JP6188390B2 - Method for measuring MMP-3 - Google Patents

Description

  The present invention relates to a method for measuring MMP-3 using a monoclonal antibody that reacts specifically with human MMP-3 and has the same reactivity with human MMP-3 in the presence of EDTA and in the absence of EDTA, and The present invention relates to a measurement reagent / measurement reagent kit used in the measurement method. The present invention also relates to the monoclonal antibody, a hybridoma that produces the monoclonal antibody, and a method for producing the monoclonal antibody.

MMP-3 (matrix metalloproteinase 3) is produced in synovial cells and chondrocytes, and its specific substrate is proteoglycan constituting cartilage, and also has an action of activating other MMPs. It is known as an enzyme that plays a central role in the destruction of joint tissues during rheumatoid arthritis (RA).
In general, as the pathological condition of rheumatoid arthritis progresses, the synovium proliferates by causing an inflammatory reaction. It is considered that MMP-3 is produced in the synovial surface cells as the synovial membrane grows, accumulates in the synovial fluid, and moves into the blood via blood vessels and lymphatic vessels. From this, blood MMP-3 concentration is widely known for its clinical usefulness as an index of diagnosis assistance and therapeutic effect, disease activity, and joint destruction of rheumatoid arthritis. As human MMP-3 measuring reagents and measuring reagent kits, reagents as shown in the following non-patent documents are known.

In the reagents and kits shown in Non-Patent Documents 1-4, since the detected MMP-3 concentration is lower than the MMP-3 concentration to be detected in EDTA plasma, the use of EDTA plasma is not recommended. Have been described. Furthermore, in Non-Patent Document 5, examination of sample measurement values derived from various blood collection tubes was carried out using the kit described in Non-Patent Document 4, and MMP-3 measurement values were lowered when EDTA was used. An experiment report has been made.
In addition, in the kit shown in Non-Patent Document 6, data obtained by measuring a normal sample are described. However, as described in Item 8, the EDTA plasma measurement value is a measured value compared with other sample types. It is clearly shown that the price tends to decrease.
Thus, there has never been an example referring to the accuracy of MMP-3 concentration measurements in EDTA plasma. However, in order to use MMP-3 as an index for diagnosis of rheumatoid arthritis, evaluation of therapeutic effect, disease activity, and joint destruction, it is extremely important to obtain an accurate measurement value, regardless of the type of specimen. Therefore, a clinical test drug that can obtain an accurate measurement value is extremely useful.

Panaclear MMP-3 plate package insert (Daiichi Fine Chemical) Panaclear MMP-3 latex package insert (Daiichi Fine Chemical) Document attached to ELISA kit KAC1541 (Invitrogen) Quantikine ELISA Human Total MMP-3 Immunoassay (DMP300) (R & D SYSTEMS) Clinical chemistry 54: 4 (2008) Impact of Blood Sampling on the Circulating Metalloproteinase 1,2,3,7,8, and 9 Amersham Matrix metalloproteinase-3 Human Biotrack ELISA System (RPN2613) Product Booklet (GE Healthcare) The Journal of Biological Chemistry Vol 261, No30 (1986) A Metalloproteinase from HumanRheumatoid Synthetic FibraststatDigestsContests

The present invention provides an anti-MMP-3 monoclonal antibody characterized by reacting specifically with human MMP-3 and having the same reactivity with human MMP-3 in the presence of EDTA and in the absence of EDTA, It is an object of the present invention to provide a functional fragment derived from the monoclonal antibody and a hybridoma that produces the monoclonal antibody.
Another object of the present invention is to provide an immunoassay method, an immunoassay reagent and an immunoassay kit for accurately measuring the MMP-3 concentration in a sample containing EDTA.

