Affinity solid phase for lead ion bond

Abstract

To provide an affinity carrier that can selectively capture lead, and is applicable to detection or quantitative determination of lead ion in a lead-containing sample of lower concentration.SOLUTION: An affinity solid phase for lead ion bond including a polypeptide made of a specific amino acid sequence and a solid phase material in which the polypeptide is immobilized can selectively capture lead, and is capable of detecting or quantitatively determining lead ion in a lead-containing sample of lower concentration.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an affinity solid phase for binding lead ions. [Background technology]

[0002] Industrial wastewater may contain high concentrations of toxic heavy metals or heavy metal compounds, and when such wastewater contaminates soil or water, it is necessary to remove such heavy metals or heavy metal compounds.

[0003] These heavy metals or heavy metal compounds may contain rare earths, which are highly valuable, and therefore techniques for separating and recovering such metals have been proposed.

[0004] For example, a method for recovering precious metals has been proposed (Patent Document 1), which includes the steps of mixing a peptide with an aqueous solution containing precious metal ions under acidic conditions, adjusting the aqueous solution to an alkaline state, and separating the precious metal captured by the peptide from the aqueous solution.

[0005] Also proposed is a method for recovering metals using a carrier for metal recovery which is a non-living organism whose outer layer is composed of a phospholipid bilayer membrane, in which a porin derived from a gram-negative bacterium penetrates the phospholipid bilayer membrane, and in which a metal ion-binding peptide motif is introduced into a region of the amino acid sequence of the porin which does not impair the porin function in the gram-negative bacterium, and in which the metal ion-binding peptide motif is presented on the surface of the phospholipid bilayer membrane (Patent Document 2).

[0006] Meanwhile, among heavy metals, lead in particular has a history of being used in various industrial products, and its release into the environment has caused water and air pollution. Furthermore, lead is absorbed into the human body directly from such polluted water and air, or indirectly through its concentration in animals and plants (Non-Patent Document 1). Lead poisoning has been reported for a long time, and it is now known that continued intake of low concentrations of lead over a long period of time has adverse effects on human reproduction and development. In particular, exposure of children to lead, which has been reported in many areas, mainly in developing countries, has become a serious problem as it weakens the next generation.

[0007] Furthermore, as part of efforts to combat environmental pollution caused by lead, research has begun into the ecological impact on birds of prey, including wild birds (Non-Patent Document 1). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP 2007-56308 A [Patent Document 2] International Publication No. 2018 / 203539 [Non-patent literature]

[0009] [Non-Patent Document 1] Document 1: Lead contamination of birds and the status of efforts to combat it, [online], March 22, 2022, Ministry of the Environment [Retrieved July 1, 2023], Internet<URL:https: / / www.env.go.jp / nature / choju / effort / effort14 / effort14-01 / mat1.pdf> Summary of the Invention [Problem to be solved by the invention]

[0010] The methods described in Patent Documents 1 and 2 do not target lead as a metal to be recovered, and selective recovery of lead is not possible. In addition, both methods are disclosed only as being applicable to samples containing the target metal at high concentrations, and it is not expected that they will be applied to the detection or quantification of lead ions in samples with low metal concentrations, such as environmental samples or biological samples for investigating lead contamination.

[0011] Therefore, an object of the present invention is to provide an affinity support that can selectively capture lead and is also applicable to the detection or quantification of lead ions in samples containing lower concentrations of lead. [Means for solving the problem]

[0012] As a result of intensive research, the present inventors have found that by immobilizing a peptide that selectively binds lead ions onto a solid phase material, lead can be selectively captured and the peptide can be used as an affinity carrier that can be applied even to samples containing lower concentrations of lead. The present invention was completed through further research based on this finding.

[0013] That is, the present invention provides the following aspects. Item 1. An affinity solid phase for binding lead ions, comprising a polypeptide consisting of an amino acid sequence represented by at least the 2nd to 8th positions of SEQ ID NO: 1, and a solid phase material to which the polypeptide is immobilized. Item 2. The affinity solid phase according to Item 1, wherein the solid phase material is polyacrylamide and / or polystyrene. Item 3. An affinity solid phase according to Item 1 or 2, wherein the solid phase material has a polyalkylene glycol chain, and the polypeptide is directly or indirectly bound to the polyalkylene glycol chain. Item 4. The affinity solid phase according to any one of Items 1 to 3, wherein the polypeptide is immobilized on the solid phase material via a linker residue consisting of 1 to 9 amino acid residues. Item 5. The affinity solid phase according to any one of Items 1 to 4, wherein a protecting group is bound to at least the end of the polypeptide opposite to the end bound to the solid phase material. Item 6. A method for producing an affinity solid phase for binding lead ions, comprising a step of synthesizing a polypeptide having an amino acid sequence represented by SEQ ID NO:1 on a solid phase material by solid-phase peptide synthesis, but not including a step of separating the polypeptide from the solid phase material. Item 7. A method for separating lead ions, comprising the step of contacting a sample solution containing lead ions with the affinity solid phase for binding lead ions according to any one of Items 1 to 5, thereby capturing the lead ions on the affinity solid phase. Item 8. The method according to Item 7, wherein the concentration of the lead ions contained in the sample solution is 100 nM to 100 μM. Item 9. The method according to Item 7 or 8, wherein the sample liquid is a biological sample liquid for testing, a water quality sample liquid, an environmental sample liquid other than water, an industrial product sample liquid, or a food or beverage sample liquid. Item 10. A method for capturing lead ions on the affinity solid phase by contacting a sample solution containing lead ions with the affinity solid phase for binding lead ions according to claim 1; contacting the affinity solid phase on which the lead ions have been captured with a chelating agent to form a lead chelate complex, thereby liberating the lead ions from the affinity solid phase; and determining the amount of lead ions from the lead chelate complex. Item 11. The method according to Item 10, wherein the chelating agent is ethylenediaminetetraacetic acid, and the quantification is carried out based on the absorbance of the lead chelate complex. Item 12. The method according to item 10, wherein the chelating reagent is a metal indicator and the quantification is carried out by chelometric titration. Item 13. A kit for quantifying lead ions, comprising the lead ion-binding affinity solid phase according to any one of Items 1 to 5 and a chelating reagent. Item 14. A step of contacting blood collected from an animal suffering from lead poisoning with an affinity solid phase for binding lead ions described in any one of Items 1 to 5; and recovering the blood that has come into contact with the affinity solid phase. Methods for producing autologous blood products. Effect of the Invention

