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Resin-based micropipette tip for immunochromatographic assays in urine samples
Journal of Immunological Methods 312 (2006) 54 – 60 www.elsevier.com/locate/jim
Research paper
Resin-based micropipette tip for immunochromatographic assays in urine samples Teruko Yuhi a , Naoki Nagatani a , Tatsuro Endo b , Kagan Kerman b , Masayuki Takata c , Hiroyuki Konaka c , Mikio Namiki c , Yuzuru Takamura b , Eiichi Tamiya b,⁎ a
b
Japan Science and Technology Agency (JST) Innovation Plaza Ishikawa, 2-13 Asahidai, Nomi City, Ishikawa 923-1211, Japan School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City, Ishikawa 923-1292, Japan c Department of Integrative Cancer Therapy and Urology, Graduate School of Medical Sciences, Kanazawa University, 13-1 Takaramachi, Kanazawa City, Ishikawa 920-8641, Japan Received 16 August 2005; received in revised form 13 February 2006; accepted 15 February 2006 Available online 30 March 2006
Abstract A novel bioanalysis system based on immunochromatography was developed in connection with a nitrocellulose resin modified micropipette tip, such as ZipTip®. The sandwich-type immunoassay was applied to our bioanalysis system. The simple handling of the micropipette enabled us to increase the sample volume and detect low concentrations of target antigens in urine samples. In addition, the washing procedure could also be performed easily to reduce the background signal levels. For analytical evaluations, the color intensity was captured by a flatbed scanner, and processed by a software. We have achieved the detection of human chorionic gonadotropin (hCG) and prostate-specific antigen (PSA). The detection limit of hCG was 0.5 ng/ml (0.05 ng/tip), which is comparable to that of other conventional immunochromatographic systems. Moreover, the detection of PSA was greatly improved over the existing systems with the application of different sample volumes, such as 1 ng/ml (0.2 ng/tip) in a 200 μl sample volume, and 1 ng/ml (0.3 ng/tip) in 300 μl sample volume. Our bioanalysis system is a promising candidate for application to point-of-care tests with its simple handling and high sensitivity. © 2006 Elsevier B.V. All rights reserved. Keywords: Immunochromatography; Human chorionic gonadotropin (hCG); Prostate-specific antigen (PSA); Gold colloidal nanoparticles; Biosensor
1. Introduction
Abbreviations: hCG, human chorionic gonadotropin; Mab-hCG, monoclonal antibody to human chorionic gonadotropin; PSA, prostate specific antigen; TPSA, total prostate specific antigen; Mab-TPSA, monoclonal antibody to total prostate specific antigen; f-PSA, free prostate specific antigen; PSA/ACT, antichymotrypsin complexed with prostate specific antigen; Au naps, colloidal gold nanoparticles. ⁎ Corresponding author. Tel: +81 761 51 1660; fax: +81 761 51 1665. E-mail address: [email protected] (E. Tamiya). 0022-1759/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2006.02.011
In the past decade, numerous tests based on immunochromatographic assays have been introduced in the field of biomedical diagnostics, because they are simple to carry out, sensitive, cost-effective for mass fabrication, and, perhaps, most important, can be performed at the point-of-care (Price et al., 1997). The lateral flow-based sandwich immunoassay technique, with one of the antibodies immobilized in a thin line on a porous membrane, has been applied for many assays
T. Yuhi et al. / Journal of Immunological Methods 312 (2006) 54–60
(Ekins, 1983). The analyte is accumulated by the immobilized antibody in a thin detection zone and will thus after the reaction with the labeled antibody give a high density of label. Particles with a unique color, such as colloidal gold nanoparticles (Au naps), carbon black, and blue polystyrene have been used as labels that can be visually monitored. Quantum dot-biotin conjugates were used as labels for the detection of staphylococcal enterotoxin type-B with a detection limit of 10 ng/ml (Lingerfelt et al., 2003). Upconverting phosphor reporters were also applied in an immunochromatographic assay for the detection of human chorionic gonadotropin hormone (hCG) (Hampl et al., 2001). The quantitative detection of immunoglobulin E in the attomole range was achieved by using immunochromatography in connection with a flatbed scanner (Lönnberg and Carlsson, 2001). Several detection kits based on immunochromatography have become commercially available for the detection of the cancer marker, prostate-specific antigen (PSA) (An et al., 2001; Sato et al., 2002). PSA is an intracellular glycoprotein (34,000 Da kallikrein-like protease) synthesized only by the prostate gland. PSA is a normal constituent of prostatic tissue, and is also found in metastatic prostatic carcinoma, prostatic fluid, and seminal plasma (Knoll et al., 2002). PSA in serum exists both as free PSA (fPSA) and antichymotrypsin complexed with PSA (ACT-PSA) (Wesseling et al., 2003; Fernandez-Sanchez et al., 2004; Balk et al., 2003). Total PSA (TPSA) is defined as the combination of both fPSA and ACT-PSA. The cut-off limit of TPSA between prostate hyperplasia and cancer is 4 ng/ml (Oberpenning et al., 2003). The pregnancy marker, hCG, is a glycoprotein hormone produced at very high concentrations by placental trophoblasts. The glycoprotein hormone family also comprises luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). All members are heterodimers consisting of an α- and β-subunit. The α-subunit, which is common to all glycoprotein hormones, contains 92 amino acids. The molecular weight of hCG is about 37,500, that of hCGβ is 23,500 and that of hCGα is 14,000 (Pierce and Parsons, 1981; Birken et al., 2003). The β-subunit, a 132-amino acid sequence, is unique to hCG and specific tests for it are not subject to hormonal cross-reactivity (Robinson et al., 1985). A woman normally produces 25 milli-international units per milliliter (mIU/ml) of hCG 10 days after the conception. Generally, hCG levels doubles every 2 to 3 days after the conception. Accordingly, the concentration of hCG rises rapidly, frequently exceeding 100 mIU/ml by the first
