JP6992262B2 - Manufacturing method of denaturing antibody measurement reagent - Google Patents

Description

The present invention relates to a method for producing a reagent for measuring the amount of denatured antibody contained in a solution. In particular, the present invention relates to a method for producing the reagent in a freeze-dried form. The present invention also relates to reagents produced by this method.

Antibodies used for pharmaceutical purposes are usually produced by culturing cells capable of expressing the antibody, and then performing purification operations such as centrifugation, filtration, and column chromatography on the obtained culture solution. At that time, the obtained antibody may contain aggregates of the antibody, which are generated by being denatured due to stress during production or the like. Since antibody aggregates may have immunogenicity (Non-Patent Document 1), it is necessary to accurately detect the aggregates, especially when used in pharmaceutical applications.

As a method for detecting an antibody aggregate, a method using size exclusion chromatography or field flow fractionation is known. However, these methods are methods for detecting antibody aggregates based on the size of the protein, not methods based on the structure and properties. Further, as a precise analysis method for the higher-order structure of an antibody, methods using X-ray crystal structure analysis and NMR are known, but these methods are extremely time-consuming methods, such as purity control during production. In recent years, molecules that specifically recognize an antibody that has been modified by stress or the like, regardless of its molecular weight, have been reported (Patent Documents 1 to 3). And non-patent document 2). However, the denatured antibody measurement method using the molecule described in these documents is a method using surface plasmon resonance, and in order to carry out the method, an expensive dedicated device such as Biacore (manufactured by GE Healthcare) is required. do.

Further, when manufacturing / using the measurement reagent, the storage stability of the measurement reagent should be high. As a method for stably storing the reagent, a cryopreservation method, a cold storage method, a freeze-drying method, or the like is generally used. The cryopreservation method is a method in which a reagent is frozen and stored at a temperature below freezing point (preferably −20 ° C. or lower) to prevent deactivation due to protein denaturation and the like, and to enable long-term storage. However, repeated freezing and thawing may denature (inactivate) the protein. The cold storage method is a method in which an additive such as glycerin is added to a reagent and then cooled and stored at a temperature below freezing point (preferably −20 ° C. or lower), similar to the cryopreservation method. However, there are known examples in which the additive affects the enzyme activity contained in the reagent. Further, in the cryopreservation method and the cold storage method, it is always necessary to freeze or cool the reagent below the freezing point, which is a heavy burden when transporting the reagent or storing it in the place of consumption.

On the other hand, the freeze-drying method is a method in which reagents can be stably and for a long period of time by storing them in a refrigerator (about 4 to 10 ° C.) after freeze-drying, and is a method widely used at present as a reagent stabilizing method. .. However, depending on the conditions for freezing and drying, proteins (antigens, antibodies, enzymes, etc.) and nucleic acids (DNA, RNA, etc.) contained in the reagent may be denatured (inactivated. Therefore, the freeze-drying method may be used. When stabilizing the reagent, it is necessary to optimize the components contained in the reagent, freeze-drying conditions (temperature / time), and the like.

International Publication No. 2014/10203 International Publication 2014/115229 International Publication No. 2008/0504030

S. K. Singh, J. et al. Pharm. Sci. , 100,354-387,2011 H. Watanabe et al., J. Mol. Biol. Chem. , 289,3394-3404, 2014

The present invention has been made in view of the problems of the prior art, and is a method for producing a reagent for measuring a denatured antibody contained in a solution, while maintaining the performance of the reagent. It is an object of the present invention to provide a method for producing a reagent capable of long-term storage in a refrigerator.

In order to solve the above problems, the present inventors have reached the present invention as a result of repeated studies on the stabilizer contained in the reagent during freeze-drying.

That is, the first aspect of the present invention is
A method for producing reagents for measuring denatured antibodies.
A step of adding a solution containing a stabilizer to a carrier to which an oligopeptide recognizing a denatured antibody is bound, and then freeze-drying the added carrier and the solution is included.
A method in which the oligopeptide is an oligopeptide containing the amino acid sequence set forth in SEQ ID NO: 1 and the stabilizer is a nonionic surfactant and / or a cationic surfactant.

A second aspect of the present invention is polysorbate 20, 0.005-0.1% (v / v) octylphenol ethoxylate and 0 with a stabilizer of 0.00005 to 0.02% (v / v). The method according to the first aspect, wherein the surfactant is at least one selected from the group consisting of .005 to 0.1% (w / v) cetyltrimethylammonium bromide.

Further, a third aspect of the present invention is 0.00005 to 0.02% (v / v) of polysolvate 20, 0.005 to 0.1% (v / v) of octylphenol ethoxylate and 0.005 to 0. The oligopeptide binding carrier to which a solution containing at least one surfactant selected from the group consisting of 1% (w / v) cetyltrimethylammonium bromide is added is lyophilized and said oligopeptide. The binding carrier contains the amino acid sequence set forth in SEQ ID NO: 1, and is a reagent for measuring the amount of denatured antibody.

