JP2006132946A - Method of measuring iga bondable alkaline phosphatase 6 and kit used for it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of measuring IgA bondable alkaline phosphatase 6 in alkaline phosphatase 6, and a kit used for it. <P>SOLUTION: In the measuring IgA bondable alkaline phosphatase 6 in a specimen, IgA bondable alkaline phosphatase 6 in the specimen, an alkaline phosphatase region recognizing material for recognizing an alkaline phosphatase region being a constitutent component of IgA bondable alkaline phosphatase 6 and an immunoglobulin A region recognizing material for recognizing the immunoglobulin A region being a constitutent component of IgA bondable alkaline phosphatase 6 are reacted to obtain an alkaline phosphatase A region recognizing material. IgA bondable alkaline phosphatase 6 is measured from the label of a bonded material of the alkaline phosphatase A region recognizing material IgA bondable alkaline phosphatase 6 and the immunoglobulin a region recognizing material. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for measuring IgA-binding alkaline phosphatase 6 (hereinafter sometimes referred to as ALP 6-IgA) and a kit used therefor. More specifically, the present invention does not require complicated operations such as determination of alkaline phosphatase 6 by electrophoresis and visual determination, and a method capable of measuring the value of ALP 6-IgA as an accurate quantitative value. It relates to the kit used. The measurement method and kit of the present invention are extremely effective in the field of clinical laboratory medicine for ulcerative bowel diseases such as ulcerative colitis, for which there has been no useful marker.

Alkaline Phosphatase (ALP) (EC.3.1.3.1) is a generic name for enzymes that hydrolyze phosphate monoesters, have an optimum pH on the alkali side, and have zinc ions at the active center. The function in the living body is considered to be involved in substance and energy transport, supply of inorganic phosphate, and bone mineralization. In serum, there are isozymes that exhibit organ specificity such as liver type, bone type, small intestine type, placenta type and tumor type (Nagao type, Reagan type, Kasahara type, fetal small intestine type, etc.). It has been widely used for diagnosis of vascular diseases, bone diseases, tumors and the like.
However, while total ALP activity in serum is widely used and effective, the measurement results alone are difficult to identify the disease. It has a problem with the previous serum isozyme. When ALP is electrophoresed, 6 and 7 bands are separated, but considering ALP from the antigenicity of the protein using antiserum, organ-specific (Tissue Specific ALP: TSALP) and organ-nonspecific (Tissue-Non Specific ALP: TNSALP) Three types of placenta type (ALP 4), small intestine type (ALP 5), and germ cell type called tissue-specific ALP (TSALP) are produced by the gene present in 2q34-37. I know that. Next, it is known that tissue-nonspecific ALP (TNSALP) is the type found in liver, bone, kidney, and leukocyte, has the same amino acid sequence, and is expressed from gene 1p34-36.1. Among them, in the liver type and bone type, two types of mRNA are produced from the same gene due to the difference in transcriptional regulation, but it is known that it has the same sequence as the enzyme matures (non-patent literature). 1). However, due to the difference in sugar chains bound to the enzyme, the liver type (ALP 2) with 3 N-type sugar chains and the bone type (ALP 3) with 2 N-type sugar chains and 1 O-type sugar chain are electrophoresed. It is thought to be divided above. Furthermore, ALP 1 is separated from the binding of membrane components and GPI anchor, and ALP 6 is separated from the binding of immunoglobulin. Since the sum of all the isozyme activities is reflected as the ALP value in serum, it is difficult for a high ALP value to have a one-to-one correspondence with the disease.

In the ALP measurement as a sum of these isozymes, the activity varies depending on the substrate of the reagent used, the type of buffer, the pH, etc., and standardization has become active in recent years, and now the International Clinical Chemistry Union (IFCC) Recommendation methods are presented by the Japan Society for Clinical Chemistry (JSCC), and standardization of total ALP measurements is progressing. However, there is still no standard detection method for isozyme itself (Non-patent Document 2).
The methods for measuring isozymes include amino acids that inhibit L-phenylalanine, liver type, bone type (ALP 2, 3), placental type with L-homoarginine, and small intestine type (ALP 4, 5). A method of measuring isozymes by utilizing (non-patent document 3), a method of specifically measuring ALP 4 while leaving only placental (ALP 4) activity by heating (non-patent document 4, non-patent document 5), A method of measuring liver type (ALP 2) and bone type (ALP 3) using lectin using the difference between the above-mentioned liver type and bone type ALP sugar chains (Non-patent document 6, Non-patent document 7) , Non-Patent Document 8, Non-Patent Document 9), a method using a specific monoclonal antibody against bone type ALP (ALP 3) (Non-Patent Document 10), etc. has been developed, and some isozymes can be measured. Yes. However, the immunoglobulin-binding ALP 6 is used for clinical diagnosis by knowing the presence or absence of its presence from electrophoresis because there is still no way to know its absolute value, although clinical significance has been advocated. It is in.

First, ALP 6 was discovered by Nagamine et al. As an ALP-conjugated immunoglobulin (Non-patent Document 11) in ulcerative colitis, which is an intractable disease designated by the Ministry of Health, Labor and Welfare (Non-patent Document 12). Kano et al. Then reported that ALP 6 increased or decreased reflecting the pathological condition (Non-patent Document 13). In addition, Kanno et al. Conducted a nationwide questionnaire in 1981 and summarized the pathological analysis (Non-patent Document 14). As a result, many cases were found to be autoantibodies. Regarding the isozyme species to which immunoglobulin binds, Crofton et al. Reported that most of ALP 6 immunoglobulin-bound ALP is TNSALP (Non-patent Document 15). Recently, Owen et al. Also reported that IgG kappa chains are bound to macro ALP (Non-patent Document 16). In general, ALP 6 expression, which is the above-mentioned polymer complex, is generally infrequent in healthy individuals, while serum containing high-activity ALP that has a total ALP activity of 1,000 units or more is frequently observed in ALP 6 It is known that ALP 6 may be elevated even in rheumatoid arthritis and tumors. In such a situation, it was strongly desired that precise measurement of ALP 6 be performed in order to further establish clinical significance.
Biochem Biophys Res Com 168,993-1000: 1990 Laboratory test 37 (9), 1041-1044: 1993 Histochem J. 13,941-951: 1981 Prenat Diagn.16,1051-1054: 1996 Enzyme Protein. 49,313-203: 1996 J Clin Pathol. 1992 45,68-71: 1992 Clin Chem. 40,1749-1756: 1994 Clin Chem. 42,1970-1974: 1996 Calcif Tissue Int. 71: 508-518: 2002 Clin Chem 44,2139-2147: 1998 Clin Chim Acta 65,39-46: 1975 Nichijou 73,162-168: 1976 Biophysical chemistry 20,325: 1977 Report on the incidence and distribution of enzyme-linked immunoglobulin in Japan: 1981 Clin Chim Acta 112,33-42: 1981 Ann Clin Biochem 39,523-5: 2002

