JPH04179477A - Luminous protein aequorin-labeled antibody - Google Patents

Abstract

PURPOSE:To obtain a protein useful as a detecting agent for detecting the target substance with luminescence due to aequorin activity thereof by binding a substance with a specific binding ability to a protein having the aequorin activity through covalent bonds using a binding means. CONSTITUTION:A substance with a specific binding ability is bound to a protein having aequorin activity through covalent bonds using a binding means. Specifically, e.g. aequorin is reacted with N,N-o-phenylenedimaleimide in a sodium phosphate buffer solution and the resultant labeling maleimide-aequorin is reacted with a reduced form antihuman TNF-alpha.rabbit IgG which is a substance having the specific binding ability to afford the objective aequorin-labeled antihuman TNF-alpha.rabbit antibody.

Description

Detailed Description of the Invention [Field of Technology] The present invention relates to a protein having aequorin activity bound to a substance having specific binding ability, a complex of the protein and a luminescent material that can be a substrate for the protein, and a complex thereof. The present invention relates to a preparation method and a method for detecting a target using the luminescent material.

[Prior art and its problems] Aequorin is a calcium-accepting photoprotein isolated from a luminescent jellyfish that lives near Friday Harbor Island, Washington, USA.

When two (or three) molecules of Ca"" ions bind to one molecule of aequorin, coelenterazine in the activated state contained in the aequorin molecule is oxidized, and it is exposed to light and Co.
Releases 2. Since Ca" ions are essential for this luminescence, it is possible to specifically and quantitatively detect Ca"" using aequorin. The detection limit for Ca2'" ions is approximately 10-"M, It is characterized by extremely high sensitivity.

However, the amount of aequorin isolated from nature is only about 200 mg from 10 tons of luminescent jellyfish, and the production volume is insufficient, and a constant supply is not guaranteed.

The present inventors used recombinant DNA techniques to clone the cDNA of apoaequorin from a luminescent jellyfish, and determined its primary structure (Japanese Patent Laid-Open No. 135-586-1986).

Next, using this cDNA and using Escherichia coli as a host, they succeeded in producing apoaequorin inside and outside the bacteria (Japanese Patent Application Laid-open No. 1962-031). Furthermore, a method for purifying apoaequorin using anion exchange chromatography was also established (Japanese Patent Application Laid-open No. 1-132.397).

In addition, for use in enzyme immunoassay, we have established a method for immobilizing proteins with aequorin activity by chemical bonding and physical adsorption.
91,489) and successfully prepared chemically modified aequorin (Japanese Patent Application No. 1-309,478).

Furthermore, by binding a protein having aequorin activity to a target substance via a protein having specific binding ability, the target substance can be specifically detected by luminescence. Here, specific binding refers to binding using antigen-antibody reaction, enzyme reaction, specific binding to a receptor, specific binding between nucleic acid and protein, etc. A protein having aequorin activity bound to a protein having specific binding ability is predicted to be useful as a test agent such as a diagnostic agent based on the above-mentioned functions.

In view of the above-mentioned technical circumstances, the present inventors conducted research and found that a protein with aequorin activity that binds to a protein with specific binding ability, a method for its preparation, and a luminescent material that can be used as a substrate for the protein and the protein have a specific binding ability. We were able to develop a new detection method using the complex and a protein with aequorin activity bound to a protein with specific binding ability.

As is clear from the above description, an object of the present invention is to provide a protein bound to a protein having a specific binding ability that retains aequorin activity, and its applications or methods.

[Means for solving problems] The present invention has the following configurations (1) to (10).

(1) A method for preparing a protein having aequorin activity, which comprises covalently bonding a substance having specific binding ability to the protein having aequorin activity using a binding means.

(2) A protein having aequorin activity that binds to a substance having specific binding ability, which is prepared by the preparation method described in item (1) above.

(3) A complex of the protein and the luminescent material obtained by binding the protein according to item (2) above and a luminescent material that can serve as a substrate for the protein.

(4) The method according to item (1) above, wherein a crosslinking agent is used as the bonding method.

(5) A protein having aequorin activity bound to a substance having specific binding ability, which is prepared by the preparation method described in item (4) above.

(6) A complex of the protein and the luminescent material obtained by binding the protein according to item (5) above and a luminescent material that can serve as a substrate for the protein.

(7) A method for detecting a target substance, which comprises binding a substance with specific binding ability to a protein having aequorin activity, and binding the thus obtained bound product to the target substance.

(8) The detection method according to item (7) above, wherein the bound substance is a substance having aequorin activity and antibody binding ability.

(9) Substances with specific binding ability include enzymes, antibodies, protein^, protein G, DNA, RN8, and DN.
(7) The above-mentioned A-binding protein or receptor.
Detection method described in Section.

(IO) The detection method according to item (7) above, wherein the target substance is a substrate, a coenzyme, a prosthetic group, an antigen, an antibody, a DNA%RNA, a hormone, or a transmitter.

The configuration of the present invention will be explained in detail below.

Here, the protein having aequorin activity includes, in addition to apoaequorin protein, proteins such as fusion proteins of various functional proteins and apoaequorin, and modified apoaequorin modified with various substances, and which have aequorin activity. say something

In addition, specific binding refers to antigen-antibody reaction, as mentioned above,
This refers to binding that utilizes enzymatic reactions, specific binding to receptors, specific binding between nucleic acids and proteins, etc.

