JPH0820448B2 - Method for producing fixed antibody - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing an immobilized antibody.

b Prior Art and Problems to be Solved by the Invention An enzyme immunoassay (EI) utilizing an immune reaction is used as one of the methods for measuring a very small amount of substances existing in a living body.
A) and radioimmunoassay (RIA) play an important role in clinical examination. In particular, EIA has high specificity and high sensitivity without the use of radioactive substances, and thus has rapidly developed in recent years. In general, an antibody immobilized on an insoluble carrier (hereinafter abbreviated as “immobilized antibody”) is used in these measurement methods, and the requirements to be satisfied are high specificity, large amount of immobilized antibody and high activity. In addition, non-specific adsorption of the enzyme-labeled antibody may be small.

Conventionally, as a method for producing an immobilized antibody, a method of covalently binding an insoluble carrier and an antibody through a cross-linking agent with an amino group, a carboxyl group or the like of the insoluble carrier, a method of immobilizing in an insoluble carrier matrix, or an insoluble carrier There are methods such as physical adsorption. The covalent bond has an advantage that the antibody is firmly bound to the insoluble carrier, but the physical adsorption method is a simple operation to obtain an immobilized antibody because the antibody is likely to be adversely affected during the binding reaction and the operation is troublesome. Is mainly used.

As a general operation method of the physical adsorption method, for example, a method in which an insoluble carrier is immersed in an antibody solution and reacted at 4 ° C. overnight or at 25 ° C. for several hours is used.

However, the physical adsorption method cannot always fix a sufficient amount of the antibody to the insoluble carrier, so that the surface of the antibody is roughened to increase the surface area (JP-A-58-70164),
Attempts have been made to increase the adsorption amount by irradiating radiation to improve the surface properties (Japanese Patent Laid-Open No. 60-260857), but the sensitivity was not always sufficient. Also when fixing the antibody, short-term treatment or (E.Ishikawa et al in pH2.5 for partial denature the antibody, J.Immunoassay 1, 385
-398, 1980), and a method for improving measurement sensitivity by pre-fixing with a denaturing agent such as guanidine, thiocyanate ion, or urea (JDConnradie, J.Immunol, Meth).
od 59, 289-299,1983) have been reported.

However, in any of the above methods, the procedure is complicated because the antibody is fixed again after pretreatment. That is, in the method of Ishikawa et al., The antibody to be immobilized is treated in a solution of pH 2.5, and then the acidic solution is neutralized, and then immobilized on plastic beads. In the method of JD Connradie, which uses urea, it is necessary to pretreat the urea for one day and then remove the urea by dialysis for several days.

In view of the above-mentioned drawbacks of the prior art, the present inventors have diligently studied a method for producing an immobilized antibody by a simple operation without impairing the activity of the antibody, and within a certain range of pH without pretreatment. By directly immobilizing the antibody on an insoluble carrier, it is possible to produce an immobilized antibody without impairing the activity of the antibody, and by further heat treating the immobilized antibody obtained by such a production method, The inventors arrived at the present invention by discovering that it is possible to produce an activated immobilized antibody.

c Means for Solving the Problems That is, according to the present invention, an insoluble carrier is immersed in an antibody solution containing an antibody or antibody fragment and having a pH of 2.0 to 4.5 to immobilize the antibody or antibody fragment on the insoluble carrier. And a method for producing a fixed antibody.

The antibody used in the present invention may be either a polyclonal antibody or a monoclonal antibody, and may be an antibody derived from any animal. The antibody fragment may be F (ab ′) ₂ , Fab ′, Facb and so on. Preparation of antibody fragments such as F (ab ′) ₂ , Fab ′ and Facb can be obtained by enzymatically treating a polyclonal antibody or a monoclonal antibody by a known method.
For example, an antibody can be treated with pepsin to give F (ab ') ₂ , and this fragment can be further reduced to give Fab' (A. Nisonoff et al, Arch. Biochem. Biophys. 8 ).
9 , 230 (1960), P.Parham, J.Immonol., 131 , 2895 (1983)
Etc.).

Examples of the insoluble carrier of the present invention include polystyrene, polyester, polyethylene, polypropylene, ABS, polyvinyl fluoride, polyamine methyl vinyl ether-maleic acid copolymer, 6-nylon, plastic such as 6,6-nylon, amino acid polymer, glass. , Silica gel, etc.

