JP4535490B2 - Protein adsorption prevention method - Google Patents

Description

  The present invention relates to a method for preventing adsorption of proteins and / or polypeptides. More specifically, the protein and / or polypeptide in the sample is adsorbed or chemically bound to the surface of the material constituting the physics and chemistry instrument used for the research and development using the protein and / or polypeptide, and the phosphorylcholine group-introduced protein is adsorbed or chemically bonded. Relates to a method for preventing non-specific adsorption on the surface of the material.

  Conventionally, physics and chemistry instruments used for research and development using proteins and / or polypeptides are composed of plastics such as polystyrene and polypropylene, glass, or substrates such as metals and ceramics.

  Since proteins and / or polypeptides have a strong adsorption action on these substrates, the proteins and / or polypeptides in the specimen are placed on the surface of physics and chemistry instruments during storage, dilution, and analysis of reagents and test solutions. Adsorption causes problems such as a decrease in concentration, a change in protein and / or polypeptide structure due to adsorption, and a decrease in activity.

On the other hand, in the measurement of an immunologically active substance using an antigen-antibody reaction, in order to prevent these non-specific adsorptions, (1) a method of performing an immunoassay with a weakly acidic buffer solution at pH 5-6,
(2) A method of blocking an excess protein binding site on the solid phase with ovalbumin, bovine serum albumin, fetal bovine serum, normal serum, etc. after adsorbing antibodies etc., (3) Main component of organic acid A method using a non-specific adsorption inhibitor in which milk protein is dissolved in a buffer and sterilized is used (Patent Document 1).

  However, depending on these methods, the protein and / or polypeptide non-specific adsorption preventing ability is not sufficient. Therefore, in the field of clinical diagnosis and the like, development of a better non-specific protein adsorption inhibitor and its prevention method is desired.

  From this point of view, a method of surface treatment with a polymer having a specific phosphorylcholine group is proposed as a method capable of sufficiently suppressing nonspecific adsorption of proteins and / or polypeptides in measuring immunologically active substances. (Patent Document 2).

  However, since this method coats with a water-soluble copolymer composed of a phosphorylcholine group and a component having a high affinity for the coated substrate, it is difficult to efficiently cause only the phosphorylcholine group to appear on the surface. There is a problem that the protein adsorption cannot be completely suppressed.

JP-A-01-217266 JP-A-10-114800

  The present invention uses a high adsorption ability to a base material inherent in protein, and further covers a base material with a phosphorylcholine group efficiently, and thereby has a superior anti-adsorption effect on proteins and / or polypeptides. A non-specific adsorption preventing method is provided.

That is, the present invention has a protein as a blocking agent on the surface of a material and chemically binds a phosphorylcholine group represented by the following formula (1) to the amino group or carboxyl group of the protein, thereby Alternatively, the present invention provides a protein adsorption preventing method for preventing a polypeptide from adsorbing nonspecifically on the surface of the material.

  According to the present invention, it is possible to extremely efficiently suppress the phenomenon that a protein in a sample is non-specifically adsorbed on the surface of a base material constituting a physics and chemistry instrument used for research and development using proteins.

  The method of the present invention has the great advantage that various substrate surfaces can be modified. And it is excellent in the introduction efficiency of the phosphorylcholine group with respect to a base material. As a result, the non-specific adsorption preventing effect is larger and more excellent as compared with the adsorption preventing method by introducing a phosphorylcholine group by a polymer coating.

  The present invention is described in detail below.

  In the present invention, the protein is adsorbed or chemically bound to the surface of the substrate (for example, the surface of a material constituting a physics and chemistry instrument used for research and development using proteins and / or polypeptides, or the surface of a plate for biochemical testing). Next, the phosphorylcholine group-containing compound represented by the above formula (1) is reacted with the protein to chemically bond the phosphorylcholine group of the above formula (1) directly to the protein on the surface of the material. A blocking agent consisting of a protein with chemically bonded groups is prepared. The blocking agent prevents non-specific adsorption of proteins and / or polypeptides in the specimen used for research and development on the surface of the material.

The present invention is performed by the following steps.
Step 1: Protein is adsorbed or chemically bound to the surface of an arbitrary material by a known method or a method developed in the future.

