JP2018140961A - Biotinylated f1106-13-3 antibody, production methods and uses thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide highly stable biotinylated F1106-13-3 antibodies.SOLUTION: Disclosed herein is a biotinylated F1106-13-3 antibody comprising a biotinylating agent bound to an amino group of F1106-13-3 antibody, where the biotinylating agent is biotin N-hydroxysuccinimide ester, 6-(biotinylamino)hexanoic acid N-hydroxysuccinimide ester, 6-[6-(biotinylamino)hexanoylamino]hexanoic acid N-hydroxysuccinimide ester, biotin N-hydroxysulfosuccinimide ester, 6-(biotinylamino)hexanoic acid N-hydroxysulfosuccinimide ester, 6-[6-(biotinylamino)hexanoylamino]hexanoic acid N-hydroxysulfosuccinimide ester or N-[[2-(biotinylamino)ethyl]dithiopropynyloxy]sulfosuccinimide sodium salt.SELECTED DRAWING: None

Description

  The present invention relates to a biotinylated F1106-13-3 antibody, its production method and its use.

  CD14 molecule is a glycoprotein expressed on the membrane surface of mononuclear cells and is known to have a function as a receptor for LPS (lipopolysaccharide). There are two types of CD14 molecules, membrane-bound CD14 (mCD14) and soluble CD14 (sCD14) expressed on the cell surface. As sCD14, sCD14 having a molecular weight of about 55 kDa and about 49 kDa (hereinafter referred to as “high molecular weight sCD14”) is known, and sepsis, acquired immune deficiency syndrome (AIDS), acute respiratory distress syndrome (ARDS), systemic It has been reported to show high levels in patients' blood in many diseases such as lupus erythematosus (SLE). Therefore, these high molecular weight sCD14 are considered not to be disease-specific markers.

  On the other hand, it has been reported that sCD14-ST (also called a soluble CD14 antigen subtype, also known as preceptin) exists as a new sCD14 molecular species whose blood concentration is characteristically increased in septic patients.

  sCD14-ST (presepsin) is a sCD14 that has a molecular weight of 13 ± 2 kDa in SDS-PAGE under non-reducing conditions and retains the N-terminal part of CD14. Compared with high molecular weight sCD14, it has an amino acid sequence greatly deleted on the C-terminal side, and unlike high molecular weight sCD14, it does not have LPS binding ability. Moreover, since preceptin shows immunogenicity different from high molecular weight sCD14, both can be distinguished using an antibody. Presepsin specifically increases blood levels in patients with sepsis. In addition, compared to patients with systemic inflammatory response (SIRS), which is difficult to distinguish from sepsis, there is a report that it shows high levels in the blood of sepsis patients, and presepsin is considered to be a specific diagnostic marker for sepsis It has been.

International Publication No. 2004/44005 Pamphlet

  Patent Document 1 describes an F1106-13-3 antibody that is an anti-preceptin monoclonal antibody. However, there is no description that the F1106-13-3 antibody becomes unstable when biotin is bound to a thiol group. It is well known to add a modifying substance such as biotin to the thiol group or amino group of an antibody. Note that adding a modifying substance to a thiol group tends to be preferred because the difference between lots is reduced.

  The present inventors have found for the first time that the storage stability decreases when biotin is bound to the thiol group of the F1106-13-3 antibody.

  The above problem was solved by binding biotin to the amino group of the F1106-13-3 antibody.

That is, according to the present invention, there is provided a biotinylated F1106-13-3 antibody characterized in that biotin is bound to the amino group of the F1106-13-3 antibody.
The present invention also provides a method for producing a biotinylated F1106-13-3 antibody, which comprises the step of binding biotin to the amino group of the F1106-13-3 antibody.

  Further, according to the present invention, a first reagent containing a biotinylated F1106-13-3 antibody in which biotin is bound to an amino group of the F1106-13-3 antibody, a second reagent containing streptavidin-binding solid phase particles, And a third reagent containing an anti-preceptin antibody that recognizes an epitope different from that of the F1106-13-3 antibody, is provided.

