JP3541701B2 - Enzyme-antibody conjugate and kit for enzyme immunoassay containing the conjugate - Google Patents

Description

[0001]
[Industrial applications]
The present invention relates to an enzyme-antibody conjugate obtained by further binding an enzyme to an enzyme to which an antibody has been bonded, and a kit for enzyme immunoassay (hereinafter referred to as EIA) containing the enzyme-antibody conjugate.
[0002]
[Prior art]
Conventionally, EIA uses an enzyme labeling reagent in which an antibody is bound to an enzyme. This reagent is a conjugate of one antibody molecule and one enzyme molecule.
[0003]
In recent years, in response to the demand for higher sensitivity in immunoassay, it is necessary to measure a conjugate in which one enzyme molecule is bound to one antibody molecule as a conjugate in which two or more enzymes are bound to one antibody molecule. Is being done. Examples of a conjugate obtained by binding an enzyme to this antibody include a conjugate obtained by reacting an antibody with a polymerized enzyme, and a conjugate obtained by reacting two or more molecules of an enzyme using a plurality of thiol groups in the antibody. Have been.
[0004]
[Problems to be solved by the invention]
However, the method using a polymerized enzyme could not control the reaction between the polymerized enzyme and the antibody. Further, a conjugate utilizing a plurality of thiol groups in an antibody can be applied to some antibodies having a plurality of thiol groups, but cannot be applied to all antibodies.
[0005]
[Means for Solving the Problems]
Thus, the present inventors have conducted intensive studies, and as a result, have found an enzyme-antibody conjugate obtained by further binding an enzyme to an enzyme to which an antibody has been bound, and have completed the present invention.
[0006]
The present invention also provides an EIA kit incorporating the enzyme-antibody conjugate.
[0007]
The conjugate of the present invention is produced by further reacting an enzyme with an antibody-bound enzyme. The enzyme to which the antibody, which is the raw material, is bound is characterized in that a functional group of the enzyme and a functional group of an antibody fragment such as an IgG class antibody, Fab ′, F (ab ′) _{2 and the} like are glutaraldehyde, periodic acid, maleimide, pyridyl disulfide. And a reaction in the presence of a crosslinking reagent such as carbodiimide or a condensing reagent.
[0008]
Examples of the enzyme that can be used in the present invention include alkaline phosphatase, peroxidase, β-galactosidase, and the like.
[0009]
In order to obtain an enzyme-antibody conjugate, a reactive functional group is introduced into the enzyme to be reacted, and the reaction is carried out in the presence of the same crosslinking reagent or condensation reagent as described above. The number of enzymes to be bound can be determined by controlling the amount of the crosslinking reagent or condensation reagent. Further, the obtained enzyme-antibody conjugate is purified by a fractionation method based on a molecular weight such as a gel filtration method, and the number of bound enzymes can be determined by analyzing the molecular weight.
[0010]
The enzyme-antibody conjugate of the present invention constitutes a kit in combination with an EIA reagent such as an antigen or antibody-binding solid phase corresponding to the substance to be measured and a substrate corresponding to the enzyme, and is used for measurement of the antigen or antibody in the sample. be able to. The EIA may be any of a well-known one-step method, a one-step delay method, a sandwich method such as a two-step method, a competitive method, and the like. The EIA forms an immunocomplex on a solid phase by an immunoreaction and binds to a chromogenic substrate. The reaction can be performed by reacting a substrate such as a fluorescent substrate or a luminescent substrate, and measuring a signal generated as a result. From the signal measurement result, the enzyme activity can be determined and the measurement target can be measured.
[0011]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples.
[0012]
Example 1 Introduction of a thiol group into alkaline phosphatase Alkaline phosphatase (ALP; manufactured by Oriental) 32 mg was passed through a gel filtration column (manufactured by Pharmacia; PD-10) to give a 0.1 M phosphate buffer (pH 7.0). Was replaced with a buffer. 0.31 mg of iminothiolane (manufactured by Nacalai Tesque) was added, and the mixture was allowed to stand at room temperature for 1 hour to introduce a thiol group. This was passed through a gel filtration column (manufactured by Pharmacia; PD-10) to remove unreacted iminothiolane to obtain a thiol group-introduced ALP.
