JP5322242B2 - Immobilization of protein / peptide on diamond chip - Google Patents

Description

  The present invention relates to a method for stably immobilizing a protein / peptide on a chemically modified diamond / DLC (Diamond-like Carbon) chip without losing its function, and a protein / peptide immobilization chip obtained by the method.

  Conventionally, a technique of coating diamond on a substrate such as silicon or coating diamond-like carbon (DLC) has been widely used. For example, a DNA immobilization chip using these (for example, see Patent Documents 1 to 4), a diamond coating high density integrated protein chip by the present inventors (for example, see Non-Patent Document 1), an organic single molecule on a diamond substrate A semiconductor sensing device having an organic monomolecular film / semiconductor structure in which a film is formed as a direct detection part (see, for example, Patent Document 5) has been proposed. In addition, various types of allergens can be detected simultaneously by reacting a solid substrate with one crude protein attached to each spot, extracted from various types of allergens, with a sample obtained from an allergic disease patient. An allergy diagnostic protein chip capable of detecting the type of antibody associated with an allergic reaction caused by a specific allergen, an allergen detection method, and an allergy-inducing antibody detection method (see, for example, Patent Document 6) are known. ing.

WO99 / 40173 WO99 / 41362 WO99 / 63072 JP 2005-55365 A Japanese Patent Laid-Open No. 2004-4007 Special table 2004-533625 gazette

NEW DIAMOND, Vol.20, No.3 (2004), 30-31

  When immobilizing proteins on substrates such as various chips, special considerations are necessary to maintain the functions of proteins that are presumed to be closely related to the three-dimensional structure, since the three-dimensional structure of proteins generally changes easily. And need ingenuity. In protein immobilization, conventional macro-immobilization techniques such as affinity chromatography and other immobilization methods limit the target even if the protein is largely inactivated by immobilization. The target substance can be identified because the signal can be amplified. However, in order to produce a high-throughput high-density protein chip, it is necessary to establish a technique for immobilizing a minute amount of protein while maintaining its activity and conformation. The activity of a protein depends on a complicated three-dimensional structure, and is usually deactivated when the three-dimensional structure is destroyed by immobilizing the protein on the substrate surface. An object of the present invention is to provide a method for immobilizing a protein / peptide on a chip substrate without impairing the function of the protein, or for immobilizing the protein / peptide on the substrate without impairing the function. .

  The present inventors activated a chemically modified diamond / DLC (Diamond-like Carbon) chip with an activating reagent, spotted a protein / peptide on the chip to perform a coupling reaction, and then unreacted activity In the method of immobilizing a protein / peptide on a chip that performs group blocking operation, 30% dimethyl sulfoxide (DMSO) or 30% polyethylene glycol (PEG) was used as a spotting additive, or activated by an activating reagent. Later, after drying with MilliQ water and drying for 1 hour under reduced pressure, the protein / peptide may be immobilized on the chip substrate without damaging the function of the protein / peptide such as allergens, It has been found that it can be maintained without damage, and the present invention has been completed.

That is, the present invention includes (1) activating a chemically modified diamond / DLC (Diamond-like Carbon) chip with an activating reagent and then spotting the protein / peptide on the chip to perform a coupling reaction. a method of securing a protein / peptide, after activation by an activating reagent, have rows vacuum drying process of 30 to 120 minutes as drying of the washing water to wash the chips and, after the coupling reaction, A protein / peptide immobilization method characterized by drying at 30 to 45 ° C. for 2 to 4 hours, or (2) Washing water is MilliQ water, The protein / peptide described in (1) above And (3) using dimethyl sulfoxide (DMSO) or polyethylene glycol (PEG) as a spotting additive The protein / peptide immobilization method according to (1) or (2) above, or (4) 20-40% DMSO or 20-40% PEG is used. An immobilization method, (5) a protein / peptide immobilization method according to (4) above, wherein 30% DMSO or 30% PEG is used , and (6 ) an activation reagent comprising WSCD · HCl and NHS The protein / peptide immobilization method according to any one of (1) to ( 5 ) above, wherein ( 7 ) the coupling reaction is performed, and then the unreacted active group is blocked ( 8 ) The protein / peptide immobilization method according to any one of (1) to ( 6 ) above, wherein ( 8 ) the protein / peptide is an antigen or an antibody. 1) to ( 7 Or a protein / peptide immobilization method according to any one of the above.

