JP2016114573A - Pretreatment method of sample for oxytocin detection - Google Patents

Abstract

The present invention provides a means for more accurately detecting oxytocin present in a trace amount (ng to pg / mL) in a biological sample. A pretreatment method for quantitative detection of a receptor for advanced glycation end-products (RAGE) binding ligand in a biological sample, comprising: And the above method comprising the step of subjecting to an affinity chromatography using a column immobilized with a RAGE protein, an anti-esRAGE antibody or an anti-sRAGE antibody. Oxytocin is included as a RAGE binding ligand. [Effect] By using RAGE protein (mRAGE, esRAGE, sRAGE) and / or anti-esRAGE antibody or anti-sRAGE antibody, it becomes possible to collect and purify oxytocin in biological fluid coexisting with various components. . According to the present invention, it is possible to analyze a trace amount of oxytocin in a biological fluid with high reproducibility and high sensitivity. [Selection figure] None

Description

  The present invention relates to a sample pretreatment method for detecting oxytocin present in a biological fluid.

  Oxytocin (OT) is a 9-amino acid cyclic peptide discovered as a labor accelerator, synthesized mainly by oxytocinergic neurons in the hypothalamus and released into the circulation from the nerve endings of the posterior pituitary gland. The physiological functions of oxytocin are known to act as reproductive hormones such as uterine contraction during labor and promotion of milk secretion, and as a neurotransmitter and neuromodulator (modulator) in the central nervous system. . In the last 10 years or so, oxytocin in the brain has been attracting attention due to its important action in social behavior (trust, friendship, affection, etc.) (Non-Patent Documents 1-3). For example, brain oxytocin concentration in autistic children is lower than that in healthy individuals, and it has been reported that oxytocin spray administration from the nasal cavity improves symptoms.

  EIA (enzyme immunoassay) and RIA (radioimmunoassay) methods based on antigen-antibody reactions are used to measure oxytocin concentrations in vivo (Non-patent Document 4). Recently, high sensitivity analysis by liquid chromatography mass spectrometry (LC-MS / MS) has also been reported (Non-patent Document 5).

  On the other hand, due to the reaction between protein and sugar, that is, by non-enzymatic sugar addition reaction (Maillard reaction or glycation), via Schiff base and Amadori rearrangement product (previous product), complex such as dehydration, oxidation, condensation, etc. Through advanced reactions, advanced glycation end-products (AGE) are formed. AGE is a general term for structures produced by such glycation, and is attracting attention as being involved in diabetic complications, arteriosclerosis, neurodegenerative diseases, and aging.

  RAGE (Receptor for advanced gylcation end-products) discovered as an AGE receptor is a protein associated with various diseases represented by inflammation and vascular disorders. RAGE has two main types of splicing variants due to alternative splicing generated from one Ager gene. One is membrane-bound RAGE (membrane-bound full-length RAGE, mRAGE), and the other is endogenous secretory RAGE (endogenous secretory RAGE, esRAGE) that lacks the transmembrane region on the C-terminal side and is secreted ( Patent Document 1). Furthermore, soluble RAGE (soluble RAGE, sRAGE) of the extracellular domain cleavage form of mRAGE also exists in plasma (nonpatent literature 6).

  mRAGE was isolated as a cell surface specific receptor protein of AGE by the group of Stern et al., and its primary structure was determined (Non-patent Document 7). Currently, it is known that the molecular weight of full-length (membrane-bound) human RAGE that has undergone sugar chain modification is 55 kDa. mRAGE is a single transmembrane receptor belonging to the immunoglobulin superfamily. It has an extracellular region consisting of three immunoglobulin-like domains, V, C1, and C2, and a short intracellular region, the most N-terminal. There is an AGE ligand binding site inside the immunoglobulin variable region-like domain part (V).

