JPH07140146A - Stable peroxidase composition and stable antibody composition - Google Patents

Abstract

PURPOSE:To obtain a stabilized composition in which the activity of peroxidase can be sustained at the time of long term preservation, room temperature preservation, and freezing/drying by adding a composition comprising peroxidase, hydroxyphenyl-alkoxy silane. CONSTITUTION:Peroxidase is stabilized by adding a peroxidase composition comprising peroxidase and hydroxyphenyl-alkoxy silane or hydroxyphenyl-alkoxy silane. On the other hand, antibody is stabilized by adding an antibody composition comprising an antibody and hydroxyphenyl-alkoxy silane or hydroxyphenyl- alkoxy silane. The peroxidase may be derived from vegetable, animal or microorganism and includes hose radish peroxydase, lactperoxydase, etc. The peroxydase may be a simple substance or may be bonded to other useful substance.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to storage stabilization of peroxidase or antibody used for immunoassay.

[0002]

2. Description of the Related Art In recent years, measuring methods using immunoreactions and enzyme reactions have been widely used in the fields of clinical examination and diagnosis.

Peroxidase is an enzyme that catalyzes a reaction of oxidizing various substances by using hydrogen peroxide as a hydrogen acceptor, and can be used for a color reaction for oxidizing a chromogen and converting it into a dye, and therefore, for example, a target organism. In recent years, its importance has been increasing, for example, it is used for measurement in which hydrogen peroxide produced by reacting a component with a corresponding oxidase is detected by an enzymatic reaction, or as a labeling enzyme in an enzyme immunoassay. However, peroxidase has poor stability in a solution state, and it is extremely difficult to maintain its activity during long-term storage at room temperature. It is also known that the peroxidase activity is considerably reduced even when freeze-dried.

It is also known that the antibodies used for immunoassays decrease in activity over time, especially at low concentrations.
Further, this tendency becomes more remarkable as the temperature of the solution increases.

Therefore, it is difficult to store peroxidase and antibody for a long period of time, and it is insufficient from the viewpoint of quality assurance to use them as measuring reagents or diagnostic reagents, and there is a strong demand for their stabilization. It was Also, especially
In the case of a diagnostic reagent that uses a peroxidase-labeled antibody in which a peroxidase and an antibody are bound, it is most preferable to simultaneously stabilize the peroxidase and the antibody by the same method so that the stabilization of one does not inhibit the activity of the other. It was strongly desired.

A method known in the prior art as an attempt to stabilize peroxidase or antibody is disclosed in, for example, JP-A-59-2.
A method of adding a polyalkylene glycol described in 5683, a method of adding a sugar or a sugar alcohol described in JP-A-60-188067, or a benzene derivative having simultaneously a hydroxyl group and an amide group described in JP-A-2-72982. Although a method of doing so is known, it is still insufficient for stabilization, and its improvement is being sought.

[0007]

In view of the above situation, a first object of the present invention is to provide a peroxidase composition containing a stabilized peroxidase, and a second object thereof is to stabilize the composition. It is intended to provide an antibody composition containing a modified antibody.

[0008]

Means for Solving the Problems As a result of intensive investigations by the present inventors on a method for suppressing the activity reduction of peroxidase and antibody, a peroxidase composition comprising peroxidase and hydroxyphenyl-alkoxysilane, and hydroxyphenyl-alkoxysilane. It was found that this was achieved by a method of stabilizing peroxidase by adding

As another embodiment, the above object is achieved by an antibody composition containing an antibody and hydroxyphenyl-alkoxysilane or a method for stabilizing an antibody by adding hydroxyphenyl-alkoxysilane.

The hydroxyphenyl-alkoxysilane is preferably a compound represented by the following general formula (1), and more preferably p- or o-hydroxyphenyl-alkoxysilane.

[0011]

[Chemical 3]

In the formula, R ₁ , R ₂ and R ₃ represent a lower alkyl group having 5 or less carbon atoms, each of which may have a substituent.
Furthermore, the alkyl group is preferably a methyl group or an ethyl group.