  In a situation where it was strongly desired that precise measurement of ALP 6 be performed in order to establish clinical significance, the present inventors used healthy human serum and high ALP serum as specimens, and used antibodies as antibodies. Using an anti-human organ non-specific alkaline phosphatase monoclonal antibody and an anti-human IgG monoclonal antibody, the amount of ALP 6 was precisely measured, and the results were found to be in good agreement with the results of ALP 6 determination by electrophoresis. In this regard, a patent application has already been filed as Japanese Patent Application No. 2003-131941. This time, ALP 6 positive sample by electrophoresis was used as the sample, and the antibody was a combination of anti-human organ non-specific alkaline phosphatase monoclonal antibody and anti-human IgG monoclonal antibody, or anti-human organ non-specific alkaline phosphatase monoclonal antibody and anti-human organ. When the amount of ALP 6 was precisely measured using a combination with a human IgA monoclonal antibody, unexpectedly, when a combination of an anti-human organ non-specific alkaline phosphatase monoclonal antibody and an anti-human IgA monoclonal antibody was used. Can be measured with high sensitivity, and in samples of patients with ischemic bowel disease, the measurement value obtained by the combination of anti-human organ non-specific alkaline phosphatase monoclonal antibody and anti-human IgA monoclonal antibody is anti-human. Organ non-specific arca Compared to the measured value obtained by using a combination of phosphatase monoclonal antibodies and anti-human IgG monoclonal antibodies, the ischemic bowel disease diagnosis and were useful. Therefore, measurement of IgA-binding alkaline phosphatase 6 using a combination of an anti-human organ non-specific alkaline phosphatase monoclonal antibody and an anti-human IgA monoclonal antibody is effective for the diagnosis of ischemic bowel disease, and IgA-binding alkaline phosphatase. 6 was found to be an effective marker for ischemic bowel disease. The present invention has been completed based on such findings.

(1) The present invention relates to a method for measuring IgA-binding alkaline phosphatase 6 in a specimen, which recognizes IgA-binding alkaline phosphatase 6 in a specimen and an alkaline phosphatase site that is a constituent of IgA-binding alkaline phosphatase 6 The alkaline phosphatase site recognition substance that reacts with the immunoglobulin site recognition substance that recognizes the immunoglobulin A site that is a constituent of IgA-binding alkaline phosphatase 6, and the resulting alkaline phosphatase A site recognition substance and IgA-binding alkaline phosphatase This is a method for measuring IgA-binding alkaline phosphatase 6, characterized in that IgA-binding alkaline phosphatase 6 is measured from the level of a conjugate of 6 and an immunoglobulin A site recognition substance.
(2) More specifically, the present invention relates to a method for measuring IgA-binding alkaline phosphatase 6, which comprises reacting an IgA-binding alkaline phosphatase 6 with an antibody that reacts with non-human organ-specific alkaline phosphatase. Subsequently, the antigen-antibody reaction product thus obtained is reacted with an immunoglobulin A site recognition substance that recognizes an immunoglobulin A site, which is a constituent of IgA-binding alkaline phosphatase 6, and the resulting human organ non-specific alkaline phosphatase is reacted. A method for measuring IgA-binding alkaline phosphatase 6, characterized in that IgA-binding alkaline phosphatase 6 is measured from the level of the conjugate of the reacting antibody, IgA-binding alkaline phosphatase 6 and immunoglobulin A site recognition substance. .

(3) The present invention further relates to a method for measuring IgA-binding alkaline phosphatase 6,
i) Human organ non-specific alkaline phosphatase in a specimen is reacted with an antibody that reacts with human organ non-specific alkaline phosphatase and then antigen-antibody-reacted human organ non-specific alkaline phosphatase is converted into an enzyme substrate for alkaline phosphatase From the level of the enzyme activity that was reacted, the human organ non-specific alkaline phosphatase activity in the sample was determined,
ii) Independently of the above i), the level of IgA-binding alkaline phosphatase 6 in the sample is determined by the measurement method of (1) or (2) above,
iii) Obtain the multiplicative value of the human organ non-specific alkaline phosphatase activity value obtained in i) above and the level value of IgA-binding alkaline phosphatase 6 obtained in ii) above, and determine IgA binding in the sample from the multiplicative value. Determining the amount of alkaline phosphatase 6;
This is a method for measuring IgA-binding alkaline phosphatase 6.
(4) Furthermore, the present invention is a kit for measuring IgA-binding alkaline phosphatase 6 in a specimen,
i) a solid support,
ii) an alkaline phosphatase site recognition substance that recognizes an alkaline phosphatase site that is a constituent of IgA-binding alkaline phosphatase,
iii) a labeled immunoglobulin A site-recognizing substance that recognizes an immunoglobulin A site that is a constituent of IgA-binding alkaline phosphatase 6, and
iv) components for detecting the label,
It is a kit containing.
(5) Furthermore, the present invention obtains the level of IgA-binding alkaline phosphatase 6 in a sample from a human suspected of ischemic bowel disease by any one of the methods (1) to (3), and ischemic A method for diagnosing bowel disease.
(6) Furthermore, the present invention is an ischemic bowel disease marker comprising IgA-binding alkaline phosphatase 6 having an alkaline phosphatase site and an immunoglobulin A site as constituent components.