Furthermore, the present invention relates to (9) the protein / peptide immobilization method according to (8) above, wherein the antigen is an allergen, and (10) the allergen is an allergen epitope or a peptide containing an allergen epitope. The protein / peptide immobilization method described in (9) above, or (11) the protein described in (10) above, wherein a chemically modified peptide is used as the allergen epitope or peptide containing allergen epitope / Peptide immobilization method and (12) peptide containing an allergen epitope containing an epitope in which at least two or more amino acids are added to the N-terminal side and / or C-terminal side of the peptide part that binds to MHC class II molecules The protein / peptide according to (10) above, wherein a peptide is used The protein / peptide immobilization method according to any one of (10) to (12) above, wherein a protease-degrading peptide containing an allergen epitope is used as an immobilization method or (13) an allergen epitope peptide (14) The protein / peptide immobilization method according to (13) above, wherein the protease degrading peptide is a trypsin degrading peptide, and (15) a food allergen is used as the allergen The protein / peptide immobilization method according to any one of (10) to (14) above, or (16) food allergens such as sodium caseinate, α-casein, β-casein, κ-casein, α-lactalbumin, β 1 selected from lactoglobulin, ovomucoid, ovalbumin, conalbumin Or (15) above, which comprises using two or more allergens relates to immobilization how the protein / peptide according.

  According to the present invention, a very small amount of protein or peptide can be mounted on the chip efficiently and accurately, and the loaded sample maintains the function as a protein / peptide and is stable without peeling off the protein / peptide through a series of operations. Fixed high-throughput protein / peptide chips can be produced.

It is a figure which shows the basic operation procedure from chip activation to fluorescence detection. It is a figure which shows the examination result of the drying process conditions before spotting after an activation process. It is a figure which shows the effect of additive DMSO and PEG in the antigen chip | tip which spots protein / peptide. It is a figure which shows the effect of additive DMSO when spotting protein / peptide to a diamond-like carbon (DLC) chip | tip and a diamond coating (GENE DIA). It is a figure which shows the examination result of the drying conditions (3 hours) after spotting an antigen. It is a figure which shows the examination result of the drying conditions (overnight) after spotting an antigen. It is a figure which shows the influence of glycerol in the direct antibody binding method and the protein G indirect antibody binding method. It is a figure which shows the influence of DNSO on the antibody binding amount in the direct antibody binding method. It is a figure which shows the influence of DNSO on the TNF- (alpha) antigen antibody binding amount in the direct antibody binding method. It is a figure which shows the result of the food (milk / egg) allergy test (IgG) of a child and a healthy person. It is a figure which shows the result of the food (milk / egg) allergy test (IgG) of a child and a healthy person. It is a figure which shows the result of the food (milk / egg) allergy test (IgE) of a child and a healthy person. It is a figure which shows the outline of activation and coupling reaction of the diamond / DLC chip | tip chemically modified.

  In the protein / peptide immobilization method of the present invention, a chemically modified diamond / DLC (Diamond-like Carbon) chip is activated with an activating reagent, and then the protein / peptide is spotted on the chip for coupling reaction. Is not particularly limited as long as it is a method of immobilizing a protein / peptide on a chip and using DMSO or PEG as a spotting additive. Examples of the chemically modified diamond / DLC chip include silicon, glass, A diamond / DLC chip that has been chemically modified so that a protein / peptide can be bound by a coupling reaction or the like to a diamond / DLC chip coated with diamond or diamond-like carbon (DLC) on a substrate such as stainless steel or plastic. If special For example, as such chemical modification, a diamond / DLC chip whose surface is subjected to chlorination, amination, carboxylation, etc. by chlorination, ammonia treatment, dicarboxylic acid treatment, etc. is exemplified as such chemical modification. Among them, a carboxylated diamond / DLC chip can be preferably exemplified. A commercially available product (Toyo Kohan) may be used as the chemically modified diamond / DLC chip.

