JPH0769934A - Production of fluorescence labeled antibody - Google Patents

Abstract

PURPOSE:To produce a fluorescence labeled antibody having high sensitivity and activity by introducing a fluorescence label in high efficiency and in a larger amount into an antibody and resultingly improving the detection accuracy and keeping the antibody activity at high level. CONSTITUTION:This fluorescence labeled antibody is produced by reacting an antibody with a fluorescence labeling agent under a high pressure of 0.5-1000MPa, preferably 50-700MPa, at 0-120 deg.C, preferably at 4-40 deg.C, for 1sec to 72hr, preferably for 30sec to 1hr, in water or a buffer solution. In this reaction, the appropriate feeding ratio of the antibody to the fluorescence labeling agent is 1:1 to 1:1000, preferably 1:50 to 1:100 in terms of weight ratio and the concentration of the antibody in the medium is preferably 10-40mg/ml. Since high pressure causes a pressure deformation of a protein e.g. an antibody by the changes of the secondary and the tertiary configuration, a reactive site existing inside the molecule and concealed under the normal conditions is brought out and revealed by this pressure deformation. Accordingly, the modifying reagent reacts easily and the labeling is performed in high efficiency.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing a fluorescently labeled antibody by binding the antibody with a fluorescently labeled reagent, particularly a diagnostic agent,
The present invention relates to a method for producing a fluorescently labeled antibody that can be used as a medicine or the like.

[0002]

2. Description of the Related Art Fluorescence-labeled antibodies obtained by labeling antibodies with fluorescent substances are widely used as diagnostic agents. This makes it possible to detect an antigen by binding an antibody with a fluorescent labeling reagent to perform fluorescent labeling and measuring the fluorescence intensity of a precipitate deposited by an antigen-antibody reaction using the fluorescent labeled antibody.

[0003] Fluorescein isothiocyanate (FITC) is a typical fluorescent labeling reagent that has been conventionally used. Conventional labeling methods use such a fluorescent labeling reagent and an antibody at room temperature and atmospheric pressure. Since the reaction was carried out, the reaction efficiency was low, and the number of FITC bound to one antibody molecule was as small as about 15. Therefore, it is desired to introduce more fluorescent substances in order to achieve higher sensitivity.

Generally, a protein is denatured in a high-concentration urea solution by dissociation of its constituent domains. However, various modification operations are performed while maintaining this denatured state, and It is known that higher reaction efficiency can be obtained as compared with modification. For example, Mizutani et al. Prepared a mediator having a high modification rate and a high electron transfer mediating ability by denaturing bovine serum albumin (BSA) in the coexistence of 6 M urea and then performing Fc modification (electrochemistry, 56. 1100 (1988)).
However, this method has a problem that after the modified enzyme is prepared, a deurea treatment must be performed using a column or the like.

On the other hand, in recent years, by placing a protein under high pressure, the higher-order structure and aggregate structure of the protein are changed or denatured, and depending on the type of protein, reversible restoration to the original state is possible. Became known. Kouto et al. Attempted a reaction under high pressure in the process of introducing ferrocene into BSA and glucose oxidase by physical adsorption. As a result, it has been reported that ferrocene can be introduced rapidly under high pressure by reacting ferrocene with BSA and glucose oxidase that have undergone pressure denaturation (S. Kunugi). , et al., Int.J.Biol.Mac
romol., 14, 210 (1992)). In addition, Okamoto et al. Performed a reduction reaction with mercaptoethanol such as ribonuclease A.
By performing the reaction at 00 MPa, the carboxymethylation reaction and the pyridylethylation reaction performed thereafter are both 10
It has been reported that it can be completed up to 0% (M. Okamoto, e
t al., Colloque INSERUM, 224 , 167 (1992). However, none of them suggests fluorescent labeling of antibodies.

