JPH058678B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] Ferritin is an iron-binding protein that is found in all eukaryotic cells and consists of a protein portion with a molecular weight of about 450,000 and iron colloid. Tissue ferritin has a different subunit composition (H chain, L chain) depending on the tissue. For example, in cancerous liver ferritin, the proportion of H chain increases compared to normal liver ferritin, and there is also a large amount of ferritin in the blood of cancer patients. Since it was reported that ferritin occurs, the significance of measuring serum ferritin has become important. Factors that increase serum ferritin include iron overload (increase in ferritin as an iron storage protein);
Conversion of hemoglobin iron to storage iron (anemia other than iron deficiency anemia), increased cell destruction (increased amount of intracellular ferritin released into the blood), decreased ability to remove serum ferritin (tissue destruction due to liver and pancreatic disorders, etc.), etc. It is given. Therefore, if the state of iron storage in ferritin can be detected sensitively, it is expected to become a direct indicator of iron deficiency state and a means of reflecting pathological conditions such as liver disease, malignant tumor, and iron overload disease. The present invention relates to a monoclonal antibody useful for testing ferritin. [Prior Art] Several kits for measuring ferritin are already on the market, but these kits use polyclonal antibodies. On the other hand, regarding monoclonal antibodies, Arosio et al. (Clinica Chimica Acta vol. 134, p. 347)
(1983) created an antibody that specifically reacts with human cardiac ferritin. Monoclonal antibodies that use human spleen ferritin as an antigen and react with the heart, placenta, and spleen have also been produced. [Problems to be Solved by the Invention] Polyclonal antibodies were insufficient to be used for the above measurement purpose. In addition, the monoclonal antibody of Arozio et al. does not react with human liver, placenta, or pancreatic ferritin at all, and its specificity for the state of iron storage in tissues is not clear. The present invention was made against the above background, and particularly aims to provide a monoclonal antibody that can accurately reflect the iron storage status of each tissue and serum ferritin. [Means for Solving the Problems] The monoclonal antibody of the present invention uses human placental ferritin as an immunogen, has reactivity to each tissue ferritin of the liver, placenta, and pancreas, and serum ferritin, and has an iron metabolic behavior. It is characterized by having. Specifically, this motoclonal antibody is a monoclonal antibody and its cell line ( 1F3) and a monoclonal antibody and its cell line (3F11), which have the same level of reactivity to the above-mentioned tissue ferritin and do not reflect the state of iron storage in tissues. Next, the properties of the monoclonal antibody of the present invention will be shown. Reactivity with each tissue ferritin Monoclonal antibody 1F3 reacted most strongly with liver ferritin, followed by placental ferritin and pancreatic ferritin, each showing different reactivity. On the other hand, monoclonal antibody 3F11 exhibits almost the same level of reactivity with the above-mentioned tissue ferritin, which distinguishes these two types of antibodies and also has different characteristics from conventional monoclonal antibodies. This reactivity was determined by measurement using Sandwich enzyme-linked immunosorbent assay (ELISA), which will be described later. Iron metabolism test using the antibody of the present invention Preparation of ferritin and apoferritin and iron metabolism test using these are described by Ian et al. (Biochem J. vol. 135, p. 136, 1973) and Horin et al. , 1974)
I followed the method. (a) To release iron from placental ferritin, add 1 ml of placental ferritin (0.1 mg/ml) to a sodium acetate buffer (1 M, PH4.8) containing 3% sodium dithionite, and release the placental ferritin at room temperature. Ferritin iron was released. As a result of this operation, the amount of iron in placental ferritin was significantly decreased from 0.06 to 0.04 when treated for 2 to 6 hours as a ratio to untreated (1.0).
As a result of measuring the reactivity of monoclonal antibodies against each ferritin with different amounts of iron using the ELISA method, the reactivity of monoclonal antibody 1F3 against ferritin treated for 2 to 6 hours was approximately 75 to 55 compared to untreated (100). , which reacted well with ferritin iron content. However, monoclonal antibody 3F11 did not show such a response and was not dependent on the amount of iron in ferritin. (b) The method of iron uptake into placental ferritin is as follows:
Ferritin with different amounts of iron in ferritin was prepared by adding monoferric ammonium sulfate solutions of various concentrations and a buffer solution containing an oxidizing agent to placental apoferritin (0.1 mg/ml) and reacting for 5 minutes. The iron content in ferritin of this product is ferrous ammonium sulfate concentration 7.4mM