  In recent years, RAGE has been recognized as a multiligand receptor, and as a ligand other than AGE, β-amyloid protein that accumulates in the brain of Alzheimer's disease, its association with cancer metastasis and inflammation has been pointed out. box 1 (HMGB-1), inflammatory mediator S100 / calgranulin secreted from immune cells, Mac1 / CD11b on leukocyte cell surface, bacterial membrane component lipopolysaccharide, complement C3a, phosphati on apoptotic cells Zirserin and the like have been reported. So far, RAGE has been reported to be associated with diseases such as diabetes, diabetic vascular disorder, Alzheimer's disease, etc., and ligands such as AGE and β-amyloid bind to RAGE and signal through activation of NF-κB Is said to have undesirable effects on the living body related to various pathologies and their onset and severity.

JP2003-125786

Carter, C.S. et al., Annu. Rev. Psychol. 65, 17-39 (2014) Insel, T.R., Neuron 65, 768-779 (2010) Modi, M.E. & Young, L.J., Horm. Behav. 61, 340-350 (2012) A. Szeto et al., Psychosomatic Medicine 73, 393-400 (2011) G. Zhang et al., Anal. Biochem. 416, 45-52 (2011) Yamamoto, Y. & Yamamoto, H. J. Diabetes Invest. 2 (3) 155-157, 2011 Neeper, M. et al., J. Biol. Chem. 267, 14998-15004 (1992)

  In order to grasp the patient's condition and confirm the therapeutic effect, it is necessary to accurately measure the in vivo oxytocin concentration. However, the amount of oxytocin in the brain and blood is extremely small, and according to the conventional detection method, the actual value is that the analytical value (concentration) varies greatly by 2 to 3 digits depending on the measurement conditions. Therefore, for example, the reported values for the concentration of oxytocin in healthy human plasma were also unreliable.

  An object of the present invention is to provide a means for more accurately detecting oxytocin present in a trace amount (ng to pg / mL) in a biological sample.

  In general, it is known that peptides in biological samples such as blood have a short lifetime because they are degraded by various peptidases. Peptidase-resistant peptides include those that form complexes with proteins that are stably present in blood such as albumin and receptors, those that form multiple disulfide bonds within the molecule, or sugar chains such as sialic acid. And all of them are immune from attack by peptidases.

  Oxytocin is a peptide having a molecular weight of about 1000 having the amino acid sequence shown in SEQ ID NO: 1 and having a cyclic structure in which the cysteine residues at positions 1 and 6 are disulfide bonded. Although the half-life of oxytocin present in the brain is several hours, it is unstable in blood, and the half-life is several minutes to several tens of minutes. The present inventors examined the possibility that some component in the sample forms a complex with oxytocin as a cause of the variation in the analytical value in the detection of the oxytocin concentration.

  As a protein that binds to oxytocin in vivo, there are oxytocin receptors expressed on cell membranes of various tissues, but other proteins that bind to oxytocin to form a complex have not been known so far. It was.

  The present inventors have surprisingly discovered that oxytocin forms a complex with a late glycation end product (Receptor for advanced gylcation end-products, RAGE). And it discovered that the oxytocin in the sample which was lost in the pretreatment operation etc. by the conventional method can be detected more accurately by utilizing the binding affinity between oxytocin and RAGE.

  The present inventors further developed the above method because RAGE is a multi-ligand receptor, and analyzed the ligand binding to RAGE as a whole, including oxytocin, and used it to investigate the relationship with the disease state. We found that it can be used for RAGE focus metabolomics.

That is, the present invention provides the following (1) to (5).
(1) A pretreatment method for quantitative detection of a receptor for advanced glycation end-products (RAGE) binding ligand in a biological sample, the biological sample comprising: The method as described above, comprising the step of subjecting to affinity chromatography using a column on which RAGE protein is immobilized.
(2) A pretreatment method for quantitative detection of a RAGE-binding ligand in a biological sample, wherein the biological sample is subjected to affinity chromatography using a column to which an anti-esRAGE antibody or anti-sRAGE antibody is immobilized. A method as described above, comprising the step of subjecting to a graph.
(3) A pretreatment method for quantitative detection of RAGE-binding ligand in a biological sample, wherein the biological sample is
(A) subjecting to affinity chromatography using a column immobilized with RAGE protein, and (b) subjecting to affinity chromatography using a column immobilized with anti-esRAGE antibody or anti-sRAGE antibody, Method.
(4) The method according to any one of (1) to (3) above, wherein the RAGE-binding ligand comprises oxytocin.
(5) A method for detecting oxytocin in a biological sample, wherein the sample pretreated by the method described in any one of (1) to (3) above is used to detect oxytocin by immunological detection or mass spectrometry. Detecting the above method.

