JPWO2012008595A1 - Production method of anti-LR11 antibody and standard for immunological measurement - Google Patents

Abstract

A means for efficiently and efficiently producing anti-LR11 antibodies and LR11 standards in immunological measurements are provided. A method for producing an anti-LR11 antibody, comprising using malignant tumor cell-derived or urine-derived LR11.

Description

  The present invention relates to an efficient method for producing an anti-LR11 antibody and a standard for immunological measurement of LR11.

LR11 (also referred to as LDL receptor relative with 11 ligand-binding repeats, SorLA, SORL1) has been identified as a novel LDL receptor-like protein having a structure characteristic of the LDL receptor family (Patent Document 1, Non-patent Document 1, Patent Document 1). As a result of immunohistochemical analysis and in situ hybridization analysis, it has been reported that LR11 is specifically expressed at an intimal thickening site formed by smooth muscle cell migration and proliferation (Non-Patent Literature). 2).
The present inventors have found that soluble LR11 is present in the blood of mammals, and that the soluble LR11 concentration in the blood of patients with arteriosclerotic disease is significantly higher than that in healthy subjects ( Patent Document 2). Furthermore, the present inventors further processed LR11 in a sample with a specific surfactant to easily measure soluble LR11 in blood or bone marrow fluid using an anti-LR11 antibody (Patent Document 3, Non-Patent Document 3). As a result of developing a patent document 4) and measuring the concentration of soluble LR11 in blood or bone marrow fluid in various diseases, it has been shown that abnormally high values are observed in malignant tumors, particularly hematopoietic tumor diseases such as leukemia and malignant lymphoma. As a result, LR11 was found to be a new tumor marker (Patent Document 4).

  Although the gene sequence of LR11 has been elucidated (Patent Document 1, Non-Patent Document 1), since full-length LR11 has a large molecular weight of 250 kDa, large-scale expression of recombinant full-length LR11 protein has not been successful. In addition, cultured cells that secrete LR11, LDL receptor-deficient CHO cells that stably express the LR11 gene, and COS-7 cells into which the full-length LR11 gene has been introduced have been reported (Non-Patent Document 5). The amount was very small or required genetic engineering treatment. Thus, the source of the immunogen for producing the antibody is limited, or processing for obtaining an LR11 producing cell is necessary.

  Although anti-LR11 antibodies are already commercially available as polyclonal antibodies and monoclonal antibodies, due to the circumstances described above, they are prepared using a synthetic peptide having a partial amino acid sequence of LR11 as an immunogen. For this reason, the commercially available antibody is practically sufficient, for example, it is very weak in reactivity to LR11 in a biological sample such as full-length LR11 or reacts only when LR11 is immobilized on a solid phase. It was hard to say that the antibody has performance.

  Moreover, in order to use soluble LR11 in blood as a source of an immunogen for producing an antibody, a large amount of blood is required, which is unrealistic considering the burden on the donor.

The above-mentioned circumstances are the same even when considered as a supply source of the standard LR11 when producing a standard for immunological measurement of LR11.
As described above, as a source of an immunogen for producing an anti-LR11 antibody or a source of a standard LR11 for producing a standard for immunological measurement, it can be easily produced in a large amount by means of low invasiveness. There was a need for a source that can collect LR11 and that can collect LR11 while maintaining the presence in the biological sample.

Japanese Patent Laid-Open No. 9-163988 WO2008 / 155891 WO2009 / 116268 Japanese Patent No. 2009-285492

J. et al. Biol. Chem. 1996; 271, 24761-24768. Arterioscler. Thromb. Vasc. Biol. 1999; 19, 2687-2695. J. et al. Clin. Invest. 2008; 118, 2733-2746 Clin. Chem. 2009; 55, 1801-1808 Circ. Res. 2004; 94, 752-758.

  The object of the present invention is to provide a means for efficiently and efficiently producing anti-LR11 antibodies, particularly a means for producing anti-LR11 antibodies that react with LR11 in a biological sample, and a standard product of LR11 in immunological measurement. Is to provide.

  As a result of studying a new source of LR11, the present inventors have found that LR11 is expressed on the surface of malignant tumor cells, that LR11 is secreted into the culture supernatant of malignant tumor cells, and human urine. It was found that soluble LR11 was also present therein. Furthermore, by using LR11 derived from malignant tumor cells and human urine as an antigen for evaluating the specificity of an immunogen or antibody, an antibody that reacts with LR11 in a biological sample such as full-length LR11 was successfully produced. Further, it was found that these malignant tumor cell-derived and human urine-derived LR11 are also useful as a standard for immunological measurement of LR11. The present invention has been completed based on such findings.