The inventors of the present invention have made extensive studies to solve the above-mentioned problems, and in the selection of a monoclonal antibody against human MMP-3, the reactivity of the antibody in the presence of EDTA and in the absence of EDTA is used as an index. Thus, the inventors have found that a monoclonal antibody having the same reactivity in the presence of EDTA and in the absence of EDTA can be specially selected, and the present invention has been completed. Monoclonal antibodies obtained in this way were not specially selected by conventional methods that had never focused on reactivity in the presence of EDTA.
In addition, the present inventors combine the monoclonal antibodies having the same reactivity in the presence of the EDTA selected above and in the absence of EDTA, thereby lowering the measured value even in the sample containing EDTA. The present inventors have found that the MMP-3 concentration can be measured accurately. Therefore, another object of the present invention is to accurately obtain MMP-3 concentration even in a sample containing EDTA by combining antibodies having strong reactivity even in the presence of EDTA in an immunoassay method utilizing antigen-antibody reaction. The present invention relates to an immunoassay method, a measurement reagent, and a measurement kit.
Specifically, the present invention has the following configuration.
<1> A method for measuring MMP-3 in a sample,
A measurement method comprising a step of contacting an anti-MMP-3 monoclonal antibody having reactivity equivalent to that in the absence of EDTA in the presence of EDTA with MMP-3 in the sample.
<2> The measurement method according to <1>, wherein the anti-MMP-3 monoclonal antibody having a reactivity equivalent to that in the absence of EDTA in the presence of EDTA is two or more antibodies.
<3> The measurement method according to <2>, wherein the two or more types of antibodies are antibodies having different recognition sites for MMP-3.
<4> The measurement method according to <2> or <3>, wherein the two or more kinds of antibodies satisfy the following (1) and (2).
(1) The reaction equilibrium constant (KD value) with human MMP-3 in the presence of EDTA is less than 200% of the KD value in the absence of EDTA. (2) Amount of binding with human MMP-3 in the presence of EDTA Rmax indicating 65% or more relative to Rmax in the absence of EDTA
<5> Two or more kinds of antibodies may be deposited with the deposit numbers FERM P-22219 (82208), FERM P-22131 (82211), FERM P-22132 (82213), FERM BP-11486 (82216), FERM P-22133 (82245). The method according to any one of <2> to <4>, wherein the method is produced by a hybridoma selected from the group consisting of:
<6> The measurement method according to any one of <1> to <5>, wherein the sample contains EDTA.
<7> A measurement reagent and a measurement reagent kit for measuring MMP-3 in a sample, which are used in the measurement method according to any one of <1> to <6>.
<8> An anti-human MMP-3 monoclonal antibody having a reactivity equivalent to that in the absence of EDTA in the presence of EDTA.
<9> Anti-MMP-3 monochrome according to the above <8>, wherein the reactivity with human MMP-3 in the presence of EDTA is equivalent to that in the absence of EDTA satisfies the following (1) and (2): -Null antibody.
(1) The reaction equilibrium constant (KD value) with human MMP-3 in the presence of EDTA is less than 200% of the KD value in the absence of EDTA. (2) Amount of binding with human MMP-3 in the presence of EDTA Rmax indicating 65% or more relative to Rmax in the absence of EDTA
<10> The anti-MMP-3 monoclonal antibody according to <8> or <9>, which is selected by a method including the following steps.
Step 1: Immunize a non-human animal using MMP-3 as an immunogen Step 2: Select immunocompetent cells that produce antibodies with high reactivity to MMP-3 Step 3: Immunocompetent selected in Step 2 Step 4 for obtaining hybridoma using cells Step 4: Step for obtaining anti-MMP-3 monoclonal antibody using the hybridoma obtained in Step 3 Step 5: In any one of Steps 2 to 4, A process for evaluating reactivity to human MMP-3 in the presence of EDTA and in the absence of EDTA, and selecting clones having the same reactivity in the presence of EDTA and in the absence of EDTA
<11> By hybridoma having deposit numbers FERM P-22219 (82208), FERM P-22131 (82211), FERM P-22132 (82213), FERM BP-11486 (82216), FERM P-22133 (82245) Monoclonal antibody produced.
<12> A monoclonal antibody fragment comprising a Fab site derived from the anti-MMP-3 monoclonal antibody according to any one of <8> to <11>.
<13> A hybridoma characterized by producing the monoclonal antibody according to <8> or <9>.
<14> The above <8> or <FERM P-22219 (82208), FERM P-22131 (82211), FERM P-22132 (82213), FERM BP-11486 (82216), FERM P-22133 (82245) The hybridoma according to 9>.
<15> A method for producing the anti-MMP-3 monoclonal antibody according to <8> or <9>, comprising the following steps.
Step 1) Obtaining an anti-MMP-3 antibody-producing hybridoma using human MMP-3 as an immunogen Step 2) Contacting the antibody produced by the hybridoma obtained in Step 1 with human MMP-3 , Selecting a hybridoma that produces a highly reactive antibody, and obtaining an antibody produced by this hybridoma. Step 3) For the antibody obtained in step 2, each human in the presence of EDTA and in the absence of EDTA. A step of evaluating the reactivity to MMP-3 and selecting clones having the same reactivity in the presence of EDTA and in the absence of EDTA

  An immunoassay reagent and measurement capable of accurately measuring the concentration of MMP-3 in a sample containing EDTA by using an anti-MMP-3 monoclonal antibody having a reactivity equivalent to that in the absence of coexistence in the presence of EDTA of the present invention A kit is now available. In addition, since the method for selecting a monoclonal antibody of the present invention is selected based on the reactivity of the antibody in the absence of EDTA and in the presence of EDTA, the target monoclonal antibody can be obtained with high accuracy. Can and is efficient. By using the immunoassay reagent and kit of the present invention, the concentration of MMP-3 in EDTA plasma can also be accurately measured, and it is suitable for use as a clinical diagnostic agent for rheumatoid arthritis because it does not select the sample type. It is.

(Anti-human MMP-3 monoclonal antibody)
The anti-human MMP-3 monoclonal antibody of the present invention is an antibody that specifically reacts with human MMP-3. In obtaining anti-human MMP-3 monoclonal antibody, selection has been made by using an antigen-antibody reaction with human MMP-3 as an index, but there is no example of selection focusing on reactivity in the presence of EDTA. The present antibody having reactivity equivalent to that in the absence of EDTA even in the presence of EDTA is completely novel.
Here, having the same reactivity as in the absence of EDTA even in the presence of EDTA means that it has been reacted with human MMP-3 in the presence of EDTA, and that it has been reacted with human MMP-3 in the absence of EDTA. In some cases, the reactivity is equivalent, and as an index of reactivity, reaction equilibrium constant (KD value) indicating the strength of antibody affinity, and Rmax indicating the binding amount to human MMP-3 are used. Can be mentioned. It can also be indicated by the amount of human MMP-3 bound to the solid phase antibody or the degree of aggregation of the carrier to which the antibody is bound.