The shape of the insoluble carrier is not particularly limited, such as beads, microspheres, sticks, test tubes, films, microplates or membranes. In addition, the surface may have any shape such as a mirror surface or a rough surface.

In the present invention, the aforementioned insoluble carrier is immersed in an antibody solution containing the above-mentioned antibody or antibody fragment and having a pH of 2.0 to 4.5 to immobilize the antibody and the like.

Such a solution can be obtained by dissolving an antibody or the like in an aqueous solution having a pH of 2.0 to 4.5.

The aqueous solution is not particularly limited, but a buffer solution is preferable. As the buffer, glycine-hydrochloric acid buffer, sodium citrate-hydrochloric acid buffer, potassium hydrogen phthalate-
Hydrochloric acid buffer solution, citric acid-potassium citrate buffer solution, citric acid-disodium phosphate buffer solution, sodium acetate-hydrochloric acid buffer solution, acetic acid-sodium acetate buffer solution, veronal buffer solution, etc. that can satisfy pH 2.0 to 4.5 Any buffer may be used so long as it is preferably a citric acid-disodium phosphate buffer. The pH of the antibody solution is less than 2.0, or 4.5
If it is larger, the antibody activity after immobilization is insufficient, resulting in low sensitivity, which is not preferable. Among them, pH 2.5 to 3.5 is preferable. The salt concentration of the buffer solution is used in the range of 0.01 to 1M, preferably in the range of 0.1 to 0.5M.

The conditions such as the concentration of the antibody in the antibody solution and the fixing time are
Depending on the type of antibody or antibody fragment, a solution of 1 to 100 μg / ml is generally used, and an insoluble carrier on which the antibody is to be immobilized is immersed in this solution for 1 to 24 hours at 4 ° C, or for several hours at room temperature. Let stand.

The insoluble carrier on which the antibody was immobilized after the standing was dried as it was, stored by removing water by centrifugation, or washed several times with phosphate buffered saline (PBS). Then, leave it in a PBS solution of about 1% bovine serum albumin (hereinafter, abbreviated as BSA-PBS) at room temperature for 2 to 12 hours to perform PB.
Wash with S several times and soak in appropriate buffer for storage.

The fixed antibody thus prepared has a stronger antigen-binding ability than the fixed antibody fixed at a pH around neutrality, and the final measurement sensitivity is significantly increased.

In the present invention, there is provided a method for producing an immobilized antibody, by which the immobilized antibody obtained by the above method is further heat-treated in a solution to obtain an immobilized antibody with higher sensitivity. Such heat treatment may be carried out directly after taking out the immobilized antibody from the antibody solution, or after taking out the solid antibody and washing it, and immersing it in a BSA-PBS solution of about 1% and washing it.

The temperature and time for heat treatment vary depending on the immobilized antibody, but it is preferably 30 to 80 ° C., and the time is preferably 1 hour to several days, especially 2 to 24 hours.

The heat treatment is preferably performed in a solution. As such a solution, a buffer having a pH of 6 to 8 is preferable. Examples of such buffers include phosphate buffer, hydrogen phthalate buffer, potassium dihydrogen phosphate-borax buffer, disodium phosphate-citrate buffer, veronal buffer, Tris-HCl buffer, etc. Any buffer solution can be used as long as it can be adjusted to ~ 8, but a phosphate buffer solution is preferably used. The salt concentration of the buffer is 0.01-1M
Used in the range of.

The fixed antibody thus heat-treated has a stronger antibody activity than the fixed antibody which is not heat-treated, and the final measurement sensitivity is significantly increased.

The fixed antibody obtained by the method of the present invention is a usual EIA measurement kit, for example, hormones such as TSH, GH, LH, FSH, HCG, prolactin, parathyroid hormone, and insulin; for example, ferritin, CEA, alphafetoprotein, etc. Peptides; for example, protein C, protein S, plasmin / α2 plasminogen inhibitor complex, tissue plasminogen activator / plasminogen activator inhibitor complex, thrombin, antithrombin III complex, thrombomodulin and other fibrinolytic proteins , But is not limited to these.

Next, the present invention will be described more specifically with reference to examples.