  By the measurement method of the present invention, IgA-binding alkaline phosphatase 6 (ALP 6-IgA) in ALP 6 is measured with higher precision and sensitivity than ALP 6 measurement by electrophoresis which has been used so far. Can do. For example, anti-TNSALP monoclonal antibody is used as the first monoclonal antibody that hardly reacts with isozymes other than human organ non-specific alkaline phosphatase (TNSALP), and immunity that is a constituent of ALP 6-IgA is used as the second monoclonal antibody. By using a labeled anti-human IgA monoclonal antibody that reacts with the globulin A site, or by using a combination of these two antibodies, non-immunoglobulin isozymes (liver type, bone type, small intestine type, placenta) Type, other tumor types), and only ALP 6-IgA can be specifically measured. The measurement of ALP 6-IgA according to the present invention can be used as a clinical diagnostic agent such as ischemic bowel disease, and can contribute to the development of basic medicine such as autoantibody mechanism.

In the present invention, the specimen is not particularly limited as long as it is a liquid or mixture that may contain ALP 6 -IgA, but in vivo samples such as blood, serum, plasma, and urine are usually preferable.
In the present specification, alkaline phosphatase 6 is a protein derived from a band that has been certified as alkaline phosphatase 6 in Disk electrophoresis methods such as capillary isoelectric focusing currently used in clinical tests. And a protein in which alkaline phosphatase and immunoglobulin are bound. Alkaline phosphatase 6 is sometimes described as ALP 6, and includes IgG-binding alkaline phosphatase 6, IgA-binding alkaline phosphatase 6 and the like.
In the present specification, IgA-binding alkaline phosphatase 6 refers to a protein in which alkaline phosphatase and immunoglobulin A are bound to alkaline phosphatase 6, and may be abbreviated as ALP 6-IgA.
In this specification, IgG-binding alkaline phosphatase 6 refers to a protein of alkaline phosphatase 6 in which alkaline phosphatase and immune globulin G are bound, and may be abbreviated as ALP 6-IgG.
In the method for measuring ALP 6-IgA of the present invention, an alkaline phosphatase site recognition substance that recognizes an alkaline phosphatase site that is a component of ALP 6-IgA and an immunoglobulin A site that is a component of ALP 6-IgA are recognized. The ALP 6-IgA is measured and quantified from the level of the resulting conjugate of the alkaline phosphatase site recognition substance, ALP 6-IgA and the immunoglobulin A site recognition substance.

In the present invention, the alkaline phosphatase site recognition substance refers to a substance that can bind to the alkaline phosphatase site in ALP 6-IgA. Specifically, substances capable of binding to human organ non-specific alkaline phosphatase (TNSALP) such as liver-derived ALP and bone-derived ALP can be exemplified. Human organ non-specific alkaline phosphatase (TNSALP) is usually a protein expressed from the gene 1P34-36.1, but in general, liver type ALP (ALP 2), bone type ALP (ALP 3), kidney type ALP And white blood cell type ALP. Preferred examples of the substance capable of binding to TNSALP include an antibody against TNSALP. The antibody may be any of antiserum, polyclonal antibody, and monoclonal antibody.
In order to accurately measure ALP 6-IgA, it is preferable to use a monoclonal antibody specific for TNSALP. Specifically, as shown in Reference Example 1 described later, the ALP enzyme activity value that reacts with small intestine type ALP and the placenta when reacted with small intestine type, placenta type, liver type and bone type ALP of the same enzyme activity amount. Anti-human TNSALP monoclonal antibody whose sum of ALP enzyme activity reacting with type ALP is less than 1/10 of sum of ALP enzyme activity reacting with liver type ALP and ALP enzyme activity reacting with bone type ALP Is preferred. In particular, an anti-human TNSALP monoclonal antibody having an ALP enzyme activity value that reacts with bone type ALP is 0.7 or less of an ALP enzyme activity value that reacts with liver type ALP is more preferable. Examples of anti-human TNSALP antibodies that can be used in the present invention include monoclonal antibodies 3-29-3R, 2C5-32, and 1A5-7 shown in Production Example 1 established by the present inventor and described later. 3-29-3R and 2C5-32 are preferred. Monoclonal antibodies such as HLMS-1, HLMS-2, HLMS-3, and HLMS-4 (above, JP-A-2-35098) are also preferred, and known anti-TNSALP monoclonal antibodies similar to them can also be used. It is.

  In the present invention, the immunoglobulin A site recognition substance refers to a substance that can bind to the immunoglobulin A site in ALP 6-IgA. Preferably, it is a substance capable of binding to IgA, specifically an antibody against IgA. The antibody may be any of antiserum, polyclonal antibody, and monoclonal antibody, but anti-human IgA monoclonal antibody is particularly preferable.

For example, the antiserum, polyclonal antibody, and monoclonal antibody against TNSALP or IgA described above can be prepared by a method known per se. Commercially available antisera, polyclonal antibodies, and monoclonal antibodies against IgA can be used. The preparation will be described in detail below using a monoclonal antibody against TNSALP as an example.
The anti-TNSALP monoclonal antibody that can be used in the present invention can be obtained, for example, by using purified human blood-derived ALP 6 as an immunogen. Moreover, since ALP 6 is divided into an ALP site and an immunoglobulin site by its structure, ALP and immunoglobulin can be used as antigens, respectively. Immunization can be performed with an antigen from a cultured osteoblast cell line as an ALP antigen, but not limited thereto, ALP such as normal osteoblasts can also be used as the antigen. The use of full-length recombinants, mutants, and partial antigens prepared by genetic engineering is also a conventional method, and this is exactly the same for the production of anti-immunoglobulin antibodies and can be used.
Monoclonal antibodies are immunized with purified human ALP 6 alone or ALP and immunoglobulin separately as an immunogen, and anti-human ALP 6 antibody-producing cells, or anti-ALP antibody-producing cells or anti-immunoglobulin antibody-producing cells. And a hybridoma obtained by fusing bone marrow tumor cells.

  The hybridoma can be obtained by the following method. That is, for example, ALP as described above is mixed together using Freund's complete, incomplete adjuvant, aluminum hydroxide adjuvant, pertussis adjuvant, and the like, and a sensitive adjuvant solution is prepared and divided into several times. Animals such as rats are immunized by intraperitoneal subcutaneous or tail vein administration every 1 to 3 weeks. The amount of sensitizing antigen is between 1 μg and 100 mg, but generally about 50 μg is preferred. The number of immunizations is generally 2-7, but various methods are known. Next, antibody-producing cells derived from the spleen and the like are fused with cells having proliferation ability in a test tube such as bone marrow tumor cells (myeloma cells). Antibody-producing cells can be obtained from mice, nude mice, and rats.

