Application of Recombinant Monoclonal Antibodies from Transgenic Chicken Bioreactors in Enzyme-Linked Immunosorbent Assay

Abstract

Transgenic chicken bioreactors can efficiently produce egg whites containing large quantities of recombinant proteins. We previously developed transgenic chickens that produce recombinant monoclonal antibodies (mAbs) against epidermal growth factor receptor 2 (HER2). However, the practical applications of mAbs derived from transgenic eggs have not yet been examined. Therefore, we aimed to evaluate whether these recombinant mAbs can be used in enzyme-linked immunosorbent assay (ELISA). Recombinant HER2 mAbs from transgenic eggs were dissolved in phosphate-buffered saline and applied directly to 96-well microplates as immobilized antibodies without purification. The performance of ELISA using the unpurified recombinant HER2 mAbs from transgenic eggs was comparable to that of ELISA using commercially available purified recombinant HER2 mAbs. Moreover, ELISA using unpurified recombinant HER2 mAbs from transgenic eggs demonstrated high antigen specificity and was successfully applied to samples from cultured cell lysates derived from HER2-positive and HER2-negative cell lines. The unpurified recombinant HER2 mAbs from transgenic eggs were also efficiently used as immobilized antibodies in paper-based ELISA. In conclusion, our findings suggest that recombinant mAbs from transgenic eggs have the potential to be used to develop economic ELISA devices. To the best of our knowledge, this study is the first to use recombinant HER2 mAbs from transgenic eggs in ELISA.

INTRODUCTION

Enzyme-linked immunosorbent assay (ELISA) is widely used in biomedical monitoring, clinical diagnosis, and food safety testing.^1–4) However, ELISA is often expensive to perform and use frequently. Sensitive detection using ELISA typically requires monoclonal antibodies (mAbs) immobilized in 96-well microplates or custom devices.⁵⁾ Moreover, the production of mAbs requires expensive cell culture media and facilities, as well as laborious downstream processes, such as isolation and purification. Therefore, the development of a cost-effective approach to obtain mAbs for ELISA is important.

The use of transgenic chickens to produce exogenous recombinant proteins from egg whites has been studied to address this issue. These transgenic chickens have several advantages, such as cost-effective production of recombinant proteins, rapid recombinant flock development, and productivity of biologically complex recombinant proteins.^6,7) Previous studies have successfully generated transgenic chickens that produce egg whites containing exogenous recombinant mAbs.^8,9) In these cases, exogenous mAb genes are driven by the constitutive gene activation promoter of beta-actin or the oviduct-specific gene promoter of ovalbumin. However, the yield of recombinant mAbs in egg whites is limited, typically ranging from tens to hundreds of micrograms per milliliter.

We recently produced knock-in transgenic chickens with an mAb gene targeting epidermal growth factor receptor 2 (HER2).¹⁰⁾ In this study, we linked the genes of heavy and light chains identical to those of trastuzumab with a 2A peptide and inserted this gene construct into the ovalbumin translation initiation site using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (CRISPR/Cas9). Recombinant mAbs from transgenic eggs yielded high concentrations (approximately 1.4–1.9 mg/mL) and demonstrated antigen recognition abilities comparable to those of commercially available trastuzumab. Therefore, knock-in transgenic chickens could be a useful platform for the cost-effective production of recombinant mAbs. However, the practical applications of recombinant mAbs from transgenic eggs have not yet been explored.

Thus, this study aimed to investigate the applicability of recombinant mAbs from transgenic eggs in ELISA. These recombinant mAbs can be produced in large quantities at a low cost, and egg whites have a simple composition of water and protein. Considering these advantages, we evaluated whether unpurified recombinant mAbs from transgenic eggs could be used as immobilized antibodies in a 96-well microplate or paper-based ELISA. Our findings indicate that recombinant mAbs from transgenic eggs have the potential to reduce ELISA manufacturing costs by providing low-cost immobilized antibodies. To the best of our knowledge, this study is the first to show that recombinant mAbs from transgenic eggs can be applied in ELISA.

MATERIALS AND METHODS

Egg White Samples

Egg white samples containing recombinant monoclonal anti-HER2 antibodies were obtained from two identical lines of transgenic chickens following a previously described method.¹⁰⁾ In this study, the transgenic eggs, referred to as KI-1 and KI-2, corresponded to lines previously identified as #41 and #42, respectively.