The amount of iron in apoferritin reached approximately 20 times the initial amount. The reactivity of monoclonal antibody 1F3 to this sample by ELISA was about 120-140% compared to untreated apoferritin (100), a result that corresponded well to the iron content of ferritin. On the other hand, monoclonal antibody 3F11 did not depend on the amount of iron in ferritin. In this way, the monocrotyl antibody of the present invention is a typical antibody that captures ferritin iron metabolic behavior.
It is unique. Reactivity with serum ferritin In order to clarify the practical effectiveness of the monoclonal antibody of the present invention,
Serum ferritin levels were measured by ELISA method. (a) Even in anemic patients whose serum iron levels are within the range of serum iron levels of healthy individuals (50 to 160 μg/dl),
It showed extremely low reactivity to monoclonal antibody 1F3. (b) Serum ferritin levels in patients with liver cancer, acute hepatitis, and liver cirrhosis, whose serum ferritin levels are expected to increase, are higher when measured using monoclonal antibody 1F3 than when using 3F11. However, serum from patients with chronic hepatitis showed similar reactivity to both antibodies. These results correspond well to iron metabolic behavior and also correspond well to conventional clinical findings. The subclasses of the molar clonal antibodies of the present invention determined by the Ochtelony method are 1gG2b for 1F3 antibody,
The 3F11 antibody was IgG1. When such an antibody is compared with the above-mentioned monoclonal antibody of Arosio et al., the antibody of Arosio et al. does not react with human liver, placenta, or pancreatic ferritin, whereas the resistance of the present invention is different in that it reacts with these. There is. Furthermore, regarding monoclonal antibodies that use human spleen ferritin as an antigen, the results of inhibition tests using a combination of this antibody and the monoclonal antibody of the present invention revealed that each antibody recognizes a different antigen site. Therefore, the present inventors came to the conclusion that this antibody is novel. The monoclonal antibody of the present invention can be obtained as follows. First, mouse spleen lymphocytes immunized with human placental ferritin are fused with mouse myeloma cells P3X62-Ag8-6.5.3 (these cells are sold by Dainippon Pharmaceutical Co., Ltd., for example). Hybridomas that produce monoclonal antibodies specific to ferritin
Acquired 1F3 shares and 3F11 shares. Subsequently, the hybridoma was intraperitoneally administered to BALB/C mice to which pristane had been administered, and condensate containing a large amount of the antibody of interest was collected.The monoclonal antibodies of the present invention (1F3, 3F11) were purified by further purifying these condensates. ) can be obtained. When measuring ferritin using the monoclonal antibody of the present invention, various radioactive isotopes may be used in radioimmunoassay (RIA) and a fluorescent substance may be used in fluorescence immunoassay as labeling substances for the antibody. I can do it. Further, as a typical example using a chromophoric substance, enzyme immunoassay, especially Sandwich enzyme immunoassay (ELISA), is useful. The binding method for monoclonal antibodies and labeling enzymes for enzyme immunoassay is described by Nakane et al. (J. Histochem.
Cytochem Vol. 22, p. 1084, 1974) may be followed, and examples of the labeling enzyme include peroxidase, alkaline phosphatase, acidophosphatase, and β-galactosidase. Also, especially when used for tissue staining, peroxidase is a preferred enzyme in relation to other reagents used. [Effect] The monoclonal antibody of the present invention reacts with human liver, placenta, and pancreatic tissue ferritin as well as serum ferritin and has iron metabolic behavior. [Example] Preparation of hybridoma Human placental ferritin (100 μg/ml) was dissolved in physiological saline and mixed in equal amounts with complete Freund's adjuvant to form an emulsion. this,
BALB/C mice (female, 8-10 weeks old) from 10 to
The mice were intraperitoneally administered three times at 14-day intervals, and finally, 75 μg of human placental ferritin was intravenously injected. 3 from last immunity
Days later, mouse splenocytes were collected and centrifugally washed three times with RPMI1640 medium. Mouse myeloma P3X63Ag8-6.5.3 cells were centrifuged and washed three times with RPMI1640 medium, and mouse splenocytes and P3X63Ag8-6.5.3 cells were mixed at a ratio of 10:1.
and centrifuged. After loosening the precipitated cells well, add 45% polyethylene glycol.
Cell fusion was performed by adding 1 ml of 3350RPMI1640 solution to the cells while thoroughly mixing them. Furthermore, 15 ml of RPM1640 solution was slowly dripped onto the cells to stop the reaction. After centrifugation, remove the supernatant and add RPMI1640 containing 15% fetal bovine serum.
Splenocytes were suspended in a solution at 1×10 ^{6 cells} /0.1 ml, and 0.1 ml each was dispensed into a 96-well microplate. Starting the day after cell fusion, 0.1 ml of HAT (pipoxanthine-aminopterin-thymidine) selection medium was added to each well, and then half of the medium was replaced with fresh HAT medium every day. Growth of hybridomas was confirmed in some wells 7 to 10 days after the start of culture. Once the hybridomas have grown sufficiently, RIA