That is, this invention provides the manufacturing method of the anti- LR11 antibody characterized by using LR11 derived from a malignant tumor cell or urine.
The present invention also provides a standard for immunological measurement of LR11 containing LR11 derived from malignant tumor cells or urine.
In the present specification, “LR11” refers to a protein having reactivity with an anti-LR11 antibody unless otherwise specified. That is, it refers to proteins known as “full length LR11” or “soluble LR11” and those partially fragmented or modified.

According to the method of the present invention, malignant tumor cells expressing LR11 on the cell surface itself, soluble LR11 secreted in the culture supernatant of the malignant tumor cells, or soluble present in human urine By using LR11 as an immunogen or an antigen for specificity evaluation, it becomes possible to produce a specific antibody against LR11 in an industrially advantageous manner.
Moreover, LR11 can be accurately measured immunologically by using a standard product containing LR11 derived from malignant tumor cells or urine.

It is the figure which detected the expression of LR11 in various hematopoietic tumor cell lines by Western blotting using a cell lysis solution and a cell culture solution. It is a graph showing the result of having measured soluble LR11 in the culture solution of various hematopoietic tumor cell lines by ELISA. It is the figure which showed the result of having analyzed the expression of LR11 on the cell surface in various hematopoietic tumor cell lines by flow cytometry. It is the figure which detected the LR11 expression on the cell surface in various epithelial malignant tumor cell lines by the western blotting using the membrane fraction solution. It is a graph showing the result of having measured soluble LR11 in the culture solution of various epithelial malignant tumor cell lines by ELISA. It is the figure which compared soluble LR11 derived from human urine, soluble LR11 derived from CCRF-SB cell line, soluble LR11 derived from human serum, and soluble LR11 derived from COLO201 cell line by Western blotting. It is a graph showing the result of having measured the anti- LR11 antibody titer in the serum of the mouse | mouth which immunized intraperitoneally with NB-4 cell strain.

  In the present invention, LR11 used for preparation of an anti-LR11 antibody or a standard for immunological measurement of LR11 is derived from malignant tumor cells or urine.

  According to the knowledge of the present inventors, LR11 is remarkably expressed on the surface of malignant tumor cells, and a high concentration of soluble LR11 is secreted in the culture solution of malignant tumor cells. Therefore, malignant tumor cells expressing LR11 on the cell surface can be used directly as an immunogen, or soluble LR11 can be collected from the culture medium of the cells to evaluate the specificity of the immunogen or anti-LR11 antibody, or immunology It can be used as a standard for standard measurement.

  Examples of malignant tumor cells that can be used in the present invention are cell lines registered in the ATCC (American Type Culture Collection), the Human Science Research Resource Bank, etc., and LR11 is expressed on the cell surface. If it is, it can be used without particular limitation. Examples of the type of malignant tumor include hematopoietic tumor or epithelial tumor, and hematopoietic tumor is preferable.

  Examples of hematopoietic tumors include leukemia and malignant lymphoma. Leukemia includes acute leukemia and chronic leukemia, and malignant lymphoma includes non-Hodgkin lymphoma. Of these, acute leukemia and malignant lymphoma are preferred. More specifically, among hematopoietic tumor cells, HL-60, ML-2, and NB-4 of acute myeloid leukemia-derived strains, NALL-1, MOLT-4, CCRF-SB of acute lymphocytic leukemia-derived strains Malignant lymphoma-derived strains such as Daudi and U937 are preferable, and HL-60, ML-2, NB-4, MOLT-4, CCRF-SB and U937 are particularly preferable. In addition to the established cells, the cells can also be purified from a cell lysate or cell culture solution of tumor cells derived from leukemia patients and malignant lymphoma patients.