Kits, Reagents, and Antibodies

Sandwich ELISAs were performed using a DuoSet ELISA development kit (R&D Systems, Inc., Minneapolis, MN, U.S.A.). Human immunoglobulin G (hIgG) levels were measured using the hIgG AssayMax ELISA kit (AssayPro, St. Louis, MO, U.S.A.). Paper-based ELISAs were performed using Whatman filter paper #1. Block Ace powder (Snow Brand Milk Products KK, Sapporo, Japan) was used as the blocking reagent. ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was used as the chemiluminescent reagent. Histidine (His)-tagged HER2-extracellular domain 1 (His-tagged HER2 ECD 1), domain 4 (His-tagged HER2 ECD 4), and full-length HER1 and HER2-extracellular domain (HER1 ECD) fragments (Sino Biological Inc., Beijing, China) were used as the recombinant model antigens. A commercially available control mAb with an amino acid sequence identical to that of trastuzumab (an anti-HER2 mAb) was used (NMIJ RM 6208-a; AIST-MABS; FUJIFILM Wako Pure Chemical Corporation).¹¹⁾ Anti HER2 polyclonal antibody (Sino Biological Inc.) was used as the primary antibody for sandwich ELISA targeting full-length HER2 ECD. Immunodetection assays were conducted using an anti-His-tag mAbs-horseradish peroxidase (HRP)-DirecT antibody (MBL, Tokyo, Japan) and HRP-conjugated anti-human IgG antibodies (Bethyl Laboratories, Montgomery, TX, U.S.A.).

Purification of Recombinant mAbs

Recombinant mAbs were purified from transgenic eggs using a HiTrap™ Protein A HP (Cytiva Life Sciences, MA, U.S.A.). Transgenic egg whites were diluted 10-fold with phosphate-buffered saline (PBS), filtered through a 0.45 µM membrane, and then subjected to the column. The column was washed with 20 mM potassium phosphate (pH 7.0), and the captured mAbs were eluted with 0.1 M glycine hydrochloride (pH 3.0) and neutralized with 0.1 M tris hydrochloride buffer (pH 9.0).

Detection of Immobilized Antibodies on Microplates

Initially, egg whites of KI-1, KI-2, and wild type (WT, negative control) were diluted 10-fold with PBS. These samples and the control mAb (10 µg/mL in PBS) were further subjected to 10-fold serial dilutions. Each sample (100 µL) was added to empty 96-well microplates to serve as immobilized antibodies. The microplates were then incubated overnight at 4 °C. The wells were washed four times with 200 µL of wash buffer to remove excess antibodies and then blocked with 200 µL of Block Ace (0.8 mg/mL) for 1.5 h. After the blocking reagent was removed, 50 µL of HRP-conjugated anti-human IgG antibody was added to each well and then incubated for 1.5 h. After being washed, 50 µL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added to each well and then incubated for 5 min. Then, 50 µL of stop solution was added to each well, and the absorbance at 450 and 650 nm was measured using a microplate reader (iMark Microplate Reader; Bio-Rad, Hercules, CA, U.S.A.).

Sandwich ELISA

Subsequently, 100 µL of 10000-fold diluted egg whites (KI-1 and WT) and the control mAb (0.15 µg/mL in PBS) were added to empty 96-well microplates to serve as immobilized antibodies. The microplates were incubated overnight at 4 °C. The wells were washed four times with 200 µL of wash buffer to remove excess antibodies and then blocked with 200 µL of Block Ace (0.8 or 0.2 mg/mL) for 1.5 h. The wells were washed to remove the blocking reagent, 100 µL of His-tagged HER2d4 was added as a recombinant antigen, and then incubated for 1.5 h. The wells were washed to remove the recombinant antigen, 100 µL of HRP-conjugated anti-His-tag antibody was added, and then incubated for 30 min. The wells were washed, 100 µL of TMB substrate was added, and then incubated at room temperature depending on the relative speed and intensity of the colorimetric reaction. Then, 100 µL of stop solution was added to each well, and the absorbance was measured as described above.

Cell Lysis Buffer

The cell lysis buffer was prepared with the following final concentrations: 50 mM Tris–HCl buffer (pH 7.5), 0.15 M sodium chloride, 0.1% sodium dodecyl sulfate, and 1% Triton X-100. The components were thoroughly mixed to ensure a uniform solution.

Cell Culture and Sample Preparation

Four cell lines were used in this study: BRL, HepG2, MCF7, and SKBR3. BRL cells, which are rat derived, and HepG2 cells do not express HER2. MCF7 cells exhibit low HER2 expression, whereas SKBR3 cells exhibit high HER2 expression.¹²⁾ The cells were cultured in high-glucose Dulbecco’s modified Eagle medium (Gibco, 11995-065) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Gibco, 15140-122), and 1% L-glutamine (Gibco, 25030-081). The cultures were maintained at 37 °C in a 5% CO₂ atmosphere. Each cell line was seeded in a 10 cm dish and cultured until confluence.

The cells were then prepared as follows: First, the culture medium was removed, and the cells were washed with chilled PBS. Then, 1 mL of chilled cell lysis buffer was added to each dish, and the cells were scraped off using a cell scraper. The detached cells were collected in 1.5 mL tubes and incubated at 4 °C for 30 min with rotation. After incubation, the cell suspension was centrifuged at 15000 rpm for 15 min at 4 °C, and the supernatant was collected. The protein concentration of the collected supernatant was measured, and each sample was diluted with PBS to adjust the concentration to 1 mg/mL. These procedures resulted in cell lysates from each cell line.

