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Preliminary studies of application of eggshell membrane as immobilization platform in sandwich immunoassay
Sensors and Actuators B 140 (2009) 200–205
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Preliminary studies of application of eggshell membrane as immobilization platform in sandwich immunoassay Jieli Tang, Juan Li, Jing Kang, Liangwei Zhong, Yihua Zhang ∗ College of Chemistry, Jilin University, Changchun 130012, PR China
a r t i c l e
i n f o
Article history: Received 13 January 2009 Received in revised form 2 April 2009 Accepted 4 April 2009 Available online 15 April 2009 Keywords: Eggshell membrane Human immunoglobulin G Fluorescence immunoassay Solid phase support
a b s t r a c t A novel immunoassay using the eggshell membrane as immobilization platform for determination of human IgG (HIgG) has been developed. The immunoassay was based on a sandwich immunoreaction of rabbit anti-human IgG (primary antibody), HIgG and the goat anti-human IgG labeled with FITC (secondary antibody). Due to well permeability and highly biocompatibility of eggshell membrane, a friendly microenvironment was constructed to prolong the life-times of the immobilized proteins. Under the optimal conditions, the fluorescence intensity is linear to the concentration of HIgG in a range of 20–100 ng mL−1 (r = 0.9918). The proposed method was successfully applied to the determination of the HIgG in the standard blood serum, and the results are satisfactory compared with that obtained by general immunonephelometric method. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Since the 1960s, when the first written work described chemical analysis using antibodies or binding proteins [1], many techniques have been reported to perform immunoassays. Immunoassays are among of the most important analytical methods for clinical diagnoses because of their extremely high sensitivity and specificity. A wide variety of immunoassay methods were reported concerning the choice of label, competition format, and procedure. And these immunoassay methods can be concluded to two basic types: homogeneous and heterogeneous assay. Homogeneous immunoassays do not require separation of unbound complexes from the bound complexes, and thus are faster and easier to perform than heterogeneous immunoassays. Those have been generally applied to the measurement of small analytes such as therapeutic drugs and hormones [2,3]. However, heterogeneous immunoassay requires an extra step to remove unbound antibody or antigen from the site, usually utilizing a solid phase support such as a magnetic particle [4,5], plastic bead [6,7], and nitrocellulose membrane [8–13]. Covalent crosslinking of antibodies on chemically-activated solid surfaces is the most common and stable method for antibody immobilization. Amino groups on the antibody surface can be readily coupled with several reactive moieties, for example, aldehyde, epoxy, and N-hydroxysuccinimide on various solid surfaces. Aldehyde- and epoxy-activated solid substrates have been widely employed as
∗ Corresponding author. Tel.: +86 431 85168352 6; fax: +86 431 85168420. E-mail address: [email protected] (Y. Zhang). 0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2009.04.003
ready-to-use supports for protein (antibody) and DNA immobilization. It has been reported that some biomaterials, such as silk [14–16], collagen [17,18], bamboo inner shell membrane [19] and eggshell membrane [20–24], were employed as platforms for the immobilization of proteins and the life-times of the immobilized proteins could be prolonged. Eggshell membrane of which the thickness is about 65–96 m [25] is a light pink double-layered membrane inside the eggshell. The membrane consists of the outer membrane layer, approximately 50 to 70 m thick, and the inner membrane, approximately 15 to 26 m thick. On the other hand, eggshell membrane consists of several discontinuous layers that were discernible as shifts in fiber position or orientation and changes in fiber size. Eggshell membrane is mainly composed of biological molecules and highly cross-linked protein fibers, and possesses excellent gas and water permeability [26]. The special performance of the eggshell membrane is due to its special composition and structure. It is reported that eggshell membrane is semipermeable, through which the small molecules can be selectively permeated, and the lager ones can not, such as glucose [27].As we know, eggshell membrane is a nontoxic and natural product, which is facile and inexpensive. And most importantly, it is easy to combine with many biomolecules, such as enzymes, proteins, antibodies. Therefore, eggshell membrane can be used as an ideal bio-platform for antibody immobilization, which is helpful for antibody to keep activity. Recently, the application of eggshell membrane has attracted considerable technological interest as its unique virtue. Choi and co-workers has reported eggshell membrane based biosensors wherein enzymes catalase, glucose oxidase, myrosinase, uricase, amino acid oxidase were immobilized using
J. Tang et al. / Sensors and Actuators B 140 (2009) 200–205