  By using RAGE proteins (mRAGE, esRAGE, sRAGE) and anti-RAGE antibodies, it becomes possible to collect and purify oxytocin in biological fluids coexisting with various components. Therefore, by using this sample pretreatment method, it is possible to analyze a trace amount of oxytocin in a biological fluid with high reproducibility and high sensitivity.

The alignment of the amino acid sequence (sequence number 2-5) of mRAGE polypeptide derived from a human, a cow, a rat, and a mouse | mouth, and the V, C1, and C2 area | region in a sequence are shown. signal: signal sequence, TM: membrane binding region. FIG. 6 shows the binding of oxytocin to esRAGE immobilized on a sensor chip by surface plasmon resonance (SPR) solid phase binding assay. KA = 5.59 x 10 ^-6 M ^-1 and KD = 1.76 x 10 ^-7 M. Microtiter plate assay (n = 6). The concentration dependence of the binding of oxytocin to the immobilized 1 μg / ml esRAGE is shown. Microtiter plate assay (n = 6). The concentration dependence of the binding of esRAGE to 1 μM oxytocin immobilized is shown. Oxytocin and the stable isotope-labeled oxytocin used in the present invention will be described. The calibration curves of oxytocin (a) and [ ¹³ C, ¹⁵ N] oxytocin (b) prepared using the method of the present invention are shown. The concentration purification of oxytocin in human serum using an affinity column in which esRAGE is immobilized on beads is shown. The peak of endogenous oxytocin is shown in the LC-MS / MS chromatogram. The peak of endogenous oxytocin + spiked oxytocin (100 ng) is shown in the LC-MS / MS chromatogram. The spiked [ ¹³ C, ¹⁵ N] oxytocin peak in the LC-MS / MS chromatogram is shown. The result of the chromatogram which monitored the mode of refinement | purification by the affinity column which fix | immobilized the anti-esRAGE antibody on the surface of the Sepharose bead is shown. The result of having carried out the quantitative analysis of the human serum endogenous oxytocin in a preparative liquid (50 microliters) by LC-MS / MS (MRM) method is shown.

  We have various forms of late saccharification reaction product receptor (RAGE) proteins, namely membrane bound RAGE (mRAGE), endogenous secreted RAGE (esRAGE) and mRAGE extracellular domain cleaved forms. It was found that some sRAGEs bind to oxytocin to form a complex. Among these RAGE proteins, soluble RAGEs present in blood, that is, esRAGE and sRAGE are assumed to have a function as a protective agent that stabilizes oxytocin in blood. Therefore, in this specification, "RAGE protein" includes mRAGE, esRAGE and sRAGE.

  The present inventors have further focused on the above findings, and can efficiently collect oxytocin in a sample by utilizing the affinity between RAGE and oxytocin, and further using an antibody against RAGE. It has been found that oxytocin can be detected more accurately. The method of the present invention can be an effective means for collecting oxytocin present in a trace amount in a biological fluid.

  Furthermore, since RAGE is a multi-ligand receptor, the present invention can be used to analyze RAGE-binding ligands as a whole.

  That is, the present invention, as one embodiment, is a pretreatment method for quantitative detection of a receptor for advanced glycation end-products (RAGE) binding ligand in a biological sample. And subjecting the biological sample to affinity chromatography using a column immobilized with RAGE protein. By the method of the present invention, a RAGE binding ligand, preferably a ligand containing oxytocin, which may be present in a free form in a sample is captured on a column immobilized with RAGE protein, which is then used with an appropriate eluate. Can be eluted.

  RAGE binding ligands subject to quantitative detection in the present invention include S100B / calgranulin, AGE, amyloid β, HMGB1, low molecular weight heparin, oxytocin, vasopressin, Mac1 / CD11b, lipopolysaccharide, complement C3a, apoptotic cells The above phosphatidylserine and the like can be mentioned. In particular, the method of the present invention is characterized by including oxytocin, which has not been known to bind to RAGE.