  As a fusion method, fusion can be performed by using polyethylene glycol (PEG) according to the conventional method of Kohler and Milstein (Nature. 256, 495.1975) which is already known per se. Fusion can also be performed by Sendai virus or electrofusion. A method for selecting a hybridoma that produces an antibody that recognizes human ALP from the fused cells can be performed as follows. That is, hybridomas are selected from colonies produced by cells surviving in the HAT medium and HT medium from the fused cells by the limiting dilution method. If colony culture supernatant made of 96-well wells contains antibodies against human ALP, place the supernatant on an assay plate with human ALP immobilized on the plate, Monoclonal antibody-producing clones against human ALP can be selected by ELISA in which a secondary labeled antibody such as anti-mouse immunoglobulin-HRP labeled antibody is reacted after the reaction. In addition to HRP, enzymes such as alkaline phosphatase, fluorescent substances, radioactive substances, etc. can be used as the labeling substance for the labeled antibody. As a control, human ALP-specific antibodies can be screened by simultaneously performing ELISA using an assay plate that binds only BSA, which is a blocking agent. In other words, clones that are positive on the human ALP plate and negative on the BSA ELISA are selected. At the same time, an anti-mouse immunoglobulin antibody is spread on a microtiter plate, and the fusion cell culture supernatant is added and reacted therewith. Further, purified ALP is added to bind the active enzyme to produce an immune complex. The activity can also be measured using a chromogenic substrate such as phenyl phosphate.

  Among the hybridomas producing antibodies that can be used in the present invention, among monoclonal antibodies that specifically recognize human ALP, it reacts particularly with active human TNSALP and almost cross-reacts with human placental ALP and human small intestine ALP. Preferred are those produced by monoclonal antibodies that do not. Examples thereof include hybridomas 3-29-3R and 2C5-32 established by the present inventor. Hybridomas 3-29-3R and 2C5-32 were deposited at the Institute of Biotechnology, AIST on April 16, 2003, under the accession numbers FERM BP-8360 and FERM BP-8359, respectively.

  The hybridoma can be cultured in a medium usually used for cell culture, such as α-MEM, RPMI1640, ASF, S-clone, and the monoclonal antibody can be recovered from the culture supernatant. Alternatively, an animal from which a hybridoma is derived or a nude mouse can be treated in advance with pristane, and ascites can be retained by intraperitoneal injection of cells into the animal, and a monoclonal antibody can be recovered from the ascites. As a method for recovering the monoclonal antibody from the supernatant or ascites, a conventional method can be used. For example, salting-out methods using ammonium sulfate, sodium sulfate, etc., chromatography, ion exchange chromatography, affinity chromatography using protein A, and the like can be mentioned.

An alkaline phosphatase site obtained by reacting an alkaline phosphatase site recognition substance such as an anti-TNSALP monoclonal antibody as described above with an immunoglobulin A site recognition substance such as an anti-IgA monoclonal antibody with ALP 6-IgA in a sample. ALP 6-IgA in a sample can be measured by determining the level of a conjugate of a recognition substance, ALP 6-IgA and an immunoglobulin A site recognition substance. More specifically, for example, by ELISA, ALP 6-IgA and anti-human TNSALP monoclonal antibody in a specimen are reacted with an antigen-antibody, and then the resulting antigen-antibody reaction product is reacted with an anti-IgA monoclonal antibody. ALP 6-IgA in the specimen can be measured from the level of the conjugate of the anti-human TNSALP monoclonal antibody, ALP 6-IgA and anti-IgA monoclonal antibody.
In order to determine the level of a bound substance that has undergone an antigen-antibody reaction by ELISA, it is preferable to use a labeled immunoglobulin A site recognition substance as an immunoglobulin A site recognition substance such as an anti-IgA monoclonal antibody. The labeling method is not particularly limited as long as it can be used for labeling in an immunoassay method, but it may be an enzyme such as peroxidase, a radioisotope, a fluorescent material, a magnetic material, or a colloid.

  As a measuring method of the present invention, a specific example carried out by sandwich assay ELISA method is as follows. First, an anti-TNSALP monoclonal antibody, which is an alkaline phosphatase site recognition substance, is adsorbed on a solid support such as a plate as a primary antibody, and reacted with the alkaline phosphatase site of ALP 6-IgA in a sample such as serum. Wash the support. Next, after reacting the adsorbed immunoglobulin site of ALP 6-IgA with a secondary antibody such as a biotinylated anti-IgA monoclonal antibody, which is an immunoglobulin A site recognition substance, and reacting with peroxidase-labeled streptavidin. ALP 6-IgA can be detected by performing a peroxidase enzyme reaction followed by a color reaction. In addition, by using an anti-IgA monoclonal antibody directly labeled with peroxidase such as HRP as the secondary antibody, after reacting with ALP and the secondary antibody, a substrate for the labeled enzyme, for example, OPD (o-phenylene) ALP 6-IgA can be detected by further reacting with (diamine), DAB (diaminobenzidine), TMB (tetramethylbenzidine) and the like to cause color development. The labeling substance to be bound to the secondary labeled antibody is not limited to the enzyme depending on the measurement method, and may be a radioisotope, a fluorescent substance, a magnetic substance, a colloid, or the like.