glutaraldehyde [28–30]. Furthermore, Li et al. immobilized glucose oxidase and horseradish peroxidase on the eggshell membrane to develop a new chemiluminescence flow-through biosensor for glucose [31]. In this paper, a novel solid phase immunoassay was explored based on the eggshell membrane as a solid support of the sandwich immunoassay. First, glutaraldehyde was added as cross-linking agent to react with the amino group in eggshell membrane. Then the primary antibody was cross-linked onto the surface of the modified fresh eggshell membrane. Subsequently, the antigen reacted with the primary antibody and then the labeled secondary antibody was bond to it. The proposed method was successfully applied to the determination of HIgG in standard serum, and the results were satisfactory compared with those obtained by immunonephelometric method [32]. The immunonephelometric method is based on determining the enhancement of the light scattering caused by suspended particles of the complex, which is formed in antigen–antibody reaction. 2. Experimental 2.1. Chemicals and reagents Human immunoglobulin G (HIgG) and goat anti-human IgG/FITC were purchased from Beijing biosynthesis Biotechnology Co.,Ltd. (Beijing, China). Rabbit anti-human IgG was obtained from Beijing DingGuo Biotechnology Co.,Ltd. (Beijing, China). Standard reference serum was purchased from National Center for Clinical Laboratory (NCCL, Beijing, China), which contains 12.03 g L−1 IgG, 1.61 g L−1 IgM, 1.60 g L−1 IgA, 1.21 g L−1 C3 and 0.27 g L−1 C4 . Glutaraldehyde solution (25%, w/w) in water was obtained from Beijing Chemical Reagent Corporation (Beijing, China). Ethanolamine was purchased from Tianjin Guangfu Fine Chemical Industry Institution (Tianjin, China). All chemicals of analytical-reagent grade were used as received and aqueous solutions were prepared with deionized water. The phosphate buffer solution (PBS) for preparing antibody and antigen was 10 mmol L−1 sodium phosphate solution at pH 7.4. Chicken eggs were provided from local markets. 2.2. Instrumentations The relative fluorescence intensity was measured with RF-5301PC fluorescence spectrophotometer (Shimadzu, Japan)
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Fig. 1. The schematic diagram of the experimental arrangement.
equipped with a xenon lamp using right-angle geometry. The fluorescence emission intensity at 520 nm was collected under an excitation wavelength of 495 nm. The pH value was measured with a Delta 320 pH meter. Unless otherwise stated, all fluorescence measurements were taken under room temperature and at atmospheric pressure. The eggshell membrane with sandwich complex was placed on the surface of the quartz plate, subsequently laid on the black cardboard, and then fixed firmly on the solid sample holder for fluorescence measurements. The diagram of the experimental arrangement is shown in Fig. 1. 2.3. Preparation of eggshell membrane The chemical composition (by weight) of eggshell has been reported as follows: calcium carbonate 94%, magnesium carbonate 1%, calcium phosphate 1% and organic matter 4% [33]. Calcium carbonate (main component) can dissolve in acetic acid, so eggs were dipped in 100% acetic acid at 4 ◦ C for 6 h to obtain whole eggshell membranes easily. The obtained eggshell membranes were cut into four equal parts and cleaned with a large amount of deionized water after removing the albumen and yolk. Further cleaning steps of the eggshell membrane were necessary in order to completely remove the albumen from the eggshell membrane [34]. The cleaned eggshell membrane was finally stored in PBS until further used.
Fig. 2. Scheme of the sandwich immunoassay immobilized on the eggshell membrane with glutaraldehyde as cross-linker.
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J. Tang et al. / Sensors and Actuators B 140 (2009) 200–205
2.4. Experimental procedure The fresh eggshell membrane (10 × 20 mm) was placed on a clean small watch glass. The rabbit anti-human IgG solution was dropped onto the surface of the eggshell membrane and kept for 20 min at 4 ◦ C for adsorption. Then glutaraldehyde solution was added as a cross-linking agent and spread evenly on the membrane surface with a small glass rod. The membrane was retained for 2 h at 4 ◦ C. The immobilized membrane was then rinsed with deionized water and immersed in PBS three times alternately to remove the excessive antibody and cross-linking reagent. Ethanolamine solution as a blocking regent was added and incubated. After washing, the HIgG solution was dropped onto the eggshell membrane and incubated at 37 ◦ C, and then goat anti-human IgG/FITC was added, then incubated, washed and stored in PBS for further detection. The experimental procedure for sandwich immunoassay immobilized on the eggshell membrane is shown in Fig. 2. 3. Results and discussion 3.1. SEM images of the eggshell membrane SEM images were obtained on JEOL JSM-6700F scanning electron microscope. To know the microstructure of the eggshell membrane with and without the immobilized antibodies, the eggshell membrane with and without the immobilized antibodies were prepared for scanning electron microscope. The membranes were placed on a clean silicon wafer, coated with a thin layer of gold using a spray gun and then observed on the scanning electron microscope. The surface morphologies of eggshell membrane with and without the immobilized antibody are displayed in Fig. 3. On the cleaned eggshell membrane (Fig. 3A) the highly cross-linked protein fibers (ca. diameter 1–5 m) and cavities which make the membrane possess excellent gas- and water-permeability are observed. In Fig. 3B, some specks or clusters of sandwich complex can be clearly seen on the protein fibers, but Fig. 3A does not show the attachment of sandwich complex on the protein fibers. From the SEM images cannot visualize the individual size of antibody, the immobilized antibodies can be observed as some condensed spots or clusters on fibers. The result obviously indicates that the supporting matrix for sandwich immunoassay is successfully produced on the eggshell membrane. 3.2. Fluorescence spectrum of the eggshell membrane
Fig. 3. SEMs of the cleaned eggshell membrane (A) and the eggshell membrane with sandwich complex (B). (1) Protein fiber; (2) Protein fiber immobilized with sandwich complex.