On the other hand, as crosslinking agents, in addition to the same-reactive divalent reagents and different-reactive divalent reagents as shown in Tables 1-1 to 1-7 below,
A two-step reaction such as an avidin-biotin system can be considered.

The present invention is based on a conjugate of a substance having specific binding ability and a protein having aequorin activity, a method for preparing the conjugate, and a method for detecting a target substance using the conjugate. It can be implemented.

The present invention will be explained with reference to the accompanying drawings and tables. Fig. 1 shows a process diagram (flow sheet) for preparing maleimide-equorin for labeling. For example, sodium phosphate buffer (0, IM, pH 6,0) containing aequorin (1,79 mg) 1, OOO
μj2, N, N'-o-phenylenadema
leimideO, 19mg75 μIt N, N-d
After adding imethylformamide, 30℃
Incubate for 30 minutes.

After that, 5ephadex G-50 (1, Ox40c
Sodium phosphate buffer (0,1
Gel filtration was performed using M, pH 6,0) to separate the maleimide-equorin beak for labeling.
croconcen-trator centrico
Concentrate the fyA maleimidoquorin fraction using n-10. By the above operations, maleimide-equorin for labeling is obtained.

FIG. 2 shows a process diagram (flow sheet) for preparing a reduced anti-human TNF-α/rabbit antibody. For example anti-human TNF-α:
To 900μIt of sodium phosphate buffer (0.1M, pl(8,0) containing 0.99mg of rabbit IgG, 0.1
5vM EDTA/sodium phosphate buffer (0.1M
, pH 6,01, and incubate for 90 minutes at 37°C.

Then Sephadex G-50 (1,OX 40cm)
Gel filtration was performed using a column using 5mM EDT^/sodium phosphate buffer (0.1M, ph8.o) as a solvent, and the reduced anti-human TNF-α/rabbit antibody peak was fractionated. By the above operations, reduced anti-human TNF-α・
Rabbit IgG is obtained.

FIG. 3 shows a process diagram (flow sheet) for preparing an aequorin-labeled anti-human TNF-α/rabbit antibody. Reduced anti-human TN
F-(!-5mM containing rabbit IgG 1-lomg
EDTA/sodium phosphate buffer 7 nights (0.1M, pH 6,
0) 2.0 mIt and maleimide-equorin 10 for labeling
Sodium phosphate containing mg &! ? #Liquid (0.1M, pH6
, o) Mix Imi and incubate for 22 hours at 4°C.

After that, Sephadex G-50 (1, Ox40cm)
Sodium phosphate buffer (0.1M,
Gel filtration was performed using pH 6.5), and the aequorin-labeled anti-human TNF-α/rabbit antibody peak was fractionated. Through the above operations, an aequorin-labeled anti-human TNF-α/rabbit antibody is obtained.

Figures 4 and 5 show a process diagram (flow sheet) for quantifying human TNF-α using an aequorin-labeled anti-human NF-α/rabbit antibody and the results thereof. Lumiphotometer (TD-
Anti-human TNF at 100μ℃ was placed in a polystyrene cuvette (outer diameter 10 x 65mm) (LaboScience Inc.).
- Add the α antibody solution, cover the top of the polystyrene cuvette with rose film, and then incubate at 4°C for 72 hours.

After removing the anti-human TNF-α antibody solution, place a 300 μm Blocking Buffer in a polystyrene cuvette, cover again with parafilm, and incubate at 37°C for 2 hours. Fixed to the inner wall.

Next, remove BlockingBuffer and set 400
After washing the inner walls of the polystyrene cuvettes three times with μA Washing Buffer, add 50μ of Sample Diluen to all polystyrene cuvettes.
Add 50% of human TNF-α solution at various concentrations.
After adding μA in increments, cover with parafilm for 2 hours at 37°C.
Incubate for an hour.

Remove human TNF-α solution and wash with 400μJ2
After washing the inner wall of the polystyrene cuvette three times with ingBuffer, 100μ2 of aequorin-labeled anti-human T
Put the NF-α/rabbit antibody solution into all polystyrene cuvettes, cover with parafilm, and incubate at 37°C for 2 hours.
Incubate for an hour.

After this, excess aequorin-labeled anti-human TNF-α.

To remove the rabbit antibody, after removing the aequorin-labeled anti-human DNF-α/rabbit antibody solution, 400ufl
Wash the inner wall of the polystyrene cuvette three times with Washing Buffer.

This was followed by 1μ of 2-mercaptoethanol.

2μs/mIL coelenterazine 1μfl, 200mM
Tris-)ICI (pH 7,6), 100mM
10 mL of EDTA 1i liquid and 88 μK of distilled water were placed in a polystyrene cuvette, covered with parafilm, incubated at 4°C for 20 hours, and treated with aequorin using a Lumiphotometer (TD-4000, Lab Science). Measure activity.

Thereby, the amount of human TNF-α in the human TNF-α solution is determined. FIG. 5 shows this result. It was confirmed that the ephorin-labeled antibody has both aequorin activity and antibody activity, and that the aequorin-labeled antibody can be applied to enzyme immunoassay.