[0014] According to the present invention, there is provided an affinity support which is capable of selectively capturing lead and is also applicable to samples containing lead at lower concentrations. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows a schematic diagram of a method for separating and quantifying lead ions using the lead ion-binding affinity solid phase of the present invention. [Diagram 2] It is shown that after lead ions are captured by the lead ion-binding affinity solid phase of the present invention (Example 3), all of the captured lead ions can be released by a single EDTA treatment (Test Example 2). [Diagram 3] It is shown that the lead ion-binding affinity solid phase of the present invention (Example 3) is capable of capturing and quantifying lead ions at any lead ion concentration from 500 nM to 5 μM (Test Example 3). [Figure 4] It is shown that the lead ion-binding affinity solid phase of the present invention (Examples 2, 4, 6, 7, and 8) can capture and quantify lead ions (Test Example 4-1). [Diagram 5] It is shown that the lead ion-binding affinity solid phase of the present invention (Examples 2, 4, and 6) enables the capture and quantification of lead ions even in the presence of serum contamination (Test Example 4-2). [Figure 6] It is shown that the lead ion-binding affinity solid phase of the present invention (Example 3) enables the capture and quantification of lead ions even in the presence of higher concentrations of serum contamination (Test Example 5). [Figure 7] It will be shown (Test Example 6) that the lead ion-binding affinity solid phase of the present invention (Examples 3 and 4) can capture and quantify lead ions. [Figure 8A]It is shown that the lead ion binding affinity solid phase of the present invention (Examples 1 and 3) can quantitatively determine lead ions (Test Example 7). The horizontal axis is in μm. [Figure 8B] It is shown that the lead ion binding affinity solid phase of the present invention (Examples 1 and 3) can quantitatively determine lead ions (Test Example 7). The horizontal axis is in μm. [Figure 8C] 1 shows a calibration curve for lead ions for the lead ion binding affinity solid phase of the present invention (Example 1) (Test Example 7). [Figure 8D] 1 shows a calibration curve for lead ions for the lead ion-binding affinity solid phase of the present invention (Example 3) (Test Example 7). [Figure 9] The selectivity for lead ions of the lead ion-binding affinity solid phase of the present invention (Example 3) is shown (Test Example 8). [Figure 10A] The results of repeatedly using the lead ion-binding affinity solid phase of the present invention (Example 1) for the capture and quantification of lead ions in the presence of serum contamination are shown. [Figure 10B] The results of repeatedly using the lead ion-binding affinity solid phase of the present invention (Example 3) for the capture and quantification of lead ions in the presence of serum contamination are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] 1. Affinity solid phase for binding lead ions The affinity solid phase for binding lead ions of the present invention is characterized by comprising a polypeptide consisting of an amino acid sequence represented by at least the 2nd to 8th positions of SEQ ID NO: 1, and a solid phase material to which the polypeptide is immobilized. In the present invention, the lead ion is Pb 2+ means.

[0017] Polypeptides A polypeptide consisting of an amino acid sequence represented by at least the 2nd to 8th positions of SEQ ID NO: 1 selectively binds lead ions. Specific examples of the polypeptide include a polypeptide consisting of SEQ ID NO: 1, a polypeptide consisting of an amino acid sequence represented by the 2nd to 9th positions of SEQ ID NO: 1 (SEQ ID NO: 2), a polypeptide consisting of an amino acid sequence represented by the 2nd to 8th positions of SEQ ID NO: 1 (SEQ ID NO: 3), and a polypeptide consisting of an amino acid sequence represented by the 1st to 8th positions of SEQ ID NO: 1 (SEQ ID NO: 4). Among these polypeptides, a polypeptide consisting of an amino acid sequence represented by any of SEQ ID NOs: 1 to 3 is preferable, and a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 1 is more preferable.

[0018] The amino acids constituting the polypeptide may be L-amino acids, D-amino acids, or a combination thereof.

[0019] The polypeptide may have a protecting group bound to one or more of its constituent amino acid residues. The polypeptide preferably has a protecting group bound to at least the end opposite to the end bound to the solid phase material. Typically, the protecting group is preferably bound directly or indirectly to the N-terminal amino acid residue of the polypeptide. The binding site of the protecting group may be the N-terminal amino group of the amino acid residue or a side chain functional group. This protecting group may be provided, for example, from the viewpoint of improving the resistance of the polypeptide to decomposition by a sample solution to which the lead ion binding affinity solid phase is applied.

[0020] The type of the protecting group is not particularly limited, but examples thereof include an aryl group, an acyl group, and a substituted or unsubstituted alkyl group.

[0021] Examples of the aryl group include a phenyl group, a naphthyl group (1-naphthyl group, 2-naphthyl group), an indenyl group, a biphenylyl group, an anthryl group, and a phenanthryl group, and preferably includes a naphthyl group, and more preferably includes a 2-naphthyl group. The aryl group can be directly or indirectly bound to the N-terminal amino group, for example. When the aryl group is indirectly bound to a peptide, the linker group interposed between the aryl group and the terminal amino acid residue of the polypeptide is not particularly limited as long as it is stable to the sample solution to which the lead ion binding affinity solid phase is applied, and examples thereof include an alkylene group (e.g., carbon number 1 to 6, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2), a carbonyl group, and a group in which these are combined. A preferred example of a form having an aryl group as a protecting group is a form in which an aryl group is indirectly bound to the N-terminal amino group, and more preferably, a form in which a naphthylmethyl (NAP) carbonyl group is amide-bound to the N-terminal amino group.

[0022] Examples of the acyl group include alkyl (carbon number 1 to 6) carbonyl groups, specifically methylcarbonyl group (acetyl group), ethylcarbonyl group, n-propylcarbonyl group, n-butylcarbonyl group, n-pentylcarbonyl group, n-hexylcarbonyl group, isopropylcarbonyl group, isobutylcarbonyl group, sec-butylcarbonyl group, tert-butylcarbonyl group, isopentylcarbonyl group, etc. The acyl group can be amide-bonded to the N-terminal amino group, for example.

[0023] The substituted or unsubstituted alkyl group includes a substituted or unsubstituted alkyl group (having 1 to 6 carbon atoms), specifically, a substituted or unsubstituted methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, an n-hexyl group, an isopropyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an isopentyl group, etc. When the alkyl group has a substitution, a substituent includes an alkylamide group, and an alkylamide group includes an alkyl (having 1 to 9 carbon atoms)amide group, more specifically, an acetamide group, a propionamide group, a pentanamide group, a 2-ethylhexanamide group, etc. A preferred example of the substituted or unsubstituted alkyl group includes an unsubstituted alkyl group, and more preferably, an acetamidomethyl (Acm) group. The substituted or unsubstituted alkyl group can be thioether-bonded to a side chain thiol group, for example.

[0024] When a protecting group is bonded to two or more of the constituent amino acid residues of a polypeptide, the protecting groups may be bonded to each other, so that the polypeptide forms a cyclic structure via the mutually bonded protecting groups (also referred to as linker groups). In this case, a preferred embodiment is one in which a protecting group is bonded to two of the constituent amino acid residues of the polypeptide (preferably the N-terminal amino acid residue and the C-terminal amino acid residue of the polypeptide), and the protecting groups are bonded to each other, so that the polypeptide forms a cyclic structure via linker groups at both ends. The combination of protecting groups forming the linker group constituting the cyclic structure includes any combination of the above-mentioned protecting groups, and preferably includes a combination of an acyl group and a substituted or unsubstituted alkyl group (in this case, the alkyl group portion of the acyl group and the substituted or unsubstituted alkyl group are bonded to each other via a sigma bond to form a carbonyl alkylene group (preferably a carboxy alkylene (having 2 to 12 carbon atoms, preferably 2 to 6, more preferably 2 to 4 carbon atoms) as the linker group).

