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Development of a modified microtiter plate with a concave portion in the center
Journal of Immunological Methods 323 (2007) 132 – 138 www.elsevier.com/locate/jim
Research paper
Development of a modified microtiter plate with a concave portion in the center Kimio Katsuta a,⁎, Hideo Namiki b , Kouji Matsushima c b
a Bioarc Laboratories, Saichi-Yoyogi Bldg. 4F, 5-15-9, Sendagaya, Shibuya-ku, Tokyo 151-0051, Japan Integrative Bioscience and Bioengineering, Graduate School of Science and Engineering, Waseda University, Shinjukuku, Tokyo 169-0051, Japan c Department of Molecular Preventive Medicine, School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Received 16 December 2006; received in revised form 7 April 2007; accepted 12 April 2007 Available online 15 May 2007
Abstract We have developed a modified microtiter plate which has following advantageous features and functions to both conventional microtiter plate and protein array, such as 1) use of conventional microtiter plate reader and washer, and 2) allowance of simultaneous reaction in the same liquid for all wells. Four proteins of human serum albumin, human C-reactive protein (CRP), human plasminogen and human MIP-1α as sample proteins, were measured with the modified microtiter plate. Although the reaction liquid of each wells on the modified microtiter plate shares through concave portion, its antigen/antibody in each well is independent. The independence of the reaction is supported by the result that the above four proteins produced dose–response curves simultaneously. Unlike a conventional protein array, our plate does not need the drying process for antibody adhesion to the plate, preventing inactivation of the antibody. And one can detect the antigen/antibody reaction using the enzymatic amplification reaction (for example utilizing the biotin–streptavidine interaction) like a conventional plate. In addition to these features, our microtiter plate also has the merit of eliminating the so-called “edge effect”. © 2007 Elsevier B.V. All rights reserved. Keywords: Protein array; Microtiter plate; ELISA; Concave portion
1. Introduction At present, many researchers are trying to develop a complete protein array for simultaneous analysis of proteins. All researchers have been applying technology of gene chip to protein array. They have been trying to produce a small spot. The objectives of such microarrays Abbreviations: ELISA, Enzyme-Linked Immunosorbent Assay; CRP, C-reactive protein; PBS, Phosphate buffered saline; TMB, Tetramethylbenzidine; SD, standard deviation. ⁎ Corresponding author. Tel.: +81 3 5312 2920; fax: +81 3 5312 2965. E-mail address: [email protected] (K. Katsuta). 0022-1759/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2007.04.007
are 1) to quantify many species of proteins at a time in the same liquid layer, showing possibility of comprehensive analysis, 2) using a small quantity of sample, such as one drop of blood, 3) in a short time, usually in 1 h. Attempts to develop protein arrays aiming at these three objectives are going on in over several hundreds of laboratories (For examples; Alan, 2004; Angenendt et al., 2002; Beath, 2002; Boutell, 2004; Bussow et al., 2001; Dunham, 2001; Ham, 2001; Michaud, 2003; Peters, 2005; Uetz et al., 2000; Washburn, 2003; Washburn et al., 2001; Wildt et al., 2000 and Willis, 2003). However in the assay development of protein arrays, two problems remain to be solved. After protein adsorption
K. Katsuta et al. / Journal of Immunological Methods 323 (2007) 132–138
on the array surface, the protein molecules may lose over 90% of their activity upon drying. In addition, one cannot use an enzymatic amplification reaction, as it is not possible to discriminate a defined protein spot on the array. Does a protein array have to be as small as a gene chip? If a microtiter plate employed for standard ELISA could be used, it would also be possible to use a conventional plate reader and plate washer. The achievement of independent antigen–antibody reaction of various kinds of protein in the whole same fluid phase and the detection of each reaction of the next can express “to quantify many species of proteins at a time in the same liquid layer”. Hence, no special device would be needed. Since different proteins can be determined in each well, it can be possible to determine simultaneously, precisely and reproducibly 96 species of proteins in a 96-well plate. However, in view of the structure of a conventional 96-well microtiter plate,
133
simultaneous determination in the same liquid layer is impossible. We investigated whether conventional microtiter plates could be modified, and whether such modified microtiter plate can enables independent antibody-based detection of distinct proteins within each well, simultaneously. 2. Materials and methods 2.1. Materials Human albumin affinity purified was obtained from Biogenesis (Poole, U.K). Human C-reactive Protein (CRP) was from CHEMICON International (Temecula, CA USA). Human Glu-plasminogen was from Haematologic Technologies Inc. (Essex Junction, VT USA). Human macrophage inflammatory protein-1 alpha (human MIP-
Fig. 1. General view of modified microtiter plate. General view of modified microtiter plate with Cross section at A-A of plane view, and with Cross section at B-B of plane. 10; Bottom, 20; Sidewall part, 30; Concave portion, W; refers to each well and the number at the bottom on the left side refers to the corresponding well No.