FIG. 6 shows a process diagram (flow sheet) for preparing biotin-labeled aequorin. 0.01 containing 0.1-2 mg of aequorin.
1M Sodium Phosphate Buffer (p) 17.0.0. IMNa
Cl, 1g/L bovine serum albumin and 0.6mM S-
Add 10 μρ of 611 mM N-hydroxysuccinimide biotin/dimethyl sulfoxide to 100 μm containing ammonium amino-1-naphthalenesulfonate and incubate for 20 minutes at 20°C. Next, 10
μf of 1M Glycine-Naoo (po7.e) is added to the reaction and incubated at 20° C. for 30 minutes.

After that, 5ephadex G-50 (1, ox40c
m) column with O.1M NaCl as solvent and 1g of sodium phosphate buffer (0.1M) containing serum albumin.
, pH 7,0) to separate the biotin-labeled aequorin peak.

Figure 7 shows a process diagram (flow sheet) for preparing maleimide-biotin for labeling, 1000 μl of sodium phosphate buffer (0.1 M, pH 7.0) containing 2.4 mg of avidin.
3.3 g/L 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester/
N.

After adding 100 μm of N-dimethylformamide, 30
Incubate for 30 minutes at °C.

After that, 5ephadex G-50 (1, Ox40c
m) column as a solvent. IM Na1l and Ig
Sodium phosphate buffer containing /L bovine serum albumin (0,
1M, pH 7.0), gel filtration was performed and the maleimide-biotin peak for labeling was fractionated.
croconcentratortorcentricon-
10 to concentrate the maleimide-biotin fraction for labeling.

FIG. 8 shows the avidin-labeled anti-human TNF-α/rabbit antibody preparation process rXJ (flow sheet), Example 2
Using the reduced anti-human TNF-α/rabbit IgG obtained in step 1, an avidin-labeled anti-human TNF-α/rabbit antibody is prepared. Reduced anti-human TNF-α/rabbit IgG1-1
5mM EDTA/Sodium Phosphate & I containing 10ll1
? #Solution (0.1M, p) 18.0) 2all and 1mj2 of sodium phosphate buffer (0.1M, pH 6.0) containing 30mg of maleimide-biotin for labeling are mixed and incubated at 4°C for 24 hours.

After that, Ultragel AcA 44 (1,Ox3
0 cm) column with 0.1 M NaCl and 1 as solvent.
Sodium phosphate buffer containing g/L bovine serum albumin (
Gel filtration is performed using 0.1 M, pH 7.0), and the avidin-labeled anti-human TNF-α/rabbit antibody peak is fractionated. By the above operations, avidin-labeled anti-human DNF-
α・Rabbit antibody was obtained.

Figures 9 and 10 show human T cells using the avidin-biotin system.
Quantification process diagram (flow sheet) of NF-α and its results are shown. First, anti-human TNF-α Antibodies are immobilized on the inner walls of polystyrene cuvettes.

Next, remove Blocking Buffer and set 40
After washing the inner wall of the polystyrene cuvette three times with Washing Buffer of 0μj2, sample dil of SOμj2 was added to all polystyrene cuvettes.
After adding 50μβ of human TNF-α solutions of various concentrations, cover with parafilm.
Incubate for 2 hours at °C.

Remove human TNF-α solution and wash 400ull.
After washing the inner wall of the polystyrene cuvette three times with ingBuffer, add avidin-labeled anti-human TN at 100μ℃.
Place the F-α rabbit antibody solution into all polystyrene cuvettes, cover with parafilm, and incubate at 37°C for 2 hours. After this, in order to remove excess avidin-labeled anti-human TNF-α/rabbit antibody, after removing the avidin-labeled anti-human TNF-α/rabbit antibody solution, the inner wall of the polystyrene cuvette was washed three times with 400 al Washing Buffer. Wash.

Next, 50 μm of biotin-labeled aequorin was added to each polystyrene cuvette, covered with parafilm, and incubated at room temperature for 2 hours. After this, in order to remove excess biotin-labeled aequorin, after removing biotin-labeled aequorin mW, 400 pl of Wa
Wash the inner wall of the polystyrene cuvette three times with shing Buffer.

Next, aequorin activity was measured in the same manner as in the case of quantifying human TNF-α using an aequorin-labeled anti-human TNF-α/rabbit antibody, and the amount of human TNF-α in the human TNF-α solution was determined. Ta. This result is shown in the 10th
It is a diagram.

The biotin-labeled anti-human TNF-α/rabbit antibody clearly has aequorin activity, and the amount of luminescence increases in proportion to the amount of human TNF-α, indicating that the aequorin-antibody via the avidin-biotin system It was confirmed that the complex (hereinafter referred to as aequorin-antibody complex) also had antibody activity.

FIG. 11 shows a process diagram (flow sheet) for preparing monomeric aequorin for labeling. Sodium phosphate buffer containing 0.79 mg of aequorin (0.1 M, p) 17.0) 1.
After adding 100 μm of 3.3 g/L 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester/N,N-dimethylformamide to 000 μm, the mixture was incubated at 30° C. for 5 minutes.

After that, 5ephadax G-50 (1, Ox40c
Sodium phosphate buffer (0,1
Perform gel filtration using M, poe, o) to separate W4 common monomeric maleimide-equorin beak.
The monomeric maleimide-equorin fraction for labeling was concentrated using ntricon-10. By the above operations, a monomeric maleimide aequorin for labeling was obtained.