The chemically modified diamond / DLC chip is subjected to an activation treatment with an activation reagent. For example, as a method for immobilizing a protein / peptide on a carboxylated diamond / DLC chip, a protein / peptide having an amino group (—NH ₂ group) using a carboxyl group (—COOH group) introduced on the substrate surface is used. It is preferable to immobilize by covalent bond using 1-Etyl-3- (3-dimethylaminopropyl) -carbodiimide, hydrochloride (WSCD · HCl), N-Hydroxy-succinimide (NHS) or other chemical crosslinking agents. An outline of chip activation and coupling reaction is shown in FIG.

  The chip after the above activation treatment is washed with MilliQ water (ultra-pure water), and a peptide coupling reaction (immobilization operation) is performed. As a pretreatment for the protein / peptide immobilization operation, sufficient dehumidification is performed. Drying is an important factor for the protein / peptide immobilization amount and the uniformity of the immobilization amount. Therefore, it is highly preferable to perform a vacuum drying treatment with a vacuum desiccator for 30 to 120 minutes, preferably about 1 hour. The peptide coupling reaction is carried out by spotting the protein / peptide solution on a chip after activation treatment, preferably a chip subjected to vacuum drying treatment, at 30-42 ° C. for 2-4 hours, preferably 37 ° C. However, if DMSO or PEG is used as a spotting additive, the protein / peptide may be immobilized on the chip substrate without increasing the function of the protein / peptide, or the amount of binding may increase. It can be maintained on the substrate without impairing its function. Moreover, it is preferable to dry at 30-42 degreeC for 2 to 4 hours after a spotting reaction, especially at 37 degreeC for 3 hours. After this drying, it is preferable to perform an unreacted active group blocking operation using BSA or the like. The protein / peptide immobilization chip of the present invention can be produced by such a protein / peptide immobilization method of the present invention.

  The above DMSO and PEG are usually used by adding to a solvent (0.1 M potassium phosphate buffer, pH 6.0) for immobilizing proteins / peptides on the chip substrate surface. 10 to 50%, particularly around 30% is preferable. Further, as PEG, a PEG having a molecular weight of 200 to 400, preferably around 300 can be suitably exemplified.

  Examples of the protein / peptide targeted for the protein / peptide immobilization method of the present invention include antigens and antibodies, and examples of antigens include allergens, various cancer antigens, and cytokines. . The allergen is not particularly limited as long as it has immunogenicity, and eggs, milk, meat, fish, crustaceans and mollusks, cereals, beans and nuts, fruits, vegetables, beer Preferred examples include food allergens such as yeast and gelatin. Among them, αs1-casein, αs2-casein, β-casein, κ-casein, α-lactalbumin, and the main whey allergen as main components of milk allergen Β-lactoglobulin, ovomucoid, ovalbumin, conalbumin as the main component of egg white allergen, gliadin as the main component of wheat allergen, proteins with molecular weights of 24 kDa and 76 kDa, buckwheat, and peanut Arah1 which is a major protein of can be specifically exemplified.

  Further, as the allergen peptide, a chemically modified peptide such as a sugar chain-modified peptide, a phosphorylated peptide, an asylated peptide, an acetylated peptide, a methylated peptide, or a ubiquitinated peptide can be used. It may be a modified peptide or an artificially chemically modified peptide. Furthermore, as a peptide containing an allergen epitope, an epitope-containing peptide in which at least two or more amino acids are added to the N-terminal side and / or the C-terminal side of a peptide part such as a 7-15 amino acid size that binds to an MHC class II molecule When used, it is preferable in that it reacts with a patient antibody several times to several tens of times with high sensitivity. A peptide containing an allergen epitope such as an epitope-containing peptide in which at least two or more amino acids are added to the N-terminal side and / or the C-terminal side of the peptide part that binds to such MHC class II molecule may be prepared by peptide synthesis. However, it can also be produced as a protease-degrading peptide containing an allergen epitope. Examples of such proteases include trypsin, chymotrypsin, cathepsin, and lysyl endopeptidase. In particular, when the allergen is a food allergen, a trypsin-degrading peptide can be preferably exemplified as a protease-degrading peptide.