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missed menstrual period and peaks in the range of 30,000–200,000 mIU/ml (∼30–200 nM) by 8– 10 weeks into pregnancy. An hCG level of less than 5 mIU/ml (∼10 pM) generally indicates that one is not pregnant. We have developed a simple bioanalysis system based on immunochromatography using a resin-modified micropipette tip called ZipTip®. We have investigated the effect of the size of Au naps on the immunochromatographic tests in our recent report (Yuhi et al., in press). We determined that Au naps at 40 nm were suitable for our assay. In this report, we have optimized and applied our method for the detection of the antigens in urine samples. 2. Materials and methods 2.1. Materials Monoclonal anti-human α-subunit of follicle-stimulating hormone (FSH) and monoclonal anti-human chorionic gonadotropin (Mab-hCG) were purchased from Medix Biochemica (Kauniainen, Finland). The recombinant human chorionic gonadotropin (hCG) as a measurement subject was purchased from Rohto Pharmaceutical Co., LTD. (Tokyo, Japan). Monoclonal anti-human total prostate specific antigen (Mab-TPSA) antibody 4D10 and N0.56 were purchased from Japan Clinical Laboratories, Inc. (Kyoto, Japan). The recombinant human total prostate specific antigen (PSA) was purchased from CosmoBio Japan. For fabrication of the bioanalysis system, disodium hydrogenphosphate (Na2HPO4), sodium dihydrogenphosphate dihydrate (NaH2PO4·2H2O), sucrose, polyethylene glycol (PEG), and potassium dihydrogenphosphate (KH2PO4) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Bovine serum albumin (BSA) for blocking of antibody immobilized nitrocellulose resin, which was filled into the ZipTip® edge, was purchased from Sigma Aldrich Japan (Tokyo, Japan). Sodium azide (NaN3) was purchased from Nakarai Tesque (Kyoto, Japan) for preserving the proteins in blocking and diluting solutions. Au naps with different diameters were purchased from Tanaka Kikinzoku Kogyo K.K. (Tokyo, Japan). ZipTip®MC was obtained from Nihon Millipore (Tokyo, Japan). 2.2. Apparatus After the antigen–antibody reaction occurs on ZipTip®, the color density images were scanned by EPSON EU-34 from Seiko Epson (Nagano, Japan). The
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scanned images were then converted into gray scale readings by Adobe™ Photoshop™ 5.5. The intensities of each signal were quantified with Image SMX vol. 1.74. At a random point of color intensity, measurements were taken for more than 8 times per 1 tip. The averages of the color intensities were calculated for each concentration of hCG and TPSA. 2.3. Methods 2.3.1. Immobilization of primary antibody onto nitrocellulose resin ZipTip® is attached to the micropipette like a disposable tip for aspirating and dispensing the solution. Monoclonal anti-human α-subunit of follicule stimulating hormone (Mab-hFSH) solution at 100 μg/ml was prepared by diluting with 50 mM phosphate buffered saline (PBS, pH 7.4). By using the micropipette, 10 μl of diluted antibody solution was aspirated and dispensed
for 10 times (ZipTip®MC: absorption capacity 400 ng/ tip) (Fig. 1A). For PSA detection experiment, 100 μg/ml of Mab-TPSA solution was prepared by diluting with PBS, and treated in the same way as described above. The immobilization of antibodies was carried out simply by this pipetting operation. Afterwards, antibody immobilized ZipTip® was kept on ice for 1 h. Blocking solution (1% (w/v) BSA and 5% (w/v) sucrose in PBS) at 100 μl was aspirated, and dispensed for 10 times to block the nonspecific adsorptions (Fig. 1B). Finally, antibody-immobilized and blocked ZipTip® was kept at 4 °C before use. 2.3.2. Preparation of Au naps conjugated Mab-hCG and antigen solutions Mab-hCG solution at 50 μg/ml was prepared by diluting with 5 mM KH2PO4 solution (pH 7.5) in ultra pure water (18.3 MΩ cm) to a final volume of 250 μl. The diluted Mab-hCG antibody solution at 200 μl was
Fig. 1. Illustration for the preparation procedures of the bioanalysis system and the detection principle. (A) Aspiration of the primary antibody, (B) aspiration of the blocking solution, (C) aspiration of the secondary antibodies conjugated with Au naps and the antigen, (D) sandwich-type immunoassay allows the capture of the target antigens by both Au naps-conjugated secondary antibodies and the primary antibodies immobilized on the resin. The accumulation of Au naps on the resin caused by the immuno-recognition process results in a color density change, which can be observed easily by the naked eye.
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The Au naps conjugated antibody solutions were applied to the antigen–antibody reaction. Different concentrations (0–100 ng/ml) of TPSA solutions were also used for the optimization of the recognition reactions.
