JPH03112493A - Monoclonal antibody and production of hybridoma producing the same antibody - Google Patents

Abstract

PURPOSE:To obtain an anti-tacorotopsin monoclonal antibody useful for taking advantage of tacorotopsin as an optical sensor or optical information recognizing element without damaging photochemical reaction of tacorotopsin by recognizing a part of stable structure. CONSTITUTION:A anti-tacorotopsin monoclonal antibody is an antibody of recognizing a part of stable structure (except site having <= about 10,000 molecular weight from C end) without damaging photochemical reaction of tacorotopsin (tacorotopsin bound to antibody receiving red/blue light and absorption maximum thereof mutually moving between 475nm and 500nm). A hybridoma to produce the antigen is produced by fusing a mouse splenocyte immunized and sensitized with purified tacorotopsin with a mouse derived myeloma cell.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an anti-tacolodopsin antibody useful for utilizing tacolodopsin as an optical sensor, optical information recognition element, etc.

[Conventional technology]

Many of the substances responsible for information processing in living organisms are proteins, such as acetylcholine receptors and rhodopsin. In order to artificially express functions using these functional proteins as materials and use them as sensors or information recognition elements, the protein molecules must be immobilized without impairing their functions.

Monoclonal antibodies are attracting attention not only as probes for protein tissue distribution and functional analysis, but also as materials for immobilizing functional proteins. In JP-A-63-111428,
This method involves inserting rhodopsin into liposomes of haptenized phospholipids and arranging the liposomes two-dimensionally on a substrate using anti-habten antibodies to construct a biological device that converts external optical information into various ions and chemical substances. be. By the way, visual substances that are sensors for optical information recognition, especially tachorodopsin, become a mixture of metalrhodobuscin and rhodopsin when they receive blue light, and the maximum absorption ranges from 475 nm to 500 nm.
Move to m. And by irradiating red light, especially light of 580 nm or more.

Metalrhodobuscin can be regenerated into rhodopsin (Motoyukitsuda, Biokimikaeto Biophysical Acta (M, Ts)
uda, Biochimica et Biophys
icaActa), 578, 372-380 (1
979)) That is, in the case of tacolodopsin, the photoreaction can be easily confirmed by alternately irradiating blue/red light and measuring the shift of the absorption maximum. If tachorodopsin can be immobilized without impairing its activity, it could be applied to optical sensors and optical memories.

Regarding monoclonal antibodies against rhodopsin, those against bovine, rat, and bacteriorhodopsin have been created as described below. Colin J. Barnstable and colleagues developed a monoclonal antibody that recognizes photoreceptor tissue using rough membrane components obtained from rat retina as an antigen, and used it in combination with a bovine rhodopsin monoclonal antibody to improve photoreceptor tissue. Journal of Neurocytology (J,
of Neurocytology), 12. p7
85-803 (1983). In addition, Donald Mackenzie (D, Mackenzie) has created a monoclonal antibody that recognizes the C-terminus of bovine rhodopsin and is analyzing rhodopsin distribution and its changes in the photoreceptor disc membrane (Biochemist).
ry), 23. pp6544-6549 (198
4). ) However, anti-tachorodopsin antibodies that can be used for optical sensors and optical information recognition devices have not yet been obtained.

[Problem to be solved by the invention]

In order to use a monoclonal antibody as a material for immobilizing a functional protein, it is necessary that the antibody not inhibit the function of the protein and recognize a stable site of the structure. For this reason, anti-tacolodopsin antibodies must be selected that do not inhibit the photochemical reaction of tacolodopsin. In other words, tachorodopsin bound to the antibody is red/
When receiving blue light, its absorption maximum is 475nm and 500n.
It must be an anti-tachorodopsin antibody that alternately migrates between m. In addition, during the purification process, tachorodopsin often has a molecular weight of about 1
The 0,000 part is cut off, resulting in a molecular weight of approximately 40,000. When immobilized with an antibody that recognizes a site with a molecular weight of about 10,000 from the C-terminal side, the site with a molecular weight of about 40,000, which is the main site for photochemical reactions, is removed. Therefore, in order to stably immobilize tacolodopsin, it is necessary to select an antibody that recognizes a region other than the region having a molecular weight of about 10,000 from the C-terminal side.

