JP2520465B2 - Multi-labeled antibody - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION a. Field of Industrial Application The present invention relates to a multi-labeled antibody used in an immunoassay.

b. Problems to be Solved by Prior Art and Invention As one of the methods for measuring a minute amount of substances present in a living body, for example, immunoassays such as enzyme immunoassay (EIA) and radioimmunoassay (RIA) are clinical tests. Occupy an important position in. In particular, EIA has a high specificity and can be measured with high sensitivity without using radioactive substances, so that it has been rapidly developed in recent years. The requirements for the labeled antibody used in EIA for high-sensitivity measurement include high specificity, high affinity, high enzyme activity, and low nonspecific adsorption to the carrier. Among them, the enzyme activity is the most directly involved requirement for high-sensitivity measurement, and in order to obtain high enzyme activity, the labeled antibody is a multi-labeled antibody having a plurality of labels per antibody molecule. desirable.

Conventionally, such a multi-labeled antibody has been (1) the glutaraldehyde method or the periodate method (J. Histochemistry).
and Cytochemistry 22 , 1084, 1974), and (2) a labeled antibody obtained by labeling with a prepolymerized enzyme (JP-A-61-73067). However, in the case of the multi-labeled antibody of (1), since it is a heterogeneous labeled antibody containing a polymer, non-specific adsorption, which is one of the requirements for the labeled antibody, becomes large, and as a result, It was not suitable for high sensitivity measurement. Further, in the case of the (2) multi-labeled antibody, macromolecules were produced by the polymerized enzyme, and as a result, non-specific adsorption increased as in (1), which was unsuitable for highly sensitive measurement.

On the other hand, JP-A-58-149700 discloses a labeled antibody having an antibody: enzyme binding ratio of 1: 1 which is obtained by labeling an enzyme through the SH group and maleimide group of the Fab ′ fragment portion of the antibody. In this case, it is described that the amount of polymer of the labeled antibody produced is small, and therefore the non-specific adsorption is small, and high-sensitivity measurement is possible. However, even with this labeled antibody,
Since only one molecule of the enzyme is bound to the antibody, it cannot be said that it is practically sufficient for high-sensitivity measurement.

c. Means for Solving the Problems It is known that the hinge portion of an antibody has 1 to 5 disulfide bonds, and the number thereof varies depending on the species of the animal and the class or subclass of the antibody. Savasti et al. (Bio
chem.J., 126 , 837, 1972) has 2,4,5,2 disulfide bonds in human IgG ₁ , IgG ₂ , IgG ₃ and IgG ₄ , respectively, and mouse IgG ₁ , IgG _2a and IgG _3b It is reported that there are 3,3,4 disulfide bonds. The present inventors have paid attention to the fact that two or more SH groups are present in the Fab ′ fragment, and as a result of diligent research, as a result, a labeled antibody in which an average of 1.5 molecules or more of the labeling substance is labeled per Fab ′ fragment molecule is specific. sex,
The present invention has been achieved by finding that it has excellent affinity, high activity, and little nonspecific adsorption.

That is, the present invention provides a multi-labeled antibody in which a labeling substance is bonded on average to 1.5 molecules or more per Fab 'fragment molecule (hereinafter referred to as an average labeled amount of 1.5) through a sulfur atom of a Fab' fragment of the antibody, and such a multi-labeled antibody. Of the present invention, an immunological measurement method using such a multi-labeled antibody, and an immunological measurement kit containing the multi-labeled antibody as a labeled antibody reagent.

In the multi-labeled antibody of the present invention, examples of the antibody include a polyclonal antibody and a monoclonal antibody, but any animal-derived antibody may be used as long as it has two or more disulfide bonds in the hinge portion.

The Fab ′ fragment of the present invention can be prepared by a known method, for example, the method of Nisonov et al. (Arch. Bio.chem. Biophys. 89 , 230, 1).
960), and the method of Palm et al. (J. Immunol. 131 , 2895, 1983), the antibody is digested with pepsin to obtain an F (ab ′) ₂ fragment, which is then reduced with mercaptoethylamine or the like. .