Fig. 2. Image for the detection of hCG by using our bioanalysis system as described in Fig. 1. Red color appears darker as the concentration of hCG increases collecting more Au naps on the resin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2.3.4. Urine samples The urine samples were collected from 3 pregnant female and 2 non-pregnant female volunteers in our laboratory. Different concentrations (0, 2, 5, 10 ng/ml) of hCG spiked in non-pregnant female urine sample were prepared. Then, the urine samples were diluted 10 times with 1% (w/v) BSA in PBS. 3. Results 3.1. Detection of hCG
added in 1.8 ml of Au naps solution (0.01% w/v) and mixed immediately. The mixture was kept for 10 min at room temperature (RT) for the immobilization of antibody onto the Au naps surfaces. After the immobilization, 100 μl of 1% (w/v) PEG, which was dissolved in 50 mM KH2PO4 solution (pH 7.5) and 200 μl of 10% (w/v) BSA, which was also dissolved in 50 mM KH2PO4 solution (pH 9.0), were added for blocking the non-coated surfaces. After the immobilization and blocking procedures, Au naps conjugated Mab-hCG was obtained by centrifugal operation (8000×g for 15 min at 4 °C). The Au naps was pulse-sonicated for a few seconds, and was added to 2 ml of preserving solution (1% (w/v) BSA, 0.05% (w/v) PEG 20000, 0.1% (w/v) NaN3 and 150 mM NaCl that were dissolved in 20 mM Tris–HCl buffer, pH 8.2). After mixing, Au naps conjugated hCG antibody was collected by the same process as described above. After pulse-sonication, the Au naps conjugated Mab-hCG solution was diluted with the preserving solution to OD520 6. To compare the visual sensitivity, the Au naps with diameters of 15, 40 and 80 nm were selected. The hCG was diluted with 1% (w/v) BSA and 0.01% (w/v) NaN3 in PBS to 0–50 ng/ml. The fundamental ratio of Au naps-conjugated antibody solution to solution of hCG was 1:10 (100 μl/tip). Au naps-conjugated Mab-hCG solution and hCG were aspirated and dispensed slowly for 10 times (Fig. 1C). 2.3.3. Preparation of Au naps conjugated Mab-TPSA and antigen solution In this research, the Au naps conjugated Mab-TPSA solution was prepared by the same procedure of the preparation of the Au naps conjugated anti-hCG antibody solution. The concentration of Mab-TPSA solution used on this procedure was 65 μg/ml diluted with 5 mM KH2PO4 solution (pH 7.5) to a final volume of 250 μl.
The different diameters (15, 40 and 80 nm) of Au naps were used for the evaluation of the sensitivity for hCG. The color density increased with the increasing antigen concentration. We detected that the color density becomes higher, when the Au naps have a diameter of 40 nm (data not shown). From these results, we pursued further experiments by using 40 nm Au naps for the conjugation of Abs. (The results of hCG analysis using 40 nm diameter of Au naps are shown in Fig. 2.) The detection limit of our bioanalysis system at a concentration of 0.5 ng/ml (0.05 ng/tip) was higher than that of the commercially available test strips (Fig. 3). 3.2. Detection of TPSA We also applied our system for the detection of TPSA. The result of color density for TPSA is shown in Fig. 6. As seen in the case of hCG, the color density
Fig. 3. Color densities of each hCG concentration results. hCG was diluted by 50 mM PBS pH 7.4 containing 1% BSA to a desired final concentration. Mab-hCG was conjugated with 40 nm Au naps.
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Then, we tried to detect hCG in 3 pregnant and 2 nonpregnant female urine samples. The result of color density for urine samples is shown in Fig. 5. There was a significant difference in the color density results obtained with the urine samples from pregnant and non-pregnant female volunteers. 4. Discussion
This bioanalysis system was then applied to the detection of hCG spiked in urine samples. The Au naps at 40 nm were used for the conjugation. The detection results of hCG spiked into non-pregnant urine sample is shown in Fig. 4. Compared with the density result obtained with hCG spiked in 1% (w/v) BSA in PBS solution, we observed lower density values for hCG in the urine samples.
The sensitivity of the result is compared using gold colloids with different diameter in a case of hCG detection (data not shown). Au naps at 40 nm is also the preferred size used in the development of commercially available test strips. The result of hCG detection showed higher sensitivity compared with the conventional test strips. While the hCG is secreted in quantity in pregnant female (3 μg/ml), the detection of hCG is usually thought to be enough by an on/off format. In case of ectopic pregnancy, a report suggested the possibility of diagnosis by measuring lower level of hCG related protein (Lee et al., 2005). In such cases, lower detection limit would be required. The hCG detection result spiked in urine showed a little lower detection limit than that performed in the presence of 1% BSA in PBS solution (Fig. 4). However, there might be some hinder for the antigen–antibody reaction in urine; the hCG detection result showed clear difference between pregnant urine samples and non-pregnant urine samples. Surely, the affinities of the antibodies were different between Mab-hCG and Mab-TPSA (Fig. 6). Therefore, the sensitivity of our system was directly affected by their affinity levels. PSA exists in male serum at about 1 ng/ml, and the value of PSA increases along aging even in healthy male (Veltri et al., 2001). Clinically, PSA values 4–10 ng/ml is called as gray zone, which is suspected as a symptom of early prostate cancer (Jung et al., 2001). After all, PSA assay requires detection of low concentration antigen. Therefore, the detection limit of
Fig. 5. Color densities of 2 non-pregnant female and 3 pregnant female urine samples. Each urine samples were diluted by 50 mM PBS pH 7.4 containing 1% BSA to the desired final concentration.
Fig. 6. Color densities of several TPSA concentrations. TPSA was diluted by 50 mM PBS pH 7.4 containing 1% BSA to the desired final concentration. Mab-TPSA was conjugated with 40 nm Au naps.
Fig. 4. Color densities of each hCG concentration in urine sample. hCG was diluted by non-pregnant urine sample to the desired final concentration.
increased with the increasing antigen concentration. In TPSA detection, a significantly low detection limit is required. Our proposed bioanalysis system can be applied in such a situation by simply increasing the sample volume. The density results for different sample volumes at trace TPSA concentrations are compared in Fig. 7. We clearly observed that the detection ability significantly improved to 1 ng/ml (0.3 ng/tip), when the sample volume increased until 300 μl. 3.3. Detection of hCG in urine samples
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Research in our laboratory is in progress towards the detection of other biomolecules, such as DNA and enzymes. Furthermore, the use of enzymatic signal amplification and fluorescent dyes would increase the sensitivity and selectivity of our system. Acknowledgements
Fig. 7. Comparison of color densities for each TPSA concentration while increasing the sample volume. The density result for TPSA was improved, when the sample volume was increased up to 300 μl.
