JPH0226960B2 - - Google Patents

Description

[Detailed description of the invention]

TECHNICAL FIELD The present invention relates to monoclonal antibodies against human apolipoprotein B48. Prior Art Apolipoprotein B is a protein that plays an important role in the synthesis and secretion of human plasma triglyceride lipoproteins. Apolipoprotein B includes apolipoprotein B100 (hereinafter abbreviated as apoB100), which is produced in the liver and is involved in the synthesis of VLDL (very low density lipoprotein), and apolipoprotein B100, which is produced in the small intestine and is involved in chylomicron synthesis.
There is B48 (hereinafter abbreviated as Apo B48). Abeta-abeta, a disease caused by a genetic defect in apolipoprotein B
There are two types of lipoproteinemia: classical abetalipoproteinemia, in which both apoB100 and apoB48 are deficient;
Only those with triglyceride deficiency have normal abetalipoproteinemia. All of them exhibit serious symptoms such as red blood cell abnormalities and neuropsychiatric symptoms, but chylomicrons can be synthesized in normotriglyceride abetalipoproteinemia. In addition, lipoproteins containing apolipoprotein B are thought to have properties that induce arteriosclerosis, and since apoB100 and apoB48 have different metabolic pathways, it is important to understand the structures of both in order to understand the pathogenesis of arteriosclerosis. is important. As mentioned above, although apolipoprotein B has various functions, it is poorly soluble and consists of a high-molecular protein that is easily decomposed, so that its research has not yet been conducted in detail. For this research,
It is necessary to specifically recognize each component of apolipoprotein B. Purpose of the Invention To this end, the present invention aims to establish a means for purifying apoB48 and obtain a monoclonal antibody against purified apoB48 (hereinafter abbreviated as anti-apoB48-). Structure of the Invention The anti-apoB48-producing hybridomas of the present invention are formed by the fusion of splenocytes from a mouse previously immunized with apoB48 and cells from a mouse myeloma cell line. Such anti-apoB48 of the present invention and hybridomas producing this antibody can be created, for example, by the following four steps. 1. ApoB48 purification, 2. Immunization, 3. Cell fusion, 4. Hybridoma selection and monocloning. Anti-apoB48-1 created in this way is
As a result of the two-way diffusion method, it was found that it belonged to the IgG1a subclass. When examined by trans-blotting, anti-apoB48- binds to apoB100 and apoB48, but cannot bind to albumin, apolipoprotein E, or other apolipoproteins. Furthermore, we developed a method for measuring immunoenzyme antibodies using this antibody. For this purpose, we used an indirect method in which the sample to be measured was attached to a 96-well polystyrene plate, and a sandwich method in which a rabbit anti-apoB polyclonal antibody was attached to the plate in advance and the sample was bound to this. Using apoB48- of the present invention, it is possible to quantify apolipoprotein B in human blood with relatively high accuracy. In Japan, the patients with aβlipoproteinemia mentioned above include normal triglyceride type (Nagoya family), which is solely deficient in apoB100, and apoB100 and apoB48.
The classic type (Kanagawa family) is known, in which both are missing. Using the transblotting method and the immunoenzyme antibody method, lipoproteins and apolipoprotein B of patients of Nagoya ancestry were detected to be the anti-apoB48 of the present invention.
-, but lipoproteins and apolipoproteins from patients of Kanagawa descent showed no binding.
This means that anti-apoB48-
This supports the fact that it cannot bind to any other apolipoprotein. In summary, the anti-apoB48- of the present invention is
(1) Reacts with apoB48 in normal human blood; (2) Does not react with apolipoproteins other than apolipoprotein B; (3) Lipoproteins in the blood of patients with TG normal aβlipoproteinemia. It was found that these substances have various properties that allow them to react with each other. Next, a preferred example for producing the anti-apoB48 of the present invention and a hybridoma producing this antibody will be described. First, apoB48 is purified. For this purpose, since a large amount of chylomicron is required, chylous ascites from a patient with chylous ascites, plasma from a patient with type V hyperlipidemia, or plasma from a normal person after a fatty meal is used. Once a sample is obtained, it is treated with EDTA, NaN ₃ and kanamycin, and then separated by ultracentrifugation to obtain a VLDL+chylomicron fraction. Furthermore, this VLDL+
From the chylomicron fraction, obtain the chylomicron fraction by ultracentrifugation. The chylomicron fraction thus obtained is defatted with an organic solvent (eg, ether and ethanol, tetramethylurea, or chloroform and methanol) and dissolved in a solution containing SDS (sodium dodecyl sulfate). This solution was heated in a water jacket to create Sepharose 4B.
Gel filtrate with SDS solution using CL column. When this step is completed within two days, the decomposition of apoB48 is small and almost pure apoB48 can be obtained. Using apoB48 purified in this way,
Immunization was successful. That is, apoB48 is administered to mice, and splenocytes from the immunized mice are removed. The splenocytes of this mouse are fused with cells from mouse myeloma (NS-1) to obtain the hybridoma of the present invention. This hybridoma can be created using, for example, the method of Ohi et al. (SELECTED METHODS IN CELLULAR
IMMUNOLOGY, 351−372, Freeman, 1980). From the obtained hybridoma, the anti-apoptotic protein of the present invention is