[0025] In the present invention, typically, the C-terminus of the polypeptide is immobilized on a solid phase material.

[0026] The polypeptide is preferably immobilized on a solid phase material (solid phase carrier) via a linker residue consisting of 1 to 9 amino acid residues. Typically, the polypeptide is preferably immobilized on a solid phase material via a linker residue bound to its C-terminal amino acid residue (cysteine ​​residue).

[0027] The type of amino acid residue constituting the linker residue is not particularly limited.

[0028] When the polypeptide contains the 9th amino acid residue of SEQ ID NO: 1 (specifically, the polypeptide represented by SEQ ID NO: 1 or 2), the amino acid residue constituting the linker residue is preferably an amino acid residue that does not have a reactive group (such as a hydroxyl group, an amino group, a thiol group, or a carboxyl group) in the side chain, more preferably an amino acid residue in which the side chain is a hydrogen atom or a lower alkyl group (having 1 to 3 carbon atoms, preferably 1 to 2 carbon atoms, more preferably 1 carbon atom), still more preferably an amino acid residue in which the side chain is a hydrogen atom, even more preferably β-alanine and / or glycine, and even more preferably β-alanine.

[0029] When the polypeptide does not contain the 9th amino acid residue of SEQ ID NO:1 (specifically, the polypeptide represented by SEQ ID NO:3 or 4), the amino acid residue constituting the linker residue is preferably an amino acid residue having a reactive group (hydroxyl group, amino group, thiol group, carboxyl group, etc.) in the side chain, more preferably an amino acid residue having a carboxyl group in the side chain, and even more preferably a glutamic acid residue.

[0030] The number of amino acid residues constituting the linker residue (1 to 9) is preferably 1 to 8, 1 to 7, or 1 to 6, more preferably 1 to 5 or 1 to 4, even more preferably 1 to 3, and even more preferably 1 to 2 or 1.

[0031] 1-2.Solid phase material The solid phase material may be a polymer selected from the group consisting of polyacrylamide and polystyrene. From the viewpoint of improving the efficiency of trapping lead ions, it is preferable to use polyacrylamide as the solid phase material.

[0032] The solid phase material preferably has a polyalkylene glycol chain to which the polypeptide is directly or indirectly bound, and the polyalkylene glycol chain is preferably grafted onto a polymer selected from the group consisting of polyacrylamide and polystyrene.

[0033] Examples of polyalkylene glycols include polyalkylene glycols having two or more types of alkylene oxides (glycol units), such as polymethylene glycol, polyethylene glycol, polypropylene glycol, polybutylene glycol, polyhexamethylene glycol, and copolymers of ethylene oxide and propylene oxide, and preferably include polyethylene glycol, polypropylene glycol, and copolymers of ethylene oxide and propylene oxide, and more preferably include polyethylene glycol.

[0034] The number of alkylene oxides (glycol units) in the polyalkylene glycol is not particularly limited, and may be, for example, 2 to 200, preferably 4 to 150, more preferably 6 to 50, 7 to 30, 8 to 20, 9 to 15, or 10 to 13, or 10 to 100, 30 to 90, or 60 to 80.

[0035] The shape of the solid phase material may be appropriately selected depending on the intended use of the lead ion binding affinity solid phase, and examples include plate-like (such as a test plate), spherical (such as beads), tubular, and filter-like shapes.

[0036] 1-3.Applications etc. The use of the lead ion-binding affinity solid phase of the present invention is not particularly limited as long as it is used to capture lead ions in a sample solution containing lead ions. Specific uses include use to capture lead ions contained in a sample solution derived from a living body, an environment, an industrial product, a food or drink, etc. More specific uses include detection, separation (removal from a sample solution), recovery, quantification, etc. of lead ions. A more specific use of separation (removal from a sample solution) includes use in the production of autologous blood products.

[0037] The lead ion-binding affinity solid phase of the present invention may be used up in one use or may be used repeatedly. In the case of repeated use, the number of times of use may be, for example, 2 to 4 times, preferably 2 to 3 times, and more preferably 2 times.

[0038] The lead ion-binding affinity solid phase of the present invention can be housed in a suitable container. The container preferably has an inlet and an outlet. The inlet and outlet may be located at positions where a liquid introduced into the container from the inlet comes into contact with the lead ion-binding affinity solid phase (preferably immerses the lead ion-binding affinity solid phase) and then the liquid can be discharged from the outlet to the outside of the container. A specific example of the container is a column (see, for example, FIG. 1).

[0039] 2. Method for preparing affinity solid phase for binding lead ions The present invention also provides a method for producing the above-mentioned "1. A method for producing an affinity solid phase for binding lead ions" of the present invention. Specifically, the method for producing an affinity solid phase for binding lead ions of the present invention includes a step of synthesizing a polypeptide having an amino acid sequence represented by SEQ ID NO: 1 on a solid phase material by a peptide solid-phase synthesis method, but does not include a step of separating the polypeptide from the solid phase material.

[0040] The solid-phase peptide synthesis method is a polypeptide synthesis method in which a peptide chain is successively extended on a solid-phase material by repeating a condensation reaction of amino acids and deprotection of terminal protecting groups. In a general solid-phase peptide synthesis method, a polypeptide extended to a desired sequence is separated from the solid-phase material, but in the production method of the present invention, the extended polypeptide is not separated from the solid-phase material, and the solid-phase material used as a scaffold for peptide synthesis is used as it is as the solid-phase material for the lead ion binding affinity solid phase.

[0041] The solid phase material and the polypeptide are as specifically described above in "1. Affinity solid phase for binding lead ions."

[0042] That is, the solid phase material may be a polymer selected from the group consisting of polyacrylamide and polystyrene, and from the viewpoint of improving the efficiency of trapping lead ions, it is preferable to use polyacrylamide as the solid phase material. The solid phase material preferably has a polyalkylene glycol chain, and the polypeptide is directly or indirectly bound to the polyalkylene glycol chain. The polyalkylene glycol chain is preferably grafted to a polymer selected from the group consisting of polyacrylamide and polystyrene.

[0043] The peptide chain extending from the solid phase material may contain a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. When producing an affinity solid phase in which a polypeptide is immobilized on a solid phase material via a linker residue, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and a linker residue may be synthesized by solid phase synthesis.

[0044] The amount of polypeptide per 1 g of solid phase material (before peptide elongation, dry weight) is not particularly limited, but may be, for example, 0.05 to 1 mmol, preferably 0.08 to 0.8 mmol, 0.1 to 0.65 mmol, more preferably 0.15 to 0.5 mmol, and even more preferably 0.2 to 0.45 mmol, or 0.3 to 0.42 mmol.

[0045] When preparing an affinity solid phase having an aryl group bound directly or indirectly to the end opposite to the end bound to the solid phase material, an arylating agent capable of introducing an aryl group as a protecting group can be reacted with the N-terminus of the extended polypeptide.