When the protein / peptide immobilization chip prepared by the protein / peptide immobilization method of the present invention is used, allergen epitopes can be advantageously determined by immunoassay. Such an immunoassay is not particularly limited as long as it is a known immunoassay capable of detecting an allergen-recognizing antibody in a sample captured on a protein / peptide-immobilized chip, but an ELISA using a labeled secondary antibody is preferable, and labeled 2 Examples of secondary antibodies include fluorescently labeled secondary antibodies such as Cy3, Cy5, FITC and rhodamine, enzyme-labeled secondary antibodies such as peroxidase and alkaline phosphatase, magnetic bead-labeled secondary antibodies, and infrared-labeled secondary antibodies. As the secondary antibody, an anti-IgG antibody or an anti-IgE antibody can be exemplified, and a Cy3-labeled anti-IgG antibody or a Cy3-labeled anti-IgE antibody can be preferably exemplified. As the secondary antibody, an antibody Fab fragment, F (ab ′) ₂ fragment or the like can be used. For example, the Fab fragment can be obtained by treating the antibody with papain or the like, and F (ab ′) _2. Fragments can be prepared by treating with pepsin or the like.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

1. Experimental Materials Gene silicon C4 (3 mm × 3 mm), Gene slide DLC (Toyo Kohan), 96-well microplate U bottom (Greiner), and 384-well microplate flat bottom (NUNC) were purchased and used. Ovomucoid (OVM) (SIGMA), Freund's complete adjuvant (DIFCO), Nembutal (Dainippon Pharmaceutical), Hybond-C Gridded membranes (Amersham), skim milk, Peroxidase-F (ab ') ₂ goat anti rabbit IgG (H + L ) (ZYMED), ECL (Amersham), CNBr-cativated Sepharose 4B (Pharmacia), ImmunoPure IgG Purification Kit (PIERCE), 7M Guanigine-HCl (pH 8.6), Dithiothreitol (DTT), Trypsin (Sigma or Promega), α- Cyano-4-hydroxycinnamic acid (Sigma), Non-immunized rabbit IgG, Goat anti-rabbit IgG (H + L) Cy3, Cy5conjugate (ZYMED), anti-human IgG antibody (Fc specific) Cy3, Cy5conjugate (ZYMED), anti-human Purchase and use IgE antibody (Fc specific) Cy3, Cy5conjugate (ZYMED), WSCD / HCl (Peptide Research Laboratories), NHS (Peptide Research Laboratories), DMSO (Wako Pure Chemicals), antiseptic (0.5% thimerosal) It was.

2. Basic operation In order to examine various conditions in the detection of an antigen (allergen) using a diamond / DLC (Diamond-like Carbon) chip and the quantification of a specific antibody, the following series of operations (1) to (6) were performed. The basic operation procedure from chip activation to fluorescence detection is shown in FIG.

(1) Activation of DLC chip The DLC chip is placed in a 96-well microplate, washed once with MilliQ water, and then 50 μL of activation reagent (100 mM WSC, 20 mM NHS, 0.1 M potassium phosphate buffer; pH 6.0) Was dispensed onto a plate and allowed to react with shaking in the dark at room temperature for 30 minutes. After removing the reaction solution by suction, the chip was thoroughly washed with MilliQ water, the chip was transferred to an 8 tube, and the moisture of the chip was immediately and completely removed using a tabletop centrifuge.

(2) Coupling reaction of antigen (allergen) DMSO was added to 0.1 M potassium phosphate buffer (pH 6.0) at a concentration of 30%, and the allergen to be spotted was dissolved in this solution. Allergens were spotted using a STAMPUMAN microarrayer (Japan Laser Electronics). The chip was set on a microarrayer sampling holder and spot operation was performed. After spotting, the chip was incubated at 37 ° C. in a cool incubator: CN-25A (Mitsubishi Electric Engineering).

(3) Blocking operation of unreacted active groups The chip was transferred to a 96-well microplate, 50 mM TTBS [0.05% Tween 20, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl] was added, and a plate shaker was used. A cleaning operation was performed. In order to treat the active groups remaining after the immobilization reaction, 150 mM NaCl-containing 1M Tris-HCl (pH 8.0) was added, and the reaction was allowed to proceed at room temperature for 1 hour in a light-shielded state to completely inactivate unreacted groups. Furthermore, in order to block the nonspecific reaction of the antibody to the chip, it was reacted overnight at 4 ° C. with a PBS solution containing 0.1% BSA under light-shielding conditions.

