Patent Application
20240151722
The present invention generally relates to a monoclonal antibody or a fragment thereof targeting tetranor-PGDM, a use thereof, and the like.
A food allergy is a disease that causes symptoms such as hives, diarrhea, and vomiting due to ingested food and also cause anaphylaxis, which may lead to death. The number of patients with this disease, which is common in children, is rapidly increasing, and it is becoming a more serious problem.
As an allergy testing method, measurement of the blood IgE antibody concentration is widely used, but this test does not show whether eating food causes symptoms. Therefore, the only method to make a definitive diagnosis of a food allergy is to perform an “oral antigen load test”. However, this requires a great deal of effort and time, and has a risk of causing anaphylaxis. In addition, “oral immunotherapy” may be used as a food allergy treatment, but it is necessary to continue to eat small amounts of the antigen without causing symptoms for several years, which also has a risk of accidents. Thus, the lack of simple and accurate diagnostic methods and symptom evaluation methods has become a bottleneck in the development of methods for diagnosing and treating the increasing number of patients with a food allergy.
The inventors found that tetranor-PGDM (9α-hydroxy-11,15-dioxo-13,14-dihydro-2,3,4,5-tetranor-prostan-1,20-dioic acid), which is a metabolite of lipid prostaglandin (PG)D2, is excreted in urine that is easy to collect even from children, in a manner specific to food allergies and in proportion to the severity of symptoms (Patent Document 1). However, this concentration measurement requires use of a mass spectrometer, which is difficult to operate and expensive.
Polyclonal antibodies against tetranor-PGDM are known (https://www.caymanchem.com/product/501001/tetranor-pgdm-elisa-kit; U.S. Patent Application Publication No. 2012/0021437 (Patent Document 2)), but polyclonal antibodies are inferior to monoclonal antibodies in terms of specificity, production stability, and measurement reproducibility. In addition to these quality issues, polyclonal antibodies are not applicable for testing human diseases due to lot-to-lot differences in performance.
Therefore, the development of monoclonal antibodies against tetranor-PGDM has been desired, but since tetranor-PGDM has a small molecular weight as an antigen and is difficult for antibodies to recognize, and also exists in vivo, it is difficult to develop monoclonal antibodies against tetranor-PGDM.
The inventors succeeded for the first time in preparing a monoclonal antibody specific to tetranor-PGDM by immunizing mice that had no genes encoding two types of PGD2 synthases, a hematopoietic type and a lipocalin type and did not produce PGD2 or its metabolite tetranor-PGDM with a complex of tetranor-PGDM and keyhole limpet hemocyanin (KLH).
Here, in Patent Document 2, a method of producing monoclonal antibodies against tetranor-PGDM by injecting a complex of tetranor-PGDM and keyhole limpet hemocyanin (KLH) into wild-type mice is described, but there is no detailed description of the obtained antibodies. When the inventors performed immunization on wild-type mice in the same manner, no monoclonal antibody specific to tetranor-PGDM was obtained.
In addition, in Patent Document 2, the cross-reactivity of the antibody against tetranor-PGDM, which is thought to be a polyclonal antibody, is described (paragraph 0347), but the antibody is different from the monoclonal antibody (for example, Tetranor-PGJM 563%; Tetranor-PGDM 100%; Tetranor-PGEM 0.00%) of the present invention, which does not substantially bind to tetranor-PGEM, in that it binds not only to tetranor-PGDM but also to tetranor-PGEM (Tetranor-PGJM 376%; Tetranor-PGDM 100%; Tetranor-PGEM 0.3%). Both antibodies are common in that they bind to tetranor-PGJM, but tetranor-PGJM is hardly produced in vivo and is not excreted in urine. On the other hand, since a large amount of tetranor-PGEM is produced in the body and detected in urine, whether it binds to tetranor-PGEM is a very important difference. Such a difference is thought to be caused by, for example, a difference in CDR.
That is, the present invention encompasses the following inventions.
Using the anti-tetranor-PGDM monoclonal antibody of the present invention, tetranor-PGDM, which is conventionally measured by mass spectrometry, can be simply detected through enzyme immunoassay or the like. Such an enzyme immunoassay method is expected to be useful for quantification of tetranor-PGDM in body fluids, evaluation of animal disease models, diagnosis indicators, and treatment monitoring of food allergies and other diseases.
In a first aspect, a monoclonal antibody or a fragment thereof that specifically binds to tetranor-PGDM is provided. As used herein, this antibody hereinafter will be simply referred to as an anti-tetranor-PGDM monoclonal antibody. As used herein, “specifically binds to tetranor-PGDM” means that an antibody does not substantially bind to tetranor-PGEM, tetranor-PGFM, or tetranor-PGAM, and specifically binds to tetranor-PGDM or tetranor-PGJM. A monoclonal antibody that does not bind to tetranor-PGEM is preferable because it is excreted particularly abundantly in body fluids including urine. Here, since tetranor-PGJM is hardly produced in vivo and is not excreted in urine, it can be said that the anti-tetranor-PGDM monoclonal antibody is substantially a tetranor-PGDM-specific antibody.
Anti-tetranor-PGDM monoclonal antibodies can be produced using hybridomas obtained by fusing antibody-producing cells with myelomas. The produced antibodies may be further subjected to an additional step such as a purifying step before recovery, and for example, in order to increase the purity, it is preferable to perform salting-out, filter filtration, clarification using centrifugation, recovery by affinity, intermediate purification by ion exchange, and final purification by gel filtration.
Examples of anti-tetranor-PGDM monoclonal antibodies obtained in this manner include antibodies including at least one or more CDRs and preferably all six CDRs selected from the group consisting of the following complementarity-determining regions (CDRs):
Antibodies generally have a structure in which two heavy chains and two light chains are covalently bound by disulfide bonds, and the heavy chains are divided into a heavy chain variable region and a heavy chain constant region, and the light chains are divided into a light chain variable region and a light chain constant region. Each variable region comprises three CDRs. Here, CDRH1, CDRH2, and CDRH3 represent the complementarity-determining regions of the heavy chain in order from the amino terminal side of the heavy chain amino acid sequence, and CDRL1, CDRL2, and CDRL3 represent the complementarity-determining regions of the light chain in order from the amino terminal side of the light chain amino acid sequence.
Therefore, the anti-tetranor-PGDM monoclonal antibody or a fragment thereof may be an antibody or a fragment thereof which preferably comprises a heavy chain variable region including:
The amino acid sequence of each CDR may have deletion, substitution or addition of one or several amino acids as long as the anti-tetranor-PGDM monoclonal antibody or a fragment thereof specifically binds to tetranor-PGDM. As used herein, although it varies depending on the length of the sequence, “several” is 2 to 10, preferably 2 to 5, and more preferably 2 or 3.
The amino acid after substitution is desirably an amino acid that preserves properties of the original amino acid side chains as classified below. However, amino acid substitutions are not limited to conservative substitutions.
In one embodiment, amino acid substitutions may occur at amino acids present at corresponding positions in a natural human antibody.
In one embodiment, amino acid substitutions may occur outside of the complementarity-determining region (CDR).
In one embodiment, amino acid substitutions may occur in the heavy chain variable region or the light chain variable region.
