JP2002267665A - Method of measuring human c-peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method by which a human C-peptide can be measured with a low cross reactivity with proinsulin with high reproducibility and with high sensitivity, and to provide a kit suitable for the method. SOLUTION: In the method of measuring the human C-peptide include a process (i) in which the human C-peptide in a sample, a first antihuman C- peptide antibody or its fragment and a second antihuman C-peptide antibody immobilized on a solid phase or its fragment form an immune complex, a process (ii) in which the immune complex formed in the process (i) is separated from the unreacted antibodies and/or their fragments and the sample and a process (iii) in which the immune complex is quantitatively determined, the first antibody recognizes a first amino acid residue to a tenth amino acid residue at the terminal of a human C-peptide molecule N, the second antibody recognizes a first amino acid residue to a 16th amino acid residue at the terminal of the human C-peptide molecule N, and mutually different epitopes are recognized.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring human C-peptide and a kit for measuring human C-peptide.

[0002]

2. Description of the Related Art Human C-peptide is a peptide consisting of 31 amino acids and is a component of proinsulin, an insulin precursor. Specifically, C-peptide is a polypeptide that is released simultaneously as a degradation product when proinsulin is cleaved by endopeptidase and insulin is released into the blood. The primary structures of human proinsulin and human C-peptide are shown in Sequence Listings 1 and 2. Human proinsulin is a polypeptide consisting of 86 amino acids,
It is mainly composed of the 1st to 30th insulin B chains, the 33rd to 63rd C-peptides, and the 66th to 86th insulin A chains.The C-peptide and insulin are 31, 32nd Arg, 64th Lys, 65th.
Each is connected via Arg. As described above, the C-peptide is released into the blood at the same time that proinsulin is processed and insulin is released. Therefore, C-peptide plays a role as an indicator of insulin secretion kinetics, and the kinetics of blood C-peptide can be an important indicator for examining endogenous insulin secretion ability of diabetic patients and the like. In fact, the measurement of C-peptide is used for diagnosis or treatment of diabetes, and is also useful for diagnosis of insulinoma, insulin autoimmune syndrome and the like.

Normally, the ratio of C-peptide to proinsulin is 1: 0.6 to 1.3 in healthy subjects, 1: 0.3 to 0.5 in obese subjects, and about 1: 5 in patients with islet adenoma of Langerhans. It has been reported that C-peptide excess and proinsulin excess can vary with diet (PN
AS, 67,148-155, 1970). In general, when the amount of the cross-reactive substance is small, even if the cross-reactivity of the measurement system is high, it may not be a big problem, but the presence of the cross-reactive substance (proinsulin in the case of C-peptide) When the amount is large and the abundance ratio of both greatly fluctuates, there has been a problem in the reliability of the measured value as an indicator of the disease state or health condition. For example, when monitoring the blood concentration of C-peptide by regular diagnosis and linking it to treatment,
The influence of the presence of the cross-reactive substance proinsulin cannot be ignored. Therefore, a C-peptide measurement system having low cross-reactivity with proinsulin is strongly desired.

Conventionally, a method based on the principle of competition using a polyclonal antibody has been mainly used for measuring C-peptide. For example, Japanese Patent Publication No. 57-44663 describes a measurement method by a competition method using a radiolabeled C-peptide, and JP-A-1-165962 describes an enzyme-labeled C-peptide.
A measurement method by a competition method using a peptide is described. However, the C-peptide detection sensitivity in these methods is not high, and further improvement such as high cross-reaction with proinsulin and low reproducibility has been required. As an improvement of the measurement system using such a competition method, a C-peptide measurement system by a sandwich method using a monoclonal antibody is described in JP-A-4-177166, and although the detection sensitivity is slightly improved by this method, Was not enough. Further, no attempt has been made to improve the cross-reactivity, and only a measurement system having a high cross-reactivity remains.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for measuring human C-peptide with low cross-reactivity with proinsulin, high reproducibility and high sensitivity, and
An object is to provide a kit suitable for measurement according to the method.

SUMMARY OF THE INVENTION The present invention is a sandwich immunoassay using first and second anti-human C-peptide antibodies that recognize different epitopes on human C-peptide molecules.