  Step 2: An aldehyde or hydrate obtained by oxidative cleavage of glycerophosphorylcholine is adsorbed or chemically bound to a protein, and the phosphorylcholine group of formula (1) is converted to a reductive amination reaction. Add to the material surface. Alternatively, a phosphorylcholine derivative having an amino group or a carboxyl group is amide-bonded by an ordinary method to an adsorbed or chemically bound protein, and the phosphorylcholine group is added to the protein on the surface of the material.

  The material used in the present invention is a material that can constitute a physics and chemistry instrument, such as plastic, rubber, glass, ceramic, and metal.

"Concrete example"
Specifically, the method (step 1) for adsorbing or chemically binding proteins to the surface of these materials is performed as described in 1 and 2 below.

1. A phosphate buffer solution (1%) of bovine serum albumin was added to a 96-well plate (polystyrene) (300 μl / well) and allowed to stand overnight at room temperature. Then, the solution was discarded and excess bovine serum albumin was phosphorylated. Wash with acid buffer to obtain a well plate with bovine serum albumin adsorbed on the surface. Various proteins such as casein, lysozyme, cytochrome C, fibrinogen, and transferrin can be used instead of bovine serum albumin.

2. A phosphate buffer solution (1%) of bovine serum albumin was added (300 μl / well) to a 96-well plate (active ester surface) and allowed to stand at room temperature overnight, then the solution was discarded and excess bovine serum albumin Is washed with a phosphate buffer to obtain a well plate in which bovine serum albumin is chemically bound to the surface. Various proteins such as casein, lysozyme, cytochrome C, fibrinogen, and transferrin can be used instead of bovine serum albumin.
It is also possible to chemically bond the protein and the plate by using a plate having an amino group surface or a carboxyl group surface instead of the active ester surface and forming an amide bond by a conventional method.

  Next, the method (step 2) for introducing a phosphorylcholine group onto the surface of an adsorbed or chemically bound protein is specifically carried out as 3 or 4 below.

3. Phosphatidyl glyceraldehyde (phosphate buffer solution) is added to the 96-well plate treated by the method 1 or 2, and left at room temperature for 6 hours. Then, sodium cyanoborate is added at room temperature and left overnight at room temperature to introduce a phosphorylcholine group into the amino group. The material is washed with a phosphate buffer and then dried to obtain a material having phosphorylcholine groups directly on the surface. Any reaction solvent can be used as long as it is a protic solvent such as methanol, water, ethanol, and 2-propanol in addition to the phosphate buffer, but the introduction rate when methanol is used tends to be high. Even if it omits the process of adding said sodium cyanoboronate as needed, there is no influence on an effect.

ｎ＝１〜１２までの整数

ｎ＝１〜１２までの整数 4). A phosphorylcholine derivative aqueous solution and a coupling agent represented by the following formula (2) or (3) are added to the 96-well plate treated by the above method 1 or 2, and left at room temperature for 6 hours. After washing with water and drying, a material having phosphorylcholine groups directly on the surface is obtained. As the reaction solvent, tetrahydrofuran, N, N-dimethylformamide, dimethyl sulfoxide, and a mixture thereof can be used in addition to water.

n = integer from 1 to 12

n = integer from 1 to 12

As described above, the protein in the specimen is non-specifically attached to the surface of the material by adsorbing or chemically binding the protein and producing a material having the phosphorylcholine group of formula (1) added to the surface. A method for preventing protein adsorption is performed to prevent adsorption to the protein.
That is, protein adsorption prevention is achieved by setting a protein blocking agent in which the phosphorylcholine group represented by the following formula (1) is chemically bonded to the amino group or carboxyl group of the protein on the surface of the material. This protein blocking agent can be produced by a method for preparing a protein blocking agent having a step of chemically bonding a phosphorylcholine group represented by the following formula (1) to an amino group or a carboxyl group of a protein as a blocking agent. The spacer for chemically bonding the protein and the phosphorylcholine group of the formula (1) is arbitrary, and may be an methylene chain, an oxyethylene chain or the like, or an alkylene chain containing one or more amino groups.

  By the method of the present invention, a physics and chemistry instrument that prevents nonspecific adsorption to the surface of the material is manufactured. The method of the present invention has a great advantage that the phosphorylcholine group introduction efficiency is good and the surface of various materials can be modified.

