 1, 5, 15, and 30 ng/mL IGF-1 standards, biotinylated antibodies, and enzyme-labeled reagents were sequentially added to the wells; then, the plates were incubated at 37 °C for 30 min and washed with 50 mM PBS (pH = 7) buffer. The optimal fluorescence working solution prepared in Section 2.3.2 was added to the wells and placed in a dark environment at 37 °C for 15 min before 100 μL of termination solution was added. Finally, the mixture of fluorescence working solution and termination solution was transferred to a microcuvette for testing. The fluorescence intensity was measured by the fluorescence spectrometer at an excitation wavelength of 570 nm and emission wavelength of 585 nm [33]. The LOD of the fluorescent sandwich ELISA was calculated from the method reported by Meng et al. [32]:
y = background + 3SD
where background is the mean value of the background fluorescence intensity and SD is the standard deviation of the background fluorescence intensity. The LOD of the fluorescent sandwich ELISA method corresponds to the IGF-1 concentration at the above calculated fluorescence intensity.

2.3.4. Specificity Study

A total of 10 ng/mL of whey protein, lactose, and BSA were added to replace IGF-1 standards in wells, and the rest of the experimental process was the same as in Section 2.3.3 (2).

2.3.5. Pretreatment of Bovine Colostrum Powders

Colostrum powders were weighed and dissolved in pure water to form a colostrum suspension. Three pretreatment methods were used, as follows (Figure 2). Method I: the colostrum suspension was diluted directly with PBS without any other treatments. Method II: the above diluted liquid colostrum obtained in Method I was then centrifuged at 3000 rpm for 15 min at room temperature, and after that, the intermediate-layer solution was separated. Method III: The pH value of the above intermediate layer was adjusted to 4.6 and then the solution was centrifuged at 5000 rpm for 10 min at room temperature. Finally, the supernatant was adjusted to pH 6.4 with 1 M NaOH. The traditional ELISA method was employed to evaluate the above pretreatments of bovine colostrum powders.

2.3.6. Real Samples Detection

After the optimal pretreatment, bovine colostrum samples were diluted by 2 and 5 times for the traditional and fluorescent sandwich ELISA methods, respectively. Diluted samples without and with three different concentrations of IGF-1 standards were detected by both the traditional and fluorescent sandwich ELISA methods. The IGF-1 concentration in the samples could be determined based on the standard curve and the measured value. The amount of IGF-1 in the colostrum powders was calculated based on the concentration of IGF-1 and the number of dilutions.

3. Results and Discussion

3.1. Optimizing Conditions

ADHP is non-fluorescent; however, after the addition of H2O2 and HRP, strong excitation and emission signals are displayed, as shown in Figure 3a, proving that resorufin generated by ADHP produces an effective fluorescent signal in the HRP enzymatic reaction. Therefore, developing an ELISA method on the basis of ADHP fluorogenic substrate is feasible. Before developing the fluorescent sandwich ELISA method, the effects of ADHP concentration, the total volume of ADHP and H2O2, temperature, and pH on the fluorescent sandwich ELISA working solution were studied to examine the fluorescent conditions. As shown in Figure 3b, with an increasing concentration of ADHP, the fluorescence intensity was significantly enhanced and was then kept stable, indicating that the fluorescence intensity finally reached saturation in the case of excessive ADHP. The fluorescence intensity reached the highest point with 250 μM ADHP, so this concentration of ADHP was selected to prepare the fluorescent working solution. When the total volume of ADHP and H2O2 increased, a similar trend was shown and the fluorescence intensity values were slightly different at a total volume between 40 and 80 μL (Figure 3c). To save fluorescent substrate and ensure high signal intensity, the fluorescent working solution with a total volume of 40 μL was finally selected for fluorescent sandwich ELISA determination.
The results of the enzymatic reaction of ADHP were also greatly influenced by environmental factors. Incubation at 37 °C gave a stronger fluorescence signal than incubation at room temperature because appropriate heating had a positive effect on the enzymatic reaction (Figure 3d). However, the fluorescence intensity decreased after the temperature continued rising to 50 °C, which was because high temperature reduced the activity of HRP, leading to a decrease in the enzymatic reaction rate and thus affecting the intensity of the fluorescence signal. Therefore, 37 °C was chosen as the reaction temperature of ADHP. Moreover, the effect of pH given in Figure 3e displays that the fluorescence intensity was higher at a pH of 7 than at a pH of 5 or 9. This is probably because alkaline buffer accelerates the decomposition of H2O2, thereby reducing the content of substrates in enzymatic reactions. Meanwhile, ADHP exists mainly as non-deprotonated neutral molecules under acidic conditions, making it difficult for oxidation reactions to occur [33]. In addition, HRP had the best enzymatic activity at a pH of 7. Therefore, the optimal working temperature and pH for the fluorescent sandwich ELISA working solution were 37 °C and a pH of 7, respectively.