[0006] In diseases such as non-insulin-dependent diabetes mellitus, insulinoma, and familial hyperproinsulinemia, split-type proinsulin in which proinsulin or any of amino acids 31-32 or 64-65 is deleted. It is known that insulin is often released into the bloodstream. C-
Since the primary structure of the peptide is completely included in the primary structure of proinsulin, it has been considered difficult to avoid high proinsulin cross-reactivity in a C-peptide immunoassay system. However, if the end portion of the antigen molecule is exposed, and there is an antibody that can identify the vicinity of the end of the molecule, it is possible to establish a measurement system that can distinguish C-peptide and polypeptide containing C-peptide sequence It is. This is because in such an antibody, a sequence that completely encapsulates the C-peptide molecule, such as a naturally occurring proinsulin or a modified C-peptide, changes in structure and charge around the recognition site. Occurs, resulting in a significant decrease in recognizability or no recognition at all. The present inventors have paid attention to this point, and
And the second antibody is an antibody that recognizes a different epitope and can simultaneously recognize human C-peptide,
A first anti-human C-peptide antibody that recognizes an epitope present in a region consisting of amino acid residues 1 to 10 at the N-terminus of a human C-peptide, and a human C-peptide N-terminal immobilized on a solid phase By using a sandwich immunoassay using a second anti-human C-peptide antibody that recognizes an epitope present in the region consisting of amino acid residues 1 to 16 of
The present inventors have found a measurement system capable of specifically measuring human C-peptide while avoiding the cross-reaction of human proinsulin, and have accomplished the present invention. The present invention also provides a kit for performing such a measurement.

[0007] That is, the method of the present invention comprises the steps of (i) the step of immobilizing a human C-peptide, a first anti-human C-peptide antibody or an antigen-binding fragment thereof on a solid phase, (Iii) forming an immune complex with the anti-human C-peptide antibody or an antigen-binding fragment thereof, and (ii) converting the immune complex formed in (i) into an unreacted antibody and / or an antigen thereof. A method for measuring human C-peptide, comprising: separating from a binding fragment and a sample; and (iii) quantifying the immune complex, wherein the first antibody has an N-terminal of the human C-peptide molecule. Recognizing an epitope present in a region consisting of amino acid residues 1 to 10, the second antibody recognizes an epitope present in a region consisting of amino acid residues 1 to 16 at the N-terminal of the human C-peptide molecule. The first and second anti-human The C-peptide antibody recognizes different epitopes and can bind to the human C-peptide at the same time, which is a method for measuring the human C-peptide.

The kit of the present invention comprises (a) a first container containing a labeled first anti-human C-peptide antibody or an antigen-binding fragment thereof, and (b) a solid phase immobilized on a solid phase. A solid phase having a second anti-human C-peptide antibody or an antigen-binding fragment thereof or a second container comprising a solid phase, wherein the first antibody comprises N-terminal amino acid residues of a human C-peptide molecule Recognizing an epitope present in a region consisting of Nos. 1 to 10, the second antibody recognizes an epitope present in a region consisting of amino acid residues 1 to 16 at the N-terminal of the human C-peptide molecule, A kit for measuring human C-peptide, wherein the first and second anti-human C-peptide antibodies recognize different epitopes and can simultaneously bind to the human C-peptide.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In the method of the present invention, human C-
An immunoassay using two kinds of antibodies recognizing different epitopes of the peptide, particularly a sandwich method is used.
In the method of the present invention, a one-step reaction (one-step sandwich method, hereinafter referred to as one-step method) in which two kinds of antibodies (for example, an immobilized antibody and a labeled antibody described below) are simultaneously reacted with a sample to be measured, or Sample to be measured and first anti-human C-peptide antibody (eg, immobilized antibody)
To form an immune complex after a certain period of incubation, followed by reaction with a second anti-human C-peptide antibody (for example, a labeled antibody) (two-step sandwich method, hereinafter referred to as a two-step method). ) Can be used.

The first anti-human used in the assay of the present invention
The C-peptide antibody recognizes an epitope present in the amino acid residues 1 to 10 at the N-terminal of the human C-peptide molecule. The first antibody preferably recognizes an epitope present in the region of amino acid residues 1 to 8, and is present in the region of positions 1 to 8 including at least a part of amino acid residues 1 to 3. More preferably, it recognizes an epitope. As used herein, "recognizing an epitope" means that an antibody reacts with the epitope by antigen-antibody reaction by binding to the epitope. The first antibody is preferably Arg-Arg- attached to the N-terminus of the human C-peptide.
It does not substantially recognize human C-peptide having a sequence (hereinafter sometimes referred to as “Arg-Arg-C-peptide”). This “substantially no recognition” means that the cross-reactivity of the first antibody to Arg-Arg-C-peptide is 10% or less when the concentration of Arg-Arg-C-peptide is 5 ng / ml. It means there is. The cross-reactivity is preferably 5% or less, more preferably 1% or less.