Further, a fourth aspect of the present invention is a method for measuring the amount of denatured antibody, which comprises the steps shown in (1) and (2) below.
(1) An oligopeptide-binding carrier by contacting a sample containing a test antibody, the reagent according to the third aspect, and a labeled antibody to which a labeling substance is bound to an antibody that recognizes the test antibody. Step of forming a complex containing the denatured antibody and the labeled antibody (2) A step of measuring the amount of the denatured antibody in the sample by detecting the labeled substance in the complex formed in (2) and (1). ..

In the present invention, a reagent containing a stabilizer is added to a carrier to which an oligopeptide that recognizes a denatured antibody is bound, and then the solution is freeze-dried to recognize the denatured antibody when a modified antibody measurement reagent is produced. The oligopeptide containing the amino acid sequence set forth in SEQ ID NO: 1 is used as the oligopeptide, and the stabilizer is a nonionic surfactant and / or a cationic surfactant. INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a reagent capable of long-term storage in a refrigerator while maintaining the reagent performance before freeze-drying (liquid reagent).

It is a figure which showed the result of Example 1 in the case of freeze-drying treatment without adding a stabilizer. It is a figure which showed the result of Example 1 when 0.005% (v / v) Tween 20 was added as a stabilizer and freeze-dried. It is a figure which showed the result of Example 1 when 0.1 mol / L sucrose was added as a stabilizer and freeze-dried. It is a figure which showed the result of Example 1 when 0.01 mol / L sucrose was added as a stabilizer and freeze-dried. It is a figure which showed the result of Example 1 when 1% (w / v) collagen peptide was added as a stabilizer and freeze-dried. It is a figure which showed the result of Example 1 when 0.1% (w / v) collagen peptide was added as a stabilizer and freeze-dried. It is a figure which showed the result of Example 1 when 10 mmol / L histidine was added as a stabilizer and freeze-dried. It is a figure which showed the result of Example 1 when 1 mmol / L histidine was added as a stabilizer and freeze-dried. It is a figure which showed the result of Example 1 in the case where the stabilizer was not added and freeze-drying treatment was not performed. It is a figure which showed the result of Example 2 when 0.0001% (v / v) Tween 20 was added as a stabilizer and freeze-dried. It is a figure which showed the result of Example 2 when 0.001% (v / v) Tween 20 was added as a stabilizer and freeze-dried. It is a figure which showed the result of Example 2 when 0.01% (v / v) Tween 20 was added as a stabilizer and freeze-dried. It is a figure which showed the result of Example 2 when 0.01% (v / v) Triton X-100 was added as a stabilizer and freeze-dried. It is a figure which showed the result of Example 2 when 0.05% (v / v) Triton X-100 was added as a stabilizer and freeze-dried. It is a figure which showed the result of Example 2 when 0.01% (w / v) cetyltrimethylammonium bromide (CTAB) was added as a stabilizer and freeze-dried. It is a figure which showed the result of Example 2 when 0.05% (w / v) CTAB was added as a stabilizer and freeze-dried. It is a figure which showed the result of Example 2 when 0.1% (w / v) sodium dodecyl sulfate (SDS) was added as a stabilizer and freeze-dried. It is a figure which showed the result of Example 2 when 0.5% (w / v) SDS was added as a stabilizer and freeze-dried. It is a figure which showed the result of Example 2 in the case where 0.1% (w / v) CHAPS (3-[(3-Cholamidotropyl) dimethyllammonio] scavengerfonate) was added as a stabilizer and freeze-dried. It is a figure which showed the result of Example 2 when 0.5% (w / v) CHAPS was added as a stabilizer and freeze-dried.

Hereinafter, the present invention will be described in detail.

<Method of manufacturing reagents for measuring denatured antibodies>
The method for producing a reagent for measuring the amount of a denatured antibody of the present invention is to add a solution containing a stabilizer to a carrier to which an oligopeptide that recognizes the denatured antibody is bound, and then add the carrier. And the step of lyophilizing the solution, the oligopeptide is an oligopeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, and the stabilizer is a nonionic surfactant and / or a cationic surfactant. Is the method.