  Furthermore, according to the present invention, biotinylated F1106-13-3 antibody in which biotin is bound to the amino group of F1106-13-3 antibody and streptavidin-bound solid phase particles are mixed, and biotinylated F1106-13- A step of obtaining a 3-antibody-streptavidin-bound solid phase particle complex (first complex), and the resulting first complex and a biological sample are mixed, and presepsin-biotinylated F1106-13-3 antibody- A step of obtaining a streptavidin-bound solid phase particle complex (second complex), a second complex obtained, and an anti-preceptin antibody that recognizes an epitope different from the F1106-13-3 antibody are mixed. Obtaining an anti-preceptin antibody-preceptin-biotinylated F1106-13-3 antibody-streptavidin-bound solid phase particle complex (third complex) that recognizes an epitope different from the F1106-13-3 antibody, and The signal obtained from the target substance in the obtained third complex A method for measuring presepsin.

  A biotinylated F1106-13-3 antibody excellent in stability can be provided.

It is a figure which shows an example of the reagent kit of this embodiment. It is a figure which shows an example of the reagent kit of this embodiment. It is a graph which shows the result of the preceptin detection performed using F1106-13-3 antibody which alkaline phosphatase (ALP) couple | bonded with the thiol group. It is a graph which shows the result of the preceptin detection performed using F1106-13-3 antibody which the biotin couple | bonded with the thiol group. It is a graph which shows the result of the preceptin detection performed using F1106-13-3 antibody which biotin couple | bonded with the amino group.

The biotinylated F1106-13-3 antibody of this embodiment is characterized in that biotin is bound to the amino group of the F1106-13-3 antibody.
As described in WO2004 / 44005 A1, the F1106-13-3 antibody is an antibody that binds to the amino acid sequence (CNFSEPQPDW; SEQ ID NO: 2) 17 to 26 from the N-terminus of CD14 represented by SEQ ID NO: 1. is there. In general, presepsin is considered to be a protein having the amino acid sequence from 1 to 64 of CD14 (SEQ ID NO: 3). In the scene of preceptin detection, the F1106-13-3 antibody specifically recognizes and binds to amino acids 17 to 26 of preceptin. The F1106-13-3 antibody is available from Mochida Pharmaceutical.

  Alternatively, the F1106-13-3 antibody can also be obtained by a method known in the art, for example, as described in WO2004 / 44005 A1. That is, a method for purifying antibodies by immunizing mammals such as horses, sheep, rabbits, goats, rats, mice, etc. using a protein consisting of 1 to 285 amino acids of CD14 as an antigen, and collecting serum from the immunized animals Can be obtained using. Alternatively, antibody-producing cells collected from the immunized mouse can be obtained by a method of obtaining an antibody from a hybridoma obtained by fusing with a mouse tumor cell, for example.

An amino group means a primary amino group represented by —NH ₂ . A plurality of amino groups are considered to exist in the F1106-13-3 antibody. In the present embodiment, “to bind biotin to the amino group of F1106-13-3 antibody” means to bind biotin to at least one of the amino groups considered to be present in the F1106-13-3 antibody. means.

Biotin is a compound of CAS number 58-85-5, IUPAC name 5-[(3aS, 4S, 6aR) -2-oxohexahydro-1H-thieno [3,4-d] imidazol-4-yl] pentanoic acid It is.
The method for binding biotin to the amino group of the F1106-13-3 antibody is not particularly limited. For example, the biotin can be bound to the amino group of the F1106-13-3 antibody using a cross-linking agent.

  The crosslinking agent is not particularly limited, and those known to those skilled in the art can be used. The cross-linking agent may be a biotin labeling agent having biotin on its own. Examples of such biotin labeling agents include biotin N-hydroxysuccinimide ester, 6- (biotinylamino) hexanoic acid N-hydroxysuccinimide ester, 6- [6- (biotinylamino) hexanoylamino] hexanoic acid N-hydroxysuccinimide. Ester, biotin N-hydroxysulfosuccinimide ester, 6- (biotinylamino) hexanoic acid N-hydroxysulfosuccinimide ester, 6- [6- (biotinylamino) hexanoylamino] hexanoic acid N-hydroxysulfosuccinimide ester, and N- And the sodium salt of [[2- (biotinylamino) ethyl] dithiopropynyloxy] sulfosuccinimide.

  The biotinylated F1106-13-3 antibody of the present embodiment can be used for preceptin detection by ELISA measurement. The biotinylated F1106-13-3 antibody in which biotin is bound to the amino group of the present embodiment is superior in stability to the conventional F1106-13-3 antibody in which biotin is bound to a thiol group. It can be stored for a long time in the state of a reagent.