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Fig. 2. How to use a modified microtiter plate. A) As a conventional microtiter plate with normal wells. B) As a protein array with concave portion.
1α), Polyclonal rabbit anti-human albumin, polyclonal anti-human C-reactive protein, polyclonal anti-human plasminogen and polyclonal anti-human MIP-1α was from DAKOCytomation (Kyoto, Japan). Immuno pure avidin horseradish peroxidase conjugated and EZ-link sulfo-NHS-biotin (Sulfosuccinimide biotin) were from Pierce Corporation (Rockford, IL USA). Skimmed milk was from Yotuba Nyugyou Co. Ltd (Otofuke Tokachi, Japan). Polyoxyethylene (20) sorbitan monolaurate (Tween 20) was from Wako Pure Chemical Industries. Ltd (Osaka Japan). Block Ace powder UK-B80 was From Dainihon Seiyaku Ltd (Osaka Japan). TMB substratechromogen was from DAKO Cytomation (Kyoto, Japan). 2.2. Labeling with biotin Biotin (manufactured by DAKO) was dissolved in 0.1 M NaHCO3 (pH 8.5) buffer solution at a concentration of 2 mg/ml. Then, the target protein was labeled with the biotin solution, whose molar concentration was 100 times that of the target protein (about 1 mg protein/ml), for 2 h in an ice-cold bath. After labeling, the solution was dialyzed for 3 h using a dialysis tube (Pierce); the external solution
(PBS) was exchanged 3 times during the dialysis. After dialysis, the resultant solution was stored until used. 3. Results Modified microtiter plate is designated to unite functions of both conventional microtiter plate and protein array. A modified microtiter plate is shown in Fig. 1, in which the height of the sidewall and the location and position of each well are the same as in conventional microtiter plate. However, unlike a conventional titer plate, the center of this microtiter plate is concave and the height of each well is 1/3 that of wells in the conventional standard microtiter plate. Furthermore, a volume of 70 ml is provided in the concave portion in the center, for antigen–antibody reactions in each well bottoms. This modified plate allows washing with a conventional washer and determination using a conventional plate reader. Moreover, the bottom of each well was irradiated using a 260-nm/187 nanometer U-shaped ultraviolet lamp (200 W) for 60–80 s from a distance of 40 mm. However, since the height of each well is made 1/3 of that of conventional microtiter plate, it was
Fig. 3. Method for adhering four antibody's on a modified microtiter plate. We divided one piece of plate 4 into a division as shown in Fig. 3. One line of column and seven lines were blanks. Abbreviation, anti-Alb: polyclonal anti-human albumin, anti-CRP: polyclonal anti-human C-reactive protein, anti-Plg: polyclonal anti-human plasminogen and anti-MIP-1α: polyclonal anti-human MIP-1α.
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difficult to homogenously disperse the protein-containing solution in the bottom of the wells. This problem was settled using polystyrene including oil of 0.5% (1/10 of oil content of normal polystyrene) that led each wells into no-leak of oil from the well wall. As a result, a small amount of solution (30 μl/well) could be dispersed in the well bottom. Bubbles that generated occasionally in the well bottom of the microtiter plate and attach there resulted in erroneous measurement. However, when modified microtiter plates made of polystyrene containing 0.5% oil, bubbles did not attach to the bottom, they were easily detached (patent pending). After solving the various problems described above, we succeeded in producing a modified microtiter plate – Biocheck – which can also be used as an conventional microtiter plate. By taking advantage of the concave portion of the plate, it is possible to fill this portion with about 70 ml of buffer solution containing a few ng proteins of the antigen or antibody specimen, to supply the above buffer solution in each well bottom and to perform the antigen–antibody reaction of various kinds of proteins in a each well bottom. The solution within each well can interchange via the concave portion. The general experimental method using the modified microtiter plate was explained as follows.
B) A method with biotinylated-second antibody (sandwich method)
A) A method with biotinylated antigen
A usage of the modified microtiter plate is illustrated in Fig. 2A) and B). The treatment for adhesion of proteins is conducted within each well in the same manner with a conventional microtiter plate (Fig. 2 A) and the treatment for antigen/antibody reaction and blocking are conducted within the whole concave portion of the modified microtiter plate (Fig. 2 B).