nearly retain initial fluorescence intensity excitated by 495 nm. It is indicated in Fig. 5 that the eggshell membrane can be kept for over a month at 4 ◦ C. The shelf-life of eggshell membrane is long enough to complete the whole experiment. The fact that the antibody bonded on eggshell membrane shows long term activity is not surprising, as the net-veined structure and the gas-permeable hydrophilic property of eggshell membrane can provide an excel-
For the successful immunoassay, the fluorescence spectra of fresh eggshell membrane, sandwich complex on membrane and solution of the goat anti-human IgG/FITC are shown in Fig. 4. It is found that the background interference of eggshell membrane is very low at the same excitation wavelength of FITC. As shown in Fig. 4, the emission peaks of the fluorescence probe of FITC are localized near 520 nm. It can also be seen that the peak of the sandwich complex is essentially consistent with that of FITC, which displays that the sandwich complex is coupled with the eggshell membrane, and the use of FITC as probe to measure the concentration of the detected antigen is practicable. 3.3. Performance comparison of fresh or stored eggshell membrane The performances of eggshell membrane obtained from different eggs were basically similar in intrinsic fluorescence with the excitation wavelength of 280 nm, so in this study the effect of different membranes can be ignored. The shelf-life of membrane with and without the immobilized antibody was tested periodically as shown in Fig. 5. After a period storage at 4 ◦ C, the membrane could
Fig. 4. Fluorescence spectra of fresh eggshell membrane, sandwich complex on membrane and the solution of goat anti-human IgG/FITC.
J. Tang et al. / Sensors and Actuators B 140 (2009) 200–205
Fig. 5. The shelf-life of eggshell membrane with and without the immobilized antibody.
lent biological micro-environment for the antibody to survive and maintain its activity. 3.4. Choice of the blocking reagents To achieve the better result of the sandwich, the effect of different blocking reagents, such as PBS, BSA-PBS and ethanolamine-PBS were examined. The results were indicated that compared with BSA-PBS, the ethanolamine-PBS utilized as blocking agent was better. Ethanolamine can block the excess binding sites, and the membrane sensor makes a good response to the analytes, which help enhancing the sensitivity of the immunoassay. It can be concluded that the ethanolamine-PBS was the best blocking reagent to avoid the cross-reactions of secondary antibodies and membrane.
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Fig. 6. Effect of glutaraldehyde concentration on the fluorescence intensity. Primary antibody, 1:8; HIgG, 100 ng mL−1 ; secondary antibody, 1:8. The error bars denote the standard deviation of the values with the three same assays.
The fluorescence intensity goes up considerably when the dilution ratio is not more than 1:8, and then rapidly dropped with more primary antibody. Since antigen–antibody reactions occur via noncovalent bonds, including hydrogen bonds, electrostatic bonds, and Van der Waals forces, they are by their nature reversible. When the quantity of the primary antibody is in excess of optimum ratio, the fluorescence intensity of the probe gradually decreases, because the complex of antigen–antibody dissociates reversely into the free antigen and antibody. As Fig. 7 depicted, the sandwich immunoassay showed the highest sensitivity when the dilution ratio of primary antibody is 1:8. Therefore, 1:8 was used as the optimal dilution ration of primary antibody for the sandwich immunoassay. 3.7. Effect of dilution ratio for secondary antibody
3.5. Effect of concentration of glutaraldehyde Covalent bonding can be used to achieve the immobilization of antibodies on the membrane matrix. These methods are based on the cross-linking reaction between the terminal functional groups of the biological molecules and reactive groups on the solid surface. In this system, the amino groups of the eggshell membrane and the antibody can react with glutaraldehyde. As a result, the concentration of glutaraldehyde is an important factor effecting experiment results. Fig. 6 displays the fluorescence intensity after adding the same amount of HIgG standard (100 ng mL−1 ) when the primary antibody was cross-linked upon the membrane by various concentrations of glutaraldehyde solutions. The result shows that the fluorescence intensity of the immuno-sandwich membrane enhances as the increase of glutaraldehyde concentration when glutaraldehyde is in the concentration range of 0.5–2.5% (w/w). But in the range of 2.5–7.5% (w/w) the fluorescence intensity of the membrane decreases appreciably. As a result, 10 L glutaraldehyde solution of 2.5% (w/w) was chosen as the optimum amount of cross-linking agent for the primary antibody immobilization on the membrane.