In addition, the antibody having the same reactivity as the anti-MMP-3 monoclonal antibody of the present invention refers to an antibody having the following characteristics when KD value and Rmax are used as indices.
1) The reaction equilibrium constant KD value with human MMP-3 in the presence of EDTA is less than 200% with respect to the reaction equilibrium constant KD value in the absence of EDTA. An antibody having
2) Rmax indicating the amount of binding to human MMP-3 in the presence of EDTA is 70% or more of Rmax in the absence of EDTA, and the amount of binding to human MMP-3 to the solid phase antibody is expressed as absorbance. 3) The absorbance in the presence of EDTA is 80 to 120% of the absorbance in the absence of EDTA.
When the degree of aggregation between the carriers bound with the antibody is used as an index, it means an antibody having the following characteristics: 4) Reactivity of aggregation caused by the aggregation reaction between carriers bound with the antibody in the presence of EDTA Aggregation signal is 80-120% relative to the aggregation signal in the absence of EDTA.
The technique for producing the anti-human MMP-3 monoclonal antibody of the present invention is not particularly limited, but a method for producing a mouse hybridoma is common. In addition, regarding structural modification and modification by genetic manipulation techniques after preparation of the anti-human MMP-3 antibody of the present invention, in particular, unless the reaction characteristics of the human MMP-3 antibody with human MMP-3 are greatly impaired. There is no limit.

  Specific examples of the anti-MMP-3 monoclonal antibody of the present invention include FERM P-22219 (82208), FERM P-22131 (82211), FERM P-22132 (82213), and FERM BP-11486 (82216). Monoclonal antibodies produced by the hybridoma, FERM P-22133 (82245).

(Monoclonal antibody fragment)
Regardless of the functional fragment containing the Fab portion of the monoclonal antibody obtained by enzymatic digestion or the functional fragment containing the Fab portion of the monoclonal antibody produced by gene recombination, the present invention includes Any functional fragment containing a Fab portion derived from a null antibody can be used. Therefore, the meaning of “functionality” of the functional fragment of the present invention specifically means having the ability to bind to human MMP-3.

(Sample to be measured)
Examples of EDTA (ethylenediaminetetraacetic acid) described in the present invention include sodium salts and lithium salts that are generally used as anticoagulants added to, or applied to, blood collection tubes, but are not limited thereto. is not. Samples that can be measured by the immunoassay method of the present invention include not only samples containing EDTA that could not be measured by conventional methods, more specifically, EDTA plasma collected using EDTA as an anticoagulant. It is also possible to measure biological samples such as blood, serum, plasma, urine, and joint fluid.

(Hybridoma)
The hybridoma used for producing the anti-human MMP-3 antibody of the present invention specifically reacts with human MMP-3 and can produce an anti-MMP-3 antibody having high reactivity in the presence of EDTA. The anti-MMP-3 monoclonal antibody of the present invention is produced through the following steps 1) -4).
Step 1) Obtaining an anti-human MMP-3 antibody-producing hybridoma using purified human MMP-3 as an immunogen Step 2) From the antibodies produced by the hybridoma obtained in Step 1), MMP-3 Step 3) Select antibodies with high reactivity against human MMP-3 in the presence of EDTA and in the absence of EDTA for the antibodies obtained in Step 2, and in the presence of EDTA and in the absence of EDTA The process of selecting clones with the same reactivity.
Step 4) Step of obtaining the hybridoma selected in Step 3) The antibody obtained through such a selection step reacts specifically with MMP-3, and in the presence of EDTA and in the absence of EDTA. Has equivalent reactivity.
Examples of the antibody selected in the above step include FERM P-22219 (82208), FERM P-22131 (82211), FERM P-22132 (82213), FERM BP-11486 (82216), and FERM P-22133 (82245). An antibody to be produced is mentioned.

(Use)
If the MMP-3 monoclonal antibody of the present invention is used, the MMP-3 concentration can be accurately measured regardless of the presence or absence of EDTA in the sample.
The MMP family is known to coordinate zinc at the active center and require calcium for structural stabilization. MMP-3 also has four calcium-binding sites, and it has been reported that calcium and EDTA affect enzyme activity (Non-patent Document 7). From these facts, it is considered impossible to produce an anti-MMP-3 antibody that is not affected by EDTA, and there is no immunoassay system that is not actually affected by EDTA.
In order to quantify the monovalent antigen MMP-3 by the sandwich method using an antigen-antibody reaction, it is necessary to use at least two kinds of antibodies, and when either one is susceptible to EDTA, the measurement system However, it will be affected by EDTA. According to the present invention for evaluating the reactivity of antibodies against human MMP-3 in the presence of EDTA and in the absence of EDTA, it becomes possible to efficiently select antibodies having equivalent reactivity in the presence or absence of EDTA, It has become possible to construct a measurement system using only at least two types of clones having reactivity equivalent to non-coexistence in the presence of EDTA. In this way, an immunoassay system that is not affected by EDTA has been constructed.
In addition, the immunoassay reagent using this antibody can accurately measure the concentration of MMP-3 in EDTA-containing samples, for example, EDTA plasma, and can be used as a clinical test for rheumatoid arthritis because it does not select a specimen type. Suitable for use.

  Hereinafter, a method for producing an anti-MMP-3 monoclonal antibody, a method for searching for antibodies having the same reactivity in the presence or absence of EDTA from the monoclonal antibodies, and a combination of the antibodies. Some examples of the immunological measurement method will be described in detail, but the present invention is not limited thereto.

[Comparative Example 1]
Using a commercially available human MMP-3 measurement kit, MMP-3 concentrations were measured for serum, EDTA plasma, and heparin plasma collected from the same healthy donor (Table 1).
The kit used is shown below.
<1> Enzyme linked Immunosorbent Assay Kit For Matrix Metalloproteinase 3
(USCN Life Science Inc)
<2> Blue Gene ELISA kits Human Stromalysin ST-1 ELISA kit
(Life Sciences Advanced Technologies Inc)
<3> Panaclear MMP-3 plate (Daiichi Fine Chemical Co., Ltd.)