3.2. Detection of IGF-1

In traditional ELISA, after TMB substrate is added, a blue color develops in proportion to the amount of IGF-1, and the color turns different degrees of yellow with the addition of termination solution. IGF-1 was detected by a colorimetric assay with the OD value monitored at 450 nm, and the standard curve is shown in Figure 4a. The OD value gradually increased with the increase in IGF-1 concentration, revealing an excellent linear relationship between the OD values and IGF-1 concentrations in the range of 1.25–100 ng/mL. The regression equation was y = 0.00463x + 0.05547, the regression coefficient was 0.9998, and the LOD was calculated to be 1.018 ng/mL.
In the fluorescent sandwich ELISA, the addition of the optimized fluorescent working solution initiated a gradual color transition from colorless to faint pink, and the color became deep pink with increasing concentrations of IGF-1. After adding the termination solution, the reaction mixtures maintained their developed color without further visible changes. The fluorescence intensities measured at the emission wavelength of 585 nm were analyzed by a four-parameter logistic regression model, which is usually performed in ELISA methods [32]. The standard curve was constructed and is shown in Figure 4b, in which all IGF-1 concentrations are closely located on the four-parameter fitting curve (R2 = 0.9968), with a detection range in the lower concentration region of 0.1–30 ng/mL and a much lower LOD of 77.29 pg/mL.
Compared with traditional ELISA, the fluorescent sandwich ELISA method has a faster experimental process within 1 h, while commercial ELISA kits usually need at least 3 h, so the fluorescent sandwich ELISA method saves time and obtains test results quickly. The LOD of this fluorescent sandwich ELISA method is reduced by about 10 times, which is suitable for measuring ultra-low concentrations of IGF-1. In addition, the signals of traditional ELISA are ineffective within 15 min after the addition of the termination solution, whereas the ADHP reaction obtains a stable fluorescent signal for up to 1 h after termination, which helps in saving results for longer time. Moreover, a comparison between the present method and other reported methods for IGF-1 determination with regard to the detection range and LOD is summed up in Table 1. It demonstrates that liquid chromatograph–mass spectrometer (LC-MS) methods usually have higher LOD values and are suitable for the detection of IGF-1 with high concentrations. ELISA methods tend to be specific to lower concentrations of IGF-1, especially the fluorescent ELISA method proposed in this work, which is ultrasensitive in IGF-1 detection, reaching a magnitude of pg/mL.

3.3. Specificity of IGF-1 Detection

Selectivity is a very important parameter to evaluate the performance of new established methods, so a highly selective response to the target over other potentially competing species is necessary. Whey protein, lactose, and BSA were chosen as the interfering substances to determine the selectivity in the established fluorescent sandwich ELISA method (Figure 5). Both whey protein and lactose widely exist in colostrum; thus, they were useful to evaluate the interference in the real samples. BSA is one of the most common proteins, which was helpful to check the specific recognition in the “sandwich” structure. Compared with lactose and BSA, whey protein showed a relative higher fluorescence intensity due to the presence of trace amounts of IGF-1 in whey protein. However, all fluorescence intensities of the interfering substances were extremely low, with intensities at least 11 times lower than those of the detection of IGF-1, indicating that the interferences were tolerable. Therefore, the fluorescent sandwich ELISA developed in this work has good specificity for IGF-1 detection.

3.4. 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 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.