FIG. 12 shows a process diagram (flow sheet) for preparing anti-human TNF-α/rabbit Fab'. Reaction solution 1 mj2 containing 0.99 mg of anti-human TNF-α/rabbit IgG,
Mix with pepsin-immobilized agarose gel and hold at 37°C.
Incubate for 8 hours. The reaction solution was separated from the gel using a separator, and then added to a 1.5ml Bin.
The gel is washed with d-ing Buffer and separated from the gel in the same manner as the reaction solution, and the separated washing solution is mixed with the previously separated reaction solution.

Next, this mixed solution was poured into an agarose gel on which Protein A was immobilized, and then 6 mil of Bind-ing was applied.
F(ab') is eluted with Buffer. Then, the eluted F(ab'12) was added to a sodium phosphate buffer (0
, 1M.

Dialyze against pH 7.0). Then m1cro
con-centrator centricon-1
0 and concentrated using anti-human TNF-a/Rabbit F (a
I got b'L.

Next, the above anti-human TNF-α rabbit F (ab′L solution) was added to 450 μm of 0.1 M 2-mercabutethylamine 15+aM EDTA/sodium phosphate buffer (Q, IM) to 50 μm.

pH 6.0) and incubate for 30 minutes at 37°C. After that, Ultragel^cA 44 (1
, Ox30cm) with 5mM EDT as solvent.
Sodium phosphate buffer containing A (0.1M, pH 6.0)
Gel filtration is performed using a filter, and the Fab' peak is fractionated.

And m1croconcentrator cen
The tab' fraction was concentrated using Tricon-30 to obtain anti-human TNF-α/rabbit Fab'.

Figure 13 shows monomeric aequorin-labeled anti-human TNF.
-α・Rabbit Fab′ preparation process diagram (flow sheet) showing anti-human TNF-a・Rabbit Fab′ 1 to 10m
5mM EDT^/sodium phosphate buffer (0,
Mix 1 ml of sodium phosphate buffer (0.1 M, IIHli, 0) containing 1M, pH 6,0)2IllIL and 10 mg of monomeric maleimide-equorin for labeling,
Incubate for 22 hours at 4°C.

After that, Ultragel Ac formula 44 (1,0x3
Sodium phosphate buffer (
Gel filtration was performed using monomeric aequorin-labeled anti-human TNF-α/rabbit F
Aliquot the ab°beak. By the above operations, monomeric aequorin-labeled anti-human TNF-α/rabbit Fab'
was gotten.

Figures 14 and 15 show a process diagram (flow sheet) for quantifying human TNF-α using monomeric aequorin-labeled anti-human TNF-α rabbit Fab' and the results. First,
Anti-human TN
The F-α antibody is immobilized on the inner wall of a polystyrene cuvette.

Next, remove Blockjng Buffer and set 40
After washing the inner walls of the polystyrene cuvettes three times with 0 μJ2 Washing Buffer, a 50 μm sample diluent was added to all polystyrene cuvettes.
After adding human TNF-α solutions of various concentrations to 50 μm each, cover with parafilm at 37°C.
Incubate for 5 hours at 4°C and overnight at 4°C.

The human TNF-α solution was removed and the inner wall of the 400μ polystyrene cuvette was washed three times with sodium phosphate buffer (0.01M, p 17.0) containing O.1M NaCl. So, 1v Na(:I
, 150 ml of sodium phosphate buffer (0.0 IM, pH 7.0) containing 1 g/L bovine serum albumin and monomeric aequorin-labeled anti-human TNF-α/rabbit Fab'.
Incubate μk for 4 hours at 20°C. After removing the above solution, add sodium phosphate 1 containing 0.1 M NaCl
1 buffer solution (0, OIM.

p) 17.0) and incubated twice at 30°C for 20 minutes each, and then incubated with 0.1M Na in the same manner as above.
Sodium phosphate buffer containing C1 (0, OIM, pH 7,
0) on the inner wall of the polystyrene cuvette at 400 μm.
Wash twice.

Next, aequorin activity was measured in the same manner as in the case of quantifying human TNF-α using an aequorin-labeled anti-human TNF-α/rabbit antibody, and human T in the human TNF-α solution was measured.
The amount of NF-α was determined.

FIG. 15 shows this result. Monomeric aequorin-labeled anti-human TNF-α/rabbit anti-Fab'
clearly has aequorin activity, and human T
Since the amount of luminescence increases in proportion to the amount of NF-α, monomeric aequorin-labeled anti-human TNF-α rabbit Fa
It was confirmed that b' also has antibody activity.

Furthermore, the aequorin-labeled anti-human TNF-α・
Compared to using rabbit antibodies, the measurement limit is approximately 1,
This has improved up to 1,000 times, making it possible to detect trace substances with higher sensitivity.

[Effects of the Invention] According to the method of the present invention, it is possible to bind a substance having specific binding ability to a protein having aequorin activity. The usefulness of the protein having this and its preparation method is as follows:
It is obvious to those skilled in the art. By utilizing the specific binding ability and luminescence ability of the binding substance, it can be applied to methods for detecting ultratrace amounts of various substances.

The above disclosure enables any person skilled in the art to practice the claimed invention. However, in order to increase the understanding of this invention, the procedures used to produce and identify aequorin conjugated to anti-human TNF-α antibodies with aequorin activity important to the invention are set forth below.

