JPH0577398B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to an anti-tacolodopsin antibody useful for utilizing tacolodopsin in optical sensors, optical information recognition elements, and the like. [Prior Art] 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 living 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. JP-A-63-111428
The publication describes a biological device in which rhodopsin is inserted into liposomes of haptenized phospholipids, and the liposomes are two-dimensionally arranged on a substrate using anti-hapten antibodies to convert external optical information into various ions and chemical substances. Here's how to build it. By the way, visual substances, especially tachorodopsin, which is a sensor for optical information recognition, become a mixture of metalrhodopsin and rhodopsin when it receives blue light, and the maximum absorption starts from 475 nm.
Move to 500nm. and red light, especially 580nm.
Metalrhodopsin can be regenerated into rhodopsin by irradiation with the above light.
Tsuda, Biochimica et Biophysica Acta),
578, 372-380 (1979)}. 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 can 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 Jaye Barnstable (CJ
Barnstable et al. created a monoclonal antibody that recognizes photoreceptor tissue using rough membrane components obtained from rat retina as an antigen, and analyzed the structure of photoreceptor tissue using it in combination with a bovine rhodopsin monoclonal antibody. J.of Neurocytology, 12, p785−803
(1983). In addition, Donald 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 {Biochemistry, 23, pp6544−6549 (1984). }However, anti-tachorodopsin antibodies that can be used in 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 does not inhibit the function of the protein and that it recognizes a stable site of the structure. be done. For this reason, anti-tacolodopsin antibodies must be selected that do not inhibit the photochemical reaction of tacolodopsin. That is, the anti-tachorodopsin antibody must be such that the antibody-bound tachorodopsin receives red/blue light and its absorption maximum shifts between 475 nm and 500 nm. In addition, during the purification process, tachorodopsin often has a molecular weight of about 40,000 due to a portion of the molecular weight of about 10,000 being cut off from the C-terminus due to the action of proteases.
become. 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 with 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 for Solving the Problems] In order to select an anti-tacolodopsin monoclonal antibody that does 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 tachorodopsin that has not reacted with the antibody, accurate determination cannot be made. 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 tachorodopsin sample as a test sample and using an ultrafiltration method, and we separated tachorodopsin that was not bound to antibodies using an ultrafiltration membrane with a molecular weight cutoff of 200,000. investigated.
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 purified tachorodopsin formed aggregates due to the low surfactant concentration.
That is, when supplemented with a surfactant and subjected to ultrafiltration,
Tachorodopsin migrated to the low molecular weight fraction. By the way, surfactants generally inhibit or break the binding between antibodies and antigens. However, when we examined the presence or absence of any effects using nonionic surfactants such as digitonin, octyl glucoside, heptyl glycoside, and sugar ester, which are membrane protein extractants,
It turned out 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 weight and low molecular weight fractions showed specific absorption of tachorodopsin. At this time, when the low-molecular-weight fraction was electrophoresed in SDS 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 is possible to separate the tacolodopsin that has bound to the antibody from the unbound tacolodopsin. I found out that it is possible. 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 about 10,000 from the C-terminus was selected by immunoblotting of protease-treated tacolodopsin. Tachorodopsin has a molecular weight of approximately 50,000,
In trypsin treatment, the molecular weight of the C-terminal side is approximately
10,000 is cut to give a molecular weight of about 40,000, which is then cut into a molecular weight of about 28,000 and a molecular weight of about 16,000.