キット〈1〉、〈3〉を用いた場合ＥＤＴＡ血漿は低値化、キット〈2〉を用いた場合ＥＤＴＡ血漿は高値化した。キット〈1〉、〈3〉は抗MMP−３抗体を用いたサンドイッチELISAを測定原理としており、抗体の反応性が低下すると、測定値が低値化すると考えられる。
一方キット〈2〉の測定原理は競合法であり、抗体の反応性が低下すると、測定値が高値化すると考えられる。
以上のことから、いずれのキットを用いても、ＥＤＴＡ血漿の値を正確に測定することはできないものと考えられた。

When kits <1> and <3> were used, EDTA plasma was lowered, and when kit <2> was used, EDTA plasma was raised. The kits <1> and <3> use a sandwich ELISA using an anti-MMP-3 antibody as the measurement principle, and it is considered that the measured value decreases as the reactivity of the antibody decreases.
On the other hand, the measurement principle of kit <2> is a competitive method, and it is considered that the measured value increases as the reactivity of the antibody decreases.
From the above, it was considered that the value of EDTA plasma could not be accurately measured with any kit.

[Comparative Example 2] Impact assessment with antibodies used in existing reagents)
Among the reagents whose measured values were lowered in Comparative Example 1, using a kit <3> Panaclear MMP-3 plate (first fine chemical) product kit, the solid-phase antibody and the label used in the reagent The reactivity of each antibody in the presence of EDTA was verified by the following method.

1) Evaluation of effects on solid phase antibody EDTA 0 mM: Dilute a sample with known MMP-3 concentration (50, 100, 200 ng / mL) with the buffer in the product kit, and follow the method described in the package insert for other steps. It was.
EDTA 3 mM: Dilute a sample with known MMP-3 concentration (50, 100, 200 ng / mL) using the buffer in the product kit to which EDTA has been added to give a final concentration of 3 mM. Followed the method.
EDTA 30 mM: Dilute a sample with known MMP-3 concentration (50, 100, 200 ng / mL) in the product kit with EDTA added to a final concentration of 30 mM. Followed the method.

About the absorbance (OD450) at a measurement wavelength of 450 nm obtained by measuring the above sample, the reactivity (%) in the presence of EDTA relative to the absence of EDTA was calculated based on the following formula, and the antibody reactivity was evaluated (Table) 2).

Antibody reactivity in the presence of EDTA (%)
= OD450 in the presence of EDTA ÷ OD450 in the absence of EDTA × 100

When the reactivity in the presence of EDTA was 80% or more relative to the reactivity in the absence of coexistence, it was judged to be equivalent, and in the case of less than that, it was judged that the reactivity decreased due to the influence of EDTA.

２）標識抗体への影響評価
測定試料にはＭＭＰ−３濃度既知試料（５０、１００、２００ｎg/mL）を用い、製品キットの二次抗体を以下の手順で調製した以外は製品キットの添付資料記載の方法に従った。
ＥＤＴＡ０ｍＭ ：酵素標識抗体液を、製品キット中の緩衝液で希釈し、そのほかの工程は添付文書記載の方法に従った。
ＥＤＴＡ３ｍＭ ：酵素標識抗体液を、終濃度３ｍＭとなるようＥＤＴＡを添加した製品キット中の緩衝液を用いて希釈し、そのほかの工程は添付文書記載の方法に従った。
ＥＤＴＡ３０ｍＭ：酵素標識抗体液を、終濃度３０ｍＭとなるようＥＤＴＡを添加した製品キット中の緩衝液を用いて希釈し、そのほかの工程は添付文書記載の方法に従った。
上記二次抗体溶液を用いて測定して得られた測定波長４５０ｎｍの吸光度（ＯＤ４５０）について、下式に基づいてＥＤＴＡ無添加に対するＥＤＴＡ共存下での反応性（％）を算出し、抗体反応性を評価した（表３）。

ＥＤＴＡ共存下での抗体の反応性（％）
＝ＥＤＴＡ共存下でのＯＤ４５０ ÷ ＥＤＴＡ非共存下でのＯＤ４５０ × １００
ＥＤＴＡ共存下での反応性が非共存化での反応性に対して８０％以上となった場合、同等と判断し、それ以下の場合はＥＤＴＡの影響を受けて反応性が低下すると判断した。

2) Evaluation of effects on labeled antibody A sample attached to the product kit, except that a sample with a known MMP-3 concentration (50, 100, 200 ng / mL) was used as the measurement sample and the secondary antibody of the product kit was prepared according to the following procedure. The described method was followed.
EDTA 0 mM: The enzyme-labeled antibody solution was diluted with the buffer solution in the product kit, and other steps were performed according to the method described in the package insert.
EDTA 3 mM: The enzyme-labeled antibody solution was diluted with a buffer solution in a product kit to which EDTA was added to a final concentration of 3 mM, and other steps were performed according to the method described in the package insert.
EDTA 30 mM: The enzyme-labeled antibody solution was diluted with a buffer solution in a product kit to which EDTA was added to a final concentration of 30 mM, and other steps were performed according to the method described in the package insert.
For the absorbance (OD450) at a measurement wavelength of 450 nm obtained by measurement using the above secondary antibody solution, the reactivity (%) in the presence of EDTA relative to the absence of EDTA was calculated based on the following formula, and the antibody reactivity Were evaluated (Table 3).