Procedure
Volume of a reaction liquid
1) Adhere to well with antibody, stand at room temperature for 2 h. 2) Washing in PBS buffer solution containing 0.05% Tween20 with washer. 3) Blocking with 0.4% skimmed milk in PBS buffer solution. 4) One hour later, dispose of the solution with manual labor. 5) Washing in PBS buffer solution containing 0.05% Tween20 with washer⁎. 6) Antigen/antibody reaction with biotinylated solution. 7) After standing at 37 °C for 15 min, dispose of the solution with manual labor. 8) Washing in PBS buffer solution containing 0.05% Tween20 with washer. 9) Reaction with avidin horseradish peroxidase conjugate(6 ng/34 μl). 10) Washing in PBS buffer solution containing 0.05%Tween20 with washer. 11) Color development with TMB for 15 to 30 min. 12) Stop of color development at 1 M H2SO4. 13) Measurement by a wavelength of 460 nm (Biotrack2 Plate reader, Amersham Bioscience).
34 μl/well 100 μl/well 25 ml/plate
Procedure
Volume of a reaction liquid
1) Adhere to well with antibody, stand at room temperature for 2 h. 2) Washing in PBS buffer solution containing 0.05%Tween20 with washer. 3) Blocking with 0.4% skimmed milk in PBS buffer solution. 4) One hour later, dispose of the solution with manual labor. 5) Washing in PBS buffer solution containing 0.05%Tween20 with washer. 6) Antigen/antibody reaction with solution containing antigens. 7) One hour later, dispose of the solution with manual labor. 8) Washing in PBS buffer solution containing 0.05%Tween20 with washer. 9) Antigen/second antibody reaction with biotinylated secondary antibody solution. 10) Washing in PBS buffer solution containing 0.05%Tween20 with washer. 11) Reaction with avidin horseradish peroxidase conjugate. 12) Washing in PBS buffer solution containing 0.05%Tween20 with washer. 13) Color development with TMB for 15 to 30 min. 14) Stop of color development at 1 M H2SO4. 15) Measurement by a wavelength of 460 nm.
34 μl/well 100 μl/well 25 ml/plate
100 μl/well 25 ml/plate
100 μl/well 34 μl/well 100 μl/well 34 μl/well 100 μl/well 30 μl/well 30 μl/well
C) Accomplishment of a method of A) and B), with the same modified microtiter plate at the same time 100 μl/well 25 ml/plate
100 μl/well 34 μl/well 100 μl/well 30 μl/well 30 μl/well
⁎Washer: Well wash Plus (Thermo ELECTRONCORPORATION)
1) Adhere to well with antibody (about 1 mg protein/ml solution, antialbumin, antiCRP, anti-plasminogen and anti-MIP-1α) at about 1 mg protein/ml of undiluted solution for 100–1600 fold dilution with 0.1 M NaHCO3 pH 9.6 shown in Fig. 3 a). 34 μl of this was added to each well. Stand at room temperature for 2 h. 2) Washing in PBS buffer solution containing 0.05% Tween20 with washer. 3) Blocking with 0.4% skimmed milk solution. 4) One hour later, dispose of the solution with manual labor. 5) Washing in PBS buffer solution containing 0.05% Tween20 with washer. 6) Antigen/antibody reaction with biotinylated antigens (biotinalbumin:1 ng protein/ml and biotin-CRP:40 ng protein/ml) and non-labeled-antigen (plasminogen: 40 ng protein/ and MIP-1α: 40ngprotein/ml) solution. 7) After stand at 37 °C for 15 min, dispose of the solution with manual labor.
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8) Washing in PBS buffer solution containing 0.05% Tween20 with washer. 9) Antigen/second antibody reaction with biotinylated secondary antibody (biotin-plasminogen and biotin-MIP-1α about 5 ng/ml solution in PBS buffer containing 0.4% Block Ace). 10) After stand at 37 °C for 15 min, dispose of the solution with manual labor. 11) Washing in PBS buffer solution containing 0.05% Tween20 with washer. 12) Reaction with avidin horseradish peroxidase conjugate. 13) Washing in PBS buffer solution containing 0.05%Tween20 with washer. 14) Color development with TMB for 15 to 30 min. 15) Stop of color development at 1 M H2SO4. 16) Measurement by a wavelength of 460 nm.