(4) Antigen (allergen) recognition antibody capture reaction The blocking buffer is aspirated and the primary antibody is diluted to various concentrations with antibody dilution buffer [60 mg / mL BSA, PBS (pH 7.4), 0.05% Tween 20]. After that, the solution was dispensed to a plate containing chips and reacted while shaking at room temperature for 5 hours under light-shielding conditions.

(5) Reaction of secondary antibody The primary antibody reaction solution was removed by suction, a washing buffer was added, and washing was performed using a plate shaker. Next, the fluorescently labeled secondary antibody is diluted to 1.5 μg / mL with a PBS solution containing 0.1% BSA, dispensed onto a plate containing the chip, and shaken at room temperature for 1 hour under light-shielded conditions. And reacted.

(6) Detection of antibody captured on antigen chip After reaction with the secondary antibody, the reaction solution was removed by suction, the chip was washed with a washing buffer and MilliQ water, and spin-down was performed to remove moisture. Next, the fluorescence intensity was measured using a FLA-8000 fluorescence scanner: FLA-8000 (FUJIFILM), and the fluorescence intensity of the spots obtained from each chip was digitized.

  A method of immobilizing a protein / peptide on a chip by spotting on a chemically modified protein / peptide diamond / DLC (Diamond-like Carbon) chip chip, performing a coupling reaction, and then blocking unreacted active groups The optimum conditions for immobilizing the protein / peptide on the chip substrate without increasing the function of the protein / peptide, increasing the binding amount, or maintaining the function on the substrate without impairing the function were studied.

1. Examination of pretreatment conditions for spotting protein / peptide After the chip activation reaction in the basic operation (1), the chip is washed with MilliQ water, and then the protein / peptide is immobilized by the coupling reaction in the basic operation (2). As the pretreatment for the protein / peptide immobilization operation, the influence of sufficient dehumidification and drying on the protein / peptide immobilization amount and the uniformity of the immobilization amount was examined.

  The conditions for drying the chips after centrifugal dehydration in the basic operation (1) were examined. As an antigen, various concentrations (50, 75, 100, 200 fmol / spot) of ovomucoid are used, 10-fold diluted patient serum is used as the primary antibody, and 1.5 μg / ml of Cy3 label is used as the secondary antibody. Goat anti-human IgE antibody was used. As the drying treatment, 1) vacuum drying treatment with a vacuum desiccator for 1 hour, 2) heating drying treatment at 37 to 42 ° C. for 1 hour, and 3) drying treatment at room temperature (RT) for 1 hour were respectively compared. The results are shown in FIG. From these results, optimal conditions were obtained in the increase in the amount of protein / peptide immobilization and homogeneity after drying under reduced pressure for 1 hour.

2. Examination of Solvent for Immobilizing Protein / Peptide The activity of protein / peptide depends on complex three-dimensional structure. Solvent for immobilizing protein / peptide on the substrate surface (0.1M potassium phosphate buffer) The additive added to the liquid, pH 6.0) is important. Therefore, as additives, 1) 10-30% glycerol, which is generally used as a protein stabilizer, 2) 20-40% DMSO, 3) 20-40% PEG (molecular weight 300) are used respectively. As for the immobilization amount of protein / peptide and the effect on the uniformity of the immobilization amount, the substrate type [Diamond coating (GENE DIA), diamond-like carbon (DLC) chip], substrate material (silicon, glass, (Stainless steel) was also examined.

  As an antigen (allergen), 100 fmol / spot α-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin, ovomucoid, ovalbumin, and conalbumin were used. Further, 10-fold diluted patient serum was used as the primary antibody, and 1.5 μg / ml of Cy3-labeled goat anti-human IgE antibody was used as the secondary antibody.

  First, as a result of using 10-30% glycerol instead of 30% DMSO in the above basic operation (2), 10-30% glycerol was used as a protein / peptide spotting additive as a substrate type (diamond It turned out to be ineffective in all cases, regardless of coating, diamond-like carbon chips) and substrate materials (silicon, glass, stainless steel).