  The RAGE protein used in the method of the present invention may be any of mRAGE, esRAGE, or sRAGE. Examples of the mRAGE polypeptide that can be used in the present invention include a human-derived mRAGE polypeptide represented by the amino acid sequence of SEQ ID NO: 2, a mouse-derived mRAGE polypeptide represented by the amino acid sequence of SEQ ID NO: 3, and the amino acid of SEQ ID NO: 4. Examples thereof include a rat-derived mRAGE polypeptide represented by the sequence and a bovine-derived mRAGE polypeptide represented by the amino acid sequence of SEQ ID NO: 5. Examples of the esRAGE polypeptide that can be used in the present invention include a human-derived esRAGE represented by the amino acid sequence of SEQ ID NO: 6 and a mouse-derived esRAGE polypeptide represented by the amino acid sequence of SEQ ID NO: 7. Furthermore, as described above, sRAGE includes those in the cleaved form of the extracellular domain of mRAGE, but more broadly includes soluble peptides containing the RAGE V region, and soluble peptides containing the V region and C1 region. be able to.

  In the method of the present invention, the RAGE protein is added to a protein consisting of the amino acid sequence represented by SEQ ID NOs: 2 to 7, and 1 to several, for example, 10 or less, A protein having an addition, deletion, or substitution of 3 or less amino acids and having binding ability to oxytocin is included.

The binding between oxytocin and the RAGE protein can be confirmed qualitatively and quantitatively by, for example, a surface plasmon resonance method, a plate binding assay or the like. In the surface plasmon resonance method, oxytocin bound to the immobilized recombinant esRAGE, and in the test conducted by the present inventors, the apparent dissociation rate constant (K _D ) was 179 nM (FIG. 2a). The concentration dependence and selectivity of binding was confirmed by plate assay (Figures 2b and 2c). As used herein, “binding ability” refers to binding defined by a dissociation constant of 10 μM or less, preferably 3 μM or less, more preferably 1 μM or less, even more preferably 500 nM or less, and most preferably 300 nM or less with respect to oxytocin. It means having strength.

  The RAGE protein that can be used in the present invention may be a fragment as long as it has the ability to bind to oxytocin. For example, in the amino acid sequence of SEQ ID NO: 6 showing the amino acid sequence of human-derived esRAGE, any fragment containing 62 amino acids or more that essentially contains the amino acids at positions 38 to 99 can be suitably used in the present invention.

  FIG. 1 shows an alignment of the amino acid sequences (SEQ ID NOs: 2 to 5) of the above-described human, bovine, rat and mouse-derived mRAGE polypeptides. As shown in the figure, the amino acids at positions 38 to 99 in the human mRAGE amino acid sequence (SEQ ID NO: 2) are the V region, the amino acids at positions 144 to 208 are the C1 region, and the amino acids at positions 258 to 301 are the amino acids in the C2 region. Equivalent to. Those skilled in the art can synthesize necessary peptide fragments based on this sequence information.

  The amino acid sequence and function of the soluble RAGE protein are disclosed in, for example, Japanese Patent Application Laid-Open No. 2003-125786, Japanese Patent Application Laid-Open No. 2003-128700, and the like, and can be suitably used in the present invention by those skilled in the art. Fragments can be easily recognized.

  Affinity chromatography techniques are well known in the art, and those skilled in the art can prepare an affinity column that can be used in the present invention based on the description of the present specification. As the column and packing material for affinity chromatography, for example, those manufactured by GE Healthcare Japan Co., Ltd., Tosoh Corporation, Showa Denko Co., Ltd., etc. can be obtained as commercial products. The affinity column used in the method of the present invention can be prepared, for example, by binding RAGE protein to a filler such as Sepharose via a spacer.

  The method of the present invention includes the step of subjecting a biological sample to affinity chromatography using the column. A biological sample is a sample taken from a subject that is assumed to have a RAGE-binding ligand, such as a body fluid such as plasma, serum, saliva, pleural effusion, ascites, cerebrospinal fluid, etc. obtain. This step captures the RAGE binding ligand present in the sample, particularly the RAGE binding ligand containing oxytocin, on the column.

