JPH04365497A - Anti-lentinan monoclonal antibody - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a monoclonal antibody against the polysaccharide lentinan and a fused cell producing the monoclonal antibody.

Lentinan, which is currently on the market as an anticancer drug, has been studied in order to express its anticancer activity more strongly by analyzing its distribution in the body after administration, determining the sensitivity of individual cancer patients to lentinan, or investigating the cells that bind to lentinan. More detailed analysis, such as identification of If these facts become clear, it may become easier to select more appropriate administration timing and dosage depending on each individual cancer patient. Anti-lentinan antibodies can be a useful means to solve these problems, and can also be applied to methods for predicting post-administration effects.

0003

[Prior Art] Conventionally, antigenic polysaccharides have been used to immunize animals using themselves as immunogens;
Polysaccharides such as lentinan, which have extremely low antigenicity, were used to form complexes with proteins and then immunized to obtain antisera (Article from the 49th Japanese Cancer Society General Meeting).
345 pages, 1990). However, the amount of antiserum against lentinan is limited, and the types and titers of antibodies induced by individual animals vary, so detailed studies cannot be conducted under the same conditions. Ta.

[0004] In order to solve these problems, it is conceivable to obtain fusion cells that produce monoclonal antibodies against polysaccharides as a method for stably and reproducibly producing the same antibody. However, the antibody detection method using immunoprecipitation, which is used to prepare lentinan antiserum, requires several hundred μl of antibody in terms of serum volume, and monoclonal antibodies in the fusion cell culture supernatant are The problem is that it is impossible to detect.

[0005]Also, it is possible to apply an enzyme immunoassay method in which an antigen is immobilized, which is used in the production of monoclonal antibodies against proteins, but unlike proteins, polysaccharides such as lentinan are immobilized. Due to the problem of difficulty in phase formation, it is difficult to detect antibodies against lentinan using such enzyme immunoassay. Therefore, it is impossible to produce monoclonal antibodies unless a highly sensitive detection method for antibodies against lentinan is established.

Lentinan, which is currently on the market as an anticancer agent, is desired to develop a better method for predicting its effects. For example, a method can be considered to determine a patient's sensitivity to lentinan based on the strength of reactivity between the patient's blood-derived cells and lentinan. For this purpose, it is necessary to detect lentinan by some method. the current,
There is a method for detecting lentinan using the Limulus test, but there are various problems such as specificity, differences in reagent lots, and differences in reactivity due to differences in lentinan lots, and only lentinan in serum can be detected. detection at the cellular or material level is not possible. Therefore, if antibodies against lentinan can be obtained, it can be an effective means to solve the above problems.

[0007] Therefore, diagnosis of patients using antibodies against lentinan is considered to be of great benefit to patients. In this case, it is desirable that each patient be diagnosed under the same conditions, but this is difficult with antisera against lentinan because the types of antibodies, titers, etc. differ. Therefore, it is desired to obtain fused cells that produce anti-lentinan monoclonal antibodies that can stably and reproducibly produce antibodies having the same reactivity to lentinan.

[0008]

OBJECTS TO BE SOLVED BY THE INVENTION The problem to be solved by the present invention is to produce fused cells that stably and reproducibly produce antibodies having the same reactivity to lentinan, and to achieve this. In order to do this, we established a highly sensitive detection method that can detect antibodies in the culture supernatant of fused cells, which was not possible with conventional methods, and used this method to detect fused cells that produce monoclonal antibodies. The objective is to obtain monoclonal antibodies by cloning.

[0009]

[Means for Solving the Problems] As a result of intensive studies in order to solve the above problems, the present inventors oxidatively cleaved lentinan with periodic acid or its salt, introduced an aldehyde group, and obtained the aldehyde. After bonding the amino group of a protein (albumin, hemoglobin, etc.) to the base in the form of a Schiff base, it is reduced with sodium borate hydride to form lentinan.
A protein complex was obtained. Next, by immobilizing the lentinan-protein complex on a plastic plate, we established a method for detecting anti-lentinan antibodies that is more than 10,000 times more sensitive than the immunoprecipitation method conventionally used to detect antibodies. Successful. Applying this method and cloning monoclonal antibody-producing fusion cells using an antibody detection method characterized by utilizing a combination with a binding competition method in the presence of lentinan to obtain antibody-producing fusion cells. , and succeeded in producing monoclonal antibodies.

The present invention will be explained in detail below.