  Therefore, 1) 30% DMSO, 2) 30% PEG, and 3) blank (-) were examined in the same manner. The results are shown in FIG. As a result, by adding 30% DMSO and 30% PEG as protein / peptide spotting additives, the reactivity between the binding amount of the protein / peptide and the antibody was maintained, and high reactivity was realized. It was found that between 30% DMSO and 30% PEG, only 30% PEG had a higher spotting efficiency. In this case, it was found that the substrate type (diamond coating, diamond-like carbon chip) and the substrate material (silicon, glass, stainless steel) were common.

Next, when 30% DMSO is added as a protein / peptide spotting additive, the amount of protein / peptide binding and antibody in each of silicon coated diamond gene (GENE DIA) and diamond-like carbon (DLC) chips The reactivity of was investigated. After activation of the chip in the above basic operation (1), rabbit IgG or anti-mouse TNF-α antibody was diluted to 1.5 μg / ml with 30% DMSO / 0.1 M MES (pH 4.5), and the room temperature or 37 The coupling reaction was carried out at 1 ° C for 1 hour with light shielding. Unreacted protein was removed by washing 10 times with TBS containing 0.05% Tween20. After adding the cleaning solution, the solution was allowed to stand for 30 seconds and removed by suction. For blocking, 1 M Tris-HCl (pH 8.0) containing 150 mM NaCl was added, and the mixture was reacted at room temperature for 1 hour under light-shielded conditions, and then reacted overnight at 4 ° C. in a PBS solution containing 0.1% BSA. . As the secondary antibody, an F (ab ′) ₂ fragment of goat against alkaline phosphatase (AP) -labeled anti-rabbit IgG (H + L) was used. As a substrate for AP, pNPP was adjusted to 2 mg / ml with 0.1 M 2-amino-2-methyl-1,3-propanediol buffer (pH 10.3) and reacted at room temperature for 5 minutes. The reaction was stopped with .75N NaOH and the absorbance at 405 nm was measured. The results are shown in FIG.

  FIG. 4 shows the measurement after spotting with and without addition of 30% DMSO, followed by a coupling reaction at room temperature (RT) or 37 ° C., and then reacting with an enzyme-labeled anti-IgG antibody for 1 hour. Results are shown. As a result, it was found that adding 30% DMSO and spotting increased the amount of antibody binding. In 10% DMSO, the reaction temperature hardly affected the reaction. In addition, common results were obtained regardless of the substrate material (silicon, glass, stainless steel).

3. Examination of reaction temperature when spotting protein / peptide When DLC / gene diamond was immobilized, the binding amount was higher at room temperature or 37 ° C than at 4 ° C. Between room temperature and 37 ° C., the amount of immobilization was somewhat higher at 37 ° C. (FIG. 4).

4). Examination of the time for spotting the protein / peptide When immobilizing the protein / peptide, DLC and Gene Diamond examined the optimum conditions for the reaction time of the immobilization reaction (1 hr, 3 hr, overnight). As a result, the reaction was performed for 3 hours at a reaction temperature of 37 ° C. (up to 42 ° C.), and the amount of protein / peptide binding on the chip was the largest and could be stably bound without variation.

5. Examination of drying treatment conditions after spotting After spotting ovomucoid at various concentrations (50, 75, 100, 200 fmol / spot) as an antigen, the chip was placed at 4 ° C, room temperature (RT), and 37 ° C. Dry for 3 hours. After blocking with 1M Tris-HCl (pH 8) / 150 mM NaCl and BSA, the patient pool serum was diluted 10-fold with a PBS / 0.05% Tween 20 solution containing 60% BSA and reacted at room temperature for 5 hours. About 1.5 μg / mL of Cy3-labeled goat anti-human IgE antibody was used as a secondary antibody for 1 hour to detect fluorescence. The results are shown in FIG. Further, fluorescence was detected in the same manner except that the drying treatment for 3 hours was replaced with the drying treatment overnight. The results are shown in FIG.

  From the results shown in FIGS. 5 and 6, the optimum treatment conditions after spotting were obtained at 37 ° C. for 3 hours in terms of increase in protein / peptide immobilization amount and uniformity.

  It was examined in detail whether a small amount of protein can be immobilized while maintaining biological function by adding glycerol or dimethyl sulfoxide (DMSO) as a spotting additive.