Example 1 10 μg of a monoclonal antibody against human ciliated gonadotropin (HCG) in solutions having different pH (pH 1 to 9)
The solution was dissolved in the solution at a concentration of / ml, and a polystyrene stick was immersed in the solution and incubated at 4 ° C. for 18 hours. Then, stick this stick in 10 mM PBS (pH7.2) to 3
It was washed twice to give an antibody-immobilized stick. This antibody-immobilized stick, 0mIU / ml, 10mIU / ml or 100mIU / ml
It was immersed for 20 minutes in a mixed solution of 0.25 ml of HCG and 0.75 ml of peroxidase-labeled anti-HCG antibody. Next, the stick was washed with tap water, and in a solution prepared by mixing 0.17% H ₂ O ₂ 7 parts (volume part) and 0.06% 3,3 ′, 5,5′-tetramethylbenzidine 3 parts (volume part). Incubated. After 10 minutes, the stick was removed, and the absorbance of the remaining solution was measured with a spectrophotometer at a measurement wavelength of 650 nm. The results thus obtained are shown in FIG. As is clear from the figure, at pH 2.5 to 4.0, the absorbance at 0 mIU / ml does not increase, the absorbance at 10 mIU / ml and 100 mIU / ml is extremely high, and the sensitivity is eventually increased. I understand.

Example 2 A monoclonal antibody against LH was used in place of the monoclonal antibody against HCG, and the result of measuring LH in place of HCG in the same procedure as in Example 1 is shown in FIG. H
It can be seen that, as in the case of CG, the sensitivity is increased by about 1.5 to about 2 times or more at pH 2.0 to 4.0.

Example 3 In the same manner as in Example 1, in solutions with different pH (pH 1 to
Polystyrene beads (Sekisui Chemical # 80) were dipped in a solution prepared by dissolving a monoclonal antibody against protein C, which is a coagulation system protein, in 9) at 10 μg / ml, and
Incubated at ~ 8 ° C for 20 ± 4 hours. The beads were then washed with PBS. The beads thus obtained were used as antibody-immobilized beads. The antibody-immobilized beads were incubated in a mixed solution of standard human serum diluted with PBS 51-fold or PBS alone and peroxidase-labeled anti-protein C antibody, washed, and then developed in the same manner as in Example 1 to give an absorbance. Was measured. FIG. 3 shows the results. As is clear from the figure, it is found that the sensitivity is increased about 4 to 5 times at pH 2.0 to 3.0.

Example 4 A mouse monoclonal antibody (JTI-4) against a human plasminogen activator inhibitor was dissolved in 0.1 M phosphate / citrate buffer (pH 3.0) to a concentration of 20 μg / ml. Beads (Sekisui Chemical # 8
0) was soaked and left at 4 ° C. overnight. After washing with PBS, soak in 1% BSA-PBS solution at room temperature for 2 hours, wash with PBS, and heat-treat in PBS at 50 ° C on Mizutani for 1-6 hours,
Stored at 4 ° C.

Heat-treated beads were added to 0.3 ml of PBS solution of human tissue plasminogen activator-plasminogen activator inhibitor complex (O, 20 ng / ml),
Incubated at 2 ° C for 2 hours. Tween20 0.05%
After washing with dissolved PBS (hereinafter abbreviated as PBS-T), Fab 'fragment PBS-T solution (0.6 μg / ml of 0.3 ml) of peroxidase-labeled mouse anti-human tissue plasminogen activator monoclonal antibody (JTA-1) And add 37 ℃
For 30 minutes.

After washing with PBS, 2.5 mM H ₂ O ₂ -0.025% 3,3 ′, 5,5 ′ tramethylpentidine solution (0.3 ml) was added, and color was developed at 37 ° C. for 30 minutes. The color reaction was stopped with 1N sulfuric acid (1 ml), and the absorbance at 450 nm was measured. The results thus obtained are shown in FIG.

As is clear from the figure, by heat treatment, 0 ng /
The OD of ml did not increase, but the OD of 20 ng / ml increased, and the OD was about doubled when the heating time was 2 hours or longer.

The mouse anti-human plasminogen activator inhibitor monoclonal antibody (JTI-4) is described in Japanese Patent Application No. 63-81366 (Invention title “Monoclonal antibody against plasminogen activator inhibitor”, April 4, 1988). Obtained by the method described in (Application). That is, Van Mourich JA et al., J. Biol. Chem., 259 ,
Spleen cells obtained by immunizing mice with plasminogen activator inhibitor (PAI) isolated and purified from the culture supernatant of human vascular wall endothelial cells by applying the method of 14914-14921 (1984) et al. Hybridoma 4 clones (JTI1 to 4) producing anti-PAI monoclonal antibody were obtained by cell fusion according to a conventional method. JTI1 to 4 all recognize PAI and t-PA-PAI complex, the subclass is IgG ₁ and K, and JTI-4 does not have the ability to neutralize PAI inhibitory activity,
It bound to both quiescent PAI and SDS activated PAI.