Alternatively, the amino acid sequence of each CDR may be consisting of amino acid sequences having a homology of 80% or more, and preferably identity of 80% or more with respect to the sequence shown in each SEQ ID NO as long as it specifically binds to tetranor-PGDM. The homology or identity of the sequence is preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
A or more, and more preferably 99% or more.
In one embodiment, the anti-tetranor-PGDM monoclonal antibody may be an antibody or a fragment thereof that binds to an epitope to which an antibody or a fragment thereof binds, which comprises a variable region including at least one or more CDRs selected from the group consisting of the following:
As used herein, “epitope” means a partial structure of tetranor-PGDM to which an anti-tetranor-PGDM monoclonal antibody or a fragment thereof binds. As such a partial structure, carbon chains at positions 7 to 11 of tetranor-PGDM represented by the following chemical formula:
particularly the 11-keto group, can be considered. The 7th to 11th positions are numbered based on the carbon number of PGD2, and the 1st to 4th positions are missing numbers.
In one embodiment, the anti-tetranor-PGDM monoclonal antibody may be an antibody or a fragment thereof which comprises a heavy chain variable region including the following:
In one embodiment, the heavy chain variable region in the anti-tetranor-PGDM monoclonal antibody or a fragment thereof may comprise at least 109 consecutive amino acids of the amino acid sequence shown in any of SEQ ID NOs: 9, 22, 35, 48, 61, and 74.
As used herein, “at least 109 consecutive amino acids of the amino acid sequence shown in any of SEQ ID NOs: 9, 22, 35, 48, 61, and 74” indicates 109, 110, 112 or more consecutive amino acids in the amino acid sequence shown in any of SEQ ID NOs: 9, 22, 35, 48, 61, and 74. Such consecutive amino acids preferably comprise amino acid sequences of the above CDRH1, CDRH2 and CDRH3.
In one embodiment, the heavy chain variable region in the anti-tetranor-PGDM monoclonal antibody or a fragment thereof may comprise amino acid sequences with deletion, substitution or addition of one or several amino acids in the amino acid sequence shown in any of SEQ ID NOs: 9, 22, 35, 48, 61, and 74 and the amino acid sequence shown in any of SEQ ID NOs: 9, 22, 35, 48, 61, and 74, or amino acid sequence or amino acid sequences with at least 80% identity to the amino acid sequence shown in any of SEQ ID NOs: 9, 22, 35, 48, 61, and 74.
In one embodiment, the anti-tetranor-PGDM monoclonal antibody may be an antibody or a fragment thereof which comprises a light chain variable region including the following:
In one embodiment, the anti-tetranor-PGDM monoclonal antibody may be an antibody or a fragment thereof which comprises a variable region including the following:
In one embodiment, when Xaa2 in CDRH1 is N, Xaa1 may be F, and Xaa3 may be A; or when Xaa2 is R, Xaa1 may be F, A or S.
Xaa1 is preferably F. Xaa2 is preferably N. Xaa3 is preferably A. CDRH1 in which Xaa1 is F, Xaa2 is N, and Xaa3 is A is preferable.
In one embodiment, when Xaa4 in CDRH2 is N, Xaa5-Xaa6-Xaa7- Xaa8 may be FNDG; or when Xaa4 is Y, Xaa5-Xaa6-Xaa7 may be GDG, and Xaa8 may be N or D.
Xaa4 is preferably N. Xaa5-Xaa6-Xaa7-Xaa8 is preferably FNDG. CDRH2 in which Xaa4 is N, and Xaa5-Xaa6-Xaa7-Xaa8 is FNDG is preferable.
In one embodiment, when Xaa10 in CDRH3 is Y, Xaa9 may be R, Xaa11 may be Y, Xaa12 may be Y, Xaa13 may be Y, Xaa14 may be G, Xaa15 may be S, Xaa16 may be K, Xaa17 may be A, Xaa18 may be M, Xaa19 may be D, and Xaa20 may be Y; when Xaa10 is V, Xaa9 may be S or T, Xaa11 may be absent, Xaa12 may be absent, Xaa13 may be absent, Xaa14 may be S, Xaa15 may be R, Xaa16 may be W, Xaa17 may be F, Xaa18 may be F, and Xaa19 may be A, Xaa20 may be V.
Xaa9 is preferably R. Xaa10 is preferably Y. It is preferable that Xaa11 be present and particularly be Y. It is preferable that Xaa12 be present and particularly be Y. It is preferable that Xaa13 be present and particularly be Y. Xaa14 is preferably G. Xaa15 is preferably S. Xaa16 is preferably K. Xaa17 is preferably A. Xaa18 is preferably M. Xaa19 is preferably D. Xaa20 is preferably Y. CDRH3 in which Xaa9 is R, Xaa10 is Y, Xaa11 is Y, Xaa12 is Y, Xaa13 is Y, Xaa14 is G, Xaa15 is S, Xaa16 is K, Xaa17 is A, Xaa18 is M, Xaa19 is D, and Xaa20 is Y is preferable.
In one embodiment, when Xaa22 and Xaa23 in CDRL1 are each T, Xaa21 may be T or S, Xaa24 may be N, Y or K, and Xaa25 may be Y or H; or when Xaa22 is R and Xaa23 is I, Xaa21 may be T, and Xaa24 may be N, and Xaa25 may be Y.
Xaa21 is preferably T. Xaa22 is preferably T or R. Xaa23 is preferably T or I. Xaa24 is preferably N. CDRL1 in which Xaa21 is T, Xaa22 is T or R, Xaa23 is T or I, Xaa24 is N, and Xaa25 is Y is preferable. CDRL1 in which Xaa21 is T, Xaa22 is R, Xaa23 is I, Xaa24 is N, and Xaa25 is Y is more preferable.
In one embodiment, when Xaa26 in CDRL2 is G, Xaa27 may not be D. Xaa26 is preferably G. Xaa27 is preferably N. CDRL2 in which Xaa26 is G, and Xaa27 is N is preferable.
In one embodiment, when Xaa28 in CDRL3 is Y, Xaa29 may be V or I; when Xaa28 is F, Xaa29 may be V; or when Xaa28 is N, Xaa29 may be V. CDRL3 in which Xaa28 is Y or N, and Xaa29 is V is preferable.
In one embodiment, the light chain variable region in the anti-tetranor-PGDM monoclonal antibody or a fragment thereof may comprise at least 99 consecutive amino acids of the amino acid sequence shown in any of SEQ ID NOs: 95 to 99.
As used herein, “at least 99 consecutive amino acids of the amino acid sequence shown in any of SEQ ID NOs: 16, 29, 42, 55, 68, and 81” indicates 99, 100, 101, 102 or more consecutive amino acids in the amino acid sequence shown in any of SEQ ID NOs: 16, 29, 42, 55, 68, and 81. Such consecutive amino acids preferably comprise the above CDRL1, CDRL2 and CDRL3.
In one embodiment, the light chain variable region in the anti-tetranor-PGDM monoclonal antibody or a fragment thereof may comprise amino acid sequence with deletion, substitution or addition of one or several amino acids in the amino acid sequence shown in any of SEQ ID NOs: 16, 29, 42, 55, 68, and 81 and the amino acid sequence shown in any of SEQ ID NOs: 16, 29, 42, 55, 68, and 81 or amino acid sequences with at least 80% identity to the amino acid sequence shown in any of SEQ ID NOs: 16, 29, 42, 55, 68, and 81.