Yamada Conference XXIon Molecular
Physiology of Retinal Proteins), pp167−172
(1988)}. Therefore, the molecular weight on the C-terminal side is approximately 10,000
In order to select antibodies that do not recognize this site, it is sufficient to select an antibody that recognizes a degradation product with a molecular weight of about 40,000. However, it was found that some antibodies do not recognize degradation products with a molecular weight of about 40,000, but recognize degradation products with a molecular weight of about 16,000 or about 28,000. Therefore, it has been found that in order to select an antibody that does not recognize a site with a molecular weight of approximately 10,000 on the C-terminal side, it is sufficient to select an antibody that recognizes a degradation product with a molecular weight of approximately 16,000 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 spleen cells immunized in vivo or in vitro with rod-sensitized rods or purified tacolodopsin with mouse-derived myeloma 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. Further, a step of performing immunoblotting with trypsin-limited degradation products of tacolodopsin to select antibodies that recognize degradation products with a molecular weight of about 16,000 or about 28,000. 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 myeloma cell, and cloning the one that produces an anti-tachorodopsin antibody. The antibody can be obtained by growing this in the mouse peritoneal cavity or in a culture container. Mouse spleen cells immunized with tacolodopsin are used as antibody-producing cells. Tachorodopsin, which serves as an immunizing antigen, can be used as a crude or purified preparation. 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 Shugar 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. For immunization, administer 40 to 100 μg of protein per subcutaneous, intravenous, or intraperitoneal injection of the antigen.
It can be done by giving 2 to 4 injections every 2 to 3 weeks. Note that some membrane protein extractants such as digitonin and sugar ester 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
The amount should be 0.5 mg or less, preferably 0.1 mg or less. Furthermore, if the antigen contains an extractant that is highly toxic to mice, an in vitro immunization method may be used. Cell fusion can be performed according to the method of Kohler and Milstein et al. The fusion partner is Mouth Bulb Sea (BALB/c)
Myeloma cells such as X63 cells (X63), P3U1 cells, NS-1 cells, and SP2 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.
Thoroughly loosen the pellets and add 30 to 50% polyethylene glycol (molecular weight 1000
~4000) and react at 30-37℃. Then,
Stop the reaction by mixing dropwise with serum-free medium. Furthermore, after adding a large amount of serum-free medium, the cells are collected by centrifugation. the cell
Suspend in HAT (containing hypoxanthine and aminopterin thymidine) medium and dispense into 96-well plate.
Incubate at 37°C. 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 hybridomas have grown sufficiently, those that produce anti-tachorodopsin antibodies are selected by immunoassay (Enzyme Linked Immunosorbent Assay, hereinafter referred to as ELISA). Then, cloning is performed by the limiting dilution method to obtain an anti-tachorodopsin antibody-producing clone. The clone was previously administered pristane.
Antibodies can be obtained by transplanting into the peritoneal cavity of BALB/c mice and collecting ascites fluid 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 purified tachorodopsin and antibody are mixed in the presence of a nonionic surfactant such as diochitonin or sugar ester, and incubated at 37°C for 1 to 4 hours. The concentration of the surfactant may be 2% or less, preferably 0.05 to 0.2%. 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 is not limited to a membrane that blocks the passage of molecules having a molecular weight of 100,000 or more, but may be any membrane that can separate tachorodopsin and antibodies. This fractionated solution with a molecular weight of 100,000 or more contains a surfactant.
PBS may be added to restore the original solution volume 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 irradiated with red light of 560 nm or more and blue light of 350 nm to 460 nm for about 10 to 60 seconds, and the total absorption curve is measured between 200 nm and 650 nm using a spectrophotometer. . Then, find the absorption maximum value when red/blue light is irradiated alternately, and choose one in which the absorption maximum around 475 nm changes alternately for red light irradiation, and the absorption maximum around 500 nm for blue light irradiation. , 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 immunoblotting. Mix purified tacolodopsin and trypsin at a ratio of 50 to 200:1 and incubate at a temperature of 37℃ for 5 minutes.
Incubate for ~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 contains mercaptoethanol.
Mix with SDS treatment solution and heat at 100°C for 1 to 5 minutes.