[0046] 3. Lead ion separation method The present invention also provides a method for separating lead ions using the above-mentioned "1. Affinity solid phase for binding lead ions". Specifically, the method for separating lead ions of the present invention includes a step of contacting a sample solution containing lead ions with the above-mentioned "1. Affinity solid phase for binding lead ions" (see FIG. 1(i)) to allow the affinity solid phase to capture the lead ions (see FIG. 1(ii)) (hereinafter also referred to as a "capturing step").

[0047] The concentration of lead ions contained in the sample solution is not particularly limited, and may be, for example, 100nM to 100μM. The lead ion binding affinity solid phase used in the present invention has excellent selective binding properties for lead ions, so that even low concentrations of lead ions can be effectively separated. From this perspective, suitable concentrations of lead ions contained in the sample solution include 100nM to 10μM, 100nM to 6μM, 100nM to 3μM, 100nM to 900nM, 100nM to 700nM, 100nM to 600nM, 300nM to 600nM, or 400nM to 600nM.

[0048] The sample solution may contain any component other than lead ions and water. The lead ion-binding affinity solid phase used in the present invention has excellent selective binding ability to lead ions, so the sample solution may contain impurities such as proteins. A specific example of the impurities is serum. The content of serum in the sample solution may be, for example, 0.3 v / v% or more, preferably 0.5 v / v% or more, more preferably 0.8 v / v% or more, even more preferably 0.9 v / v% or more, 2 v / v% or more, 4 v / v% or more, 6 v / v% or more, 8 v / v% or more, or 9 v / v% or more, and the upper limit may be, for example, 20 v / v% or less, preferably 15 v / v% or less, more preferably 12 v / v% or less, even more preferably 10 v / v% or less, 8 v / v% or less, 6 v / v% or less, 4 v / v% or less, 2 v / v% or less, or 1.5 v / v% or less.

[0049] Specific examples of sample liquids include sample liquids derived from living organisms for testing, water quality sample liquids, environmental sample liquids other than water, sample liquids derived from industrial products, and sample liquids derived from food and beverages.

[0050] Examples of sample liquids derived from living organisms include body fluid samples (particularly blood, urine, etc.), tissue (hair, stool, etc.) homogenates, etc. Examples of water quality sample liquids include drinking water, industrial water, wastewater, environmental water (water from public water bodies such as rivers, lakes, and seas, and groundwater, etc.) Examples of sample liquids derived from environments other than water include soil-derived sample liquids (supernatants of mixtures of soil and water, etc.), and air-derived sample liquids (air is passed through a container containing an absorbing liquid to absorb air components, etc.). Examples of sample liquids derived from industrial products include the supernatants of mixtures of crushed industrial products or their waste products and water, etc. Examples of samples derived from food and beverages include liquids obtained by dissolving or suspending beverages and foods in water, and the supernatants of mixtures of crushed food products and water, etc.

[0051] In the capture step, when the sample solution containing lead ions is brought into contact with the affinity solid phase, there is no particular need to control the pH of the sample solution. The specific pH (25°C) of the sample solution when the sample solution containing lead ions is brought into contact with the affinity solid phase is, for example, 5 to 7, preferably 5.5 to 6.5, and more preferably 5.7 to 6.3.

[0052] In the capture step, the temperature and time for contacting the sample liquid containing lead ions with the affinity solid phase are not particularly limited, and examples include room temperature (preferably 15 to 30°C) and for, for example, 1 to 48 hours, preferably 6 to 36 hours, and more preferably 15 to 30 hours.

[0053] The lead ion separation method of the present invention may include any step other than the capture step, such as a separation step of recovering the sample liquid after contacting with the affinity solid phase from the affinity solid phase that has captured lead by solid-liquid separation.

[0054] 4. Method for quantifying lead ions and kit for quantifying lead ions The present invention also provides a method for quantifying lead ions using the above-mentioned "1. Affinity solid phase for binding lead ions". Specifically, the method for quantifying lead ions of the present invention includes a step of contacting a sample solution containing lead ions with the above-mentioned "1. Affinity solid phase for binding lead ions" (see FIG. 1(i)) to capture the lead ions on the affinity solid phase (see FIG. 1(ii)) (hereinafter also referred to as a "capturing step"); a step of contacting the affinity solid phase (see FIG. 1(iii)) that has captured the lead ions with a chelating reagent to generate a lead chelate complex (see FIG. 1(iv)) to release the lead ions from the affinity solid phase (hereinafter also referred to as a "releasing step"); and a step of quantifying the lead ions from the lead chelate complex (hereinafter also referred to as a "quantifying step"). The quantitative determination method of the present invention may further include a separation step between the capturing step and the releasing step.

[0055] 4-1. Capture process Details of the sample solution containing lead ions and the capture step are as described above in "3. Lead ion separation method." In addition, the sample solution after contacting with the affinity solid phase is subjected to solid-liquid separation from the affinity solid phase that has captured lead (see Wash out in FIG. 1(ii)), whereby the affinity solid phase that has captured lead can be recovered (see FIG. 1(iii)).

[0056] 4-2.Release process In the release step, when the affinity solid phase capturing lead ions is brought into contact with a chelating reagent to generate a lead chelate complex (see FIG. 1 (iv)), there is no particular need to control the pH of the liquid phase containing the affinity solid phase and the chelating reagent. The pH (25° C.) of the liquid phase may be the same as the pH (25° C.) in the capture step, and specifically, may be, for example, 5 to 7, preferably 5.5 to 6.5, and more preferably 5.7 to 6.3.

[0057] In the release step, the temperature and time for contacting the affinity solid phase capturing lead ions with the chelating reagent are not particularly limited, and examples include room temperature (preferably 15 to 30°C) and 10 to 60 minutes, preferably 20 to 40 minutes.

[0058] The chelating reagent can be appropriately selected depending on the quantification method adopted in the quantification step. When the absorbance of the lead chelate complex is measured in the quantification step (see UV-Vis measurement in FIG. 1(iv)) (hereinafter also referred to as "quantification mode A"), ethylenediaminetetraacetic acid can be used as the chelating reagent. When chelating titration is performed in the quantification step (hereinafter also referred to as "quantification mode B"), a metal indicator for lead ions is used as the chelating reagent.

[0059] In the release step, since the efficiency of chelating the lead ions with the chelating reagent is excellent, the lead ions can be released efficiently without repeating the capture step and the release step. Therefore, although the quantitative method of the present invention does not exclude the inclusion of a combination of the capture step and the release step two or more times, it is preferable that the combination is included only once.

[0060] 4-3.Release process The liquid phase containing the lead chelate complex produced in the release step can be recovered from the affinity solid phase by solid-liquid separation.