The effect of dilution ratio for the secondary antibody (initial concentration is 0.6 mg mL−1 ) was studied as shown in Fig. 8. The effect of dilution ratio for secondary antibody was tested in range of 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64 in PBS. It can be found that the fluorescence intensity is related to the dilution ratio of secondary antibody. As the increase of dilution ratio of secondary antibody, which means that the amount of formed sandwich
3.6. Effect of dilution ratio for primary antibody The effect of primary antibody concentration (0.5 mg mL−1 ) with the series dilution ratio of 1:1, 1:2, 1:4, 1:8, 1:32, 1:64 in PBS was investigated by adding 100 ng mL−1 HIgG standard onto the eggshell membrane. It is obvious in Fig. 7 that the fluorescence intensity of eggshell membrane has undergone dramatic change.
Fig. 7. Effect of dilution ratio for primary antibody on the fluorescence intensity. Glutaraldehyde, 2.5% (w/w); HIgG, 100 ng mL−1 ; secondary antibody, 1:8.
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J. Tang et al. / Sensors and Actuators B 140 (2009) 200–205 Table 1 The comparison of different methods to determine the HIgG in the standard reference serum.
Fig. 8. Effect of dilution ratio for secondary antibody on the fluorescence intensity. Glutaraldehyde, 2.5% (w/w); HIgG, 100 ng mL−1 ; primary antibody, 1:8.
complex increases, the fluorescence intensity could be enhanced. However, when the dilution ratio of secondary antibody is higher than 1:8, the fluorescence intensity decreased with further increase of the dilution ratio of secondary antibody as the dissolution of sandwich complex. The ratio between the antigen and antibody influences the detection of antigen–antibody complexes, because the size of the complexes formed is related to the concentration of the antigen and antibody. As a result, the dilution ratio of secondary antibody 1:8 was chosen for the further study. 3.8. Calibration curve for the HIgG Under the selected conditions mentioned above, the fluorescence intensity is in proportion to the concentration of HIgG and the linear range is from 20 to 100 ng mL−1 , the detection limit is 15 ng mL−1 . The regression equation is F = 214.458 + 1.064C (where F is fluorescence intensity and C is the HIgG concentration, ng mL−1 ) with a correlation coefficient of 0.9918 (n = 7). The corresponding calibration curve is shown in Fig. 9. 3.9. Analysis of the standard serum In order to test the feasibility of the proposed method, the standard reference serum was analyzed. The concentration of the HIgG
Methods
HIgG (g L−1 )
SD
The immunonephelometric method The proposed method Certified value
11.07 12.30 12.03
1.17 1.38 1.17
in the standard reference serum was 12.03 g L−1 . The standard reference serum sample was serially diluted with PBS to yield a testing sample solution. The HIgG in the testing sample was directly determined by the proposed method, which is depicted in Table 1. From Table 1, it can be seen that the content of HIgG in the sample determined by the proposed method was 12.30 g L−1 which is consistent with the standard value, and those foreign substances of sample had little effect on the determination of HIgG. In addition, comparison of the proposed method and the immunonephelometric method were also studied listed in Table 1. The result indicated that compared with immunonephelometric method, the proposed method was more accurate, and this technique may be applied in many types of antibody–antigen system. 4. Conclusion In this study, eggshell membrane sensor was successfully constructed and applied to determine the concentration of HIgG in standard serum. As advantages of eggshell membrane such as cheap and easily available, highly biocompatibility, well gas and water permeability, this method could provide a simple, high-specific immobilization process, and construct a friendly environment for keeping the activity of protein. A microstructure of the eggshell membrane is revealed and antibody immobilized on the eggshell membrane could be clearly seen by the scanning electron micrographs. In addition, a sandwich structure was constructed to improve the sensitivity of immunoassay and the detection limit of HIgG is 15 ng mL−1 . It can be concluded that eggshell membrane utilized as a new solid phase support in immunoassay has practical applications in biochemistry analysis. Acknowledgements The work described in this paper was partially supported by a grant from the Key Lab for Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, P.R. China. We are also thankful to the Second Hospital of Jilin University. References
Fig. 9. Standard curve for the determination of HIgG.