Example 1 [Preparation of maleimide-equorin for labeling Conifolin 0
．． Sodium phosphate M solution containing 79 mg (0.1 M, pH
Fi, O) to 1,000 μft, N, N-o-phe
nyl-enedemaleimide O, 19mg
15 μj2, N, N-dimethyl-fo
After adding rIIlawide, it was incubated at 30°C for 30 minutes.

Apoequorin used at this time was prepared by the following method. Apoequorin (Patent Application No. 61-249,098) produced by recombinant DNA method was treated by anion exchange chromatography method.
91,640), purified by coarse reverse phase HPLC.

Reversed phase 1 (PLC is Cosmosil 10C4-300 (10
A water/acetonitrile (both containing 0.1% trifluoroacetic acid) system was used as the solvent system in a column of 150 mm x 150 mm, a gradient was performed from 20 to 80%, and the apoaequorin peak was fractionated. This apoaequorin fraction was freeze-dried to prepare apoaequorin, which was stored at -20°C and used.

After that, 5ephadex, G-50 (1, Ox40
cm) column with sodium phosphate & 1gX solution (
0.1M, pH 8.0) was used to perform gel filtration, and the maleimide-equorin peak for labeling was fractionated.
The maleimide-aequorin fraction for labeling was concentrated using icon-10. By the above operations, maleimide-equorin for labeling was obtained.

Example 2 [Preparation of reduced anti-human TNF-α/rabbit IgG] Anti-human TNF-α/rabbit IgG Sodium phosphate m solution containing 0.99+ag (0.1 M, p) 16.0) 900
μm, 0.1M 2-mercaptoethyla
SmM EDTA/Sodium Phosphate 11 containing mine?
Add 100μm of #solution (0.1M, pH 8.0),
Incubation was carried out for 90 minutes at 7°C.

After that, 5ephadex G-50 (1, Ox 40
Gel filtration was performed using a column of 1.0 cm) using SaM EDTA/Sodium Phosphate M buffer (0, IM, p) 16.0) as a solvent, and the reduced anti-human TNF-α/rabbit antibody peak was fractionated. By 0 or more operations, reduced anti-human TN
F-α rabbit IgG was obtained.

Example 3 [Preparation of aequorin-labeled anti-human TNF-α/rabbit antibody] Using the labeling maleimide-aequorin obtained in Examples 1 and 2 and reduced anti-human TNF-α/rabbit IgG,
Aequorin-labeled anti-human TNF-α/rabbit antibody was prepared.

5mM EDTA/sodium phosphate N solution (0.1M
, p) 18.0) Sodium phosphate buffer (0.1M
, pH 6,0) was mixed and incubated at 4°C for 22 hours.

After that, 5ephadex G-50 (1, Ox 40
sodium phosphate buffer (0, cm) as a solvent.
Gel filtration was performed using 1M, p) 16.5), and the aequorin-labeled anti-human TNF-α/rabbit antibody peak was fractionated. By the above operations, aequorin-labeled anti-human TNF
-α rabbit antibody was obtained.

Example 4 [Quantification of human TNF-α using aequorin-labeled anti-human TNF-α/rabbit antibody] BI (l KINE TN
F Te5tHit(T Ce1l 5sciences
, Inc.).

Polystyrene cuvette (outer diameter 10 x 85 cm) of Lumiphotometer (TD-4000, Lab Science)
A 100 μC anti-human TNF-α antibody solution was added to the cuvette, and the top of the polystyrene cuvette was covered with parafilm, followed by incubation at 4°C for 72 hours.

After removing the anti-human TNF-α antibody solution,
Place the Blocking Buffer in a polystyrene cuvette, cover again with parafilm, and incubate at 37°C.
The anti-human TNF-α antibody was immobilized on the inner wall of the polystyrene cuvette.

Next, remove BlockingBuffer and set 400
After washing the inner walls of the polystyrene cuvettes three times with μ Meng Washing Buffer, add 50 μm Sample Diluen into all polystyrene cuvettes.
Add 50% of human TNF-α solution at various concentrations.
After adding μl at a time, cover with parafilm and store at 37℃ for 2 hours.
Incubated for hours.

Remove the human TNF-α solution and insert a 400 μm Washi
Inner wall of polystyrene cuvette with ngBuffer
After washing twice, aequorin-labeled anti-human T
Put the NF-α/rabbit antibody solution into all polystyrene cuvettes, cover with parafilm, and incubate at 37°C for 2 hours.
Incubated for hours.

After this, in order to remove excess aequorin-labeled anti-human TNF-α/rabbit antibody, aequorin-labeled anti-human TNF-α
- After removing the α rabbit antibody solution, 400 μl of Wa
The inner wall of the polystyrene cuvette was washed three times with shing Buffer.

Subsequently, 2-nermaptoethane 1 μm,
2 ug/mA coelenterazine 1 μm1, 20
0mM Tris-)1cI (pH 7J).

100mM EDTA buffer 10μjZ and distilled water 88
μl was placed in a polystyrene cuvette, covered with parafilm, incubated at 4° C. for 200 hours, and aequorin activity was measured using a Lumiphotometer (TD-4000, Labo Science).

Thereby, the amount of human TNF-α in the human TNF-α solution was determined. FIG. 5 shows this result. The aequorin-labeled anti-human TNF-α/rabbit antibody clearly has aequorin activity, and the amount of luminescence increases in proportion to the amount of human TNF-α.
It was confirmed that the anti-human TNF-α/rabbit antibody also has antibody activity.

