JPH0373853A - Preparation of labeled antibody - Google Patents

Abstract

PURPOSE:To easily obtain the labeled IgM antibody by introducing a specific functional group into immune globulin M. CONSTITUTION:The IgM antibody is dissolved in, for example, a phosphoric acid buffer soln., etc., and thereafter, succinic anhydride is added thereto. The SH group is introduced into the IgM antibody by making such operation. N- succinimidyl3-(2'-pyridyldithio) propionate is then added to alkaline phosphatase, etc. to obtain modified enzyme (PDP-ALP). The PDP-ALP obtd. in such a manner and the IGM antibodyhaving the SH group are brought into reaction to obtain the ALP labeled antibody. The labeled IgM antibody is easily prepd. in such a manner.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing a labeled antibody of immunoglobulin M (hereinafter referred to as 1 gM) type antibody.

[Prior Art] In the standard immuno-AIIJ method, it is very important to produce a labeled antibody. For example, various preparation methods using crosslinking reagents have been considered for enzyme-labeling antibodies (Eiji Ishikawa et al., Enzyme Immunoassay Method, 3rd Edition, Igaku Shoin). However, all of these methods require that the antibody class is Ig.
It is a G-type antibody and cannot be applied to IgM-type antibodies.

Up to now, examples of enzymatic labeling of IgM include introducing a thiol (hereinafter referred to as 911) reactive group (i.e., a group that can react with the 911 group), such as a maleimide group, into IgH, and using enzymes with free SH groups, such as There was a method of reacting with β-D-galactosidase (Tsunehiro Kitayo et al., Journal of Immunological Methods).
Logial Methods volume 112, page 77,
(1988)).

[Problems to be Solved by the Invention] However, this method has the drawback that only enzymes having free SH groups can be labeled. An object of the present invention is to provide a method for easily producing labeled antibodies.

[Means and effects for solving the problem] The present inventors have discovered that a highly sensitive labeled antibody can be easily produced by introducing an SH group into an IgM antibody, and have completed the present invention. .

That is, the present invention provides a method for labeling an IgM antibody in which S
) A method for producing a labeled antibody, which comprises reacting IgM introduced with an I group and a label introduced with an SH reactive group.

The IgM type antibodies used in the present invention are preferably polyclonal antibodies obtained from antiserum of an animal immunized with the antigen, or obtained by fusing antibody-producing cells of the animal with myeloma cells. Hybridomas that produce monoclonal antibodies that recognize antigens can be obtained, and then the hybridomas and/or cell lines derived therefrom can be cultured and harvested from the culture. Ibridomas that produce monoclonal antibodies that recognize antigens can be produced by cell fusion method CG, which is a well-known method.
Xohler and C. Milstein
1stein), Nature,
258, p. 495, 1975).

Moreover, the IgM type antibody may be fragmented. The fragmented IgM type antibody is not particularly limited, and includes, for example, Fab.

F(abo)2. Examples include IgG.

Examples of reagents used to fragment IgM include enzymes and reducing agents. The enzyme is not particularly limited as long as it decomposes proteins, and examples thereof include trypsin, pepsin, papain, etc., and reducing agents include cysteine, dithiothreitol, mercaptoethanol, etc.

S) to the antibody! As a reagent for introducing a group, S)! There is no particular limitation as long as it is a reagent that can introduce a group, for example, S-
Examples include acetylmercaptosuccinic anhydride, 2-iminothiolane, methyl-3-mercaptoprobionimidate, and the like.

In addition, as a reagent for introducing an SH reactive group into a label, Sl
! There is no particular limitation as long as the reagent can introduce a group capable of reacting with the group, and for example, N-succinimidyl-4-maleimidobutyrate, N-succinimidyl-3-(2°-pyridylthio)propionate, etc. are used.

Examples of labels include enzymes and fluorescent dyes. - By way of example, enzymes such as peroxidase, alkaline phosphatase, etc. can be used.