As shown in Fig. 3, it was measured simultaneously at the same plate, CRP and albumin by a method of A) and also measured plasminogen and MIP-1α by a method of B). The color development (460 nm) obtained from four proteins (biotin-albumin, biotin-CRP, and plasminogen and MIP-1α) were dependent on concentration of each antibody as shown in Fig. 4, and was able to measure reproducibly and simultaneously all four targets within one modified microtiter plate. In addition to these features, modified microtiter plate also has extraordinary feature that is elimination of “edge effect” (Table 1a and b). Table 1b measured human albumin with the conventional ELISA. All the reagent concentration between each well should be same in the same measured value entirely, but as for the well to be located in circumstance of the microtiter plate, measured values were significantly different (P b 0.01). This phenomenon is known as an edge effect. However, the edge was not observed as had shown in Table 1a. This feature will give extra benefit for modified microtiter plate not only by
Fig. 4. The simultaneous measurement of albumin, CRP, plasminogen and MIP-1α with modified microtiter plate. The experiment was shown in C) of a result.
increasing number of usable wells (80 wells or less to all 96 wells) but also by improving intra plate precision. 4. Discussion We developed a modified plate, which satisfied three following conditions. 1. Drying process of the plate for protein adhesion is not needed. Therefore, the protein molecules cannot be inactivated. 2. Since the modified microtiter plate have wells with concave portion, one can use enzymatic amplification reaction such as biotin system for the detection of protein. 3. By existence of concave portion on the modified microtiter plate, one can accomplish a lot of independent antigen/antibody reaction in the same fluid phases at the same time. Hence, it has two functions of a protein array and a conventional microtiter plate. For a solution, we tried to improve a conventional microtiter plate. We established a concave portion in the central part of a conventional microtiter plate and the height of the well was 1/3 of the well of a standard plate. The capacities of each well decreased to 100 μl from 430 μl. The problem to occur by having made height 1/3 usually solved it in polystyrene by reducing as oil ingredient included in to 0.5% from 5%. The modified microtiter plate developed was able to use a conventional leader and washer. The precision for measurement was same as conventional microtiter plate. In addition, we were able to carry out various complicated experiments as described. We were able to accomplish a sandwich method and other methods at the same time. But the number of simultaneous measurement is limited to 96 wells because it is the simultaneous measurements by improvement of conventional microtiter plate. It will be to 384 wells in future even if it improves. There is little it if we will surely study protein generated by one cell comprehensively. However, we developed it in the clinical field for the purpose of offering a modified microtiter plate to be able to use to correct diagnosis and treatment. Precision, sensitivity and the shortness of measurement time are demanded from this purpose. In this paper we could show that our modified microtiter plate meets such demands in the clinical field. We are studying the simultaneous measurement of some proteins helping correct diagnosis and therapy for early stage of renal disease. In addition to these features, the modified microtiter plate has the advantageous feature of elimination of
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Table 1a An edge effect does not appear in the measurement with modified microtiter plate
Table 1b An edge effect does not appear in the measurement with modified microtiter plate
a) Microtiter plate made by plasma-irradiation treatment, instead of U-V lamp was tested for edge effect. b) Modified microtiter plate was tested also. Both plates were tested as follows. Human serum albumin (about 1 mg protein/ml) was diluted in 100 fold of NaHCO3 buffer solution (ph 9.6). 34 μl of this was added in each well of vertical lane 1 to 10 on the plate. And vertical lane 11 and 12 were added the buffer only as control. Other procedures were same as described in general procedure, except 1 μl of human serum albumin (1 mg protein/ml) as antigen solution was diluted in 25 ml of PBS solution containing 0.4% Block Ace and used.
edge effect, as shown in Table 1a). However, it cannot be understood the cause for the elimination of an edge effect.
Murakoshi, Dr. Takashi Yoneda, Mr. Takeshige Ogawa, Mr. Naoya Harano, and Mr. Shigeru Kanamori. References
Acknowledgements We would like to extend our deep appreciation to Dr Takeshi Kinebuchi of Tokyo Institute for Molecular Medicine who allowed us to work at his laboratory and gave valuable advice. We are also grateful to those who supported this experiment including Ms. Aiko
Alan, D., 2004. Analyzing proteins on arrays defies expectations. Genomics & Proteomics. 31 July /August. Angenendt, P., Gloker, J., Murphy, D., Lehrach, H., Calilla, D.J., 2002. Toward optimized antibody microarrays: a comparison of current microarray support materials. Analytical Biochemistry 309, 253. Beath, M., 2002. Protein microarrays and proteinmics. Nature Genetics Supplement 32, 526.
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Boutell, J.M. 2004. Functional protein microarrays for pallalel characterization of P53 mutants, Proteomics 2000, 4 c7, 2004. 50-58. Bussow, K., Konthur, Z., Lueking, A., Lehvach, H., Walter, G., 2001. Protein array technology. American Journal of Pharmacogenomics 1(1), 1175-2203/01/0001-0000/0. Dunham, H.B., 2001. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solution. Genome Biology 2, 1. Ham, M.J.H.D., Brown, P.O., 2001. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solution. Genome Biology 2, 1. Michaud, G.A., 2003. Protease microarrays. Nature Biotechnology 21, 1509. Peters, J.K., Spek, P.J.V.d., 2005. Growing applications and advancements in microarray technology and analysis tools. Cell Biochemistry and Biophysics 43, 149.