  According to the findings of the present inventors, oxytocin may exist by binding to esRAGE or sRAGE present in blood to form a stable complex. Therefore, the method of the present invention uses a (second) affinity column in which an anti-esRAGE antibody or an anti-sRAGE antibody is immobilized in addition to or separately from the above (first) affinity chromatography. Performing chromatography.

  Accordingly, another embodiment of the present invention is a pretreatment method for quantitative detection of RAGE binding ligand in a biological sample, wherein the biological sample is immobilized with anti-esRAGE antibody or anti-sRAGE antibody. It is the said method including the step which uses for the affinity chromatography using the activated column. According to the method of the present invention, a ligand that forms a complex by binding to the RAGE protein in the sample is captured on the column to which the anti-esRAGE antibody or the anti-sRAGE antibody is immobilized, and then this is used as an appropriate eluate. And can be eluted.

  The anti-esRAGE antibody or anti-sRAGE antibody can be prepared by a method known to those skilled in the art using the above esRAGE or sRAGE as an antigen. More specifically, for example, an antibody against soluble RAGE can be prepared by, for example, the method described in JP-A-2003-128700.

  If an anti-esRAGE antibody or an anti-sRAGE antibody is immobilized on the bead surface and packed into a column, an affinity column for these complexes can be prepared. Alternatively, if the antibody is immobilized on the surface of the magnetic beads, the complex can be easily collected, washed and eluted by turning the magnetic field on and off. Thereafter, oxytocin can be released from the receptor. The advantages of magnetic bead-immobilized antibodies are described in the VERITAS “Dynabeads” document (http://www.veritastk.co.jp/attached/4911/Dynabeads_catalog), which is faster and easier to operate. There are some points.

Another embodiment of the present invention is a pretreatment method for quantitative detection of RAGE binding ligand in a biological sample, the biological sample comprising:
(A) subjecting to affinity chromatography using a column immobilized with RAGE protein, and (b) subjecting to affinity chromatography using a column immobilized with anti-esRAGE antibody or anti-sRAGE antibody, Is the method. Here, steps (a) and (b) may be carried out first.

  By the above step (a), a RAGE-binding ligand, preferably a ligand containing oxytocin, which may be present in a free form in the sample, is captured on the column on which the RAGE protein is immobilized, and this is then used as an appropriate eluate. And can be eluted.

  In addition, in step (b) above, the ligand that forms a complex by binding to the RAGE protein in the sample is captured on the column to which the anti-esRAGE antibody or anti-sRAGE antibody is immobilized, and then this is appropriately used. Can be eluted using a simple eluate.

  By combining the above steps (a) and (b), it is possible to reliably detect both the ligand that forms a complex with the RAGE protein in the sample and the ligand that exists in a free form. It becomes like this.

  For example, in the (first) affinity chromatography in step (a), the free form of the RAGE-binding ligand is captured by binding to the RAGE protein immobilized on the column. However, the RAGE-binding ligand that has already formed a complex with esRAGE or sRAGE in the sample may elute without being captured by the column. The complexed RAGE-binding ligand eluted here is then bound to the anti-esRAGE antibody or anti-sRAGE antibody immobilized on the column by subjecting it to the (second) affinity chromatography in step (b). Can be captured.

  Alternatively, the order of step (a) and step (b) can be reversed. That is, by first subjecting the biological sample to the affinity chromatography in step (b), the RAGE binding ligand that has already formed a complex with esRAGE or sRAGE in the sample becomes an anti-esRAGE antibody or anti-sRAGE antibody. Captured on an immobilized column. In this case, the RAGE-binding ligand present in a free form in the sample may be eluted without being captured by the column. Therefore, the free form of the RAGE-binding ligand eluted here can be bound to and captured by the RAGE protein immobilized on the column by subjecting it to the affinity chromatography in step (a).