In the measurement method of the present invention, the level of ALP 6-IgA in the sample is determined by the above-described measurement method, and independently of that, the human organ non-specific alkaline phosphatase (TNSALP) activity is determined in the sample, A multiplicative value of the ALP 6-IgA level value and the human TNSALP activity value in the sample can be obtained, and the amount of ALP 6-IgA in the sample can be obtained from the multiplicative value. In the case of a method using this multiplicative value, an antibody against ALP 6-IgA in the sample and human TNSALP which is the primary antibody is used to determine the level of ALP 6-IgA in the sample by the measurement method described above. The effect of competitive reaction with other ALPs other than ALP 6 present in the sample can be taken into account in the antigen-antibody reaction of the sample, so the above multiplicative value is the actual amount of ALP 6-IgA present in the sample. The value more reflects
In the measurement method using this multiplicative value, human TNSALP activity in a sample is obtained by reacting human TNSALP in the sample with an antibody that reacts with human TNSALP and then reacting the antigen-antibody-reacted human TNSALP with an alkaline solution. It can be carried out by reacting with an enzyme substrate for phosphatase and determining the human TNSALP activity in the sample from the level of the enzyme activity reacted. As the enzyme substrate for alkaline phosphatase, a commonly used substrate can be used. For example, human TNSALP in a sample can be obtained by causing a color developing substrate such as phenyl phosphate to undergo an enzymatic reaction with antigen-antibody-reacted human TNSALP to develop color, and measuring the absorbance.

In the present invention, as described above, when ALP 6-IgA in a sample is measured, for example, by ELISA, it can be carried out using an ELISA kit. When the measurement method of the present invention is carried out by ELISA, for example, a kit for measuring ALP 6-IgA, comprising i) a solid phase support, ii) an alkaline phosphatase site recognition substance such as a monoclonal antibody against TNSALP Iii) ALP 6-IgA can be efficiently measured by using a kit containing a labeled immunoglobulin A site recognition substance such as an anti-IgA monoclonal antibody and iv) a component for detecting the label.
The component for detecting the label is a component for measuring the labeled antibody. When the label is biotin, it contains peroxidase-labeled streptavidin, tetramethylbenzidine peroxidase enzyme substrate, and hydrogen peroxide solution. When the label is peroxidase, it is a reagent containing a peroxidase substrate such as OPD (o-phenylenediamine), DAB (diaminobenzidine), TMB (tetramethylbenzidine) and the like. This kit may contain a detergent solution as required. In the present invention, when using this kit, it is preferable to include a washing solution in order to remove components that have not been adsorbed to the solid support after binding ALP 6-IgA in the specimen to the primary antibody. . As the cleaning liquid, for example, a Tris buffer containing a surfactant can be used. Furthermore, the kit of the present invention may contain a sample diluent as necessary. As the sample dilution solution, for example, a buffer solution such as Tris can be used. If necessary, a chelating agent such as EDTA · 2Na, an inorganic salt such as sodium chloride, or an animal protein such as BSA may be added to the buffer to prevent nonspecific adsorption of the protein.

Ischemic bowel disease can be diagnosed by the ALP 6-IgA measurement method and kit used therefor described in detail above. Specifically, the ALP 6-IgA level in an in vivo sample such as blood, serum, plasma, urine obtained from a human suspected of ischemic bowel disease is determined by the measurement method and kit of the present invention, and the level If it is higher than that of a normal person, it can be diagnosed as ischemic bowel disease.
As is apparent from the above description, ALP 6 -IgA having alkaline phosphatase and immunoglobulin A as constituent components among ALP 6 can be used as a marker for ischemic bowel disease.

EXAMPLES Hereinafter, although a manufacture example, a reference example, and an Example demonstrate this invention further in detail, this invention is not limited to these examples at all.
Production Example 1
Manufacture of anti-human TNSALP monoclonal antibody (1) Selection and preparation of antigen for monoclonal antibody production The present inventors are known to express TNSALP as an antigen for preparing anti-human TNSALP monoclonal antibody. ALP from the blast cell line Saos-2 was purified. The purification method is described below.
Saos-2 (HTB-85) was obtained from ATCC (American Type Culture Collection) and cultured in α-MEM (Dainippon Pharmaceutical) containing 10% fetal calf serum (FCS). When about 80% of the cells became confluent, the dish was washed 3 times with 50 mM Tris buffer (pH 7.5), and 50 mM Tris buffer (pH 7.5) containing 0.5% adekatol SO-135 was added. The cells were collected using a rubber policeman and homogenized for 1 minute on ice with an ultrasonic homogenizer. The cell lysate was centrifuged (22,000 rpm 30 min RT), and the supernatant was fractionated with 40% ammonium sulfate. Centrifugation (10,000 rpm 30 min RT) was performed, and ammonium sulfate was further dissolved in the supernatant to adjust to 60% ammonium sulfate, followed by re-fractionation. This 40-60% ammonium sulfate fraction precipitate was redissolved in 50 mM Tris buffer (pH 7.5) and dialyzed against 50 mM Tris buffer (pH 7.5). The dialyzed sample was applied to a DEAE column (Amersham Bioscience), and the adsorbed protein was eluted with a linear concentration gradient (0-0.5 M NaCl) of the above Tris buffer containing NaCl.
The peak ALP activity was measured using an N-assay ALP-LS reagent (Nittobo) using a Hitachi 7150 automatic analyzer. The active fraction was concentrated by ultrafiltration and purified by S-300 column (Amersham Bioscience). When this active fraction was concentrated in the same manner as DEAE and confirmed by SDS-PAGE, TNSALP was almost the major, and this was used as an antigen for immunization.

(2) Immunopurification Dilute TNSALP with 50 mM Tris acid buffer (pH 7.5) to 1 mg / ml, take 50 μg (50 μl) and mix well until emulsified with 50 μl Freund complete adjuvant (WAKO). did. The prepared suspension was intraperitoneally administered to Balb / c 6-week-old female mice (Clear Japan) under diethyl ether anesthesia. Two weeks later, the same amount of TNSALP (50 μg / ml) was mixed with Freund's incomplete adjuvant (Wako Pure Chemical Industries, Ltd.) to make an emulsified suspension by the same procedure as Freund's complete adjuvant, Made. Thereafter, the same procedure was performed 2 weeks later. In the fourth round, 50 μg / ml of TNSALP was prepared as a final immunization in 50 mM tris-acid buffer (pH 7.5) and administered by tail vein injection of mice.