First, in order to establish a highly sensitive method for detecting antibodies against lentinan, we will examine the application of enzyme immunoassay, which is used to detect antibodies against proteins. Unlike proteins, polysaccharides such as lentinan cannot be immobilized on plastic plates, so they form a complex with lentinan and proteins, and utilize the binding ability of proteins to plastic plates. Lentinan is immobilized in a protein-mediated manner. Specifically, the polysaccharide lentinan is oxidatively cleaved with sodium metaperiodate to introduce an aldehyde group, and the amino group of a protein is bonded to the aldehyde group in the form of a Schiff base, which is then reduced with sodium hydroxide borate. , to obtain a lentinan-protein conjugate. The protein used here is not particularly limited, but bovine serum albumin (hereinafter referred to as BSA) or human hemoglobin (hereinafter referred to as Hb) is suitable. This lentinan-protein complex is then purified by high performance liquid chromatography to remove unreacted raw materials. Thereafter, this lentinan-protein complex is added to a plastic plate and left at room temperature to solidify on the plastic plate.

Next, the establishment of a highly sensitive antibody detection method using a solid-phase lentinan-protein complex will be explained. Anti-lentinan/protein complex mouse antiserum obtained by immunizing a mouse (any animal species) with the lentinan/protein complex prepared by the above method was added to phosphate buffered saline (hereinafter referred to as PBS). After dilution, add to the solid phase on a plastic plate.

[0013] Here, the protein of the lentinan-protein complex used as an immunogen for mice is preferably different from the protein of the lentinan-protein complex used for immobilization on the plastic plate, for example. It is preferable to use a lentinan/BSA complex as a mouse immunogen and to use a lentinan/Hb complex for immobilization on the plastic plate. The reason why complexes of different proteins are used in this way is that this mouse antiserum also contains antibodies against BSA, so if a complex using the same protein (BSA) is immobilized on a plastic plate, This is because antibodies against BSA are thought to react with the addition of mouse antiserum, and antibodies against lentinan cannot be specifically detected. After adding the mouse antiserum, a commercially available enzyme, fluorescent or biotin-labeled anti-mouse immunoglobulin antibody diluted with PBS is further added to detect the mouse-derived anti-lentinan antibody bound to lentinan. As a result, it was found that it was possible to detect antibodies even at a 10,000-fold dilution at the serum level, and the detection sensitivity was more than 10,000 times higher than that of the immunoprecipitation method that has traditionally been used with lentinan's antibody detection method. succeeded in doing so.

[0014] The above detection method is considered to be fully applicable to the production of monoclonal antibodies against the polysaccharide lentinan, but the immobilized lentinan and the lentinan used for immunization are was treated with sodium metaperiodate to partially convert β-(1
→6) Since the bond branches are oxidatively cleaved and then reduced and converted into polyalcohols, it is possible that antibodies that recognize the processed polyalcoholized portions are also detected. Therefore, in the process of this detection method, when adding an antibody solution or a culture supernatant of fused cells, an untreated lentinan solution is added to check whether binding to the immobilized lentinan/Hb complex is inhibited. We apply a combination of binding competitive inhibition methods using By using this, if an antibody that recognizes the polyalcohol moiety exists, it will react only with the immobilized lentinan/Hb complex and not with the added untreated lentinan, thereby preventing binding inhibition. Furthermore, if there is an antibody that reacts with lentinan itself, it will also react with the added untreated lentinan, and therefore the amount of antibody that binds to the immobilized lentinan/Hb complex is thought to decrease.

[0015] Using the above detection method, a monoclonal antibody against lentinan is produced. First, mice are immunized with a lentinan/BSA complex in which BSA is bound to lentinan using the method described above. The spleen of the mouse was removed and a single cell suspension was prepared using mouse myeloma cells P3×63Ag8.
．． Perform cell fusion with 653. The fused cells are treated with RPMI containing, for example, hypoxanthine, aminopterin, and thymidine.
-1640 medium (hereinafter referred to as HAT medium) in a 96-well plastic plate, and the culture fluid in which colonies were observed was examined for the presence or absence of antibodies using the above-mentioned detection method. Colonies positive for antibody titer are diluted by limiting dilution and cloned to obtain a monoclonal antibody against lentinan and its antibody-producing fusion cells. Monoclonal antibodies are then obtained from the antibody-producing fused cells thus obtained.

The present invention will be explained in more detail below based on examples. Of course, the invention is not limited to these examples.