[0011] The cross-reactivity of the first antibody to the Arg-Arg-C-peptide is first determined by different concentrations of human known as samples.
Using a C-peptide solution, a standard curve is prepared according to the method of the present invention in a conventional manner, and is determined by measuring with an Arg-Arg-C-peptide solution set to an actual concentration of 5 ng / ml. Ar
The cross-reactivity of the immunoassay to g-Arg-C-peptide is the percentage of the measured concentration measured based on a standard curve against the actual Arg-Arg-C-peptide concentration. However, the actual concentration is calculated by multiplying the actual concentration by the molecular weight ratio.
The difference in molecular weight between C-peptide and Arg-Arg-C-peptide is corrected (see Example 4 below). The cross-reactivity of the first antibody to the Arg-Arg-C-peptide was determined by adding L to the C-terminal.
It should be noted that the measurement can be performed by using an Arg-Arg-C-peptide to which ys-Arg- is bound ("modified C-peptide" shown in Example 3 below). Because the first
No. 1 recognizes the epitope present at amino acid residues 1-10, and the unambiguous structure at the C-terminal end of the peptide does not affect the binding of the first antibody.
The first antibody is preferably a monoclonal antibody from the viewpoint of reproducibility and specificity. An antigen-binding fragment such as Fab or F (ab ') 2 of the first antibody can be used in place of the first antibody. The first antibody or the antigen-binding fragment thereof is used without being immobilized and not bound.

[0012] The first antibody can be produced by methods known in the art. For example, anti-human C-peptide antibodies (preferably monoclonal antibodies) can be produced by methods generally known in the art, and the produced antibodies have been tested for binding to the corresponding epitope. The epitope of the corresponding antibody can be determined by an inhibition reaction using a series of fragments of the human C-peptide as exemplified in Example 5 below. If the antigen-antibody reaction between the tested antibody and the human C-peptide is inhibited in a concentration-dependent manner by adding a known amount of the polypeptide fragment, the tested antibody is the added polypeptide. Recognizes the epitope present on the fragment.

As described above, by using a polypeptide fragment consisting of at least 5 consecutive amino acids in the region of amino acid residues 1 to 10 of the human C-peptide as a competitor in the inhibition test, An antibody that can be used as an antibody can be selected. Among the anti-human C-peptide antibodies, an antibody that recognizes an epitope present in the region of amino acid residues 1 to 10 of the human C-peptide is selected according to the method described above, and is used as the first antibody in the method of the present invention. can do. A first antibody that does not recognize human C-peptide having Arg-Arg- attached to the N-terminal can be selected in a similar manner. That is, such an antibody uses, as a competitor, a polypeptide fragment in which Arg-Arg- is bonded to the N-terminal of human C-peptide, and Arg-Arg- which is bonded to the N-terminal in the competitive immunoassay. Human C with
-Can be selected by eliminating antibodies that recognize the peptide. Further, the first antibody uses an immunogenic peptide having at least 5 consecutive amino acids in the region of amino acid residues 1 to 10 bound to a protein carrier such as KLH or BSA and used as an immunogen. It can be obtained by selecting an antibody that recognizes the obtained peptide fragment.

A commercially available anti-human C-peptide monoclonal antibody can be used. Among commercially available monoclonal antibodies, amino acid residue 1 of human C-peptide
An antibody that recognizes the # 10 to # 10 epitope is conveniently used as the first antibody of the present invention. Commercially available monoclonal antibodies used as the first antibody in the method of the present invention include Medix Biochemical Inc.'s anti-human
The C-peptide monoclonal antibody 9101 is exemplified.
The antigen-binding fragment of the first antibody can be produced by a method generally known in the art. That is, Fab
The fragment can be produced by digesting the first antibody with papain, and the F (ab ') 2 fragment can be produced by digesting the first antibody with pepsin.

[0015] The second antibody used in the sandwich immunoassay of the present invention comprises amino acid residue 1 of human C-peptide.
Recognize the epitope present at the No. 16 to No. 16. As shown in the Examples below, by using an antibody that recognizes the epitope present at amino acid residues 1 to 16 of the human C-peptide, the C-peptide of the human C-peptide was used as the second antibody.
Cross-reactivity with human proinsulin can be extremely reduced as compared to an antibody that recognizes an epitope present in the half of the side terminal. This phenomenon was first discovered by the present inventors. It is preferable to use a monoclonal antibody as the second antibody from the viewpoint of reproducibility and specificity. To perform a sandwich immunoassay, a second
Must recognize an epitope different from the epitope recognized by the first antibody, and the first antibody and the second antibody need to be able to bind simultaneously to the human C-peptide. If two antibodies can bind to the human C-peptide simultaneously, the two antibodies are considered to recognize different epitopes, even though each of the epitope regions may partially overlap.