In the above method, the compound containing an aldehyde obtained by oxidative cleavage reaction of glycerophosphorylcholine causes oxidative cleavage of a known glycerophosphorylcholine group by a known method. is there. In this reaction, 1,2-diol is oxidized with periodic acid, periodate, or bismuth trioxide to cleave the bond to obtain two aldehydes. Produces phosphorylcholine aldehyde and formaldehyde. The reaction is usually carried out in water or an organic solvent containing water. The reaction temperature is from 0 ° C. to room temperature. Aldehydes may undergo hydration through an equilibrium reaction in water, but do not affect the subsequent reaction with amines. Scheme 1 for preparing a monofunctional aldehyde compound containing a phosphorylcholine group is shown below.

  The aldehyde obtained by the above scheme reacts with the amino group of the protein to form a bond.

  The reductive amination reaction in which the aldehyde (or hydrate) obtained by oxidative cleavage of glycerophosphorylcholine is bound to the amino group of the protein can be easily performed by standing in a solvent or shaking. . In this reaction, both are dissolved or dispersed in water or alcohol (the third organic solvent may be mixed) to form an imine, which is then reduced with a reducing agent to obtain a secondary amine. It is. The reducing agent is preferably a mild reducing agent such as sodium cyanoborate, but other reducing agents can be used as long as phosphorylcholine is stable. The reaction is usually performed at 0 ° C. to room temperature, but may be heated in some cases. Moreover, since the bond is formed without performing reduction with sodium cyanoborate or the like, there is no problem even if it is omitted as necessary.

  By the above method, a material for a physics and chemistry instrument or a plate for a biochemical test containing an arbitrary amount of hydrophilic phosphorylcholine groups can be easily obtained.

The physics and chemistry instrument which consists of the material processed by the method of this invention prevents protein and / or polypeptide adsorb | sucking to the surface nonspecifically. The type of physics and chemistry equipment does not matter. For example, laboratory instruments such as beakers, flasks, pipettes and various containers, analyzers and columns, and packing materials thereof.
And the physics and chemistry instrument which consists of the material processed by the method of the present invention has very little adsorption of protein and / or polypeptide. For example, it was obtained by separating proteins in human serum or digesting proteins with trypsin. It can be preferably used for research such as separation of polypeptides contained in a sample.

  Next, the present invention will be described in more detail based on examples. The present invention is not limited to these examples.

Synthesis Example 1 Aldehyde compound containing phosphorylcholine group L-α-glycerophosphorylcholine (450 mg) is dissolved in 15 ml of distilled water and cooled in an ice-water bath. Add sodium periodate (750 mg) and stir for 2 hours. Add more ethylene glycol (150 mg) and stir overnight. The reaction mixture is concentrated under reduced pressure, dried under reduced pressure, and the desired product is extracted with methanol.
The structural formula and NMR spectrum are shown in FIG.

"Comparison between bovine serum albumin bound with Example 1 phosphorylcholine group and the technology described in Patent Document 2 (Japanese Patent Laid-Open No. 10-114800)"
A bovine serum albumin phosphate buffer solution (1%) was added to a 96-well plate (active ester, Sumitomo Bakelite) (300 μL / well), adsorbed at room temperature for 6 hours, washed with phosphate buffer, and synthesis example Example 1 was obtained by adding a phosphate buffer solution (1%) of 1 aldehyde compound and allowing it to stand overnight at room temperature, followed by washing with a phosphate buffer. A blocking agent N102 manufactured by Nippon Oil & Fats Co., Ltd., for which the technology of JP-A-10-114800, has been commercialized, is diluted 5-fold with a phosphate buffer and added to a 96-well plate (active ester, Sumitomo Bakelite Co., Ltd.) (300 μL / well). ), Adsorbed overnight at room temperature, washed with phosphate buffer, and used as Comparative Example 1. To Example 1 and Comparative Example 1, horseradish peroxidase 40 μg / ml phosphate buffer solution 100 μL / well was added, left at room temperature for 2 hours, and washed with phosphate buffer, peroxidase coloring kit (manufactured by Sumitomo Bakelite) was used. The amount of horseradish peroxidase adsorbed by the color development test (reaction time 30 minutes) was compared. The results are shown in FIG. The vertical axis in FIG. 2 represents absorbance, and the larger the value, the more protein adsorption has occurred, confirming that the effect of the present invention is high.

