Patent Application
20250155453
The present invention relates to an immunological detection method and an immunological detection kit.
Priority is claimed on Japanese Patent Application No. 2022-050846, filed Mar. 25, 2022, the contents of which are incorporated herein by reference.
Type I procollagen N-propeptide (hereinafter referred to as “P1NP” or “PINP”) is a polypeptide with a molecular weight of approximately 35 kDa that is cleaved from the N-terminus when type I collagen, the main component of bone matrix protein, is produced from the precursor type I procollagen. PINP released into the blood is known to be a bone formation marker that sensitively reflects early stage of bone formation. Therefore, blood PINP concentration is useful for determining and monitoring the therapeutic effects of osteoporosis drugs such as PTH (parathyroid hormone) preparations and bisphosphonate preparations. The measurement of PINP (total PINP) sometimes results in detection of monomers as well as intact PINP as a trimer.
As type I procollagen-N-propeptide kit (generic name), for example, Procollagen Intact PINP (product name, manufactured by Fujirebio Inc., Non-Patent Document 1) and Elecsys® Reagent Total P1NP (product name, Roche Diagnostics, Non-Patent Document 2) are commercially available.
Patent Document 1 discloses a method and a test kit for determining the presence of amino-terminal propeptides of type I procollagen in individual persons.
Patent Literature 2 discloses an assay method for the α1-homotrimer amino-terminal propeptide which is an intact trimer of type I procollagen or the heterotrimer amino-terminal propeptide which is an intact classical type I procollagen, and an assay kit suitable for use in the assay method.
Non-Patent Literature 3 discloses that a trimeric PINP and a monomeric PINP have different clearance pathways (trimer: liver, monomer: kidney), and that patients with renal disease tend to lack ability of form clearance of the monomer and have high monomer concentrations in the blood.
Non-Patent Literature 4 discloses that a monomeric PTNP reflects the degradation product of pN-collagen rather than the thermal denaturation product of the intact propeptide.
Procollagen Intact PINP (hereinafter abbreviated as “Intact PINP”) described in Non-Patent Literature 1 can detect only the trimer of the α-chain of type I collagen N-terminal propeptide. Intact PINP is a measurement kit based on radioimmunoassay method, and uses an antibody labeled with a radioisotope (125I). Therefore, the measurement needs to be performed in a radiation controlled area and requires about 3-hour measurement time, which is cumbersome. In addition, since the half-life of the radioisotope (125I) is as short as 59.4 days, the shelf life of the kit is also as short as 6 weeks at 2 to 8° C.
The Elecsys reagent total P1NP (hereinafter abbreviated as “total P1NP”) described in Non-Patent Literature 2 can detect the trimer and monomer of the α-chain of type I collagen N-terminal propeptide. The total P1NP is a measurement kit based on electrochemiluminescence immunoassay (ECLIA), and is widely used because it is easy to handle with no need for a radiation control area and a measurement time of about 20 minutes. The kit also has a long shelf life of about 18 months at 2 to 8° C.
There is a good correlation between the Intact PINP and the total P1NP, and it is assumed that there is no significant difference in performance between these products.
However, in patients with renal disease, clearance of the α-chain monomer of type I collagen N-terminal propeptide cannot be formed, so that the PINP value of samples from patients with renal disease is measured to be high when using total P1NP.
The present invention has been made in consideration of the above circumstances, and one of its objects is to provide an immunological detection method and an immunological detection kit that are easy to handle and can specifically (selectively) measure the trimer in biological samples containing the trimer and monomer of the α-chain of PINP.
The present invention can provide an immunological detection method and an immunological detection kit that are easy to handle and can specifically (selectively) measure the trimer in biological samples containing the trimer and monomer of the α-chain of PINP.
Hereinbelow, embodiments of the present invention are described, but the present invention is not limited to such embodiments, and various modifications can be made as long as such modifications do not deviate from the substance of the present invention.
In the present invention, the one-letter abbreviations for amino acids follow those described in IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN), Nomenclature and Symbolism for Amino Acids and Peptides Recommendations 1983, European Journal of Biochemistry, 1984, Vol. 138, pp. 9-37. The amino acid sequence is described in the order of from N-terminus to C-terminus.