Determination of Optimal Antibody Concentration

To achieve a high signal-to-noise ratio, we determined the optimal antibody concentration that would not interfere with the reaction. First, transgenic egg-derived antibodies or control antibodies were applied at 15 ng/well for immobilization, followed by blocking. To determine the optimal secondary antibody concentration, we added serially diluted anti-rabbit HRP-conjugated antibody at 100 µL/well after washing away the excess reagent. After 30 min, the excess reagent was washed away, and 100 µL of TMB substrate was added. After an additional 30 min, the stop solution was added, and the absorbance was measured. The highest concentration of secondary antibody that produced no difference from the background was determined to be the optimal concentration.

Next, the optimal primary antibody concentration was determined. Wells immobilized with antibodies and blocked were treated with 100 µL of serially diluted anti-HER2 antibodies per well. After 1.5 h, the excess solution was washed away, and the secondary antibody at the previously determined optimal concentration was added at 100 µL/well. After 30 min, the excess solution was washed away, and 100 µL of TMB substrate was added. After another 30 min, the stop solution was added, and the absorbance was measured. The highest concentration of primary antibody that produced no difference from the background was determined to be the optimal concentration.

Sample Preparation

The samples used in this study for Sandwich ELISA targeting the full-length HER2 ECD included recombinant HER1 at concentrations of 80, 40, 20, and 10 ng/mL and recombinant HER2 domain 1 (HER2d1) at concentrations of 80, 40, 20, and 10 ng/mL. Additionally, BRL, HepG2, MCF7, and SKBR7 cell lysates were prepared. The total protein concentrations of the BRL, HepG2, and MCF7 cell lysates were adjusted to 500, 250, 125, and 67.5 µg/mL, whereas the total protein concentration of the SKBR7 cell lysates was adjusted to 500, 250, 125, 67.5, 33.8, 16.9, 8.4, 4.2, 1.1, 0.5, and 0.3 µg/mL.

Sandwich ELISA for Full-Length HER2 ECD

Subsequently, 100 µL of 10000-fold diluted egg whites (KI-1 and WT) and the control mAb (0.15 µg/mL in PBS) were added to empty 96-well microplates to serve as immobilized antibodies. The microplates were incubated overnight at 4 °C. The wells were washed four times with 200 µL of wash buffer to remove excess antibodies and then blocked with 200 µL of Block Ace (0.2 mg/mL) for 1.5 h. The wells were washed to remove the blocking reagent, 100 µL of His-tagged HER2d4 as a recombinant antigen was added, and then incubated for 1.5 h. The wells were washed to remove the recombinant antigen, 100 µL of HRP-conjugated anti-His-tag antibody was added, and then incubated for 30 min. The wells were washed, 100 µL of TMB substrate was added, and then incubated at room temperature depending on the relative speed and intensity of the colorimetric reaction. Then, 100 µL of stop solution was added to each well, and the absorbance was measured as described above.

Paper-Based ELISA

Whatman filter paper #1 was used to develop a paper-based ELISA as previously described.¹³⁾ The filter paper was cut into 10 cm rectangles, and lines were drawn in a grid pattern with 7.5 mm spaces using a thin paraffin block. Subsequently, the paper was heated at 60 °C for 15 min to dissolve the paraffin and create a detection area surrounded by a hydrophobic barrier by the dissolved paraffin. After cooling to room temperature, the mixture was used in subsequent experiments.

To each detection area, 4 µL of 100-fold diluted egg whites of KI-1 and WT, as well as the control mAb (15 µg/mL in PBS), were added to serve as immobilized antibodies and then incubated overnight at 4 °C. Each detection area was washed twice with 20 µL of wash buffer to remove excess antibodies and then blocked with 4 µL of Block Ace for 1.5 h. After the blocking reagent was removed, 5 µL of HRP-conjugated anti-human IgG antibody (diluted 1 : 1000) was added to the detection area and then incubated for 10 min. Each detection area was washed and 5 µL of ImmunoStar LD was added. Luminescence was detected using a WSE-6100 LuminoGraph I (ATTO Corporation, Tokyo, Japan).

Additionally, 5 µL of His-tagged HER2d4 and the immobilized antibodies were added to detection area, blocked with Block Ace as described above, and then incubated for 10 min. Excess His-tagged HER2d4 was removed, and then the wells were washed, 5 µL of HRP-conjugated His-tag antibodies was added, and then incubated for 10 min. Each detection area was washed and then 5 µL of luminescence reagent was added. Luminescence was detected as described above.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 10 statistical software (GraphPad Software, Inc., San Diego, CA, U.S.A.). Between-group and multiple-group comparisons were analyzed using an independent sample t-test and one-way ANOVA, respectively. Subsequently, pairwise comparisons were performed using Tukey’s multiple comparison test. Dose–response curves for the antigen of His-tagged HER2d4 were fitted with a logistic equation and subjected to a comparison-of-fit analysis. Statistically significant differences were considered at p < 0.05.