(3) Establishment of hybridoma Three days after the final immunization, the spleen that was surgically removed under anesthesia with diethyl ether from mice sensitized with TNSALP was aseptically dispersed to prepare spleen cells. Fusion is performed according to the method of Kohler and Milstein (Nature. 256, 495. 1975), and splenocytes and myeloma cells using polyethylene glycol (PEG 4000) (Merck) P3-X63-Ag8-U1 (P3U1) Fused. The fusion ratio was 5: 1 with 10 × 10 ⁷ spleen cells and 2 × 10 ⁷ myeloma cells P3-X63-Ag8-U1 (P3U1). The fused cells are dispersed in 10% FCS (Invitrogen) α-MEM (Irvine) HAT (Cosmo Bio) medium, dispensed into 96-well microtiter culture plates (Sumitomo Bakelite), and cultured at 37 ° C and 5% CO ₂ conditions. did.

(4) Screening Screening was performed after confirming the growth of colonies after about 2 weeks. The screening method is described below.
To prepare the screening plate, TNSALP purified in (1) above was dissolved in 50 mM tris-acid buffer and dispensed into 96-wells (NUNK) at 0.5 μg / 100 μl / well. . The plate is allowed to stand at 4 ° C for 2 nights, then washed 3 times with Tris buffer containing 0.05% Tween 20, and 200 µl of 1.5% BSA solution is added to suppress non-specific reaction. I left still overnight. The completed plate was washed 3 times with Tris buffer containing 0.05% Tween 20, and then 100 µl of the culture supernatant was reacted. After further washing, the secondary antibody, HRP-labeled anti-mouse immunoglobulin antibody (Zymed) was added. In addition, it was made to react. After washing, add 100 μl of 3 mg / ml o-phenylenediamine (OPD) (Nacalai Tesque) citric acid coloring solution, which is a color developing substrate of HRP, and after a certain period of color development, add another 100 μl of 1 N sulfuric acid as a stop solution, Absorbance was measured at a measurement wavelength of 492 nm. Clones that became positive as described above were recloned by limiting dilution and the supernatant was checked again.

(5) Confirmation of antibody
The reactivity with purified TNSALP was confirmed by ELISA, and the inventors selected that clones 3-29-3R, 2C5-32, and 1A5-7 recognized TNSALP. The results of assaying the obtained antibodies with a monoclonal antibody typing kit (Amersham Biosciences) are shown in Table 1 below.

(6) Production and purification of monoclonal antibody 2 weeks after administration of 0.5 ml pristane (Aldrich) 1 × 10 ⁷ hybridoma 3-29-3R, 2C5-32, 1A5-7 cells obtained in (5) above Balb / c mice (CLEA Japan, Inc.), 10 weeks old, were administered intraperitoneally to females, and ascites collected in the abdominal cavity of mice about 2 weeks later was surgically collected under diethyl ether anesthesia. When the ascitic fluid was used as a sample and confirmed by serial dilution by ELISA, a high concentration of monoclonal antibody was contained. This ascites was treated with 40% ammonium sulfate and dialyzed against PBS. 3-29-3R and 1A5-7 were purified with a protein G column (Amersham Biosciences) and confirmed by SDS-PAGE. 2C5-32 was purified using S-300. As a result, 3-29-3R and 1A5-7 showed a single band with a molecular weight of about 150,000 in non-reduction, and a band with a molecular weight of about 50,000 and two bands of 25,000 in reduction with mercaptoethanol. 2C5-32 showed a single band with a molecular weight of about 900,000 when it was not reduced, and two bands with a molecular weight of about 70,000 and 25,000 when it was reduced with mercaptoethanol. The purified monoclonal antibodies 3-29-3R, 1A5-7, and 2C5-32 were each about 10 mg or more per mouse and were sufficient for industrial use.

Reference example 1
Specificity assay of anti-human TNSALP monoclonal antibody To investigate the specificity of monoclonal antibodies 3-29-3R, 2C5-32, 1A5-7, various isozymes, namely TNSALP derived from Saos-2, liver ALP (ordinary light), The following experiments were performed using bone-type ALP (Tokomitsu), small intestine-type ALP (Tokomitsu), and placenta-type ALP (Sigma). The measurement method is as follows.
The purified monoclonal antibody was dispensed on a solid layer plate (NUNC) to 2 μg / 100 μL PBS / well and allowed to stand at 4 ° C. for 2 days. After washing 3 times with a 20 mM Tris buffer (pH 7.0) washing solution containing 0.05% Tween 20, 200 μl of 1.5% BSA Tris buffer (pH 7.0) was added and blocked overnight at 4 ° C. The plate thus prepared was washed once with the previous washing solution and reacted with various isozymes. The enzyme activity of the isozyme was measured using the Kind-King modified reagent (formazan method, using Nittobo N-Test ALP KK kit), and 100 μl was added to the antibody-binding plate in accordance with 25 IU / L. The reaction was allowed to proceed at room temperature for 2 hours, and after the completion of the reaction, the plate was washed 3 times with the above washing solution, and the enzyme activity was measured according to the Kind-King modified method (formazan method). That is, ALP enzyme bound to the antibody plate was added with 100 μl of a substrate solution (a solution containing disodium phenylphosphate and 4-aminoantipyrine solution) and reacted at 37 ° C. for 1 hour to capture the ALP enzyme captured by the antibody. Then, a color developing reagent (solution containing sodium metaperiodate) was added to cause color development, and the absorbance was measured at 492 nm. The data at this time is shown in Table 2. At this time, a blank value obtained by the same operation without adding ALP is obtained, and a value obtained by subtracting the blank value from each measured value becomes the activity value of each ALP against the antibody.

As can be seen from Table 2, antibodies 3-29-3R and 2C5-32 react only with Saos-2-derived TNSALP, liver and bone, and show little cross-reactivity with other isozymes. It was found to be a highly specific monoclonal antibody. That is, antibodies 3-29-3R and 2C5-32 are specific to TNSALP.For example, the sum of small intestine type ALP activity value and placental type ALP activity value is the difference between liver type ALP activity value and bone type ALP activity value. It was found to be less than 1/10 of the sum.
1A5-7 reacted well with all isozymes including TNSALP derived from Saos-2. In other words, 1A5-7 was found to react with any ALP isozyme.
In addition, the liver type and bone type reaction ratio (liver: bone reaction ratio) at 25 IU / L of the two monoclonal antibodies was 1: 29.30 for 3-29-3R and 1: 0.93 for 2C5-32. . By the way, in general, ALP 6 tends to appear when the liver type becomes stronger than the bone type in the electrophoresis result of the high ALP sample. Therefore, antibody 3-29-3R is more effective when used in the present invention because it has a bone type ALP activity of 0.7 or less of liver type ALP activity.