In the present invention, the denatured antibody means an antibody in which the entire structure or a part of the structure of the antibody is changed by stress due to acid, alkali, heating, freezing and thawing, stirring and the like. The type of the denatured antibody is not particularly limited as long as it has an Fc region, and examples thereof include immunoglobulin isotypes (IgG, IgA, IgD, IgE, IgM, etc.) and subclasses of those isotypes, which are more specific. In particular, human immunoglobulins include nine classes of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. The origin of the denatured antibody is not particularly limited, and examples thereof include antibodies derived from humans, mice, rats, rabbits, goats, etc., and antibodies derived from humans are particularly preferable. Further, the denatured antibody is not limited to such an antibody derived from a single animal species, and may be an antibody to which fragments of an antibody derived from a different animal species are bound. Examples of such antibodies include sequences (variable regions, CDRs, etc.) derived from chimeric antibodies (Fc region of human antibody and antibodies derived from other animals (eg, mouse antibody, rat antibody, rabbit antibody, goat antibody)). Chimera antibody, etc.), which may be a humanized antibody. Further, the antibody according to the present invention may be a monoclonal antibody or a polyclonal antibody.

In the present invention, the oligopeptide that recognizes a denatured antibody is an oligopeptide containing the amino acid sequence set forth in SEQ ID NO: 1. As such an oligopeptide, an oligopeptide consisting only of the amino acid sequence set forth in SEQ ID NO: 1 may be used, or a number such as a linker peptide for binding to a carrier described later on the N-terminal side or the C-terminal side of the oligopeptide. An oligopeptide to which an oligopeptide consisting of several tens of amino acid residues is added may be used. The length of the oligopeptide according to the present invention is not particularly limited as long as the denatured antibody is recognized, but is usually 50 to 25 amino acids, preferably 40 to 25 amino acids, and more preferably 30 to 25 amino acids. It is particularly preferably 25 amino acids (only the amino acid sequence set forth in SEQ ID NO: 1). Further, the oligopeptide is usually composed of natural amino acids linked by peptide bonds, but may contain non-natural amino acids in the present invention, or may be modified. There may be.

In the present invention, the carrier on which the oligopeptide is immobilized is not particularly limited, and examples thereof include plates (including plates having a plurality of recesses (including well plates, microplates, etc.)), beads, gels, and the like. The carrier material includes plastics (for example, styrene resin such as polystyrene, vinyl polymer such as polyvinyl chloride, acrylic resin such as polymethyl (meth) acrylate, polyolefin resin such as polyethylene, polyethylene terephthalate and the like. Polyethylene resin, polycarbonate resin, etc.), metals (gold, silver, aluminum, stainless steel, etc.), oxides (glass, silica, alumina, titania, zirconia, indium tin oxide (ITO), etc.), agar, and the like. More specifically, well plates used in the ELISA (Enzyme-Linked Polymer Assay) method, glass beads, magnetic beads, metal particles, silica beads, zirconia beads, cellulose gel, agarose gel, TOYOPEARL (registered trademark, Tosoh stock). Polymer gels such as (manufactured by the company) can be mentioned.

The carrier to which the oligopeptide used in the present invention is bound (oligopeptide-binding carrier) can be obtained by chemically or physically immobilizing the oligopeptide on the above-mentioned carrier. The method for immobilizing the oligopeptide according to the present invention on the carrier is not particularly limited, and examples thereof include a method using physical adsorption, electrostatic interaction, hydrophobic interaction, a cross-linking agent, and the like. The concentration of the oligopeptide according to the present invention at the time of immobilization may be appropriately adjusted according to the material, shape, immobilization method, etc. of the carrier, and examples thereof include a concentration of 5 to 50 μg / mL, preferably 10. It is ~ 40 μg / mL. Further, at that time, it is preferable to perform a blocking operation with a blocking agent on the carrier after oligopeptide immobilization because non-specific adsorption is suppressed and the background at the time of measurement is also suppressed. The blocking agent may be appropriately selected from agents usually used in the field of measurement using an antigen-antibody reaction such as the ELISA method, and one embodiment of a preferable blocking agent is collagen peptide.

In the present invention, the stabilizer contained in the solution added to the above-mentioned oligopeptide binding carrier is a nonionic surfactant and / or a cationic surfactant. The nonionic surfactant and the cationic surfactant may be appropriately selected from the reagents usually used in the measurement reagent utilizing the antigen-antibody reaction. Examples of nonionic surfactants include octylphenols such as polysorbate 20 (trade name: Tween 20), polysorbate 80 (trade name: Tween 80), Triton X-100 (trade name), and Triton X-114 (trade name). Ethoxylate, Brij 58 (trade name), Brij 35 (trade name), n-octyl-β-D-glucopyranoside, n-octyl-β-D-thioglucopyranoside, n-dodecyl-β-D-maltopyranoside, n- Dodecyl-α-D-maltopyranoside, n-dodecyl-N, N-dimethylamine-N-oxide, sucrose monododecanoic acid, n-octyl-β-D-glucopyranoside, n-dodecyl-β-D-maltopyranoside, n- Tridecyl-β-D-maltopiranoside can be mentioned. Examples of the cationic surfactant include cetyltrimethylammonium bromide (CTAB), cetyldimethylammonium bromide, cetyltrimethylammonium chloride, and myristyltrimethylammonium bromide. The concentration of the surfactant may also be appropriately determined in consideration of the critical micelle concentration and the concentration range usually used in the measuring reagent utilizing the antigen-antibody reaction. As an example, when polysorbate 20, which is a nonionic surfactant, is used as a stabilizer, it may be between 0.00005% (w / v) and 0.02% (w / v), 0.0001. It is preferably between% (w / v) and 0.01% (w / v), and more preferably between 0.0005% (w / v) and 0.008% (w / v). When octylphenol ethoxylate, which is a nonionic surfactant, is used as the stabilizer, it is preferably between 0.005% (v / v) and 0.1% (v / v), preferably 0.01% (v / v). It is more preferably between v / v) and 0.05% (v / v). Further, when CTAB, which is a cationic surfactant, is used as a stabilizer, it may be between 0.005% (w / v) and 0.1% (w / v), and may be 0.01% (w). It is preferably between / v) and 0.05% (w / v).