IgM introduced with SH groups obtained as above,
A labeled antibody is produced by reacting with a label into which an SH reactive group has been introduced. The reaction at this time depends on the reagent used.
-8 exchange reaction, maleimide method, etc. are performed.

[Effects of the Invention] According to the method of the present invention, an unprecedented labeled IgM antibody can be easily produced, and this can be used for treatment and diagnosis of diseases.

In addition, particularly when a fragmented IgM antibody is used, nonspecific adsorption peculiar to IgM antibodies can be suppressed to a lower level.

[Examples] Hereinafter, the present invention will be explained in detail with reference to Examples. However, the present invention is not limited to these examples.

(Example 1) 1. Preparation of enzyme-labeled antibody 1) Introduction of 811 groups into antibody CA 19-9-specific IgM monoclonal antibody 2
mg to 0.15M phosphate buffer (pH 7,5) 1 m
1 and incubated at 37°C. 100 μl of 50 mM S-acetylmercaptosuccinic anhydride dissolved in dioxane was added thereto, and the mixture was incubated at 37° C. for 1 hour. After that, O, 1M phosphate buffer (pH 7)
, 0) to obtain an antibody into which SH groups had been introduced.

2) Introduction of 811 reactive group into enzyme Alkaline phosphatase derived from bovine small intestine (hereinafter referred to as ALP. [EC3,1,3,1]) 2 mg to 0.1
Dissolve in 1 ml of 5M phosphate buffer (pH 7.5),
12μ of 7,5d N-succinimidyl 3-(2′-pyridyldithio)propionate dissolved in dioxane.
1 was added and stirred at 4° C. for 18 hours. After that, 0.1H
The modified enzyme (hereinafter referred to as PDP-ALP) was obtained by dialysis against a phosphate buffer (pf 47°0).

3) Preparation of enzyme-labeled antibody Add FDP-ALP prepared in 2> to the SH group-introduced antibody prepared in 1), and add 50% of 1M hydroxylamine.
μm was added and left at 4° C. for 24 hours.

The reaction product obtained as described above was transferred to TSK Gel G40.
It was purified by high performance liquid chromatography using 0Qsw (manufactured by Tosoh Corporation, trade name) to obtain an ALP-labeled antibody.

2. Preparation of immobilized antibodies Add 0.01M sodium carbonate buffer (pH 9.6,
200 μm of an IgM type monoclonal antibody (same as that used in 1) specific for CAL9-9 (1 μg/ml dissolved in 1 μg/ml) was added and incubated at 4° C. for 20 hours. Next, remove the solution from each well and add 0.04% Tw.
A PBS (0.0 M phosphate buffer containing 0.85% NaCl, pH 7.0) solution (hereinafter referred to as P
After washing three times with 1% BSA (referred to as BS-T solution)
300 μl of PBS-T solution containing (bovine serum albumin) was added, and blocking treatment was performed at 4° C. for 20 hours.

3. Quantification of CA19-9 by enzyme immunoassay The antibody-immobilized plate prepared in 2. was returned to room temperature, washed with PBS-T solution, and the purified CA19-9 was incubated at O~240 U.
50 μi of 0.1% BSA-containing PBS solution containing 0.1% BSA/ml was added to each well as a standard. Then add 1 to each well. ALP-labeled antibody prepared in 0.1 mg/m
A 25-fold diluted solution of 0.1% BSA-containing PBS solution was added in 200 μm portions, and after incubation at room temperature for 3 hours, the solution was removed and the plate was washed three times with PBS-T solution.

Additionally, 0,5M 2-amino-2-methylpropano-containing 10mM varanitrophenyl phosphate
200 μm of roux acetate-acid buffer (pH 10) was added to each well and reacted for 30 minutes at room temperature, and then 100 μm of 1M sodium hydroxide solution was added to stop the reaction. After stopping the reaction, wavelength 405 nm, control wavelength 6 for each well.
The absorption intensity of 00r+m was measured using an automatic microtiter plate reader (manufactured by Tosoh Corporation, trade name MPR-A4), and the concentration and absorption intensity of the standard substance were plotted to obtain the results shown in Table 1, and a calibration curve was created. .