  Examples of epithelial malignant tumors include stomach cancer, liver cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, esophageal cancer, breast cancer, cervical cancer, ovarian cancer, colon cancer, colon cancer, kidney cancer, gallbladder cancer, nerve tumor (Glioma), melanoma (melanoma). In particular, among epithelial malignant tumors, gastric cancer-derived strains MKN1, GCIY, liver cancer-derived strains HLE, HepG2, HuH-7, JHH-6, pancreatic cancer-derived strains AsPC-1, BxPC-3, lung cancer-derived strains Oka-C-1, prostate cancer-derived strain PC-3, bladder cancer-derived strain KMBC-2, esophageal cancer-derived strain T. Tn, breast cancer-derived strain YMB-1, cervical cancer-derived strain HeLa, ovarian cancer-derived strain OVISE, colon cancer / colon cancer-derived strain COLO201, kidney cancer-derived strain Caki-1, and gallbladder cancer-derived strain OCUG -1, neuronal tumor-derived strain KINGS-1, melanoma-derived strain G-361, and the like are preferable. In addition to the established cells, the cells can also be used after purification from a cell lysate or cell culture solution of tumor cells derived from patients with epithelial malignant tumors.

  The present inventors have also found that soluble LR11 is present in urine at a high concentration. As urine, human urine is particularly preferable. Urine is generally suitable as a supply source of LR11 in the present invention because it has few contaminating proteins, is substantially non-invasive, and can be easily collected in large quantities.

As described above, malignant tumor cells expressing LR11 on the cell surface can be used directly as an immunogen. On the other hand, it is preferable to use LR11 collected from a lysate or culture solution of malignant tumor cells, or urine as a standard for immunogen, anti-LR11 antibody specificity evaluation and immunological measurement.
In order to collect LR11 from a lysate or culture solution of malignant tumor cells or urine, for example, soluble LR11 in urine is adsorbed on an insoluble carrier carrying an LR11 affinity substance such as RAP (receptor-related protein). After washing with an appropriate buffer, soluble LR11 adsorbed on the insoluble carrier may be eluted.

  These LR11s may be further purified and used by ion exchange chromatography, hydrophobic chromatography, gel filtration, specific antibody affinity, and the like.

Since the LR11 derived from the malignant tumor cell or human urine can be produced in a large amount and stably, it is useful as a standard for immunological measurement of LR11, and is also useful as an immunogen when producing anti-LR11 antibody, or as anti-LR11. This is useful for evaluating the specificity of antibodies.
For immunogen and specificity evaluation at the time of anti-LR11 antibody production, LR11 of different origins may be used in combination. For example, when malignant tumor cells expressing LR11 on the cell surface are used as the immunogen, In the evaluation of the specificity of the anti-LR11 antibody, the soluble LR11 derived from urine is used, and when the soluble LR11 derived from urine is used as an immunogen, the specificity of the anti-LR11 antibody is evaluated by the soluble LR11 secreted from malignant tumor cells. It is conceivable to use malignant tumor cells themselves in which LR11 is expressed on the cell surface. Further, even when a synthetic peptide having a partial amino acid sequence of LR11 is used as an immunogen, by using LR11 derived from malignant tumor cells or human urine for specificity evaluation, LR11 in a biological sample such as full-length LR11 can be used. A highly reactive antibody can be obtained. As described above, the source of the immunogen or the standard product in the present invention is very close to the so-called natural state, both in the form of a biological sample and in the form of existing LR11. It can be said that it is particularly advantageous from the viewpoint of preserving the properties of LR11 with respect to conventional sources such as genetic recombination.

  The LR11 can be an immunogen for both monoclonal and polyclonal antibodies. The antibody can be prepared by a known method. For example, for the production of polyclonal antibodies, mice, rats, hamsters, rabbits, goats, sheep, chickens and the like are used as animals to be immunized. The antiserum containing an anti-LR11 polyclonal antibody can administer the LR11 once or multiple times subcutaneously, intradermally or abdominally in an animal. Further, immunization of a mixture with a replacement fluid having an immunostimulatory effect is more preferable.

For the production of monoclonal antibodies, known monoclonal antibody production methods such as Kamon Nagamune and Hiroko Terada, “Monoclonal Antibody” Yodogawa Shoten (1990), James W., et al. Golding, “Monoclonal Antibody”, 3rd edition, Academic Press, (1996).
The monoclonal antibody can be produced by culturing a hybridoma prepared according to a conventional method and separating it from the culture supernatant, or by administering the hybridoma to a compatible mammal and collecting it as ascites.

  The anti-LR11 antibody can be purified and used as necessary. Methods for purifying and isolating antibodies include conventionally known methods such as salting out such as ammonium sulfate precipitation, gel filtration using Sephadex, ion exchange chromatography, affinity purification using protein A column, and the like. is there.