  As another embodiment, the present invention provides a method for detecting oxytocin in a biological sample, wherein the sample pretreated by any of the above methods is used to detect oxytocin by immunological detection or mass spectrometry. The above method is provided.

  The immunological detection of oxytocin was performed using a commercially available oxytocin ELISA kit (Enzo Life Sciences, NY, USA), for example, using a previously reported pretreatment (Jin D. et al., Nature 446, 41-45 (2007) ) Can be quantified without performing.

  In the present invention, mass spectrometry can be performed by liquid chromatography tandem mass spectrometry (LC-MS / MS) combined with high performance liquid chromatography, and is widely used in the art for quantitative detection of peptides. It can be done by multiple reaction monitoring (MRM). The tandem MS (MRM) analysis is for selecting a precursor ion derived from a target peptide and further subjecting it to mass spectrometry, and the peptide can be identified and quantified by the generated product ion.

  EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples, and various modifications can be made within the scope of the claims. .

The material used in this example was obtained as follows. Oxytocin was obtained from Peptide Institute. High pressure liquid chromatography mass spectrometer (LC-MS) grade water, acetonitrile (ACN), formic acid, trifluoroacetic acid (TFA) and trichloroacetic acid (TCA) were purchased from Wako Pure Chemical Industries, Ltd. Phosphate buffered saline (PBS) was purchased from Sigma-Aldrich (St. Louis, USA). The stable isotope oxytocin uses [ ¹³ C × 5, ¹⁵ N × 1] proline and [ ¹³ C × 6, ¹⁵ N × 1] leucine, and oxytocin 7-position proline and 8-position leucine are each [ ¹³ C , ¹⁵ N] proline and [ ¹³ C, ¹⁵ N] leucine were synthesized. The obtained oxytocin isotope is referred to as [ ¹³ C, ¹⁵ N] OT in the present specification. The purity of [ ¹³ C, ¹⁵ N] OT TFA salt was> 95% as confirmed by HPLC. The theoretical molecular weight was 1020.2, higher than 1007.2 for natural oxytocin.

  In enzyme immunoassays, the level of oxytocin immunoreactivity was determined using a commercially available oxytocin ELISA kit (Enzo Life Sciences, NY, USA) using a previously reported pretreatment (Jin D. et al., Nature 446, 41 -45 (2007)). The same lot number kit was used in all experiments (catalog number AD1901-153). Cerebrospinal fluid (CSF) samples (5 μl) were thawed and diluted 1:20 with assay buffer. Plasma samples (100 μl) were thawed on ice and assayed according to the instructions. The sensitivity of the oxytocin assay was 5 pg / ml and the coefficient of variation between and within the assay was less than 15%.

[Example 1]
Oxytocin binding to RAGE In order to examine the binding of oxytocin to RAGE, surface plasmon was used using purified recombinant esRAGE (purified with affinity column from COS cell expression strain, Biochem. J. (2003) 370, 1097-1109). Resonance and plate binding assays were performed.

  First, purified human esRAGE with a ligand binding domain was immobilized to a BIAcore CM5 research grade sensor chip at a density of about 5,000 response units (RU) using an amine coupling kit. Oxytocin binding to esRAGE bound to immobilized LPS was measured using a BIAcore 2000 system (GE Healthcare, Japan). The flow buffer contains 10 mM HEPES (pH 7.4), 0.15 M NaCl, 3 mM Na-EDTA and 0.005% (V / V) surfactant P-20. Association and dissociation were measured at 25 ° C. and a flow rate of 20 μL / min.

As a result, physical direct binding was observed between esRAGE and oxytocin (FIG. 2a). The apparent dissociation (K _D ) rate constant calculated for oxytocin binding to esRAGE was 179 nM.

  Binding of oxytocin to RAGE and competition between various RAGE ligands and oxytocin-RAGE binding in plate-binding assays has been demonstrated in oxytocin-coated 96-well plates, human esRAGE protein and HRP-labeled anti-human RAGE antibody (B-Bridge International, Inc. esRAGE ELISA Kit). Coat 100 μM oxytocin with 100 μl overnight on a 96-well plate for ELISA, then wash, block with 1% BSA / PBS solution, then wash, shake the esRAGE protein at a varying concentration for 2 hours at room temperature. Incubation followed by washing was performed to quantify the amount of esRAGE protein bound by the human RAGE antibody.