Using this calibration curve, the concentration of each measurement sample could be determined.

Table 1 (Example 2) 1. Preparation of enzyme-labeled antibody i) Fragmentation of antibody by enzyme treatment CA 19-
3 mg of IgM type monoclonal antibody specific for 50
It was dissolved in 1.5 ml of mM Tris-HCl buffer (pH 8,0), and cysteine was added to the solution to make it 10 mM. Add 1/50th amount of trypsin to it, and after replacing with nitrogen,
Incubated for 4 hours at 7°C. Next, add trypsin inhibitor in twice the amount of trypsin, and store at 37°C for 15 minutes.
Incubated for minutes.

The reaction product obtained as described above was added to TSK gel G3Q.
It was purified by high performance liquid chromatography using OOsw (manufactured by Tosoh Corporation, trade name) to obtain a 1 gM fragment.

2) Introduction of SH group into antibody 1 g of fragment 2II prepared in 1) was treated in the same manner as in Example 1 to obtain a fragment into which an SH group was introduced.

3) Introduction of SR reactive group to enzyme The same treatment as in Example 1 was performed to obtain PDP-ALP.

4) Preparation of enzyme-labeled antibody The SH group-introduced fragment prepared in 2) and 3)
The FDP-ALP prepared in Example 1 was treated in the same manner as in Example 1, except that for purification, TSK gel
(manufactured by Tosoh ■, trade name) to obtain an ALP-labeled fragment.

2. Preparation of immobilized antibody An immobilized antibody was prepared in the same manner as in Example 1.

3. Quantification of CA 19-9 by enzyme immunoassay Using the ALP-labeled fragment prepared in 1 and the phased antibody prepared in 2, CAL9-9 was quantified by enzyme immunoassay in the same manner as in Example 1. Ta. The results are shown in Table 2. A calibration curve was created from this result, and the concentration of each measurement sample could be determined using it.

Table 2 (Example 3) 1. Preparation of enzyme-labeled antibodies ALP-labeled antibodies and ALP-labeled fragments were obtained in the same manner as in Examples 1 and 2.

In addition, 3 mg of IgM type monoclonal antibody specific to CA 19-9 was added to 501!1M Tris-HCl buffer (pHs,
o) Dissolved in 1.5 ml and added cysteine to 1 OmM.

After nitrogen substitution, the mixture was incubated at 37°C for 4 hours.

This reaction product was purified by high performance liquid chromatography using TSK Gel G3000sw (manufactured by Tosoh Corporation, trade name) to obtain a 1 gM fragment. Thereafter, SH groups were introduced in the same manner as in Example 2, and reacted with FDP-ALP to obtain an ALP-labeled fragment.

2. Preparation of immobilized antibody An immobilized antibody was prepared in the same manner as in Example 1.

3. Quantification of CA19-9 by enzyme immunotherapy APJ method Example 1
In the same manner as above, CAL9-9 was obtained by the enzyme-immunized δpj standard method.
was quantified. However, the initial rate of enzyme reaction was determined by adding 4-methylumbelliferyl phosphate as a substrate and then measuring the fluorescence intensity of 4-methylumbelliferone produced by alkaline phosphatase. The results are shown in Table 3. A calibration curve was created from this result, and using this it was possible to determine the concentration of each measurement sample.

Note) a, b, and c in the table respectively indicate a: ALP-labeled antibody not subjected to fragment treatment, b = ALP-labeled fragment treated with residue C, ALP-labeled fragment treated with ligistin and trypsin.

Table 3

Claims (2)

[Claims] (1) A method for producing a labeled antibody, which comprises reacting immunoglobulin M into which a thiol group has been introduced with a label into which a thiol-reactive group has been introduced, in the method for labeling immunoglobulin M. (2) The method according to claim 1, wherein the immunoglobulin M is fragmented.