[0017]

【Example】

(1) Preparation of lentinan-protein complex Lentinan 2m
g/ml was subjected to oxidative cleavage with 3 mM sodium metaperiodate (this concentration was found to be optimal as a result of various studies) at 4°C overnight in the dark. Excess sodium metaperiodate was decomposed by adding ethylene glycol, and the reaction solution was dialyzed against distilled water at 4°C overnight. 1/4 amount of BSA or Hb (17 mg/ml) was added to the dialysed fluid, the pH was adjusted to 9.5 with a sodium hydroxide solution, and the mixture was stirred at 4°C all day and night to bind lentinan and protein. After the binding reaction, a sufficient amount of sodium borohydride was added and the mixture was reduced at 4° C. with stirring all day and night. Thereafter, excess sodium borohydride was decomposed with hydrochloric acid to neutralize the pH of the solution, and the reaction solution was further dialyzed against distilled water at 4°C overnight.

In order to remove unreacted BSA or Hb from the reaction solution, gel filtration was performed using high performance liquid chromatography. The column is TSKgelG300
The analysis was carried out using OSW analytical and preparative columns and PBS as the elution solvent. For detection and analysis, a differential refractometer and an ultraviolet detector (wavelength 280 nm) were used.
It must not have ultraviolet absorption at 0 nm (hereinafter referred to as UV absorption) and be eluted at the exclusion limit, while BSA, Hb
It was confirmed that all of them had UV absorption and were not eluted at the exclusion limit, but were eluted after a delay. Next, as a result of analyzing the reaction solution, it was found that the peak eluted at the exclusion limit had UV absorption, indicating that protein was bound to lentinan. Later, unreacted BSA or Hb
was eluted. FIG. 1 shows chromatograms of a lentinan/BSA reaction solution, a lentinan solution, and a BSA solution in the preparation of a lentinan/BSA complex using a gel filtration method using high-speed chromatography. The fraction eluted at the exclusion limit of this reaction solution was separated and purified using a preparative column. Among these synthesized lentinan/protein complexes, the lentinan/BSA complex was used as an immunogen, while the lentinan/Hb complex was used to establish a highly sensitive detection method for antibodies as described below.

(2) Preparation of anti-lentinan monoclonal antibody and its antibody-producing fused cells Add Freund's complete adjuvant (manufactured by Difco) to the lentinan/BSA complex solution, mix and emulsify, and adjust the amount of lentinan to 1 mg/kg (BSA Mice were immunized by subcutaneous injection at a dose of 2 mg/kg once every two weeks for a total of two times. Every week from the first day of immunization, blood was collected from the fundus of the eye to obtain serum. Antibodies against lentinan in these serum samples were examined using the highly sensitive antibody detection method described below.
An increase in antibody titer was confirmed within the first week. FIG. 2 shows that an increase in antibody titer was observed.

Whole blood was collected from the heart of a mouse with increased antibody titer to obtain serum, which was used as a positive control (hereinafter referred to as mouse anti-lentinan antiserum) for antibody detection. Furthermore, the spleen of this mouse was removed, a single cell suspension was prepared, and cell fusion was performed with mouse myeloma P3x63Ag8.653 according to a conventional method using polyethylene glycol. This cell suspension was cultured in a 96-well plate in HAT medium, and the culture supernatant of the wells in which colony formation was observed was examined using an antibody detection method. As a result, the antibody-positive colonies were diluted by the limiting dilution method, and secondary culture was performed in RPMI-1640 medium using mouse thymocytes as feeder cells. Thereafter, anti-lentinan antibodies were detected in the culture supernatants of cells in which colony formation was observed after secondary culture using an antibody detection method to obtain anti-lentinan monoclonal antibody-producing fused cells. This anti-lentinan monoclonal antibody-producing fusion cell has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (FERM P-12284). FIG. 3 shows the results of detecting an anti-lentinan monoclonal antibody using a combination of a highly sensitive anti-lentinan antibody detection method and a binding competitive inhibition test (described later).

[0021] The fused cells thus obtained were cultured in large quantities and cryopreserved according to a conventional method using dimethyl sulfoxide. Furthermore, the fused cells were intraperitoneally inoculated into mice that had been previously intraperitoneally inoculated with pristane (manufactured by Sigma), and then ascites was collected, and the ascites cells were cryopreserved in the same manner. The obtained monoclonal antibody was examined using a mouse monoclonal antibody isotyping kit (manufactured by Amersham) and was found to be immunoglobulin M (IgM).