The stabilizer used in the present invention may contain at least one of a nonionic surfactant and a cationic surfactant, and may contain only one or a plurality of nonionic surfactants, or may contain cations. Only one or more surfactants may be contained, or one or more nonionic surfactants may be combined with one or more nonionic surfactants.

The liquid for dissolving the stabilizer used in the present invention is not particularly limited, and examples thereof include a buffer solution (for example, a buffer solution having a pH of 6 to 8 (phosphate buffered saline (PBS))).

Then, in the present invention, a modified antibody measuring reagent can be produced by adding a solution containing such a stabilizer to the above-mentioned oligonucleotide-binding carrier and then freeze-drying the solution.

The solution containing the stabilizer may be added by using a known method as appropriate according to the type and form of the carrier to be used. Further, the conditions for freeze-drying may be any conditions as long as the solution can be frozen and the water can be sublimated at a temperature suitable for subsequent drying, depending on the composition of the denaturing antibody measuring reagent, the type and size of the carrier, and the like. The temperature at the time of freezing is usually −10 ° C. or lower (for example, −50 ° C. to −10 ° C., preferably −40 ° C. to −20 ° C.), although it may vary depending on the temperature. The freezing time is usually 30 minutes or more (for example, 0.5 to 48 hours, preferably 1 to 24 hours, more preferably 2 to 10 hours). When freezing is performed in multiple stages, the freezing temperature and the freezing time may be the same or different in each stage. Further, in the subsequent drying, the drying temperature may be the same as the freezing temperature, and is a temperature higher than the freezing temperature (for example, −10 ° C. to 40 ° C., preferably −20 ° C. to 25 ° C.). The drying time is, for example, 0.5 to 72 hours (preferably 1 to 72 hours, more preferably 2 to 48 hours). When the drying after freezing is performed in multiple stages, the drying temperature and the drying time may be the same or different in each stage. Further, in freeze-drying, the drying pressure is usually 300 mm Torr or less, preferably 200 mm Torr or less, more preferably 100 mm Torr or less (for example, 50 to 100 mm Torr). As a more specific example of the freeze-drying conditions in the present invention, after freezing by leaving at −40 ° C. for 30 minutes or more, the primary order is left at −20 ° C. for 2 hours under vacuum conditions (100 mmtorr or less). It may be dried and left at 25 ° C. for 2 hours for secondary drying.

When the carrier is a plate such as a well plate, the oligopeptide is bound to a holding portion (well or the like) provided on the plate, and then a solution containing a stabilizer is added to the holding portion to obtain the solution. Freeze-dried together with the plate (Conditions: As an example, freeze by leaving at -40 ° C for 30 minutes or more, and then leave at -20 ° C for 2 hours under vacuum conditions (100 mmtorr or less) for primary drying, 25. The modified antibody measuring reagent of the present invention can be produced by subjecting it to secondary drying at ° C. for 2 hours). When the carrier is beads, after binding the oligopeptide to the beads, the obtained oligopeptide-binding beads and a solution containing a stabilizer are contained in one container, and the solution is freeze-dried together with the container. (Conditions: As an example, after freezing by leaving at -40 ° C for 30 minutes or more, primary drying by leaving at -20 ° C for 2 hours under vacuum conditions (100 mmtorr or less), and leaving at 25 ° C for 2 hours. By performing secondary drying), the denaturing antibody measuring reagent of the present invention can be produced.

<Method of measuring the amount of denatured antibody>
As described above, according to the method of the present invention, it is possible to provide a reagent capable of long-term storage in a refrigerator while maintaining its performance. Then, the reagent is suitably used for measuring the amount of denatured antibody. Therefore, the method for measuring the amount of the denatured antibody of the present invention is a method including the steps shown in (1) and (2) below.
(1) By contacting a sample containing a test antibody, a reagent for measuring the amount of the denatured antibody of the present invention, and a labeled antibody in which a labeling substance is bound to an antibody that recognizes the test antibody. By detecting the labeled substance in the complex formed in the steps (2) and (1) of forming a complex containing the oligopeptide binding carrier, the denatured antibody and the labeled antibody, the denatured antibody in the sample is detected. The process of measuring the amount.