We searched for the desired hybridoma. 100μ of the supernatant from the wells where the hybridomas were growing was taken and dispensed into test tubes. Next, ¹²⁵ I-labeled placental ferritin (5000 cpm/1
tube) was added and the primary reaction was carried out at 37°C for 2 hours. After the reaction, 100μ of 1% normal mouse serum and 100μ of anti-mouse immunoglobulin (secondary antibody) were added, and a secondary reaction was performed at 37°C for 2 hours. After the secondary reaction, each tube was centrifuged at 3000 rpm for 15 minutes to remove the supernatant, and the precipitate was
- Measured using a counter. Wells to which culture supernatant producing antibodies with antigen-binding ability have been added can be easily identified by the above procedure, and hybridomas can be searched for. Hybridomas that reacted with placental ferritin were selected by the RIA method, and cloning was performed three times using the limiting dilution method to obtain a single clone antibody-producing hybridoma. As a result, hybridoma strains 1F3 and 3F11, which produce antibodies specific to placental ferritin, were obtained. Production of monoclonal antibodies Hybridoma strains 1F3 and 3F11 were cultured in RPMI1640 medium containing 10% fetal bovine serum while scaling up in 96-well plates, 12-well plates, and 75 cm flasks.・14-Tetramethylpentedecane) was administered intraperitoneally to BALB/C mice to produce a large amount of antibodies. Hybridomas form ascites tumors in 10 to 14 days,
High concentrations of antibodies were produced in ascites fluid. Ascites is 50
After fractionation with % ammonium sulfate, highly pure monoclonal antibodies 1F3 and 3F11 were obtained by purification by DEAE cellulose ion exchange chromatography. Enzyme immunoassay and measurement of ferritin in each tissue Human placenta, liver, and pancreatic ferritin are measured using gel filtration method, Sepharose 6B column method, CM, DEAE
It was purified by operations such as cellulose column method. The reactivity of these tissue ferritins was determined by ELISA using the antibody of the present invention and the corresponding peroxatase-labeled antibody of the present invention. That is, the antibody of the present invention was dissolved in advance at 10 μg/ml in sodium bicarbonate buffer (0.1 M, PH9.5), and 100 μg/ml was added to a 96-well plate for ELISA.
A solid phase was prepared by adding the mixture in batches and leaving it at room temperature overnight. Next, the above antibody was discarded, and after washing three times with phosphate buffered saline (PBS), 200μ of PBS containing 1% bovine serum albumin (BSA) was added and left at room temperature for 1 hour to allow antigen binding. parts that are not
Blocked with BSA. Next, PBS containing 1% BSA
After washing three times with PBS containing 0.05% Tween 20, each tissue ferritin was prepared at a known concentration (each tissue ferritin was sequentially prepared at a concentration from 2 to 250 ng/ml in PBS containing 1% BSA).
Add 100μ each and leave at room temperature for 3 hours, then add 0.05
Washed three times with PBS containing % Tween 20. Then, 100μ of peroxidase-labeled resistor of the present invention corresponding to the solid phase was added, left at room temperature for 2 hours, and washed 3 times with PBS containing 0.05% Tween 20.
Enzyme substrate (orthophenylenediamine)
Add 200μ each to develop color, then add 50μ of 6N sulfuric acid.
The reaction was stopped. As a result, when monoclonal crosslinker 1F3 was used, as shown in Figure 1, it reacted most strongly with liver ferritin, followed by placental ferritin and pancreatic ferritin, with different reactivity depending on the tissue ferritin. Shown. Furthermore, when monoclonal antibody 3F11 was used, as shown in FIG. 2, equivalent reactivity was shown with liver, placenta, and pancreatic ferritin.
In FIGS. 1 and 2, the black circles indicate the results of measuring liver ferritin, the double circles indicate the results of measuring placental ferritin, and the open circles indicate the results of measuring pancreatic ferritin. Iron metabolism test (a) The method for releasing iron in placental ferritin is to add placental ferritin (0.1 mg) to a sodium acetate buffer (1M, PH4.8) containing 3% sodium dithionite and incubate at room temperature. Placental ferritin iron was released by reacting for 0.1 to 6 hours. After release, each placental ferritin was dialyzed against distilled water overnight to remove free iron. For each of the above placental ferritins, when the amount of iron in ferritin was measured using the absorbance of a spectrophotometer OD = 420 mm (E ¹ % ^Fe cm = 100), as shown in Table 1, the amount of iron in ferritin decreased over time. . Using each of the apoferritins obtained by such operations as materials, the reactivity with two types of monoclonal antibodies obtained by the methods of and was comparatively investigated. These reactivities were determined by the ELISA method described in . As a result, as shown in Table 1, in the case of monoclonal antibody 3F11, its reactivity towards any ferritin did not change at all, regardless of the amount of iron in ferritin. Furthermore, in the case of monoclonal antibody 1F3, its reactivity decreased as the amount of iron stored in the ferritin decreased.