(3) Immobilization of lentinan on a plastic plate In the process of producing an anti-lentinan monoclonal antibody described in (2) above, an enzyme immunoassay method ( In order to establish the ELISA method (hereinafter referred to as ELISA method), lentinan was immobilized on a plastic plate. After diluting the lentinan/Hb complex solution described in (1) with PBS to a lentinan concentration of 5 μg/ml (Hb concentration of 10 μg/ml),
It was added to a 96-well plastic plate at 0 μl/well and left at room temperature for 2 hours or more. Then, 150 μl/well of P
The plate was washed with BS, 300 μl/well of Block Ace (manufactured by Snow Brand Co., Ltd.) diluted 4 times with PBS was added, and after leaving it at room temperature for 1 hour, the portion of the plate that had not been immobilized was blocked. Whether or not the lentinan/Hb complex was immobilized was confirmed as follows. That is, lentinan/BSA prepared by the method described in (1)
Rabbit anti-lentinan antiserum obtained by immunizing rabbits with the complex (presence of anti-lentinan antibodies has been confirmed by immunoprecipitation method) was diluted 100 to 1000 times with PBS, and diluted with 50 μl/well. After adding and leaving at room temperature for 1 hour,
Plates were washed three times with PBS containing 0.5% BSA. To detect the rabbit-derived anti-lentinan antibody bound to the immobilized lentinan-Hb complex, a commercially available alkaline phosphatase-labeled anti-rabbit immunoglobulin antibody (manufactured by Zymet) was diluted 200 times with PBS and added at 50 μl/well. did. After leaving for 1 hour at room temperature, P containing 0.5% BSA
After washing 3 times with BS, add 10ml to 1mg/ml.
0.05M sodium bicarbonate buffer containing M magnesium chloride, pH = 9.5 (hereinafter referred to as PNPP buffer)
The commercially available alkaline phosphatase substrate (manufactured by Sigma) prepared above was added at 100 μl/well, and after 20 to 30 minutes, the absorbance at 405 nm of the yellow colored solution was measured using a microplate reader. In order to eliminate the possibility of non-specific binding of the anti-lentinan antibody to the Hb portion of the immobilized lentinan/Hb complex, a similar study was performed using a plate immobilized with only Hb (10 μg/ml). . FIG. 4 shows the results of an investigation using anti-lentinan antiserum with the lentinan/Hb complex immobilized (a) and only Hb immobilized (b).

[0023] From the results shown in Fig. 4, it was revealed that only the plate on which the lentinan/Hb complex was immobilized was bound to the anti-lentinan antibody and did not non-specifically bind to Hb. Based on the above results, by using a lentinan/Hb complex, we succeeded in immobilizing lentinan on a plastic plate in a Hb-mediated manner. (4) Establishment of a highly sensitive detection method for anti-lentinan antibodies using immobilized lentinan-Hb complexes using the method described in (3) to detect anti-lentinan antibodies. A highly sensitive detection method was established. As a positive control for mouse anti-lentinan antibody (
Mouse anti-lentinan antiserum obtained by the method described in 2) was used. After immobilizing the lentinan/Hb complex and blocking with Block Ace, mouse anti-lentinan antiserum or normal mouse serum was diluted 5000 times or more with PBS and added at a volume of 50 μl/well. After leaving it at room temperature for 2 hours, it was washed 3 times with PBS containing 0.5% BSA.
A commercially available biotin-labeled anti-mouse immunoglobulin antibody (manufactured by Zymet) diluted 200 times with PBS was added at 50 μl/well. After being left at room temperature for 1 hour, the wells were washed three times with PBS containing 0.5% BSA, and 50 μl/well of commercially available streptavidin alkaline phosphatase conjugate (manufactured by Amersham) diluted 1000 times with PBS was added. After leaving for 1 hour at room temperature and washing 3 times with PBS containing 0.5% BSA, add 100 μl of a substrate solution (same as described in (3)) prepared to 1 mg/ml with PNPP buffer.
After 20 to 30 minutes, absorbance (405 nm) was measured using a plate reader. Figure 5 shows the results. Although FIG. 5 shows an example in which the serum was diluted 5,000 times, it was sufficiently detectable even when the serum was diluted 10,000 times. From this result, whereas the immunoprecipitation method conventionally used to detect polysaccharide antibodies requires several hundred μl of serum stock solution, this detection method requires 50 μl of serum when diluted 10,000 times.
We succeeded in improving the detection sensitivity by more than 10,000 times using only a microliter amount.