In the present invention, the terms “react,” “recognize,” and “bind” are used synonymously in relation to the reactivity of an antibody with an antigen. When an antibody disclosed herein is described as not reactive with respect to a certain compound, it means that the antibody does not substantially react with the certain compound. For example, in the Examples described below, the antibody is described as not reactive when the measured value is below the detection limit.
In the present invention, the terms “detection” and “measurement” are used in a sense encompassing the verification of the presence and/or quantification of trimeric type I collagen N-terminal propeptide.
The immunological detection method of the present invention is an immunological detection method for trimeric type I collagen N-terminal propeptide in a biological sample, and uses a first antibody that binds to a first specific portion of a pro-al chain in the trimeric type I collagen N-terminal propeptide and a second antibody that binds to a second specific portion of a pro-al chain in the trimeric type I collagen N-terminal propeptide.
The biological sample that can be used in the immunological detection method of the present invention is not particularly limited as long as it is a sample derived from a living body, but is preferably a liquid sample such as a body fluid, more preferably at least one selected from the group consisting of blood, plasma, and serum, and even more preferably any one selected from the group consisting of blood, plasma, and serum. The living body is preferably a human or a mammal (e.g., a mouse, a guinea pig, a rat, a monkey, a dog, a cat, a hamster, a horse, a cow, and a pig), and more preferably a human. The biological sample may be one collected or prepared during the implementation of the present invention, or may be one collected or prepared in advance and stored. The biological sample may usually contain both the trimer and monomer of type I collagen N-terminal propeptide, or may contain only one of the trimer and the monomer, or may contain neither the trimer nor the monomer.
The biological sample is preferably one derived from a subject with impaired renal function. Examples of subjects with impaired renal function include patients with chronic kidney disease. Renal function can be evaluated, for example, by eGFR value (estimated glomerular filtration rate, unit: mL/min/1.73 m2)
Trimeric type I collagen N-terminal propeptide is a polypeptide with a molecular weight of approximately 35 kDa, which is cleaved from the N-terminus during the formation of type I collagen from its precursor, i.e., type I procollagen. This polypeptide is a heterotrimer consisting of two al chains (pro-al chains) with a molecular weight of approximately 15 kDa and one α2 chain (pro-α2 chain) with a molecular weight of approximately 5 kDa.
The first specific portion of the pro-al chain is a portion having a specific primary structure (amino acid sequence) to which the first antibody binds. The primary structure (amino acid sequence) of the first specific portion of the pro-al chain is, for example, QEEGQVEGQD (SEQ ID NO: 1) shown in
The second specific portion of the pro-al chain is a portion having a specific primary structure (amino acid sequence) to which the second antibody binds. The primary structure (amino acid sequence) of the second specific portion of the pro-al chain is, for example, PDGSESPTDQETT (SEQ ID NO: 2) shown in
The first antibody is not particularly limited as long as it binds to the first specific portion of the pro-al chain, but is preferably an antibody that binds to the primary structure (amino acid sequence) of QEEGQVEGQD (SEQ ID NO: 1) shown in
The first antibody is preferably a monoclonal antibody. A monoclonal antibody can be produced, for example, by fusing a B lymphocyte with an immortalized cultured cell to form a hybridoma. Monoclonal antibodies are preferable because they are specific and are expected to produce significantly lower background signals than polyclonal antibodies.
The anti-ClA1-01 antibody is an antibody prepared by the method of the Examples described below using an immunogen obtained by binding ovalbumin, a carrier protein, to a peptide having a primary structure represented by SEQ ID NO: 3: QEEGQVEGQDC. The hybridoma producing the antibody 524206R shown in
The second antibody is not particularly limited as long as it binds to the second specific portion of the pro-α1 chain, but is preferably an antibody that binds to the primary structure (amino acid sequence) of PDGSESPTDQETT (SEQ ID NO: 2) shown in
The anti-C1A1-02 is an antibody prepared by the method of the Examples described below using an immunogen obtained by binding ovalbumin, a carrier protein, to a peptide having a primary structure represented by SEQ ID NO: 4: PDGSESPTDQETTC. The hybridoma producing the antibody S24217 shown in
It is preferable that either the first antibody or the second antibody, or neither the first antibody nor the second antibody, binds to a monomer of type I collagen N-terminal propeptide derived from a patient with renal disease.