An antigen-binding fragment such as a Fab fragment or F (ab ') 2 fragment of the first antibody can be used in place of the second antibody. The fragments can be produced according to methods known in the art as described above. The second antibody can be obtained according to the following. First, an antibody that recognizes an epitope present at amino acid residues 1 to 16 of a human C-peptide molecule is obtained. These antibodies are used as competitors to bind the inhibitory reaction or as immunogens bound to protein carriers, from amino acid residues 1 to 16 of the human C-peptide.
The first antibody can be obtained in the same manner as described above for obtaining the first antibody by using a polypeptide fragment consisting of at least 5 contiguous amino acids in the number region. The epitope need not be determined accurately, and candidate antibodies for the second antibody can be selected by simply determining whether the antibody recognizes the region of amino acid residues 1-16. Further, an antibody capable of binding to the human C-peptide simultaneously with the first antibody can be selected from these candidate antibodies as the second antibody. This can be performed, for example, by measuring and comparing a conventional sandwich immunoassay using a first antibody and a candidate antibody as the second antibody for human C-peptide.

Among the commercially available monoclonal antibodies, an antibody that recognizes the epitope at amino acid residues 1 to 16 of the human C-peptide and binds to the human C-peptide simultaneously with the first antibody is the present invention. Can be conveniently used as the second antibody in the above method. For example, the first antibody is an anti-human antibody commercially available from Medix BioChemica.
When the C-peptide monoclonal antibody 9101 is used, the anti-human C-peptide monoclonal antibody 9103 commercially available from Medix BioChemica and the anti-human C-peptide monoclonal antibody CPT3F11 commercially available from Dako may be used as the second antibody. it can. The use of the above-mentioned first antibody and second antibody is characteristic. Otherwise, the sandwich immunoassay of the present invention can be carried out according to a usual method. In the following description (excluding the following examples)
Apart from specific statements or statements that are clear from the context,
"Antibody" includes an antibody and an antigen-binding fragment. The second antibody is immobilized on a solid phase. As shown in the examples below,
The cross-reactivity of the immunoassay for human insulin is higher than when the first antibody is immobilized and the second antibody is not bound.
It is smaller when the second antibody is immobilized and the first antibody is not bound.

It is preferable that one of the two kinds of antibodies used in the method of the present invention is immobilized on an appropriate solid phase. Such an immobilized antibody can be produced by a known appropriate method. As the solid phase, polystyrene, polyethylene, Sepharose (Pharmacia), cellulose or the like can be used, and the physical shape is not essential. The solid phase to be used is preferably one that can easily immobilize the antibody on its surface and can easily separate the immune complex formed during the measurement from unreacted sample components and antibodies. In particular,
Plastic plates and magnetic particles used for ordinary immunoassays are preferred. From the viewpoint of ease of handling, storage and separation, it is most preferable to use magnetic particles of the above-mentioned materials.

In general, the other antibody which is not immobilized is labeled for easy detection. The labeling method is not particularly limited, but is generally a radioisotope,
Enzymes, fluorescent dyes and the like are used. As the radioisotope, for example, ¹²⁵ I, ¹³¹ I, etc. can be used.As the enzyme, peroxidase, β-galactosidase, alkaline phosphatase, etc. can be used, and as the substrate, O-nitrophenyl-β-D-galactopyrase can be used. Noside, p-nitrophenyl phosphate, AMPPD ((3-2'-spiroadamantane)-
4-methoxy-4- (3``-phosphoryloxy) phenyl-1,2-
Dioxetane) can be used. Fluorescein thiocyanate (FITC) or the like can be used as the fluorescent dye. The method for detecting these labels depends on the label used, but methods generally known in the art can be used. In a preferred embodiment of the present invention, an alkaline phosphatase-conjugated antibody is used as the enzyme-labeled antibody and the chemiluminescent substance AMPPD is used as the substrate.