[0013]
Example 2 Preparation of Enzyme-Antibody Conjugate ALP-labeled anti-HBs antibody (1: 1) produced by the maleimide method and thiol group-introduced ALP produced in Example 1 were mixed at a molecular weight of 1: 2 and left for 1 hour. did. As a result, the maleimide group present on the ALP and the thiol group of the ALP reacted to obtain an enzyme-antibody conjugate. This is pooled with a gel filtration column (Pharmacia; Superose 200) to fractions of ALP: Fab '= 3: 1 having a molecular weight of around 500,000, and is conjugated with an enzyme-antibody (hereinafter referred to as ALP-labeled anti-HBs antibody (3: 1)). Got.
[0014]
Example 3 Preparation of Anti-HBs Polyclonal Antibody-Binding Particles 300 mg of ferrite particles (manufactured by Nippon Paint Co., Ltd.) were washed three times with a 0.4% sodium chloride solution, suspended in 1.5 ml of purified water, and then EDC (manufactured by Nacalai Tesque, Inc.) ) Add 1.5 ml of 40 mg / ml aqueous solution and leave at room temperature for 5 minutes. Add 15 mg / 7.5 ml of the anti-HBs polyclonal antibody, and stir at 25 ° C. for 1 hour end-over-end. After washing the particles, 20 ml of post-coat buffer was added and stirred overnight at 37 ° C end-over-end. After washing, the particle concentration was adjusted to 1.5% to obtain anti-HBs polyclonal antibody-bound particles.
[0015]
Example 4 Measurement of HBs Antigen Using ALP-Labeled Anti-HBs Antibody (1: 1) and ALP-Labeled Anti-HBs Antibody (3: 1) 0.5 μg / ALP-Labeled Anti-HBs Antibody (1: 1) as a Raw Material of Example 2 The HBs antigen subtype ad (concentration: 0.05 to 0.4 ng / ml) was measured using 0.5 ml / ml or the ALP-labeled anti-HBs antibody (3: 1) 0.5 μg / ml prepared in Example 2. . Using the anti-HBs polyclonal antibody-bound particles prepared in Example 3, measurement was performed by a two-step sandwich method using a chemiluminescence enzyme immunoassay device (Lumipulse f; manufactured by Fujirebio), and the amount of luminescence for 5 seconds was measured. . The result is shown in FIG.
[0016]
Similarly, measurement was performed for the HBs antigen subtype ay (concentration: 0.05 to 0.4 ng / ml). The result is shown in FIG.
[0017]
【The invention's effect】
The enzyme-antibody conjugate of the present invention is a compound in which a predetermined number of enzymes are bound to an antibody to increase the specific activity of the enzyme. Therefore, in the EIA using the enzyme-antibody conjugate, a trace substance can be measured with high sensitivity.
[Brief description of the drawings]
FIG. 1 is a diagram showing the results when the HBs antigen subtype ad at each concentration was measured with an ALP-labeled anti-HBs antibody (1: 1) and an ALP-labeled anti-HBs antibody (3: 1).
FIG. 2 is a diagram showing the results when the HBs antigen subtype ay at each concentration was measured with an ALP-labeled anti-HBs antibody (1: 1) and an ALP-labeled anti-HBs antibody (3: 1).

Claims (4)

A method for producing an enzyme-antibody conjugate, comprising binding a desired number of enzymes to one molecule of enzyme to which one molecule of antibody has been bound,
(A) producing an enzyme-labeled antibody comprising one molecule of an antibody and one molecule of an enzyme,
(B) further binding an enzyme to the enzyme in the labeled antibody,
(C) Purifying the enzyme-antibody conjugate to which a predetermined number of enzymes are bound by a fractionation method
Manufacturing method comprising: (A ') reacting one molecule of the antibody with one molecule of the enzyme into which a plurality of first functional groups have been introduced to produce an enzyme-labeled antibody comprising one molecule of the antibody and one molecule of the enzyme;
(B ′) reacting the first functional group present in the enzyme in the labeled antibody with the enzyme into which the second functional group has been introduced, thereby further binding the enzyme to the enzyme in the labeled antibody;
(C) Purifying the enzyme-antibody conjugate to which a predetermined number of enzymes are bound by a fractionation method
The method according to claim 1, comprising: The method according to any one of claims 1 to 2, wherein the antibody is a Fab 'fragment of IgG. The method according to any one of claims 1 to 3, wherein the enzyme is alkaline phosphatase.

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