In the present invention, the sample is not particularly limited as long as it contains the above-mentioned antibody regardless of whether it is denatured or undenatured, and is usually a solution containing an antibody, and a sample containing a human antibody is particularly preferable. Further, the solution is not particularly limited as long as it does not inhibit the binding between the denatured antibody and the above oligopeptide.

In the present invention, the antibody that recognizes the test antibody may be any antibody that can recognize the test antibody including the denatured antibody, and any of IgG, IgA, IgD, IgE, IgM, IgA, IgY and the like. It may be an isotype. Further, the form of the antibody is not particularly limited, and the antibody that recognizes the test antibody may be a functional fragment thereof. However, even if the antibody that recognizes the test antibody is denatured, the denatured recognition antibody is difficult to bind to the oligopeptide binding carrier, and only the denatured antibody in the test antibody is the oligopeptide binding carrier. The antibody that recognizes the test antibody is preferably a functional fragment of the antibody that does not contain the Fc region from the viewpoint of facilitating binding to. Functional fragments of such Fc region-free antibodies include, for example, Fab, Fab', F (ab') 2, Fv, scFv, and diabody. Further, it is preferable that the test antibody recognizing the test antibody and the test antibody containing the denatured antibody are derived from different species. For example, when the test antibody containing the denatured antibody is a human-derived antibody. Examples of the antibody that recognizes the test antibody include antibodies derived from non-human animals (for example, mouse antibody, rat antibody, rabbit antibody, goat antibody).

The labeling substance that binds to the antibody that recognizes the test antibody used in the present invention may be appropriately selected from substances usually used in the field of measurement using an antigen-antibody reaction, and as an example, a fluorescent substance such as fluorescein or a fluorescent substance may be used. , Enzymes such as alkaline phosphatase, and radioactive substances.

Further, the binding between the antibody that recognizes the test antibody and the labeling substance is usually carried out by a cross-linking reaction via a linker (for example, NHS ester, maleimide) or the like because of the convenience in detecting the substance described later, but labeling is performed. It can also be carried out using a secondary antibody to which a substance is bound. Here, the secondary antibody is an antibody that exhibits specific binding property to an antibody that recognizes a test antibody. Then, by utilizing this specific binding property, the labeling substance can be indirectly bound to the antibody that recognizes the test antibody via the secondary antibody. When a secondary antibody to which this labeling substance is bound is used, the labeling substance is added to the antibody that recognizes the antibody via the secondary antibody after forming a complex with the oligopeptide binding carrier and the denatured antibody. It may be combined.

In the present invention, the conditions for contacting the labeled antibody with the reagent containing the reagent for measuring the amount of the denatured antibody of the present invention and the test antibody are the above-mentioned reagent and the denatured antibody in the sample. It suffices as long as it is a condition that can bind to and that the test antibody and the labeled antibody can bind to each other, and is usually contact at a temperature of 0 to 45 ° C, preferably at a temperature of 0 to 40 ° C. The contact is more preferably at a temperature of 4 to 37 ° C, and even more preferably at a temperature of 25 ° C to 37 ° C. The contact time is also not particularly limited and may be appropriately adjusted depending on the type of the oligopeptide-binding carrier in the reagent, the concentrations of the antibody and the labeled antibody in the sample, the type of the labeling substance, etc., but usually 5 minutes or more. It is 6 hours, preferably 10 minutes to 2 hours, and more preferably 20 minutes to 1 hour.

Further, in order to remove the antibody that could not bind to the reagent, the labeled antibody that could not bind to the test antibody, and the like after this contact, a washing step may be provided in the method of the present invention. The washing solution used for the washing is any one that does not inhibit the binding between the reagent and the denatured antibody and the binding between the test antibody and the labeled antibody, and can wash the antibody non-specifically adsorbed on the carrier or the like. Often, for example, buffers (pH 6-8), more specifically Tris buffers, phosphate buffers, HEPES buffers are mentioned. Further, these buffer solutions may appropriately contain salts, surfactants, proteins, sugars, zwitterionic compounds and the like. Further, a cleaning solution (for example, E-test "TOSOH" II cleaning solution, manufactured by Tosoh Corporation) included in a commercially available reagent kit for immune reaction is also preferably used.