  To evaluate the specificity of the anti-LR11 antibody using LR11, immunostaining (Western blotting), enzyme-linked immunosorbent assay (ELISA), flow cytometry and the like can be mentioned.

Moreover, the immunological measurement method of LR11 using LR11 as a standard product is not particularly limited, but is measured by an immunological method using an anti-LR11 antibody, a method using affinity such as RAP, and a method combining them. It is desirable to do. Examples of immunological methods include immunostaining (Western blotting), enzyme-linked immunosorbent assay (ELISA), immunoturbidimetry (TIA and LTIA), enzyme immunoassay, chemiluminescence immunoassay, fluorescent immunoassay, etc. A sandwich ELISA using an antibody and a substance having affinity for LR11 such as RAP can also be used.
The ELISA (Patent Document 3 and Non-Patent Document 4) constructed by the present inventors for pretreatment of a specimen with a surfactant is particularly preferable because it can be measured very easily and accurately.

  Hereinafter, the present invention will be described specifically by way of examples. However, the present invention is not limited to these examples.

Reference Example 1 Measurement of soluble LR11 by ELISA Anti-LR11 monoclonal antibody (M3: Patent Document 3) was diluted to 10 μg / mL with PBS, added to a microplate (manufactured by NUNC) at 100 μL per well, and allowed to stand at room temperature for 2 hours. I put it. After washing with PBS containing 0.05% Tween 20 (PBST), 200 μL of PBST containing 1% BSA (BSA-PBST) was added per well and blocked at room temperature for 1 hour. The sample to be measured was diluted with a sample treatment solution in which 7% MEGA-9 (manufactured by Dojin Chemical Co., Ltd.) and HBR (manufactured by Scantibodies Laboratory) were mixed at a ratio of 3: 1. Soluble LR11 purified from rabbit serum was also serially diluted with the sample treatment solution to prepare a calibrator (0 to 4 ng / mL). These diluted specimens were added at 100 μL per well and allowed to react overnight at room temperature, and then biotin-labeled anti-LR11 monoclonal antibody (R14: Patent Document 3) was diluted with BSA-PBST to 0.4 μg / mL. 100 μL was added per well and reacted at room temperature for 4 hours. After washing with PBST, peroxidase-labeled streptavidin (manufactured by PIERCE) was diluted with BSA-PBST to add 0.2 μg / mL, 100 μL per well, and allowed to react at room temperature for 1 hour. After washing with PBST, 100 μL of TMB substrate solution was added per well and reacted at room temperature for 30 minutes, and 100 μL of 1.5 N sulfuric acid was added per well to stop the reaction. The measurement was performed using a microplate reader (Abs. 450 nm). The concentration of soluble LR11 in the sample was calculated from the calibration curve by the calibrator and multiplied by the dilution factor to obtain the soluble LR11 concentration in the sample.

Example 1 Measurement of Soluble LR11 in Human Urine Urine was collected at random from healthy volunteers (10 subjects) for several days and diluted 4-fold with the sample treatment solution described in Reference Example 1. In the same manner as the ELISA method described in Reference Example 1, the concentration of soluble LR11 in human urine was measured. The concentration distribution was 0.1 to 8.4 ng / mL, and the average was 3.1 ng / mL. It was found that soluble LR11 is present in human urine at a concentration comparable to the blood concentration in the normal range (according to Non-Patent Document 4 etc.).