Image Analysis

Image analysis was performed using ImageJ (National Institutes of Health, U.S.A.). Brightness was standardized across all images obtained from paper-based ELISA to ensure consistency.

LOD Analysis

To evaluate the limit of detection (LOD) of His-tagged HER2d4 ECD and full-length HER2 ECD ELISAs, we conducted measurements using standards of known concentrations to generate a standard curve.¹⁴⁾ Blank samples containing zero analyte concentrations were measured to assess background noise. The mean and standard deviation of the blank sample measurements were calculated. The LOD was determined using the following formula:

  

RESULTS

Immobilization of Recombinant mAbs on 96-Well Microplates

We evaluated whether the recombinant mAbs secreted into the transgenic egg whites can be immobilized in 96-well microplates. A schematic of the sandwich ELISA procedure is shown in Fig. 1A. Hereafter, the recombinant anti-HER2 mAbs secreted to the transgenic egg whites are referred to as “egg mAbs,” whereas commercially available recombinant anti-HER2 mAb with an amino acid sequence identical to trastuzumab are referred to as “control mAb.”

Fig. 1. Antibody Immobilization in 96-Well Microplates

A) Schematic of detection for immobilized antibodies. B) Color development of immobilized antibodies in a 96-well microplate. This panel shows visible differences in color development across serial dilutions of immobilized unpurified egg mAbs (KI-1 and KI-2) and control mAb. C–E) Absorbance response curves of immobilized antibodies in 96-well microplates. Immobilized control mAb (C), KI-1 unpurified mAb (D), and KI-2 unpurified mAb (E) are detected using HRP-conjugated anti-human IgG. The four-parameter logistic model was used for the nonlinear regression analysis. The EC₅₀ of each immobilized antibody is indicated using a dotted line. The baseline response is indicated by a dotted line. F) Analysis of the negative control of WT egg white. G) Interpolation of absorbance response curves of immobilized antibody in 96-well microplates. The four-parameter logistic model was used for the nonlinear regression analysis. The baseline response is indicated by a dotted line. H) Comparison of interpolated log EC₅₀ values of immobilized antibodies in 96-well microplates. Symbols: KI-1 unpurified egg mAb (■), KI-2 unpurified egg mAb (▲), control mAb (●), and WT egg white (□). Each point represents the mean ± standard error of the mean (n = 4). HRP; Horseradish peroxidase, TMB; 3,3′,5,5′-tetramethylbenzidine, Abs; absorbance, cont mAb; control mAb, KI-1; KI-1 unpurified egg mAb, KI-2; KI-2 unpurified egg mAb, WT; WT egg white, n.s.; not significant.

At the dilution range of 10²–10⁶, the unpurified egg mAbs from KI-1 and KI-2 and the control mAb exhibited coloration (Fig. 1B). By contrast, the WT egg whites showed no coloration. Based on the IgG concentration (Table 1), each absorbance response (%) was plotted against the logarithmic concentration of IgG, and the half-maximal effective concentration (log EC₅₀) values were calculated (Figs. 1C–1F). The response curves of the unpurified egg mAbs were compared by fitting a linear regression analysis against the control mAb (Fig. 1G). No significant differences in log EC₅₀ values were found between the unpurified egg mAbs and the control mAb (Fig. 1H, Table 2). These results indicated that compared with the control mAb, the unpurified egg mAbs were immobilized in 96-well microplates.

Table 1. Concentrations of IgG in Unpurified Egg mAbs

	Concentration
Unpurified egg mAb from KI-1	1.589 mg/mL in egg white
Unpurified egg mAb from KI-2	0.995 mg/mL in egg white

Table 2. Comparison of Log EC₅₀ Values for Control mAb and Unpurified Egg mAbs Immobilized in 96-Well Microplates Using Tukey’s Multiple Comparison Test

Tukey’s multiple comparison test	Mean diff.	95.00% CI of diff.	Adjusted p-value
Control mAb (interpolated) vs. KI-1 (interpolated)	0.8791	−8.028 to 9.786	0.9697
Control mAb (interpolated) vs. KI-2 (interpolated)	1.302	−7.605 to 10.21	0.9348
KI-1 (interpolated) vs. KI-2 (interpolated)	0.4228	−8.484 to 9.330	0.9929

Antigen-Recognition Ability of Recombinant mAbs in Sandwich ELISA

We evaluated whether the unpurified egg mAbs immobilized in 96-well microplates can recognize antigens. A schematic of the sandwich ELISA procedure is shown in Fig. 2A. His-tagged HER2d4 was used as the model antigen to evaluate the antigen recognition ability in sandwich ELISA. When the immobilized antibody concentration of the control mAb reached 150 ng/mL, the color response plateaued (Fig. 1C). Therefore, 150 ng/mL was selected as the optimal concentration of the immobilized antibodies in this experiment. This optimal concentration corresponded to a 1 : 10000 dilution of KI-1 unpurified egg mAb. The diluted control mAb and unpurified egg mAbs were immobilized in 96-well microplates and blocked with 200 µL of 0.8 mg/mL Block Ace. A sandwich ELISA was performed to detect His-tagged HER2d4.