Uetz, P., Giot, L., Cang, G., Mansfield, T.A., Judson, R.S., Knight, J.R., Lockshon, D., Navyan, V., Svinivasan, M., Pochart, P., QureshiEmill, A., Li, Y., Godwin, B., Conover, D., Kalbfleish, K., Vyayadamosder, G., Yang, M., Johnston, M., Field, S., Rthber, J.M., 2000. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae, Nature 403, 403,10. Washburn, M., 2003. Soft landing for protein chips. Nature Biotechnology 21 (10). Washburn, M.P., Wolters, D., Yates, J.R., 2001. Large-scale analysis of the yeast proteome by multi dimentional protein identification technology. Nature Biotechnology 19, 242. Wildt, R., Mundy, C., Gorick, B., Tomlinson, I., 2000. Antibody arrays for high throughput screening of antibody–antigen interactions. Nature Biotechnology 18, 989. Willis, R.C., 2003. Protein chips: bound to deliver. American Chemical Society January Today's Chemist at Work, p. 29.
Research paper
Development of a modified microtiter plate with a concave portion in the center Kimio Katsuta a,⁎, Hideo Namiki b , Kouji Matsushima c b
a Bioarc Laboratories, Saichi-Yoyogi Bldg. 4F, 5-15-9, Sendagaya, Shibuya-ku, Tokyo 151-0051, Japan Integrative Bioscience and Bioengineering, Graduate School of Science and Engineering, Waseda University, Shinjukuku, Tokyo 169-0051, Japan c Department of Molecular Preventive Medicine, School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Received 16 December 2006; received in revised form 7 April 2007; accepted 12 April 2007 Available online 15 May 2007
Abstract We have developed a modified microtiter plate which has following advantageous features and functions to both conventional microtiter plate and protein array, such as 1) use of conventional microtiter plate reader and washer, and 2) allowance of simultaneous reaction in the same liquid for all wells. Four proteins of human serum albumin, human C-reactive protein (CRP), human plasminogen and human MIP-1α as sample proteins, were measured with the modified microtiter plate. Although the reaction liquid of each wells on the modified microtiter plate shares through concave portion, its antigen/antibody in each well is independent. The independence of the reaction is supported by the result that the above four proteins produced dose–response curves simultaneously. Unlike a conventional protein array, our plate does not need the drying process for antibody adhesion to the plate, preventing inactivation of the antibody. And one can detect the antigen/antibody reaction using the enzymatic amplification reaction (for example utilizing the biotin–streptavidine interaction) like a conventional plate. In addition to these features, our microtiter plate also has the merit of eliminating the so-called “edge effect”. © 2007 Elsevier B.V. All rights reserved. Keywords: Protein array; Microtiter plate; ELISA; Concave portion
1. Introduction At present, many researchers are trying to develop a complete protein array for simultaneous analysis of proteins. All researchers have been applying technology of gene chip to protein array. They have been trying to produce a small spot. The objectives of such microarrays Abbreviations: ELISA, Enzyme-Linked Immunosorbent Assay; CRP, C-reactive protein; PBS, Phosphate buffered saline; TMB, Tetramethylbenzidine; SD, standard deviation. ⁎ Corresponding author. Tel.: +81 3 5312 2920; fax: +81 3 5312 2965. E-mail address: [email protected] (K. Katsuta). 0022-1759/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2007.04.007
are 1) to quantify many species of proteins at a time in the same liquid layer, showing possibility of comprehensive analysis, 2) using a small quantity of sample, such as one drop of blood, 3) in a short time, usually in 1 h. Attempts to develop protein arrays aiming at these three objectives are going on in over several hundreds of laboratories (For examples; Alan, 2004; Angenendt et al., 2002; Beath, 2002; Boutell, 2004; Bussow et al., 2001; Dunham, 2001; Ham, 2001; Michaud, 2003; Peters, 2005; Uetz et al., 2000; Washburn, 2003; Washburn et al., 2001; Wildt et al., 2000 and Willis, 2003). However in the assay development of protein arrays, two problems remain to be solved. After protein adsorption
K. Katsuta et al. / Journal of Immunological Methods 323 (2007) 132–138
on the array surface, the protein molecules may lose over 90% of their activity upon drying. In addition, one cannot use an enzymatic amplification reaction, as it is not possible to discriminate a defined protein spot on the array. Does a protein array have to be as small as a gene chip? If a microtiter plate employed for standard ELISA could be used, it would also be possible to use a conventional plate reader and plate washer. The achievement of independent antigen–antibody reaction of various kinds of protein in the whole same fluid phase and the detection of each reaction of the next can express “to quantify many species of proteins at a time in the same liquid layer”. Hence, no special device would be needed. Since different proteins can be determined in each well, it can be possible to determine simultaneously, precisely and reproducibly 96 species of proteins in a 96-well plate. However, in view of the structure of a conventional 96-well microtiter plate,
133
simultaneous determination in the same liquid layer is impossible. We investigated whether conventional microtiter plates could be modified, and whether such modified microtiter plate can enables independent antibody-based detection of distinct proteins within each well, simultaneously. 2. Materials and methods 2.1. Materials Human albumin affinity purified was obtained from Biogenesis (Poole, U.K). Human C-reactive Protein (CRP) was from CHEMICON International (Temecula, CA USA). Human Glu-plasminogen was from Haematologic Technologies Inc. (Essex Junction, VT USA). Human macrophage inflammatory protein-1 alpha (human MIP-
Fig. 1. General view of modified microtiter plate. General view of modified microtiter plate with Cross section at A-A of plane view, and with Cross section at B-B of plane. 10; Bottom, 20; Sidewall part, 30; Concave portion, W; refers to each well and the number at the bottom on the left side refers to the corresponding well No.