The present invention provides a monoclonal antibody that does not inhibit the photochemical reaction of tacolodopsin and recognizes a structurally stable site.

[Means to solve the problem]

In order to select anti-tacolodopsin monoclonal antibodies that do not inhibit the photochemical reaction of tacolodopsin, it is necessary to bind the antibody to tacolodopsin and measure the photochemical reaction. However, if there is tacolodopsin that has not reacted with the antibody, I can't make a correct judgment. For this purpose, it is necessary to remove the tacolodopsin that is not bound to the antibody while maintaining the binding between the antibody and tacolodopsin. Therefore, we thought that the above conditions could be satisfied by using a purified tacolodopsin sample as a test sample and using an ultrafiltration method. We considered separation. First, ultrafiltration was performed using only purified tachorodopsin. However, tachorodopsin did not migrate to the low molecular weight fraction and remained in the high molecular weight fraction. As a result of examining the cause of this, it was found that the purified tachorodopsin formed aggregates due to the low surfactant concentration. That is, when the surfactant was supplemented and ultrafiltration was performed, tachorodopsin migrated to the low molecular weight fraction.

By the way, surfactants generally inhibit or break the binding between antibodies and antigens. However, digitonin, octyl glucoside, which is an extractant for membrane proteins.

When we examined whether nonionic surfactants such as heptyl glucoside or sugar ester would have an effect, we found that there was no problem. That is, the polymer fraction was returned to the volume before ultrafiltration with phosphate buffered saline (PBS) containing the surfactant, and an antibody was added to cause an antigen-antibody reaction and ultrafiltration was performed. However, there was no specific absorption of tacolodopsin in the low-molecular fraction, and specific absorption of tacolodopsin was observed in the high-molecular fraction. This was due to binding between the antibody and tacolodopsin. Furthermore, when the antibody was reacted under conditions with excess tacolodopsin and ultrafiltered, both the high-molecular and low-molecular fractions showed specific absorption of tachorodopsin.

At this time, when the low-molecular-weight fraction was subjected to electrophoresis in a Niss Deinis polyacrylamide gel, only the tachorodopsin band was detected, and no antibodies were detected. Therefore, by reacting tacolodopsin with an antibody in the presence of a nonionic surfactant and performing ultrafiltration in the presence of the surfactant,
It was found that it was possible to distinguish between tacolodopsin that bound to antibodies and tachorodopsin that did not. The antibody-bound tacolodopsin thus obtained was irradiated with red/blue light alternately to examine the presence or absence of a photochemical reaction, and an anti-tacolodopsin monoclonal antibody that did not inhibit the photochemical reaction was selected. Furthermore, an antibody that does not recognize a region with a molecular weight of 10,000 from the C-terminus was selected by immunoplot of protease-treated tacolodopsin.

Tachorodopsin has a molecular weight of approximately 50,000, and when treated with trypsin, the molecular weights to and ooo on the C-terminal side are first cut off, creating a molecular weight of 40,000, which is then reduced to a molecular weight of 28,000 and a molecular weight of 16,000. (M, Tsuda, Proceedings of the Yamada Conference 21 Onmolecular Physiology of Retinal Proteins)
he YamadaConference
an Molecular Physiology
fRetinal Proteins), pp167
-172 (1988)) a Therefore, in order to select antibodies that do not recognize the site with a molecular weight of 10,000 on the C-terminal side, it is sufficient to select an antibody that recognizes a degradation product with a molecular weight of about 40,000. .

However, without recognizing the degradation products with a molecular weight of 40,000, the molecular weight of 16,000 or 2
It was also found that some antibodies recognize the degradation products of 8.000. Therefore, the molecular weight of the C-terminal side is 10,00
To select antibodies that do not recognize the 0 site, the molecular weight 1
It has been found that it is sufficient to select one that recognizes a decomposition product with a molecular weight of 6,000 or 28,000.

Based on the above results, a monoclonal antibody that serves as a sequence material for tacolodopsin can be obtained through the following steps.

A step of fusing mouse visceral cells immunized in vivo or in vitro with rod sensitizers or purified tacolodopsin with mouse-derived myeloid cells to create a hybridoma that produces antibodies against tachorodopsin. Next, the hybridoma is grown in vivo or in vitro, and the antibody is purified from ascites or culture fluid. A step of mixing the purified antibody and purified tachorodopsin in the presence of a surfactant to react with the antibody and tachorodopsin, and then collecting the antibody-bound tachorodopsin. A step of alternately irradiating the antibody-bound tachorodopsin solution with red light and blue light and measuring a total absorption curve.