Reference example 2
Measurement of serum TNSALP level by immunoenzyme activity measurement method Monoclonal antibodies 3-29-3R and 1A5-7 prepared and purified by the above method were applied to Nunc plates in the same manner as in Reference Example 1 at 2 μg / 100 μL. An antibody-binding plate was prepared by dispensing and blocking with PBS. Prior to use, the plate was washed once with 200 μL of 100 mM Tris buffer (pH 7.5) containing 0.05% Tween 20, and 10 μL of informed consent volunteers and patient samples and 100 mM Tris were added. 90 μL of buffer solution (pH 7.5) was added and reacted at room temperature for 1 hour. After completion of the reaction, the plate was washed 3 times with 200 μL of 100 mM Tris buffer (pH 7.5) containing 0.05% Tween 20. Next, add 100 μl of N-test ALP Kind-King reagent (Nittobo) substrate solution, that is, a solution containing disodium phenylphosphate and 4-aminoantipyrine to the plate, and further add an additional 1 hour at 37 ° C, pH 10. The enzyme reaction was performed. Further, 100 μL of a coloring reagent (solution containing sodium metaperiodate) was added to the reaction solution, and the TNSALP value was determined by colorimetrically comparing the coloring value at 492 nm. High-value samples exceeding the measurement range were diluted and remeasured.
Table 3 shows the results of TNSALP values obtained using monoclonal antibodies 3-29-3R and 1A5-7. These results were compared with the total ALP activity value (JSCC compliant value).

  The TNSALP value obtained using monoclonal antibody 3-29-3R and the total ALP activity value are strongly different from each other in the sample 15 data. On the other hand, there is a strong correlation between the TNSALP value obtained using monoclonal antibody 1A5-7 and the total ALP activity value (JSCC compliant value), including the sample 15 data. In other words, the TNSALP activity value obtained using antibody 3-29-3R differs from the total ALP activity value in that it does not include the placental type ALP or small intestine type ALP activity values. ALP increases due to increased ALP and increased ALP due to small intestine type secretion (ALP) after meals of blood group O and B. Therefore, use of antibody 3-29-3R as the primary antibody is considered to be more advantageous when ALP 6-IgA is measured by ELISA.

Example 1
Comparison of ALP 6-IgA and ALP 6-IgG values by ELISA and sensitivity of ALP6 measurement by disk electrophoresis
ALP 6-IgA and ALP 6-IgG values were determined by ELISA using two human sera 80-21 and 81-23 that were positive for ALP 6 by disk electrophoresis. Next, the sensitivity of the ALP 6 measurement method by disk electrophoresis was compared with those measurement methods. This will be described in detail below.
In the ELISA method, a 3-29-3R plate was used as a primary antibody, and a 1 / 200-diluted HRP-labeled anti-human IgA monoclonal antibody and IgG monoclonal antibody (Zymed) were used as secondary antibodies that are immunoglobulin binding substances. First, the antibody 3-29-3R plate was washed once with 200 μL of 100 mM Tris buffer (pH 7.5) containing 0.05% Tween 20. Next, ALP 6 positive specimens are diluted 1-fold, 10-fold, 20-fold, 40-fold, 80-fold to these plates, and 50 μL of each sample and 50 μL of 100 mM Tris buffer (pH 7.5) are added at room temperature. The reaction was performed for 1 hour. After the primary antibody reaction, the plate was washed three times with 200 μL of 100 mM Tris buffer (pH 7.5) containing 0.05% Tween 20, and 100 μl of secondary labeled antibody HRP-labeled anti-human IgA monoclonal antibody or IgG monoclonal antibody ( Zymed) was added, and the mixture was further reacted at room temperature for 1 hour. After washing for 1 hour, react with the substrate solution (OPD solution) for labeled HRP. After a certain time, add 100 μL of color stop reagent 1 N sulfuric acid and colorize the color value at 492 nm. -IgA and ALP 6-IgG values were determined.
On the other hand, Disk electrophoresis used ALP isozyme detection kit “Alfour” (Joko). The same ALP 6 positive patient specimens used in ELISA as specimens were diluted 1-fold, 10-fold, 20-fold, 40-fold, 80-fold, and 25 μL each was used. The operating method was carried out according to the ALP isozyme detection kit “Alfour” (Joko) 's document.

  The result of electrophoresis is shown in FIG. 1, and the result of ELISA is shown in FIG. As a result, ALP 6 was confirmed only by using the sample stock solution as the sample by electrophoresis, but the value was confirmed by the ELISA method until the sample was diluted 40 to 80 times. Compared with electrophoresis, ELISA showed that ALP 6-IgA was detected with 40 to 80 times higher sensitivity, and good dilution linearity was observed. On the other hand, ALP 6-IgG did not reach this level. This indicates that the ELISA method can detect and quantify a trace amount of ALP 6-IgA that could not be detected by electrophoresis.

Example 2
Diagnosis of ischemic bowel disease by multiplicative ALP 6-IgA measurement Using antibody 3-29-3R as the primary antibody and HRP-labeled anti-IgA monoclonal antibody and IgG monoclonal antibody as the secondary antibody, sample by ELISA ALP 6-IgA and ALP 6-IgG values in the sample, and ALP 6-IgA and ALP 6-IgG values in the sample by multiplicative values, can be applied to the diagnosis of ischemic bowel disease investigated. As specimens, we used 69 specimens of healthy volunteers who gave informed consent and 98 specimens of serum from patients with ischemic bowel disease. ALP 6-IgA and ALP 6-IgG values determined by ELISA were determined by the same method as in Example 1. Further, the TNSALP value was measured by the method described in Reference Example 2 using monoclonal antibody 3-29-3R. The ALP 6-IgA value or ALP 6-IgG value based on the multiplicative value was obtained by calculating the multiplicative value between the “ALP 6-IgA value or ALP 6-IgG value based on the ELISA method” and the “TNSALP value”.
Table 4 shows the results of the average values of ALP 6-IgA value and ALP 6-IgG value by ELISA method.