As long as the anti-tetranor-PGDM monoclonal antibody comprises the above CDRs, other structures, for example, the structures of variable regions and constant regions other than CDRs, are not particularly limited, and examples of amino acid sequences encoding the heavy chain variable region containing the above CDRH1, CDRH2 and CDRH3 and the light chain variable region containing the CDRL1, CDRL2 and CDRL3 comprise amino acid sequences of SEQ ID NOs: 9, 22, 35, 48, 61, and 74 and amino acid sequences of SEQ ID NOs: 16, 29, 42, 55, 68, and 81.
In a preferable embodiment, the anti-tetranor-PGDM monoclonal antibody or a fragment thereof may comprise a heavy chain variable region having an amino acid sequence of SEQ ID NOs: 9, 22, 35, 48, 61, and 74 and a light chain variable region having an amino acid sequence of any of SEQ ID NOs: 16, 29, 42, 55, 68, and 81.
In a preferable embodiment, the humanized anti-tetranor-PGDM monoclonal antibody or a fragment thereof may comprise a heavy chain variable region having an amino acid sequence of any of SEQ ID NOs: 9, 22, 35, 48, 61, and 74 and a light chain variable region having an amino acid sequence of any of SEQ ID NOs: 16, 29, 42, 55, 68, and 81.
As used herein, “antibody” refers to one that occurs naturally or a wholly or partially synthetically produced artificial immunoglobulin or a fragment thereof. Antibodies may be of any isotype of IgG, IgM, IgA, IgE, and IgD. Nine types of classes (isotypes) of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM are known as human immunoglobulins.
Without limitation on the origin of antibodies, any species of mammals, for example, humans, and non-human animals such as mice, rats, guinea pigs, hamsters, rabbits, goats, sheep, and camels, may be used. Not only human antibodies but also recombinant antibodies such as humanized antibodies and chimeric antibodies can be used. As used herein, “humanized antibody” is one in which the CDRs of a human antibody are replaced with CDRs derived from a non-human animal antibody. In addition to the CDRs, some framework amino acid residues may be grafted into the human antibody. When CDRs derived from non-human animal antibodies are used in humans, they are preferably combined with constant regions of human antibodies.
A chemical or biological modification may be performed on the anti-tetranor-PGDM monoclonal antibody as long as a desired effect is obtained. Chemical modification components include attachment of chemical moieties to the amino acid framework, chemical modification components of N-linked or O-linked carbohydrate chains and the like. Biological modification components may include post-translational modifications (for example, N-linked or O-linked glycosylation, N-term inal or C-terminal processing, deamidation, aspartic acid isomerization, and methionine oxidation) and addition of a methionine residue to the N terminal by expression using a prokaryotic host cell.
Modification components may further include deletion components in which one or two amino acids are deleted at the heavy chain carboxyl terminal and deletion components that are amidated.
The two heavy chains constituting the anti-tetranor-PGDM monoclonal antibody may include a combination of a full length chain and any one heavy chain selected from the group consisting of the above deletion components or a combination of any two heavy chains.
As used herein, a “fragment” of an antibody is a functional fragment that exhibits at least a part of the function exhibited by the antibody before it is fragmented. Examples thereof include Fab, F(ab′)2, scFv, Fab′, and single chain antibodies (scFv). The fragment is not limited to these molecules, and may be any fragment as long as it specifically binds to tetranor-PGDM. For example, the anti-tetranor-PGDM monoclonal antibody may be an antibody having a single heavy chain variable region and having no light chain sequence such as a single domain antibody or nanobody.
The anti-tetranor-PGDM monoclonal antibody or a fragment thereof may comprise a linker depending on the purpose, and for example, when a plurality of variable regions constituting an antigen-binding site of an antibody are bound as in a single chain antibody, a linker may be interposed therebetween. For example, a single chain antibody is obtained by linking a heavy chain variable region and a light chain variable region via a linker. The linker sequence can be appropriately determined by those skilled in the art.
The fragment of the anti-tetranor-PGDM monoclonal antibody can be obtained by treating full-length molecules of antibody proteins with enzymes such as papain and pepsine. Alternatively, a gene encoding a desired fragment may be expressed in suitable host cells to produce the fragment. When host cells are transformed, the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector or can be inserted into separate expression vectors. Host cells may be either prokaryotic cells or eukaryotic cells. Examples of prokaryotic cells include bacterial cells such as E. coli and bacillus subtilis. When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. Animal cells, plant cells, or fungal cells can be used. Examples of animal cells include the following cells.
In a second aspect, a polynucleotide encoding the anti-tetranor-PGDM monoclonal antibody or a fragment thereof of the present invention (polynucleotide of the present invention) is provided.
The polynucleotide of the present invention may be a single- or double-stranded polynucleotide (DNA including cDNA and the like or RNA including mRNA, cRNA and the like) including nucleotide sequence encoding an amino acid sequence of an anti-tetranor-PGDM monoclonal antibody or a fragment thereof and/or its complementary chain.
The heavy chain variable region (or heavy chain) and the light chain variable region (or light chain) of the anti-tetranor-PGDM monoclonal antibody or a fragment thereof may be encoded on the same polynucleotide, encoded on separate polynucleotides and provided as a combination thereof. The “polynucleotide encoding an anti-tetranor-PGDM monoclonal antibody or a fragment thereof” also includes a combination of a polynucleotide encoding a heavy chain variable region (or heavy chain) and a polynucleotide encoding a light chain variable region (or light chain).
Examples of polynucleotides encoding an anti-tetranor-PGDM monoclonal antibody or a fragment thereof include those having a base sequence encoding at least one or more CDRs and preferably a base sequence encoding all six CDRs selected from the group consisting of the following:
Examples of VH base sequences including the base sequences (a) to (c) include SEQ ID NOs: 8, 21, 34, 47, 60, and 73. Examples of VL base sequences including the base sequences (d) to (f) include SEQ ID NOs: 15, 28, 41, 54, 67, and 80. A polynucleotide encoding a gene for an antibody or a fragment thereof may have substantially the same base sequence as a base sequence shown in any of the above SEQ ID Nos. Substantially the same base sequence means one, in a polynucleotide composed of a sequence having a homology of 80% or more, preferably an identity of 80% or more, for example, 81 0/0, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and preferably an identity of 99% or more, with respect to the base sequence shown in each SEQ ID NO, or alternatively in a polynucleotide that can hybridize under stringent conditions with a polynucleotide composed of a sequence complementary to a base sequence shown in each SEQ ID NO, a protein encoded with the polynucleotide has a desired function, for example, specifically binding to tetranor-PGDM.
Here, stringent conditions are hybridization conditions that are easily determined by those skilled in the art, and are generally empirical experiment conditions depending on the base length of the nucleic acid, the washing temperature, and the salt concentration. Generally, when the base is longer, the temperature for appropriate annealing is higher, and when the base is shorter, the temperature is lower. Hybrid formation generally depends on the ability of reannealing of the complementary chain in an environment of which the temperature is slightly lower than the melting point.