[0061] 4-4.Quantitative process In the quantitative determination step, in the case of quantitative embodiment A, the lead chelate complex (ethylenediaminetetraacetic acid-lead chelate complex) is quantified. As a specific method for the quantitative determination, any method capable of quantifying the ethylenediaminetetraacetic acid-lead chelate complex can be used. Since the ethylenediaminetetraacetic acid-lead chelate complex has absorption around 240 nm, it is preferable to use ultraviolet-visible spectroscopy to measure the amount of light of the absorption (absorbance), and to perform quantitative determination based on the level of absorbance.

[0062] In the quantitative determination step, in the case of quantitative determination mode B, ethylenediaminetetraacetic acid can be added dropwise to the recovery solution containing the lead chelate complex to thereby perform titration of lead ions.

[0063] 4-5.Application examples When a sample liquid containing lead ions is a biological sample liquid for testing, a water quality sample liquid, an environmental sample liquid other than water, a sample liquid derived from an industrial product, or a sample liquid derived from food and beverages, the quantification method of the present invention can be used for testing for lead poisoning, investigating lead contamination of water quality, investigating lead contamination of the environment, investigating lead contamination of industrial products (inspection of the lead content of industrial products, investigation of lead contamination of medical equipment, etc.), and investigating the lead content of food and beverages (inspection).

[0064] 4-6.Kit The present invention also provides a lead ion quantification kit for carrying out the above-mentioned lead ion quantification method. Specifically, the lead ion quantification kit of the present invention includes the above-mentioned "1. Affinity solid phase for binding lead ions" and a chelating reagent as items. The affinity solid phase for binding lead ions may be contained in a container having an inlet and an outlet described in the above "1. Affinity solid phase for binding lead ions".

[0065] 5. Methods for producing autologous blood products The present invention also provides a method for producing an autologous blood product using the above-mentioned "1. Affinity solid phase for binding lead ions." Specifically, the method for producing an autologous blood product of the present invention includes a step of contacting blood collected from an animal suffering from lead poisoning with the above-mentioned "1. Affinity solid phase for binding lead ions," and a step of recovering the blood that has come into contact with the affinity solid phase.

[0066] There is no particular limitation on the type of animal that is poisoned by lead, and examples include mammals such as humans, monkeys, cows, horses, sheep, dogs, and cats, as well as birds.

[0067] The blood product produced by the production method of the present invention has lead ions selectively removed and can be returned to the body of the animal from which the blood was taken. By returning the production method of the present invention and the blood product produced thereby to the body of the original lead-poisoned animal, it becomes possible to provide causal therapy for lead poisoning. EXAMPLES

[0068] The present invention will be described in more detail below with reference to examples, but each configuration and their combinations in each embodiment are merely examples, and addition, omission, substitution, and other modifications of the configurations are possible as appropriate within the scope of the present invention. The present invention is not limited to the embodiments, but is limited only by the scope of the claims.

[0069] [Test Example 1] The lead ion-binding affinity solid phases (hereinafter also referred to as affinity solid phase carriers) shown in Table 1 were synthesized. [Table 1]

[0070] The polypeptides used in Examples 1 to 8 were synthesized from the C-terminus side by Fmoc solid-phase synthesis. Among the Fmoc amino acid derivatives used in solid-phase synthesis, those with protected side chains were Fmoc-Cys(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Thr(tBu)-OH. The Fmoc amino acid derivatives are collectively referred to as "Fmoc-AA-OH". In addition, all of the Fmoc amino acid derivatives used were L-form.

[0071] The resin (solid phase material) for solid phase synthesis has a linker that cannot be excised. In Examples 1, 3, and 5, Amino PEGA resin (amino group introduction rate: 0.39 mmol / g), which is a PEG-grafted polyacrylamide (PEG-polyacrylamide), was used, and in Examples 2, 4, and 6 to 8, TentaGel S NH2 resin (amino group introduction rate: 0.25 mmol / g), which is a PEG-grafted polystyrene (PEG-polystyrene), was used.

[0072] <Examples 1 and 2> A resin (solid phase material) equivalent to a 50 μmol scale was weighed. (The PEG-grafted polystyrene (TentaGel S NH2resin) used in the synthesis of Example 2 was swollen in 1-methyl-2-pyrrolidone (NMP) for one day, and the NMP was removed.) The resin was then washed five times with NMP, and 10 equivalents of Fmoc-AA-OH, 10 equivalents of HBTU, and 10 equivalents of HOBt·H2O were dissolved in NMP relative to the amount of peptide that could be introduced into the resin, and 15 equivalents of DIEA were added and reacted at 37°C for 30 minutes. This operation was repeated twice. The deprotection of the Fmoc group after coupling was performed as follows. A 20% PPD / NMP solution was added and washed once. 20% PPD / NMP was added again and reacted for 4 minutes while stirring. The Fmoc group on the resin surface was removed by reacting for 4 minutes while stirring in the same manner again. This was washed five times with NMP. The above procedure was repeated to obtain the peptide immobilized on the resin.

[0073] Then, a cocktail of m-cresol:1,2-ethanedithiol:thioanisole:TFA=3:8:8:81 was added to the resin and reacted for 2 hours. After the reaction was completed, the resin was washed five times with a solution of the same composition as that used for deprotection, and then washed five times each with MilliQ 100% / TFA 0.1% and NMP to completely remove the protecting groups. This resulted in the affinity solid phase carriers [undried] of Examples 1 and 2.

[0074] The affinity solid phase carrier [undried] of Example 1 was stored at 4° C. without drying. The affinity solid phase carrier [undried] of Example 2 was further washed five times with chloroform and then vacuum dried for 90 minutes, and the completely dried one (affinity solid phase carrier [dried] of Example 2) was stored at 4° C.

[0075] <Examples 3 and 4> An operation of coupling 2-naphthylacetic acid was further performed on the affinity solid phase carriers [undried] of Examples 1 and 2. Specifically, 4 equivalents of 2-naphthylacetic acid, HATU, and 8 equivalents of DIEA were added to the amount of peptide that can be introduced into the resin (solid phase material), and coupling was performed at 37°C for 30 minutes. After the reaction was completed, a Kaiser test was performed, and if there was no blue resin, coupling was performed again under the same conditions, and a second Kaiser test was not performed. If there was a blue resin, coupling was performed again, and a second Kaiser test was also performed.

[0076] Then, a cocktail of m-cresol:1,2-ethanedithiol:thioanisole:TFA=3:8:8:81 was added to the resin and reacted for 2 hours. After the reaction was completed, the resin was washed five times with a solution of the same composition as that used for deprotection, and then washed five times each with MilliQ 100% / TFA 0.1% and NMP to completely remove the protecting groups. This resulted in the affinity solid phase carriers [undried] of Examples 3 and 4.

[0077] The affinity solid phase carrier [undried] of Example 3 was stored at 4° C. without drying. The affinity solid phase carrier [undried] of Example 4 was further washed five times with chloroform and then vacuum dried for 90 minutes, and the completely dried one (affinity solid phase carrier [dried] of Example 4) was stored at 4° C.