The detection method is not particularly limited as long as it is an immunological method, but is preferably any one selected from the group consisting of an electrochemiluminescence method, latex agglutination immunoturbidimetry, and enzyme-linked immunosorbent assay (ELISA).
For detection, it is preferable that at least one of the first antibody and the second antibody is labeled with a labeling substance. Examples of the labeling substance include enzymes, fluorescent substances, chemiluminescent substances, biotin, avidin, gold colloid particles, and colored latex.
In the detection method of the present invention, the following embodiments (1) or (2) are preferred.
The method for detecting a signal derived from a labeling substance can be appropriately selected depending on the respective labeling substances.
The immunological detection kit of the present invention is an immunological detection kit for trimeric type I collagen N-terminal propeptide in a biological sample, and includes a first antibody that binds to a first specific portion of a pro-al chain in the trimeric type I collagen N-terminal propeptide, and a second antibody that binds to a second specific portion of a pro-al chain in the trimeric type I collagen N-terminal propeptide.
The biological sample, trimeric type I collagen N-terminal propeptide, the first antibody, and the second antibody are as described for the immunological detection method of the present invention.
The immunological detection kit of the present invention may further include a protocol, a detection device, an analysis device, etc.
The immunological detection kit of the present invention is preferable for carrying out the above-mentioned immunological detection method of the present invention.
Hereinbelow, the present invention will be described with reference to Examples which, however, should not be construed as limiting the present invention.
The following peptides were bound to the carrier protein ovalbumin (OVA) to prepare immunogens.
C1A1-01-OVA, and C1A2-01-OVA were used as immunogens, and were mixed at 1:1 with Freund's Complete Adjuvant (Difco Laboratories) for the first immunization and Freund's Incomplete Adjuvant (Difco Laboratories) for the second and subsequent immunizations. 6-week-old (female) Balb/c mice (CLEA Japan, Inc.) or F344/Jcl rats (CLEA Japan, Inc.) were subcutaneously immunized every 2 weeks with 40 μg of the immunogen for the first immunization and 20 g for the second and subsequent immunizations. The immunization was continued for 8 weeks. Antigen-immobilized ELISA was performed according to the following procedure to determine the antibody titer in the blood.
Individuals showing a sufficient increase in antibody titer were intraperitoneally immunized with 50 μg of each immunogen diluted in PBS 1 to 3 days before dissection. Then, spleen cells, iliac lymph node cells and inguinal lymph node cells were collected and fused with myeloma cell SP2/0 by electrofusion method. The fused cells were cultured in a 96-well plate, and the culture supernatant was collected 7 or 8 days after the fusion. The above-mentioned antigen-immobilized ELISA was performed while replacing the antiserum solution with the culture supernatant, and cell lines producing antibodies that reacted with the immunogens were selected to obtain the following 3 antibody clones.
The samples were measured using Procollagen Intact PINP (Fujirebio Inc.) and Elecsys Reagent Total P1NP (Roche Diagnostics K.K.) by an outside contractor.
10 mg of magnetic particles (manufactured by Sekisui Medical Co., Ltd.) with a diameter of 3 μm were washed with PBS to remove the solution. A S24217 antibody solution or S24219 antibody solution diluted with PBS to give an absorbance of 0.5 at 280 nm was added to the magnetic particles and the resulting was stirred at room temperature for 20 hours. The antibody solution was removed, and the magnetic particles were washed three times with 1 mL of blocking solution (0.1% BSA, 50 mM HEPES, 150 mM NaCl, 0.01% Tween-20, 0.05% ProClin 300, pH 7.2). 500 μL of the blocking solution was added, and after stirring at room temperature for 16 hours, the resulting was diluted with the blocking solution to 0.5 mg/mL to obtain an S24217 antibody-immobilized magnetic particle suspension and an 824219 antibody-immobilized magnetic particle suspension.
S24206R antibody was prepared in PBS to a concentration of 2.0 mg/mL, followed by adding, with stirring, 20 equivalents of bis(2,2′-bipyridine) [4′-methyl-4-(4-NHS-4-oxobutyl)-2,2′bipyridine] ruthenium (II) bis(hexafluorophosphate) (hereinafter referred to as Ru complex NHS, manufactured by Tokyo Chemical Industry Co., Ltd.) per mole of antibody, which had been dissolved in a small amount of DMSO, and the resulting was stirred for 30 minutes at room temperature in the dark. 2M glycine was added while stirring so that the molar ratio of glycine to Ru complex NHS was 2:1, and the resulting was stirred for 20 minutes in the dark. Using Sephadex G-25 equilibrated with PBS, Ru complexes not bound to the antibody were removed from the resulting solution, and the solution was diluted to 1 μg/mL with a blocking solution to obtain a Ru complex-labeled S24206R antibody solution.