Antibody reactivity in the presence of EDTA (%)
= OD450 in the presence of EDTA ÷ OD450 in the absence of EDTA × 100
When the reactivity in the presence of EDTA was 80% or more relative to the reactivity in the absence of coexistence, it was judged to be equivalent, and in the case of less than that, it was judged that the reactivity decreased due to the influence of EDTA.

１）、２）の結果から、既存試薬に使用されている抗体は、ＥＤＴＡ添加によって、反応性は８０％以下となり、いずれの抗体もＥＤＴＡの影響を受けて反応性が低下していることがわかった。

From the results of 1) and 2), the reactivity of the antibodies used in the existing reagents is reduced to 80% or less by the addition of EDTA, and the reactivity of each antibody is affected by EDTA. all right.

(Evaluation Example 1 Establishment of MMP-3 Antibody)
1. Preparation of anti-MMP-3 monoclonal antibody (1) Preparation of hybridoma a. Materials • Human MMP-3: purified from culture supernatant of normal human fibroblasts NB1RGB, RIKEN RCB, # RCB0222 • Freund complete adjuvant: Wako Pure Chemical Industries, 014-09541
・ Myeloma cells (SP2 / O)
RPMI1640, GlutaMAX: GIBCO, 61870-036
・ Fetal Bovine Serum (FBS): manufactured by Biologic I NDUSTRIES, 04-001-1A
-Polyethylene glycol solution (PEG): SIGMA, P7306
HAT medium: Cosmo Bio, 16213004
96-well plate: NUNC, 167008
HRP-labeled goat anti-mouse IgG (γ) antibody: manufactured by Southern Biotech, 1030-05

b. Method (immunity to animals)
Using an emulsion prepared by mixing equal amounts of human MMP-3 and Freund's complete adjuvant, 30 μg per mouse was injected into the peritoneal cavity or foot pad of male BALB / c mice. Further, the emulsion injection was repeated 2-7 times at one week intervals. The antibody titer in the antiserum obtained by collecting blood from the mouse fundus vein was measured by the antigen-immobilized ELISA method described later.

(Confirmation of antibody titer (antigen-immobilized ELISA method))
The presence of the anti-MMP-3 antibody in the above-mentioned mouse antiserum was confirmed by ELISA method (antigen-immobilized ELISA method) in which human MMP-3 was immobilized. Details of the antigen-immobilized ELISA method are as follows.
First, human MMP-3 was dissolved in a 10 mM phosphate buffer solution (pH 7.2; hereinafter referred to as PBS) containing 150 mM sodium chloride so as to be 1 μg / mL to prepare an MMP-3 solution, and 50 μL of the solution was added. The solution was dispensed into each well of a 96-well microplate and allowed to stand overnight at 4 ° C.
Each well was washed three times with 300 μL of PBS containing 0.05% Tween (registered trademark) 20 (hereinafter referred to as PBST), and then 300 μL of PBST containing 1% bovine serum albumin (hereinafter referred to as BSA-PBST) was added. Blocking was performed at room temperature for 1 hour.
After each well was washed 3 times with BSA-PBST, 50 μL of mouse antiserum diluted 10-fold to 100-fold with BSA-PBST was added to each well and allowed to stand at room temperature for 1 hour.
Each well was washed three times with PBST, and then 50 μL of HRP-labeled goat anti-mouse IgG (γ) diluted 5000 times was dispensed into each well and allowed to stand at room temperature for 1 hour.
Each well was washed 3 times with PBST, 50 μL of citrate buffer solution (pH 5.0) containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide was added, and the mixture was allowed to stand at room temperature for 10 minutes. The enzyme reaction was stopped by adding 50 μL of 5N sulfuric acid, and the absorbance at a wavelength of 492 nm was measured. As a result of the measurement, spleen or lymph node was extracted from a mouse having a high antibody titer, and spleen-derived cells or lymph node-derived cells were prepared and used for cell fusion.

(Cell fusion)
Either the spleen-derived cells or lymph node-derived cells and myeloma cells were mixed at a cell ratio of 6: 1, and PEG was added to effect cell fusion. The fused cells were suspended in HAT medium and cultured in a CO ₂ incubator at 37 ° C. and 5% CO _{2 for} 8 days to obtain fused cells (hybridomas).

(2) Selection of hybridoma (selection by ELISA using solid phase antigen)
In the antigen-immobilized ELISA method described above, the same method was performed except that the culture supernatant of the fused cells was used instead of the mouse antiserum. As a result of the measurement, a well having a high absorbance was selected as a well in which an anti-MMP-3 antibody-producing hybridoma was present (positive well), and an anti-MMP-3 antibody-producing strain was selected.

(Selection by competitive ELISA method)
In the above-described antigen-immobilized ELISA method, a competitive ELISA method was performed in the presence of human MMP-3.
As a result of the measurement, a well having a high absorbance was selected as a well in which an anti-MMP-3 antibody-producing hybridoma was present (positive well), and an anti-MMP-3 antibody-producing strain was selected.
Using the hybridoma selected as an anti-MMP-3 antibody-producing strain by both of the above-mentioned two kinds of selection methods, single cloning of the hybridoma and purification of the monoclonal antibody were performed.