Fig. 2. Antigen Recognition Ability in Sandwich ELISA

A) Schematic of detection for the model antigen, His-tagged HER2d4, by immobilized egg mAb. B) Color development of His-tagged HER2d4 in a 96-well microplate. This panel shows visible differences in color development across serial dilutions of His-tagged HER2d4 in a sandwich ELISA using control mAb and unpurified egg mAbs. C) Absorbance response curves of the antigen recognition ability of control mAb and unpurified egg mAbs against His-tagged HER2d4. The four-parameter logistic model was used for the nonlinear regression analysis. The baseline response is indicated by a dotted line. Symbols: control mAb (●), unpurified egg mAb (KI-1) (■), and WT egg white (□). Each point represents the mean ± standard error of the mean (n = 4).

In the sandwich ELISA using the unpurified egg mAbs, coloration was observed at the His-tagged HER2d4 concentration range of 8–0.125 µg/mL in 96-well microplates, and it was comparable to that observed in the sandwich ELISA using the control mAb (Fig. 2B). By contrast, no coloration was exhibited by the WT egg whites 10000-fold diluted with PBS. The plot of the absorbance response (%) against the logarithmic concentration of His-tagged HER2d4 shows that the response curves of the unpurified egg mAbs were comparable to that of the control mAb (Fig. 2C). No significant difference in log EC₅₀ values was found between the unpurified egg mAbs and the control mAb (Table 3). These data indicated that the unpurified egg mAbs, when used as immobilized antibodies in sandwich ELISA, can recognize antigens comparable to the control mAb.

Table 3. Comparison of Log EC₅₀ Values for His-Tagged HER2d4 ECD Detected by Sandwich ELISA Using Control mAb and Unpurified Egg mAbs

Comparison of fits	95% CI log EC50 value profile likelihood	p-Value	F (DFn, DFd)
Control mAb	−6.157 to −5.684	0.3835	0.7714 (1, 56)
Unpurified egg mAbs	−6.179 to −5.963

Effect of Egg White Contaminants on Sandwich ELISA Sensitivity

To evaluate whether the presence of egg white contaminants with immobilized antibodies affects the sensitivity of sandwich ELISA, we compared the sensitivity of sandwich ELISAs using unpurified and purified egg mAbs.

In the sandwich ELISA using the unpurified egg mAbs, coloration was observed at the His-tagged HER2d4 concentration range of 8–0.125 µg/mL in 96-well microplates, and this result was comparable to that observed in the sandwich ELISA using the purified egg mAbs (Fig. 3A). By contrast, no coloration was exhibited by the WT egg whites 10000-fold diluted with PBS. The plot of the absorbance response (%) against the logarithmic concentration of His-tagged HER2d4 shows that the response curves of the unpurified egg mAbs were comparable to that of the purified egg mAbs (Fig. 3B). No significant difference in log EC₅₀ values was found between the unpurified and purified egg mAbs (Table 4).

Fig. 3. Effect of Egg White Contaminants on Sandwich ELISA Sensitivity

A) Color developments of His-tagged HER2d4 in a 96-well microplate using unpurified and purified egg mAbs. This panel shows visible differences in color development across serial dilutions of His-tagged HER2d4 in a sandwich ELISA. B) Absorbance response curves of His-tagged HER2d4 with sandwich ELISA using unpurified and purified egg mAbs. The four-parameter logistic model was used for the nonlinear regression analysis. The baseline response is indicated by a dotted line. C) Color developments of His-tagged HER2d4 in a 96-well microplate using control mAb with or without egg white contaminants. This panel shows visible differences in color development across serial dilutions of His-tagged HER2d4 in a sandwich ELISA. D) Absorbance response curves of His-tagged HER2d4 with sandwich ELISA control mAb with or without egg white contaminants. The four-parameter logistic model was used for the nonlinear regression analysis. The baseline response is indicated by a dotted line. Symbols: unpurified egg mAb (■), purified egg mAb (), control mAb (●), control mAb with egg white contaminants (), and WT (□). Each point represents mean ± standard error of the mean (n = 4).