Using this ALP 6-IgA value, a test was conducted on the difference between the mean values of healthy subjects and patients with ischemic bowel disease. A p-value of 0.0006 in the t-test showed a significant difference between the two groups. From this result, it became clear that ischemic bowel disease can be diagnosed more specifically by using an HRP-labeled anti-human IgA monoclonal antibody as the secondary antibody (positive rate 51%). On the other hand, in the ALP 6-IgG value, there was almost no difference in the average value between normal subjects and patients.
From the above results, it is possible to precisely measure ALP 6-IgA using the reaction system example of the present invention (primary antibody: human TNSALP-recognizing monoclonal antibody, secondary labeled antibody: HRP-labeled anti-human IgA monoclonal antibody). Thus, it has been found that it can be an index for diagnosis of ischemic bowel disease.

  In the measurement method of the present invention, ALP 6-IgA that could not be measured conventionally can be easily measured. Furthermore, conventionally, there has been no useful diagnostic marker for ischemic bowel disease using a blood sample, but in the present invention, ischemic bowel disease can be diagnosed by measuring ALP 6-IgA in blood.

The results of disk electrophoresis are shown. As shown by the arrow, the ALP 6 band can be confirmed only when the sample stock solution is used. The dilution linearity of ALP 6-IgA and ALP 6-IgG in the sample is shown. The vertical axis indicates the absorbance, and the horizontal axis indicates the diluted concentration when the stock solution is 1.0. In the measurement of ALP 6-IgA, even a sample diluted 80 times can be detected as a numerical value.

Claims (17)

  A method for measuring IgA-binding alkaline phosphatase 6 in a sample, comprising an IgA-binding alkaline phosphatase 6 in a sample and an alkaline phosphatase site recognition substance that recognizes an alkaline phosphatase site that is a constituent of IgA-binding alkaline phosphatase 6 , Reacting with an immunoglobulin A site recognition material that recognizes an immunoglobulin A site, which is a component of IgA-binding alkaline phosphatase 6, and obtaining the resulting alkaline phosphatase site recognition material, IgA-binding alkaline phosphatase 6 and immunoglobulin A site recognition A method for measuring IgA-binding alkaline phosphatase 6, comprising measuring IgA-binding alkaline phosphatase 6 from the level of a substance-bound substance.   2. The method according to claim 1, wherein the alkaline phosphatase site recognition substance is a substance capable of binding to human organ non-specific alkaline phosphatase.   The method according to claim 2, wherein the substance capable of binding to human organ non-specific alkaline phosphatase is an anti-human organ non-specific alkaline phosphatase monoclonal antibody.   4. The method according to claim 1, wherein the immunoglobulin A site recognition substance is a substance capable of binding to IgA.   The method according to claim 4, wherein the substance capable of binding to IgA is an anti-human IgA monoclonal antibody.   A method for measuring IgA-binding alkaline phosphatase 6 in a specimen, comprising reacting IgA-binding alkaline phosphatase 6 in a specimen with an antibody that reacts with human organ non-specific alkaline phosphatase, and then antigen antibody obtained The reaction product reacts with an immunoglobulin A site-recognizing substance that recognizes the immunoglobulin A site, which is a component of IgA-binding alkaline phosphatase 6, and the antibody reacting with non-human organ-specific alkaline phosphatase is obtained. A method for measuring IgA-binding alkaline phosphatase 6, characterized in that IgA-binding alkaline phosphatase 6 is measured from the level of a conjugate with an immunoglobulin A site recognition substance that is a constituent of alkaline phosphatase 6.   The method according to claim 6, wherein the immunoglobulin A site recognition substance is a substance capable of binding to IgA.   The method according to claim 7, wherein the substance capable of binding to IgA is an anti-human IgA monoclonal antibody. A method for measuring IgA-binding alkaline phosphatase 6 in a sample, comprising:
i) Human organ non-specific alkaline phosphatase in a specimen is reacted with an antibody that reacts with human organ non-specific alkaline phosphatase and then antigen-antibody-reacted human organ non-specific alkaline phosphatase is converted into an enzyme substrate for alkaline phosphatase From the level of the enzyme activity that was reacted, the human organ non-specific alkaline phosphatase activity in the sample was determined,
ii) Independently of the above i), the level of IgA-binding alkaline phosphatase 6 in the sample is determined by the method according to any one of claims 6 to 8,
iii) Obtain the multiplicative value of the human organ non-specific alkaline phosphatase activity value obtained in i) above and the level value of IgA-binding alkaline phosphatase 6 obtained in ii) above, and determine IgA binding in the sample from the multiplicative value. Determining the amount of alkaline phosphatase 6;
A method for measuring IgA-binding alkaline phosphatase 6 characterized by the above. A kit for measuring IgA-binding alkaline phosphatase 6 in a specimen,
i) a solid support,
ii) an alkaline phosphatase site recognition material that recognizes an alkaline phosphatase site that is a constituent of IgA-binding alkaline phosphatase 6;
iii) a labeled immunoglobulin A site-recognizing substance that recognizes an immunoglobulin A site that is a constituent of IgA-binding alkaline phosphatase 6, and
iv) components for detecting the label,
Including kit.   11. The kit according to claim 10, wherein the alkaline phosphatase site recognition substance is a substance capable of binding to human organ non-specific alkaline phosphatase.   The kit according to claim 11, wherein the substance capable of binding to human organ non-specific alkaline phosphatase is an anti-human organ non-specific alkaline phosphatase monoclonal antibody.   The kit according to any one of claims 10 to 12, wherein the immunoglobulin A site recognition substance is a substance capable of binding to IgA.   The kit according to claim 13, wherein the substance capable of binding to IgA is an anti-human IgA monoclonal antibody.   The kit according to any one of claims 10 to 14, which is used for diagnosis of ischemic bowel disease.   A method for diagnosing ischemic bowel disease by determining the level of IgA-binding alkaline phosphatase 6 in a sample from a human suspected of being ischemic bowel disease according to any one of claims 1-9. A marker for ischemic bowel disease comprising IgA-binding alkaline phosphatase 6 having an alkaline phosphatase site and an immunoglobulin A site as constituent components.