Further, the phenyl group may have a substituent.

The compound represented by the general formula (1) is, for example, o-hydroxyphenyltrimethoxysilane o-hydroxyphenyltriethoxysilane o-hydroxyphenyltributoxysilane p-hydroxyphenyltrimethoxysilane p-hydroxyphenyltriethoxysilane. p-hydroxyphenyltributoxysilane o-hydroxyphenyldimethoxyethoxysilane o-hydroxyphenyldiethoxymethoxysilane o-hydroxyphenyldimethoxybutoxysilane p-hydroxyphenyldimethoxyethoxysilane p-hydroxyphenyldiethoxymethoxysilane p-hydroxyphenyldimethoxybutoxysilane Silanes such as o-hydroxyphenyltripropoxysilane and p-hydroxyphenyltripropoxysilane are preferred, and p- or o-hydroxy is preferred. It is E sulfonyl trimethoxysilane. These compounds have 0.03
mM concentration or higher, preferably 0.3 mM or higher, more preferably 1 to
3 mM concentration is added.

The peroxidase according to the present invention may be derived from any of plant origin, animal origin, microorganism origin and the like. Examples thereof are horseradish peroxidase, lactoperoxidase, glutathione peroxidase, myeloperoxidase, cytochrome C.
Examples thereof include peroxidase, but horseradish peroxidase is particularly preferable.

The peroxidase according to the present invention may be a non-bonding simple substance, or may be a conjugate with another useful substance. It may also be a complex composed of peroxidase and anti-peroxidase antibody.

The peroxidase conjugate according to the present invention is not particularly limited as long as it is a conjugate of peroxidase with other useful substances, but a conjugate with an immunoactive substance is preferable. Examples of the immunologically active substance include antigens, antibodies, haptens, protein A and the like. Examples of the antigen or hapten include proteins, sugars, lipids, glycoconjugates, polysaccharides, nucleic acids, enzymes, hormones, haptens, antibiotics, bacterial antigens, virus antigens, cancer antigens, avidin, biotin and the like. The antibody according to the second aspect of the present invention or the antibody capable of binding to peroxidase is prepared by immunizing a mammal such as rabbit, goat, mouse or the like or a bird such as chicken by immunizing the antigen or hapten by a conventionally known method. Antibodies obtained from serum, and monoclonal antibodies obtained from plasma cells transformed by hybridomas and viruses produced by cell fusion and the like, and also fragments thereof, such as Fab, F (ab ') ² etc. included.

The antibody according to the second aspect of the present invention may be bound to a substance useful for measurement using the same, for example, a labeling substance or an immunoactive substance. Examples of the labeling substance include radioisotopes, peroxidase, alkaline phosphatase, enzymes such as β-galactosidase, fluorescent substances, luminescent substances, biotin, avidin, chelates, metal colloids, and toxins.
Examples include antibodies and haptens.

The binding between peroxidase and a useful substance or the binding between an antibody and a useful substance may be a physical bond such as adsorption, an ionic bond, a chemical bond such as a covalent bond, and a covalent bond is preferred. As a method of binding, various known methods may be used, for example, a method of binding an amino group and an amino group using glutaraldehyde as a cross-linking agent or an aldehyde group formed by treatment of a sugar chain of peroxidase with a periodic acid. Examples include a method of binding an amino group of a substance.

In order to enhance the effect of the composition of the present invention, animal serum such as horse serum, bovine serum, sheep serum, rabbit serum,
Human serum or serum components, for example, bovine albumin, human albumin, etc. are preferably contained. In the case of a solution composition, the content is 1 to 50 ( ^w / _v )% for serum and 0.1 to 10 for serum components. 5 ( ^W / _V )% is preferred.

If necessary, other substances, for example, proteins such as gelatin, ovalbumin, casein, etc., water-soluble polymers such as polyalkylene glycol, saccharides such as sucrose, preservatives such as thimerosal, etc. may be contained. Is also good.

