JP2013217848A - Protein adsorption evaluation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a protein adsorption evaluation method that is simple and inexpensive without requiring a special device.SOLUTION: After the protein fluorescence-labeled by a fluorescence label is brought into contact with a material, the fluorescence intensity of the protein adsorbed to the target material is measured. The fluorescence label has a fluorescein group. After the protein fluorescence-labeled by the fluorescence label is brought into contact with a material, the concentration of the fluorescence-labeled protein solution is measured.

Description

The present invention relates to a method for measuring the adsorption of biologically related substances such as various proteins to parts, instruments, containers and the like used in, for example, clinical diagnosis and biochemical experiments.

In recent years, therapeutic agents containing protein as an active ingredient are increasing, and their effective use is required. However, there is a decrease in active ingredients due to adsorption to containers, and countermeasures are required. In addition, for the purpose of early detection of diseases and the like, it is required to increase the sensitivity of examinations and to improve the sensitivity of diagnostic agents, but sufficient sensitivity is not obtained.
The reason for this is that in the diagnosis of detecting specific substances in the presence of biomolecules such as serum, coexisting biomolecules, secondary antibodies, luminescent substrates, etc. adsorb nonspecifically to instruments and containers, resulting in noise. This is because it increases and prevents high sensitivity. In order to take these measures, a technique for measuring the amount of adsorption is required, and a simple method is required. However, the measurement of the amount of protein in the container is insensitive and requires time, such as using liquid chromatography.

A method has been proposed in which a protein having enzyme activity is adsorbed and the amount of adsorption is determined using the enzyme reaction (Patent Document 1). Although this method has an advantage of high sensitivity, it requires work and time to perform an enzyme reaction, and a reagent is necessary to perform the reaction. For this reason, there exists a fault which a running cost becomes high.

As a method for directly measuring proteins, a method using X-ray photoelectron spectroscopy (hereinafter abbreviated as XPS) has been proposed (Patent Document 2). XPS has the disadvantage that the device itself is expensive and the method of analyzing with high vacuum cannot speed up the work. Moreover, although analysis is performed using nitrogen atoms as an index, there is a drawback that the analysis becomes difficult if the substance to be subjected to adsorption evaluation contains nitrogen atoms.

JP 2009-216572 A Special table 2008-526377

This inventor made it the subject to provide the method of enabling the evaluation of the quantity which protein adsorb | sucks easily and cheaply without requiring a special apparatus.

[1] A protein adsorption measurement method in which a fluorescently labeled protein with a fluorescent label is brought into contact with the material, and then the fluorescence of the material is measured.
[2] The adsorption measurement method according to [1], wherein the fluorescent label has a fluorescein group.
[3] The measurement method according to [1] or [2], wherein a protein fluorescently labeled with a fluorescent label is brought into contact with a material, and then the concentration of the fluorescently labeled protein solution is measured.

According to the present invention, simple and quick protein adsorption evaluation can be performed, and the development of containers and materials with suppressed protein adsorption can be rapidly advanced. Since proteins are used in pharmaceuticals and research reagents, improvements are desired in terms of concentration reduction and quantitativeness due to adsorption.

The evaluation method of the present invention is a method of measuring fluorescence of a protein component adsorbed on a target material after bringing the fluorescently labeled protein into contact with the target material.
It is also possible to measure the amount of protein contained in the supernatant after it has been adsorbed after contacting the material.
The material in contact with the fluorescently labeled protein can be subjected to fluorescence measurement as it is, or it can be washed with a buffer or water, the excess fluorescent protein can be removed, and the adsorbed protein component can be detected.

The contact condition may be adjusted according to the protein solution composition, and pH, salt concentration, etc. can be freely selected. The contact temperature is also an evaluation item and can be set freely, but if it exceeds 60 ° C., it must be considered that protein denaturation has occurred. Further, since the solution freezes and mass transfer hardly occurs at −20 ° C. or lower, it is preferably performed at −10 ° C. to 60 ° C.

The protein to be used is not particularly limited, and those having a molecular weight of 3000 to 300,000 can be easily used. The type of protein that is desired to be adsorbed may be selected, and insulin, immunoglobulin, albumin, collagen, globulin, fibrinogen, etc. are often used and are easily available. Moreover, these mixtures may be used and the inside of serum may be used as a mixture. Moreover, milk protein, soybean protein, etc. used as food can also be used.

Protein fluorescent labeling can be made by conventional methods. For example, a kit can be used. More specifically, it is also possible to use Fluorocein Labeling Kit, HiLyte Fluor 555 Labeling Kit-NH2, HiLyte Fluor 647 Labeling Kit-NH2, HiLyte Fluor 750 Labeling Kit-NH2 manufactured by Dojindo Laboratories Co., Ltd. . There is no particular limitation on the structure of the substituent to be fluorescentized. Excitation light and detection light may be selected according to the purpose of use.

Fluorescent agents include fluorescein isothiocyanate, 5- or 6- (N-succinimidyloxycarbonyl) -fluorescein 3 ', 6'-diacetate, N-ethyl-N'-[5- (N "- Succinimidyloxycarbonyl) pentyl] indocarbocyanine chloride, N-ethyl-N '-[5- (N ”-succinimidyloxycarbonyl) pentyl] -3,3,3', 3'-tetramethyl- 2,2'-Indocarbocyanine chloride, 1H, 5H ,, 11H, 15H-xenzenol [2,3,4-i]: 5,6,7-i'j '] diquinolizine-18-nium, 9- ( 2-sulfo-4-chlorosulfophenyl) -2,3,6,7,12,13,16,17-octahydro-intramolecular salts and the like are typical examples. Fluorescein isothiocyanate, which is particularly often used, is preferred.