For example, the following two methods can be used to create B48-. Method 1 is a method in which hybridomas are cultured in an appropriate culture medium (in vitro) and antibodies produced in the supernatant are collected. Another method is to inject hybridomas into mice (in vivo), incubate them in vivo, and collect the antibodies produced in the ascites of the mice. The former method yields antibodies with low antibody titers but high purity, while the latter methods yield slightly inferior antibodies but with very high antibody titers. Which method to choose depends on the purpose. Hereinafter, the present invention will be explained in more detail based on Examples. Example 1 In this example, apoB48 is purified. Chylomicron is purified using various samples, but here it is purified from chyle ascites.
Immediately after collecting approximately 2 pieces of chyle ascites, add 1mM
Prepare by adding EDTA and 0.02% NaN ₃ and ultracentrifuge at 27,000 rpm for 20 hours in a Beckman SW 27 rotor to obtain the chylomicron + VLDL fraction. Furthermore,
This fraction was transferred to a Beckmann 40.3 rotor at 20,000 rpm.
Ultracentrifuge for 30 minutes and collect the floating chylomicron fraction. This chylomicron fraction is defatted for 10 hours in a 20-fold volume of ether and ethanol (1:3 vol/vol) solution. The precipitate was collected by centrifugation at 2000 rpm for 5 minutes, and mixed with approximately 10 c.c. of 10% SDS and 1% 2-
Dissolve in mercaptoethanol solution. Of this, 5c.c. was 2cm x 2cm with a water jacket at 50℃.
Gel-filter through a 100 cm Sepharose 4B/CL column. At that time, 0.5% SDS was added as the effluent.
and 0.01 M Tris HCl adjusted to pH 7.2 with 0.15 M NaCl at a flow rate of 20 ml/hour. The result is nearly pure ApoB48. Degreasing is almost complete. Example 2 In this example, a monoclonal antibody (anti-apoB48-) of the invention and a hybridoma for producing it are generated. 1 Immunization 100 μg of purified apoB48 in SDS solution was injected subcutaneously with Freund's complete adjuvant into male Bulb/c mice (6 weeks old). 3
After a week, 10 μg of the apoB48 was intraperitoneally injected together with the adjuband. 2 Cell Fusion Three days after the second immunization, the spleen of the mouse is removed and used as a suspension of splenocytes. 1 x 10 ⁸ splenocytes were fused with 3 x 10 ⁷ 8-azaguanine resistant myelocytoma NS-1 using polyethylene glycol #4000. Cells were distributed into five 96-well microculture plates. After 24 hours, half of the supernatant was mixed with HAT medium (hypoxanthine 1×10 ^-4 M, aminopterin 4×
10 ^-7 M, thymidine 1.6×10 ^-3 M).
HAT resistant cells (hybridomas) are observed to grow in most media after 2-3 weeks. 3 Hybridoma selection and monocloning 10μ of culture supernatant in microplate
is placed in an assay plate coated with triglyceride lipoprotein containing apoB48 obtained from post-fat meal plasma, left at room temperature for 1 hour, and then washed three times. Add peroxidase-labeled anti-mouse IgG diluted 1000 times to this,
After reacting for 1 hour at room temperature and washing three times, a coloring solution was added and the colors were compared using a photometer. Hybridomas secreting antibodies that strongly react with triglyceride lipoproteins were selected. Monocloning was performed by subjecting the selected hybridoma twice to the limiting dilution method using mouse thymocytes as feeder cells. 4. Creation of anti-apoB48- (a) In vitro method The hybridoma obtained in the above step is cultured in an appropriate culture medium (for example, containing 10% fetal bovine serum).
RPMI1640 medium), and the culture supernatant was collected. Antibodies secreted into the supernatant are
A highly pure product was obtained that could be used as is without purification. (b) In vivo method 2,6,10,14-tetramethylpentadecane is intraperitoneally injected into the mouse to which the hybridoma is to be injected (four days before). Next, 5×10 ⁶ antibody-producing hybridomas per mouse are intraperitoneally injected.
Four to 10 days after the injection, ascites containing a high concentration of antibodies begins to form in the abdominal cavity of the mouse. This ascites fluid can be used as is as an antibody. Example 3 In this example, the specificity of anti-apoB48- was investigated. The binding between anti-apoB48− and apolipoprotein is
The results were investigated using the transblotting method. First, each apolipoprotein was determined using the method of Yutani et al. (J.
Biochem 94 1241-1245, 1983)
SDS-polyacrylamide gradient gel electrophoresis was performed, followed by the method of Tobin et al. (Proc.
Natl.Acad.Sci.USA, 76 4350−4354, 1977)
transferred to cellulose nitrate paper. The cellulose nitrate paper was incubated with anti-apoB48 for 1 hour at room temperature and then treated with peroxidase-labeled anti-mouse IgG (by the method of Tsang et al.
Methods in Enzymology, 92 377−390,
Academic Press, New York, 1983), or
¹²⁵ Detected by protein A. Table 1 summarizes the results. Table 1 shows the binding between various apolipoproteins and anti-apoB48-. From this table, it can be seen that anti-apoB48- can bind to apoB48 and apolipoprotein B of a patient with normal TG aβlipoproteinemia of the Nagoya pedigree.

【table】

[Table] As described above, since apoB48 can be purified and monoclonal antibodies can be obtained according to the present invention, these can be used for qualitative and quantitative blood analysis, clinical treatment, disease diagnosis, etc. be.

Claims (1)

[Claims] 1 Produced by a hybridoma formed by the fusion of mouse splenocytes previously immunized with human apolipoprotein B48 and cells from a mouse myeloma cell line, Reacts with protein B48, (2) cannot react with apolipoproteins other than apolipoprotein B, and (3) has the following properties that can react with lipoproteins in the blood of patients with normal TG aβlipoproteinemia. human apolipoprotein B48
Monoclonal antibodies against.