In one embodiment, a polynucleotide encoding an anti-tetranor-PGDM monoclonal antibody or a fragment thereof comprises the following:
In one embodiment, a polynucleotide encoding an anti-tetranor-PGDM monoclonal antibody or a fragment thereof comprises the following:
In one embodiment, a polynucleotide encoding a gene for an anti-tetranor-PGDM monoclonal antibody or a fragment thereof comprises the following:
In a third aspect, an expression vector containing a polynucleotide encoding an anti-tetranor-PGDM monoclonal antibody or a fragment thereof and host cells transfected with the expression vector are provided. The host cells transfected with the expression vector can produce an anti-tetranor-PGDM monoclonal antibody or a fragment thereof.
A polynucleotide encoding a heavy chain variable region (or heavy chain) of an anti-tetranor-PGDM monoclonal antibody or a fragment thereof and a polynucleotide encoding a light chain variable region (or light chain) of an anti-tetranor-PGDM monoclonal antibody or a fragment thereof may be contained in the same expression vector or may be incorporated into separate expression vectors and provided as a combination. The “expression vector containing a polynucleotide encoding an anti-tetranor-PGDM monoclonal antibody or a fragment thereof” also include a combination of an expression vector containing a polynucleotide encoding a heavy chain variable region (or heavy chain) (expression vector of a heavy chain variable region (or heavy chain)) and an expression vector containing a polynucleotide encoding a light chain variable region (or light chain) (expression vector of a light chain variable region (or light chain)).
An expression vector containing a polynucleotide encoding an anti-tetranor-PGDM monoclonal antibody or a fragment thereof and host cells transfected with the expression vector can be obtained by a method known to those skilled in the art. For example, a polynucleotide encoding a single-chain anti-tetranor-PGDM monoclonal antibody is incorporated into a vector suitable for its expression and introduced into hosts, and thus the anti-tetranor-PGDM monoclonal antibody or a fragment thereof is expressed. The expression vector preferably contains a promoter, a terminator and the like suitable for expressing an anti-tetranor-PGDM monoclonal antibody or a fragment thereof in host cells. The expression vector can further contain other appropriate control sequences such as an enhancer.
The vector can be appropriately selected from among expression vectors well-known to those skilled in the art in consideration of the type of host cells and the expression efficiency of the anti-tetranor-PGDM monoclonal antibody or a fragment thereof, and the type may be a plasm id vector or of a virus vector derived from retrovirus or adenovirus.
Host cells are not limited as long as they express an anti-tetranor-PGDM monoclonal antibody or a fragment thereof from an expression vector, and include prokaryotic cells such as E. coli, mammal-derived cells such as CHO, COS, NIH3T3, HEK293, HEK293T, and COS-7, insect-derived cells such as F9, and eukaryotic cells such as yeast cells. Examples of mammals include primates such as humans and chimpanzees and rodents such as mice, rats, and hamsters.
Transfection of the expression vector into host cells can be performed using, for example, a method well-known to those skilled in the art such as an electroporation method, a calcium phosphate method, a lipofection method, or a DEAE dextran method, or a method using a commercially available reagent.
The host cells (transfectants) transfected with an expression vector containing a polynucleotide encoding an anti-tetranor-PGDM monoclonal antibody or a fragment thereof include
In a fourth aspect, a hybridoma that produces an anti-tetranor-PGDM monoclonal antibody is provided.
Hybridomas producing an anti-tetranor-PGDM monoclonal antibody can be obtained by immunizing an animal to be immunized with tetranor-PGDM according to a general method and fusing immune cells obtained from the immunized animal with known myeloma cells according to a general cell fusion method. However, tetranor-PGDM has a low-molecular weight, and is also present in vivo, and thus antibodies are unlikely to be produced. Therefore, it is preferable to use mice in which genes encoding two types of PGD2 synthases, a hematopoietic and lipocalin type, are deleted by a gene modification technique, and which do not produce PGD2 or its metabolite tetranor-PGDM, or the like as an animal to be immunized, and conjugate the immunogen tetranor-PGDM with a carrier protein such as keyhole limpet hemocyanin (KLH). Mice that do not produce PGD2 or its metabolite tetranor-PGDM can be obtained by, for example, replacing cysteine 65 in the PGD2 production site present in L-PGDS exon II with serine using a technique such as a CRISPR/Cas system. The 65th cysteine in the amino acid sequence (SEQ ID NO: 84) of L-PGDS is the center of enzyme activity (J Biol Chem. 2009 14; 284(33): 22344-52.), and if this cysteine is replaced with other amino acids, for example, serine, the PGD synthase activity disappears and PGD2 cannot be produced. Cells producing an anti-tetranor-PGDM monoclonal antibody are screened from among such hybridomas. Examples of animals to be immunized include rodents such as mice, rats, hamsters, and rabbits, and mammals such as monkeys.
In a fifth aspect, a method of producing an anti-tetranor-PGDM monoclonal antibody or a fragment thereof is provided. An anti-tetranor-PGDM monoclonal antibody or a fragment thereof may be produced by culturing the above host cells, transfectants and hybridomas or produced using a recombinant DNA technique.
Examples of antibodies produced as recombinant proteins by a recombinant DNA technique include chimeric antibodies, humanized antibodies and human antibodies. These antibodies can be produced, for example, by culturing host cells transformed with a recombinant vector encoding the antibody or its heavy chain or light chain. Recombinant antibodies can also be produced using transgenic animals into which a gene encoding a desired antibody is introduced.
Culture conditions for host cells, transfectants and hybridomas can be appropriately determined by those skilled in the art according to a desired anti-tetranor-PGDM monoclonal antibody or a fragment thereof and the type of host cells. The production method may further include a step of recovering an antibody or a fragment thereof that specifically binds to tetranor-PGDM from the cultured product obtained in the culturing step.
The produced antibody or fragment thereof may be further subjected to an additional step such as an additional purifying step before recovery, and for example, in order to increase purity, it is preferable to perform salting-out, filter filtration, clarification using centrifugation, recovery by affinity, intermediate purification by ion exchange, and final purification by gel filtration.
In a sixth aspect, various applications of anti-tetranor-PGDM monoclonals, for example, diagnostic agents for tetranor-PGDM-related disease, comprising the antibody or fragment thereof, are provided.