Regarding the step (1) according to the present invention, as described above, the reagent for measuring the amount of the denatured antibody of the present invention, the sample containing the test antibody, and the labeled antibody are brought into contact with each other all at once. Although the embodiment of forming a complex containing the reagent, the denatured antibody, and the labeled antibody has been described, the step (1) according to the present invention is not limited to the above embodiment. For example, by first contacting the reagent with a sample containing the test antibody, a complex containing the reagent and the denatured antibody is formed, and then by further contacting the labeled antibody, the antibody denatured with the reagent is formed. And a labeled antibody may be formed. Further, in the step (1) according to the present invention, the sample containing the test antibody and the labeled antibody are first brought into contact with each other to form a complex containing the denatured antibody and the labeled antibody, and then the reagent is further added. By contacting them, a complex containing the reagent, a modified antibody and a labeled antibody may be formed. The contact conditions for each substance are as described above. Further, in order to remove the antibody that could not bind to the reagent, the labeled antibody that could not bind to the test antibody, and the like even after the contact of each of the substances, the above-mentioned washing step may be appropriately provided. Further, in such an embodiment, from the viewpoint that the detection sensitivity of the denatured antibody can be more easily improved, in the step (1) according to the present invention, the reagent and the sample containing the test antibody are first brought into contact with each other, and then the sample is brought into contact with the test antibody. The step of contacting with the labeled antibody is preferable.

Next, in the step (2) of the method of the present invention, the amount of the denatured antibody in the sample is measured by detecting the labeling substance in the complex formed through the contact in the above-mentioned step (1). do.

In the present invention, the detection of a labeling substance means the detection of a signal derived from the labeling substance, and the detection can be performed according to a method known in the art. For example, when a fluorescent substance is used as a labeling substance, the fluorescence emitted by the fluorescent substance can be detected and quantified using a plate reader or the like. When an enzyme is used as a labeling substance, the color development, luminescence, etc. can be detected and quantified using a plate reader or the like by adding a substrate that decomposes by the action of the enzyme and develops color, emits light, etc. can. When a radioactive substance is used as the labeling substance, the radiation dose emitted by the substance can be detected and quantified by a scintillation counter or the like.

In the present invention, as shown in Examples described later, the intensity of the signal derived from the labeling substance detected in this manner (absorbance, fluorescence intensity, etc.) and the amount of the denatured antibody in the sample are highly correlated. Show sex. Therefore, the method of the present invention is carried out using a standard sample containing a denatured antibody having a known concentration, and correlation data with the measured value obtained using the sample containing the test antibody is obtained, and the correlation data is used. Based on this, the amount of denatured antibody present in the test sample can be measured (quantified). The correlation data is, for example, a calibration curve.

<Reagent for measuring the amount of denatured antibody>
As described above, in the present invention, an oligopeptide containing the amino acid sequence set forth in SEQ ID NO: 1 is used as an oligopeptide for recognizing a denatured antibody, and a nonionic surfactant and / or a cationic surfactant is used as a stabilizer. By using a solution containing an agent, it is possible to obtain a freeze-dried reagent that can be stored for a long period of time in a refrigerator while maintaining the reagent performance before freeze-drying (liquid reagent).

Therefore, the reagent for measuring the amount of the denatured antibody of the present invention is
The oligopeptide-binding carrier to which a solution containing a nonionic surfactant and / or a cationic surfactant is added is freeze-dried, and the oligopeptide-binding carrier is the amino acid sequence set forth in SEQ ID NO: 1. A reagent for measuring the amount of a denatured antibody, which is a carrier to which an oligopeptide that recognizes a denatured antibody is bound.

Further, as a preferred embodiment thereof,
0.00005 to 0.02% (v / v) polysolvate 20, 0.005 to 0.1% (v / v) octylphenol ethoxylate and 0.005 to 0.1% (w / v) cetyl The oligopeptide-binding carrier to which a solution containing at least one surfactant selected from the group consisting of trimethylammonium bromide is added is freeze-dried, and the oligopeptide-binding carrier is set forth in SEQ ID NO: 1. Examples thereof include a reagent for measuring the amount of a denatured antibody, which is a carrier to which an oligopeptide for recognizing a denatured antibody is bound, which comprises the amino acid sequence of the above.

Further, in the present invention, a kit for measuring the amount of denatured antibody, which comprises the reagent and a labeled antibody in which a labeling substance is bound to the antibody that recognizes the antibody, can be taken. In addition to these, the kit of the present invention stops the above-mentioned liquid for dissolving or diluting the test antibody, a blocking agent, a washing liquid, a substrate that decomposes by the action of an enzyme to develop color, emit light, etc., and the color development thereof is stopped. It may contain a stop solution for the purpose, a standard sample containing a denatured antibody having a known concentration, and the like. Further, the reagent and kit of the present invention can include instructions for use of the oligopeptide binding carrier and the like in carrying out the method of the present invention.

Hereinafter, the present invention will be described in detail based on Examples, but the present invention is not limited to these Examples.