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Biographies Jieli Tang is currently a PhD student in College of Chemistry, Jilin University. Her current research is the construction of an immunosensor and its application in biochemistry analysis. Juan Li is currently a PhD student in College of Chemistry, Jilin University. Jing Kang is currently a graduate student in College of Chemistry, Jilin University. Liangwei Zhong is graduated from College of Chemistry, Jilin University in 2008. Yihua Zhang is a professor of College of Chemistry, Jilin University. She graduated from Jilin University in 1977. Her research area is spectrochemical analysis, included fluorescence immunoassay, resonance light-scattering, and chemiluminescence assay.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Preliminary studies of application of eggshell membrane as immobilization platform in sandwich immunoassay Jieli Tang, Juan Li, Jing Kang, Liangwei Zhong, Yihua Zhang ∗ College of Chemistry, Jilin University, Changchun 130012, PR China
a r t i c l e
i n f o
Article history: Received 13 January 2009 Received in revised form 2 April 2009 Accepted 4 April 2009 Available online 15 April 2009 Keywords: Eggshell membrane Human immunoglobulin G Fluorescence immunoassay Solid phase support
a b s t r a c t A novel immunoassay using the eggshell membrane as immobilization platform for determination of human IgG (HIgG) has been developed. The immunoassay was based on a sandwich immunoreaction of rabbit anti-human IgG (primary antibody), HIgG and the goat anti-human IgG labeled with FITC (secondary antibody). Due to well permeability and highly biocompatibility of eggshell membrane, a friendly microenvironment was constructed to prolong the life-times of the immobilized proteins. Under the optimal conditions, the fluorescence intensity is linear to the concentration of HIgG in a range of 20–100 ng mL−1 (r = 0.9918). The proposed method was successfully applied to the determination of the HIgG in the standard blood serum, and the results are satisfactory compared with that obtained by general immunonephelometric method. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Since the 1960s, when the first written work described chemical analysis using antibodies or binding proteins [1], many techniques have been reported to perform immunoassays. Immunoassays are among of the most important analytical methods for clinical diagnoses because of their extremely high sensitivity and specificity. A wide variety of immunoassay methods were reported concerning the choice of label, competition format, and procedure. And these immunoassay methods can be concluded to two basic types: homogeneous and heterogeneous assay. Homogeneous immunoassays do not require separation of unbound complexes from the bound complexes, and thus are faster and easier to perform than heterogeneous immunoassays. Those have been generally applied to the measurement of small analytes such as therapeutic drugs and hormones [2,3]. However, heterogeneous immunoassay requires an extra step to remove unbound antibody or antigen from the site, usually utilizing a solid phase support such as a magnetic particle [4,5], plastic bead [6,7], and nitrocellulose membrane [8–13]. Covalent crosslinking of antibodies on chemically-activated solid surfaces is the most common and stable method for antibody immobilization. Amino groups on the antibody surface can be readily coupled with several reactive moieties, for example, aldehyde, epoxy, and N-hydroxysuccinimide on various solid surfaces. Aldehyde- and epoxy-activated solid substrates have been widely employed as
∗ Corresponding author. Tel.: +86 431 85168352 6; fax: +86 431 85168420. E-mail address: [email protected] (Y. Zhang). 0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2009.04.003
ready-to-use supports for protein (antibody) and DNA immobilization. It has been reported that some biomaterials, such as silk [14–16], collagen [17,18], bamboo inner shell membrane [19] and eggshell membrane [20–24], were employed as platforms for the immobilization of proteins and the life-times of the immobilized proteins could be prolonged. Eggshell membrane of which the thickness is about 65–96 m [25] is a light pink double-layered membrane inside the eggshell. The membrane consists of the outer membrane layer, approximately 50 to 70 m thick, and the inner membrane, approximately 15 to 26 m thick. On the other hand, eggshell membrane consists of several discontinuous layers that were discernible as shifts in fiber position or orientation and changes in fiber size. Eggshell membrane is mainly composed of biological molecules and highly cross-linked protein fibers, and possesses excellent gas and water permeability [26]. The special performance of the eggshell membrane is due to its special composition and structure. It is reported that eggshell membrane is semipermeable, through which the small molecules can be selectively permeated, and the lager ones can not, such as glucose [27].As we know, eggshell membrane is a nontoxic and natural product, which is facile and inexpensive. And most importantly, it is easy to combine with many biomolecules, such as enzymes, proteins, antibodies. Therefore, eggshell membrane can be used as an ideal bio-platform for antibody immobilization, which is helpful for antibody to keep activity. Recently, the application of eggshell membrane has attracted considerable technological interest as its unique virtue. Choi and co-workers has reported eggshell membrane based biosensors wherein enzymes catalase, glucose oxidase, myrosinase, uricase, amino acid oxidase were immobilized using
J. Tang et al. / Sensors and Actuators B 140 (2009) 200–205