As used herein, “tetranor-PGDM-related disease” means a disease for which tetranor-PGD serves as a diagnostic marker such as a food allergy (WO 2016/021704 (mentioned above); Maeda S, Nakamura T, Harada H, Tachibana Y, Aritake K, Shimosawa T, et al. Prostaglandin D2 metabolite in urine is an index of food allergy. Sci Rep. 2017; 7(1): 17687. Epub 2017/12/17. doi: 10.1038/s41598-017-17798-w. PubMed PMID: 29247205; PubMed Central PMCID: PMCPMC5732293.; Inagaki S, Maeda S, Narita M, Nakamura T, Shimosawa T, Murata T, et al. Urinary PGDM, a prostaglandin D2 metabolite, is a novel biomarker for objectively detecting allergic reactions of food allergy. J Allergy Clin Immunol. 2018; 142(5): 1634-6.e10. Epub 2018/07/10. doi: 10.1016/j.jaci.2018.06.032. PubMed PMID: 29981807.), anaphylactic reaction, muscular dystrophy such as Duchenne muscular dystrophy (Takeshita E, Komaki H, Tachimori H, Miyoshi K, Yamamiya I, Shimizu-Motohashi Y, et al. Urinary prostaglandin metabolites as Duchenne muscular dystrophy progression markers. Brain Dev. 2018; 40(10): 918-25. Epub 2018/07/15. doi: 10.1016/j.braindev.2018.06.012. PubMed PMID: 30006121.), and aspirin intolerant asthma (Higashi et al., “Urinary tetranor-PGDM concentrations in aspirin-intolerant asthma and anaphylaxis”, J Allergy Clin Immunol. 2012 Feb; 129(2): 557-9, 559.e1-2. doi: 10.1016/j.jaci.2011.09.019. Epub 2011 Oct 22.).
In a seventh aspect, a kit comprising an anti-tetranor-PGDM monoclonal antibody or a fragment thereof is provided.
The kit is intended to be used for tetranor-PGDM measurement, for example, a qualitative test or quantitative test. As a method of measuring tetranor-PGDM, for example, an immunoassay using an antigen-antibody reaction with tetranor-PGDM in a specimen is preferable.
For the immunoassay, a detectably labeled anti-tetranor-PGDM monoclonal antibody or an antibody (secondary antibody) to a detectably labeled anti-tetranor-PGDM monoclonal antibody is used. Depending on the antibody labeling method, methods are classified into enzyme immunoassay (EIA or ELISA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), fluorescence polarization immunoassay (FPIA), chemiluminescence immunoassay (CLIA) and the like, and any of these can be suitably used in the kit. The above measurement methods are only examples, and the above antibodies can also be used in other known methods known to those skilled in the art, for example, an aggregation method, chemiluminescence immunoassay (CLIA), electrochemiluminescence immunoassay (ECLIA) and the like.
Antibodies labeled with enzymes such as peroxidase and alkaline phosphatase are used in the ELISA method, antibodies labeled with radioactive substances such as 125I, 131I, 35S, and 3H are used in the RIA method, antibodies labeled with fluorescent substances such as fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine isothiocyanate, and near-infrared fluorescent materials are used in the FPIA method, and antibodies labeled with luminescent substances such as luciferase, and luciferin, aequorin are used in the CLIA method. In addition, antibodies labeled with nanoparticles such as colloidal gold, and quantum dots can be detected.
In addition, in the immunoassay, an anti-tetranor-PGDM monoclonal antibody can be labeled with biotin and bound with avidin or streptavidin labeled with an enzyme or the like for detection.
Among immunoassays, in an ELISA method using an enzyme label, it is possible to measure an antigen easily and rapidly. The ELISA method includes a competitive method and a sandwich method. In the competitive method, an anti-tetranor-PGDM monoclonal antibody is immobilized on a solid phase carrier such as a microplate, and a urine specimen and an enzyme-labeled tetranor-PGDM are added to induce an antigen-antibody reaction. After washing once, a reaction with an enzyme substrate is performed to develop a color and the absorbance is measured. When the level of tetranor-PGDM in the urine specimen is higher, the color development becomes weaker, and when the tetranor-PGDM level in the urine specimen is lower, the color development becomes stronger, and thus the tetranor-PGDM level can be determined using a calibration curve.
In the sandwich method, an anti-tetranor-PGDM monoclonal antibody is immobilized on a solid phase carrier, a urine sample is added and reacted, and another enzyme-labeled anti-tetranor-PGDM monoclonal antibody is then additionally added and reacted. After washing, a reaction with an enzyme substrate is performed to develop a color, the absorbance is measured, and thus the tetranor-PGDM level can be determined. In the sandwich method, after the antibody immobilized on the solid phase carrier and tetranor-PGDM in the urine specimen are reacted, an unlabeled anti-tetranor-PGDM monoclonal antibody (primary antibody) is first added, and an antibody (secondary antibody) to this unlabeled anti-tetranor-PGDM component may be labeled with an enzyme and then additionally added.
When the enzyme is a peroxidase, 3,3′-diaminobenzidine (DAB), 3,3′,5,5′-tetramethylbenzidine (TMB), o-phenylenediamine (OPD) or the like can be used as the enzyme substrate, and when the enzyme is an alkaline phosphatase, p-nitropheny phosphate (NPP) or the like can be used as the enzyme substrate.
As used herein, “solid phase carrier” is not particularly limited as long as it can immobilize an antibody, and examples thereof include microtiter plates made of glass, a metal, a resin or the like, substrates, beads, nitrocellulose membranes, nylon membranes, and PVDF membranes, and target substances and antibodies can be immobilized on these solid phase carriers according to a known method.
Although the form of the kit is not limited, a form of a stick comprising an anti-tetranor-PGDM monoclonal antibody and in which the tetranor-PGDM level in the specimen can be visualized with a colored line or the like is preferable. A known kit can be used for such a stick-like kit. For example, a stick using an immunochromatographic method is known, and a configuration in which an antibody storage unit in which a first anti-tetranor-PGDM monoclonal antibody labeled with colloidal gold or the like is stored and a determination unit in which a second anti-tetranor-PGDM monoclonal antibody that recognizes another epitope of tetranor-PGDM is immobilized in a line on a cellulose membrane or the like are connected via a narrow groove can be used. When a specimen is added to such a stick, the labeled antibody and tetranor-PGDM are bound in the antibody storage unit to form a tetranor-PGDM-labeled antibody complex, and the complex moves to the determination unit through the groove due to a capillary phenomenon. When the complex is captured by the immobilized second anti-tetranor-PGDM monoclonal antibody, a red line appears in the determination unit due to a plasmon effect of the colloidal gold.
A control line on which an antibody to the labeled antibody is immobilized may be provided in the determination unit of the stick. Since the control line develops a color with or without tetranor-PGDM, it is possible to check whether the test is performed correctly.
The stick may further comprise absorbent paper for absorbing a urine specimen, a desiccant and the like.
In the stick, the labeled anti-tetranor-PGEM antibody is also stored in the antibody storage unit, and the anti-tetranor-PGEM antibody that recognizes another epitope of tetranor-PGEM is immobilized in a line in the determination unit, and thus tetranor-PGDM and tetranor-PGEM can be measured at the same time.
With such a stick, the tetranor-PGDM level can be easily measured even at home, and the risk of developing a food allergy and its degree can be evaluated.
The kit can be used not only for diagnosing a tetranor-PGDM-related disease, for example, a food allergy or Duchenne muscular dystrophy, but also for evaluating activation of mast cells in a food allergy.
In an eighth aspect, a method for detecting tetranor-PGDM using an anti-tetranor-PGDM monoclonal antibody or a fragment thereof is provided.
The method for detecting tetranor-PGDM can be used not only for diagnosing a food allergy or Duchenne muscular dystrophy but also for evaluating activation of mast cells in a food allergy.
In a ninth aspect, a method for diagnosing a food allergy using an anti-tetranor-PGDM monoclonal antibody or a fragment thereof is provided.