A fully automated electrochemiluminescence immunoassay device PicoLumi III (manufactured by Sekisui Medical Co., Ltd.) was used for the measurement. 100 μL of blocking solution containing mouse IgG and 5 μL of the measurement sample were added to a reaction tube. 25 μL of the S24217 antibody-immobilized magnetic particle suspension or the S24219 antibody-immobilized magnetic particle suspension was added and reacted at 28° C. for 5 minutes. After separation of BF using the BF separation solution specified for the device, 100 μL of Ru complex-labeled S24206R antibody solution was added and the resulting was reacted for 3 minutes. After the BF separation, the magnetic particles were suspended in the substrate (tripropylamine) solution specified for the device, and electricity was applied at a predetermined position on the device.
The samples used were a mixture of equal amounts of sample 1 and sample 2, as well as sample 1, as shown in the table below. Sample 1 contains a large amount of monomeric P1NP.
Monomeric P1NP and trimeric P1NP in the sample were separated by size exclusion chromatography using Prominence (LC-20A Series manufactured by Shimadzu Corporation) and TSKgel G2000SW XL (Tosoh Corporation). PBST (phosphate-buffered saline with Tween®) was delivered at a flow rate of 1.0 mL/min, and 20 μL of the sample was injected. 500 μL fractions were collected in test tubes to which 100 μL of 1% BSA/PBST had been added beforehand.
From the elution times of the molecular weight marker MW-Marker (HPLC) (Oriental Yeast Co., Ltd.), the trimeric P1NP was mainly contained in fraction No. 7 and the monomeric P1NP was contained in fraction No. 10. Each fraction was measured by electrochemiluminescence using a combination of S24217 as the solid-phase antibody and S24206R as the Ru-labeled antibody, and by Elecsys reagent total P1NP. In the former, serial dilutions of serum from patients with cancer bone metastasis with high P1NP levels, valued with the Elecsys reagent total P1NP, were used as standard to calculate the measured values. Both S24217 and S24206R are antibodies that bind to the al chain, but when the fraction containing monomeric P1NP was measured by electrochemiluminescence using the combination of these antibodies, no significant increase in the measured value was observed. Therefore, the antibody combination of S24217 and S24206R can specifically detect trimer P1NP.
<Measurement of Samples from Osteoporosis Patients and Healthy Subjects by ELISA>
15 Samples from healthy subjects and 5 samples from osteoporosis patients without renal disease were used.
Sandwich ELISA was performed using the antibody combinations shown in Table 3. The same samples were also measured with Procollagen Intact PINP and Elecsys Reagent Total P1NP.
The results are shown in
<Measurement of Samples from Patients with Osteoporosis and Renal Disease Using Electrochemiluminescence Method>
10 samples from patients with renal disease and 7 samples from patients with osteoporosis were used. Serial dilutions of serum from patients with cancer bone metastasis with high P1NP levels, valued with the Elecsys reagent total P1NP, were used as standard, and P1NP measurements were calculated by electrochemiluminescence method using the antibody combinations shown in Table 4. The same samples were also measured with Procollagen Intact PINP and Elecsys Reagent Total P1NP.
The results are shown in
<Measurement of Samples from Patients with Renal Disease Using ELISA>
3 Samples from healthy individuals, 10 samples from osteoporosis patients without renal disease, and 75 samples from patients with renal disease were used. Sandwich ELISA was performed using the antibody combinations shown in Table 3. The same samples were also measured with Procollagen Intact PINP and Elecsys Reagent Total P1NP.
The results are shown in
The relationship between renal disease (chronic renal disease) stage and eGFR (unit: mL/min/1.73 m2) is as follows:
“NPMD” is an acronym for the National Institute of Technology and Evaluation, Patent Microorganism Depositary (Room 122, 2-5-8 Kazusakamatari, Kisarazu, Chiba Prefecture, Japan).
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022-050846 | Mar 2022 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2023/011188 | 3/22/2023 | WO |