  The binding between oxytocin and esRAGE was dependent on the oxytocin concentration and esRAGE concentration (FIGS. 2b and 2c). Two similar peptides with 9 amino acids were also tested and found to be specific for oxytocin. Arginine-vasopressin bound to RAGE with low affinity and no bradykinin binding was observed (data not shown).

[Example 2]
Preparation of calibration curves for oxytocin and [ ¹³ C, ¹⁵ N] oxytocin Using cerebrospinal fluid as a matrix, five different concentrations of oxytocin (OT, 0.0-40 ng / mL) and stable isotope substitution ([ ¹³ C, ¹⁵ N] ) A standard sample of oxytocin (0.0-30.2 ng / mL) (FIG. 3) was prepared. Stock solutions of OT and [ ¹³ C, ¹⁵ N] OT were prepared from OT (100 μg) and [ ¹³ C, ¹⁵ N] OT (75 μg) using 0.1% TFA (1 mL) and stored at −80 ° C. . Standard samples were prepared by diluting the stock solution with 0.1% TFA as needed.

  These standard samples were analyzed repeatedly by liquid chromatography tandem mass spectrometry. The LC-MS / MS system uses a system consisting of Shimazu UFLC (Shimadzu Corporation) and AB Sciex 4000 QTRAP (AB Sciex), and samples are separated on a ZORBAX 300SB-C8 column (2.1 x 150 mm, 5 μm). , Detected in the multiple reaction monitoring (MRM) mode of the MS / MS system (hereinafter referred to as the LS-MS / MS (MRM) method).

Tables 1 and 2 show the peak area, average area (Ave), standard deviation (SD), and relative SD (RSD%) for each of oxytocin and [ ¹³ C, ¹⁵ N] oxytocin. A calibration curve was prepared by optimal fitting by the least square method of all data. From the calibration curve and MRM chromatogram (Figure 4), the correlation coefficient (oxytocin: 0.997 and [ ¹³ C, ¹⁵ N] oxytocin: 0.998) and the lower limit of quantification (oxytocin: 0.3 ng / mL and [ ¹³ C, ¹⁵ N] oxytocin : 0.06 ng / mL).

[Example 3]
First affinity chromatography purified esRAGE (0.5mg) immobilized on NHS-activated Sepharose beads surface (inner diameter 1.6mm, length 2.5mm, trade name HiTrap NHS-activated HP (manufactured by GE Healthcare)) ) Was produced. Low molecular weight fraction (MW <3,000, Amicon Ultracel3K) derived from human serum (50 mL) spiked or not spiked with a known amount of oxytocin (100 ng) on a column pre-equilibrated with PBS (HiTrap-esRAGE column) Applied. The column was washed with 50 mL of PBS solution and then eluted from the column with 10 mM Tris-HCl (pH 7.4) and 2 M NaCl.

  FIG. 5a is a chromatogram recorded by monitoring the state of affinity column purification with a UV (280 nm) detector. The peak fraction indicated by the arrow was collected and assayed by EIA to confirm that the peak had immunoreactivity with oxytocin. That is, endogenous oxytocin and synthetic oxytocin spiked in human serum (100 ng) both bound to the immobilized esRAGE and could then be eluted.

  From the results of this example, it is possible to purify and concentrate oxytocin that may be present in a sample using the binding affinity between oxytocin and RAGE protein, and RAGE protein as a pretreatment method for quantitative detection of oxytocin It has been demonstrated that affinity chromatography with immobilized can be used.