  The scope of the present invention also includes a method for producing a biotinylated F1106-13-3 antibody, which comprises the step of binding biotin to the amino group of the F1106-13-3 antibody.

  The scope of the present invention includes a first reagent containing a biotinylated F1106-13-3 antibody in which biotin is bound to the amino group of the F1106-13-3 antibody, a second reagent containing streptavidin-bound solid phase particles, A reagent kit for measuring presepsin is also included, which includes a third reagent containing an anti-preceptin antibody that recognizes an epitope different from the F1106-13-3 antibody.

The biotinylated F1106-13-3 antibody is as described above.
Streptavidin-bound solid phase particles are particles in which streptavidin is bound to the surface of the solid phase particles. The material of the solid phase particle is not particularly limited as long as it is a normal material used for immunoassay. Examples thereof include polymer materials such as latex, rubber, polyethylene, polystyrene, and polymethacrylate; agarose; gelatin; silica gel; inorganic materials such as glass, inert alumina, and a magnetic substance. You may combine these 1 type, or 2 or more types.

The solid phase particles are preferably magnetic particles. The magnetic particles are particles containing a magnetic material as a base material. Such magnetic particles are known in the art, and those using, for example, Fe ₂ O ₃ and / or Fe ₃ O ₄ , cobalt, nickel, ferrite, magnetite and the like as a base material are known. For the purpose of binding proteins or the like to the surface of the magnetic particles, a substrate whose surface is coated with a polymer or the like is more preferable.

  A method for binding a biotinylated F1106-13-3 antibody to solid phase particles via biotin in the antibody is known in the art. For example, it can be performed by a physical adsorption method, a covalent bond method, an ionic bond method, or a combination thereof.

  The anti-preceptin antibody that recognizes an epitope different from the F1106-13-3 antibody is an antibody that specifically binds to preceptin and is not particularly limited as long as it recognizes an epitope different from the F1106-13-3 antibody. . Examples of such antibodies include S68 polyclonal antibody, S68 monoclonal antibody, and F1146-17-2 antibody.

The anti-preceptin antibody that recognizes an epitope different from the F1106-13-3 antibody is labeled with a detectable labeling substance. Such a labeling substance is not particularly limited as long as it is a labeling substance that can be used in a normal immunoassay, and examples thereof include enzymes, fluorescent substances, and radioisotopes. Examples of the enzyme include alkaline phosphatase (ALP), peroxidase, glucose oxidase, tyrosinase, and acid phosphatase. Examples of the fluorescent substance include fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), and luciferin. Examples of radioactive isotopes include ¹²⁵ I, ¹⁴ C, and ³² P. The labeling substance is preferably alkaline phosphatase.

  In the present embodiment, a container containing the above various reagents may be packed in a box and provided to the user. This box may contain a package insert of the reagent kit. The package insert preferably describes, for example, the configuration of the reagent kit, the preceptin measurement protocol, and the like. FIG. 1A shows an example of the appearance of the reagent kit of this embodiment. In the figure, 110 shows a reagent kit, 111 shows a first container containing a first reagent, 112 shows a second container containing a second reagent, and 113 contains a third reagent. A third container is shown, 115 is a package insert, and 116 is a packing box.

  The reagent kit for presepsin measurement is 2-chloro-5- (methoxyspiro [1,2-dioxetane-3,2 ′-(5′-chloro) tricyclo [3.3.1.13,7] decan] -4-yl) phenyl A fourth reagent containing disodium phosphate may be further included. FIG. 1B shows an example of the appearance of the reagent kit of the present embodiment. In the figure, 210 indicates a reagent kit, 211 indicates a first container that stores a first reagent, 212 indicates a second container that stores a second reagent, and 213 stores a third reagent. The third container is shown, 214 is the fourth container containing the fourth reagent, 215 is the package insert, and 216 is the packing box.

  The scope of the present invention also includes a method for measuring preceptin using a biotinylated F1106-13-3 antibody. The measurement method is not particularly limited, and methods known to those skilled in the art such as enzyme immunoassay (EIA), for example, ELISA, Western blotting, etc. can be used.