Furthermore, immunoplotting was performed using a trypsin-limited cleavage product of tacolodopsin, and the molecular weight was 16,000 or about 2.
Step of selecting antibodies that recognize 8,000 degradation products.

Anti-tachorodopsin monoclonal antibodies can be produced by culturing hybridomas created by the so-called cell fusion method. An anti-tachorodopsin monoclonal antibody-producing hybridoma can be established by constructing a hybridoma between an antibody-producing cell and a myeloid cell, and cloning the hybridoma that produces an anti-tachorodopsin antibody. The antibody can be obtained by growing this in the mouse peritoneal cavity or in a culture container.

Antibody-producing cells were obtained from mice immunized with tacolodopsin #
Uses visceral cells. Tachorodopsin, which serves as an immunogenic antigen, is
Crude or purified preparations are available. The crude preparation is a protein extract extracted from photoreceptor rods of the retina of the eyeball or the rods using a membrane protein extractant such as digitonin or sugar ester.

A purified sample is obtained by treating the protein extract with ion exchange and affinity chromatography. It is then mixed with an adjuvant to immunize mice. Immunization can be carried out by subcutaneously, intravenously, or intraperitoneally injecting 40 to 100 μg of the antigen in protein amount 2 to 4 times every 2 to 3 weeks. Note that some membrane protein extractants such as digitonin and sugar esters are toxic, so it is necessary to test them in advance to determine the permissible amount of toxic substances. For example, digitonin exhibits hemolytic and inotropic effects. When administering an antigen containing digitonin, the dose of digitonin is 0.5 mg or less,
Preferably, the amount should be 0.1 mg or less.

Furthermore, when the antigen contains an extractant that is highly toxic to individual mice, it is preferable to employ an in vitro immunization method.

Cell fusion can be performed according to the method of Kohler and Milstein. The fusion partners are X63 cells (X63) derived from mouse BALB/c, B3N1 (P
Myeloma cells such as 3IJI) ill cells, Nisniswan (MS-1) cells, and SP2 (SF3) cells can be used. B cells from the mouse spleen are mixed 2 to 10 times the pre-cultured myeloma cells and centrifuged, and the supernatant is completely removed by suction to obtain a mixed pellet of myeloma cells and B cells. Loosen this pellet well,
Add 30-50% polyethylene glycol (molecular weight 1000-4000) pre-warmed at 37°C and mix at 30-37°C.
React with. The reaction is then stopped by dropwise mixing with serum-free medium. Furthermore, after adding a large amount of serum-free medium, the cells are collected by centrifugation. The cells were suspended in HAT (containing hypoxanthine and aminopterin thymidine) medium, dispensed into a 96-well plate, and incubated at 37°C.
Cultivate with After 3 to 4 days of culture, half of the culture medium is removed by suction every two days and fresh HAT medium is added to allow only hybridomas to grow.

After the hybridoma has grown sufficiently, immunoassay (E
nzyme Linked Immunosorben
t As5ay, hereinafter.

Those that produce anti-tachorodopsin antibodies are selected by ELISA) method. Then, cloning is performed by the limiting dilution method to obtain an anti-tachorodopsin antibody-producing clone.

The clone was treated with BAL [l/c
Antibodies can be obtained by transplanting into the abdominal cavity of a mouse and collecting ascites 10 to 14 days later. Antibodies can also be produced by culturing the clone in an animal cell culture device or the like. Antibodies are then purified from ascites fluid or culture fluid through steps such as ammonium sulfate fractionation and ion exchange chromatography.

The above purified tachorodopsin and antibody are mixed in the presence of a nonionic blushing activity such as digitonin or sugar ester.
Incubate for 1-4 hours at 7°C. The concentration of the surfactant may be 2% or less, but preferably 0.05 to 0.2%.
% is good. After incubation, unreacted tachorodopsin is separated using an ultrafiltration membrane that does not allow molecules with a molecular weight of 100,000 or more to pass through. The ultrafiltration membrane has a molecular weight of ioo,
It is not limited to membranes that block the passage of molecules larger than ooo.