glutaraldehyde [28–30]. Furthermore, Li et al. immobilized glucose oxidase and horseradish peroxidase on the eggshell membrane to develop a new chemiluminescence flow-through biosensor for glucose [31]. In this paper, a novel solid phase immunoassay was explored based on the eggshell membrane as a solid support of the sandwich immunoassay. First, glutaraldehyde was added as cross-linking agent to react with the amino group in eggshell membrane. Then the primary antibody was cross-linked onto the surface of the modified fresh eggshell membrane. Subsequently, the antigen reacted with the primary antibody and then the labeled secondary antibody was bond to it. The proposed method was successfully applied to the determination of HIgG in standard serum, and the results were satisfactory compared with those obtained by immunonephelometric method [32]. The immunonephelometric method is based on determining the enhancement of the light scattering caused by suspended particles of the complex, which is formed in antigen–antibody reaction. 2. Experimental 2.1. Chemicals and reagents Human immunoglobulin G (HIgG) and goat anti-human IgG/FITC were purchased from Beijing biosynthesis Biotechnology Co.,Ltd. (Beijing, China). Rabbit anti-human IgG was obtained from Beijing DingGuo Biotechnology Co.,Ltd. (Beijing, China). Standard reference serum was purchased from National Center for Clinical Laboratory (NCCL, Beijing, China), which contains 12.03 g L−1 IgG, 1.61 g L−1 IgM, 1.60 g L−1 IgA, 1.21 g L−1 C3 and 0.27 g L−1 C4 . Glutaraldehyde solution (25%, w/w) in water was obtained from Beijing Chemical Reagent Corporation (Beijing, China). Ethanolamine was purchased from Tianjin Guangfu Fine Chemical Industry Institution (Tianjin, China). All chemicals of analytical-reagent grade were used as received and aqueous solutions were prepared with deionized water. The phosphate buffer solution (PBS) for preparing antibody and antigen was 10 mmol L−1 sodium phosphate solution at pH 7.4. Chicken eggs were provided from local markets. 2.2. Instrumentations The relative fluorescence intensity was measured with RF-5301PC fluorescence spectrophotometer (Shimadzu, Japan)
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Fig. 1. The schematic diagram of the experimental arrangement.
equipped with a xenon lamp using right-angle geometry. The fluorescence emission intensity at 520 nm was collected under an excitation wavelength of 495 nm. The pH value was measured with a Delta 320 pH meter. Unless otherwise stated, all fluorescence measurements were taken under room temperature and at atmospheric pressure. The eggshell membrane with sandwich complex was placed on the surface of the quartz plate, subsequently laid on the black cardboard, and then fixed firmly on the solid sample holder for fluorescence measurements. The diagram of the experimental arrangement is shown in Fig. 1. 2.3. Preparation of eggshell membrane The chemical composition (by weight) of eggshell has been reported as follows: calcium carbonate 94%, magnesium carbonate 1%, calcium phosphate 1% and organic matter 4% [33]. Calcium carbonate (main component) can dissolve in acetic acid, so eggs were dipped in 100% acetic acid at 4 ◦ C for 6 h to obtain whole eggshell membranes easily. The obtained eggshell membranes were cut into four equal parts and cleaned with a large amount of deionized water after removing the albumen and yolk. Further cleaning steps of the eggshell membrane were necessary in order to completely remove the albumen from the eggshell membrane [34]. The cleaned eggshell membrane was finally stored in PBS until further used.
Fig. 2. Scheme of the sandwich immunoassay immobilized on the eggshell membrane with glutaraldehyde as cross-linker.
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2.4. Experimental procedure The fresh eggshell membrane (10 × 20 mm) was placed on a clean small watch glass. The rabbit anti-human IgG solution was dropped onto the surface of the eggshell membrane and kept for 20 min at 4 ◦ C for adsorption. Then glutaraldehyde solution was added as a cross-linking agent and spread evenly on the membrane surface with a small glass rod. The membrane was retained for 2 h at 4 ◦ C. The immobilized membrane was then rinsed with deionized water and immersed in PBS three times alternately to remove the excessive antibody and cross-linking reagent. Ethanolamine solution as a blocking regent was added and incubated. After washing, the HIgG solution was dropped onto the eggshell membrane and incubated at 37 ◦ C, and then goat anti-human IgG/FITC was added, then incubated, washed and stored in PBS for further detection. The experimental procedure for sandwich immunoassay immobilized on the eggshell membrane is shown in Fig. 2. 3. Results and discussion 3.1. SEM images of the eggshell membrane SEM images were obtained on JEOL JSM-6700F scanning electron microscope. To know the microstructure of the eggshell membrane with and without the immobilized antibodies, the eggshell membrane with and without the immobilized antibodies were prepared for scanning electron microscope. The membranes were placed on a clean silicon wafer, coated with a thin layer of gold using a spray gun and then observed on the scanning electron microscope. The surface morphologies of eggshell membrane with and without the immobilized antibody are displayed in Fig. 3. On the cleaned eggshell membrane (Fig. 3A) the highly cross-linked protein fibers (ca. diameter 1–5 m) and cavities which make the membrane possess excellent gas- and water-permeability are observed. In Fig. 3B, some specks or clusters of sandwich complex can be clearly seen on the protein fibers, but Fig. 3A does not show the attachment of sandwich complex on the protein fibers. From the SEM images cannot visualize the individual size of antibody, the immobilized antibodies can be observed as some condensed spots or clusters on fibers. The result obviously indicates that the supporting matrix for sandwich immunoassay is successfully produced on the eggshell membrane. 3.2. Fluorescence spectrum of the eggshell membrane
Fig. 3. SEMs of the cleaned eggshell membrane (A) and the eggshell membrane with sandwich complex (B). (1) Protein fiber; (2) Protein fiber immobilized with sandwich complex.