Examples of the labeling substance of the present invention include enzymes, fluorescent substances, luminescent substances and the like. Examples of enzymes include lysozyme,
Malate dehydrogenase, glucose-6-phosphate dehydrogenase, peroxidase, glucose oxidase, alkaline phosphatase, beta-galactosidase, etc .; as fluorescent substances, for example, fluorescein, rhodamine, umbelliferone, lanthanide chelate, etc .; as luminescent substances Examples include luminol, acrilydium ester, luciferin and the like. Of these, alkaline phosphatase and peroxidase are preferable.

The maleimidation of the labeling substance is a known method, for example, succinimidyl 4- (N-maleimidomethyl) cyclohexane carbonate (SMCC), sulfosuccinimidyl 4-.
(N-maleimidomethyl) cyclohexane carbonate (sulfo SMCC), succinimidyl-metamaleimidobenzoate (MBS), succinimidyl 6-maleimidohexanoate (EMCS), etc. (edited by Eiji Ishikawa "Enzyme Immunoassay", Medical Institute) You can

The multi-labeled antibody of the present invention is produced by reacting the Fab ′ fragment of the antibody with a maleimidated labeling substance.

The reaction between the Fab ′ fragment and the maleimidated labeling substance is generally carried out by the method described above (“Enzyme Immunoassay” edited by Eiji Ishikawa, Medical Institute), for example, reaction temperature 4 to 30 ° C., Fab ′ fragment and maleimidated labeling substance in moles. The ratio can be 1: 1 and the reaction time is 20 hours. In this case, a mixture of one molecule of the labeling substance labeled with one molecule of the Fab ′ fragment and a labeled antibody labeled with two or more molecules can be obtained. In such a reaction, the reaction temperature is set to 25 ° C. or higher as a reaction condition, or the molar ratio of the Fab ′ fragment to the maleimidated labeling substance is 1: 4 or more, and the reaction time is 24 hours or more, the yield is high and the labeled antibody is high. Is preferred, because it can be obtained. The multi-labeled antibody thus obtained can be separated and purified from the reaction solution by using, for example, gel chromatography. For example, the carrier used in gel chromatography is Ultrogel AcA44 (LKB
Company), TSK GelG-3000SW (Tosoh),
HPLC using DIOL-200 (Yamamura Chemical Laboratory) column etc.
Is preferred because it is superior in separation. In this case, the single-molecule labeled antibody and the multi-labeled antibody of two or more molecules may not always be separated cleanly, so an antibody containing a small amount of the single-molecule labeled antibody is also within the scope of the present invention.

The number of labels of the labeling substance in the separated and purified multi-labeled antibody can be determined by measuring the molecular weight, the absorbance, the fluorescence intensity, the enzyme activity and the like. For example, when the labeling substance is peroxidase, the number of labels can be determined by measuring the absorbance at 280 nm derived from the Fab ′ fragment and peroxidase and the absorbance at 403 nm derived from peroxidase. (E. Ishikawa e
tal, J. Immunoassay., 4 , 209-327, (1983), p243) If the average labeled amount of the multi-labeled antibody of the present invention is 1.5 or more,
The high sensitivity of the measurement of the present invention can be achieved. Below 1.5, the amount of enzyme bound is small and the activity is low, so it is not suitable for highly sensitive measurement. It is preferably 1.8 or more. The upper limit is not particularly limited, but is preferably up to 5.

Any buffer may be used as the buffer for the reaction as long as it does not inhibit the reaction, but 0.01 to 0.5M phosphate buffer pH
5.5 to 8.0 is preferably used, and it is better to include about 1 to 10 mmol of EDTA for stabilizing the Fab ′ fragment.

Since the multi-labeled antibody of the present invention has no Fc portion, non-specific adsorption to the carrier is small, and since 1.5 molecules or more of the labeling substance is bound, it is useful for highly sensitive measurement. In addition, since it has no Fc portion, it is a useful reagent for immunoassays, which also has the property of being less affected by rheumatoid factors.

The multi-labeled antibody of the present invention can be widely used in an immunoassay using a labeled antibody. Examples of such immunological assay methods include hormones, peptides, and other biological components such as plasmin / α2 plasmin inhibitor complex, protein C, tissue plasminogen activator / plasminogen activator inhibitor complex, thrombin / Examples of the substances to be measured include antithrombin III complex and protein S. Furthermore, it can be used in the field of clinical tests such as tissue staining of estrogen receptor and progesterone receptor, and food tests using immunoassay. The present invention will be described in detail below with reference to examples and examples.