Table 4. Comparison of Log EC₅₀ Values for His-Tagged HER2d4 Detected by Sandwich ELISA Using Unpurified and Purified Egg mAbs and Control mAb with or without Egg White Contaminants

Comparison of fits	95% CI log EC50 value profile likelihood	p-Value	F (DFn, DFd)
Unpurified egg mAbs	−6.254 to −6.063	0.3339	0.9566 (1, 40)
Purified mAbs	−6.349 to −6.108
Control mAb	−6.228 to −6.095	0.5439	0.3747 (1, 40)
Control mAb + WT	−6.275 to −6.101

Additionally, we compared the sensitivity of sandwich ELISAs using the control mAb with or without egg white contaminants. For this experiment, the control mAb was added to the WT egg white corresponding to the protein concentration of the 10000-fold-diluted WT egg white. The control mAb with or without egg white contaminants was immobilized in 96-well microplates, and a sandwich ELISA was performed to detect His-tagged HER2d4. Coloration was observed at the His-tagged HER2d4 concentration range of 8–0.125 µg/mL in 96-well microplates in sandwich ELISAs using the control mAb with or without egg white contaminants (Fig. 3C). The response curve of the control mAb without egg white contaminants was comparable to that of the control mAb with egg white contaminants (Fig. 3D). No significant difference in log EC₅₀ values was observed between the control mAb with or without egg white contaminants (Table 4). These results suggested that the sensitivity of sandwich ELISA was hardly affected by the presence of egg white contaminants in the immobilized antibodies.

LOD in Sandwich ELISA for Model Antigen of His-Tagged HER2d4

We also evaluated the LOD for low antigen concentrations in sandwich ELISA following the procedure described in Materials and Methods. In this experiment, transgenic egg whites containing the recombinant anti-HER2 mAbs and control mAb were adjusted to an IgG concentration of 150 ng/mL, immobilized in 96-well microplates, and blocked with 200 µL of 0.2 mg/mL Block Ace. Sandwich ELISA was performed to detect His-tagged HER2d4. Coloration was observed at the His-tagged HER2d4 concentration range of 1.875–120 ng/mL in sandwich ELISAs using unpurified egg mAbs (Fig. 4A). The response curve of the egg mAbs was plotted (Fig. 4B). No coloration was exhibited by the PBS and WT egg white. The WT egg white corresponded to the total protein concentration of the unpurified egg mAbs. Similarly, the coloration using control mAbs was observed under the same conditions (Fig. 4C). The response curve of the control mAbs was plotted (Fig. 4D). The response curve of the egg mAbs was comparable to that of the control mAb (Table 5). The LOD was calculated as 3.30 ng/mL for the unpurified egg mAbs and 9.85 ng/mL for the control mAb. These results suggested that the sandwich ELISA using the unpurified egg mAbs can detect low antigen concentrations with sensitivity comparable to that of the sandwich ELISA using the control mAb.

Fig. 4. Limit of Detection Sensitivity of Sandwich ELISA Using Unpurified Egg mAbs and Control mAb

A, C) Color developments of His-tagged HER2d4 in a 96-well microplate. This panel shows visible differences in color development across serial dilutions of His-tagged HER2d4 in a sandwich ELISA using immobilized antibodies from unpurified egg mAbs (A) and control mAb (C). B, D) Absorbance response of His-tagged HER2d4 with sandwich ELISA using unpurified egg mAbs (B) and control mAb (D). Linear regression analysis was used. R² quantifies the goodness of fit. The baseline response is indicated by a dotted line. Symbols: unpurified egg mAbs (■), control mAb (●), and WT or PBS (□). * means p < 0.05. Each point represents the mean ± standard error of the mean (n = 5).

Table 5. Comparison of Detection Sensitivity of His-Tagged HER2d4 by ELISA Using Unpurified Egg mAbs or Control mAb as Immobilized Antibodies

F test to compare variances	F, DFn, Dfd	p-Value
Control mAb vs. unpurified egg mAb	1.071, 100, 100	0.7330

Validation of Tg Egg-Derived Recombinant Antibodies for HER2 Detection in Sandwich ELISA

The main objective of this study was to demonstrate that egg mAbs can function as capture antibodies in a HER2 sandwich ELISA. Previous experiments have revealed that these antibodies recognize the extracellular HER2 domain 4. However, to validate their performance under conditions closer to practical use, we developed a detection system that uses the full-length extracellular domain of the HER2 protein (Fig. 5A). As detailed in Materials and Methods, we determined the optimal dilution for both primary and secondary antibodies in the HER2 sandwich ELISA (Supplementary Fig. 1).

Fig. 5. Validation of Egg mAbs in HER2 Sandwich ELISA

A) Schematic of HER2 sandwich ELISA using egg mAbs. B) Dose-dependent response of full-length HER2 ECD detection using immobilized egg mAbs. This panel shows visible differences in color development across serial dilutions of full-length HER2 ECD. C) Absorbance response of full-length HER2 ECD with sandwich ELISA using unpurified egg mAbs. A linear regression analysis was performed. R² quantifies the goodness of fit. The baseline response is indicated by the dotted line. D) Cross-reactivity assessment of the unpurified egg mAbs with HER1 and HER2 domain 1 (HER2d1). No significant color development was observed in the wells with each dilution of HER1 ECD and HER2d1. E) Detection of HER2 expression in lysates of BRL, HepG2, MCF7, and SKBR3 cells using immobilized egg mAbs. Color development was observed in wells with lysates from SKBR3 cells, whereas no significant color development was observed in the wells with lysates from BRL, HepG2, or MCF7 cells. F) Quantification of HER2 expression levels in HER1, HER2d1, BRL, HepG2, MCF7, and SKBR3 cells. Symbols: Unpurified egg mAbs (KI-1) (■). Each point represents mean ± standard error of the mean (n = 4).