Any material that can separate tacolodopsin and antibodies may be used. The buffer solution may be added to this fractionated solution having a molecular weight of 100,000 or more, and the original volume may be returned to the original volume using PBS containing a surfactant, and ultrafiltration may be performed again. The method for separating antibody-bound and unbound tachorodopsin may be not only ultrafiltration but also carrier-free electrophoresis, as long as it does not destroy the bond between the antibody and tachorodopsin. Immediately after ultrafiltration, the fractionated liquid is exposed to red light of 560 nm or more and 350 nm to 46 nm.
Irradiate with 0 nm blue light for about 10 to 60 seconds, and measure the total absorption curve between 200 nm and 650 nm using a spectrophotometer. Then, find the absorption maximum value when red/blue light is irradiated alternately, and select one in which the absorption maximum around 475 nm changes alternately when irradiating red light, and the absorption maximum around 500 nm changes alternately when irradiating blue light. , it is possible to select antibodies that do not inhibit the photoreaction of tachorodopsin.

Next, the ability of the selected antibody to recognize a trypsin-limited cleavage product of tacolodopsin is examined by immunoplotting. Purified tacolodopsin and trypsin at 50 to 200
: Mix at 1:1 and incubate at 37°C for about 5 to 40 minutes. Note that the mixing ratio with trypsin, reaction temperature, and reaction time are not particularly limited, and the optimum conditions may be selected each time. The reaction solution is mixed with a mercaptoethanol-containing SDS treatment solution and heated at 100° C. for 1 to 5 minutes. Then, after electrophoresis in Niss Dei Nis polyacrylamide gel, it is transferred to nitrocellulose pus. Then, the selected antibody was applied according to the ELISA method to examine its ability to recognize trypsin degradation products, and the molecular weight of 16,
000 or a decomposition product with a molecular weight of 28,000, and
Select antibodies that do not recognize this site.

Through the above steps, an antibody that does not inhibit photochemical reactions and recognizes a structurally stable site can be created as a monoclonal antibody for immobilizing tacolodopsin.

〔Example〕

EXAMPLES The present invention will be specifically explained below with reference to Examples, but the present invention is not limited to these Examples.

[Example 1] 1) Purification of antigen Rhodopsin is a membrane protein present in rod-sensitive cells of photoreceptor cells forming the retina.

First, we fractionated the rod-like clones. Note that the following operations were performed in a dark room.

Mizuda=l (Paroctopus deflein
i) The retinas detached and collected from 100 eyeballs were soaked in buffer solution (1
0mM MOPS-NaOH, pH 7.4,
After suspending in 20 μM p-aminophenylmethasulfonyl fluoride hydrochloride (P-APASF) 1.2Q,
Large solids were removed with gauze. Then 40,00
The cells were centrifuged at 0 x g and 4°C for 30 minutes, and photoreceptor cell segments constituting the retina were collected. 34% of the segment
Suspended in 300 mQ of sucrose-containing buffer,
Sucrose floating centrifugation was performed at xg for 1 hour, and the supernatant was collected to obtain rod-sensitive clones. Add buffer to this supernatant and
00 mΩ and 40,000Xg.

A rod-sensitive pellet was obtained by centrifugation at 4°C for 20 minutes. The pellet was then resuspended in buffer and centrifuged under similar conditions. This operation was performed three times for washing. The rod sensitive body was dissolved in digitonin solution (2% digitonin, 1%
00mM Na2HP04-KH2PO4, pHL8)
The suspension was suspended in water and shaken for 1 hour to extract rhodopsin.

Then centrifugation I! (25,000×g, 1 hour) to obtain an extract from which the residue was removed. The residue was extracted with digitonin again, and unextracted rhodopsin was recovered.

DEAE-5epha was used to remove ommochrome proteins from the digitonin extract.
Ion exchange chromatography was performed using cel, Pharmacia H). DEAE-5ephacel5
Fill the column with mQ and add buffer (0.1% digitonin,
50mM Na2HP04-KH, PO4, pH6,8
), the extract was charged and passed through the buffer to obtain fractions with absorption at 280 nm and 480 nm. Next, Connie Sepharose (Can A-5eph
Rhodopsin was isolated by affinity chromatography using arose* (manufactured by Pharmacia). Cony Sepharose (Con A-5epharose)
) Fill the column with 10aIl and add buffer (0.2% digitonin, 50mM Tris-HCl2).