PSA is pursued (Harma et al., 2001). From these reasons, the sample volume was increased in order to be able to detect PSA under 5 ng/ml concentration (Fig. 7). By increasing the sample volumes, a visible increase in the color density was observed with 5 ng/ml PSA. In our system, it is quite easy to increase the sample volumes, in contrast to using conventional test strips. It is noteworthy that there was only a negligible change in the background density level, even when the sample volume was increased significantly. The detection limit of our bioanalysis system at 1 ng/ml was comparable with that of the conventional test strips. The advantage of our bioanalysis system is the short fabrication time. According to the protocol of ZipTip®, the whole fabrication procedure involves the adsorption of the primary antibodies on the resin inside of ZipTip®, which takes place rapidly by a simple pipetting. Additionally, one ZipTip® requires only 10 μl of 100 μg/ml of antibody solution for immobilization onto the resin surface. Thus, the sample volume can also be modified, since ZipTip® is attachable to a micropipette. In the case of measurements with low concentration solutions like environmental and/or clinical samples, aspiration of a high volume of the sample solution would make visual judgment easy. Furthermore, the washing procedure can also be performed by pipetting. By using other types of ZipTip® with less resin filled inside, we are planning to detect antigens in less volume of sample solutions. 5. Conclusions This study demonstrates the applicability of our immunochromatographic assay tip (ZipTip®) for antigen detection in real samples. This novel bioanalysis system is easily applicable in clinical diagnosis, environmental measurements, and forensic research.
This works was funded by Japan Science and Technology Agency (JST). We are deeply grateful to Nihon Millipore, Tokyo, Japan for technical suggestions. We would like to thank Japan Clinical Laboratories, Inc., Kyoto, Japan for providing the antibody against TPSA used in this study. We would also like to express our gratitude to Tanaka Kikinnzoku Kogyo K. K., Tokyo, Japan for providing the colloidal gold nanoparticles. References An, C.D., Yoshiki, T., Lee, G., Okada, Y., 2001. Evaluation of a rapid qualitative prostate specific antigen assay, the One Step PSA™ test. Cancer Lett. 162, 135. Balk, P.S., Ko, Y., Burbley, G., 2003. Biology of prostate-specific antigen. J. Clin. Oncol. 21, 383. Birken, S., Berger, P., Bidart, J.M., Weber, M., Bristow, A., Norman, R., 2003. Preparation and characterization of new WHO reference reagents for human chorionic gonadotropin and metabolites. Clin. Chem. 49, 144. Ekins, R.P., 1983. In: Hunter, W.M., Corrie, J.E.T. (Eds.), Immunoassays for Clinical Chemistry. Churchill Livingstone, Edinburgh, p. 76. Fernandez-Sanchez, C., McNeil, J.C., Rawson, K., Nilsson, O., 2004. Disposable noncompetitive immunosensor for free and total prostate-specific antigen based on capacitance measurement. Anal. Chem. 76, 5649. Hampl, J., Hall, M., Mufti, N.A., Yao, Y.-M.M., MacQueen, D.B, Wright, W.H., Cooper, D.E., 2001. Upconverting phosphor reporters in immunochromatographic assays. Anal. Biochem. 288, 176. Harma, H., Soukka, T., Lovgren, T., 2001. Europium nanoparticles and time-resolved fluorescence for ultrasensitive detection of prostatespecific antigen. Clin. Chem. 47, 561. Jung, K., Stephan, C., Elgeti, U., Lein, M., Brux, B., Kristiansen, G., Rudolph, B., Hauptmann, S., Schnorr, D., Loening, A.S., Sinha, P., 2001. Molecular forms of prostate-specific antigen in serum with concentrations of total prostate-specific antigen <4 mg/L: are they useful tools for early detection and screening of prostate cancer? Int. J. Cancer 93, 759. Knoll, S., Vogel, R.F., Niessen, L., 2002. Identification of fusarium graminearum in cereal samples by DNA detection Test Strips™. Lett. Appl. Microbiol. 34, 144. Lee, J., Oh, M., Shin, J., Lee, K., Nam, J., Cha, J., Chang, J., Cho, D., Kang, I., Lee, I.P., 2005. Clinical effectiveness of urinary human chorionic gonadotropin related protein (hCGRP) quantification for diagnosis of ectopic pregnancy. J. Korean Med. Sci. 20, 461. Lingerfelt, B.M., Mattoussi, H., Goldman, E.R., Mauro, J.M., Anderson, G.P., 2003. Preparation of quantum dot-biotin
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conjugates and their use in immunochromatographic assays. Anal. Chem. 75, 4043. Lönnberg, M., Carlsson, J., 2001. Quantitative detection in the attomole range for immunochromatographic tests by means of a flatbed scanner. Anal. Biochem. 293, 224. Oberpenning, F., Hetzel, S., Weining, C., Brandt, B., Angelis, A.G., Heinecke, A., Lein, M., Fornara, P., Schmid, H., Hertle, L., Semjonow, A., 2003. Semi-quantitative immunochromatographic test for prostate specific antigen in whole blood: tossing the coin to predict prostate cancer? Eur. Urol. 43, 478. Pierce, J.G., Parsons, T.F., 1981. Glycoprotein hormones: structure and function. Annu. Rev. Biochem. 50, 465. Price, C.P., Thorpe, G.H.G., Hall, J., Bunce, R.A., 1997. In: Price, C. P., Newman, D.J. (Eds.), Principles and Practice of Immunoassay, 2nd ed. Macmillan, London, p. 580. Robinson, G.A., Hill, H.A., Philo, R.D., Gear, J.M., Rattle, S.J., Forrest, G.C., 1985. Bioelectrochemical enzyme immunoassay of human choriogonadotropin with magnetic electrodes. Clin. Chem. 31, 1449. Sato, I., Sagi, M., Ishiwari, A., Nishijima, H., Ito, E., Mukai, T., 2002. Use of the “SMITEST” PSA card to identify the presence of
prostate-specific antigen in semen and male urine. Forensic Sci. Int. 127, 71. Veltri, W.R., Miller, C., O'dowd, J.G., Partin, W.A., 2001. Impact of age on total and complexed prostate-specific antigen cutoff in a contemporary referral series of men with prostate cancer. Urology 60, 47. Wesseling, S., Stephan, C., Semjonow, A., Lein, M., Brux, B., Sinha, P., Loering, A.S., Jung, K., 2003. Determination of non-aantichymotrypsin-complexed prostate-specific antigen as an indirect measurement of free prostate-specific antigen: analytical performance and diagnostic accuracy. Clin. Chem. 49, 887. Yuhi, T., Nagatani, N., Endo, T., Kerman, K., Takata, M., Konaka, H., Namiki, M., Takamura, Y., Tamiya, E., in press. Gold nanoparticle based immunochromatography using a resin modified micropipette tip for rapid and simple detection of human chorionic gonadotropin hormone and prostate-specific antigen. Sci. Tech. Adv. Mater. (Available online 2 March 2006).