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K. Katsuta et al. / Journal of Immunological Methods 323 (2007) 132–138
Fig. 2. How to use a modified microtiter plate. A) As a conventional microtiter plate with normal wells. B) As a protein array with concave portion.
1α), Polyclonal rabbit anti-human albumin, polyclonal anti-human C-reactive protein, polyclonal anti-human plasminogen and polyclonal anti-human MIP-1α was from DAKOCytomation (Kyoto, Japan). Immuno pure avidin horseradish peroxidase conjugated and EZ-link sulfo-NHS-biotin (Sulfosuccinimide biotin) were from Pierce Corporation (Rockford, IL USA). Skimmed milk was from Yotuba Nyugyou Co. Ltd (Otofuke Tokachi, Japan). Polyoxyethylene (20) sorbitan monolaurate (Tween 20) was from Wako Pure Chemical Industries. Ltd (Osaka Japan). Block Ace powder UK-B80 was From Dainihon Seiyaku Ltd (Osaka Japan). TMB substratechromogen was from DAKO Cytomation (Kyoto, Japan). 2.2. Labeling with biotin Biotin (manufactured by DAKO) was dissolved in 0.1 M NaHCO3 (pH 8.5) buffer solution at a concentration of 2 mg/ml. Then, the target protein was labeled with the biotin solution, whose molar concentration was 100 times that of the target protein (about 1 mg protein/ml), for 2 h in an ice-cold bath. After labeling, the solution was dialyzed for 3 h using a dialysis tube (Pierce); the external solution
(PBS) was exchanged 3 times during the dialysis. After dialysis, the resultant solution was stored until used. 3. Results Modified microtiter plate is designated to unite functions of both conventional microtiter plate and protein array. A modified microtiter plate is shown in Fig. 1, in which the height of the sidewall and the location and position of each well are the same as in conventional microtiter plate. However, unlike a conventional titer plate, the center of this microtiter plate is concave and the height of each well is 1/3 that of wells in the conventional standard microtiter plate. Furthermore, a volume of 70 ml is provided in the concave portion in the center, for antigen–antibody reactions in each well bottoms. This modified plate allows washing with a conventional washer and determination using a conventional plate reader. Moreover, the bottom of each well was irradiated using a 260-nm/187 nanometer U-shaped ultraviolet lamp (200 W) for 60–80 s from a distance of 40 mm. However, since the height of each well is made 1/3 of that of conventional microtiter plate, it was
Fig. 3. Method for adhering four antibody's on a modified microtiter plate. We divided one piece of plate 4 into a division as shown in Fig. 3. One line of column and seven lines were blanks. Abbreviation, anti-Alb: polyclonal anti-human albumin, anti-CRP: polyclonal anti-human C-reactive protein, anti-Plg: polyclonal anti-human plasminogen and anti-MIP-1α: polyclonal anti-human MIP-1α.
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difficult to homogenously disperse the protein-containing solution in the bottom of the wells. This problem was settled using polystyrene including oil of 0.5% (1/10 of oil content of normal polystyrene) that led each wells into no-leak of oil from the well wall. As a result, a small amount of solution (30 μl/well) could be dispersed in the well bottom. Bubbles that generated occasionally in the well bottom of the microtiter plate and attach there resulted in erroneous measurement. However, when modified microtiter plates made of polystyrene containing 0.5% oil, bubbles did not attach to the bottom, they were easily detached (patent pending). After solving the various problems described above, we succeeded in producing a modified microtiter plate – Biocheck – which can also be used as an conventional microtiter plate. By taking advantage of the concave portion of the plate, it is possible to fill this portion with about 70 ml of buffer solution containing a few ng proteins of the antigen or antibody specimen, to supply the above buffer solution in each well bottom and to perform the antigen–antibody reaction of various kinds of proteins in a each well bottom. The solution within each well can interchange via the concave portion. The general experimental method using the modified microtiter plate was explained as follows.