After equilibration at pH 7.0), the fractionated solution was charged.

After passing through the buffer several times, rhodopsin was eluted with a buffer containing IMα-methylglucoside.

2) Immunization of mice The purified tacolodopsin, which had been made to have a digitonin concentration of 0.1% or less using Centricon 30K (manufactured by Amicon), was diluted with physiological saline to prepare a tacolodopsin solution with a protein concentration of 200 μg/mp. . This was diluted and injected into the abdominal cavity of BALB/c female mice in the following manner to sensitize them to tacolodopsin.

Table 1 Sensitization method number of times Protein amount Adjuvant amount Injection volume 1st time
100μg 15μg 0.5mQ2-3rd 20μg 15μg 0.5n+fi
Final round 40 pg 30 μg O, 5 mQ interval 1st round 12 weeks -> 2nd round (-2 weeks -> 3rd round 12 weeks -> Final round Adjuvant: ENU Acetyl Mura Miruel - 7 Raniludi Isoglutamine (N -acetylmur
amyl-L-alanyl-D-isoglutam
ine, manufactured by Sigma) 3) Cell fusion Three days after the final immunization, the spleen was removed from the mouse and subjected to cell fusion. The spleen was loosened in a 1:1 mixture of RAM medium (manufactured by Oral Pharmaceuticals) and IMDM medium (manufactured by Sigma) (hereinafter referred to as HI medium), and then centrifuged (1600 rpm, 5 mL).
Spleen cells were collected using L/C. HI the cell pellet again
Suspend in medium, add spleen cell suspension and 10% fetal bovine serum.
Myeloma cells (P3U1) cultured in HI medium containing
strain) were collected by centrifugation for 1 m (1000 rpm, 5 minutes), then resuspended in serum-free HI medium and centrifuged. This operation was performed twice to prepare a myeloma cell suspension from which serum components had been removed.

Both cell suspensions were mixed to give 2 x 10" spleen cells and 4 x 10' myeloma cells, and centrifuged (180
0 rpm, 5 minutes) to obtain a mixed pellet. After completely removing the supernatant, the pellet was mixed. 3 in advance for this
5 inch polyethylene glycol 1500 heated at 7°C (
(manufactured by Boehringer Mannheim) was added dropwise over 1 minute. After stirring for 1 minute, 1 mQ of HI medium was added dropwise over 1 minute. Further, 8 mfl of HI medium was added dropwise over 3 minutes, and then the cells were collected by centrifugation (1000 rpm, 5 minutes).

The cell pellet was suspended in 40 mQ of HAT medium (HI medium supplemented with HAT solution (manufactured by Dai-Nippon Pharmaceutical Co., Ltd.)) containing 20% tallow serum, and placed in each well of a 966 microplate (manufactured by Cong Co., Ltd.). 100 μΩ was dispensed. After 4 days, 100 μQ of the HAT medium was added and thereafter replaced with fresh HAT medium at a rate of 50% every 2 days. After the 10th day of culture, H containing 10% beef tallow serum was added every 2 days.
The medium was replaced with T medium (HI medium to which HT solution (manufactured by Dai Nippon Kanyaku) was added) at a rate of 50%.

4) Selection of hybridomas Supernatants were sampled from wells of large hybridoma colonies, and hybridomas producing anti-tachorodopsin antibodies were selected by ELISA.

Purified tachorodopsin (0.01 mg/ml) was added to each well of a microtiter plate previously coated with poly-L-lysine.
m Q )50 μQ was dispensed and left at 4° C. for about 18 hours.

Then, 50 μQ of 1% glutaraldehyde was dispensed, and 1 minute later, the plate was washed three times with 200 μQ of phosphate buffered saline (PBS). Furthermore, 200 μQ of blocking solution (Block Ace (manufactured by Dainippon Pharmaceutical Co., Ltd.) diluted with PBS to 4 min. 100 μΩ of the supernatant was added, and the mixture was left standing at 37°C for 1 hour.0.1% Tween
After washing three times with PBS containing 20, biotinylated anti-mouse TgG antibody (ABC kit manufactured by Vectorstin)
100 μQ of the solution was added and allowed to stand at 37° C. for 1 hour. P.E.
After washing three times with S, 100 μQ of a mixed solution of biotinylated peroxidase and avidin was added, and the mixture was incubated at 37° C. for 30 minutes. After washing three times with PBS, 0.1M < citric acid buffer (pl+ 5.4) of orthophenylenediamine containing 0.001% hydrogen peroxide solution was added, and the absorbance at a wavelength of 412 nm was measured.