(Example 1) Examination of stabilizer (No. 1)
The stabilizer to be added when the freeze-dried product of the denaturing antibody reagent was produced was examined by the following method.
(1) In this example, an anti-CD20 antibody was used as the human antibody according to the present invention. Then, a 10 mg / mL anti-CD20 antibody solution diluted with a phosphate buffer (pH 7.4) to 1 mg / mL was denatured by dialysis with 100 mmol / L glycine buffer (pH 2.0) overnight. A human antibody solution was prepared.
(2) Magnetic beads (ethylene-kneaded with ferrite) of an oligopeptide (hereinafter, also referred to as "2A1") containing the amino acid sequence set forth in SEQ ID NO: 1 and recognizing a denatured human antibody by the method shown below. Immobilization on particles) with a vinyl acetate copolymer (EVA) was performed.
(2-1) 4% (v / v) glutaraldehyde was added to the magnetic beads, and after stirring at 37 ° C. for 2 hours, 2A1 was added and left at 37 ° C. for 2 hours to immobilize the magnetic beads. ..
(2-2) 1% (w / v) collagen peptide (collagen peptide with weight average molecular weight: 3000 to 5000, obtained by hydrolyzing collagen or gelatin derived from fish scale or fish skin, product name: Ikuos HDL, A carbon dioxide buffer (pH 9.6) containing (manufactured by Nitta Gelatin Co., Ltd.) was added at 300 μl / well, and the mixture was stirred at 37 ° C. for 2 hours to perform blocking treatment.
(3) Magnetic beads in which an oligopeptide having the amino acid sequence set forth in SEQ ID NO: 1 (hereinafter referred to as 2A1) is bound to a measuring container for a fully automatic enzyme immunoassay device AIA-600II (manufactured by Tosoh Corporation) and the following (hereinafter referred to as 2A1). After adding any of the solutions shown in A) to (H), the measuring container was freeze-dried using a freeze-dryer (VirTis Genesis 25XL, manufactured by SP Scientific) to produce a freeze-drying reagent. As the conditions for freeze-drying, after freezing by leaving at -40 ° C for 30 minutes or more, primary drying is performed by leaving at -20 ° C for 2 hours under vacuum conditions (100 mmtorr or less), and then left at 25 ° C for 2 hours. It was done by secondary drying.

As a comparison target, a reagent (without freeze-drying, reagent (I)) in which only magnetic beads having 2A1 bonded to the measuring container for AIA-600II was added was also produced.
(A) Phosphate buffer (pH 7.4) (without additives)
(B) Phosphate buffer (pH 7.4) containing 0.005% (v / v) Tween 20 (trade name).
(C) Phosphate buffer containing 0.1 mol / L sucrose (pH 7.4)
(D) Phosphate buffer containing 0.01 mol / L sucrose (pH 7.4)
(E) Phosphate buffer containing 1% (w / v) collagen peptide (pH 7.4)
(F) Phosphate buffer containing 0.1% (w / v) collagen peptide (pH 7.4)
(G) Phosphate buffer containing 10 mmol / L histidine (pH 7.4)
(H) Phosphate buffer containing 1 mmol / L histidine (pH 7.4)
(4) The denatured human antibody solution prepared in (1) was added to the reagent prepared in (3), and the mixture was stirred at 37 ° C. for 20 minutes.
(5) After removing free components by washing with E-test "TOSOH" II cleaning solution (manufactured by Tosoh Corporation), alkaline phosphatase-labeled goat-derived anti-human IgG F (ab') 2 (manufactured by Jackson Immunoresearch, catalog number:) A solution containing 109-056-097) was added and stirred at 37 ° C. for 20 minutes.
(6) After removing excess labeled human antibody by washing with E-test "TOSOH" II washing solution (manufactured by Tosoh Corporation), 4-methylumbelliferyl phosphate as a substrate is added, and fluorescence generated by an enzymatic reaction is generated. The amount of denatured human antibody bound to the magnetic beads was measured by measuring the production rate (rate value) of the substance (4-methylumbelliferone) with a fully automatic enzyme immunoassay device AIA-600II (manufactured by Tosoh Corporation).

The rate value measurement results when the denatured human antibody solutions having various concentrations were added are shown in FIGS. 1A to 1H. When 0.005% (v / v) of Tween 20 is added as a stabilizer (FIG. 1B), the detection sensitivity is equivalent to that of the reagent not freeze-dried (FIG. 1I), and the linearity of the calibration curve is also good. Was (R ² = 0.997). On the other hand, when a solution containing no stabilizer or containing a stabilizer other than the nonionic surfactant was added and freeze-dried, the detection sensitivity decreased (FIGS. 1A and CH). For this reason, when a freeze-dried product of a denatured human antibody reagent is produced, a nonionic surfactant is added and freeze-dried to maintain the reagent performance before freeze-drying (liquid reagent) while refrigerating. It can be seen that reagents that can be stored for a long period of time can be provided.

