JPH085919B2 - Saliva-α-amylase activity-inhibiting monoclonal antibody and pancreas-α-amylase fractional quantification method using the same - Google Patents

Description

The present invention relates to a monoclonal antibody that inhibits only saliva-α-amylase activity in body fluid and pancreas using the same.
The present invention relates to a fractional quantification method for α-amylase.

(Prior Art) α-Amylase is an enzyme that randomly hydrolyzes α-1,4-glycoside bonds of starch, polysaccharides, glycogen and the like. The content of α-amylase in body fluid varies remarkably due to various diseases, so it is important for diagnosis, but α-amylase in blood and urine is derived from completely different organs of pancreas and salivary gland. Since it exists as an isozyme, the definite diagnosis is difficult even if the total α-amylase activity is measured. Therefore, it is necessary to limit the origin of α-amylase, that is, it is necessary to perform differential quantification of α-amylase isozyme, and in particular, pancreatic α-amylase is extremely important as a marker for pancreatic disease.

Conventionally, the fractionation method of α-amylase isozymes is generally performed by an electrophoresis method and a wheat-derived inhibitor method, but the former has a drawback that the operation is complicated and takes time, and the latter is α-amylase -It has the drawback that the selectivity for amylase isozyme is not sufficient and that its inhibition rate changes depending on the lot, the addition amount, and the reaction time. In addition, an enzyme immunoassay (EI) using a monoclonal antibody against pancreas-α-amylase
Although a pancreas-α-amylase assay method by the method A) was developed, it still takes a long time to obtain the results.

On the other hand, JP-A-58-183098 discloses, as an example, the measurement of pancreatic-α-amylase using a soluble starch as a substrate by an immunoinhibition method using one kind of monoclonal antibody. However, it is difficult to obtain a hybridoma producing the monoclonal antibody used here, and further, a measuring reagent using this antibody has not been put on the market. In addition, recently, a method for measuring α-amylase isozymes by an immunoinhibition method using two types of monoclonal antibodies has been developed (Japanese Patent Publication No. 63-2600). This is because the first monoclonal antibody that specifically inhibits salivary enzyme by less than 97%, the second monoclonal antibody that inhibits this enzyme by less than 10%.
It is a specific assay method for pancreatic-α-amylase characterized by being used in combination with a monoclonal anti-saliva-α-amylase-antibody, and a measurement reagent utilizing this method is currently on the market. However, with this method, inhibition of saliva-α-amylase is less than 97.5%.

(Problems to be Solved by the Invention) Although the α-amylase isozyme fractional quantification by the immunoinhibition method is considered to be the best at present, even in the case of this method, as described above, the inhibition against saliva-α-amylase is 97.5. % Or less, the effect of saliva-α-amylase cannot be said to be sufficiently avoided, and the remaining 2.5
A salivary-α-amylase activity of greater than or equal to% often causes diagnostic confusion.

In the measurement of α-amylase isozymes by the immunoinhibition method, when the antibody used is a combination of two or more kinds of monoclonal antibodies, those antibodies have hitherto been obtained by directly modifying saliva-α-amylase or chemically modifying them. By immunizing animals with each of them, they are individually selected from monoclonal antibodies that partially inhibit saliva-α-amylase activity, and therefore, antibodies that can suppress enzyme activity almost completely are selected. It takes a lot of work to find a combination, and
The combination is contingent.

By the way, MMBenkiran et al.
e et al.), Molecular. Immunology (Mol.Immuno
l), 1987 24 (12) p1309-1315, immunize an animal with an immune complex of human-TSH and anti-human-TSH monoclonal antibody to prepare a monoclonal antibody against the immune complex, and then perform a radioimmunoassay (RIA). Used for.

The present inventor has found that, by developing this method, a monoclonal antibody having a higher degree of inhibition of saliva-α-amylase activity can be obtained, and has completed the present invention.