[0078] <Examples 5 and 6> A polypeptide of sequence number 2 was solid-phase synthesized on a resin (solid-phase material), 4-bromobutyric acid was coupled to the N-terminus, and a cyclization reaction was performed between Br coupled to the N-terminus and SH of the C-terminus residue side chain of the polypeptide. The cyclization reaction was performed by adding 1.5 mL of 5% DIEA in NMP to a 5 μmol scale peptide-immobilized resin, and reacting for 24 hours at room temperature while shaking at 1500 rpm. Then, a cocktail of m-cresol: 1,2-ethanedithiol: thioanisole: TFA = 3: 8: 8: 81 was added to the resin, and reacted for 2 hours. After the reaction was completed, the resin was washed five times with a solution of the same composition as that used for deprotection, and then washed five times each with MilliQ 100% / TFA 0.1% and NMP in that order to completely remove the protecting groups. This resulted in the affinity solid-phase carriers [undried] of Examples 5 and 6.

[0079] The affinity solid phase carrier [undried] of Example 5 was stored at 4° C. without drying. The affinity solid phase carrier [undried] of Example 6 was further washed five times with chloroform and then vacuum dried for 90 minutes, and the completely dried one (affinity solid phase carrier [dried] of Example 6) was stored at 4° C.

[0080] <Example 7> A 50 μmol scale equivalent of resin (solid phase material) was weighed out, swollen in 1-methyl-2-pyrrolidone (NMP) for one day, and the NMP was removed. The resin was then washed five times with NMP, and 10 equivalents of Fmoc-AA-OH, 10 equivalents of HBTU, and 10 equivalents of HOBt·H2O were dissolved in NMP for the amount of peptide that could be introduced into the resin, and 15 equivalents of DIEA were added and reacted at 37°C for 30 minutes. This operation was repeated twice. The deprotection of the Fmoc group after coupling was performed as follows. A 20% PPD / NMP solution was added and washed once. 20% PPD / NMP was added again and reacted for 4 minutes while stirring. The Fmoc group on the resin surface was removed by reacting again for 4 minutes while stirring. This was washed five times with NMP. The above operations were repeated to obtain a peptide immobilized on the resin.

[0081] Then, a cocktail of m-cresol:1,2-ethanedithiol:thioanisole:TFA=3:8:8:81 was added to the resin and reacted for 2 hours. After the reaction was completed, the resin was washed five times with a solution of the same composition as that used for deprotection, and then washed five times with MilliQ 100% / TFA 0.1% and NMP in that order to completely remove the protecting groups. This resulted in the affinity solid phase carrier [undried] of Example 7. Furthermore, the affinity solid phase carrier [undried] of Example 7 was washed five times with chloroform and then vacuum dried for 90 minutes, and the completely dried one (affinity solid phase carrier [dry] of Example 7) was stored at 4°C.

[0082] <Example 8> A 50 μmol scale equivalent of resin (solid phase material) was weighed out, swollen in 1-methyl-2-pyrrolidone (NMP) for one day, and the NMP was removed. The resin was then washed five times with NMP, and 10 equivalents of Fmoc-AA-OH, 10 equivalents of HBTU, and 10 equivalents of HOBt·H2O were dissolved in NMP for the amount of peptide that could be introduced into the resin, and 15 equivalents of DIEA were added and reacted at 37°C for 30 minutes. This operation was repeated twice. The deprotection of the Fmoc group after coupling was performed as follows. A 20% PPD / NMP solution was added and washed once. 20% PPD / NMP was added again and reacted for 4 minutes while stirring. The Fmoc group on the resin surface was removed by reacting again for 4 minutes while stirring. This was washed five times with NMP. The above operations were repeated to obtain a peptide immobilized on the resin.

[0083] Then, a cocktail of m-cresol:1,2-ethanedithiol:thioanisole:TFA=3:8:8:81 was added to the resin and reacted for 2 hours. After the reaction was completed, the resin was washed five times with a solution of the same composition as that used for deprotection, and then washed five times with MilliQ 100% / TFA 0.1% and NMP in that order to completely remove the protecting groups. This resulted in the affinity solid phase carrier [undried] of Example 8. Furthermore, the affinity solid phase carrier [undried] of Example 8 was washed five times with chloroform and then vacuum dried for 90 minutes, and the completely dried one (affinity solid phase carrier [dry] of Example 8) was stored at 4°C.

[0084] The structures of the binding sites between the PEG-grafted polyacrylamide and the polypeptide in Examples 1, 3 and 5 are as shown below (n=11).

[0085] [ka]

[0086] In addition, in Examples 1, 3, and 5, since a large excess of substrate amino acids was used in the solid-phase peptide synthesis, the peptide incorporation rate into the solid phase was equivalent to the amino group incorporation rate into the raw material PEG-grafted polyacrylamide.

[0087] The structures of the binding sites between the PEG-grafted polystyrene and the polypeptide in Examples 2, 4, and 6 to 8 are as shown below (n=70).

[0088] [ka]

[0089] Moreover, the peptide introduction rate to the solid phase in Examples 2, 4, and 6 to 8 was 0.1 mmol / g.

[0090] [Test Example 2] The affinity solid phase carrier of Example 3 was prepared so that the amount of polypeptide on the solid phase carrier was 1×10 -7 The solid-phase carrier was weighed out to 100 mol and shaken at 1500 rpm at room temperature for 24 hours in 1 mL of a sample solution containing lead ions ([Pb(NO3)2] = 100 μM, [MES (pH 6.0)] = 25 mM). After that, EDTA was added to the affinity solid-phase carrier washed five times with MilliQ to a concentration of 1 mM, and the solid-phase carrier was shaken at 1500 rpm at room temperature for 30 minutes to obtain a Pb eluate (first EDTA treatment). Further, EDTA was added to a concentration of 1 mM, and the solid-phase carrier was shaken at 1500 rpm at room temperature for 30 minutes to obtain a Pb eluate (second EDTA treatment). The Pb eluate (first EDTA treatment) and the Pb eluate (second EDTA treatment) were subjected to UV-Vis measurement.

[0091] The results are shown in Figure 2. As shown in Figure 2, the Pb eluate (first EDTA treatment) showed absorption around 240 nm due to the EDTA-Pb(II) complex, whereas the Pb eluate (second EDTA treatment) showed no absorption around 240 nm due to the EDTA-Pb(II) complex. In other words, it was revealed that a single EDTA treatment could release all of the Pb ions captured by the affinity solid support.

[0092] [Test Example 3] Using the affinity solid phase support of Example 3, a sample solution not containing lead ions (no Pb) and a sample solution containing lead ions (Pb ion concentration 500 nM to 5 μM) were treated, and the release of Pb ions by EDTA and the detection of EDTA-Pb(II) complexes were performed by UV-Vis measurement.

[0093] Specifically, [Peptide] = 1 × 10 -7 The affinity solid support was weighed out so that the total amount was 100 mol, and the sample solution was shaken at 1500 rpm at room temperature for 24 hours under the conditions of [Pb(NO3)2] = 0μM or 0.5-5 μM, [MES (pH 6.0)] = 25 mM. At that time, the reaction was carried out with 20 mL for 0.5 μM, 10 mL for 1 μM, 5 mL for 2 μM, and 2 mL for 5 μM. After washing five times with MilliQ, the solution was shaken at 1500 rpm at room temperature for 30 minutes under the condition of [EDTA] = 1 mM, and the obtained solution was measured by UV-Vis.