(3) Purification of monoclonal antibody Monocloning is performed by a conventional method (limit dilution method), positive wells are selected by the same method as the above-mentioned two kinds of ELISA methods, and finally 26 anti-MMP-3 monoclonal antibodies are selected. A production hybridoma was obtained.
About 10 ⁵ cells were administered to the abdominal cavity of mice pretreated with pristane, and the ascites produced were collected. Insoluble matter was removed from each collected ascites by centrifugation, an equal amount of saturated ammonium sulfate solution was added, and the mixture was allowed to stand overnight with stirring, and then the precipitate was collected by centrifugation. The recovered precipitate was dissolved in 20 mM Tris buffer (pH 8.0) and dialyzed against the same buffer. After each dialysis content was separately adsorbed on a DEAE-Sepharose column equilibrated with the same buffer, the IgG fraction obtained by elution with a concentration gradient of 0 to 300 mM sodium chloride in the same buffer was added to 50 mM glycine. Dialyzed against buffer, 26 antibodies were obtained.

(4) Evaluation of antibody combinations Among the above 26 types of clones, combinations of antibodies were further evaluated by the following method.
The anti-MMP-3 monoclonal antibody of each clone was diluted to 2 μg / mL in PBS to prepare an MMP-3 antibody solution, and 50 μL of the lysate was dispensed into each well of a 96-well microplate. And left at 4 ° C. overnight.
Each well was washed 3 times with 400 μL of PBST, and then 100 μL of BSA-PBST was added, followed by blocking at room temperature for 1 hour.
50 μL of antigen diluted to 100 ng / mL with BSA-PBST was added to each well and allowed to stand at room temperature for 1 hour.
Each well was washed 3 times with PBST, and each monoclonal antibody previously labeled with biotin was diluted to 1.5 μg / mL with PBST, dispensed in 50 μL to each well, and allowed to stand at room temperature for 1 hour. .
Each well was washed 3 times with PBST, 50 μL of citrate buffer solution (pH 5.0) containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide was added, and the mixture was allowed to stand at room temperature for 10 minutes. The enzyme reaction was stopped by adding 50 μL of 5N sulfuric acid, and the absorbance at a wavelength of 492 nm (OD492) was measured. From these, 10 antibodies capable of being combined with an OD492 of 0.5 or more were selected. Table 4 shows the combinations in which the absorbance was recognized.
For each combination, the OD492 was determined to be 0.2 or higher: +, 0.5 or higher: ++, 1.0 or higher: ++.

(Evaluation Example 2 Selection of antibodies having reactivity equivalent to that in the absence of EDTA (evaluation using a commercially available polyclonal antibody as the secondary antibody))
The anti-MMP-3 monoclonal antibody of each clone was diluted in PBS to 2 μg / mL to prepare an MMP-3 antibody solution, and 50 μL of the lysate was dispensed into each well of a 96-well microplate. And left at 4 ° C. overnight.
Each well was washed 3 times with 400 μL of PBST, and then 100 μL of BSA-PBST was added, followed by blocking at room temperature for 1 hour.
An antigen solution of 4000 ng / mL diluted to 400 ng / mL with BSA-PBST or BSA-PBST containing 3 mM EDTA was added to each well, and allowed to stand at room temperature for 1 hour.
Each well was washed 3 times with PBST, and then 50 μL of rabbit anti-MMP-3 polyclonal antibody (Santacruz sc6839-R) prepared to 0.2 μg / mL was dispensed into each well and allowed to stand at room temperature for 1 hour. I put it.
HRP-labeled anti-rabbit antibody (BIO-RAD Laboratories. Inc. Goat anti-Rabbit IgG (H + L) -HRP conjugate # 172-1019) diluted 3000 times was dispensed into each well and allowed to stand at room temperature for 1 hour.
Each well was washed 3 times with PBST, 50 μL of citrate buffer (pH 5.0) containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide was added, and the mixture was allowed to stand at room temperature for 10 minutes. The enzyme reaction was stopped by adding 50 μL of 5N sulfuric acid, and the absorbance OD492 at a wavelength of 492 nm was measured.
Based on the obtained OD492, the reactivity (%) of the antibody in the presence of EDTA was calculated based on the following formula.
Antibody reactivity in the presence of EDTA (%)
= OD492 in the presence of EDTA ÷ OD492 × 100 in the absence of EDTA

The clones having a high reactivity of 80 to 120% in the absence of EDTA were determined as +, and the clones having a reactivity as low as 80% or less were determined as-(Table 5). Among them, the reactivity of clones 82216 and 82245 could not be confirmed even in the absence of EDTA. Since these antibodies may interfere with the reactivity between the commercially available rabbit polyclonal antibody and the antigen, the influence of EDTA in the sandwich ELISA in which the monoclonal antibodies of the present invention were continuously combined, including the antibody, was evaluated.

(Evaluation Example 3 Selection of an antibody having reactivity equivalent to that in the absence of EDTA (evaluation using a monoclonal antibody as a secondary antibody))
) Evaluation of the effect of EDTA in the solid phase Primary screening <2>
In the above (Evaluation Example 2), antibodies that did not show reactivity were re-evaluated by using a combination of antibodies in which sandwich ELISA was established in the preliminary examination between the monoclonal antibodies shown in Evaluation Example 1.
As a specific method, the rabbit anti-MMP-3 polyclonal antibody used in Evaluation Example 2 is replaced with a clone that can form a sandwich with the evaluation clone (82208, 82211, and 82213 are used here). Except for the above, it was the same as (Evaluation Example 2).
It was confirmed that both 82216 and 82245 have the same reactivity in the presence of EDTA and in the absence of EDTA.