Research paper
Resin-based micropipette tip for immunochromatographic assays in urine samples Teruko Yuhi a , Naoki Nagatani a , Tatsuro Endo b , Kagan Kerman b , Masayuki Takata c , Hiroyuki Konaka c , Mikio Namiki c , Yuzuru Takamura b , Eiichi Tamiya b,⁎ a
b
Japan Science and Technology Agency (JST) Innovation Plaza Ishikawa, 2-13 Asahidai, Nomi City, Ishikawa 923-1211, Japan School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City, Ishikawa 923-1292, Japan c Department of Integrative Cancer Therapy and Urology, Graduate School of Medical Sciences, Kanazawa University, 13-1 Takaramachi, Kanazawa City, Ishikawa 920-8641, Japan Received 16 August 2005; received in revised form 13 February 2006; accepted 15 February 2006 Available online 30 March 2006
Abstract A novel bioanalysis system based on immunochromatography was developed in connection with a nitrocellulose resin modified micropipette tip, such as ZipTip®. The sandwich-type immunoassay was applied to our bioanalysis system. The simple handling of the micropipette enabled us to increase the sample volume and detect low concentrations of target antigens in urine samples. In addition, the washing procedure could also be performed easily to reduce the background signal levels. For analytical evaluations, the color intensity was captured by a flatbed scanner, and processed by a software. We have achieved the detection of human chorionic gonadotropin (hCG) and prostate-specific antigen (PSA). The detection limit of hCG was 0.5 ng/ml (0.05 ng/tip), which is comparable to that of other conventional immunochromatographic systems. Moreover, the detection of PSA was greatly improved over the existing systems with the application of different sample volumes, such as 1 ng/ml (0.2 ng/tip) in a 200 μl sample volume, and 1 ng/ml (0.3 ng/tip) in 300 μl sample volume. Our bioanalysis system is a promising candidate for application to point-of-care tests with its simple handling and high sensitivity. © 2006 Elsevier B.V. All rights reserved. Keywords: Immunochromatography; Human chorionic gonadotropin (hCG); Prostate-specific antigen (PSA); Gold colloidal nanoparticles; Biosensor
1. Introduction
Abbreviations: hCG, human chorionic gonadotropin; Mab-hCG, monoclonal antibody to human chorionic gonadotropin; PSA, prostate specific antigen; TPSA, total prostate specific antigen; Mab-TPSA, monoclonal antibody to total prostate specific antigen; f-PSA, free prostate specific antigen; PSA/ACT, antichymotrypsin complexed with prostate specific antigen; Au naps, colloidal gold nanoparticles. ⁎ Corresponding author. Tel: +81 761 51 1660; fax: +81 761 51 1665. E-mail address: [email protected] (E. Tamiya). 0022-1759/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2006.02.011
In the past decade, numerous tests based on immunochromatographic assays have been introduced in the field of biomedical diagnostics, because they are simple to carry out, sensitive, cost-effective for mass fabrication, and, perhaps, most important, can be performed at the point-of-care (Price et al., 1997). The lateral flow-based sandwich immunoassay technique, with one of the antibodies immobilized in a thin line on a porous membrane, has been applied for many assays
T. Yuhi et al. / Journal of Immunological Methods 312 (2006) 54–60
(Ekins, 1983). The analyte is accumulated by the immobilized antibody in a thin detection zone and will thus after the reaction with the labeled antibody give a high density of label. Particles with a unique color, such as colloidal gold nanoparticles (Au naps), carbon black, and blue polystyrene have been used as labels that can be visually monitored. Quantum dot-biotin conjugates were used as labels for the detection of staphylococcal enterotoxin type-B with a detection limit of 10 ng/ml (Lingerfelt et al., 2003). Upconverting phosphor reporters were also applied in an immunochromatographic assay for the detection of human chorionic gonadotropin hormone (hCG) (Hampl et al., 2001). The quantitative detection of immunoglobulin E in the attomole range was achieved by using immunochromatography in connection with a flatbed scanner (Lönnberg and Carlsson, 2001). Several detection kits based on immunochromatography have become commercially available for the detection of the cancer marker, prostate-specific antigen (PSA) (An et al., 2001; Sato et al., 2002). PSA is an intracellular glycoprotein (34,000 Da kallikrein-like protease) synthesized only by the prostate gland. PSA is a normal constituent of prostatic tissue, and is also found in metastatic prostatic carcinoma, prostatic fluid, and seminal plasma (Knoll et al., 2002). PSA in serum exists both as free PSA (fPSA) and antichymotrypsin complexed with PSA (ACT-PSA) (Wesseling et al., 2003; Fernandez-Sanchez et al., 2004; Balk et al., 2003). Total PSA (TPSA) is defined as the combination of both fPSA and ACT-PSA. The cut-off limit of TPSA between prostate hyperplasia and cancer is 4 ng/ml (Oberpenning et al., 2003). The pregnancy marker, hCG, is a glycoprotein hormone produced at very high concentrations by placental trophoblasts. The glycoprotein hormone family also comprises luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). All members are heterodimers consisting of an α- and β-subunit. The α-subunit, which is common to all glycoprotein hormones, contains 92 amino acids. The molecular weight of hCG is about 37,500, that of hCGβ is 23,500 and that of hCGα is 14,000 (Pierce and Parsons, 1981; Birken et al., 2003). The β-subunit, a 132-amino acid sequence, is unique to hCG and specific tests for it are not subject to hormonal cross-reactivity (Robinson et al., 1985). A woman normally produces 25 milli-international units per milliliter (mIU/ml) of hCG 10 days after the conception. Generally, hCG levels doubles every 2 to 3 days after the conception. Accordingly, the concentration of hCG rises rapidly, frequently exceeding 100 mIU/ml by the first