(Means for Solving the Problems) The present invention does not inhibit pancreatic-α-amylase activity at all,
A second monoclonal antibody which can be obtained by immunizing an animal with an immune complex of a saliva-α-amylase and a salivary-α-amylase, which comprises IgG ₁ It belongs to the subclass, is precipitated with 50% saturated ammonium sulfate, is dialyzed against physiological saline, and is then mixed with 1.5 M glycine-3.0 M NaCL (pH 9.0) solution in an equal amount to bind to protein A and bind to protein A. Elution with a 0.1 M citric acid (pH 3.0) solution in column chromatography alone did not inhibit pancreatic-α-amylase activity at all, and only saliva-α-amylase activity was inhibited by 85% or less. When combined with one monoclonal antibody, a saliva-α-amylase activity-inhibiting monoclonal antibody that inhibits only saliva-α-amylase activity by 99% or more is provided. . In addition, the present invention provides a method for separately quantifying pancreatic isozymes in a sample containing human-α-amylase salivary (S) and pancreatic (P) isozymes by inhibiting the activity of salivary isozymes. Also provided is a method for fractionating and quantifying pancreatic-α-amylase, which comprises using one monoclonal antibody in combination with the second monoclonal antibody of the present invention.

The first monoclonal antibody may be any monoclonal antibody that does not inhibit pancreatic-α-amylase activity at all and only partially inhibits saliva-α-amylase activity. And the antibody-producing cells of the animal and the myeloma cells of the animal are fused according to a conventional method. As the myeloma cells, mouse myeloma cells FO are preferable, but other cells can also be used. Examples of the cell fusion method include polyethylene glycol method, HVJ method and electrofusion method. The obtained hybridoma is cultured in, for example, an appropriate medium, and the antibody in the culture solution is allowed to act on saliva-α-amylase, and then the remaining α-amylase activity is measured to produce an appropriate monoclonal antibody. Hybridomas to be selected. The first monoclonal antibody thus obtained is preferably, for example, one that does not inhibit pancreatic-α-amylase activity at all and inhibits saliva-α-amylase activity only by 10% or more, but alone measures isozyme. Anything that cannot be used for

The monoclonal antibody of the present invention (second monoclonal antibody) is administered to an animal by using an immune complex of the first monoclonal antibody and saliva-α-amylase as an immunogen,
It is obtained from a hybridoma producing a second monoclonal antibody produced in the same manner as the first monoclonal antibody. The second monoclonal antibody can be selected by examining the inhibitory effect on the residual enzyme activity of the immune complex used as the immunogen. As a result, it is possible to more effectively select the second monoclonal antibody to be combined with the first monoclonal antibody at the stage of hybridoma production, as compared with the conventional method of separately producing the first and second monoclonal antibodies. Becomes

The obtained hybridoma is cultured in, for example, an appropriate medium, and the second monoclonal antibody is separated and purified from the culture supernatant. Alternatively, the hybridoma is intraperitoneally administered to a compatible mammal to grow it, and the second monoclonal antibody is separated and purified from the ascites of the animal. Examples of the medium used in the former method include Dulbecco's modified Eagle medium, RPMI 1640 medium, and commercially available serum-free medium. Separation and purification can be performed by salting out with ammonium sulfate, ion exchange chromatography, affinity chromatography and the like.

The second monoclonal antibody of the present invention thus obtained belongs to the IgG ₁ subclass and is precipitated with 50% saturated ammonium sulfate. Also, this precipitate was dissolved in physiological saline and dialyzed against physiological saline, and an equal amount of 1.5M glycine-3.0M NaCL (pH
9.0) When mixed with a solution, it binds to protein A and becomes eluted with 0.1 M citric acid solution (pH 3.0) in protein A column chromatography. The second monoclonal antibody alone does not inhibit pancreatic-α-amylase activity at all, and only 85% salivary-α-amylase activity.
Although it inhibits only the following, when combined with the first monoclonal antibody, only 99% saliva-α-amylase activity is obtained.
It inhibits more.