Example 5 [Preparation of biotin-labeled aequorin] Sodium phosphate buffer containing 0.1 to 2 mg of aequorin (
pl (containing 7,0,0,1M NaCl, 1g/L bovine serum albumin and 0.6mM ammonium 8-amino-1-N naphthalenesulfonate) in 100μJZ
μ m of 66 mM N-hydroxysuccinimide biotin/dimethyl sulfoxide was added and incubated at 20° C. for 20 minutes.

followed by 10 μm of 1M glycine-NaOH (p)17
．． 6) was added to this reaction solution and incubated at 20°C for 30 minutes.

Apoequorin used at this time was prepared by the following method. Apoequorin (Japanese Unexamined Patent Publication No. 63-102695) produced by recombinant DNA method was treated by anion exchange chromatography method.
397), which was further purified by reverse phase HPLC.

Reverse phase HPLC was performed using Cosmocil 10C4-300 (IOX
Using a water/acetonitrile (both containing 0.1% trifluoroacetic acid) system as the solvent system in a 150 mm) column, a gradient was performed from 20 to 80%, and the apoaequorin peak was fractionated.

Apoequorin was prepared by freeze-drying this apoaequorin fraction, and the one stored at -20°C was used.

After that, 5ephadex G-50 (1, Ox40c
m) column with 0.1M NaCl and Ig/
Sodium phosphate buffer containing L bovine serum albumin (0,1
M,ph? , o) to perform gel filtration and fractionate the biotin-labeled aequorin beak.

Example 6 [Preparation of avidin-labeled antibody] Sodium phosphate solution containing avidin 2.4B (0.1 M
, ph7.0) to 1000 μk, 3.38/L4-(
After adding 1000 μl of N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester/N,N-dimethylformamide, the mixture was incubated at 30° C. for 30 minutes.

After that, 5ephadex G-50 (1, Ox 40
cm) column as a solvent. IM NaC1 and Ig
Sodium phosphate buffer containing /L bovine serum albumin (0,
1M, ph7. Gel filtration was performed using o) to separate a maleimide-biotin beak for labeling. Then m1
crococentrator centricon-1
0 was used to concentrate the maleimide-biotin fraction for labeling.

Next, using the reduced anti-human TNF-α rabbit IgG obtained in Example 2, avidin-labeled anti-human TNF-α
Rabbit antibodies were prepared. 5mM EDT^/sodium phosphate buffer (0.1M, pH 6.0j2+IL containing 1-10mg of reduced anti-human TNF-a/rabbit IgG) and sodium phosphate buffer (0.1M, containing maleimide-biotin 30+ag for labeling) ph6.O) l+jl were mixed and incubated at 4°C for 24 hours.

After that, Ultragel AcA 44 (1,Ox
30 cm) column with O, 1M NaCl as solvent.
Sodium phosphate IJ containing Ig/L bovine serum albumin
Gel filtration was performed using i buffer (0.1M, pH 7.o), and the avidin-labeled anti-human TNF-α/rabbit antibody beak was fractionated. Antibodies were obtained.

Example 7 [Quantification of human TNF-α using an aequorin-antibody complex] For the quantification of human TNF-α using an aequorin-antibody complex, BIG was equipped with the INE TNF Te5t Kit (T
Ce1l 5cien-ces, Inc) was utilized.

First, an anti-human TNF-α antibody was immobilized on the inner wall of a polystyrene cuvette in the same manner as in the case of quantifying human TNF-α using an Equon-labeled anti-human TNF-α/rabbit antibody.

Next, remove Blocking Buffer and set 40
After washing the inner wall of the polystyrene cuvette three times with 0 μm Washing Buffer, add 50 μl of Sample Dilue to every polystyrene cuvette.
nt, and then added human TNF-α solutions of various concentrations for 5 minutes.
After adding 0 μm each, the cells were covered with parafilm and incubated at 37° C. for 2 hours.

Remove human TNF-α solution, 400μj! Wash
After washing the inner wall of the polystyrene cuvette three times with ingBuffer, add 100μ of avidin-labeled anti-human TN.
The F-α rabbit antibody solution was placed in all polystyrene cuvettes, covered with parafilm, and incubated at 37°C for 2 hours.

After this, in order to remove excess avidin-labeled anti-human TNF-α/rabbit antibody, avidin-labeled anti-human TNF-α
・After removing the rabbit antibody solution, wash 400 μL
The inner wall of the polystyrene cuvette was washed three times with ingBuffer.

Next, 50 μL of biotin-labeled aequorin was added to each polystyrene cuvette, covered with parafilm, and incubated at room temperature for 2 hours. After this, in order to remove excess biotin-labeled aequorin, after removing the biotin-labeled aequorin m solution, 400 μf of Wa
The inner wall of the polystyrene cuvette was washed three times with shing Buffer.

Next, aequorin activity was measured in the same manner as in the case of quantifying human TNF-α using an aequorin-labeled anti-human TNF-α/rabbit antibody, and human T in the human TNF-α solution was measured.
The amount of NF-α was determined.

This result is shown in Fig. 1θ. The biotin-labeled anti-human TNF-α/rabbit antibody clearly has aequorin activity, and the amount of luminescence increases in proportion to the amount of human TNF-α, indicating that the aequorin-antibody complex also has antibody activity. It was confirmed that this was the case.