B) A method with biotinylated-second antibody (sandwich method)
A) A method with biotinylated antigen
A usage of the modified microtiter plate is illustrated in Fig. 2A) and B). The treatment for adhesion of proteins is conducted within each well in the same manner with a conventional microtiter plate (Fig. 2 A) and the treatment for antigen/antibody reaction and blocking are conducted within the whole concave portion of the modified microtiter plate (Fig. 2 B).
Procedure
Volume of a reaction liquid
1) Adhere to well with antibody, stand at room temperature for 2 h. 2) Washing in PBS buffer solution containing 0.05% Tween20 with washer. 3) Blocking with 0.4% skimmed milk in PBS buffer solution. 4) One hour later, dispose of the solution with manual labor. 5) Washing in PBS buffer solution containing 0.05% Tween20 with washer⁎. 6) Antigen/antibody reaction with biotinylated solution. 7) After standing at 37 °C for 15 min, dispose of the solution with manual labor. 8) Washing in PBS buffer solution containing 0.05% Tween20 with washer. 9) Reaction with avidin horseradish peroxidase conjugate(6 ng/34 μl). 10) Washing in PBS buffer solution containing 0.05%Tween20 with washer. 11) Color development with TMB for 15 to 30 min. 12) Stop of color development at 1 M H2SO4. 13) Measurement by a wavelength of 460 nm (Biotrack2 Plate reader, Amersham Bioscience).
34 μl/well 100 μl/well 25 ml/plate
Procedure
Volume of a reaction liquid
1) Adhere to well with antibody, stand at room temperature for 2 h. 2) Washing in PBS buffer solution containing 0.05%Tween20 with washer. 3) Blocking with 0.4% skimmed milk in PBS buffer solution. 4) One hour later, dispose of the solution with manual labor. 5) Washing in PBS buffer solution containing 0.05%Tween20 with washer. 6) Antigen/antibody reaction with solution containing antigens. 7) One hour later, dispose of the solution with manual labor. 8) Washing in PBS buffer solution containing 0.05%Tween20 with washer. 9) Antigen/second antibody reaction with biotinylated secondary antibody solution. 10) Washing in PBS buffer solution containing 0.05%Tween20 with washer. 11) Reaction with avidin horseradish peroxidase conjugate. 12) Washing in PBS buffer solution containing 0.05%Tween20 with washer. 13) Color development with TMB for 15 to 30 min. 14) Stop of color development at 1 M H2SO4. 15) Measurement by a wavelength of 460 nm.
34 μl/well 100 μl/well 25 ml/plate
100 μl/well 25 ml/plate
100 μl/well 34 μl/well 100 μl/well 34 μl/well 100 μl/well 30 μl/well 30 μl/well
C) Accomplishment of a method of A) and B), with the same modified microtiter plate at the same time 100 μl/well 25 ml/plate
100 μl/well 34 μl/well 100 μl/well 30 μl/well 30 μl/well
⁎Washer: Well wash Plus (Thermo ELECTRONCORPORATION)
1) Adhere to well with antibody (about 1 mg protein/ml solution, antialbumin, antiCRP, anti-plasminogen and anti-MIP-1α) at about 1 mg protein/ml of undiluted solution for 100–1600 fold dilution with 0.1 M NaHCO3 pH 9.6 shown in Fig. 3 a). 34 μl of this was added to each well. Stand at room temperature for 2 h. 2) Washing in PBS buffer solution containing 0.05% Tween20 with washer. 3) Blocking with 0.4% skimmed milk solution. 4) One hour later, dispose of the solution with manual labor. 5) Washing in PBS buffer solution containing 0.05% Tween20 with washer. 6) Antigen/antibody reaction with biotinylated antigens (biotinalbumin:1 ng protein/ml and biotin-CRP:40 ng protein/ml) and non-labeled-antigen (plasminogen: 40 ng protein/ and MIP-1α: 40ngprotein/ml) solution. 7) After stand at 37 °C for 15 min, dispose of the solution with manual labor.