Example 2 Purification of Soluble LR11 from Human Urine Escherichia coli DH5α transformed by introducing a pGEX2T (manufactured by GE Healthcare) vector incorporating a human RAP gene was cultured, and the cells were collected by centrifugation. The cells recovered from 3 L of the culture solution were suspended in PBS containing lysozyme and Triton X-100, and the cells were disrupted by ultrasonic treatment. This disrupted solution was passed through 200 mL of Glutathione Sepharose 4 FF (manufactured by GE Healthcare) to adsorb the RAP / GST fusion protein in the centrifugal supernatant, and then washed with PBS to prepare a RAP-Sepharose resin.
As a batch, 5-8 L of human volunteer pool urine was passed through 200 mL of this RAP-Sepharose resin. After washing with PBS, human urine-derived soluble LR11 was eluted with 50 mM citrate buffer (pH 5.0) containing 150 mM NaCl. After concentration, dialyzed against PBS. Further, this solution was passed through 5 mL of rProtein Sepharose A FF (manufactured by GE Healthcare), and an anti-soluble LR11 monoclonal antibody (M3) binding resin was added to the obtained non-adsorbed fraction, followed by stirring overnight. After washing the resin with PBS, human urine-derived soluble LR11 was eluted with 50 mM citrate buffer (pH 3.0) containing 150 mM NaCl. Further, the eluate was separated and purified with PBS by gel filtration chromatography (Superdex 200; manufactured by GE Healthcare). The soluble LR11-eluted fraction was collected and concentrated to obtain purified human urine-derived soluble LR11. The LR11 content was calculated based on the method described in Reference Example 1. As a result of the final purification process for 12 batches, about 0.8 μg of purified human urine-derived soluble LR11 was obtained per liter of human urine on average. In Reference Example 1, the highest concentration of the calibrator was 4 ng / mL, and it was found that a sufficient amount of soluble LR11 can be easily secured from human urine as a standard for immunological measurement.

Example 3 Confirmation of LR11 Expression in Various Hematopoietic Tumor Cell Lines Anti-LR11 monoclonal antibody (A2-2) prepared by immunizing a peptide having a partial amino acid sequence of LR11 with LR11 expression in various hematopoietic tumor cell lines 3: Confirmed by Western blot using Patent Document 3). Cell lines are 3 strains derived from acute myeloid leukemia (HL-60, ML-2, NB-4), 3 strains derived from acute lymphocytic leukemia (NALL-1, MOLT-4, CCRF-SB), 2 strains derived from lymphoma A total of 8 types (Daudi, U937) were used. These cell lines were suspended in HT medium (RPMI-1640 containing 15% fetal bovine serum, HT supplement, penicillin / streptomycin), cultured at 37 ° C. in a 5% carbon dioxide incubator, and grown. The number of cells of each cell line was counted, sorted to 1 × 10 ⁷ , and RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM) containing 1% protease inhibitor (Sigma Aldrich; P8340). NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) was added to lyse the cells. After removing the insoluble matter by centrifugation, the protein concentration in the cell lysate was measured, and 20 μg per lane was boiled under reducing conditions containing SDS. Each of SDS-PAGE (2-15%) Applied to the lane and electrophoresed. After completion of the electrophoresis, the protein was transferred from the gel to a PVDF membrane, reacted with an anti-LR11 monoclonal antibody (A2-2-3), and LR11 was detected using a mouse IgG ABC kit (manufactured by Vector).
On the other hand, the detection of soluble LR11 secreted in the culture supernatant of each cell line was performed as follows. In the same manner as described above, each cell line cultured in HT medium was separated to 1 × 10 ⁷ cells, and further cultured in 2 mL of RPMI-1640 medium for 14 hours. Soluble LR11 in the culture supernatant was measured by Western blotting and the same ELISA as in Reference Example 1.
In the Western blot using the cell lysate of each cell line, two bands having slightly different molecular weights were detected around the molecular weight of 230 kDa (FIG. 1). Further, it was confirmed that soluble LR11 was also present in the culture supernatant except for NALL-1, and as shown in FIG. 2, HL-60, ML-2, NB-4, CCRF-SB, Daudi, U937. In these 6 strains, the amount of soluble LR11 secreted exceeded 1 ng per 2 mL of culture supernatant. In particular, the secretion amount of CCRF-SB exceeded 5 ng / 2 mL, and it was found that soluble LR11 can be easily obtained by culturing hematopoietic tumor cells.

Example 4 Flow cytometry of various hematopoietic tumor cell lines For the eight cell lines used in Example 3, whether or not LR11 was expressed on the cell surface was analyzed by flow cytometry. Each cell line was suspended in 50 μL Staining Medium (1% FBS / PBS) so as to be 5 × 10 ⁵ cells. After centrifuging at 1500 rpm for 5 minutes, the supernatant was removed, and 3 μL of 1 mg / mL FITC-labeled anti-LR11 monoclonal antibody (M3) was added to the pellet and reacted at 4 ° C. for 60 minutes. After the cells were washed twice with Staining Medium, flow cytometry analysis was performed using JSAN (manufactured by Bay bioscience) according to the instruction manual. As a negative control of FITC-labeled anti-LR11 monoclonal antibody, normal mouse IgG (BD pharmingen) was used. The results are shown in FIG. Among the seven strains in which secretion of soluble LR11 was confirmed in Example 3, six strains of HL-60, ML-2, NB-4, MOLT-4, CCRF-SB, and U937 were strongly positive to weakly positive. Reaction was observed, and it was found that LR11 was remarkably expressed on the surface of these cells.