After electrophoresis in SDS polyacrylamide gel, it is transferred to a nitrocellulose membrane. Then, the selected antibodies were activated according to the ELISA method to examine their ability to recognize trypsin degradation products, and those that recognized degradation products with a molecular weight of about 16,000 or about 28,000 were selected, and those that recognized degradation products with a molecular weight of about 10,000 on the C-terminal side were selected. Select antibodies that do not recognize the site. Through the above steps, it is possible to create an antibody that does not inhibit photochemical reactions and recognizes a structurally stable site as a monoclonal antibody for immobilizing tacolodopsin. [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. 100 eyes of the water octopus (Paroctopus defleini)
The retina detached and collected from the individual was suspended in a buffer solution {10mM MOPS-NaOH, PH7.4, 20μM p-aminophenylmethasulfonyl fluoride hydrochloride (P-APASF)} 1.2, and then wrapped in gauze. to remove large solids. Then 40000×
g. Centrifugation was performed at 4°C for 30 minutes to collect the outer segments of photoreceptor cells that constitute the retina. the outer clause
Suspended in 300 ml of 34% sucrose-containing buffer, 40,000x
g. Sucrose suspension centrifugation was performed for 1 hour, and the supernatant was collected to obtain rod-sensitive clones. Buffer was added to this supernatant to make 600 ml, and the mixture was heated at 40,000
Centrifugation was performed under conditions of 10 minutes to obtain rod-sensitive pellets. The pellet was then resuspended in buffer and centrifuged under similar conditions. Perform this operation 3
I went around and washed it. The rod sensitive body was dissolved in digitonin solution (2% digitonin, 100mM Na ₂ HPO ₄ −
Rhodopsin was extracted by suspending it in KH ₂ PO ₄ , pH 6, 8) and shaking for 1 hour. Next, an extract was obtained from which residues were removed by centrifugation (25,000 xg, 1 hour). The residue was extracted with digitonin again,
Unextracted rhodopsin was collected. In order to remove ommochrome proteins from the digitonin extract, ion exchange chromatography was performed using DEAE-Sephacel (manufactured by Pharmacia).
Fill the column with 5 ml of DEAE-Sephacel and add buffer (0.1% digitonin, 50mM Na ₂ HPO ₄ -
After equilibration with KH ₂ PO ₄ , pH 6, 8), the extract was charged and passed through the buffer at 280 nm.
A fraction with absorption at 480 nm was obtained. Rhodopsin was then isolated by affinity chromatography using Con A-Sepharose (manufactured by Pharmacia). Con A-Sepharose 10
ml was packed into a column, equilibrated with a buffer solution (0.2% digitonin, 50mM Tris-HCl, PH7.0), and then the fractionated solution was charged. After passing through the buffer several times, rhodopsin was eluted with a buffer containing 1M α-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/ml. This was diluted and injected into the abdominal cavity of BALB/c female mice using the method described below to sensitize them to tacolodopsin.

[Table] Interval 1st <--2 weeks--> 2nd <--2 weeks--> 3rd <--2 weeks--> Final adjuvant: N-acetylmouramil-
El-alanyl-dei-isoglutamine (N-
acetylmuramyl-L-alanyl-D-
isoglutamine, 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 cultured in a 1:1 mixture of HAM medium (Nissui Pharmaceutical Co., Ltd.) and IMDM medium (Sigma Co., Ltd.) (hereinafter referred to as HI).
After loosening the cells in a medium (referred to as medium), spleen cells were collected by centrifugation (1600 rpm, 5 min).
The cell pellet was resuspended in HI medium, and spleen cell suspension and HI containing 10% fetal bovine serum were added.
Myeloma cells (strain P3U1) cultured in the medium were collected by centrifugation (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. 2 x 10 ⁸ spleen cells, 4 x 10 ⁷ myeloma cells
Both cell suspensions were mixed to give a mixed pellet by centrifugation (1800 rpm, 5 minutes). After completely removing the supernatant, the pellet was mixed. To this, 1 ml of 50% polyethylene glycol 1500 (manufactured by Boehringer Mannheim), which had been preheated at 37°C, was added dropwise over 1 minute. and 1
After stirring for a minute, 1 ml of HI medium was added dropwise over a period of 1 minute. Further, 8 ml of HI medium was added dropwise over 3 minutes, and the cells were collected by centrifugation (1000 rpm, 5 minutes). The cell pellet contained 20% fetal bovine serum.