nearly retain initial fluorescence intensity excitated by 495 nm. It is indicated in Fig. 5 that the eggshell membrane can be kept for over a month at 4 ◦ C. The shelf-life of eggshell membrane is long enough to complete the whole experiment. The fact that the antibody bonded on eggshell membrane shows long term activity is not surprising, as the net-veined structure and the gas-permeable hydrophilic property of eggshell membrane can provide an excel-
For the successful immunoassay, the fluorescence spectra of fresh eggshell membrane, sandwich complex on membrane and solution of the goat anti-human IgG/FITC are shown in Fig. 4. It is found that the background interference of eggshell membrane is very low at the same excitation wavelength of FITC. As shown in Fig. 4, the emission peaks of the fluorescence probe of FITC are localized near 520 nm. It can also be seen that the peak of the sandwich complex is essentially consistent with that of FITC, which displays that the sandwich complex is coupled with the eggshell membrane, and the use of FITC as probe to measure the concentration of the detected antigen is practicable. 3.3. Performance comparison of fresh or stored eggshell membrane The performances of eggshell membrane obtained from different eggs were basically similar in intrinsic fluorescence with the excitation wavelength of 280 nm, so in this study the effect of different membranes can be ignored. The shelf-life of membrane with and without the immobilized antibody was tested periodically as shown in Fig. 5. After a period storage at 4 ◦ C, the membrane could
Fig. 4. Fluorescence spectra of fresh eggshell membrane, sandwich complex on membrane and the solution of goat anti-human IgG/FITC.
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Fig. 5. The shelf-life of eggshell membrane with and without the immobilized antibody.
lent biological micro-environment for the antibody to survive and maintain its activity. 3.4. Choice of the blocking reagents To achieve the better result of the sandwich, the effect of different blocking reagents, such as PBS, BSA-PBS and ethanolamine-PBS were examined. The results were indicated that compared with BSA-PBS, the ethanolamine-PBS utilized as blocking agent was better. Ethanolamine can block the excess binding sites, and the membrane sensor makes a good response to the analytes, which help enhancing the sensitivity of the immunoassay. It can be concluded that the ethanolamine-PBS was the best blocking reagent to avoid the cross-reactions of secondary antibodies and membrane.
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Fig. 6. Effect of glutaraldehyde concentration on the fluorescence intensity. Primary antibody, 1:8; HIgG, 100 ng mL−1 ; secondary antibody, 1:8. The error bars denote the standard deviation of the values with the three same assays.
The fluorescence intensity goes up considerably when the dilution ratio is not more than 1:8, and then rapidly dropped with more primary antibody. Since antigen–antibody reactions occur via noncovalent bonds, including hydrogen bonds, electrostatic bonds, and Van der Waals forces, they are by their nature reversible. When the quantity of the primary antibody is in excess of optimum ratio, the fluorescence intensity of the probe gradually decreases, because the complex of antigen–antibody dissociates reversely into the free antigen and antibody. As Fig. 7 depicted, the sandwich immunoassay showed the highest sensitivity when the dilution ratio of primary antibody is 1:8. Therefore, 1:8 was used as the optimal dilution ration of primary antibody for the sandwich immunoassay. 3.7. Effect of dilution ratio for secondary antibody
3.5. Effect of concentration of glutaraldehyde Covalent bonding can be used to achieve the immobilization of antibodies on the membrane matrix. These methods are based on the cross-linking reaction between the terminal functional groups of the biological molecules and reactive groups on the solid surface. In this system, the amino groups of the eggshell membrane and the antibody can react with glutaraldehyde. As a result, the concentration of glutaraldehyde is an important factor effecting experiment results. Fig. 6 displays the fluorescence intensity after adding the same amount of HIgG standard (100 ng mL−1 ) when the primary antibody was cross-linked upon the membrane by various concentrations of glutaraldehyde solutions. The result shows that the fluorescence intensity of the immuno-sandwich membrane enhances as the increase of glutaraldehyde concentration when glutaraldehyde is in the concentration range of 0.5–2.5% (w/w). But in the range of 2.5–7.5% (w/w) the fluorescence intensity of the membrane decreases appreciably. As a result, 10 L glutaraldehyde solution of 2.5% (w/w) was chosen as the optimum amount of cross-linking agent for the primary antibody immobilization on the membrane.