"Examples 2 to 7 Protein adsorption inhibitory effects of various proteins bound with phosphorylcholine groups"
Similarly, phosphate buffer for bovine serum albumin (Comparative Example 2), casein (Comparative Example 3), transferrin (Comparative Example 4), fibrinogen (Comparative Example 5), lysozyme (Comparative Example 6), and cytochrome C (Comparative Example 7) The solution (1%) was added to a 96-well plate (active ester manufactured by Sumitomo Bakelite Co., Ltd.) (300 μL / well), adsorbed at room temperature for 6 hours, washed with phosphate buffer, and used as comparative examples. In addition, to each of the above comparative examples, a phosphate buffer solution (1%) of the aldehyde compound of Synthesis Example 1 was added, left overnight at room temperature, then washed with phosphate buffer to obtain each example (bovine serum albumin ( Example 2), casein (Example 3), transferrin (Example 4), fibrinogen (Example 5), lysozyme (Example 6), cytochrome C (Example 7), whereas horseradish peroxidase 40 μg / Add 100 μL / well of phosphate buffer solution, leave at room temperature for 2 hours, and wash with phosphate buffer, adsorbed by horseradish puroxidase adsorbed by color development test (reaction time 10 minutes) using peroxidase coloring kit (Sumitomo Bakelite) The results are shown in Fig. 3. In each case, a comparative example using only protein as a blocking agent As a result, the protein adsorption suppression effect of the surface treatment with the phosphorylcholine group was improved.

"Examples 8 to 9: Protein adsorption inhibitory effect of proteins bound with phosphorylcholine groups"
A phosphate buffer solution (1%) of bovine serum albumin was added to a 96-well plate (active ester, manufactured by Sumitomo Bakelite) (300 μL / well), adsorbed at room temperature for 6 hours, washed with phosphate buffer, and Comparative Example It was set to 8. On the other hand, a phosphorylcholine derivative represented by the formulas (2) and (3) 1% aqueous solution 200 μL / well, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1% aqueous solution) 100 μL were added, and room temperature was added. Then, the sample was allowed to stand for 6 hours and washed with a phosphate buffer to obtain Examples 8 and 9. On the other hand, horseradish peroxidase 40 μg / ml phosphate buffer solution 100 μL / well was added, allowed to stand at room temperature for 2 hours, washed with phosphate buffer, color development test using peroxidase coloring kit (Sumitomo Bakelite) (reaction time 10) The amount of horseradish peroxidase adsorbed by (min) was compared. The results are shown in FIG. Phosphorylcholine treatment improved the protein nonspecific adsorption inhibitory effect of protein bovine serum albumin bound with phosphorylcholine group.

Since the physics and chemistry instrument made of the material processed by the protein adsorption preventing method of the present invention can efficiently prevent the protein adsorption action, the protein adsorption is performed during the storage, dilution and analysis of the reagent and test solution containing the protein. Problems such as denaturation due to concentration changes and adsorption do not occur.
In particular, it is extremely useful to carry out the method of the present invention on the material surface of a physics and chemistry instrument used for measurement of an immunologically active substance using an antigen-antibody reaction.

It is a structural formula and NMR spectrum of the aldehyde body containing the phosphorylcholine group of Synthesis Example 1. It is explanatory drawing which compared the effect of the method of this invention, and the method of patent document 2 (Example 1). It is explanatory drawing which showed that the protein nonspecific adsorption | suction suppression effect of various proteins (blocking agent) which couple | bonded the phosphorylcholine group has improved with the method of this invention (Examples 2-7). It is explanatory drawing which showed that the protein nonspecific adsorption | suction suppression effect of the bovine serum albumin couple | bonded with the phosphorylcholine group of this invention has improved (Examples 8-9).

Claims (1)

A protein and / or polypeptide in a specimen has a protein as a blocking agent on the surface of the material, and is chemically bonded to a phosphorylcholine group represented by the following formula (1) to the amino group or carboxyl group of the protein. A protein adsorption prevention method for preventing nonspecific adsorption on a surface.

Images