1. Method (1) Protein Coupling, Washing and Blocking After activating the chip with 100 mM WSC and 20 mM NHS in a 1.5 ml flat-bottomed assist tube, protein coupling in the direct antibody binding method or protein G indirect antibody binding method, respectively. Went. As a direct antibody binding method, a rabbit IgG or anti-mouse TNF-α antibody was diluted with 0.1M MES (pH 4.5) to 1.5 μg / mL, and then a coupling reaction was performed. In addition, as a protein G indirect antibody binding method, protein G was diluted to 20 μg / mL with 0.1 M MES (pH 4.5), and protein G molecules were coupled. Unreacted protein was removed by washing 10 times with TBS containing 0.05% Tween20. For washing, the washing solution was added and left still for 30 seconds, followed by suction removal. For blocking, 1M Tris-HCl (pH 8.0) containing 150 mM NaCl was added and reacted at room temperature for 1 hour, and then reacted overnight at 4 ° C. in a PBS solution containing 0.1% BSA. Further, in the case of protein G indirect antibody binding method, rabbit IgG or anti-mouse TNF-α antibody is diluted with PBS containing 0.1% BSA at 0.5 μg / mL and shaken at room temperature for 1 hour on the chip. Antibody molecules were immobilized.

(2) Detection of antibody molecules immobilized on the chip The amount of antibody molecules immobilized on the chip was quantitatively calculated from the calibration curve by measuring the activity of the enzyme labeled with the enzyme-labeled secondary antibody. As the secondary antibody, a goat F (ab ′) 2 fragment against AP-labeled anti-rabbit IgG (H + L) was used. As a substrate for AP, pNPP was prepared to 2 mg / mL with 0.1 M 2-amino-2-methyl-1,3-propandiol buffer (pH 10.3), reacted at room temperature for 5 minutes, The reaction was stopped with 75N NaOH and the absorbance at 405 nm was measured.

(3) Detection of TNF-α antigen captured on antibody chip The binding amount of the antigen molecule captured by the antibody molecule immobilized on the chip was measured using a mouse TNF-α immunoassay kit manufactured by Biosource. The antigen solution was prepared by preparing mouse TNF-α with a blocking solution containing 0.05% Tween 20 to 6250 pg / mL, reacting with the antibody chip at room temperature for 90 minutes, and then reacting with biotinylated anti-mouse TNF-α antibody by the ABC method. Detected.

2. Results (1) Effect of glycerol First, the effect of glycerol addition during antibody coupling in the direct antibody binding method was examined. In the case of DLC, the amount of antibody binding decreased in the presence of 30% glycerol, whereas the amount of antibody binding increased when Gene Diamond was used (see FIG. 7). This difference is presumed to be due to the physicochemical characteristics of the DLC and gene diamond substrate surfaces, but further investigation is required. Furthermore, in the protein G indirect antibody binding method, the amount of antibody binding decreased in the presence of 30% glycerol in the case of DLC (see FIG. 7). Therefore, it was considered that the addition of glycerol is not appropriate for protein stabilization.

(2) Effect of DMSO Next, the effect of adding DMSO instead of glycerol during antibody coupling in the direct antibody binding method was examined. In both DLC and Gene Diamond, the amount of antibody binding increased dramatically when 30% DMSO was added (see FIG. 8). Although no data is shown, 10% DMSO had little effect. However, there was a difference in reaction temperature between DLC and Gene Diamond, and DLC had a higher amount of antibody binding at 37 ° C. than room temperature, whereas Gene Diamond gave the opposite result. Furthermore, in order to examine whether not only the antibody binding amount but also the activity of the antibody is maintained, it was analyzed whether the anti-TNF-α antibody immobilized by the direct antibody binding method can capture the antigen TNF-α. As a result, it was confirmed that not only the antibody but also the antigen binding amount increased in the presence of 30% DMSO (see FIG. 9). From this, it has been clarified that the antigen-capturing ability is increased by increasing the amount of immobilization even with a small amount of antibody in the presence of 30% DMSO.