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missed menstrual period and peaks in the range of 30,000–200,000 mIU/ml (∼30–200 nM) by 8– 10 weeks into pregnancy. An hCG level of less than 5 mIU/ml (∼10 pM) generally indicates that one is not pregnant. We have developed a simple bioanalysis system based on immunochromatography using a resin-modified micropipette tip called ZipTip®. We have investigated the effect of the size of Au naps on the immunochromatographic tests in our recent report (Yuhi et al., in press). We determined that Au naps at 40 nm were suitable for our assay. In this report, we have optimized and applied our method for the detection of the antigens in urine samples. 2. Materials and methods 2.1. Materials Monoclonal anti-human α-subunit of follicle-stimulating hormone (FSH) and monoclonal anti-human chorionic gonadotropin (Mab-hCG) were purchased from Medix Biochemica (Kauniainen, Finland). The recombinant human chorionic gonadotropin (hCG) as a measurement subject was purchased from Rohto Pharmaceutical Co., LTD. (Tokyo, Japan). Monoclonal anti-human total prostate specific antigen (Mab-TPSA) antibody 4D10 and N0.56 were purchased from Japan Clinical Laboratories, Inc. (Kyoto, Japan). The recombinant human total prostate specific antigen (PSA) was purchased from CosmoBio Japan. For fabrication of the bioanalysis system, disodium hydrogenphosphate (Na2HPO4), sodium dihydrogenphosphate dihydrate (NaH2PO4·2H2O), sucrose, polyethylene glycol (PEG), and potassium dihydrogenphosphate (KH2PO4) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Bovine serum albumin (BSA) for blocking of antibody immobilized nitrocellulose resin, which was filled into the ZipTip® edge, was purchased from Sigma Aldrich Japan (Tokyo, Japan). Sodium azide (NaN3) was purchased from Nakarai Tesque (Kyoto, Japan) for preserving the proteins in blocking and diluting solutions. Au naps with different diameters were purchased from Tanaka Kikinzoku Kogyo K.K. (Tokyo, Japan). ZipTip®MC was obtained from Nihon Millipore (Tokyo, Japan). 2.2. Apparatus After the antigen–antibody reaction occurs on ZipTip®, the color density images were scanned by EPSON EU-34 from Seiko Epson (Nagano, Japan). The
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scanned images were then converted into gray scale readings by Adobe™ Photoshop™ 5.5. The intensities of each signal were quantified with Image SMX vol. 1.74. At a random point of color intensity, measurements were taken for more than 8 times per 1 tip. The averages of the color intensities were calculated for each concentration of hCG and TPSA. 2.3. Methods 2.3.1. Immobilization of primary antibody onto nitrocellulose resin ZipTip® is attached to the micropipette like a disposable tip for aspirating and dispensing the solution. Monoclonal anti-human α-subunit of follicule stimulating hormone (Mab-hFSH) solution at 100 μg/ml was prepared by diluting with 50 mM phosphate buffered saline (PBS, pH 7.4). By using the micropipette, 10 μl of diluted antibody solution was aspirated and dispensed
for 10 times (ZipTip®MC: absorption capacity 400 ng/ tip) (Fig. 1A). For PSA detection experiment, 100 μg/ml of Mab-TPSA solution was prepared by diluting with PBS, and treated in the same way as described above. The immobilization of antibodies was carried out simply by this pipetting operation. Afterwards, antibody immobilized ZipTip® was kept on ice for 1 h. Blocking solution (1% (w/v) BSA and 5% (w/v) sucrose in PBS) at 100 μl was aspirated, and dispensed for 10 times to block the nonspecific adsorptions (Fig. 1B). Finally, antibody-immobilized and blocked ZipTip® was kept at 4 °C before use. 2.3.2. Preparation of Au naps conjugated Mab-hCG and antigen solutions Mab-hCG solution at 50 μg/ml was prepared by diluting with 5 mM KH2PO4 solution (pH 7.5) in ultra pure water (18.3 MΩ cm) to a final volume of 250 μl. The diluted Mab-hCG antibody solution at 200 μl was
Fig. 1. Illustration for the preparation procedures of the bioanalysis system and the detection principle. (A) Aspiration of the primary antibody, (B) aspiration of the blocking solution, (C) aspiration of the secondary antibodies conjugated with Au naps and the antigen, (D) sandwich-type immunoassay allows the capture of the target antigens by both Au naps-conjugated secondary antibodies and the primary antibodies immobilized on the resin. The accumulation of Au naps on the resin caused by the immuno-recognition process results in a color density change, which can be observed easily by the naked eye.