  In the present embodiment, a method for measuring preceptin using an ELISA using a biotinylated F1106-13-3 antibody is provided. More specifically, a biotinylated F1106-13-3 antibody in which biotin is bound to the amino group of the F1106-13-3 antibody and streptavidin-bound solid phase particles are mixed to obtain a biotinylated F1106-13-3 antibody. A step of obtaining a streptavidin-bound solid phase particle complex (first complex), and the resulting first complex and a biological sample are mixed, and presepsin-biotinylated F1106-13-3 antibody-streptavidin A step of obtaining a bound solid phase particle complex (second complex), an obtained second complex, and an anti-preceptin antibody that recognizes an epitope different from the F1106-13-3 antibody, and F1106 A step of obtaining an anti-preceptin antibody-preceptin-biotinylated F1106-13-3 antibody-streptavidin-bound solid phase particle complex (third complex) recognizing an epitope different from the -13-3 antibody, and A step of measuring a signal obtained from the target substance in the third complex A method for measuring presepsin is provided.

The biotinylated F1106-13-3 antibody, streptavidin-bound solid phase particles, and the anti-preceptin antibody that recognizes an epitope different from the F1106-13-3 antibody are as described above.
The biological sample is not particularly limited as long as it is suitable for preceptin measurement, but it is preferable to use a blood sample. Examples of such blood samples include whole blood, serum, and plasma.

  The method for measuring the signal obtained from the labeling substance itself is known in the art. In the present embodiment, a measurement method corresponding to the type of signal derived from the labeling substance may be selected as appropriate. For example, when the labeling substance is an enzyme, it can be performed by measuring signals such as light and color generated by reacting a substrate for the enzyme with a known device such as a spectrophotometer.

  The measurement method of this embodiment may be used to determine whether the biological sample is from a sepsis patient. For example, from the measurement value (signal intensity value) of the signal obtained by the measurement method of the present embodiment, the preceptin concentration value in the biological sample is obtained, and based on the concentration value, whether the biological sample is of a septic patient It can be determined whether or not. More specifically, when the obtained preceptin concentration value is higher than a predetermined threshold value, it is determined that the biological sample is that of a septic patient. It can be determined that it is not a thing. The above determination result can be used to assist the doctor in diagnosing whether or not the subject suffers from sepsis.

  As the preceptin concentration value, for example, the signal intensity value itself or a value obtained from the intensity value can be used. Examples of the value obtained from the signal intensity value include a value obtained by subtracting the signal intensity value or background value of the negative control sample from the signal intensity value. The negative control sample can be appropriately selected, and examples thereof include a blood sample obtained from a healthy person.

  The predetermined threshold is not particularly limited and can be set as appropriate. For example, the predetermined threshold may be set empirically by accumulating data associated with sepsis, including preceptin concentration values in blood samples for septic patients. In setting the threshold, it is preferable to consider sensitivity, specificity, positive predictive value, negative predictive value, and the like.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited by these Examples.

Example 1: Labeling of thiol group with alkaline phosphatase (ALP) (1) Preparation of reagent containing ALP-labeled antibody
(1-1) Presepsin detection reagent
The F1106-13-3 antibody is available from Mochida Pharmaceutical. The F1106-13-3 antibody was dissolved in a phosphate buffer, and the EDTA concentration of the resulting solution was adjusted to 1 mM. An appropriate amount of 2-mercaptoethylamine hydrochloride solution was added to the antibody solution and reacted at 37 ° C. for a predetermined time. Thereafter, the antibody solution was desalted using a PD-10 column (GE Healthcare Japan) and adjusted to a predetermined concentration.
A maleimide crosslinking agent as an ALP labeling agent was dissolved in N, N-dimethylformamide (DMF) to prepare a maleimide crosslinking agent solution. An appropriate amount of the above maleimide crosslinker solution was added to the ALP solution and allowed to stand at 37 ° C. for a predetermined time to introduce maleimide groups. Thereafter, the solution was desalted using a PD-10 column (GE Healthcare Japan) and adjusted to a predetermined concentration.
The above antibody solution and the above ALP solution were mixed at a predetermined ratio and placed in a refrigerator to cause a coupling reaction. Thereafter, 2-mercaptoethylamine hydrochloride solution was added to stop the reaction. The ALP antibody solution was gel filtered and the desired fraction was collected.
The obtained desired fraction was dissolved in a buffer solution containing BSA to a predetermined concentration, and placed in a 37 ° C. constant temperature bath. Sampling was performed on the 0th, 1st, and 4th days, and these were used as preceptin detection reagents described in the following item (3-1).