(5) Establishment of a detection method for anti-lentinan antibodies using a combination with a binding competition test method Although it is thought that the method described in (4) can detect anti-lentinan antibodies with high sensitivity, it is possible to detect anti-lentinan antibodies with high sensitivity. In order to obtain antibodies, lentinan has extremely low antigenicity, so it is necessary to immunize as a lentinan-protein (BSA) complex. In the case of polysaccharides with other antigenic properties, the polysaccharide itself acts as an immunogen, so
Only antibodies against lentinan itself will be induced, but when immunized with lentinan/BSA complex, antibodies against BSA and lentinan can be generated by oxidative cleavage and reduction of a part of lentinan during complex synthesis. Antibodies against the polyalcoholized moiety may be induced. In addition, as mentioned in (3), the lentinan-Hb complex solidified for use in the detection method can also be used as a lentinan-B complex.
Since it is treated in the same way as the SA complex, some of the lentinan is polyalcoholized. Therefore, if antibodies against the polyalcoholized portion of lentinan are induced by immunization with the lentinan/BSA complex, there is a possibility that the antibodies will react with the detection method shown in (4). Therefore, as a method for detecting only antibodies against untreated lentinan, (4
), when adding anti-lentinan antiserum or culture supernatant, an untreated lentinan solution is added to react with an antibody against lentinan, thereby binding the antibody to the immobilized lentinan/Hb complex. We have found that a combination of competitive binding inhibition assays reduces the By combining these methods, antibodies against the partially polyalcoholized portion of lentinan do not react with untreated lentinan, and binding to the immobilized lentinan/Hb complex is not inhibited. Only antibodies can be detected. As mentioned earlier,
In the case of immunization with the antigenic polysaccharide itself, a combination of competitive binding inhibition assays may not be necessary.

The results obtained using the above method are shown in FIG. Antibody detection samples include the mouse anti-lentinan antiserum described in (2), the culture supernatant that was positive by the method described in (4), and lentinan with a significant degree of polyalcoholization.
A BSA complex was synthesized and mice were immunized with it.
Using an antiserum against the polyalcoholized portion of lentinan, we investigated the binding to the immobilized lentinan/Hb complex in the presence or absence of untreated lentinan solution and in the presence of polyalcoholized lentinan. . the result,
The anti-lentinan antiserum and the positive culture supernatant of (4) are mixed with lentinan by coexisting with untreated lentinan.
Binding to the Hb complex was inhibited. On the other hand, the binding of antiserum to the polyalcoholized moiety to the lentinan-Hb complex was not inhibited even when untreated lentinan was added, but the binding was inhibited only when polyalcoholized lentinan was added. It was demonstrated that the anti-lentinan antibody detection method combined with this binding competition test method can only detect antibodies against lentinan itself.

[0026]

[Effects of the Invention] As detailed above, there is a highly sensitive detection method for antibodies against lentinan that utilizes technology to immobilize lentinan on plastic through protein mediation, and the coexistence of untreated lentinan during antibody detection. By using a combination of competitive binding inhibition testing methods, it has become possible to detect antibodies with a sensitivity that is more than 10,000 times higher than that of conventional immunoprecipitation methods.

[0027] Furthermore, by using this lentinan antibody detection method, it is possible to detect antibodies in the culture supernatant of monoclonal antibody-producing fusion cells, which was not possible using conventional immunoprecipitation methods. As a result, a lentinan monoclonal antibody and a fused cell producing it were obtained.

Lentinan, which is currently on the market as an anticancer drug, needs monitoring to predict its anticancer effect by determining its more detailed biodistribution or individual patient's sensitivity to the drug, in order to enhance its effect. Development of a method is desired. To this end, as mentioned above, antibodies against lentinan may be an effective means. However, the conventionally produced antiserum against lentinan has a quantitative limitation and each antiserum has a different titer, so there is a problem that many patients cannot be diagnosed under the same conditions. The monoclonal antibody that specifically reacts with lentinan obtained by the present invention is considered to be useful for selecting a more effective administration method for lentinan, determining a patient's sensitivity to lentinan, and the like.

[Brief explanation of drawings]

FIG. 1 is a graph showing a chromatogram of a raw material and a reaction solution obtained by high performance liquid chromatography using a gel filtration method after synthesis of a lentinan-protein (BSA) complex.

FIG. 2 is a graph showing an increase in antibody titer after immunizing mice with a lentinan/BSA complex.

FIG. 3 is a graph showing the results of mouse anti-lentinan monoclonal antibody detection.

FIG. 4 is a graph showing the binding of an anti-lentinan antibody regarding immobilization of a lentinan-protein (Hb) complex onto a plastic plate.

[Fig. 5] In a highly sensitive detection method of anti-lentinan antibody,
It is a graph showing the absorbance when mouse anti-lentinan antiserum and normal mouse serum are used.

FIG. 6 is a graph showing the absorbance when using each antiserum in an anti-lentinan antibody detection method using a combination of a high-sensitivity antibody detection method and a binding competitive inhibition test method.

Claims (2)

[Claims] Claim 1: A monoclonal antibody against the polysaccharide lentinan. 2. A fused cell producing a monoclonal antibody against the polysaccharide lentinan according to claim 1.