First, we evaluated whether immobilized chicken-derived recombinant antibodies could recognize the full-length HER2 ECD antigen. Detection of the entire extracellular domain of the full-length HER2 protein using wells coated with immobilized egg-derived mAb showed a dose-dependent response (Fig. 5B). Moreover, the response was linear (Fig. 5C). The LOD was calculated as 3.55 ng/mL for the unpurified egg mAbs. These data indicate that the immobilized chicken-derived recombinant antibodies recognize the full-length HER2 protein.

Next, to assess whether the immobilized unpurified egg mAbs retained their antigen recognition ability after immobilization, we evaluated their cross-reactivity using the full-length extracellular domain proteins of HER1 and HER2d1. HER1 has a different structure from HER2, and HER2d1 is a partial structure of HER2 that does not include the trastuzumab antigen recognition site. ELISA using the unpurified egg mAbs did not detect these proteins (Fig. 5D). This result suggests that the unpurified egg mAbs retained their high selectivity and antigen recognition ability after immobilization.

Furthermore, to evaluate antibody performance under conditions closer to clinical applications, we analyzed HER2 expression in various cultured cells. The cells were prepared as described in Materials and Methods. The BRL, HepG2, MCF7, and SKBR3 cell lysates were used to detect HER2 expression. HER2 expression was not observed in the BRL, HepG2, or MCF7 cells. By contrast, HER2 was dose-dependently and linearly expressed in the SKBR3 cells (Fig. 5E, Supplementary Fig. 2). The expression levels of HER2 in the cell lysates were quantified (Fig. 5F). The results indicated that the assay can reliably detect HER2 expression levels in cell lysates with high specificity and sensitivity.

Application of Unpurified Egg mAbs for Paper-Based ELISA

Finally, to explore the broad applicability of the unpurified egg mAbs, we tested their use as immobilized antibodies in paper-based ELISA. A schematic of the preparation of the filter paper and antibody immobilization is displayed in Fig. 6A. The diluted transgenic egg whites and control mAb were immobilized on the detection area on the filter paper and blocked with 4 µL of 0.8 mg/mL Block Ace. The immobilized antibodies were detected using HRP-conjugated anti-human IgG. Signals were detected within the detection area (Fig. 6B). By contrast, no signal was exhibited by the WT egg whites 100-fold diluted in PBS. These data demonstrated that similar to the control mAb, the unpurified egg mAbs can be immobilized in the detection area.

Fig. 6. Application of Unpurified Egg mAbs as Immobilized Antibodies in Paper-Based ELISA

A) Schematic of preparation of filter paper in paper-based ELISA. B) Detection of immobilized antibodies in a filter paper. This panel shows immobilized antibodies from unpurified egg mAbs and control mAb in the filter paper are detected using HRP-conjugated anti-human IgG. C) Detection of His-tagged HER2d4 in paper-based ELISA. This panel shows the normalize visible response of His-tagged HER2d4.

Subsequently, a paper-based ELISA was performed using filter paper that immobilized the unpurified egg mAbs and control mAb to detect His-tagged HER2d4. Signals were observed at the His-tagged HER2d4 concentration range of 1–5 µg/mL in paper-based ELISAs using the unpurified egg mAbs and control mAb (Fig. 6C). By contrast, no signal was exhibited by the WT egg whites 100-fold diluted in PBS. These findings indicated that similar to the control mAb, the unpurified egg mAbs can be used as immobilized antibodies in paper-based ELISA.

DISCUSSION

In this study, we demonstrated that recombinant mAbs from transgenic chicken egg whites can be effectively used as immobilized antibodies in ELISA. The performance of the ELISA using unpurified egg mAbs was comparable to that of the ELISA using a highly purified commercially available mAb. Moreover, ELISA using egg-derived mAbs exhibited high specificity for HER2 detection. To the best of our knowledge, this study is the first to apply recombinant mAbs from transgenic eggs to ELISA.