[0006]

The object of the present invention is to efficiently introduce a fluorescent label into an antibody and to introduce a large amount of a fluorescent label to enhance detection accuracy and to maintain high antibody activity. Another object of the present invention is to propose a method for producing a fluorescently labeled antibody capable of producing a highly sensitive and highly active fluorescently labeled antibody.

[0007]

The present invention provides 0.5% antibody.
A method for producing a fluorescently labeled antibody, which comprises binding with a fluorescently labeled reagent under a high pressure of MPa to 1000 MPa.

The antibody used in the present invention is mainly IgG derived from all animal species such as cow, goat, rabbit, mouse, chicken and human.
It may be an immunoglobulin belonging to any class such as IgM, IgD and IgE, and may be a polyclonal or monoclonal antibody. It may also be various antibody-derived segments such as Fab and F (ab) ′.

The type of fluorescent labeling reagent used in the present invention is not particularly limited as long as it can chemically or physically bind to an antibody to perform a fluorescent labeling function. Examples of such fluorescent labeling reagents include fluorescein isothiocyanate (FITC) such as fluorescein-4-isothiocyanate, 7-benzylamino-4-nitrobenzoxadiazole, and 4
-Chloro-7-nitrobenzofurazan, 4-fluoro-
Examples thereof include 7-sulfobenzofurazan ammonium salt and sulforhodamine 101 acid chloride. In addition, Wako Pure Chemical Industries, Ltd. catalog “LIFE SCIENCE”
The fluorescent substances described in "Reagents" can also be used.

The mode of binding between these fluorescent labeling substances and the antibody is not particularly limited, and chemical binding to amino acid residues having a thiol group, an amino group, a carboxylic acid group, a hydroxyl group, etc. in the antibody by a covalent bond, a coordinate bond, etc. Any binding type may be used, such as a method of binding to the above or a method of binding by electrostatic or hydrophobic physical adsorption. Among these, those capable of binding in an aqueous medium, particularly those capable of chemical bonding in an aqueous medium are preferred.

Although the antibody and the fluorescent labeling reagent can be bound in any medium, it is preferably bound by a chemical reaction in an aqueous medium. As the aqueous medium used here,
In addition to water or a buffer solution, a mixed solution of water or a buffer solution and an organic solvent such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, tetrahydrofuran or dioxane can be used. When mixing an organic solvent,
In order to suppress the inactivation of the antibody, the concentration of the organic solvent in the mixed solution is preferably 20% by weight or less.

The charging ratio for binding (reaction) between the antibody and the fluorescent labeling reagent is not particularly limited, but the weight ratio is 1: 1 to 1.
A ratio of 1: 1000, preferably 1:50 to 1: 100 is suitable. The antibody concentration in the medium at the time of binding (reaction) is also not particularly limited, but is 10 to 40 mg / ml.
Is preferred.

In the present invention, when the antibody and the fluorescent labeling reagent are bound, they are bound under a high pressure of 0.5 MPa to 1000 MPa. When the antibody and the fluorescent labeling reagent are reacted, it is preferable to react them in water or a buffer solution in order to minimize the inactivation of the antibody, and 0.5 MPa to 1000 M.
Pa, preferably 0 to 120 ° C., preferably 4 to 40 ° C. and 1 second to 72 under a pressure of 50 MPa to 700 MPa.
It is preferable to react for a time, preferably 30 seconds to 1 hour. By carrying out labeling in the above aqueous medium under such conditions, the inactivation of the antibody can be minimized, and the labeling can be introduced with high efficiency.

Generally, a protein such as an antibody is 0.5 M
Under a high pressure of Pa to 1000 MPa, pressure denaturation due to changes in secondary structure and tertiary structure occurs. As a result, in the normal state, the reactive site that is hidden inside the protein is exposed to the outside by pressure denaturation, and the modifying reagent easily acts, so that labeling is performed with high reaction efficiency. In the present invention, the antibody in this pressure-denatured state is treated with a fluorescent labeling reagent to perform labeling with high efficiency to obtain a fluorescently labeled antibody.