Reference Example 1 <Preparation of Fab ′ fragment of mouse monoclonal antibody> Mouse anti-human tissue plasminogen activator antibody (JTA-1) in 0.01M PBS solution (1mg / ml) was added to 10M in 1M.
Citrate buffer (pH 3.5) was added little by little to pH 3.75. To this solution, pepsin in citrate buffer (1 mg / m
l) was added in an amount of 0.2 ml and incubated at 37 ° C. for 1 hour. After adjusting the pH of the reaction solution to approximately 6.0 with 1N NaOH, it was concentrated to approximately 1 ml using an ultrafiltration device (Filtron Omega Cell: Fuji Filter Co.) and then subjected to HPLC (column TSK Gel G-3000S).
W (Tosoh), eluent 5M EDTA-containing 0.1M phosphorus buffer pH 6.
0) to give 5.9 mg of F (ab ') ₂ fragment.

To 2.0 ml of a phosphate buffer solution containing 5.9 mg of this F (ab ') ₂ fragment, 0.2 ml of a 0.1 M 2-mercaptoethylamine phosphate buffer solution was added, and the mixture was incubated at 37 ° C for about 90 minutes. HPLC (above conditions) yielded 5.3 mg of Fab 'fragment.

The mouse anti-human tissue plasminogen activator antibody (JTA-1) is described in Japanese Patent Application No. 63-81367 (invention title "monoclonal antibody against plasminogen activator", filing date April 4, 1988). The one obtained by the described method was used. Specifically, it is one of four monoclonal antibodies obtained by the conventional method by the method of Khler and Milstein using purified human tissue plasminogen activator (tPA) as an antigen,
Recognizes the heavy chain of tPA or tPA-plasminogen activator inhibitor (PAI) complex,
_It has the properties of ₁ and k.

Reference Example 2 <Preparation of Maleimidated Peroxidase> Peroxidase (Toyobo) 40 mg 0.1 M phosphate buffer
0.5 ml of a dimethylformamide solution of succinimidyl 4- (N-maleimidomethyl) cyclohexane carbonate (33.4 mg / ml) was added to pH 7.06 ml, and the mixture was stirred at 30 ° C. for 1 hour. After centrifuging the reaction mixture, Sephadex G-25
Gel filtration was performed using a column (eluent 0.1 M phosphate buffer pH 7.0) to obtain 39 mg of maleimidated peroxidase.

Example 1 <Preparation of Fab ′ fragment / peroxidase 1: 2 binding enzyme labeled antibody> Fab ′ fragment (1.0 mg) obtained in Reference Example 1 and maleimidated peroxidase (3.2 m obtained in Reference Example 2)
g) was reacted at 25 ° C for 24 hours and separated and purified by HPLC with TSK Gel G-3000SW (eluent 0.01M PBS).
0.35 mg of a labeled antibody having a molecular weight of about 130,000 was obtained. Based on the absorbance at 280 nm and 403 nm of the obtained labeled antibody, the binding ratio of Fab ′ fragment to peroxidase was 1: 2. Fab ′ below
-(HRP) ₂ is abbreviated.

The reaction yield of this labeled antibody over time was as shown in FIG.

As shown in Table 1, when the Fab ′ fragment and the maleimidated peroxidase were changed in molar ratio and reaction temperature and reacted in the same manner as above, the labeled antibody yield was
It was as in the table.

Example 2 <Preparation of Fab ′ fragment / peroxidase 1: 1.5-conjugated enzyme-labeled antibody> The Fab ′ fragment and maleimidated peroxidase were reacted under the same conditions as in Example 1. Ultrogel AcA44
(LKB) column (separated with 0.1 M phosphate buffer as an eluent, the binding ratio of Fab ′ fragment to peroxidase was 1.5 based on the absorbances of the obtained labeled antibody at 280 nm and 403 nm.
Met. Hereinafter, it is abbreviated as Fab ′-(HRP) _1.5 .

Comparative Example 1 Preparation of Fab ′ fragment / peroxidase 1: 1 binding enzyme-labeled antibody Fab ′ fragment (1.0 mg) obtained in Reference Example 1 and maleimidated peroxidase (0.8 m) obtained in Reference Example 2
g) was reacted at 4 ° C for 24 hours and purified by HPLC.
0.61 mg (as Fab ') of a labeled antibody having a molecular weight of about 90,000 in which the b'fragment and the peroxidase were bonded one-to-one was obtained.