(1-2) Insulin detection reagent
A commercially available anti-insulin monoclonal antibody was ALP-labeled as described in (1-1) above, except that a commercially available anti-insulin monoclonal antibody was used in place of the F1106-13-3 antibody. The obtained fraction of ALP-labeled anti-insulin antibody was dissolved in a buffer solution containing BSA to a predetermined concentration and placed in a constant temperature bath at 40 ° C. Sampling was performed on days 0 and 4, and these were used as insulin detection reagents described in the following item (3-2).

(1-3) pro-GRP detection reagent
A commercially available anti-proGRP monoclonal antibody was ALP-labeled as described in (1-1) above, except that a commercially available anti-proGRP monoclonal antibody was used instead of the F1106-13-3 antibody. The obtained fraction of ALP-labeled anti-proGRP antibody was dissolved in a buffer solution containing BSA to a predetermined concentration and placed in a constant temperature bath at 35 ° C. Sampling was performed on day 0 and day 3, and these were used as pro-GRP detection reagents described in the following item (3-3).

(1-4) PIVKA-II detection reagent
A commercially available anti-PIVKA-II monoclonal antibody was ALP-labeled as described in (1-1) above, except that a commercially available anti-PIVKA-II monoclonal antibody was used instead of the F1106-13-3 antibody. The obtained fraction of ALP-labeled anti-PIVKA-II antibody was dissolved at a predetermined concentration in a buffer solution containing BSA and placed in a constant temperature bath at 40 ° C. Sampling was performed on the 0th and 3rd days, and these were used as PIVKA-II detection reagents described in the following item (3-4).

(1-5) KL-6 detection reagent
A commercially available anti-KL-6 monoclonal antibody was ALP-labeled as described in (1-1) above, except that a commercially available anti-KL-6 monoclonal antibody was used instead of the F1106-13-3 antibody. The obtained fraction of ALP-labeled anti-KL-6 antibody was dissolved in a buffer solution containing BSA to a predetermined concentration and placed in a constant temperature bath at 40 ° C. Sampling was performed on day 0 and day 3, and these were used as reagents for detecting KL-6 described in the following item (3-5).

(2) Preparation of sample A sample for preceptin measurement was prepared by diluting recombinant antigen (Mochida Pharmaceutical) to 10000 pg / mL with a buffer containing BSA. For measurement items other than preceptin, a commercially available calibrator was used as a sample.

(3) Measurement
(3-1) Preceptin detection HISCL 2000i (manufactured by Sysmex Corporation) was used for preceptin detection.
As a preceptin capturing antibody, a biotin-conjugated anti-preceptin antibody in which biotin was bound to a commercially available anti-preceptin antibody was used. A buffer solution (50 μl) containing this biotin-conjugated anti-preceptin antibody and a sample (10 μL) were mixed, and the biotin-conjugated anti-preceptin antibody and preceptin were reacted specifically. A buffer solution (30 μL) containing streptavidin-bound magnetic particles was added thereto to bind the antibody-preceptin complex to the streptavidin-bound magnetic particles. The reagent for preceptin detection (30 μL) obtained in the above item (1-1) was added, and the ALP-labeled antibody was specifically reacted with preceptin in the complex. After removing unreacted solution by washing 9 times with 300 μL of HISCL washing solution, two types of HISCL luminescent substrate sets (50 μL and 100 μL, respectively) were added, and the luminescence intensity was measured.

(3-2) Insulin detection HISCL 2000i (manufactured by Sysmex Corporation) was used for insulin detection. The insulin detection reagent (100 μL) obtained in item (1-2) above was used instead of the preceptin detection reagent (30 μL), and the buffer solution (50 μL) containing the biotin-conjugated anti-preceptin antibody was used. Then, the luminescence intensity was measured in the same manner as the preceptin detection except that a buffer solution (50 μL) containing a biotin-conjugated anti-insulin antibody in which biotin was bound to a commercially available anti-insulin antibody was used.

(3-3) proGRP detection
HISCL 2000i (manufactured by Sysmex Corporation) was used for pro-GRP detection. The pro-GRP detection reagent (30 μL) obtained in item (1-3) above was used instead of the preceptin detection reagent (30 μL), and it was replaced with a buffer solution (50 μL) containing a biotin-conjugated anti-preceptin antibody. Using a buffer solution (50 μL) containing a biotin-conjugated anti-pro-GRP antibody in which biotin is bound to a commercially available anti-pro-GRP antibody, and in the same manner as the preceptin detection except that the sample was 30 μL, The luminescence intensity was measured.