The pancreas-α-amylase differential quantification method of the present invention is a saliva-α-sample in a sample prepared by mixing a test sample such as blood and urine with the first and second monoclonal antibodies according to a known immunoinhibition method.
It can be carried out by a two-step method including a first step for inactivating the amylase activity and then a second step for measuring the enzyme activity. In addition, since the first-step antigen-antibody reaction proceeds rapidly under the conditions for measuring enzyme activity (temperature, pH, salt concentration, etc.), conventional automatic analyzers capable of measuring enzyme activity can be used.
Can be easily applied. As the system for measuring α-amylase activity, all known methods can be used. For example, a commercially available α-amylase measurement reagent (autopack α-amylase, West Germany Boehringer
This is performed by adding an appropriate amount of purified monoclonal antibody to R1 solution (mannheim). This reagent can be applied to automatic analyzers such as Hitachi-705 and Hitachi-7150. Commercially available α-amylase isozyme standard (calibzyme AMY [P, S], international reagents,
By measuring the product number 7892) or saliva-α-amylase preparation (Sigma, USA, product number A0521), inhibition of each isozyme can be known.

(Example) Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1 Preparation of First Monoclonal Antibody 100 μg of a human-saliva-α-amylase preparation (Sigma, USA, product number A0521) was subcutaneously administered to 8-week-old male mice together with Freund's complete adjuvant (FCA) on the back. Immunization started. One month later, 50 μg of saliva-α-amylase was subcutaneously administered to the back together with Freund's incomplete adjuvant (FIA) for booster immunization,
One month later, 50 μg of saliva-α-amylase was intraperitoneally administered for the final immunization. Three days later, the spleen was extracted and fused with mouse myeloma cells FO by a conventional method using polyethylene glycol (PEG) 4000 as a cell fusion promoter. After selecting HAT for the fused cells, saliva-α-amylase activity-inhibiting monoclonal antibody-producing cells were selected. For the selection, a commercially available α-amylase assay reagent (Nescort AMY-V2, manufactured by Nippon Shoji Co., Ltd.) using a synthetic substrate parantrophenylmaltopeptaoside was used. First, about 50
The same amount of the hybridoma culture supernatant was added to 0 IU / l of the isoamylase standard solution (P-type and S-type α-amylase, manufactured by Boehringer Mannheim, West Germany, product number 836176) and reacted for about 30 minutes. After this reaction, 10 μl of α-
After adding 300 μl of a solution of amylase measuring reagent R1 (containing α-D-glucosidase) and incubating for 5 minutes, R2
A solution (containing p-nitrophenyl-α-D-maltoheptaoside) (25 μl) was added, and the change in absorbance was measured at a main wavelength of 415 nm and a sub-wavelength of 480 nm. As a control, the myeloma culture supernatant was used instead of the hybridoma culture supernatant. Saliva
A hybridoma producing an α-amylase activity-inhibiting monoclonal antibody was cloned by the limiting dilution method. A hybridoma producing a monoclonal antibody that partially inhibits saliva-α-amylase activity but does not inhibit pancreatic-α-amylase activity at all was selected from the obtained clones, and named NHAMY-2. This was mass-cultured in Dulbecco's modified Eagle medium. Ammonium sulfate was added to the culture supernatant to 50% saturation for salting out, and the precipitate was centrifuged. This was dissolved in physiological saline and dialyzed at 4 ° C. for 20 hours using physiological saline as an external solution. The dialyzed solution thus obtained was mixed with an equal amount of a 1.5 M glycine-3.0 M-NaCl (pH 9.0) solution, subjected to protein A column chromatography, and washed with the same solution. Then, it was eluted with a 0.1 M citric acid (pH 3.0) solution, and the protein fraction was collected while monitoring the eluate at an absorbance of 280 nm to obtain a purified first monoclonal antibody belonging to the IgG ₁ subclass. α-amylase assay reagent (Autopack α-amylase, West Germany Boehringer
Mannheim's) R1 solution, the purified monoclonal antibody was added at a concentration of 60 μg / ml, saliva- and pancreas-α-amylase standard (calibzyme AMY [P, S], using a Hitachi-7150 automatic analyzer). International Reagent, Product Number 789
The activity of 2) was measured. At the same time, the degree of inhibition of saliva- and pancreas-α-amylase of the first monoclonal antibody was measured by using the same measurement reagent without addition of antibody as a control. The results are shown in Table 1.