In parallel, an ELISA method was performed using a 0.1% digitonin solution.

As a result, out of 405 samples, 35 samples recognized tachorodopsin, and 6 of them recognized digitonin.

The hybridomas in the wells that recognized only tacolodopsin were cloned by limiting dilution method, and the hybridoma cell line B6°C'l,
C6, C11, C15, D9°Dll, and D24 were obtained.

5) Preparation of monoclonal antibodies To facilitate antibody purification, the hybridoma was subcultured every 3 days in a serum-free medium (E-RDF medium: Kyokuto Seiyaku) to obtain a stable strain capable of producing antibodies in serum-free culture. were selected.

Both strains grew in the serum-free medium. However, as shown in Figure 1, B6. C1゜C1l, C1
The antibody production ability of the 5, D9,011 strain was unchanged from that at the early stage of passage, but the production ability of the C6 and 024 strains decreased. From this result, B6.
C1, C1l.

C15, D9,011 strains were selected.

Each strain was cultured in suspension using a tapping culture flask (manufactured by Ike Suirika).

The flask capacity was 200 mQ, and the charging capacity was 7° mQ. Then, the temperature was set to 37°C, the number of shaking of the tapping stirrer was set to 350 times per minute, and the gas phase in the flask was replaced with 5% C○2 air. Then, the medium was replaced with fresh medium every day.

As an example of the culture results, the culture progress of strain 011 is shown in FIG.

Each of the above culture solutions was filtered through an ultrafiltration membrane C with a molecular weight cutoff of 200,000.
; manufactured by Toyo Atopantique Co., Ltd.) for about 5 minutes, salting out with ammonium sulfate and dialysis against PBS to obtain a crude antibody solution. Then, it was purified using a monoclonal antibody fractionator (manufactured by Bio-Rad).

6) Characteristics of monoclonal antibodies Class and subclass The class and subclass of each antibody was tested using a mouse monoclonal antibody isotyping kit (manufactured by Amarjam).

As a result, B6. The class of the antibodies produced by strains C1, C15, and Dl1 was IgG, and their respective subclasses were IgG2a for the antibodies produced by strain B6 and IgG1 for the others.

In addition, the class of antibodies produced by C1l and D9 strains is IgM
Met.

■Influence of antibodies on the photochemical reaction of tacolodopsin Purified tacolodopsin was bound to the above antibody and alternately irradiated with red/blue light to examine changes in absorption maximum.

Purified tachorodopsin and antibodies (B6.C1, C15, C1
1, antibodies produced by strains D9 and D11) were mixed at a molar ratio of 3:2 with PBS containing 0.1% digitonin.
Incubated at ℃ for 2 hours. Next, an ultrafiltration membrane (molecular weight cut off 200.000, manufactured by Toyo Atpantic Co., Ltd.)
Unreacted rhodopsin was removed. Immediately remove the supernatant to 0.
．． Red/blue light filters (Toshiba glass filter 0-58.V-44) were brought back to original volume with PBS containing 1% digitonin and set on a slide objector in a dark room.
The supernatant was placed at a distance of 1 cm from the tube and irradiated with light for 10 seconds. Then, measure the total absorption curve with a spectrophotometer (Hitachi Layer),
The absorption maximum value after red/blue light irradiation was investigated.

Table 21 shows changes in absorption maximum values after red/blue light irradiation.

Tachorodopsin (OR) only system and bovine serum albumin (BSA) as controls Table 2 Effect of antibodies on photochemical reaction of tacolodopsin Sample Absorption maximum (nm) Red light irradiation Blue light irradiation 475 506 476 502 475 500 477 502 474 503 477 503 474 501 474 501 OR OR+BSA OR+ Antibody produced by strain 86 OR+ Antibody produced by CI strain OR+ Antibody produced by C11 strain OR+ Antibody produced by C15 strain OR+ Antibody produced by strain 09 OR+Antibody produced by strain 011 was mixed in a system. For all antibodies, the absorption maximum values shifted alternately after irradiation with red/blue light, almost the same as in the case of tacolodopsin alone. Therefore, it was found that these antibodies do not inhibit the photochemical reaction of tacolodopsin.