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The Au naps conjugated antibody solutions were applied to the antigen–antibody reaction. Different concentrations (0–100 ng/ml) of TPSA solutions were also used for the optimization of the recognition reactions.
Fig. 2. Image for the detection of hCG by using our bioanalysis system as described in Fig. 1. Red color appears darker as the concentration of hCG increases collecting more Au naps on the resin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2.3.4. Urine samples The urine samples were collected from 3 pregnant female and 2 non-pregnant female volunteers in our laboratory. Different concentrations (0, 2, 5, 10 ng/ml) of hCG spiked in non-pregnant female urine sample were prepared. Then, the urine samples were diluted 10 times with 1% (w/v) BSA in PBS. 3. Results 3.1. Detection of hCG
added in 1.8 ml of Au naps solution (0.01% w/v) and mixed immediately. The mixture was kept for 10 min at room temperature (RT) for the immobilization of antibody onto the Au naps surfaces. After the immobilization, 100 μl of 1% (w/v) PEG, which was dissolved in 50 mM KH2PO4 solution (pH 7.5) and 200 μl of 10% (w/v) BSA, which was also dissolved in 50 mM KH2PO4 solution (pH 9.0), were added for blocking the non-coated surfaces. After the immobilization and blocking procedures, Au naps conjugated Mab-hCG was obtained by centrifugal operation (8000×g for 15 min at 4 °C). The Au naps was pulse-sonicated for a few seconds, and was added to 2 ml of preserving solution (1% (w/v) BSA, 0.05% (w/v) PEG 20000, 0.1% (w/v) NaN3 and 150 mM NaCl that were dissolved in 20 mM Tris–HCl buffer, pH 8.2). After mixing, Au naps conjugated hCG antibody was collected by the same process as described above. After pulse-sonication, the Au naps conjugated Mab-hCG solution was diluted with the preserving solution to OD520 6. To compare the visual sensitivity, the Au naps with diameters of 15, 40 and 80 nm were selected. The hCG was diluted with 1% (w/v) BSA and 0.01% (w/v) NaN3 in PBS to 0–50 ng/ml. The fundamental ratio of Au naps-conjugated antibody solution to solution of hCG was 1:10 (100 μl/tip). Au naps-conjugated Mab-hCG solution and hCG were aspirated and dispensed slowly for 10 times (Fig. 1C). 2.3.3. Preparation of Au naps conjugated Mab-TPSA and antigen solution In this research, the Au naps conjugated Mab-TPSA solution was prepared by the same procedure of the preparation of the Au naps conjugated anti-hCG antibody solution. The concentration of Mab-TPSA solution used on this procedure was 65 μg/ml diluted with 5 mM KH2PO4 solution (pH 7.5) to a final volume of 250 μl.
The different diameters (15, 40 and 80 nm) of Au naps were used for the evaluation of the sensitivity for hCG. The color density increased with the increasing antigen concentration. We detected that the color density becomes higher, when the Au naps have a diameter of 40 nm (data not shown). From these results, we pursued further experiments by using 40 nm Au naps for the conjugation of Abs. (The results of hCG analysis using 40 nm diameter of Au naps are shown in Fig. 2.) The detection limit of our bioanalysis system at a concentration of 0.5 ng/ml (0.05 ng/tip) was higher than that of the commercially available test strips (Fig. 3). 3.2. Detection of TPSA We also applied our system for the detection of TPSA. The result of color density for TPSA is shown in Fig. 6. As seen in the case of hCG, the color density
Fig. 3. Color densities of each hCG concentration results. hCG was diluted by 50 mM PBS pH 7.4 containing 1% BSA to a desired final concentration. Mab-hCG was conjugated with 40 nm Au naps.
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Then, we tried to detect hCG in 3 pregnant and 2 nonpregnant female urine samples. The result of color density for urine samples is shown in Fig. 5. There was a significant difference in the color density results obtained with the urine samples from pregnant and non-pregnant female volunteers. 4. Discussion
This bioanalysis system was then applied to the detection of hCG spiked in urine samples. The Au naps at 40 nm were used for the conjugation. The detection results of hCG spiked into non-pregnant urine sample is shown in Fig. 4. Compared with the density result obtained with hCG spiked in 1% (w/v) BSA in PBS solution, we observed lower density values for hCG in the urine samples.
The sensitivity of the result is compared using gold colloids with different diameter in a case of hCG detection (data not shown). Au naps at 40 nm is also the preferred size used in the development of commercially available test strips. The result of hCG detection showed higher sensitivity compared with the conventional test strips. While the hCG is secreted in quantity in pregnant female (3 μg/ml), the detection of hCG is usually thought to be enough by an on/off format. In case of ectopic pregnancy, a report suggested the possibility of diagnosis by measuring lower level of hCG related protein (Lee et al., 2005). In such cases, lower detection limit would be required. The hCG detection result spiked in urine showed a little lower detection limit than that performed in the presence of 1% BSA in PBS solution (Fig. 4). However, there might be some hinder for the antigen–antibody reaction in urine; the hCG detection result showed clear difference between pregnant urine samples and non-pregnant urine samples. Surely, the affinities of the antibodies were different between Mab-hCG and Mab-TPSA (Fig. 6). Therefore, the sensitivity of our system was directly affected by their affinity levels. PSA exists in male serum at about 1 ng/ml, and the value of PSA increases along aging even in healthy male (Veltri et al., 2001). Clinically, PSA values 4–10 ng/ml is called as gray zone, which is suspected as a symptom of early prostate cancer (Jung et al., 2001). After all, PSA assay requires detection of low concentration antigen. Therefore, the detection limit of
Fig. 5. Color densities of 2 non-pregnant female and 3 pregnant female urine samples. Each urine samples were diluted by 50 mM PBS pH 7.4 containing 1% BSA to the desired final concentration.