Examples and comparative examples
Based on the above evaluation results, serum and plasma (heparin, EDTA) pair specimens collected from the same healthy donor were measured by combining antibodies that were sandwiched and were not easily affected by EDTA. Based on the measured value of the serum sample and the measured value of the plasma sample, the ratio (%) to the measured value of serum was calculated based on the following formula. Note that the measurement was determined to be accurate when the measured value ratio (%) was 80% to 120%.
Comparative Example 1 With the combination of the 82212 antibody and the 82216 antibody, the measurement value ratio (%) to serum was 26.8-71.3%, indicating that the plasma measurement value was low in any sample. On the other hand, as shown in Example 1-6, when antibodies having the same reactivity in the presence of EDTA and in the absence of EDTA are combined, the measurement ratio of serum is 80 to 120%, and the plasma sample is accurately I found that I was able to measure.
From the above, by the method evaluated in Evaluation Example 1-4, by selecting an antibody having reactivity equivalent to that in the absence of EDTA in the presence of EDTA, and using a measurement system that combines this clone, It was confirmed that the measured value was obtained accurately even with the EDTA-containing sample.

(Evaluation Example 4) Analysis of interaction between biomolecules using BIA method (Biacore) The antibodies used in the above examples and comparative examples were analyzed in the presence of EDTA and in the absence of coexistence using the biomolecule interaction analysis method. Biacore T100 (GE Healthcare) was used for the measurement in which the affinity between each monoclonal antibody and MMP-3 antigen was evaluated. In accordance with the recommended protocol of Human Antibody Capture Kit, anti-human IgG antibody was immobilized on Sensor Chip CM5 and used for measurement.
The KD value of the antibody was calculated by the following method using an HBS buffer consisting of 10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% Tween 20, or an HBS buffer containing 3 mM EDTA as a running buffer. First, each monoclonal antibody microchannel system prepared to 5 μg / mL with HBS was flowed to capture the immobilized anti-IgG antibody.
Subsequently, the MMP-3 antigen prepared to 0, 2.5, and 5 μg / mL with a running buffer was passed through the microchannel system and captured by the anti-MMP-3 antibody. At this time, the surface plasmon resonance RU was measured, and the binding measurement constants ka and Rmax were measured. Subsequently, only the running buffer was passed, the captured antigen was released for a while, and the dissociation rate constant kd value was calculated. The antibody-antigen dissociation constant KD value was calculated from the shape of the drawn sensor-gram based on the following equation.
KD = kd / ka
The ka, kd, KD, and Rmax are automatically calculated by the Biacore T100. At this time, the molecular weight of MMP-3 required was set to a molecular weight of 57,000 of latent MMP-3. Thereafter, the playback buffer was flowed to regenerate the chip, and the chip was repeatedly used.
From the KD and Rmax obtained in the EDTA non-coexistence and EDTA coexistence obtained as described above, the ratio to the non-coexistence in the presence of EDTA was calculated as follows.

Ratio of non-coexistence in the presence of EDTA%
= Value at each EDTA concentration ÷ Value in the absence of EDTA x 100

When the KD value indicating the strength of antibody affinity and the Rmax indicating the amount of antigen binding are less than 200% and 65% or more when EDTA is added to EDTA-free, respectively, the antibody is present in the presence of EDTA. It was judged to show equivalent reactivity. The results are shown in Table 8.

この判定結果は評価例２、３での評価結果と合致する結果となり、ＥＤＴＡ非共存下とＥＤＴＡ共存下で反応性が同等の抗体は、
１）ＥＤＴＡ共存下での、ヒトＭＭＰ−３との反応平衡定数ＫＤ値が、ＥＤＴＡ非共存下での反応平衡定数ＫＤ値に対して２００％未満。
２）ＥＤＴＡ共存下での、ヒトＭＭＰ−３との結合量を示すＲmaxが、ＥＤＴＡ非共存下でのＲｍａｘに対して６５％以上。
と特徴付けられることを確認した。

This determination result is a result that agrees with the evaluation results in Evaluation Examples 2 and 3, and antibodies having the same reactivity in the absence of EDTA and in the presence of EDTA are as follows:
1) The reaction equilibrium constant KD value with human MMP-3 in the presence of EDTA is less than 200% of the reaction equilibrium constant KD value in the absence of EDTA.
2) Rmax indicating the amount of binding with human MMP-3 in the presence of EDTA is 65% or more relative to Rmax in the absence of EDTA.
It was confirmed that

Since the monoclonal antibody of the present invention is selected based on the reactivity with human MMP-3 in the presence of EDTA, the MMP-3 concentration can be accurately quantified even in an EDTA-containing sample.
Also provided are a human MMP-3 immunoagglutination measurement reagent and measurement kit for accurately quantifying the concentration of MMP-3 in an EDTA-containing sample, for example, EDTA plasma, using the monoclonal antibody of the present invention.
In the immunological measurement method, immunological measurement reagent and measurement kit using the monoclonal antibody of the present invention, the MMP-3 concentration of serum, heparin plasma, and EDTA plasma can be accurately measured with one reagent. Thus, it becomes possible to provide a more accurate MMP-3 measurement value as a clinical diagnostic agent for rheumatoid arthritis.