■ Recognition ability of tacolodopsin for limited trypsin digest When purified tacolodopsin is limitedly degraded with trypsin, tacolodopsin (molecular compact 50,000) has a molecular compact of 40,000 with the molecular compact of 10,000 on the C-terminal side removed.
0, plus about 16,000, and 28,000
A product with a molecular weight of 0 is produced. The ability of each antibody to recognize these limited degradation products was examined by immunoplot.

Purified tacolodopsin and trypsin (manufactured by Worthington Biochemical) were mixed at a ratio of 100:1 and incubated at 37°C for 30 minutes. Immediately mix with varnish treatment solution.

It was heated at 100°C for 5 minutes. Then, the varnish treated solution was electrophoresed on SDS Far-Stgel (manufactured by Pharmacia) using a First System electrophoresis apparatus (manufactured by Pharmacia).

Next, a wet nitrocellulose membrane was placed on the gel and heated from the bottom of the gel at a temperature of 70° C. for 20 minutes to transfer rhodopsin and its trypsin-degraded product. The nitrocellulose membrane was blocked with a blocking agent (manufactured by Dai-Nippon Pharmaceutical Co., Ltd.), washed with PBS containing 0.1% Tween-20, and then the antibody was allowed to act. From now on, ELISA
The procedure was carried out according to the procedure of the law, and finally color was developed with 4-chloro-naphthol.

Figure 3 shows the results of immunoplot. Molecular Covenant 40.
000 is recognized by B6゜C1 and D9
The antibodies for strains C11, C15, and D11 did not recognize the antibodies. Antibodies with a molecular weight of 28,000 were recognized by the B6°C1, C15 and Dl1 strains.

Furthermore, the D11 strain antibody recognized a molecular weight of 16,000.

From this result, it was found that the antibody of the C11 strain recognized a site with a molecular weight of about 10,000 on the C-terminal side. Also, those with a molecular weight of about 16,000 and about 28,000,
Since it is a decomposition product with a molecular weight of approximately 40,000, C
It was found that the antibodies of the 1l and Dll strains recognized a degradation product with a molecular weight of about 40,000, similar to the antibodies of the B6°C1 and D9 strains.

Through the above steps, we were able to create an antibody that does not inhibit photochemical reactions and recognizes a structurally stable site as a monoclonal antibody for immobilizing tacolodopsin.

Example 2 The above antibody was covalently bonded to a carrier, tacolodopsin was immobilized thereon, and red/blue light was irradiated to examine the presence or absence of a photochemical reaction.

The antibody produced by the Dll strain was covalently bound to 30 mg of activated microspheres (about 3 μm in diameter; manufactured by Funakoshi Pharmaceutical Co., Ltd.), and the carrier was recovered by centrifugation at 39 minutes (3000 rpm, 10 minutes). This was suspended in PBS and centrifuged twice for washing. The antibody-bound carrier was mixed with 0.1% digitonin-containing tachorodopsin solution (1.1 mg/m n
) was suspended in S and incubated at 37° C. for 2 hours with gentle stirring, that is, allowed to react. After centrifugation, the carrier was recovered by centrifugation, suspended in PBS containing 0.1% digitonin, and washed by centrifugation.

The washed carrier was colored pale orange and rhodopsin was immobilized thereon.

This was suspended in PBS containing 0.1% digitonin and dropped onto a quartz glass plate. After irradiating red/blue light for 20 seconds through an optical fiber in a dark room, the absorption was detected using an instantaneous multi-photometric detector (manufactured by Otsuka Electronics). Changes were measured.

As a result, when irradiated with red light, an absorption maximum value of 475 nm was obtained, and with blue light, an absorption maximum value of 502 nm was obtained.
Both absorption maxima moved alternately by irradiating red/blue light alternately. Since there is no change in the maximum absorption value when only the antibody-bound carrier is similarly irradiated with red/blue light, the above change is due to a photochemical reaction of tachorodopsin immobilized by the antibody.