The urinary tetranor-PGDM level increases according to the increase in severity of food allergy symptoms, and the number and activation of mast cells in tissues, and decreases according to inhibition of symptoms by administration of a food allergy treatment agent. Therefore, when the urinary tetranor-PGDM level is measured, it is possible to evaluate not only whether a subject has a food allergy but also its severity. In addition, in the urine of the subject with a food allergy, a high tetranor-PGDM concentration may be observed without performing an antigen stimulation. Therefore, it is possible to measure the urinary tetranor-PGDM level in the subject who has not developed a food allergy and to evaluate a risk of developing a food allergy.
Such an evaluation method may comprise a step of detecting the urinary tetranor-PGDM level and a step of assisting in determining that, when the urinary tetranor-PGDM level is higher, food allergy symptoms are severe or have become severe, or a risk of developing symptoms is higher or has become higher. The threshold value used in such determination may vary depending on factors such as a subject age. Therefore, although not intended to be limiting, the urinary tetranor-PGDM concentration is 1.5±0.3 ng/mg creatinine (Cre) in healthy individuals, but the urinary tetranor-PGDM concentration may be 2.25 ng/mg Cre or more in subjects who have or have developed severe food allergy symptoms or who are or have become at high risk of developing symptoms.
As used herein, “food allergy” refers to various allergy reactions that occur when allergens contained in food are taken into the body. As food allergies, allergies to chicken eggs, milk, crustaceans, wheat, fruit, nuts, seafood, buckwheat and the like are well known, but allergies to allergens other than these food components are also included. Symptoms occur in the skin, mucous membranes, digestive organs, respiratory organs and the like and are typically diarrhea, vomiting, skin inflammation and the like.
As used herein, “subject” includes a person who may have a food allergy, a person who has actually developed a food allergy, a person who is undergoing a food allergy treatment, a person who is taking a food allergy treatment agent, a person who is unsure of whether he/she has a food allergy and the like. In addition, “subject” is not limited to humans, and may be mammals such as mice, rats, rabbits, cats, dogs, monkeys, pigs, sheep, cows, and horses.
In one aspect of the food allergy test method according to the present invention, when the urinary tetranor-PGDM level is higher, it is determined that symptoms are more severe or a development risk is higher. Regarding whether the urinary tetranor-PGDM level is higher, the tetranor-PGDM level may be determined by measuring the tetranor-PGDM levels in urine collected from the same individual a plurality of times and comparing the results. In this case, it is possible to evaluate changes in the severity and the development risk in the same individual. In addition, whether the urinary tetranor-PGDM level is higher may be determined by measuring the tetranor-PGDM levels in urine collected from a plurality of persons and comparing the results. In this case, it is possible to evaluate whether the severity or the development risk is higher than those of other people.
In one aspect of the food allergy test method according to the present invention, when the urinary tetranor-PGDM level is higher than a predetermined value, it is determined that symptoms are severe or a development risk is high, and when the level is lower than a predetermined value, it is determined that symptoms are mild or a development risk is low. The predetermined value (cutoff value) can be determined by comparing the tetranor-PGDM level in urine specimens of a plurality of healthy subjects or non-food allergy patients with the tetranor-PGDM level in urine specimens of a plurality of food allergy patients by a general method.
In one aspect, the food allergy test method according to the present invention is used to evaluate a treatment agent administered to food allergy patients and a treatment method to be performed on patients. For example, the tetranor-PGDM level in the urine specimen collected at any time between before the treatment starts and after the treatment starts is measured, and when the tetranor-PGDM level decreases, it is determined that the treatment agent or the treatment method is effective, and when the tetranor-PGDM level does not decrease but increases, it is determined that the treatment agent or the treatment method is not effective. In this case, the decrease or increase in the tetranor-PGDM level may not be a significant decrease or increase, and may be to the extent that those skilled in the art can determine that there is a tendency to decrease or increase.
In one aspect, the food allergy test method according to the present invention may be used to evaluate the effect of agents in animal experiments for development of food allergy treatment agents.
In one aspect, the food allergy test method according to the present invention involves examining a sample collected from a subject in order to obtain information necessary for diagnosis. Such a test method is performed by, for example, a test company.
For urine to be used in the test of the present invention, a sample collected by a general method can be used. It may be a single urine collection or two or more urine collections. The collected urine may be stored at room temperature during the test or stored at −40° C. or lower, for example, −80° C. Frozen urine can be rapidly thawed and used for measurement.
In addition to the urinary tetranor-PGDM level in subjects, the level of tetranor-PGEM (9,15-dioxo-11α-hydroxy-13,14-dihydro-2,3,4,5-tetranor-prostan-1,20-dioic acid) which is a metabolite of PGE2, may be measured.
The urinary tetranor-PGDM level behaves differently in a food allergy than in other inflammatory diseases. Therefore, when the urinary tetranor-PGDM level is measured, it is possible to distinguish symptoms from other inflammatory diseases (for example, enteritis, asthma, allergic dermatitis). In addition, the urinary tetranor-PGEM level also behaves differently in a food allergy than in other inflammatory diseases. Therefore, when both the urinary tetranor-PGDM level and the urinary tetranor-PEGM level are measured, it is possible to distinguish symptoms more accurately.
More specifically, when the urinary tetranor-PGDM level remains elevated for a certain period of time and the urinary tetranor-PGEM level transiently increases and then immediately decreases, it can be determined that there is a food allergy. Here, a certain period of time is, for example, 3 days or longer, 5 days or longer, 7 days or longer, 10 days or longer, or 20 days or longer after a substance that causes a food allergy is taken. Transiently increasing and then immediately decreasing means, for example, decreasing within 5 days, 4 days, or 3 days after increasing.
The urinary tetranor-PGEM level can be measured using a known method, for example, a mass spectrometer.
Activation of mast cells in a food allergy can be evaluated using an anti-tetranor-PGDM monoclonal antibody or a fragment thereof.
As used herein, “activation of mast cells” means an increase in the number of mast cells and an increase in activities including degranulation. As shown in examples to be described below, the urinary tetranor-PGDM level increases as the number of mast cells increases, but does not increase under inhibition of degranulation. In addition, the urinary tetranor-PGEM level also increases as the number of mast cells increases. Therefore, activation of mast cells can be evaluated using the urinary tetranor-PGDM level and the urinary tetranor-PGEM level as indicators.
For example, it is known that allergic rhinitis causes symptoms in mast cell-deficient mice, but a food allergy causes almost no symptoms in mast cell-deficient mice, and it is known that a food allergy is highly dependent on mast cells, and an increase in the number of mast cells and activities thereof are important for the development and progress of a food allergy. As a method for detecting activation of the mast cells (particularly gastrointestinal mucosal mast cells) specific to a food allergy, the only method is to measure the blood histamine concentration so far. However, since blood histamine disappears in 15 minutes, there is no actual method for detecting activation of mast cells.
In a tenth aspect, a method of treating a subject diagnosed with an anti-tetranor-PGDM-related disease is provided.
A necessary treatment is appropriately performed on a subject diagnosed with a tetranor-PGDM-related disease according to the above diagnosis method. For example, a known treatment method such as a hyposensitization treatment can be applied to a subject diagnosed with a food allergy.