Fluorescently labeled proteins can be used without purification, or can be used after purification that removes fluorescent low molecular weight components (fluorescent agents or their reaction products). . For purification, a column, an adsorbent, dialysis, lyophilization, salting out, etc. can be used.

The fluorescence can be detected by visual observation, a fluorescence spectrometer, a fluorescence microscope, a fluorescence scanner, a camera with a fluorescence filter, or the like. Although the wavelength used for fluorescence is not limited, it is preferable because excitation at 495 nm and detection of fluorescence at 520 nm are the same as fluorescein in which filters and the like are widely used. Moreover, it becomes possible to investigate with high precision by measuring the absorbance or fluorescence of the protein that has not been adsorbed at the same time.

As the material to be evaluated, glass, plastic, metal, a composite material thereof or the like can be used, and the change in adsorptivity can be examined by surface treatment or the like.

In order to suppress the adsorption of proteins to glass, plastics, etc., it is possible to use skim milk, albumin, globulin, collagenase, and serum as other proteins. Further, pyrroloquinoline quinone can be used as the low molecular weight compound. The pyrroloquinoline quinone can also be used as an alkali metal salt or an ammonium salt.

  The present invention will be described more specifically with reference to examples and comparative examples, but the present invention is not limited to these examples.

Example 1 Synthesis of Fluorescent Labeled Protein (FITC-Insulin) 50 mg of Sigma-Aldrich insulin is dissolved in 0.5 ml of water in which 2.8% NaHCO ₃ and 1.7% Na ₂ CO ₃ are dissolved. To this, 11 mg of fluorescein isothiocyanate (FITC) manufactured by Dojindo Laboratories Co., Ltd. dissolved in dimethyl sulfoxide is added. This was reacted at room temperature for 1 day. This solution was placed in a Thermo dialysis machine Slide LyzerMz 3000 and dialyzed for one week. The solution from which the low molecules had been removed was lyophilized and redissolved with water to prepare a FICT-insulin solution.

Example 2 Glass container 10 ml of an invitrogen phosphate buffer (PBS pH 7.4) was added to a 9 cm diameter borosilicate glass petri dish. 1 ml of a solution prepared by diluting FICT-insulin prepared in Example 1 to 0.5 mg / ML with a phosphate buffer was added. This was left at 30 ° C. for 2 days. The supernatant was taken and the container was washed 3 times with 10 ml of phosphate buffer.
A TE-2000S fluorescent microscope (NIKON) was used for this container, and a fluorescent filter GFP block (NIKON) was attached to a high-pressure mercury lamp as a light source. A fluorescent photograph was taken at this time. This was color-analyzed with the paint software attached to Windows XP, and the green numerical value (G component) was converted into a numerical value as the amount of adsorption.
The supernatant was measured for absorption at 490 nm using a HITACHI U-2000 Spectrophotometer. The recovery rate was measured from this. The results are shown in Table 1.
According to the present invention, protein adsorption can be easily measured.

Example 3 In order to suppress the adsorption of surface-treated glass protein by skim milk, it was decided to suppress the adsorption of insulin by coating the surface with another protein, DIFCO skim milk. DIFCO skim milk 0.5 g was suspended in 50 ml of water. 5 ml of this solution was placed in the same petri dish as in Example 2 and treated at 37 ° C. for 1.5 hours. The solution was removed and washed with 5 ml of phosphate buffer. FITC-insulin was added to the petri dish in the same manner as in Example 2 and the amount of adsorption was measured. The results are shown in Table 1. Skim milk, known as protein adsorption blocking, inhibited the adsorption of fluorescently labeled insulin.

Example 4 Surface treatment with pyrroloquinoline quinone 0.1 g of pyrroloquinoline quinone was suspended in 50 ml of water. 5 ml of this solution was placed in the same petri dish as in Example 2 and treated at 37 ° C. for 1.5 hours. The solution was removed and washed with 5 ml of phosphate buffer. FITC-insulin was added to the petri dish in the same manner as in Example 2 and the amount of adsorption was measured. The results are shown in Table 1. Pyrroloquinoline quinone had the effect of suppressing protein adsorption.

Comparative Examples 1 and 2 As an untreated glass container, the experiment of Example 2 was conducted without adding FICT-insulin. This is referred to as Comparative Example 1. Further, an experiment conducted without adding FICT-insulin under the conditions of Example 3 as the surface-treated glass is referred to as Comparative Example 2.

The present invention facilitates the development of materials that suppress or promote protein adsorption, and development fields such as container materials for food, biocompatible materials such as artificial organs, various manufacturing equipment materials, laboratory instrument materials, etc. It is effective for.

Claims (3)

A protein adsorption measurement method in which a fluorescently labeled protein with a fluorescent label is brought into contact with the material, and then the fluorescence of the material is measured. The adsorption measurement method according to claim 1, wherein the fluorescent label has a fluorescein group. The method according to claim 1 or 2, wherein the concentration of the fluorescently labeled protein solution is measured after contacting the fluorescently labeled protein with a fluorescent label in contact with the material.