Example 5 Confirmation of LR11 protein expression in various epithelial malignant tumor cell lines The LR11 protein expression in various epithelial tumor cell lines was determined by Western blotting using the anti-LR11 monoclonal antibody (A2-2-3) used in Example 1. confirmed. Cell lines include HLE (derived from liver cancer), MKN1 (derived from stomach cancer), T. Tn (derived from esophageal cancer), COLO201 (derived from colon cancer), AsPC-1 (derived from pancreatic cancer), Caki-1 (derived from kidney cancer), Oka-C-1 (derived from lung cancer), PC-3 (derived from prostate cancer) , KMBC-2 (derived from bladder cancer), HeLa (derived from cervical cancer), OVISE (derived from ovarian cancer), YMB-1 (derived from breast cancer), KINGS-1 (derived from neuronal tumor), G-361 (derived from melanoma) 14) in total. These cell lines are suspended in a designated medium (for example, RPMI-1640 containing 10% fetal bovine serum, penicillin / streptomycin), cultured in a 5% carbon dioxide incubator at 37 ° C. for an appropriate time, and allowed to grow. It was. Recovery of the proliferated cells from the culture flask was performed using a trypsin-EDTA solution (Invitrogen, 25200-072) after the cells were washed several times with PBS. The number of cells in each cell line was counted, sorted to 1 × 10 ⁷ cells, suspended in an appropriate amount of PBS, and the cells were collected by centrifugation. This operation was repeated 3 times to wash the cells. Thereafter, 100 μL of PBS containing 1% protease inhibitor (manufactured by Sigma Aldrich, P8340) was added to the collected cells, and the cells were disrupted with an ultrasonic vibrator (homogenizer). The cell fragments were removed by centrifugation, and then the supernatant was precipitated by ultracentrifugation (100,000 g, 10 minutes). After removing the supernatant and washing the precipitate with PBS, 100 μL of PBS containing 1% protease inhibitor (manufactured by Sigma-Aldrich; P8340) and 1% MEGA-9 was added, and the membrane was removed using an ultrasonic vibrator (homogenizer). The fraction was suspended and dissolved, and the insoluble membrane fraction was removed again by ultracentrifugation (100,000 g, 10 minutes) to obtain a soluble membrane fraction solution.
The soluble membrane fraction solution of each cell line was boiled under a reducing condition containing SDS at 2 μL per lane, and each treatment solution was applied to each lane of SDS-PAGE (2-15%) and separated. After completion of the electrophoresis, the protein is transferred from the gel to a PVDF membrane and reacted with an anti-LR11 monoclonal antibody (A2-2-3) as a primary antibody. After that, Biotinylated anti-mouse IgG polyclonal antibody (manufactured by DAKO) and HRP labeling Streptavidin was combined and finally developed with diaminobenzidine to detect LR11 protein. As a result, LR11 protein bands were detected in all 14 cell lines (FIG. 4).
On the other hand, the detection of soluble LR11 secreted in the culture supernatant of each cell line was performed as follows. HLE, COLO201, AsPC-1, KINGS-1, PC-3, and KMBC-2 are selected from the cell lines in which the expression of LR11 has been confirmed as described above, and each of them is designated using a 75 cm ² culture flask. The culture medium was cultured until it became confluent at 40 mL. Moreover, COLO201 was separately seeded in 20 mL of serum-free RPMI1640 medium at 1 × 10 ⁶ cells / mL and cultured for 2 days. Soluble LR11 in the culture supernatant was measured by ELISA as in Reference Example 1. As a result, it was confirmed that soluble LR11 was present in the culture supernatants of all cell lines (FIG. 5). Among the cell lines examined, the COLO201 cell line had a secretion amount exceeding 5 ng per mL when cultured in a serum-free medium. Thus, when cultured cells are used, the yield can be increased by devising the culture conditions, and LR11 can be obtained efficiently and simply under conditions where there are no contaminants such as serum components. It is effective as a means for preparing an immunogen or a standard for antibody production.