HAT medium {HAT solution in HI medium (manufactured by Dai Nippon Pharmaceutical)
suspended in 40 ml and added to each well of a 96-well microplate (Kong).
Dispense 100μ. After 4 days, add 100μ of the HAT medium.
and then replaced with fresh HAT medium at a rate of 50% every 2 days thereafter. After the 10th day of culture,
The medium was replaced every 2 days at a rate of 50% with HT medium containing 10% fetal bovine serum (HI medium supplemented with HT solution (manufactured by Dai-Nippon Pharmaceutical Co., Ltd.)). (4) Selection of hybridomas Supernatants were sampled from wells of large hybridoma colonies, and hybridomas producing anti-tachorodopsin antibodies were selected by ELISA. Dispense 50μ of purified tacolodopsin (0.01mg/ml) into each well of a microtiter plate previously coated with poly-L-lysine, and incubate at 4°C for approximately 18 hours.
Let it stand for a while. Then, 50μ of 1% glutaraldehyde was dispensed, and after 1 minute, the plate was washed three times with 200μ of phosphate buffered saline (PBS). Furthermore, add 200μ of blocking solution {Block Ace (manufactured by Dainippon Pharmaceutical) diluted to one-fourth with PBS,
The mixture was allowed to stand at 37°C for 1 hour, and the adsorbed portion of each well was blocked. Wash 3 times with PBS and remove the supernatant.
100μ was added and left at 37°C for 1 hour. 0.1%
After washing 3 times with PBS containing Tween 20, 100μ of biotinylated anti-mouse IgG antibody (ABC kit manufactured by Vector Stain) was added and incubated at 37℃ for 1 hour.
Let it stand for a while. After washing three times with PBS, add a mixed solution of biotinated peroxidase and avidin.
100μ was added and left at 37°C for 30 minutes. and
After washing three times with PBS, a 0.1M citrate buffer (PH5.4) of orthophenylenediamine containing 0.001% hydrogen peroxide was added, and the absorbance at a wavelength of 412 nm was measured. In parallel, ELISA 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. Hybridomas in wells that recognized only tacolodopsin were cloned by limiting dilution method and hybridoma cell line B
6, C1, C6, C11, C15, D9, D1
1, D24 was obtained. (5) Preparation of monoclonal antibodies To facilitate antibody purification, the hybridoma was cultured in a serum-free medium (E-RDF medium: Kyokuto Seiyaku).
Subculture was carried out every 3 days, and strains with stable antibody production ability were selected by serum-free culture. Both strains grew in the serum-free medium. However, as shown in Figure 1, B6, C
1, C11, C15, D9, and D11 strains had the same antibody production ability as at the early stage of passage;
The productivity of strains 6 and D24 decreased. From this result, B6,
C1, C11, C15, D9, and D11 strains were selected. Each strain was cultured in suspension using a tapping culture flask (manufactured by Ikemoto Rika). The flask capacity was 200 ml and the charging capacity was 70 ml. The temperature was 37℃, the number of shaking of the tapping stirrer was 350 times, and the gas phase in the flask was 5%.
_CO2 was replaced with air. Then, the medium was replaced with fresh medium every day. As an example of the culture results, the culture progress of the D11 strain is shown in FIG. Each of the above culture solutions was filtered through an ultrafiltration membrane (molecular weight cutoff).
200000; manufactured by Toyo Advantake Co., Ltd.) approximately 5
After concentrating to one-fold, a crude antibody solution was obtained by salting out with ammonium sulfate and dialysis against PBS.