The hybridoma NHAMY-2 thus obtained was 1989 10
Contract number FE to Institute of Microbiology, Institute of Industrial Science, dated 25th March
Deposited as RM P-11067.

Example 2 Preparation of Second Monoclonal Antibody An antigen-antibody complex of the first monoclonal antibody produced by hybridoma NHAMY-2 and saliva-α-amylase was prepared. That is, the first monoclonal antibody and a saliva-α-amylase preparation were mixed in a physiological phosphate buffer (PBS) at a molar ratio of 1: 2, and a complex was formed by an antigen-antibody reaction. The antigen-antibody complex is cefacryl
Purified by S-200 column chromatography. 200 μg of the purified complex was added to the Libi adjuvant system (R
AS) and was intraperitoneally administered to 8-week-old male mice to start immunization. One month later, 50 μg was intraperitoneally administered for booster immunization, and one month later, 50 μg was intraperitoneally administered for final immunization. Three days later, the spleen was removed, and PEG4000 was used as a cell fusion promoter.
Fused with FO. For the fused cells, a hybridoma producing the first monoclonal antibody / saliva-α-amylase complex-inhibiting activity against the first monoclonal antibody obtained from NHAMY-2 was selected and the hybridoma was named NHAMY-3. The obtained hybridoma was mass-cultured in Dulbecco's modified Eagle medium, ammonium sulfate was added to the culture supernatant until 50% saturation, salting out, and the precipitate was centrifuged. This was dissolved in physiological saline and dialyzed at 4 ° C. for 20 hours using physiological saline as an external solution. The dialyzed solution obtained was mixed with an equal volume of 1.5 M glycine-3.0 M-NaC.
l (pH 9.0) solution, mixed with protein A column chromatography, and washed with the same solution. Then 0.
Elution was performed with a 1 M citric acid (pH 3.0) solution, and the protein fraction was collected while monitoring the eluate at an absorbance of 280 nm to obtain a purified second monoclonal antibody belonging to IgG ₁ . An α-amylase assay reagent (Autopack α-amylase, manufactured by Boehringer Mannheim, West Germany) R1 solution to which the purified second monoclonal antibody was added at a concentration of 60 μg / ml, and the first monoclonal antibody 60μ
The activity of saliva- and pancreas-α-amylase standards (Calibuzyme AMY [P, S], International Reagent, Product No. 7892) was measured by Hitachi-7150 automatic analyzer using the one added at the concentration of g / ml. did. At the same time, as a control, the activity of the α-amylase standard was measured using the same assay reagent containing no antibody. Each degree of inhibition was calculated from these measured values. Table 2 shows the results.

The hybridoma NHAMY-3 thus obtained was 1989 10
Contract number FE to Institute of Microbiology, Institute of Industrial Science, dated 25th March
Deposited under RM P-11068.

Example 3 Saliva-α-amylase, pancreas-α-amylase standard (Calibuzyme AMY [P, S], International Reagent, Product No. 7892) was dissolved in distilled water to obtain standard solutions of 1431 and 1211 IU / L, respectively. Prepared. In addition, both standard solutions should be mixed in a prescribed ratio (5:
0, 4: 1, 3: 2, 2: 3, 1: 4 and 0: 5) were mixed to prepare a standard solution having a known α-amylase isozyme content.