In this case, if the labeling is carried out under high pressure, the labeling rate will be faster than that under normal pressure, the amount of the introduced label will be large, and the activity of the antibody will be kept high. Although such a phenomenon also occurs in physical labeling such as physical adsorption, it is excellent in the case of a chemical reaction, and is particularly remarkable when the labeling is performed by reacting with a water-soluble labeling reagent in an aqueous medium. A high rate of high activity antibody is obtained.

The labeled antibody thus obtained has no aggregation or denaturation, and has a high labeling rate and activity. Therefore, it can be used as it is or in dialysis, ultrafiltration, affinity column,
It can be purified by a gel filtration column, an ion exchange column or the like and used as an immunological diagnostic agent, which enables highly sensitive diagnosis. Further, it can be dried by freeze-drying or the like to be used as an immunological diagnostic agent, or can be used as a medicine or the like.

[0017]

INDUSTRIAL APPLICABILITY In the present invention, since the antibody and the fluorescent labeling reagent are bound under high pressure for labeling, the fluorescent label can be efficiently introduced into the antibody, and at the same time, a large amount of the fluorescent label can be introduced to improve the detection accuracy. And maintain high antibody activity,
It is possible to produce a highly sensitive and highly active fluorescently labeled antibody.

[0018]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example 1 A goat-derived anti-human albumin antibody was dissolved in 0.25 M sodium carbonate buffer (pH = 9.0) to give 20 mg /
Prepared to ml. To 1 ml of this antibody solution was added 1 g of fluorescein-4-isothiocyanate, and the pressure was 400 MPa.
The reaction was carried out at 4 ° C for 3 hours under the pressure of. The resulting reaction mixture was purified by Sephadex G25 and DEAE-cellulose column to obtain FITC labeled antibody.
The fluorescence intensity in 1 ml of the obtained labeled antibody 10 mg / ml solution was measured at an excitation wavelength of 495 nm and a measurement wavelength of 520 nm, and the result was converted to the number of moles of the fluorescent substance per antibody molecule, resulting in 46 mol / mol.

Example 2 In the same manner as in Example 1, except that the pressure was changed to 200 MPa, the number of moles of the fluorescent substance per molecule of the obtained labeled antibody was determined, and the result was 28 mol / mol.

Example 3 In the same manner as in Example 1, except that the pressure was changed to 50 MPa, the number of moles of the fluorescent substance per molecule of the obtained labeled antibody was determined, and the result was 19 mol / mol.

Example 4 In the same manner as in Example 1, except that 7-benzylamino-4-nitrobenzoxadiazole was used as the fluorescent labeling reagent and the reaction pressure was 500 MPa to obtain a labeled antibody. The fluorescence intensity in 1 ml of the obtained labeled antibody 10 mg / ml solution was measured at an excitation wavelength of 464 nm and a measurement wavelength of 532 nm, and the result was converted to the number of moles of the fluorescent substance per antibody molecule, resulting in 54 mol / mol.

Example 5 A labeled antibody was obtained in the same manner as in Example 1 except that 4-chloro-7-nitrobenzofurazan was used as the fluorescent labeling reagent and the reaction pressure was 200 MPa. The fluorescence intensity in the obtained labeled antibody 10 mg / ml solution 1 ml was measured by the excitation wavelength 46
It was measured at 4 nm and a measurement wavelength of 512 nm, and the result was converted to the number of moles of the fluorescent substance per antibody molecule, 28 mol / mol
Met.

Example 6 As in Example 1, except that 4-fluoro-7-sulfobenzofurazan ammonium salt was used as the fluorescent labeling compound and 0.1 M borate buffer (pH = 9.
5) Use 1 mM EDTA and set the reaction pressure to 100
A labeled antibody was obtained as MPa. Obtained labeled antibody 10m
The fluorescence intensity in 1 ml of the g / ml solution was measured using an excitation wavelength of 385n.
m was measured at a measurement wavelength of 515 nm, and the result was 22 mol / mol when converted into the number of moles of the fluorescent substance per antibody molecule.