(3-4) PIVKA-II detection
HISCL 2000i (manufactured by Sysmex Corporation) was used for PIVKA-II detection. The PIVKA-II detection reagent (120 μL) obtained in the above item (1-4) was used instead of the preceptin detection reagent (30 μL), and a buffer solution (50 μL) containing a biotin-conjugated anti-preceptin antibody Instead of this, the luminescence intensity was measured in the same manner as the preceptin detection except that a buffer solution (100 μL) containing a biotin-conjugated anti-PIVKA-II antibody in which biotin was bound to a commercially available anti-PIVKA-II antibody was used.

(3-5) KL-6 detection
For detection of KL-6, HISCL 2000i (manufactured by Sysmex Corporation) was used. The KL-6 detection reagent (50 μL) obtained in item (1-5) above was used instead of the preceptin detection reagent (30 μL), and the buffer solution (50 μL) containing the biotin-conjugated anti-preceptin antibody was used. The luminescence intensity was measured in the same manner as in the preceptin detection except that a buffer solution (100 μL) containing a biotin-conjugated anti-KL-6 antibody in which biotin was bound to a commercially available anti-KL-6 antibody was used.

(4) Results The measurement results are shown in FIG. As shown in FIG. 2, the luminescence intensity seen in preceptin detection showed similar intensity to that seen in other molecule measurements. This indicates that the F1106-13-3 antibody in which the thiol group is ALP-labeled is as stable as other ALP-labeled antibodies.

Example 2: Biotin labeling of thiol group (1) Preparation of reagent
(1-1) Preceptin-capturing reagent Instead of alkaline phosphatase (ALP), biotin-labeled F1106-13-3 was used in the same manner as in Example 1 except that the thiol group of F1106-13-3 antibody was labeled with biotin. Antibody was obtained.
Biotin labeling was performed as follows. First, F1106-13-3 antibody was dissolved in a phosphate buffer, and the EDTA concentration was adjusted to 1 mM. An appropriate amount of 2-mercaptoethylamine hydrochloride solution was added to the solution and reacted at 37 ° C. for a predetermined time. Thereafter, the solution was desalted using PD-10 (GE Healthcare Japan) and adjusted to a predetermined concentration.
A biotin labeling agent solution was prepared by dissolving the biotin labeling agent in dimethyl sulfoxide (DMSO).
The F1106-13-3 antibody solution and the biotin labeling agent solution were mixed at a predetermined ratio and placed at 37 ° C. to introduce a maleimide group. The mixture was gel filtered and the desired fraction was collected.
The obtained desired fraction was dissolved in a buffer solution containing BSA to a predetermined concentration, and placed in a 37 ° C. constant temperature bath. Sampling was performed on days 0, 1 and 4, and these were used as preceptin-capturing reagents described in the following item (3-1).

(1-2) Insulin Capture Reagent A biotin-labeled anti-insulin antibody was obtained in the same manner as in Example 1 except that the thiol group of the anti-insulin antibody was labeled with biotin instead of alkaline phosphatase (ALP). The obtained biotin-labeled anti-insulin antibody was dissolved in a buffer solution, and the obtained solution was placed in a constant temperature bath at 40 ° C. Sampling was performed on the 0th and 4th days, and these were used as reagents for capturing insulin described in the following item (3-2).

(1-3) Reagent for pro-GRP capture Biotin-labeled anti-pro-GRP in the same manner as in Example 1 except that the thiol group of the anti-pro-GRP antibody was labeled with biotin instead of alkaline phosphatase (ALP). Antibody was obtained. The obtained biotin-labeled anti-pro-GRP antibody was dissolved in a buffer solution, and the obtained solution was placed in a constant temperature bath at 40 ° C. Sampling was performed on the 0th, 1st and 3rd days, and these were used as pro-GRP capturing reagents described in the following item (3-3).

(1-4) Reagent for PIVKA-II capture In the same manner as in Example 1 except that the thiol group of the anti-PIVKA-II antibody was labeled with biotin instead of alkaline phosphatase (ALP), biotin-labeled anti-PIVKA-II Antibody was obtained. The obtained biotin-labeled anti-PIVKA-II antibody was dissolved in a buffer solution, and the obtained solution was placed in a constant temperature bath at 40 ° C. Sampling was performed on days 0 and 3, and these were used as PIVKA-II capture reagents described in the following item (3-4).