The effect of dilution ratio for the secondary antibody (initial concentration is 0.6 mg mL−1 ) was studied as shown in Fig. 8. The effect of dilution ratio for secondary antibody was tested in range of 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64 in PBS. It can be found that the fluorescence intensity is related to the dilution ratio of secondary antibody. As the increase of dilution ratio of secondary antibody, which means that the amount of formed sandwich
3.6. Effect of dilution ratio for primary antibody The effect of primary antibody concentration (0.5 mg mL−1 ) with the series dilution ratio of 1:1, 1:2, 1:4, 1:8, 1:32, 1:64 in PBS was investigated by adding 100 ng mL−1 HIgG standard onto the eggshell membrane. It is obvious in Fig. 7 that the fluorescence intensity of eggshell membrane has undergone dramatic change.
Fig. 7. Effect of dilution ratio for primary antibody on the fluorescence intensity. Glutaraldehyde, 2.5% (w/w); HIgG, 100 ng mL−1 ; secondary antibody, 1:8.
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J. Tang et al. / Sensors and Actuators B 140 (2009) 200–205 Table 1 The comparison of different methods to determine the HIgG in the standard reference serum.
Fig. 8. Effect of dilution ratio for secondary antibody on the fluorescence intensity. Glutaraldehyde, 2.5% (w/w); HIgG, 100 ng mL−1 ; primary antibody, 1:8.
complex increases, the fluorescence intensity could be enhanced. However, when the dilution ratio of secondary antibody is higher than 1:8, the fluorescence intensity decreased with further increase of the dilution ratio of secondary antibody as the dissolution of sandwich complex. The ratio between the antigen and antibody influences the detection of antigen–antibody complexes, because the size of the complexes formed is related to the concentration of the antigen and antibody. As a result, the dilution ratio of secondary antibody 1:8 was chosen for the further study. 3.8. Calibration curve for the HIgG Under the selected conditions mentioned above, the fluorescence intensity is in proportion to the concentration of HIgG and the linear range is from 20 to 100 ng mL−1 , the detection limit is 15 ng mL−1 . The regression equation is F = 214.458 + 1.064C (where F is fluorescence intensity and C is the HIgG concentration, ng mL−1 ) with a correlation coefficient of 0.9918 (n = 7). The corresponding calibration curve is shown in Fig. 9. 3.9. Analysis of the standard serum In order to test the feasibility of the proposed method, the standard reference serum was analyzed. The concentration of the HIgG
Methods
HIgG (g L−1 )
SD
The immunonephelometric method The proposed method Certified value
11.07 12.30 12.03
1.17 1.38 1.17
in the standard reference serum was 12.03 g L−1 . The standard reference serum sample was serially diluted with PBS to yield a testing sample solution. The HIgG in the testing sample was directly determined by the proposed method, which is depicted in Table 1. From Table 1, it can be seen that the content of HIgG in the sample determined by the proposed method was 12.30 g L−1 which is consistent with the standard value, and those foreign substances of sample had little effect on the determination of HIgG. In addition, comparison of the proposed method and the immunonephelometric method were also studied listed in Table 1. The result indicated that compared with immunonephelometric method, the proposed method was more accurate, and this technique may be applied in many types of antibody–antigen system. 4. Conclusion In this study, eggshell membrane sensor was successfully constructed and applied to determine the concentration of HIgG in standard serum. As advantages of eggshell membrane such as cheap and easily available, highly biocompatibility, well gas and water permeability, this method could provide a simple, high-specific immobilization process, and construct a friendly environment for keeping the activity of protein. A microstructure of the eggshell membrane is revealed and antibody immobilized on the eggshell membrane could be clearly seen by the scanning electron micrographs. In addition, a sandwich structure was constructed to improve the sensitivity of immunoassay and the detection limit of HIgG is 15 ng mL−1 . It can be concluded that eggshell membrane utilized as a new solid phase support in immunoassay has practical applications in biochemistry analysis. Acknowledgements The work described in this paper was partially supported by a grant from the Key Lab for Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, P.R. China. We are also thankful to the Second Hospital of Jilin University. References
Fig. 9. Standard curve for the determination of HIgG.
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Biographies Jieli Tang is currently a PhD student in College of Chemistry, Jilin University. Her current research is the construction of an immunosensor and its application in biochemistry analysis. Juan Li is currently a PhD student in College of Chemistry, Jilin University. Jing Kang is currently a graduate student in College of Chemistry, Jilin University. Liangwei Zhong is graduated from College of Chemistry, Jilin University in 2008. Yihua Zhang is a professor of College of Chemistry, Jilin University. She graduated from Jilin University in 1977. Her research area is spectrochemical analysis, included fluorescence immunoassay, resonance light-scattering, and chemiluminescence assay.