JPH08262021A - Pyrocypris luciferase-labeled antibody and its preparation - Google Patents

Abstract

PURPOSE: To obtain a pyrocypris luciferase-labeled antibody whose yield is high and whose lumininescent-activity residual rate is excellent by a method wherein the antibody is prepared by an antibody to be labeled, by an anti- pyrocypris luciferase antibody and by pyrocypris luciferase. CONSTITUTION: An antibody to be labeled is reduced it is treated with an excess amount of a cross-linking agent which comprises maleimide groups at both terminals, the cross-linking operation of identical antibodies is suppressed, and the meleimide groups are introduced. Then, an anti-pyrocypris luciferase antibody is reduced, and it is reacted with the antibody which has introduced the maleimide groups, and the complex of the antibody is prepared. At this time, monoclonal antibody which is composed of a subclass unity is especially preferable as the antibody to be labeled and the anti-pyrocypris luciferase antibody. Then, the prepared complex antibody and a pyrocypris luciferase are reacted at an equal mole, and a labeled antibody is prepared.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a Cypridina luciferase-labeled antibody used as a highly sensitive immunoassay reagent.

[0002]

2. Description of the Related Art Conventionally, horseradish oxidase (HRP) alkaline phosphatase, β-galactosidase and the like have been generally used as an enzyme for an enzyme-labeled antibody.

As the enzyme labeling method for antibodies, the glutaraldehyde method, carbodiimide method, cyanogen bromide method, periodate oxidation method, maleimide hinge method and the like are generally used. However, labeled antibodies prepared by these methods, excluding the maleimide hinge method, are poor in storage stability because of the low yield and the majority of the polymers in which multiple molecules are bound intricately. The background is high in the measurement. On the other hand, the maleimide hinge method has a high yield and is known as a method in which a polymer is difficult to form. In addition, Cypridina luciferase is expected as a labeling enzyme because of its high quantum yield.

However, the preparation of Cypridina luciferase-labeled antibody was attempted by the maleimide hinge method, but the yield was low, and the luminous activity of Cypridina luciferase was also 1/10.
Fell to. This is because treatment of Cypridina luciferase with a binder caused a change in the structure showing activity, and if the treatment conditions for the binder were further relaxed, the yield was further reduced.

[0005] Cypridina luciferase can be produced from a recombinant form of its gene.
The price is higher than that of RP and alkaline phosphatase, and they have not been widely used as labeling enzymes.

[0006]

In order to prepare a labeled antibody of an immunoassay reagent with high sensitivity, and to prepare it at a cost as low as or better than conventional methods, Cypridina luciferase, which enables high sensitivity measurement, is used. One of the challenges is to label while maintaining the luminescence activity. Another object is to provide a method for preparing a labeled antibody with high efficiency of labeling with Cypridina luciferase.

[0007]

[Means for Solving the Problems] The present invention has been achieved as a result of studies to solve the above-mentioned problems.

According to the present invention, Cypridina luciferase was efficiently labeled while its luminescent activity was largely maintained.
The present invention relates to a Cypridina luciferase-labeled antibody and a method for preparing the same. That is, the antibody to be labeled,
The present invention relates to a Cypridina luciferase-labeled antibody comprising an anti-Cypridina luciferase antibody and a Cypridina luciferase and a method for preparing the same.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In order to bind Cypridina luciferase to an antibody in a state where the luminescent activity is maintained, a chemical modification that impairs the reaction or activity of the binder to the luminous active center of Cypridina luciferase. Must be avoided. In fact, in the trial using the maleimide hinge method, a large decrease in luminescence activity was observed despite the mild treatment conditions of the binder.

Therefore, the present inventors have conducted an immunological reaction with an anti- Cypridina luciferase antibody to determine Cypridina luciferase.
The present invention was conceived to be possible by efficiently labeling Cypridina luciferase according to the amount of antibody without causing any damage to Cypridina luciferase itself by binding and labeling the cypress.

That is, first, the antibody to be labeled was reduced and treated with an excessive amount of a cross-linking agent having a maleimide group at both ends to suppress cross-linking between the same antibodies to introduce a maleimide group. Then, the anti- Cypridina luciferase antibody was reduced and reacted with the maleimide group-introduced antibody to prepare an antibody complex. Further, the antibody to be labeled and the anti-cypridina luciferase antibody used in the present invention may be either a monoclonal antibody or a polyclonal antibody, but preferably at least one, and more preferably both antibodies are subclasses. Monoclonal antibodies are desirable due to their unity. Also,
Each of the antibodies may be used as it is, but it is preferable to use at least one antibody, and more preferably both antibodies in Fab form. Further, the cross-linking agent is not particularly limited as long as it has maleimide groups at both ends. Then, the prepared composite antibody is reacted with Cypridina luciferase to prepare a labeled antibody.
In this case, the complex antibody and Cypridina luciferase are preferably reacted in equimolar amounts, but the ratio may be changed depending on the case.

[0012]

The present invention will be described in detail with reference to the following examples.