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 Amasyam). As a result, B6, C1, C15 and D11
The class of the antibodies produced by the strain was IgG, and their respective subclasses were IgG2a for the antibodies produced by the B6 strain and IgG1 for the others. Furthermore, the class of antibodies produced by C11 and D9 strains was IgM. Effect 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,
Antibodies produced by strains C15, C11, D9 and D11) were mixed at a molar ratio of 3:2 in PBS containing 0.1% digitonin and incubated at 37°C for 2 hours. Next, an ultrafiltration membrane (molecular weight cutoff 200000: manufactured by Toyo Advantake Co., Ltd.)
Unreacted rhodopsin was removed. Immediately, the supernatant was brought back to its original volume with PBS containing 0.1% digitonin and filtered through a red/blue light filter (Toshiba glass filter 0-58, V-44) set on a slide objector in a dark room.
The supernatant was placed at a distance of cm and irradiated with light for 10 seconds. Then, the total absorption curve was measured using a spectrophotometer (manufactured by Hitachi), and the maximum absorption value after irradiation with red/blue light was investigated. Table 2 shows changes in absorption maximum values after red/blue light irradiation. Tachorodopsin (OR) only system and bovine serum albumin (BSA) as controls

【table】

〔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 produced. Using this antibody, it is possible to construct optical sensors using tacolodopsin, as well as devices such as optical memories.

[Brief explanation of the drawing]

Figure 1 shows B6, C1, C6, C11, C1
5. Diagram showing antibody production ability in serum-free culture of D9, D11 and D24 cell lines, Figure 2 is D11
Diagram showing the results of tapping culture of the strain, Figure 3 is B
6, C1, C11, C15, D9, and D11 strain immunoblot results.

Claims (1)

[Scope of Claims] 1. A monoclonal antibody that does not inhibit the photochemical reaction of tacolodopsin and recognizes a region other than a region having a molecular weight of about 10,000 from the structurally stable C-terminal side. 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. Hybridomas that produce antibodies against tacolodopsin are produced 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. A step of producing the hybridoma, and a step of growing the hybridoma in vivo or in vitro.An antibody purified from ascites or culture fluid and tacolodopsin extracted and purified with a surfactant are mixed in the presence of a surfactant to form the antibody and octopus. After reacting with rhodopsin, the photochemical reaction of tachorodopsin is inhibited by having a step of collecting antibody-bound tachorodopsin, and a step of alternately irradiating the tachorodopsin solution with red light and blue light and measuring the total absorption curve. A method for producing a hybridoma that produces a monoclonal antibody that does not. 5. Producing the monoclonal antibody according to claim 4, wherein in the step of collecting the antibody-bound tacolodopsin, high molecular components are fractionated using an ultrafiltration membrane that does not pass molecular weights of 100,000 or more in the presence of a surfactant. Method for producing hybridomas. 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 method for producing a hybridoma producing a monoclonal antibody according to claim 4, characterized in that 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. . 9 Mouse spleen cells immunized in vivo or in vitro with tacolodopsin extracted and purified using rod-sensitive clones or surfactants are fused with mouse-derived myeloid cells to generate hybridomas that produce antibodies against tacolodopsin. A step of producing the hybridoma, and a step of growing the hybridoma in vivo or in vitro.An antibody purified from ascites or culture fluid and tacolodopsin extracted and purified with a surfactant are mixed in the presence of a surfactant to form the antibody and octopus. Tachorodopsin produced by reacting with rhodopsin, collecting antibody-bound tacolodopsin, and measuring the total absorption curve by alternately irradiating the tacolodopsin solution with red light and blue light. A hybridoma that produces monoclonal antibodies that do not inhibit photochemical reactions. 10. Producing the monoclonal antibody according to claim 9, 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. hybridoma. 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 the amount of digitonin injected is 0.5 mg or less per mouse when immunizing in vivo with tacolodopsin extracted and purified with the digitonin.