A mouse anti-human tissue plasminogen activator monoclonal antibody (JTA-1) is disclosed in Japanese Patent Application No. 63-813.
No. 67 (Invention title “monoclonal antibody against plasminogen activator”, filed on April 4, 1988). That is, from the human melanoma cell culture supernatant, the method of Rijken DC et al. (Rijken D.C. et al,
J. Biol. Chem., 256 , 7035 to 7041 (1981) was applied to the isolated and purified tPA as an antigen to immunize mice, and spleen cells obtained were used for cell fusion according to a conventional method to obtain anti-tPA. Hybridomas producing monoclonal antibodies were cloned to obtain 4 clones producing anti-tPA monoclonal antibodies JTA1-4.

JTA1~4 Both bind to tPA and tPA-PAI complex, JTA-l, 2,4 binds to the H chain of tPA, JTA-3 does not bind, either the subclass IgG _1, K Met.

The above mouse anti-human tPA monoclonal antibody was added to A. Nison
Processing was performed by the method of off et al. to obtain a Fab 'fragment of the antibody.

Example 5 A rabbit antibody against bovine chondrocalcin was dissolved in 0.1 M phosphate / citrate buffer (pH 3.0) at 20 μg / ml, and polystyrene beads (Sekisui Chemical, # 8) were dissolved therein.
0) was soaked and left at 4 ° C. overnight. After washing with PBS, soak it in 1% BSA-PBS solution for 2 hours at room temperature, then use PBS.
After washing with PBS, it was heat-treated in PBS in a water bath at 50 ° C for 3 hours to 6 days, and stored at 4 ° C.

Heat treated beads in bovine chondrocalcin PBS
Solution (0,5ng / ml) (0.2ml) and rabbit antibody against bovine chondrocalcin labeled with peroxidase (0.2m
It was put in the mixed solution of l) and allowed to stand at 37 ° C. for 2 hours. After washing with PBS-T, 2.5 mM H ₂ O ₂ -0.025% 3,3 ', 5,5'-tetramethylbenzidine mixture (0.4 ml) was added, and the mixture was incubated at 37 ° C for 30 minutes.
Colored for minutes. Stop the color reaction with 1N sulfuric acid (1 ml), and
Absorbance at 50 nm was measured.

The rabbit antibody against bovine chondrocalcin is
The method of Choi et al. (HU Choi et al, J. Biol. Chem. 258,655
-661, 1983), bovine chondrocalcin was purified from an antiserum obtained by immunizing a rabbit.

 The results thus obtained are shown in FIG.

As is clear from the figure, the OD of Ong / ml did not change by the heat treatment, and the OD of 5 ng / ml showed about 1.6 times the OD by heating.

[Brief description of drawings]

FIG. 1 shows the measurement results of HCG when the immobilized antibody immobilized in the HCG antibody solution of pH 1 to 9 was used. FIG. 2 shows the LH measurement results when the immobilized antibody immobilized in the LH antibody solution of pH 1 to 9 was used. FIG. 3 shows the measurement results of protein C when the immobilized antibody immobilized in the protein C antibody solution of pH 1 to 9 was used. FIG. 4 shows a case where a fixed antibody immobilized with a human plasminogen activator inhibitor MCA antibody solution having a pH of 3.0 is further treated with heat for 1 to 6 hours.
The measurement result of human-tissue plasminogen activator-plasminogen activator inhibitor complex is shown. Fig. 5 shows the measurement results of chondrocalcin when the fixed antibody immobilized with the rabbit anti-bovine chondrocalcin PCA antibody solution of pH 3.0 was used, which was further heat-treated by changing the heating time. Show.

Claims (2)

[Claims] 1. A pH 2. containing an antibody or antibody fragment.
A method for producing an immobilized antibody, which comprises immersing an insoluble carrier in an antibody solution of 0 to 4.5 to immobilize the antibody or antibody fragment on the insoluble carrier. 2. The method for producing an immobilized antibody according to claim 1, wherein the antibody or the fragment is immobilized on the insoluble carrier, and the obtained immobilized antibody is heat-treated in a solution. Method for producing fixed antibody.