[0094] The results are shown in Figure 3. As shown in Figure 3, absorption at around 240 nm originating from the EDTA-Pb(II) complex was confirmed at all Pb ion concentrations from 500 nM to 5 μM.

[0095] [Test Example 4] [Test Example 4-1] The amount of Pb ions captured by chelometric titration was calculated using the affinity solid phase carriers of Examples 2, 4, 6, 7, and 8. As a control, an acetylated PEG-grafted polystyrene resin (Ac-PS resin) having the structure (n=70) shown in the following formula was used. The synthesis of Ac-PS resin was performed according to the following procedure. First, TentaGel S NH2 was swollen in NMP at room temperature for 24 hours before synthesis. Then, 20 equivalents of acetic anhydride and 10 equivalents of DIEA, which correspond to the number of amino groups present in the resin, were directly added to the column, and the reaction was carried out at 37°C for 30 minutes. This operation was repeated twice. Then, the column was washed five times with NMP and chloroform, in that order. After washing, the resin was completely dried by vacuum drying for 90 minutes and then stored at 4°C.

[0096] [ka]

[0097] The affinity solid phase carrier of the embodiment or the control solid phase carrier that had been stored was left to stand until it returned to room temperature, and then weighed directly into a column for peptide synthesis to be used as a reaction vessel. The peptide (in the case of the affinity solid phase carrier of the embodiment) or acetyl group (in the case of the control solid phase carrier) was 1×10 -7 NMP was added to the solid phase carrier weighed out to 1000 mol, and the resin was allowed to swell at room temperature for 24 hours. The weighed solid phase carrier was washed with methanol and MilliQ, in that order, five times each. The reaction solution was then added to the column containing the solid phase carrier, and the column was shaken at 1500 rpm at room temperature for 24 hours.

[0098] The sample solution containing lead ions was prepared by diluting the solution with MilliQ to a final concentration of 1 mL, with [Pb(NO3)2] = 100 μM (final concentration) and [MES (pH = 6.0)] = 25 mM (final concentration). Pb ions were captured using affinity solid support or Ac-PS resin, and chelate titration was performed using xylenol orange (XO). The amount of Pb ions captured was calculated from the amount of EDTA added and the amount of Pb ions charged.

[0099] The results are shown in Figure 4. It was shown that 81.7% of Pb ions could be separated in Example 2, 52.5% in Example 4, 37.5% in Example 6, 26.7% in Example 7, and 23.3% in Example 8. These results suggest that although Pb ions can be quantified in all Examples, the peptide sequence having an SH group of Cys residue (Examples 2 and 4) has higher sensitivity and is more suitable for quantification.

[0100] [Test Example 4-2] Using the affinity solid phase carriers of Examples 2, 4, and 6, chelate titration was performed in the same manner as in Test Example 4-1, except that the sample solution containing lead ions was prepared to contain [Pb(NO3)2] = 100 μM and serum-containing medium (89% D-MEM, 10% fetal bovine serum, 1% penicillin) 10% (the content of fetal bovine serum in the sample solution containing lead ions is 1 v / v%).

[0101] The results are shown in Figure 5. This suggests that Pb ions can be separated and quantified even when serum is mixed into a sample solution containing lead ions.

[0102] [Test Example 5] Using the affinity solid phase carrier of Example 3 or the following control solid phase carrier, a sample solution containing lead ions was prepared containing [Pb(NO3)2] = 100 μM and serum-containing medium (89% D-MEM, 10% fetal bovine serum, 1% penicillin) 99% (the content of fetal bovine serum in the sample solution containing lead ions is 9.9 v / v%) (Pb+ medium) or a sample solution not containing lead ions (medium only). In the same manner as in Test Example 3, the sample solution was treated, Pb ions were released by EDTA, and EDTA-Pb(II) complex was detected by UV-Vis measurement.

[0103] As a control solid phase carrier, acetylated PEG-grafted polyacrylamide resin (Ac-PEGA resin) with the structure shown in the formula below (n=11) was used. Ac-PEGA resin was synthesized by directly adding 20 equivalents of acetic anhydride and 10 equivalents of DIEA, based on the number of amino groups present in the PEG-grafted polyacrylamide resin (Amino PEGA resin), to the column and reacting for 30 minutes at 37°C, twice. The resin was then washed five times with NMP and stored at 4°C.

[0104] [ka]

[0105] The results are shown in Figure 6. It was suggested that Pb ions can be separated and quantified even if a sample solution containing lead ions is mixed with serum at a higher concentration than in Test Example 4-2.

[0106] [Test Example 6] The affinity solid phase supports of Examples 3 and 4 that had been stored were left to stand until they returned to room temperature, and were then weighed directly into a column for peptide synthesis to be used as a reaction vessel. The affinity solid phase support of Example 3 was washed five times with NMP, after which the peptide was weighed out at a concentration of 1×10 -7 The affinity solid phase carrier of Example 4 was weighed out and swollen with NMP for 24 hours after weighing. The affinity solid phase carrier was washed five times with methanol and MilliQ in that order. Thereafter, a sample solution containing lead ions was added to the column containing the affinity solid phase carrier, and the column was shaken at 1500 rpm at room temperature for 24 hours. The sample solution containing lead ions was prepared by diluting with MilliQ so that [Pb(NO3)2]=100 μM (final concentration), [MES (pH=6.0)]=25 mM (final concentration), and the total volume was 1 mL. After the reaction, the column was washed five times with 1 mL of MilliQ, 1 mL of 1 mM EDTA was added, and the column was shaken at 1500 rpm at room temperature for 30 minutes. The reaction solution was then collected, UV-Vis measurement was performed, and the absorbance at 240 nm was confirmed.

[0107] The results are shown in Figure 7. It was confirmed that the affinity solid phase supports of Examples 3 and 4 were both capable of trapping and quantifying Pb ions.

[0108] [Test Example 7] Using the affinity solid phase carrier of Example 1 or 3, Pb ions at various concentrations were separated and detected. First, the affinity solid phase carrier that had been stored was left to stand until it returned to room temperature, and then weighed directly into a column for peptide synthesis to be used as a reaction vessel. The affinity solid phase carrier was washed five times with NMP, and then the peptide was weighed out at a concentration of 1×10 -7 The affinity solid phase carrier was weighed out so that it was 100 mol. The weighed affinity solid phase carrier was washed five times each with methanol and MilliQ. Then, a sample solution containing lead ions was added to the column containing the affinity solid phase carrier, and the column was shaken at room temperature for 24 hours at 1500 rpm. As the sample solution containing lead ions, a solution containing [Pb(NO3)2] = 0.5 to 100 μM (final concentration) and [MES (pH = 6.0)] = 25 mM (final concentration) was used, and the total volume was adjusted to 20 mL in the case of [Pb(NO3)2] = 0.5 μM (final concentration), 10 mL in the case of [Pb(NO3)2] = 1 μM (final concentration), 5 mL in the case of [Pb(NO3)2] = 2 μM (final concentration), and 2 mL in the case of [Pb(NO3)2] = 5 μM (final concentration) by measuring up with MilliQ. Sample solutions under other concentration conditions were prepared so that the reaction volume was 1 mL. After the reaction, the mixture was washed five times with 1 mL of MilliQ, 1 mL of 1 mM EDTA was added, and the mixture was shaken at 1500 rpm at room temperature for 30 minutes. The reaction solution was then collected and subjected to UV-Vis measurement to confirm the absorbance at 240 nm. The absorbance was corrected based on the ratio of the volume (1-20 mL) when the Pb ions were separated to the volume (1 mL) when the EDTA was treated.