3.5. Detection of IGF-1 in Bovine Colostrum Powders

After the multi-step pretreatment of bovine colostrum powders, real samples were measured by both the traditional and fluorescent sandwich ELISA methods. Since the LOD of the fluorescent sandwich ELISA method is much lower than that of the traditional ELISA method, real samples were diluted five times and two times, respectively, to obtain similar results, which further proved the high sensitivity of the fluorescent sandwich ELISA method. As shown in Table 2, an IGF-1 content of about 1125.00 ng/g (CV = 3.81%) was detected via the fluorescent sandwich ELISA method, and 1026.56 ng/g (CV = 3.12%) of IGF-1 was detected by the traditional ELISA method in comparison. The very similar values reveal the accuracy of the proposed fluorescent sandwich ELISA method. Recovery tests were also carried out to further demonstrate the availability of the proposed fluorescent sandwich ELISA method. We added 1, 5, and 15 ng/mL of IGF-1 standards to five-fold-diluted bovine colostrum samples and the solutions were measured by the fluorescent sandwich ELISA method. The recovery rates were in the range of 91.71–102.32% (CV < 5.54%), and the final calculated IGF-1 content was about 1096.68 ng/g. CV values could be further decreased when maintaining a constant environmental temperature and humidity and, moreover, avoiding photobleaching caused by long-term exposure to light. For comparison, a similar standard addition method was used to detect IGF-1 concentration by the traditional ELISA method. Since the linear range and LOD are much higher in traditional ELISA than fluorescent sandwich ELISA, lower dilutions (two-fold-diluted bovine colostrum samples) and larger amounts of standards (25, 60, and 80 ng/mL of IGF-1 standards) were applied in traditional ELISA. The average recoveries were in the range of 100.69% to 109.75% with a CV of 2.50% to 5.89%, and the IGF-1 content was calculated as about 1087.72 ng/g. The similar contents between the two methods demonstrate that the proposed fluorescent sandwich ELISA method had high accuracy, precision, and reproducibility in the detection of IGF-1. Furthermore, the good recovery results further prove that the pretreatment of bovine colostrum with Method III effectively avoided the influence of the matrix in the colostrum powder, thus ensuring that IGF-1 was specifically recognized. However, the specific detection of IGF-1 in other milk foods still remains challenging due to differences in bioactive component profiles compared to bovine colostrum and potential amino acid sequence variations in IGF-1 from different sources [40]. These factors limit the use of the proposed multi-step pretreatment and fluorescent sandwich ELISA.

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.

Author Contributions

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.

Funding

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).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

We are grateful to Shiyun Lai (Hangzhou Popide Technology Co., Ltd., Hangzhou, 310063, China) for help with the methodology.

Conflicts of Interest

The authors declare no conflicts of interest.