[Reference to deposited biological materials]
(1) Hybridoma 82208 producing 82208 antibody
The name and address of the depository institution that deposited the biological material National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center 1-chome, 1-chome, East 1-chome, Tsukuba, Ibaraki, Japan (zip code 305-8586)
Date of deposit of biological materials at the depository in Loi January 27, 2012 (January 27, 2012)
Deposit number FERM P-22219 (82208) attached to the depositary of the high
(2) Hybridoma 82211 that produces the 82211 antibody
The name and address of the depository institution that deposited the biological material National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center 1-chome, 1-chome, East 1-chome, Tsukuba, Ibaraki, Japan (zip code 305-8586)
Date of deposit of biological materials at Loi depository June 16, 2011 (June 16, 2011)
Deposit number FERM P-22131 (82211) attached to the depositary of the high
(3) Hybridoma 82213 producing 82213 antibody
The name and address of the depository institution that deposited the biological material National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center 1-chome, 1-chome, East 1-chome, Tsukuba, Ibaraki, Japan (zip code 305-8586)
Date of deposit of biological materials at Loi depository June 16, 2011 (June 16, 2011)
Deposit number FERM P-22132 (82213) attached to the depository by the high depository
(4) Hybridoma 82216 producing 82216 antibody
The name and address of the depository institution that deposited the biological material National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center 1-chome, 1-chome, East 1-chome, Tsukuba, Ibaraki, Japan (zip code 305-8586)
Date of deposit of biological materials at the depository in Loi April 27, 2010 (April 27, 2010) (original deposit date)
May 25, 2012 (May 25, 2012) (Transfer date to the deposit under the Budapest Treaty by the original deposit)
Deposit number FERM BP-11486 (82216) attached by the depository institution of high
(5) Hybridoma 82245 producing 82245 antibody
The name and address of the depository institution that deposited the biological material National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center 1-chome, 1-chome, East 1-chome, Tsukuba, Ibaraki, Japan (zip code 305-8586)
Date of deposit of biological materials at Loi depository June 16, 2011 (June 16, 2011)
Deposit number FERM P-22133 (82245) attached to the depository by the depository in Hai

Claims (15)

A method for measuring MMP-3 in a sample, comprising:
A measurement method comprising a step of contacting an anti-MMP-3 monoclonal antibody having a binding force equivalent to that in the absence of EDTA in the presence of EDTA with MMP-3 in the sample. The measurement method according to claim 1, wherein the anti-MMP-3 monoclonal antibody having a binding force equivalent to that in the absence of EDTA in the presence of EDTA is two or more antibodies. The measurement method according to claim 2, wherein the two or more types of antibodies are antibodies having different recognition sites for MMP-3. The measurement method according to claim 2 or 3, wherein the two or more kinds of antibodies satisfy the following (1) and (2).
(1) The reaction equilibrium constant (KD value) with human MMP-3 in the presence of EDTA is less than 200% of the KD value in the absence of EDTA. (2) Amount of binding with human MMP-3 in the presence of EDTA Rmax indicating 65% or more relative to Rmax in the absence of EDTA Two or more antibodies consist of deposit numbers FERM P-22219 (82208), FERM P-22131 (82211), FERM P-22132 (82213), FERM BP-11486 (82216), FERM P-22133 (82245). The measurement method according to any one of claims 2 to 4, which is produced by a hybridoma selected from the group. The measurement method according to any one of claims 1 to 5, wherein the sample contains EDTA. A measuring reagent and a measuring reagent kit for measuring MMP-3 in a sample, which are used in the measuring method according to claim 1. An anti-human MMP-3 monoclonal antibody having a binding force equivalent to that in the absence of EDTA in the presence of EDTA. 9. The anti-MMP-3 monoclonal antibody according to claim 8, wherein the binding force with human MMP-3 equivalent to that in the absence of EDTA in the presence of EDTA satisfies the following (1) and (2).
(1) The reaction equilibrium constant (KD value) with human MMP-3 in the presence of EDTA is less than 200% of the KD value in the absence of EDTA. (2) Amount of binding with human MMP-3 in the presence of EDTA Rmax indicating 65% or more relative to Rmax in the absence of EDTA The anti-MMP-3 monoclonal antibody according to claim 8 or 9 selected by a method comprising the following steps.
Step 1: Immunize a non-human animal using MMP-3 as an immunogen Step 2: Select an immunocompetent cell that produces an antibody having a high binding power to MMP-3 Step 3: The immunocompetent selected in Step 2 Step of obtaining hybridoma using cells Step 4: Step of obtaining anti-MMP-3 monoclonal antibody using the hybridoma obtained in Step 3.
Step 5: In any one of the steps 2 to 4, the binding strength to human MMP-3 in each of the conditions in the presence of EDTA and in the absence of EDTA is evaluated, and the binding strength is determined in the presence of EDTA and in the absence of EDTA. The process of selecting equivalent clones Produced by hybridomas with deposit numbers FERM P-22219 (82208), FERM P-22131 (82211), FERM P-22132 (82213), FERM BP-11486 (82216), FERM P-22133 (82245) Monoclonal antibody. A monoclonal antibody fragment comprising a Fab site derived from the anti-MMP-3 monoclonal antibody according to any one of claims 8 to 11. A hybridoma which produces the monoclonal antibody according to claim 8 or 9. The monochrome of claim 8 or 9, which is FERM P-22219 (82208), FERM P-22131 (82211), FERM P-22132 (82213), FERM BP-11486 (82216), FERM P-22133 (82245). A hybridoma producing a null antibody . A method for producing an anti-MMP-3 monoclonal antibody according to claim 8 or 9, comprising the following steps.
Step 1) Obtaining an anti-MMP-3 antibody-producing hybridoma using human MMP-3 as an immunogen Step 2) Contacting the antibody produced by the hybridoma obtained in Step 1 with human MMP-3 , Selecting a hybridoma that produces an antibody having a strong binding force, and obtaining an antibody produced by this hybridoma. Step 3) For the antibody obtained in step 2, each human in the presence of EDTA and in the absence of EDTA A process of evaluating the binding strength to MMP-3 and selecting clones having the same binding strength in the presence of EDTA and in the absence of EDTA