(Example 2) Examination of stabilizer (Part 2)
The freeze-dried product was prepared in the same manner as in Example 1 except that the solution to be added to the AIA-600II container in Example 1 (2) was any of the solutions shown in (A) to (K) below. The stabilizer to be added during production was examined.
(A) Phosphate buffer (pH 7.4) containing 0.0001% (v / v) Tween 20.
(B) Phosphate buffer (pH 7.4) containing 0.001% (v / v) Tween 20.
(C) Phosphate buffer (pH 7.4) containing 0.01% (v / v) Tween 20
(D) Phosphate buffer (pH 7.4) containing 0.01% (v / v) Triton X-100 (trade name).
(E) Phosphate buffer (pH 7.4) containing 0.05% (v / v) Triton X-100.
(F) Phosphate buffer (pH 7.4) containing 0.01% (w / v) cetyltrimethylammonium bromide (CTAB).
(G) Phosphate buffer containing 0.05% (w / v) CTAB (pH 7.4)
(H) Phosphate buffer (pH 7.4) containing 0.1% (w / v) sodium dodecyl sulfate (SDS).
(I) Phosphate buffer containing 0.5% (w / v) SDS (pH 7.4)
(J) Phosphate buffer (pH 7.4) containing 0.1% (w / v) CHAPS (3-[(3-Cholamidotropyl) dimethyllammonio] propanesulfonate).
(K) Phosphate buffer containing 0.5% (w / v) CHAPS (pH 7.4)
The rate value measurement results when the denatured human antibody solutions having various concentrations were added are shown in FIGS. 2A to 2K. When Tween 20 was added as a stabilizer from 0.0001% (v / v) to 0.01% (v / v) (FIGS. 2A-C), Triton X-100 was added at 0.01% (v / v). When 0.05% (v / v) was added from (FIGS. 2D and E), and when CTAB was added from 0.01% (w / v) to 0.05% (w / v) (FIGS. 2F and G). ) Had the same detection sensitivity as the reagent not subjected to the freeze-drying treatment (FIG. 1I). On the other hand, when a solution containing an anionic surfactant such as SDS and an amphoteric surfactant such as CHAPS was added and freeze-dried, the detection sensitivity decreased (FIGS. 2H to K). Therefore, when producing a lyophilized product of a denaturing antibody reagent, the reagent performance before frost-drying (liquid reagent) is maintained by adding a nonionic surfactant or a cationic surfactant and lyophilizing. However, it can be seen that it is possible to provide reagents that can be stored for a long time in a refrigerator.

As described above, a reagent containing a stabilizer is added to a carrier to which an oligopeptide recognizing a denatured antibody is bound, and then the added carrier and the solution are freeze-dried to produce a modified antibody measurement reagent. By using an oligopeptide containing the amino acid sequence set forth in SEQ ID NO: 1 as an oligopeptide that recognizes a denatured antibody, and by using a nonionic surfactant and / or a cationic surfactant as a stabilizer. It is possible to provide a reagent that can be stored for a long period of time in a refrigerator while maintaining the reagent performance before freeze-drying (liquid reagent).

Then, by using the reagent, the amount of the antibody denatured by stress (for example, acid, alkali, heating, freeze-thaw, stirring) or the like can be measured quickly and easily. Therefore, the present invention is useful in quality control and the like of pharmaceutical products containing antibodies.

Claims (3)

A method for producing reagents for measuring denatured antibodies.
A step of adding a solution containing a stabilizer to a carrier to which an oligopeptide recognizing a denatured antibody is bound, and then freeze-drying the added carrier and the solution is included.
The oligopeptide is an oligopeptide consisting only of the amino acid sequence set forth in SEQ ID NO: 1, and the stabilizer is 0.01 to 0.05% (v / v) octylphenol ethoxylate and / or 0.01 to 0.01. The method, which is 0.05% (w / v) cetyltrimethylammonium bromide. At least one surfactant selected from the group consisting of 0.01-0.05% (v / v) octylphenol ethoxylates and 0.01-0.05% (w / v) cetyltrimethylammonium bromides. The oligopeptide-binding carrier to which the containing solution is added is freeze-dried, and the oligopeptide-binding carrier binds to an oligopeptide consisting only of the amino acid sequence set forth in SEQ ID NO: 1 that recognizes a denatured antibody. A reagent for measuring the amount of denatured antibody, which is a carrier. A method for measuring the amount of denatured antibody, which comprises the steps shown in (1) and (2) below. (1) Recognizing a sample containing a test antibody, the reagent according to claim 2, and the test antibody. In the complex formed in the steps (2) and (1) of contacting the antibody with the labeled antibody to which the labeling substance is bound, a complex containing the oligopeptide binding carrier, the denatured antibody and the labeled antibody is formed. A step of measuring the amount of denatured antibody in the sample by detecting the labeled substance of.

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