[Table] Displayed in %.
(b) Regarding the uptake of iron into placental ferritin and its method, 1 ml of placental apoferritin (0.1
1 ml of ferrous ammonium sulfate (0.074-7.4 mM) at various concentrations was added to the solution (mg/ml). Next, 1ml of imidazole buffer (18.5mM, PH7.45) containing potassium iodate (3.7mM) and sodium thiosulfate (14.3mM) was added to this solution and reacted for 5 minutes, and then dialyzed against distilled water.
Free iron was removed. Using placenta ferritin with different amounts of iron in ferritin obtained by this procedure as materials, the reactivity of monoclonal antibodies was comparatively investigated by ELISA method according to the method described in . As a result, as shown in Table 2, there was almost no change in the case of monoclonal antibody 3F11. Furthermore, in the case of monoclonal antibody 1F3, its reactivity increased as the amount of iron in placental ferritin increased.

It is displayed in [Table].

Measurement of serum ferritin Serum ferritin levels were investigated using monoclonal antibodies 1F3 and 3F11 in anemic patients (patients with a hemoglobin value of 12.0 or less) and various liver diseases. Serum ferritin values were measured in exactly the same manner as described in 2012, except that serum was used as the test sample instead of tissue ferritin. As standard ferritin, liver ferritin (2-
(Sequentially prepared to concentrations up to 250ng/ml)
or the same test serum was added to measure the serum ferritin level. (a) As shown in Table 3, the serum ferritin levels of anemic patients are determined using the monoclonal antibody 1F3.
It was lower than that measured using 3F11. In addition, the serum iron level measured by the vasoferontroline method shows a high value from the normal range (50
Even in anemic cases (~160 μg/dl), the ferritin values measured using monoclonal antibody 1F3 were all lower than the ferritin values measured using monoclonal antibody 3F11.

〔Effect of the invention〕

As described above, the monoclonal antibody of the present invention has various characteristics, and in particular, by utilizing the characteristics of both antibodies, it is possible to clearly investigate the storage status of ferritin in the blood of patients with anemia and iron overload, and it is also possible to investigate the storage status of ferritin in the blood of patients with anemia and iron overload. It is extremely useful as a reagent in clinical tests, as it can detect the leakage of ferritin into the blood during the acute phase of cancer and the stage of necrosis of hepatocytes and pancreatic cells.

[Brief explanation of the drawing]

FIG. 1 shows the reactivity of each tissue ferritin using monoclonal antibody 1F3, and FIG. 2 shows the reactivity of each tissue ferritin using monoclonal antibody 3F11. FIG. 3 shows serum ferritin levels measured by enzyme immunoassay using serum from various liver diseases.

Claims (1)

[Scope of Claims] 1. A monoclonal antibody using human placental ferritin as an immunogen, having reactivity with human liver, placenta, pancreatic tissue ferritin and serum ferritin, and having iron metabolic behavior. . 2. The monoclonal antibody according to claim 1, which is directly or indirectly labeled with a radioactive substance, a fluorescent substance, or a chromophoric substance.