The composition of the present invention may contain any buffering agent and salts such as sodium chloride. As the buffering agent, a commonly used buffer such as Tris-based, phosphoric acid-based, carbonic acid-based, acetic acid-based can be used. When the pH in the aqueous solution is a peroxidase composition, it is 4 to 9, and when it is an antibody composition, it is 3 to 11 Is preferable.

The order of adding each component at the time of preparing the solution composition is not particularly limited.

The peroxidase activity is measured by measuring the peroxidase composition of the present invention with hydrogen peroxide and a substrate such as
It is performed by contacting a chromogen such as o-phenylenediamine and analyzing the degree of the enzymatic reaction. When a chromogen is used, the analysis is performed by measuring the absorbance of the produced dye.

A general immunoassay can be used to measure the antibody activity. For example, agglutination method, competitive method, sandwich method, ELISA method, etc. are possible. One example is a method of reacting a test labeled antibody with a corresponding antigen immobilized on a carrier and measuring the amount of the label bound to the carrier.

The stability of the activity of peroxidase or antibody can be evaluated, for example, by examining the change in activity over time or at 4 ° C.
It can be carried out by comparing the activity between the case of holding and the case of holding at 37 ° C.

The peroxidase composition of the present invention retains the activity of peroxidase during long-term storage, room temperature storage and freeze-drying. Also, the antibody composition of the present invention is similarly excellent in storage stability. The substituted benzene ring according to the present invention is useful as a stabilizer for both peroxidase and antibody, and thus is particularly useful as a stabilizer for peroxidase-labeled antibody. The immunological measurement reagent to which the composition of the present invention is applied has excellent stability over time, and thus has extremely high utility in the fields of clinical examination and diagnosis.

[0028]

EXAMPLES The present invention will be described in more detail below with reference to examples.

Example 1 25 mM phosphate buffer containing 100 mM sodium chloride (pH 7.
4: 1 ( ^W / _V )% bovine serum albumin (hereinafter abbreviated as BSA) and 1 μg / ml horseradish peroxidase in p) and p-hydroxyphenyltrimethoxysilane or o as a stabilizer -Hydroxyphenyltrimethoxysilane was dissolved to prepare a peroxidase solution. These solutions were kept at 4 ° C or 37 ° C for 1 week. For comparison, polyethylene glycol or sucrose was added as a stabilizer and those without a stabilizer were also prepared at the same time.

The retained peroxidase solution was diluted 10 times with PBS and its peroxidase activity was measured as follows. That is, 2 μl of the diluted solution was added to 500 μl of a citric acid-phosphate buffer solution (pH 5.0) containing o-phenylenediamine at a concentration of 2 mg / ml and 0.02% hydrogen peroxide, and the mixture was reacted at 37 ° C. for 30 minutes. After that, 2 ml of 1N sulfuric acid was added, and the absorbance at 492 nm was measured.

100% of peroxidase activity immediately after preparation
Table 1 shows the peroxidase activity of the peroxidase solution retained for 1 week.

[0032]

[Table 1]

It can be seen from Table 1 that the sample to which the compound of the present invention is added stably retains the peroxidase activity at 37 ° C. for at least 1 week.

Example 2 Anti-GAT monoclonal antibody MAb8628 (Japanese Patent Laid-Open No. 3590/1993)
93) was labeled with horseradish peroxidase by the method of P108, “Enzyme-linked immunosorbent assay (3rd edition) Medical Shoin 1987” edited by Eiji Ishikawa to prepare a labeled antibody.

This labeled antibody was added to PBS containing 1% BSA.
Was diluted to 200 ng / ml, and the hydroxyphenyl-alkoxysilane shown in Table 2 was dissolved to prepare a labeled antibody solution. These labeled antibody solutions should be 4 ℃ or 37
Hold at 1 ° C for 1 week. For comparison, those with polyethylene glycol or sucrose as stabilizers and those without stabilizers were prepared at the same time.

Using this retained labeled antibody solution as a secondary antibody, peroxidase stability and antibody stability were calculated as follows.