(2) Preparation of sample A sample for preceptin measurement was prepared by diluting recombinant antigen (Mochida Pharmaceutical) to 10000 pg / mL with a buffer containing BSA. For measurement items other than preceptin, a commercially available calibrator was used as a sample.

(3) Measurement
(3-1) Preceptin detection HISCL 2000i (manufactured by Sysmex Corporation) was used for preceptin detection. As an antibody for preceptin detection, an ALP-labeled anti-preceptin antibody obtained by labeling a commercially available anti-preceptin antibody with ALP was used. The ALP labeling of the antibody was performed in the same manner as in Example 1. The biotin-labeled F1106-13-3 antibody obtained by mixing the preceptin-capturing reagent (50 μl) containing the F1106-13-3 antibody labeled with biotin-labeled thiol group obtained in (1-1) above and a sample (10 μL) Was specifically reacted with preceptin. A buffer solution (30 μL) containing streptavidin-bound magnetic particles was added thereto to bind the antibody-preceptin complex to the streptavidin-bound magnetic particles. A buffer solution (30 μL) containing the above ALP-labeled anti-preceptin antibody was added, and the ALP-labeled anti-preceptin antibody was reacted specifically with preceptin in the above complex. After removing unreacted solution by washing 9 times with 300 μL of HISCL washing solution, two types of HISCL luminescent substrate sets (50 μL and 100 μL, respectively) were added, and the luminescence intensity was measured.

(3-2) Insulin detection Instead of the presepsin capture reagent, the insulin capture reagent obtained in item (1-2) above was used, and a commercially available anti-preceptin antibody was used instead of the ALP-labeled anti-preceptin antibody. Luminescence intensity was measured in the same manner as preceptin detection except that an ALP-labeled anti-insulin antibody in which an insulin antibody was labeled with ALP was used.

(3-3) pro-GRP detection Instead of the preceptin capture reagent, the pro-GRP capture reagent obtained in item (1-3) above was used, and the ALP-labeled anti-preceptin antibody was used instead. The luminescence intensity was measured in the same manner as the preceptin detection except that an ALP-labeled anti-pro-GRP antibody obtained by labeling a commercially available anti-pro-GRP antibody with ALP was used.

(3-4) Detection of PIVKA-II In place of the reagent for capturing preceptin, the reagent for capturing PIVKA-II obtained in the above item (1-4) was used, and in place of the ALP-labeled anti-preceptin antibody. The luminescence intensity was measured in the same manner as preceptin detection except that an ALP-labeled anti-PIVKA-II antibody obtained by labeling a commercially available anti-PIVKA-II antibody with ALP was used.

(4) Results The measurement results are shown in FIG. As shown in FIG. 3, the fluorescence intensity seen in preceptin detection was significantly reduced compared to that seen in other molecular measurements. This indicates that the F1106-13-3 antibody in which a thiol group is labeled with biotin is more unstable than other biotin-labeled antibodies.

Example 3: Biotin labeling of amino group (1) Preparation of preceptin detection reagent containing F1106-13-3 with amino group biotinylated Instead of thiol group, the amino group of F1106-13-3 antibody was replaced with biotin A biotin-labeled F1106-13-3 antibody was obtained in the same manner as in Example 2 except that labeling was performed. The biotin labeling of the amino group was performed according to the Technical Manual of Biotin Labeling Kit-NH ₂ (Dojindo Laboratories). The obtained biotin-labeled F1106-13-3 antibody was dissolved in a buffer solution, and the obtained solution was placed in a constant temperature bath at 37 ° C. Sampling was performed on days 0, 2, 5, 7, and 14, and these were used as preceptin detection reagents described in the following item (3).

(2) Preparation of sample A sample for preceptin measurement was prepared by diluting recombinant antigen (Mochida Pharmaceutical) to 10000 pg / mL with a buffer containing BSA. For measurement items other than preceptin, a commercially available calibrator was used as a sample.

(3) Measurement—Preceptin detection As in Example 2 except that a buffer solution (50 μl) containing the F1106-13-3 antibody labeled with biotin in the amino group as described above was used as a reagent for capturing preceptin. The luminescence intensity was measured.