  Using a chip produced by the method of immobilizing proteins / peptides on the chip of the present invention (the basic method described above), food allergy samples were actually analyzed for children. After receiving approval from the University of Tokushima Ethics Committee and approval from the Kagawa National Children's Hospital, clinical specimens were obtained from Kagawa Children's Hospital (Prof. Michinori Ito) with sufficient informed consent. We compared the results with healthy controls without food allergies. First, in order to test allergen components of egg and milk, 10 fmol of each allergen protein per spot was spotted on a DLC chip using silicon as a substrate, and the serum was diluted 100 times to react. Detection was carried out using a Cy3-labeled goat anti-human IgG antibody. Since the dilution rate of rabbit serum immunized as strongly as possible is not helpful for human serum, it is not shown in the figure, but one sample is selected from each patient and healthy person, and 50, 100, 200, 400, 800, The reactivity with the chip was assayed by diluting 1600 times. As a result, the measurable dilution with less nonspecific reaction in the reactivity with the chip was determined to be in the range of 400-1600 times dilution.

  The measurement results obtained by diluting patient serum 1000 times are shown in FIG. Most of the patients were milk allergic and reacted with α-, β-, and κ-casein with their respective reaction intensities. Some patients also reacted with α-lactoalbumin, but the proportion was small compared to casein. However, even those who are allergic to chief complaints of milk allergies have been found to be reactive with egg components such as OVM, ovalbumin, and conalubumin (see FIG. 11). . In the above measurement system using a diamond / DLC chip, at least 18-25 items can be qualitatively and quantitatively measured with 1 microliter of serum, and the conventional test method is only qualitative test, and 250 to 500 micron per test. It was estimated that it was superior in terms of patient burden and convenience compared to the testing method that required liters.

  As in the case of IgG described above, for the purpose of testing allergen components of eggs and milk, 100 fmol of each allergen protein was spotted per spot on a DLC chip using silicon as a substrate, and the serum was diluted 10 times and reacted. The detection was performed using a goat anti-human IgE antibody labeled with Cy3 as the secondary antibody. The measurement results are shown in FIG.

Claims (16)

A method of immobilizing a protein / peptide on a chip by activating a chemically modified diamond / DLC (Diamond-like Carbon) chip with an activating reagent and then spotting the protein / peptide on the chip to perform a coupling reaction Then, after being activated with an activating reagent, a drying treatment for 30 to 120 minutes is performed as a drying treatment for washing water for washing the chip, and after the coupling reaction, 2 to 4 hours at 30 to 45 ° C. A method for immobilizing a protein / peptide characterized by drying. 2. The protein / peptide immobilization method according to claim 1, wherein the washing water is MilliQ water. 3. The protein / peptide immobilization method according to claim 1, wherein dimethyl sulfoxide (DMSO) or polyethylene glycol (PEG) is used as a spotting additive. The protein / peptide immobilization method according to claim 3, wherein 20-40% DMSO or 20-40% PEG is used. The method for immobilizing a protein / peptide according to claim 4, wherein 30% DMSO or 30% PEG is used. 6. The protein / peptide immobilization method according to any one of claims 1 to 5, wherein the activating reagent is a solution containing WSCD.HCl and NHS. The protein / peptide immobilization method according to any one of claims 1 to 6, wherein an unreacted active group is blocked after the coupling reaction. The method for immobilizing a protein / peptide according to any one of claims 1 to 7, wherein the protein / peptide is an antigen or an antibody. 9. The protein / peptide immobilization method according to claim 8, wherein the antigen is an allergen. 10. The protein / peptide immobilization method according to claim 9, wherein the allergen is an allergen epitope or a peptide containing an allergen epitope. 11. The protein / peptide immobilization method according to claim 10, wherein a chemically modified peptide is used as the allergen epitope or the peptide containing the allergen epitope. The epitope-containing peptide to which at least two or more amino acids are added to the N-terminal side and / or the C-terminal side of a peptide portion that binds to an MHC class II molecule is used as a peptide containing an allergen epitope. The protein / peptide immobilization method described. The method for immobilizing a protein / peptide according to any one of claims 10 to 12, wherein a protease-degrading peptide containing an allergen epitope is used as the peptide containing an allergen epitope. 14. The protein / peptide immobilization method according to claim 13, wherein the protease-degrading peptide is a trypsin-degrading peptide. The method for immobilizing a protein / peptide according to any one of claims 10 to 14, wherein a food allergen is used as the allergen. As a food allergen, one or more allergens selected from sodium caseinate, α-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin, ovomucoid, ovalbumin, and conalbumin are used. The method for immobilizing a protein / peptide according to claim 15.