 Hereinafter, it is abbreviated as Fab′-HRP.

Example 3 <Application to Tissue Plasminogen Activator / Plasminogen Activator Inhibitor Complex Measurement System> a. Preparation of immobilized antibody Polystyrene beads (Sekisui Chemical 6.35 mmφ # 80) were used as mouse anti-human plasminogen activator. Inhibitor antibody (JTI-4) concentration 20 μg / ml in 0.1M phosphate buffer (p
H9.0) and the mixture was allowed to stand at 4 ° C. for 20 hours.

After washing the beads 3 times with 0.01 M PBS, 1% BSA-P
After leaving it in the BS solution at room temperature for 2 hours, it was washed again with 0.01 M PBS three times to obtain a fixed antibody.

The mouse anti-human plasminogen activator inhibitor antibody (JTI-4) is described in Japanese Patent Application No. 63-81366 (the title of the invention "monoclonal antibody against plasminogen activator inhibitor", filing date: Showa
The method described by van Mourik JA et al. (J. Biol. Chem., 25) .
9 , 14914-14921 (1984)), which is one of four kinds of antibodies obtained by a conventional method using a plasminogen activator inhibitor (PAI) obtained according to It has the property of binding to both SDS-activated PAI and tPA-PAI complex and having no PAI-neutralizing activity in subclass IgG ₁ , k.

b. Preparation of calibration curve Human tissue plasminogen activator-plasminogen activator inhibitor complex was added to 0.01M phosphate-0.5M NaCl buffer containing 0.25% skim milk (p
H7.2) to prepare 25, 12.5, 6.25, 3.125, 0 ng / ml standard solution. 0.3 ml of the standard solution and fixed beads were placed in a small examiner and incubated at 37 ° C for 2 hours. Next, 0.01M PBS-0.05% Tween 20 (pH 7.
Washed 3 times in 2). The peroxidase-labeled anti-human tissue plasminogen activator inhibitor antibody Fab ′ fragment obtained in Examples 1 and 2 and Comparative Example 1 was replaced with Fab ′.
0.01M so that the fragment concentration is 0.6 μg / ml
It was adjusted with PBS-0.05% Tween 20 (pH 7.2), 0.3 ml of the labeled antibody solution was added, and the mixture was incubated at 37 ° C for 30 minutes.
Wash three times with 0.01 M PBS-0.05% Tween 20, and 0.3 ml of substrate solution for peroxidase (2.5 mM H ₂ O ₂ -0.025% 3,
Add 3 ', 5,5' tetramethylbenzidine) and add 37 ℃
Was developed for 30 minutes, the color development was stopped with 1N-sulfuric acid, and the absorbance at 450 nm was measured.

 The calibration curve obtained as a result is shown in FIG.

FIG. 2 shows that the peroxidase-labeled antibody obtained in Example 1 has about 4 times the activity of the peroxidase-labeled antibody obtained in Comparative Example 1. It can be seen that is about twice as active.

[Brief description of drawings]

FIG. 1 shows the reaction yield over time when the Fab ′ fragment was reacted with the maleimidated pperoxidase at a molar ratio of 1: 4,25 ° C. Figure 2 shows Fab'-HRP, Fab '-(HRP) _1.5 and Fab'-(H
3 shows a calibration curve in the measurement of human tissue plasminogen activator plasminogen activator inhibitor (tPA / PAI) complex when RP) ₂ is used as a labeled antibody.

Claims (5)

(57) [Claims] 1. The labeling substance is averaged per molecule of the Fab ′ fragment through the sulfur atom of the Fab ′ fragment of the antibody.
A multi-labeled antibody composed of 1.5 molecules or more. 2. The multi-labeled antibody according to claim 1, wherein the labeling substance is an enzyme, a fluorescent substance, or a luminescent substance. 3. The multi-labeled antibody according to claim 1, wherein the labeling substance is peroxidase. 4. The method for producing a multi-labeled antibody according to claim 1, wherein the Fab ′ fragment of the antibody is reacted with a maleimidated labeling substance. 5. An immunological measurement kit containing the multi-labeled antibody according to claim 1 as a labeled antibody reagent.