Example 8 [Preparation of monomeric maleimido aequorin for labeling] In order to apply it to a more sensitive detection method, firstly, in order to prepare a monomeric aequorin ttA-recognizing antibody in which apoaequorin and an antibody are bound in a 1:1 ratio, A monomeric maleimide-equorin for labeling was prepared such that the number of maleimide groups introduced into each molecule of apoaequorin was one.

Sodium phosphate M street solution containing 0.79 mg of aequorin (0
, 1M, pH 7,0) to 1000μA, 3J g/L
4-(N-maleimidomethyl)cyclohexane-1-
After adding 100 μA of carboxylic acid succinimide ester/N,N-dimethylformamide, the mixture was incubated at 30° C. for 5 minutes.

The apoaequorin used in this toyari was prepared by the following method. Apoequorin (Japanese Patent Application Laid-Open No. 1983-102-6951) produced by recombinant DNA method was treated by anion exchange chromatography method.
32.397), which was purified by rough reverse phase HPLC.

Reverse phase HPLC was performed using Cosmosil IOC4-34-300 (
A water/acetonitrile (both containing 0.1% trifluoroacetic acid) system was used as a solvent system in a 10x150 column, a gradient was performed from 20 to 80%, and the apoaequorin peak was fractionated. This apoaequorin fraction was freeze-dried to prepare apoaequorin, which was stored at -20°C and used.

After that, 5ephadex G-50 (1, Ox40c
Sodium phosphate buffer (0,1
M, pH 6.0) was used to perform gel filtration, and the monomelicutamaleimide aequorin peak for labeling was fractionated.

Then m1croconcentrator cen
The monomeric maleimide-equorin fraction was concentrated using Tricon-10. By the above operations, a monomeric maleimide aequorin for labeling was obtained.

Example 9 [Preparation of anti-human TNF-α/rabbit Fab'] Anti-human T
Immun for the preparation of NF-a/Rabbit Fab'.

Pure F(ab')2Preparation H
It (Prece) was used.

A reaction solution containing 99 mg of anti-human TNF-α/rabbit IgG O was mixed with an agarose gel on which pepsin was immobilized, and incubated at 37°C for 8 hours.The 0 reaction solution was separated from the gel using a separator, and then ,
The gel was washed with 1.5 mL of Binding Buffer and separated from the gel in the same manner as the reaction solution, and the separated washing solution was mixed with the previously separated reaction solution.

Next, this mixed solution was poured into an agarose gel on which protein A was immobilized, and then 6 ml of BindingBu
F(ab')2 was eluted with ffer. Then, the eluted F(ab') was added to sodium phosphate u1. Liquid solution (0,
1M, pH 7.0). Then m1
croconcentra-tor centrico
Concentrated using n-10, anti-human TNF-α/Rabbit F
(ab')2 was obtained.

Next, 50 μm of 0.1M 2-mercaptoethylamine/5 mM EDTA/sodium phosphate &! Street liquid (0.1M.

po6. o) was added and incubated at 37°C for 30 minutes.

After that, Ultragel Ac 8 to 4 (+, Ox3
Gel filtration was performed using a 0 cm) column using sodium phosphate solution II (0.1 M, pH 6.0) containing 5 mM EDTA as a solvent, and the Fab' peak was fractionated. and m1croconcentratortorcentri
The Fab' fraction was concentrated using con-30 and anti-human T
NF-α rabbit Fab' was obtained.

Example 1O [Preparation of monomeric aequorin-labeled anti-human TNF-α/rabbit Fab'] Monomeric maleimide-equorin for labeling prepared in Examples 9 and 10 and anti-human TNF-α/rabbit Fab'
using monomeric aequorin-labeled anti-human TNF-
a ・tI rabbit Fab'! 3 were made.

Anti-human TNF-a - 5mMEDTA/sodium phosphate buffer (0.1M,
Sodium phosphate buffer (pH 6.0) containing 2 ml of monomeric maleimide-aequorin for labeling (pH 6.0) and 10 mg of monomeric maleimide-equorin for labeling (pH 6.0)
1M, pH 6,0) 11k was mixed and incubated at 4°C for 22 hours.

After that, Ultragel Ac^44 (1,Ox3
Gel filtration was performed using a 0cm) column using sodium phosphate & IWI solution (0.1M, p) 18.5) as a solvent, and the monomeric aequorin-labeled anti-human TNF-α/rabbit Fab' peak was fractionated. . By the above operations, monomeric aequorin-labeled anti-human DingNF-α/rabbit Fa
b' was moved by 1.

Example 11 [Quantification of human TNF-α using monomeric aequorin-labeled anti-human TNF-α/rabbit Fab'] Quantification of human TNF-α using monomeric aequorin-labeled anti-human TNF-α/rabbit Fab' As in Example 4 and Habo, the BIOKINE TNF Te5t Kit was used.

First, an anti-human TNF-α antibody was immobilized on the inner wall of a polystyrene cuvette in the same manner as in the case of quantifying human TNF-α using an aequorin-labeled anti-human TNF-α/rabbit antibody.

Next, remove Blocking Buffer and set 4o
After washing the inner walls of the polystyrene cuvettes three times with Washing Buffer of
After adding 50 μA each of human TNF-α solutions at various concentrations, cover with rose film and cover with 37 μA.
The cells were incubated at 4°C for 5 hours and then overnight at 4°C.