Example 7 As in Example 1, except that sulforhodamine 101 acid chloride was used as the fluorescent labeling reagent and the reaction pressure was 50%.
A labeled antibody was obtained as MPa. Obtained labeled antibody 10m
The fluorescence intensity in 1 ml of the g / ml solution was measured with an excitation wavelength of 568n.
m was measured at a measurement wavelength of 620 nm and converted to the number of moles of the fluorescent substance per antibody molecule, the result was 18 mol / mol.

Comparative Example 1 When the reaction was carried out in the same manner as in Example 1, but under normal pressure, the number of moles of the fluorescent substance per antibody molecule of the obtained labeled antibody was 11 mol / mol.

Comparative Example 2 When the reaction was carried out in the same manner as in Example 4, but under normal pressure, the number of moles of the fluorescent substance per antibody molecule of the obtained labeled antibody was 10 mol / mol.

Comparative Example 3 When the reaction was carried out in the same manner as in Example 5, but under normal pressure, the number of moles of the fluorescent substance per antibody molecule of the obtained labeled antibody was 8 mol / mol.

Comparative Example 4 When the reaction was carried out in the same manner as in Example 6 but under normal pressure, the number of moles of the fluorescent substance per antibody molecule of the obtained labeled antibody was 11 mol / mol.

Comparative Example 5 When the reaction was carried out in the same manner as in Example 7, but under normal pressure, the number of moles of the fluorescent substance per antibody molecule of the obtained labeled antibody was 10 mol / mol.

Reference Example 1 Glass beads (diameter 3 mm, rough surface) activated by a 2% solution of γ-aminopropylsilane were shaken in a 5% glutaraldehyde aqueous solution at room temperature for 3 hours. This was thoroughly washed with physiological saline to obtain activated glass beads.
The activated glass beads were treated with anti-human albumin antibody 20
20 mM phosphate buffer (pH 7.4) containing μg / ml
The medium was shaken at room temperature for 1 hour and washed thoroughly with physiological saline.
This was shaken in a 1 M aqueous solution of glycine at room temperature for 1 hour to block residual aldehyde groups, and
Blocking was performed with% bovine serum albumin to obtain anti-human albumin antibody-supporting glass beads. Next, as an antigen solution, a 50 μg / ml human albumin solution (0.5 M
KCl, 20 mM phosphate buffer, pH 7.0) was prepared, and 0.4 ml of the solution was reacted with two anti-human albumin antibody-supporting glass beads in a test tube at room temperature for 1 hour. Then, 0.5M NaCl was washed 3 times, and further, physiological saline was washed 10 times to remove excess antigen, and human albumin-supporting glass beads were obtained.

Example 8 As a labeled antibody, 20 mM containing 100 μg / ml of the FITC-labeled anti-human albumin antibody obtained in Example 1
0.4 ml of a phosphate buffer (pH 7.4) was added to the human albumin-supporting glass beads obtained in Reference Example 1, and the mixture was reacted at room temperature for 1 hour. This was washed 3 times with 0.5 M NaCl and 10 times with physiological saline to obtain glass beads carrying a labeled antibody by the sandwich method. 4M MgCl ₂
Add 0.5 ml of the aqueous solution and shake at room temperature for 1 hour.
The antibody complex was dissociated. The supernatant containing the free labeled antibody obtained here was measured with a fluorometer equipped with a microcell. Similarly, Examples 2 and 3 and Comparative Example 1
The fluorescent intensity of the labeled antibody obtained in
The measurement was performed at 5 nm and a measurement wavelength of 520 nm. Table 1 shows the relative intensities of the labeled antibodies obtained in Comparative Example 1 in Examples 1 to 3 when the fluorescence intensity is 1.

[0033]

[Table 1]

Claims (1)

[Claims] 1. A method for producing a fluorescently labeled antibody, which comprises binding the antibody with a fluorescently labeled reagent under a high pressure of 0.5 MPa to 1000 MPa.