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8) Washing in PBS buffer solution containing 0.05% Tween20 with washer. 9) Antigen/second antibody reaction with biotinylated secondary antibody (biotin-plasminogen and biotin-MIP-1α about 5 ng/ml solution in PBS buffer containing 0.4% Block Ace). 10) After stand at 37 °C for 15 min, dispose of the solution with manual labor. 11) Washing in PBS buffer solution containing 0.05% Tween20 with washer. 12) Reaction with avidin horseradish peroxidase conjugate. 13) Washing in PBS buffer solution containing 0.05%Tween20 with washer. 14) Color development with TMB for 15 to 30 min. 15) Stop of color development at 1 M H2SO4. 16) Measurement by a wavelength of 460 nm.
As shown in Fig. 3, it was measured simultaneously at the same plate, CRP and albumin by a method of A) and also measured plasminogen and MIP-1α by a method of B). The color development (460 nm) obtained from four proteins (biotin-albumin, biotin-CRP, and plasminogen and MIP-1α) were dependent on concentration of each antibody as shown in Fig. 4, and was able to measure reproducibly and simultaneously all four targets within one modified microtiter plate. In addition to these features, modified microtiter plate also has extraordinary feature that is elimination of “edge effect” (Table 1a and b). Table 1b measured human albumin with the conventional ELISA. All the reagent concentration between each well should be same in the same measured value entirely, but as for the well to be located in circumstance of the microtiter plate, measured values were significantly different (P b 0.01). This phenomenon is known as an edge effect. However, the edge was not observed as had shown in Table 1a. This feature will give extra benefit for modified microtiter plate not only by
Fig. 4. The simultaneous measurement of albumin, CRP, plasminogen and MIP-1α with modified microtiter plate. The experiment was shown in C) of a result.
increasing number of usable wells (80 wells or less to all 96 wells) but also by improving intra plate precision. 4. Discussion We developed a modified plate, which satisfied three following conditions. 1. Drying process of the plate for protein adhesion is not needed. Therefore, the protein molecules cannot be inactivated. 2. Since the modified microtiter plate have wells with concave portion, one can use enzymatic amplification reaction such as biotin system for the detection of protein. 3. By existence of concave portion on the modified microtiter plate, one can accomplish a lot of independent antigen/antibody reaction in the same fluid phases at the same time. Hence, it has two functions of a protein array and a conventional microtiter plate. For a solution, we tried to improve a conventional microtiter plate. We established a concave portion in the central part of a conventional microtiter plate and the height of the well was 1/3 of the well of a standard plate. The capacities of each well decreased to 100 μl from 430 μl. The problem to occur by having made height 1/3 usually solved it in polystyrene by reducing as oil ingredient included in to 0.5% from 5%. The modified microtiter plate developed was able to use a conventional leader and washer. The precision for measurement was same as conventional microtiter plate. In addition, we were able to carry out various complicated experiments as described. We were able to accomplish a sandwich method and other methods at the same time. But the number of simultaneous measurement is limited to 96 wells because it is the simultaneous measurements by improvement of conventional microtiter plate. It will be to 384 wells in future even if it improves. There is little it if we will surely study protein generated by one cell comprehensively. However, we developed it in the clinical field for the purpose of offering a modified microtiter plate to be able to use to correct diagnosis and treatment. Precision, sensitivity and the shortness of measurement time are demanded from this purpose. In this paper we could show that our modified microtiter plate meets such demands in the clinical field. We are studying the simultaneous measurement of some proteins helping correct diagnosis and therapy for early stage of renal disease. In addition to these features, the modified microtiter plate has the advantageous feature of elimination of
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Table 1a An edge effect does not appear in the measurement with modified microtiter plate
Table 1b An edge effect does not appear in the measurement with modified microtiter plate
a) Microtiter plate made by plasma-irradiation treatment, instead of U-V lamp was tested for edge effect. b) Modified microtiter plate was tested also. Both plates were tested as follows. Human serum albumin (about 1 mg protein/ml) was diluted in 100 fold of NaHCO3 buffer solution (ph 9.6). 34 μl of this was added in each well of vertical lane 1 to 10 on the plate. And vertical lane 11 and 12 were added the buffer only as control. Other procedures were same as described in general procedure, except 1 μl of human serum albumin (1 mg protein/ml) as antigen solution was diluted in 25 ml of PBS solution containing 0.4% Block Ace and used.
edge effect, as shown in Table 1a). However, it cannot be understood the cause for the elimination of an edge effect.
Murakoshi, Dr. Takashi Yoneda, Mr. Takeshige Ogawa, Mr. Naoya Harano, and Mr. Shigeru Kanamori. References
Acknowledgements We would like to extend our deep appreciation to Dr Takeshi Kinebuchi of Tokyo Institute for Molecular Medicine who allowed us to work at his laboratory and gave valuable advice. We are also grateful to those who supported this experiment including Ms. Aiko
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