Polystyrene beads and anti-GAT monoclonal antibody MAb8513 at a concentration of 10 μg / ml (JP-A-3-2590)
93) in PBS overnight at 4 ° C, then 1% BS
Antibody-immobilized beads were prepared by blocking with A / PBS at 37 ° C. overnight. 50 μl of a sample (serum of cancer patient) having a GAT concentration of about 50 U / ml was diluted with 200 μl of 0.01% Tween-20-containing PBS, 1 prepared antibody-immobilized bead was added, and the mixture was reacted at 45 ° C. for 1 hour. After washing three times with PBS, 250 μl of the above-mentioned labeled antibody solution was added, and the mixture was reacted at 20 ° C. for 2 hours. PB
After washing 4 times with S, 300 μl of citric acid-phosphate buffer solution (pH 5.0) containing o-phenylenediamine at a concentration of 3 mg / ml and 0.02% hydrogen peroxide was added, and color reaction was performed at room temperature for 30 minutes. It was Thereafter, 1 ml of 1N sulfuric acid was added, and the absorbance at 492 nm was measured.

Using a labeled antibody kept at 4 ° C., a known amount of G
Using a calibration curve obtained by similarly measuring a standard product having an AT concentration, the GAT concentration when various labeled antibodies were used was calculated, and the temperature was 37 ° C against the GAT concentration when the labeled antibody retained at 4 ° C was used. The ratio of GAT concentration in the case of retention was defined as the labeled antibody stability.

Further, the peroxidase activity of the labeled antibody was measured in the same manner as in Example 1, and the peroxidase activity at 37 ° C. relative to the activity at 4 ° C.
The activity of retention was peroxidase stability.

The ratio of labeled antibody stability to peroxidase stability was defined as antibody stability, and the results are shown in Table 2.

[0041]

[Table 2]

From Table 2, it can be seen that the sample to which the compound of the present invention is added stably retains the peroxidase activity and the antibody activity at 37 ° C. for at least 1 week.

Example 3 The labeled antibody prepared in Example 2 was added to PB containing 1% BSA.
The solution was diluted with S to 200 ng / ml, the stabilizers shown in Table 3 were dissolved, and horse serum and sucrose were further added to prepare a labeled antibody solution. 0.5 ml of each of these labeled antibody solutions was dispensed into vials, frozen at -40 ° C, and then freeze-dried under reduced pressure. After the freeze-drying was completed, 0.5 ml of pure water was added to obtain the peroxidase activity and GAT concentration in the same manner as in Example 2. The stability was calculated as the ratio of the case of non-lyophilized to the case of non-lyophilized, and the results are shown in Table 3.

[0044]

[Table 3]

From Table 3, the sample to which the compound of the present invention is added controls the decrease in peroxidase activity due to the freeze-drying operation. When horse serum or sucrose coexists, the effect is remarkable. It also controls the decrease in antibody activity due to the freeze-drying operation.

[0046]

According to the present invention, a peroxidase composition containing a stabilized peroxidase and an antibody composition containing a stabilized antibody were obtained.

Claims (6)

[Claims] 1. A peroxidase composition comprising peroxidase and hydroxyphenyl-alkoxysilane. 2. A method for stabilizing peroxidase by adding hydroxyphenyl-alkoxysilane. 3. The peroxidase composition according to claim 1 or the method for stabilizing peroxidase according to claim 2, wherein the hydroxyphenyl-alkoxysilane is a compound represented by the following general formula (1). [Chemical 1] In the formula, R ₁ , R ₂ and R ₃ represent a lower alkyl group having 5 or less carbon atoms, and each may have a substituent. 4. An antibody composition comprising an antibody and hydroxyphenyl-alkoxysilane. 5. A method for stabilizing an antibody by adding hydroxyphenyl-alkoxysilane. 6. The antibody composition according to claim 4 or the method for stabilizing an antibody according to claim 5, wherein the hydroxyphenyl-alkoxysilane is a compound represented by the following general formula (1). [Chemical 2] In the formula, R ₁ , R ₂ and R ₃ represent a lower alkyl group having 5 or less carbon atoms, and each may have a substituent.