Example 6 Comparison of Various Soluble LR11 Soluble LR11 derived from human urine obtained in Example 2, soluble LR11 derived from cultured cells (CCRF-SB) obtained in Example 3, and culture obtained in Example 5 The soluble LR11 derived from cells (COLO201) was compared by Western blotting. As a control, soluble LR11 extracted from human serum (Non-Patent Document 4) was used. As a result, as shown in FIG. 6, a band was observed in the vicinity of a molecular weight of 230 kDa in all cases.

Example 7 Cellular Immunity The NB-4 strain, one of the hematopoietic tumor-derived cells in which LR11 was significantly expressed on the cell surface in Example 4 tested by flow cytometry, was expanded and cultured in HT medium. After washing twice, 1-2 × 10 ⁷ cells were suspended in PBS, injected into the abdominal cavity of BALB / c mice (female), and immunized. Immunization was performed every other week or weekly, and anti-LR11 antibody titer in mouse serum was confirmed at an appropriate time by the following method.
Purified soluble LR11 derived from human urine obtained in Example 2 was diluted to 50 ng / mL with PBS on a microplate (manufactured by NUNC), added 50 μL per well, and allowed to stand at room temperature for 2 hours. After washing with PBS containing 0.05% Tween 20 (PBST), 200 μL of PBST containing 1% BSA (BSA-PBST) was added per well and blocked at room temperature for 1 hour. Serum obtained by collecting blood from the tail of the mouse was diluted 200, 400, 800, 1600 times with BSA-PBST, added at 50 μL per well, and allowed to react at room temperature for 1 hour. After washing the wells with PBST, HRP-labeled anti-mouse IgG polyclonal antibody (manufactured by Rockland) was diluted 10,000 times, added at 50 μL per well, and allowed to react at room temperature for 1 hour. After washing with PBST, 50 μL of o-phenylenediamine substrate solution was added per well, color was allowed to develop for 20 minutes at room temperature, and the reaction was stopped by adding 50 μL of 1.5 N sulfuric acid per well, followed by microplate reader (Abs. 492 nm). ).
The result of measuring the anti-LR11 antibody titer in mouse serum after 5 immunizations is shown in FIG. In the mouse sera before immunization, no reaction was observed with the solid-phased human urine-derived purified soluble LR11, whereas in the sera of mice immunized with the NB-4 cell line, the change in absorbance corresponding to the change in dilution concentration was observed. As a result, it was confirmed that anti-LR11 antibody was produced.

Claims (14)

  A method for producing an anti-LR11 antibody, comprising using malignant tumor cell-derived or urine-derived LR11.   The method according to claim 1, wherein malignant cell-derived or urine-derived LR11 is used as an immunogen and / or for evaluating the specificity of an anti-LR11 antibody.   The method according to claim 1 or 2, wherein the malignant tumor cell-derived LR11 is expressed on the cell surface.   The method according to any one of claims 1 to 3, wherein the malignant cell-derived LR11 is secreted into the culture supernatant.   The method according to any one of claims 1 to 4, wherein the malignant tumor cells are hematopoietic tumor cells or epithelial malignant tumor cells.   6. The method according to claim 5, wherein the hematopoietic tumor cell is derived from acute leukemia or malignant lymphoma.   Epithelial malignant tumor cells are stomach cancer, liver cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, esophageal cancer, breast cancer, cervical cancer, ovarian cancer, colon cancer, colon cancer, kidney cancer, gallbladder cancer, nerve tumor, The production method according to claim 5, which is selected from the group consisting of melanoma.   The method according to claim 1 or 2, wherein the urine-derived LR11 is human urine-derived soluble LR11.   A standard product for immunological measurement of LR11 containing LR11 derived from malignant tumor cells or urine.   The standard product according to claim 9, wherein the LR11 derived from malignant tumor cells is secreted into the culture supernatant.   The standard product according to claim 9 or 10, wherein the malignant tumor cells are hematopoietic tumor cells or epithelial malignant tumor cells.   The standard product according to claim 11, wherein the hematopoietic tumor cell is derived from human acute leukemia or human malignant lymphoma.   Epithelial malignant tumor cells are stomach cancer, liver cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, esophageal cancer, breast cancer, cervical cancer, ovarian cancer, colon cancer, colon cancer, kidney cancer, gallbladder cancer, nerve tumor, The standard product according to claim 11, which is selected from the group consisting of melanoma.   The standard product according to claim 8 or 9, wherein the urine-derived LR11 is human urine-derived soluble LR11.