In traditional ELISAs, mAbs are primarily produced by hybridoma cells.¹⁵⁾ However, this production method increases the cost of obtaining mAbs owing to the need for fetal bovine serum, cell culture equipment, and labor-intensive downstream processes such as isolation and purification.^16,17) These high mAb production costs inevitably increase the manufacturing costs of ELISA. Recently, plant and silkworm-based expression systems have been used as cost-effective production systems to obtain recombinant mAbs as immobilized antibodies in ELISA.^18–20) However, the production cost of ELISA may remain high with these systems because of the laborious extraction and purification required for plant seeds, leaves, and silkworm cocoons.^19–21) By contrast, our study showed that the recombinant mAbs secreted into transgenic egg whites can be used as immobilized antibodies for ELISA by simply dissolving them in PBS. This approach potentially eliminates the need for downstream processing, which is typically required to obtain immobilized antibodies. Therefore, our findings indicate that the use of unpurified egg mAbs provides a simple and cost-effective approach for supplying immobilized antibodies for ELISA.

In this experiment, we used recombinant mAbs from transgenic eggs containing approximately 1.5 mg/mL of mAbs (Table 1). These mAbs were used as immobilized antibodies by diluting them with PBS at a ratio of 1 : 10000 and adding 100 µL to each well of a 96-well microplate. The optimal dilution of the immobilized antibodies was determined based on the lowest concentration that was sufficient to saturate the 96-well microplate surface (Fig. 3). Approximately 30 mL of egg whites was obtained from each transgenic egg, indicating that one transgenic egg could provide sufficient immobilized antibodies to prepare 30000 plates for 96-well microplate ELISAs. Additionally, 60 ng per spot of unpurified egg mAbs from transgenic eggs can be effectively used as immobilized antibodies in paper-based ELISA (Fig. 6). Therefore, one transgenic egg could provide sufficient immobilized antibodies to prepare 750000 spots for paper-based ELISA (each measuring 5 × 5 mm, covering a total detection area of 18.75 m²). Transgenic chicken bioreactors can be used to cost-effectively produce recombinant mAbs.¹⁰⁾ The production cost of commercial eggs is approximately $1 per dozen,²²⁾ which may not be significantly different from that of transgenic eggs.²³⁾ Therefore, recombinant mAbs from transgenic eggs may be used as immobilized antibodies for ELISA at a low cost and on a large scale.

The use of unpurified ascites or serum in ELISA can significantly reduce assay performance.²⁴⁾ Nevertheless, the results of the present study showed that the performance of the ELISA using unpurified recombinant mAbs from transgenic eggs was comparable to that of the ELISA using a highly purified control mAb. Supporting our results, a recent study has demonstrated that scFv produced by transgenic silkworms can be used as immobilized antibodies in ELISA, even in the presence of host-derived silk contaminants.²⁵⁾ Additionally, recombinant mAbs produced by transgenic plants can be used to develop antigen-capturing devices with a performance comparable to that of purified mAbs, even when crudely purified.¹⁹⁾ Therefore, our findings indicated that the performance of ELISA was not significantly affected by the presence of contaminants in the immobilized antibodies from transgenic eggs. The simpler composition of egg whites than that of ascites or serum may contribute to the efficient performance of recombinant mAbs in ELISA. However, background levels in the ELISA using unpurified recombinant mAbs from transgenic eggs increased when detected by the avidin-biotin reaction (data not shown). This high background level may be attributed to the natural presence of avidin in egg whites.²⁶⁾ These observations indicated that the avidin-biotin reaction cannot be used in ELISA with recombinant mAbs from transgenic eggs. Alternatively, advances in transgenic technology have led to the development of knockout chickens.^27–31) Thus, the development of avidin-knockout chickens may allow biotin detection systems to be used in ELISA with recombinant mAbs from transgenic eggs.

In conclusion, unpurified recombinant mAbs from transgenic eggs can be effectively used as immobilized antibodies in a 96-well microplate or paper-based ELISA. However, a limitation of this study was the incomplete exploration of their detection capabilities in clinical biological samples. Nevertheless, our findings suggest that transgenic eggs can supply immobilized antibodies for ELISA at a low cost and in large quantities. In applications such as food safety testing and early detection of cancer recurrence, frequent testing with ELISA can become a significant economic burden.^4,32,33) Moreover, paper-based ELISA is a promising diagnostic system for point-of-care testing,^34,35) but it requires more immobilized antibodies per detection than the traditional 96-well plate ELISA.^36,37) Therefore, using recombinant mAbs from transgenic eggs may contribute to the development of economic ELISA devices and reduction of routine testing costs.

Acknowledgments

We thank the staff of Cosmo Bio Co., Ltd. for caring for the birds and providing fertilized eggs.

Author Contributions

Conceptualization: T.M. Investigation: T.M. Methodology: K.Y. and T.M. Project administration: T.M. Supervision: T.M. and I.O. Validation: I.O. Writing—original draft: T.M. Writing—review and editing: I.O. Funding acquisition: T.M. All authors have read and agreed to the published version of the manuscript.

Conflict of Interest

This work was supported by the KAKENHI program of the Japan Society for the Promotion of Science (Grant Number: 22K18236) and Mishima Kaiun Memorial Foundation.

Data Availability

The data presented in this study are available from the corresponding author upon request.

Supplementary Materials

This article contains supplementary materials.

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