Fig. 6. Color densities of several TPSA concentrations. TPSA was diluted by 50 mM PBS pH 7.4 containing 1% BSA to the desired final concentration. Mab-TPSA was conjugated with 40 nm Au naps.
Fig. 4. Color densities of each hCG concentration in urine sample. hCG was diluted by non-pregnant urine sample to the desired final concentration.
increased with the increasing antigen concentration. In TPSA detection, a significantly low detection limit is required. Our proposed bioanalysis system can be applied in such a situation by simply increasing the sample volume. The density results for different sample volumes at trace TPSA concentrations are compared in Fig. 7. We clearly observed that the detection ability significantly improved to 1 ng/ml (0.3 ng/tip), when the sample volume increased until 300 μl. 3.3. Detection of hCG in urine samples
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Research in our laboratory is in progress towards the detection of other biomolecules, such as DNA and enzymes. Furthermore, the use of enzymatic signal amplification and fluorescent dyes would increase the sensitivity and selectivity of our system. Acknowledgements
Fig. 7. Comparison of color densities for each TPSA concentration while increasing the sample volume. The density result for TPSA was improved, when the sample volume was increased up to 300 μl.
PSA is pursued (Harma et al., 2001). From these reasons, the sample volume was increased in order to be able to detect PSA under 5 ng/ml concentration (Fig. 7). By increasing the sample volumes, a visible increase in the color density was observed with 5 ng/ml PSA. In our system, it is quite easy to increase the sample volumes, in contrast to using conventional test strips. It is noteworthy that there was only a negligible change in the background density level, even when the sample volume was increased significantly. The detection limit of our bioanalysis system at 1 ng/ml was comparable with that of the conventional test strips. The advantage of our bioanalysis system is the short fabrication time. According to the protocol of ZipTip®, the whole fabrication procedure involves the adsorption of the primary antibodies on the resin inside of ZipTip®, which takes place rapidly by a simple pipetting. Additionally, one ZipTip® requires only 10 μl of 100 μg/ml of antibody solution for immobilization onto the resin surface. Thus, the sample volume can also be modified, since ZipTip® is attachable to a micropipette. In the case of measurements with low concentration solutions like environmental and/or clinical samples, aspiration of a high volume of the sample solution would make visual judgment easy. Furthermore, the washing procedure can also be performed by pipetting. By using other types of ZipTip® with less resin filled inside, we are planning to detect antigens in less volume of sample solutions. 5. Conclusions This study demonstrates the applicability of our immunochromatographic assay tip (ZipTip®) for antigen detection in real samples. This novel bioanalysis system is easily applicable in clinical diagnosis, environmental measurements, and forensic research.
This works was funded by Japan Science and Technology Agency (JST). We are deeply grateful to Nihon Millipore, Tokyo, Japan for technical suggestions. We would like to thank Japan Clinical Laboratories, Inc., Kyoto, Japan for providing the antibody against TPSA used in this study. We would also like to express our gratitude to Tanaka Kikinnzoku Kogyo K. K., Tokyo, Japan for providing the colloidal gold nanoparticles. References An, C.D., Yoshiki, T., Lee, G., Okada, Y., 2001. Evaluation of a rapid qualitative prostate specific antigen assay, the One Step PSA™ test. Cancer Lett. 162, 135. Balk, P.S., Ko, Y., Burbley, G., 2003. Biology of prostate-specific antigen. J. Clin. Oncol. 21, 383. Birken, S., Berger, P., Bidart, J.M., Weber, M., Bristow, A., Norman, R., 2003. Preparation and characterization of new WHO reference reagents for human chorionic gonadotropin and metabolites. Clin. Chem. 49, 144. Ekins, R.P., 1983. In: Hunter, W.M., Corrie, J.E.T. (Eds.), Immunoassays for Clinical Chemistry. Churchill Livingstone, Edinburgh, p. 76. Fernandez-Sanchez, C., McNeil, J.C., Rawson, K., Nilsson, O., 2004. Disposable noncompetitive immunosensor for free and total prostate-specific antigen based on capacitance measurement. Anal. Chem. 76, 5649. Hampl, J., Hall, M., Mufti, N.A., Yao, Y.-M.M., MacQueen, D.B, Wright, W.H., Cooper, D.E., 2001. Upconverting phosphor reporters in immunochromatographic assays. Anal. Biochem. 288, 176. Harma, H., Soukka, T., Lovgren, T., 2001. Europium nanoparticles and time-resolved fluorescence for ultrasensitive detection of prostatespecific antigen. Clin. Chem. 47, 561. Jung, K., Stephan, C., Elgeti, U., Lein, M., Brux, B., Kristiansen, G., Rudolph, B., Hauptmann, S., Schnorr, D., Loening, A.S., Sinha, P., 2001. Molecular forms of prostate-specific antigen in serum with concentrations of total prostate-specific antigen <4 mg/L: are they useful tools for early detection and screening of prostate cancer? Int. J. Cancer 93, 759. Knoll, S., Vogel, R.F., Niessen, L., 2002. Identification of fusarium graminearum in cereal samples by DNA detection Test Strips™. Lett. Appl. Microbiol. 34, 144. Lee, J., Oh, M., Shin, J., Lee, K., Nam, J., Cha, J., Chang, J., Cho, D., Kang, I., Lee, I.P., 2005. Clinical effectiveness of urinary human chorionic gonadotropin related protein (hCGRP) quantification for diagnosis of ectopic pregnancy. J. Korean Med. Sci. 20, 461. Lingerfelt, B.M., Mattoussi, H., Goldman, E.R., Mauro, J.M., Anderson, G.P., 2003. Preparation of quantum dot-biotin
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