The hyposensitization treatment is performed to induce immune tolerance by administering dilute allergens to patients under the supervision of a doctor. Many allergic disease treatment methods are symptomatic treatments, but the hyposensitization treatment is being focused on because it targets radical curing by acting on the mechanisms of action of an allergic disease. The hyposensitization treatment begins with a very low dose and the dose gradually increases, but in this case, a method of appropriately evaluating the severity of allergic symptoms in patients is required in order to prevent severe symptoms from occurring due to administration of an amount of allergens exceeding a threshold value.
The administration route, dose and frequency of administration of the hyposensitization treatment are not particularly limited, and can be appropriately selected according to various conditions such as the patient's age, weight, and symptoms. Examples of administration routes include routes of administration to individuals by intradermal, subcutaneous, intramuscular, intraperitoneal, transdermal, transmucosal, oral, and inhalation routes.
While the present invention will be described below in more detail with reference to examples, the present invention is not limited thereto.
Using an offset-nicking method to which a CRISPR/Cas system was applied, L-PGDS C65S mutant-mice in which cysteine at position 65 of L-PGDS (SEQ ID NO: 84) was replaced with serine were prepared. Specifically, using exon-3 of L-PGDS of targeted embryonic stem cells as a targeting vector, after cleavage using Cas9 nickase and guide RNA, vector DNA was inserted to convert TGC (cysteine) to AGC (serine). L-PGDS C65S mutant mice were obtained by microinjection of these cells into 8-cell stage embryo of ICR strain mice.
Keyhole limpet hemocyanin (KLH) conjugated tetranor-PGDM was subcutaneously administered to L-PGDS C65S mice to perform immunization. Splenocytes of the immunized mice were fused with myeloma cells (SP2/0-Ag14) according to standard protocols [6]. Positive hybridomas were cloned by a limiting dilution method. IgG antibodies were purified from serum-free media of the hybridoma culture supernatant.
Total RNA was prepared from 1×106 hybridomas producing each antibody using TRI Reagent (Cat#TR118, commercially available from Molecular Research Center, Inc.).
Gene cloning of VH and VL of anti-tetranor-PGDM monoclonal antibody:
cDNA was prepared from 1 μg of total RNA of each hybridoma using SMARTer (registered trademark) Mouse BCR IgG H/K/L Profiling Kit (Cat#634422, commercially available from Clontech). The isotype of the obtained antibody was identified using Iso-Gold Rapid Mouse Monoclonal Antibody Isotyping κ&λ Kit (Cat# KSOT03-010, commercially available from BioAssay Works). Using the obtained cDNA as a template, BCR Primer 1V (containing a forward primer) bundled in the kit, and a reverse primer of mBCR Primer 1H (encoding a mouse IgG heavy chain constant region), mBCR Primer 1L(κ) or mBCR Primer 1L(λ) (encoding a mouse IgG light chain constant region) were used in combination, and thus cDNA sequences of VH and VL were determined.
Specifically, a PCR reaction was performed using a combination of BCR Primer 1V and mBCR Primer 1H, and VH cDNA fragments of the antibodies were amplified. In addition, PCR was performed using a combination of mouse Ig(κ)-specific primer (mBCR Primer 1L(κ)) and BCR Primer 1V or a combination of mouse Ig(λ)-specific primer (mBCR Primer 1L(λ)) and BCR Primer 1V, and VL cDNA fragments of the antibodies were amplified.
PCR was performed for 35 reaction cycles including 98° C. for 10 seconds, 60° C. for 15 seconds, and 68° C. for 45 seconds.
Each PCR amplification product was purified using FastGene Gel/PCR Extraction kit (Cat#FG-91202, commercially available from NIPPON Genetics Co., Ltd.).
dA was added to the 3′ terminal of the obtained gene fragment using TArget Clone™-Plus-(Cat#TAK-201, commercially available from TOYOBO) and inserted onto the pTA2 Vector bundled in the kit.
The obtained plasmid was introduced into E. coli DH5a stains. A plasmid was extracted from the obtained transformant using FastGene Plasmid Mini Kit (Cat#FG-90502, commercially available from NIPPON Genetics Co., Ltd.) and the base sequence was analyzed. As a result, it was confirmed that the full-length VH cDNA and VL cDNA having an ATG sequence assumed to be the start codon at the 5′ terminal of cDNA were obtained.
The reactivity of the tetranor-PGDM monoclonal antibody was examined by competitive EIA. Tetranor-PGDM monoclonal antibodies with various concentrations were incubated in an anti-mouse goat IgG antibody-immobilized 96-well plate (Cayman Chemical, Michigan, USA) for 1 hour. Next, the plate was washed three times with a phosphate buffered saline (PBS-T) containing 0.5% bovine serum albumin (BSA) and 0.05% (v/v) Tween-20 (PBS-T). Subsequently, a tetranor-PGDM-conjugated acetylcholinesterase tracer (tracer) was incubated overnight. Next, the plate was washed three times with PBS-T and incubated with an Ellman's reagent for 1 hour. The absorbance at 405 nm was measured with a microplate reader (Perkin Elmer, Massachusetts, USA). The selectivity of tetranor-PGDM with respect to a prostaglandin metabolite was examined by competitive EIA using tetranor-PGEM, tetranor-PGAM, and tetranor-PGFM.
The results are shown below.
The influence of the pH (5.0, 6.0, 6.5, 7.0, 7.5, 8.0, and 9.0) and the ionic strength (NaCl content in a 10 mM phosphate buffer solution) on the EIA performance was examined. A standard inhibition curve of tetranor-PGDM was plotted under each condition. The cross-reactivity was calculated based on the following formula.
cross-reactivity (%)=(IC50 of tetranor-PGDM/IC50 of other tetranor-PGM)×100.
Tetranor-PGDM with a known concentration was added to the assay solution for measurement. Simultaneous reproducibility was obtained from 9 measurements, and inter-day reproducibility was obtained by performing measurement for 3 days.
A data set of the analyte concentration and the absorbance was fitted with the following 4-parameter logistic formula with fitting parameters a, b, c, and d. [7, 8]
f(X)=(a−d)/(1+(X/c)b)+d
Based on the model [9] proposed by Hayashi et al., the relative standard deviation (RSD) of the entire analysis (ρT) was represented by the following formula.
ρT2=X2/(X±G)2×(ρX2+ρG2)+ρB2+ρS2+(σW/f(X)×100)2 [9]
In this formula, G was the amount of labeled antigens, ρX, ρG, ρB, and ρS were RSDs of pipette volumes of analytes labeled as the antigen, the antibody, and the substrate and ρS was two thirds of the RSD of the chromogenic substrate solution of the pipette volume. σW was the SD of the intrinsic absorbance between wells of the plate. Since the concentration of labeled antigens was unknown, the approximate formula of the uncertainty of the competitive EIA ρT2=ρB2+(σW/f(X)×100)2 was used [9]. ρB=0.782116 and σW=0.0026. ρT was calculated from the blank-subtracted measured value. For the ρB value, the weight of the liquid collected with a pipette was measured 20 times and the RSD was calculated. The σW value was calculated from the well-to-well SD of the absorbance of empty wells (n=96).