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Applsci 15 03972 g001
Figure 1. (a) Mechanism of IGF-1 detection based on the fluorescent sandwich ELISA method: (1) Coating capture antibody, (2) adding IGF-1, (3) Incubating biotinylated antibody, (4) binding HRP-streptavidin, (5) adding fluorescent sandwich ELISA working solution for enzymatic signal generation. (b) Mechanism of the ADHP enzymatic reaction.
Figure 1. (a) Mechanism of IGF-1 detection based on the fluorescent sandwich ELISA method: (1) Coating capture antibody, (2) adding IGF-1, (3) Incubating biotinylated antibody, (4) binding HRP-streptavidin, (5) adding fluorescent sandwich ELISA working solution for enzymatic signal generation. (b) Mechanism of the ADHP enzymatic reaction.
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Figure 2. Pretreatment steps for bovine colostrum powders using different methods: bovine colostrum powders (A) were dissolved and dispersed in water (B), then the solution was divided into three layers after centrifugation (C), after removing the upper and lower layers, skimmed milk was obtained (D), then the acid treatment and centrifugation followed to obtain the supernatant and precipitation (E), and finally a transparent and light-yellow solution was obtained after throwing the precipitation (F).
Figure 2. Pretreatment steps for bovine colostrum powders using different methods: bovine colostrum powders (A) were dissolved and dispersed in water (B), then the solution was divided into three layers after centrifugation (C), after removing the upper and lower layers, skimmed milk was obtained (D), then the acid treatment and centrifugation followed to obtain the supernatant and precipitation (E), and finally a transparent and light-yellow solution was obtained after throwing the precipitation (F).
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Figure 3. (a) Excitation and emission spectra of resorufin generated by ADHP in HRP enzymatic reaction. Optimization of (b) concentration of ADHP, (c) total volume, (d) temperature, and (e) pH of fluorescent sandwich ELISA working solution.
Figure 3. (a) Excitation and emission spectra of resorufin generated by ADHP in HRP enzymatic reaction. Optimization of (b) concentration of ADHP, (c) total volume, (d) temperature, and (e) pH of fluorescent sandwich ELISA working solution.
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Figure 4. Standard curves obtained by (a) traditional ELISA and (b) fluorescent sandwich ELISA methods. The error bars indicate the standard deviation of three replicate experiments.
Figure 4. Standard curves obtained by (a) traditional ELISA and (b) fluorescent sandwich ELISA methods. The error bars indicate the standard deviation of three replicate experiments.
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Figure 5. The specificity of interfering substances in the fluorescent sandwich ELISA method for IGF-1 detection.
Figure 5. The specificity of interfering substances in the fluorescent sandwich ELISA method for IGF-1 detection.
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Figure 6. OD values of IGF-1 in bovine colostrum powders of three pretreatments based on traditional ELISA method.
Figure 6. OD values of IGF-1 in bovine colostrum powders of three pretreatments based on traditional ELISA method.
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Table 1. The detection range and detection limit of some other methods for the detection of IGF-1.
Table 1. The detection range and detection limit of some other methods for the detection of IGF-1.
MethodIGF-1 SourceLOD a/LOQ b (ng/mL)Detection Range (ng/mL)Reference
Fluorescent sandwich ELISABovine colostrum powder0.07729 (LOD)0.1–30This work
Traditional ELISABovine colostrum powder1.018 (LOD)1.25–100This work
Traditional ELISAMilk0.3 (LOD)0.3–10[18]
Traditional ELISABuffalo milk0.1 (LOD)0.25–4[34]
LC-MS/MS cHuman plasma10 (LOQ)10–1000[35]
LC-MSHuman serum50 (LOQ)50–1000[36]
LC-MS/MSHuman plasma5.9 (LOQ)15–1100[37]
a: LOD represents the limit of detection. b: LOQ represents the limit of quantitation. c: LC-MS represents liquid chromatography–tandem mass spectrometry.
Table 2. Measurements of IGF-1 in pretreated bovine colostrum powders.
Table 2. Measurements of IGF-1 in pretreated bovine colostrum powders.
Added IGF-1 Concentration (ng/mL)Calculated IGF-1 Concentration (ng/mL)Recovery Rate (%)CV a
(n = 3, %)		Calculated IGF-1 Content in Bovine Colostrum Powder (ng/g)Average IGF-1 Content in Bovine Colostrum Powder (ng/g)
Fluorescent sandwich ELISA
014.06/3.811125.001096.68
115.08102.325.541126.85
518.8796.093.341109.36
1527.8291.711.991025.50
Traditional ELISA
032.08/3.121026.561087.72
2557.25100.695.891032.09
6097.93109.753.891213.86
80113.70102.022.501078.39
a: CV represents the coefficient of variation.
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Hu, T.; Liu, B.; He, S.; Teng, Y.; Pan, Z. Fluorescent Sandwich ELISA Method for Specific and Ultra-Sensitive Trace Detection of Insulin-like Growth Factor-1 in Bovine Colostrum Powders. Appl. Sci. 2025, 15, 3972. https://doi.org/10.3390/app15073972

AMA Style

Hu T, Liu B, He S, Teng Y, Pan Z. Fluorescent Sandwich ELISA Method for Specific and Ultra-Sensitive Trace Detection of Insulin-like Growth Factor-1 in Bovine Colostrum Powders. Applied Sciences. 2025; 15(7):3972. https://doi.org/10.3390/app15073972

Chicago/Turabian Style

Hu, Tianyu, Bingying Liu, Siqian He, Yuanjie Teng, and Zaifa Pan. 2025. "Fluorescent Sandwich ELISA Method for Specific and Ultra-Sensitive Trace Detection of Insulin-like Growth Factor-1 in Bovine Colostrum Powders" Applied Sciences 15, no. 7: 3972. https://doi.org/10.3390/app15073972

APA Style

Hu, T., Liu, B., He, S., Teng, Y., & Pan, Z. (2025). Fluorescent Sandwich ELISA Method for Specific and Ultra-Sensitive Trace Detection of Insulin-like Growth Factor-1 in Bovine Colostrum Powders. Applied Sciences, 15(7), 3972. https://doi.org/10.3390/app15073972

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