Next, an α-amylase measurement reagent (Nescort AMY-V2,
Hybridoma NHAMY-2 to R1 solution of Nippon Shoji Co., Ltd.
And NHAMY-3 produced monoclonal antibodies were added at concentrations of 60 μg / ml and 100 μg / ml, respectively.
An amylase measuring reagent was prepared. Using this measurement reagent, the pancreatic-α-amylase activity in the standard solution was measured by Hitachi-
It was measured with a 7150 automatic analyzer. As shown in FIG. 1, the pancreatic-α-amylase was able to be measured without being affected by the mixed saliva-α-amylase by using the antibody-added pancreatic-α-amylase measuring reagent.

Example 4 Monoclonal antibodies produced by hybridomas NHAMY-2 and NHAMY-3 were each added to R1 solution of a commercially available amylase measuring reagent (Nescort AMY-V2, manufactured by Nippon Shoji Co., Ltd.).
A pancreas-α-amylase assay reagent was prepared by adding the solution at a concentration of μg / ml. A commercially available α-amylase isozyme measuring reagent using this measuring reagent and parantrophenylmaltopeptaoside which is a synthetic substrate (isoamylase measuring reagent PNP, product number 97975 manufactured by Boehringer Mannheim)
9) was used to measure pancreatic-α-amylase in human serum with Hitachi-7150, and the measured values were compared (see FIG. 2).

(Effect of the Invention) According to the present invention, the pancreatic-α-amylase can be fractionated and quantified with high accuracy by the immunoinhibition method, and the accuracy of diagnosis of pancreatic diseases can be improved.

[Brief description of drawings]

FIG. 1 is a graph showing the results of measuring the activity of an α-amylase isozyme mixed standard solution of known concentration with a pancreas-α-amylase measuring reagent according to the method of the present invention, wherein the horizontal axis represents the pancreas (P) in the standard solution. Type and saliva (S) type α-amylase content and mixing ratio, and the vertical axis represents the measured value of each measurement reagent.
FIG. 2 shows the correlation between the measured value of pancreatic-α-amylase activity in human serum obtained by the reagent for measuring pancreatic-α-amylase according to the method of the present invention and the measured value obtained by the reagent for measuring commercially available isoamylase. It is a graph.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12P 21/08 9358-4B (C12P 21/08 C12R 1:91) (56) References 61-164161 (JP, A) JP 62-22600 (JP, A) JP 60-155134 (JP, A) JP 58-183098 (JP, A)

Claims (3)

[Claims] 1. An animal is immunized with an immune complex of saliva-α-amylase and a first monoclonal antibody that does not inhibit pancreatic-α-amylase activity at all but partially inhibits saliva-α-amylase activity. A second monoclonal antibody, which belongs to the IgG ₁ subclass, is precipitated with 50% saturated ammonium sulfate, is dialyzed against physiological saline, and is then treated with 1.5 M glycine-3.0 M NaCl.
(PH 9.0) solution was mixed with protein A to bind to protein A and eluted with 0.1 M citric acid (pH 3.0) solution in protein A column chromatography. Pancreatic-α-amylase activity alone It inhibits saliva-α-amylase activity only by 85% or less, but when combined with the first monoclonal antibody, inhibits saliva-α-amylase activity only by 99% or more. A saliva-α-amylase activity inhibiting monoclonal antibody. 2. The first monoclonal antibody is NHAMY-2 (F
ERM P-11067) hybridoma, wherein the second monoclonal antibody is NHAMY-3 (FERM P-
The monoclonal antibody according to (1), which is produced from the hybridoma of 11068). 3. The method according to claim 1, wherein the pancreatic isozyme in a sample containing human-α-amylase salivary and pancreatic isozymes is subjected to fractional quantification by inhibiting the activity of salivary isozyme. ) The above-mentioned first monoclonal antibody and a second monoclonal antibody are used in combination, and a fractional quantification method.