[Example 4]
LC-MS / MS (MRM) analysis Peak fractions indicated by arrows in Fig. 5a were collected and subjected to LC-MS / MS analysis.
Elution from HPLC uses two eluents: A (0.1% (v / v) formic acid / water) and B (0.1% (v / v) formic acid / acetonitrile). 0.0-8.0 min) and increased linearly. The HPLC pump flow rate was set at 0.3 ml / min and the column temperature was maintained at 50 ° C. MS / MS (MRM) analysis conditions are as follows: positive ion electrospray (ESI) mode (capillary voltage 5 kV); solvent removal pressure (75 V); curtain gas (nitrogen, 12 l / h, 65 psi and 600 ° C.) ), Nitrogen gas pressure (65 psi), and collision pressure (30V). The types of MRM transition ions and fragments are as follows: OT: (m / z: 1008.2, 723.2 (b6)), [ ¹³ C, ¹⁵ N] OT: (m / z: 1021.2, 723.2 (b6)). All data were acquired and analyzed with AB Sciex Analyst Version 1.4.1.

Figures 5b and 5c are the results. FIG. 5b shows the amount of endogenous oxytocin in the serum, and FIG. 5c shows the amount of oxytocin when the serum spiked with a known amount (100 ng) of oxytocin is analyzed. From the peak area values of these two data, the endogenous oxytocin concentration (1.6 ng / mL) could be calculated. FIG. 5d is a detection peak of stable isotope substitution [ ¹³ C, ¹⁵ N] OT spiked in serum as an internal standard.

  In general, analysis of complex samples such as serum shows many interference peaks even in highly selective LC-MS / MS analysis, but in samples purified and concentrated with an affinity column using the method of the present invention, It was found that there were few peaks.

[Example 5]
Affinity column (1.6 mm ID, 2.5 mm length, trade name HiTrap NHS-activated HP (GE Healthcare) with a second affinity chromatography monoclonal anti-esRAGE antibody (1.0 mg) immobilized on the surface of NHS-activated Sepharose beads Manufactured)). 70 ml of healthy human serum was applied to a column (HiTrap-anti-RAGE column) previously equilibrated with 50 mM Tris-HCl (pH 7.4) and 0.15 M NaCl. After washing with 5 bed volumes of equilibration buffer, the bound protein was eluted with 100 mM glycine-HCl buffer (pH 2.5) and aliquoted (1 mL). FIG. 6a shows the result of chromatogram recorded by monitoring the state of affinity column purification with a UV (280 nm) detector.

  In the same manner as in Example 2, oxytocin in the preparative solution (50 μL) was quantitatively analyzed by the LC-MS / MS (MRM) method. FIG. 6b shows the result.

  The results of this example suggest that a part of endogenous oxytocin is present in the blood as a complex with esRAGE, and using the binding affinity between oxytocin and RAGE protein, It was demonstrated that affinity chromatography with immobilized anti-esRAGE antibody or anti-sRAGE antibody can be used as a pretreatment method for quantitative detection.

  The method of the present invention is effective for examining relevance to various disease states by exhaustively extracting RAGE-binding ligands from biological samples and measuring the increase or decrease of each amount.

  In addition, oxytocin concentration in biological samples containing contaminants such as plasma can be quantified more accurately by finding a binding between oxytocin and RAGE, whose behavior in blood has been unknown in the past. Thus, a more accurate blood concentration before and after administration of oxytocin can be monitored.

Claims (5)

  A pretreatment method for quantitative detection of a receptor for advanced glycation end-products (RAGE) binding ligand in a biological sample, wherein the biological sample is treated with RAGE protein The method as described above, which comprises the step of subjecting to affinity chromatography using an immobilized column.   A pretreatment method for quantitative detection of a RAGE-binding ligand in a biological sample, wherein the biological sample is subjected to affinity chromatography using a column to which an anti-esRAGE antibody or anti-sRAGE antibody is immobilized. The above method comprising the steps. A pretreatment method for the quantitative detection of RAGE binding ligands in a biological sample, the biological sample comprising:
(A) subjecting to affinity chromatography using a column immobilized with RAGE protein, and (b) subjecting to affinity chromatography using a column immobilized with anti-esRAGE antibody or anti-sRAGE antibody, Method.   The method according to any one of claims 1 to 3, wherein the RAGE-binding ligand comprises oxytocin.   A method for detecting oxytocin in a biological sample, wherein oxytocin is detected by immunological detection or mass spectrometry using the sample pretreated by the method according to any one of claims 1 to 3. Method.

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