Remove the human TNF-α solution and dilute with 4 ml of sodium phosphate buffer (0.01 M, pH 7, o) containing 0.1 M NaC+.
Wash the inner wall of the polystyrene cuvette three times with 0.0 μm and 0.1 M NaCl in the polystyrene cuvette.
A solution of 150 μm of sodium phosphate solution (0, OIM, PH 7,0) containing g/L bovine serum albumin and monomeric aequorin-labeled anti-human TNF-α/rabbit Fab' was incubated at 20° C. for 4 hours.

After removing the above solution, using a sodium phosphate buffer (0.01M, pH 7°O) containing 0.1M NaCl,
Incubate twice for 20 minutes at 30°C, and
As above, the inner wall of the polystyrene cuvette was washed three times with 400 μA of sodium phosphate buffer (0, OIM, I)H, 0) containing 0.1 M Na[:l.

Next, Aequorin alli! li anti-human TNF-α・
Aequorin activity was measured in the same manner as in the case of quantifying human TNF-α using a rabbit antibody, and the amount of human TNF-α in the human TNF-α solution was determined.

FIG. 15 shows this result. Monomeric aequorin-labeled anti-human TNF-α/rabbit Fab' clearly has aequorin activity, and human TN
Since the amount of luminescence increases in proportion to the amount of F-α, monomeric aequorin-labeled anti-human TNF-α/rabbit Fab
' was confirmed to also have antibody activity.

Furthermore, the aequorin-labeled anti-human TNF-α・
Compared to using rabbit antibodies, the measurement limit is approximately 10
The lid has been improved by up to 00 times, making it possible to detect trace substances with higher sensitivity.

Combining the above results, we found that maleimide-equorin for labeling, biotin-labeled aequorin, monomeric maleimide-equorin for labeling, and aequorin-labeled anti-human TNF-α.
・Rabbit antibody, aequorin-antibody complex, and monomeric aequorin-labeled anti-human TNF-α ・Rabbit Fab' has aequorin activity, and reduced anti-human TN
F-α/rabbit IgG, avidin-labeled antibody, monomeric maleimide-equorin for labeling, aequorin-labeled anti-human TNF-α/rabbit antibody, aequorin-antibody complex, and monomeric aequorin-labeled anti-human TNF-α/rabbit Fab' It was confirmed that the antibody had antibody activity.

By using this aequorin-labeled anti-human TNF-α/rabbit antibody, the aequorin-antibody complex, and the monomeric aequorin-labeled anti-human TNF-α/rabbit Fab', a trace amount of human τNF-α was generated using luminescence. It became possible to quantify the amount. Furthermore, as shown in Example 11, by using the antibody Fab', a more sensitive measurement could be performed.

Furthermore, instead of anti-human TNF-α antibody, various enzymes,
Antibody, pronine A1 protein G, DNA, RNA,
By using DNA-binding proteins or receptors, it is possible to detect and quantify trace amounts of all kinds of substrates, coenzymes, prosthetic groups, antigens, antibodies, DNA, RNA1 hormones or transmitters by luminescence. is easily predicted.

In addition, aequorin-labeled anti-human TNF-α/rabbit antibody,
Since the aequorin-antibody complex and the monomeric aequorin-labeled anti-human TNF-α/rabbit Fab' have aequorin activity, it is obvious to those skilled in the art that modified aequorin or immobilized aequorin can be used instead of aequorin. It is.

As described above, the present invention provides a conjugate of a protein having specific binding ability and aequorin activity, a method for preparing the same, and a method for detecting a target using the conjugate.

Table 1-1-1-7 shown below shows reagents that can be used as the above-mentioned crosslinking agent.

[Brief explanation of drawings]

Figures 1 to 3, Figures 6 to 9, and Figures 11 to 14 are process diagrams (flow sheets) for explaining the method for preparing the product of the present invention, and Figure 4 shows the antibody and target according to the present invention. It is a process diagram (flow sheet) for showing the combination of things. Furthermore, Figures 5 and 10.15 are diagrams showing the relationship between the aequorin activity of the conjugate and the antibody activity. that's all

Claims (10)

[Claims] (1) A method for preparing a protein having aequorin activity, which comprises covalently bonding a substance having specific binding ability to the protein having aequorin activity using a binding means. (2) A protein having aequorin activity bound to a substance having specific binding ability, which is prepared by the preparation method according to claim (1). (3) A complex of the protein and the luminescent material obtained by combining the protein according to claim (2) with a luminescent material that can serve as a substrate for the protein. (4) The method according to claim (1), wherein a crosslinking agent is used as the bonding method. (5) A protein having aequorin activity bound to a substance having specific binding ability, which is prepared by the preparation method according to claim (4). (6) A complex of the protein and the luminescent material obtained by binding the protein according to claim (5) and a luminescent material that can serve as a substrate for the protein. (7) A method for detecting a target substance, which comprises binding a substance having specific binding ability to a protein having aequorin activity, and binding the thus obtained bound substance to the target substance. (8) The detection method according to claim (7), wherein the bound substance is a substance having aequorin activity and antibody binding ability. (9) The substance having specific binding ability is an enzyme, an antibody, protein A, protein G, DNA, RNA, a DNA binding protein, or a receptor.
Detection method described in section ). (10) The detection method according to claim (7), wherein the target substance is a substrate, a coenzyme, a prosthetic group, an antigen, an antibody, DNA, RNA, a hormone, or a transmitter.