Example 1 Anti-h-TSH antibody 3.0 mg / ml of 0.2 M sodium acetate buffer solution (pH = 4.2) was added to 500 μl of pepsin 1 m.
Add 18 μl of the same buffer solution of g / ml, and add 1 at 37 ° C.
After standing for 7 hours and neutralizing with 90 μl of 1 M Tris solution, PBS solution containing 0.5 mM EDTA by gel filtration was used to remove undigested IgG through a Protein A column equilibrated with the same buffer and concentrated to give anti-h- F (a of TSH antibody
500 μl of a solution of b ′) ₂ (1.2 mg / ml) was prepared. To this, 200 mM mercaptoethylamine was added.
1M sodium phosphate buffer (5 mM EDTA pH =
6.5) 55 μl of the solution was added, and the mixture was allowed to stand at 37 ° C. for 1 hour and then concentrated by gel filtration to prepare 600 μl of the same buffer solution (1.0 mg / ml) of Fab ′ of the anti-h-TSH antibody. 300 μl of this solution was added to N, N'-bis (3-maleimidoprop
yl) -2-hydroxy-1,3-propandiamine (1 mg) in DMF solution (30 μl) was added, and the mixture was allowed to stand at 37 ° C. for 20 minutes, and then gel filtration was performed to remove the maleimide group-introduced anti-h-TSH antibody Fab in the same buffer solution (120 μg / 2 ml was prepared.

Anti Cypridina luciferase antibody (rabbit) 4
150 μl of 0.1 M sodium phosphate buffer (5 mM EDTA pH = 6.5) solution of 25 mg / ml mercaptoethylamine in 500 μl of 20 μg / ml PBS solution
Was added, and the mixture was allowed to stand at 37 ° C. for 1 hour, and then concentrated by gel filtration to prepare 500 μl of a reduced anti-cypridina luciferase antibody in the same buffer solution (400 μg / ml).

Maleimide group-introduced anti-h-TSH antibody Fab
To 2 ml of the solution, 500 μl of a reduced anti-cypridina luciferase antibody solution was added, and the mixture was stirred at room temperature for 3 hours, 15 μl of an aqueous mercaptoethylamine solution (25 mg / ml) was added, and the mixture was stirred for 30 minutes, and then gel filtered to obtain anti-h-TSH antibody 111 of the composite antibody of the sea urchin firefly luciferase antibody
1 ml of a μg / ml PBS (0.05% NaN ₃ ) solution was prepared.

Solution of complex antibody (111 μg / ml) 28
Cypridina luciferase solution (3 mg / ml) in μl
Add 2 μl and let stand for 30 minutes at room temperature.
A Cypridina luciferase-labeled anti-h-TSH antibody solution was prepared by diluting 200 times with a g / ml PBS (0.05% NaN ₃ ) solution.

100 μl each of h-TSH standard solution was placed in polystyrene tube in which anti-h-TSH antibody was insolubilized, shaken at room temperature for 30 minutes and washed 3 times with 3 ml of washing solution, and then Cypridina luciferase labeled antibody. 50 solution
Each μl was taken, shaken at room temperature for 30 minutes, washed 3 times with 3 ml of washing solution, and then 150 mM sodium phosphate buffer solution.
(pH = 7.2) 100 μl each, and Cypridina luciferin 3.3 μg / ml solution (10 mM sodium citrate buffer pH = 4.5, 140 mM NaCl) 100 μl
10 seconds after the addition of l, photometry was performed for 10 seconds to measure the calibration value of h-TSH, and a standard solution of 0 μIU / ml was measured 5 times to determine the detection sensitivity by the 2SD method.

[0018]

[Table 1] Since the labeled antibody was prepared by directly using the anti- Cypridina luciferase antibody without being Fabized, the background at the time of measuring the calibration value was high, and although the measurement sensitivity was slightly low, the calibration value was sufficiently high and the calibration curve was linear. Showing sex
It was possible to obtain a good Cypridina luciferase-labeled antibody.

Example 2 A Cypridina luciferase solution having the same concentration as the Cypridina luciferase concentration of the labeled antibody was prepared, and the luminescence amount of each 100-fold diluted solution was measured to label the Cypridina luciferase. The change in luminescence activity due to the above was assayed.

[0020]

[Table 2] The luminescent activity of the labeled Cypridina luciferase remained almost unchanged with a residual rate of 93.3%, and almost all the Cypridina luciferase used could be labeled.