Tetranor-PGDM dissolved in distilled water was added to artificial urine (Table 2) to prepare urine samples with four different concentrations (1.25, 2.5, 5.0, and 10.0 ng/mL).
For solid phase extraction (SPE), 0.4 mL of a urine sample was diluted to 0.8 mL with 0.1% (v/v) formic acid. This was applied to an SPE cartridge column (HLBμ Elution plate, Waters, Massachusetts, USA) pretreated with 200 μL of acetonitrile and distilled water. After the column was washed with 200 μL of distilled water and 200 μL of hexane, the lipid fraction was eluted with 50 μL of acetonitrile. The eluate was recovered and dried in a vacuum, and the obtained residue was re-dissolved in 0.4 mL of an assay solution. Next, the sample solution containing tetranor-PGDM was subjected to EIA. At the same time, tetranor-PGDM was measured through LC-MS/MS [2, 3]. The recovery from SPE determined through LC-MS/MS was 77.1% (Table 3).
A hybridoma producing monoclonal antibodies against tetranor-PGDM (Mab) was obtained from the immunized mouse splenocytes. Next, 60-141 Mab was purified and applied in a competitive EIA.
Since patient urine samples had different physicochemical properties, the optimal ionic strength and the pH for Mab-based EIA were examined. As shown in
In order to determine the optimal pH of the assay buffer, the IC50 value at a pH of 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, and 9.0 was 8.6, 6.6, 5.5, 5.6, 4.8, 5.2, and 4.8 ng/mL, respectively (
Under the optimized conditions, the IC50 value was 1.79±0.36 from the competition curve of tetranor-PGDM (
Tetranor-PGDM was gradually diluted with the assay solution and the limit of detection of tetranor-PGDM was examined. The limit of detection (LOD) was 0.0498 ng/mL, and the range of quantification (ROQ) value was 0.252 to 20.2 ng/mL. The LOD and the ROQ were defined as the 30% RSD concentration and 10% RSD concentration ranges (
The inaccuracy of this EIA was evaluated using an assay solution containing tetranor-PGDM at three different concentrations. Intra-and inter-experimental CVs were 3.9% to 6.0% (n=9) and 5.7% to 10.4% (n=9) (Table 4).
The recovery of tetranor-PGDM was 89.4% to 111.7%. All recoveries and coefficients of variation of tetranor-PGDM were within the acceptable range.
In addition to tetranor-PGDM, other urinary PG metabolites such as tetranor-PGEM and tetranor-PGFM were present in urine (
Since no significant cross-reactivity with these metabolites was shown in the developed EIA method, it was suggested that urinary tetranor-PGDM could be measured with sufficient specificity using this EIA method.
A known amount of tetranor-PGDM was added to artificial urine, and recovery of tetranor-PGDM was measured by a competitive EIA method using 60-141 Mab.
The dilution linearity was an indicator of efficacy of the EIA method using an anti-tetranor-PGDM monoclonal antibody. First, an artificial urine sample containing 20 ng/mL of tetranor-PGDM was gradually diluted and the recovery of tetranor-PGDM according to the antibody was measured. The tetranor-PGDM measured value was not linearly correlated with a dilution factor. In addition, the recovery of tetranor-PGDM was less than 30.2% (
Therefore, the urine sample was subjected to solid phase extraction (SPE) in order to eliminate the negative interference. The recovery of tetranor-PGDM from SPE determined through LC-MS/MS was 77.1% (Table 2). After SPE, an artificial urine sample containing 13.9 ng/mL of tetranor-PGDM was gradually diluted and measured. After SPE, the measured value of the artificial urine showed good linearity with a dilution factor (r=0.999,
Tetranor-PGDM with different concentrations was added to artificial urine, and the recovery was determined. The recovery of tetranor-PGDM was in a range of 82.3% to 113.5%. All recoveries of tetranor-PGDM were within the acceptable range (Table 6).
The concentration of tetranor-PGDM was 0.85 (AU-1) and 5.95 (AU-2).
In this study, for tetranor-PGDM measurement, an anti-tetranor-PGDM monoclonal antibody was prepared and a highly sensitive competitive EIA method was developed. A high average recovery (89.4% to 111.7%) with a coefficient of variation of 3.9% to 10.4% was obtained in the assay solution sample. EIA ROQ for tetranor-PGDM measurement was 0.252 to 196 20.2 ng/m L. The matrix effect of the sample was eliminated by using SPE. A high average recovery (82.3% to 113.5%) was obtained in the urine sample after SPE.
Since antigen-antibody binding was characterized as weak intermolecular binding, a change in either the ionic strength or the pH could influence this interaction. Individual properties of respective urine samples could influence the reliability of analysis using the EIA method [10]. Up to a pH of 9.0, the EIA IC50 value did not change significantly. Even if the ionic strength increased to 300 mM, recognition of tetranor-PGDM did not significantly change. These results suggested that a change in either the ionic strength or the pH had had little influence on the EIA method using 60-141 antibodies.
In healthy individuals, the urinary tetranor-PGDM concentration was 1.5±0.3 ng/mg creatinine (Cre) [1]. The urinary Cre concentration was about 1 mg/mL [11]. The inventors previously performed the oral load test for food allergy patients and found that the optimal cutoff value of the urinary tetranor-PGDM was 2.25 ng/mg Cre [3]. The LOQ value was lower than the cutoff value. In other diseases, it has been reported that the urinary tetranor-PGDM concentration in Duchenne muscular dystrophy patients was 9.7 ng/mg Cre [4]. These results indicated that this optimized EIA method was acceptable for detection of urinary tetranor-PGDM in food allergy or Duchenne muscular dystrophy patients.
The urinary tetranor-PGEM and tetranor-PGFM concentrations were 8 to 15 ng/mg Cre and 11 to 59 ng/mL [12, 13]. In this competitive EIA method, the cross-reactivity with other tetranor-PG metabolites was negligible. Mice were immunized using KLH-conjugated tetranor-PGDM prepared by binding KLH to the carboxyl group of tetranor-PGDM via NHS/EDC-mediated esterification. Generally, it was thought that the antibody best recognized the hapten moiety furthest away from the conjugate bond. Therefore, the epitope of the 60-141 antibody was thought to be on the cyclopentane ring of tetranor-PGDM. In addition, the 60-141 antibody did not recognize tetranor-PGEM, tetranor-PGFM, or tetranor-PGAM without the 11-keto group.
This suggested that the epitope of the 60-141 antibody was the 11-keto group of tetranor-PGDM. That is, according to the cross-reactivity test, it was shown that the 60-141 antibody was directed almost exclusively against the tetranor-PGDM structure.
In addition, the 60-141 antibody had a sufficiently high specificity for PGDM compared to other lipids, such as 0.631% for tetranor-PGEM, 3.876% for tetranor-PGAM, and <0.003% for tetranor-PGFM.
In this study, one monoclonal antibody was successfully produced and a highly sensitive competitive EIA method was developed. This EIA method was useful for quantification of tetranor-PGDM in body fluids, evaluation of animal disease models, diagnosis indicators, and treatment monitoring of food allergies and other diseases.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-026290 | Feb 2021 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/006890 | 2/21/2022 | WO |