(4) Results The measurement results are shown in FIG. As shown in FIG. 4, the fluorescence intensity seen in preceptin detection showed similar intensity to that seen in other molecule measurements. This indicates that the F1106-13-3 antibody in which the amino group is labeled with biotin is as stable as other biotin-labeled antibodies. From the above results, it was shown that the F1106-13-3 antibody can be stabilized by biotin labeling the amino group instead of the thiol group of the F1106-13-3 antibody.

110, 210 Reagent kit 111, 211 First container 112, 212 Second container 113, 213 Third container 214 Fourth container 115, 215 Package insert 116, 216 Packing box

Claims (12)

  A biotinylated F1106-13-3 antibody, wherein biotin is bound to the amino group of the F1106-13-3 antibody.   The biotinylated F1106-13-3 antibody according to claim 1, wherein a biotin labeling agent is bound to the amino group of the F1106-13-3 antibody.   Biotin labeling agents include biotin N-hydroxysuccinimide ester, 6- (biotinylamino) hexanoic acid N-hydroxysuccinimide ester, 6- [6- (biotinylamino) hexanoylamino] hexanoic acid N-hydroxysuccinimide ester, biotin N- Hydroxysulfosuccinimide ester, 6- (biotinylamino) hexanoic acid N-hydroxysulfosuccinimide ester, 6- [6- (biotinylamino) hexanoylamino] hexanoic acid N-hydroxysulfosuccinimide ester, and N-[[2- ( The biotinylated F1106-13-3 antibody according to claim 2, which is at least one selected from the group consisting of sodium salts of biotinylamino) ethyl] dithiopropynyloxy] sulfosuccinimide.   A method for producing a biotinylated F1106-13-3 antibody, comprising a step of binding biotin to an amino group of the F1106-13-3 antibody.   The production method according to claim 4, wherein the step of binding is performed by binding a biotin labeling agent to the amino group of the F1106-13-3 antibody.   Biotin labeling agents include biotin N-hydroxysuccinimide ester, 6- (biotinylamino) hexanoic acid N-hydroxysuccinimide ester, 6- [6- (biotinylamino) hexanoylamino] hexanoic acid N-hydroxysuccinimide ester, biotin N- Hydroxysulfosuccinimide ester, 6- (biotinylamino) hexanoic acid N-hydroxysulfosuccinimide ester, 6- [6- (biotinylamino) hexanoylamino] hexanoic acid N-hydroxysulfosuccinimide ester, and N-[[2- ( The production method according to claim 5, which is at least one selected from the group consisting of sodium salts of biotinylamino) ethyl] dithiopropynyloxy] sulfosuccinimide. A first reagent containing a biotinylated F1106-13-3 antibody in which biotin is bound to the amino group of the F1106-13-3 antibody;
A second reagent comprising streptavidin-bound solid phase particles;
A third reagent comprising an anti-preceptin antibody that recognizes an epitope different from the F1106-13-3 antibody;
A reagent kit for measuring presepsin. 2-chloro-5- (methoxyspiro [1,2-dioxetane-3,2 '-(5'-chloro) tricyclo [3.3.1.13,7] decan] -4-yl) phenyl phosphate containing disodium Further comprising four reagents,
The reagent kit for preceptin measurement according to claim 7.   The reagent kit for preceptin measurement according to claim 7 or 8, wherein the anti-preceptin antibody recognizing an epitope different from the F1106-13-3 antibody is an anti-preceptin antibody labeled with a detectable labeling substance.   The reagent kit for preceptin measurement according to any one of claims 7 to 9, wherein the solid phase particles are magnetic particles.   The reagent kit for preceptin measurement according to any one of claims 7 to 10, wherein the labeling substance is alkaline phosphatase (ALP). A biotinylated F1106-13-3 antibody in which biotin is bound to the amino group of F1106-13-3 antibody and streptavidin-bound solid phase particles are mixed, and biotinylated F1106-13-3 antibody-streptavidin-bound solid phase is mixed. Obtaining a particle complex (first complex);
Mixing the obtained first complex and a biological sample to obtain a preceptin-biotinylated F1106-13-3 antibody-streptavidin-bound solid phase complex (second complex);
An anti-preceptin antibody that recognizes an epitope different from the F1106-13-3 antibody is mixed with the obtained second complex and an anti-preceptin antibody that recognizes an epitope different from the F1106-13-3 antibody. Obtaining a biotinylated F1106-13-3 antibody-streptavidin-conjugated solid phase particle complex (third complex);
And measuring a signal obtained from the target substance in the obtained third complex.

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