Example 3 Anti-h-TSH antibody 3.0 mg / ml of a 0.2 M sodium acetate buffer solution (pH = 4.2) in 500 μl of pepsin 1 m
Add 18 μl of the same buffer solution of g / ml, and add 1 at 37 ° C.
After standing for 7 hours and neutralizing with 90 μl of 1 M Tris solution, PBS solution containing 0.5 mM EDTA by gel filtration was used to remove undigested IgG through a Protein A column equilibrated with the same buffer and concentrated to give anti-h- F (a of TSH antibody
500 μl of a solution of b ′) ₂ (1.3 mg / ml) was prepared. To this, 200 mM mercaptoethylamine was added.
1M sodium phosphate buffer (5 mM EDTA pH =
6.5) 55 μl of the solution was added, and the mixture was left standing at 37 ° C. for 1 hour and then concentrated by gel filtration to prepare 600 μl of the same buffer solution of Fab ′ of the anti-h-TSH antibody (1.1 mg / ml).
300 μl of this solution was added to N, N'-bis (3-maleimidopropyl) -2-
Hydroxy-1,3-propandiamine 1mg DMF solution 30μ
After adding 1 to it and allowing it to stand at 37 ° C. for 20 minutes, 2 ml of the same buffer solution (130 μg / ml) of the maleimide group-introduced anti-h-TSH antibody Fab was prepared by gel filtration.

Anti- Cypridina luciferase monoclonal antibody 18 μl of pepsin 1 mg / ml buffer solution was added to 500 μl of 0.2 M sodium acetate buffer solution (pH = 4.2) 3.0 mg / ml, and the mixture was incubated at 37 ° C. for 17 hours. After neutralization with 90 μl of 1M Tris solution, PBS solution containing 0.5 mM EDTA by gel filtration was used to remove undigested IgG through a Protein A column equilibrated with the same buffer and concentrated to concentrate anti- Cypridina luciferase monoclonal antibody. F (ab ') ₂ solution (0.7 mg / ml)
500 μl was prepared. 200 mM mercaptoethylamine in 0.1 M sodium phosphate buffer (5 mM
55 μl of EDTA pH = 6.5) solution was added, and the mixture was allowed to stand at 37 ° C. for 1 hour, then concentrated by gel filtration, and the same buffer solution of anti-h-TSH antibody Fab ′ (0.6 mg / ml) 50 was added.
0 μl was prepared.

Maleimide group-introduced anti-h-TSH antibody Fab
To 2 ml of the solution was added 500 μl of the reduced anti-cypridina luciferase monoclonal antibody solution, and the mixture was stirred at room temperature for 3 hours, and an aqueous mercaptoethylamine solution (25 mg / ml) was added.
After adding 15 μl and stirring for 30 minutes, the solution was concentrated by gel filtration, and 400 μg / ml PBS (0.05% NaN) of a composite antibody of anti-h-TSH antibody and anti-cypridina luciferase antibody was added.
₃ ) 1 ml of the solution was prepared.

Solution of complex antibody (100 μg / ml) 30
To μl, Cypridina luciferase solution (3 mg / m
1) 2 μl was added, and the mixture was allowed to stand at room temperature for 30 minutes, diluted 200-fold with 10 mg / ml PBS (0.05% NaN ₃ ) solution of BSA, and treated with Cypridina luciferase-labeled anti-h-TSH.
An antibody solution was prepared.

100 μl each of h-TSH standard solution was placed in polystyrene tube in which anti-h-TSH antibody was insolubilized, shaken at room temperature for 30 minutes and washed 3 times with 3 ml of washing solution, and then labeled with Cypridina luciferase. 50 antibody solution
Each μl was taken, shaken at room temperature for 30 minutes, washed 3 times with 3 ml of washing solution, and then washed with 150 mM sodium phosphate buffer (p
(H = 7.2) 100 μl each, add 100 μl of Cypridina luciferin 3.3 μg / ml solution (10 mM sodium citrate buffer pH = 4.5, 140 mM NaCl), and after 10 seconds, perform photometry for 10 seconds to measure the calibration value of h-TSH. Then, the standard solution of 0 μIU / ml was measured 5 times, and the detection sensitivity was determined by the 2SD method.

[0026]

[Table 3] The anti- Cypridina luciferase antibody was used as a monoclonal antibody in the form of Fab to further improve the sensitivity.

[0027]

EFFECTS OF THE INVENTION According to the present invention, a Cypridina luciferase-labeled antibody having a high yield and an excellent residual ratio of luminescent activity can be obtained. Therefore, a high-performance Cypridina luciferase-labeled antibody can be used as a reagent for a highly sensitive immunoassay. It is possible to provide a method for efficiently and inexpensively preparing.

Claims (5)

[Claims] 1. A Cypridina luciferase-labeled antibody comprising an antibody to be labeled, an anti-Cypridina luciferase antibody and a Cypridina luciferase. 2. The Cypridina luciferase-labeled antibody according to claim 1, wherein the antibody to be labeled and / or the anti-cypridina luciferase antibody is a monoclonal antibody. 3. The Cypridina luciferase-labeled antibody according to claim 1, wherein the antibody to be labeled and / or the anti-Cypridina luciferase antibody is Fab-ized. 4. The sea urchin firefly according to claim 1, wherein a complex of an antibody to be labeled and an anti-cypridina luciferase antibody is prepared, and the complex is immunoreacted with Cypridina luciferase. A method for preparing a luciferase-labeled antibody. 5. The method for preparing a Cypridina luciferase-labeled antibody according to claim 4, wherein the antibody to be labeled is subjected to reduction treatment.