〔Effect of the invention〕

According to the present invention, a monoclonal antibody for immobilization that can be stably immobilized without inhibiting the photochemical reaction of tacolodopsin can be obtained. Using this antibody, it is possible to construct optical sensors using tacolodopsin, as well as devices such as optical memories.

[Brief explanation of drawings]

Figure 1 shows B6. CI, C6, C11, C15°D9.
Diagram showing the antibody production ability of Dll and D24 cell lines in serum-free culture. Figure 2 is a diagram showing the results of tapping culture of Dll strain. Figure 3 is B6° C1, C1l, C15, D.
FIG. 9 is a diagram showing the results of immunoplot of antibodies produced by strains 9 and D11. θ! 2 3 4 At the time of compensation Fl/I (d mountain 1) Utaiza (-) Sales - wo cliff (-) A certain figure C t rt 15 Dri 11

Claims (1)

[Claims] 1. Without inhibiting the photochemical reaction of tacolodopsin,
A monoclonal antibody characterized by recognizing a structurally stable site. 2. The monoclonal antibody according to claim 1, wherein the antibody is an anti-tachorodopsin antibody. 3. The monoclonal antibody according to claim 2, wherein the anti-tacolodopsin antibody is a trypsin-limited degradation product of tacolodopsin and recognizes a degradation product with a molecular weight of about 16,000 or about 28,000. 4. A hybridoma that produces antibodies against tacolodopsin by fusing mouse spleen cells immunized in vivo or in vitro with rod-sensitive rods or tacolodopsin extracted and purified with a surfactant and mouse-derived myeloid cells. Next, the hybridoma is grown in vivo or in vitro, and the antibody purified from ascites or culture fluid is mixed with tachorodopsin extracted and purified with a surfactant in the presence of a surfactant. a step of collecting antibody-bound tacolodopsin after reacting with tacolodopsin;
A method for producing a hybridoma that produces a monoclonal antibody that does not inhibit the photochemical reaction of tacolodopsin, the method comprising the step of alternately irradiating the tacolodopsin solution with red light and blue light and measuring a total absorption curve. 5. The monoclonal antibody according to claim 4, wherein in the step of collecting the antibody-bound tacolodopsin, high molecular components are fractionated by an ultrafiltration membrane that does not pass through a molecular weight of 100,000 or more in the presence of a surfactant. A method for producing a hybridoma that produces 6. The method for producing a hybridoma producing a monoclonal antibody according to claim 4 or 5, wherein the surfactant is a nonionic surfactant. 7. The method for producing a hybridoma producing a monoclonal antibody according to claim 4 or 5, wherein the surfactant is digitonin. 8. The hybridoma producing the monoclonal antibody according to claim 4, wherein when immunizing in vivo with tacolodopsin extracted and purified with digitonin, the amount of digitonin injected is 0.5 mg or less per mouse. Production method. 9. A hybridoma that produces antibodies against tacolodopsin by fusing mouse spleen cells immunized in vivo or in vitro with rod-sensitive rods or tacolodopsin extracted and purified with a surfactant and mouse-derived myeloma cells. Next, the hybridoma is grown in vivo or in vitro, and the antibody purified from ascites or culture fluid is mixed with tachorodopsin extracted and purified with a surfactant in the presence of a surfactant. a step of collecting antibody-bound tacolodopsin after reacting with tacolodopsin;
A hybridoma that produces a monoclonal antibody that does not inhibit the photochemical reaction of tacolodopsin, which is produced by the step of alternately irradiating the tacolodopsin solution with red light and blue light and measuring a total absorption curve. 10. The monoclonal antibody according to claim 9, wherein in the step of collecting the antibody-bound tachorodopsin, high molecular components are fractionated by an ultrafiltration membrane that does not pass through a molecular weight of 100,000 or more in the presence of a surfactant. A hybridoma that produces 11. The hybridoma producing a monoclonal antibody according to claim 9 or 10, wherein the surfactant is a nonionic surfactant. 12. The hybridoma producing a monoclonal antibody according to claim 9 or 10, wherein the surfactant is digitonin. 13. The hybridoma producing a monoclonal antibody according to claim 9, wherein when in vivo immunization is performed with tacolodopsin extracted and purified with the digitonin, the amount of digitonin injected is 0.5 mg or less per mouse.