[0109] The results are shown in Figures 8A and 8B. In the figures, the unit of the horizontal axis is μm. It was suggested that there was a linear relationship between the lead ion concentration and the absorbance at 240 nm in the range of 0.5 to 50 μM for Example 1 and 0.5 to 100 μM for Example 3. Furthermore, Figures 8C and 8D show calibration curves of Pb ions when separated and detected using the affinity solid phase carriers of Examples 1 and 3, respectively.

[0110] [Test Example 8] Metal selectivity was confirmed using the affinity solid phase carrier of Example 3. First, the affinity solid phase carrier that had been stored was left to stand until it returned to room temperature, and then weighed directly onto a column for peptide synthesis to be used as a reaction vessel. The affinity solid phase carrier was washed five times with NMP, and then the peptide was weighed out at a concentration of 1×10 -7 The resin was weighed out so that the total amount was 100 mol. The weighed affinity solid phase carrier was washed five times with methanol and then with MilliQ. Then, a sample solution containing metal ions was added to the column containing the affinity solid phase carrier, and the column was shaken at 1500 rpm at room temperature for 24 hours. The sample solution containing metal ions was prepared to contain [Pb(NO3)2] = 100 μM (final concentration), [CaCl2] = 100 μM (final concentration), or [CoCl2] = 100 μM (final concentration), and [MES (pH = 6.0)] = 25 mM (final concentration). After the reaction, the column was washed five times with 1 mL of MilliQ, 1 mL of 1 mM EDTA was added, and the column was shaken at 1500 rpm at room temperature for 30 minutes. The reaction solution was then collected and UV-Vis measurement was performed to confirm the absorbance of Ca and Co, with the absorbance of Pb at 240 nm taken as 100%.

[0111] The results are shown in Figure 9. As shown in Figure 9, the detected amount of Ca and Co was only about 6% of the detected amount of Pb ions, demonstrating that it is possible to selectively separate and detect Pb ions.

[0112] [Test Example 9] First, the stored solid phase carrier (affinity solid phase carrier of Example 1 or 3 or Ac-PEGA as a control) was left to stand until it returned to room temperature, and then weighed directly into a column for peptide synthesis to be used as a reaction vessel. The solid phase carrier was washed five times with NMP, and then the peptide (in the case of affinity solid phase carrier) or acetyl group (Ac-PEGA) was weighed in an amount of 1×10 -7 The solid phase carrier was weighed out so that it was 100 mol. The weighed solid phase carrier was washed five times with methanol and then with MilliQ. The reaction solution was then added to the column containing the solid phase carrier, and the column was shaken at 1500 rpm at room temperature for 24 hours. As a sample solution containing lead ions, 1 mL of a solution prepared so that serum-containing medium (89% D-MEM, 10% fetal bovine serum, 1% penicillin) 99% and [Pb(NO3)2] = 100 μM (final concentration) (the content of fetal bovine serum in the sample solution containing lead ions is 9.9 v / v%) was used. After the reaction, the column was washed five times with 1 mL of MilliQ, 1 mL of 1 mM EDTA was added, and the column was shaken at 1500 rpm at room temperature for 30 minutes. The reaction solution was then collected and UV-Vis measurement was performed.

[0113] The series of steps of reaction of the sample solution containing lead ions with the solid support, addition of EDTA, washing, and UV-Vis measurement was repeated three more times.

[0114] The results using the affinity support of Example 1 are shown in Figure 10A, and the results using the affinity support of Example 3 are shown in Figure 10B. It was revealed that the affinity supports of Examples 1 and 3 can withstand repeated use even when the sample solution containing lead ions contains serum.

Claims

1. An affinity solid phase for binding lead ions, comprising a polypeptide consisting of an amino acid sequence represented by at least the 2nd to 8th positions of SEQ ID NO:1, and a solid phase material on which said polypeptide is immobilized.

2. The affinity solid phase according to claim 1, wherein the solid phase material is polyacrylamide and / or polystyrene.

3. The affinity solid phase according to claim 1, wherein the solid phase material has a polyalkylene glycol chain, and the polypeptide is directly or indirectly bound to the polyalkylene glycol chain.

4. The affinity solid phase according to claim 1, wherein the polypeptide is immobilized on the solid phase material via a linker residue consisting of 1 to 9 amino acid residues.

5. The affinity solid phase according to claim 1 , wherein the polypeptide has a protecting group bound to at least the end opposite to the end bound to the solid phase material.

6. A method for producing an affinity solid phase for binding lead ions, comprising the step of synthesizing a polypeptide having an amino acid sequence represented by SEQ ID NO:1 on a solid phase material by a peptide solid phase synthesis method, and not comprising the step of separating the polypeptide from the solid phase material.

7. A method for separating lead ions, comprising the step of contacting a sample solution containing lead ions with the affinity solid phase for binding lead ions according to claim 1 to allow the affinity solid phase to capture the lead ions.

8. The method according to claim 7, wherein the concentration of the lead ions contained in the sample solution is 100 nM to 100 μM.

9. The method according to claim 7 , wherein the sample liquid is a biological sample liquid for testing, a water quality sample liquid, an environmental sample liquid other than water, an industrial product sample liquid, or a food or drink sample liquid.

10. a step of contacting a sample solution containing lead ions with the affinity solid phase for binding lead ions according to claim 1 to allow the affinity solid phase to capture the lead ions; contacting the affinity solid phase on which the lead ions have been captured with a chelating agent to form a lead chelate complex, thereby liberating the lead ions from the affinity solid phase; and determining the amount of lead ions from the lead chelate complex.

11. 11. The method of claim 10, wherein the chelating agent is ethylenediaminetetraacetic acid and the quantification is based on the absorbance of a lead chelate complex.

12. The method of claim 10, wherein the chelating reagent is a metal indicator and the quantification is performed by chelometric titration.

13. A kit for quantifying lead ions, comprising the lead ion-binding affinity solid phase according to claim 1 and a chelating agent.

14. contacting blood taken from an animal suffering from lead poisoning with the lead ion binding affinity solid phase according to claim 1; and recovering the blood that has come into contact with the affinity solid phase. Methods for producing autologous blood products.

Images