As is well known, the labeling of the first antibody is not a requirement to achieve quantification of the immune complex.
The immune complex can be quantified by reacting the immune complex on the solid phase with a labeled third antibody.
This third antibody recognizes the first antibody, which is not immobilized. Using the above-mentioned first antibody and second antibody,
A sandwich immunoassay is performed in the usual way. The sandwich method can be performed in either a one-step method or a two-step method. In the one-step method,
The first and second antibodies and the human C-peptide contained in the sample are reacted simultaneously. After washing, the immune complexes bound to the solid phase are quantified. This method has an advantage that the immune reaction is one step, and as a result, the operation is simple and the time required for the immunoassay is short. In the two-step method, the human C-peptide contained in the sample is reacted with the first immobilized second antibody. After washing, the unbound first antibody is then bound to the solid phase by a second antibody.
Reacts with C-peptide. After washing, the immune complex bound to the solid phase is quantified. This two-step method has the advantage of lower cross-reactivity with human proinsulin than the one-step method.

As can be seen from the above, the incubation time and temperature in the sandwich method may be a time and temperature sufficient for one of the antibodies and the human C-peptide to form an immune complex, for example, 37 ° C. or It can be set as needed at about 5 minutes or more at room temperature, preferably 15 minutes to 30 minutes, or at 4 ° C. overnight. Further, a longer time may be used as long as protein degradation does not significantly occur. In the above incubation and other operations, buffers commonly used in immunoassays can be used. Such buffers include:
For example, 50 mM Tris buffer (pH 7.2) with or without PBS, bovine serum albumin (BSA) or 50 mM female buffer (pH 6.8) is included. These buffers may contain about 0.1% sodium azide to prevent bacterial growth.

The sandwich immunoassay according to the present invention comprises:
It has high detection sensitivity for human C-peptide.
A method of low cross-reactivity with amino acids 1-32 or proinsulin deficient in amino acids 64-65). The cross-reactivity of this sandwich immunoassay for human proinsulin indicates that human proinsulin levels
When 50 ng / ml, it is preferably 10% or less, and 5%
It is more preferably at most 1%, more preferably at most 1%. The cross-reactivity of the human proinsulin immunoassay was determined by using a human proinsulin solution having various known concentrations as a sample and preparing a standard curve in accordance with the well-known method described in the present invention, and determining the predetermined human proinsulin concentration (50 ng). / ml). Cross-reactivity in an immunoassay for human proinsulin is the percentage of the concentration measured by the method of the present invention based on a standard curve for human proinsulin concentration. However, the difference in molecular weight between human C-peptide and human proinsulin was corrected by multiplying the actual concentration by the molecular weight ratio (see Example 4 below). The detection sensitivity of the sandwich immunoassay according to the present invention is 0.05 ng / ml or less, preferably 0.02 ng.
/ ml or less.

In the present specification, the detection sensitivity means a value of 2S / N. 2S / N is an antigen concentration that produces a signal twice as high as a signal (noise signal) at an antigen concentration of 0 mg / ml. Further, the cross-reactivity means the ratio of the cross-reactive substance measured as C-peptide, and the percentage obtained by dividing the concentration at which the cross-reactive substance is apparently measured as C-peptide by the actual cross-reactive substance concentration. It shall be represented by A cross-reactive substance refers to a substance that is recognized as a C-peptide by an antibody although it is not a C-peptide. Samples to which the method of the present invention can be applied include secretions or excretions where C-peptide is normally secreted,
For example, a sample derived from a body fluid such as serum or urine is included.

Further, in order to easily carry out the present invention, a kit containing a combination of the above-mentioned appropriate antibodies is provided. The kit of the present invention comprises a container containing a first anti-human C-peptide antibody and another container containing a second anti-human C-peptide antibody. Further, the kit of the present invention may contain one or more buffers or concentrated stock solutions thereof suitable for assay or dilution as described above, a substrate for measurement or a solution thereof, a human C-peptide or a solution thereof as a standard sample, and An assay or dilution container may be included. Such buffers include 50 mM Tris buffer pH 7.2 containing BSA, 150 mM sodium chloride and 0.1% sodium azide as a buffer for the anti-human C-peptide antibody binding particles, and a labeled anti-human Examples of the buffer for the C-peptide antibody include those obtained by adding 0.3 mM of zinc chloride to the above buffer.

In the present kit, the first antibody is preferably immobilized on a solid phase, and particularly preferably immobilized on magnetic particles. The second antibody is labeled, particularly preferably enzyme-labeled. The first and second antibodies may be present as a solution in a suitable buffer, or may be present as a lyophilized product. Each reagent included in this kit may be provided by being dispensed to a container for each sample measurement, or may be provided by including a plurality of sample measurements for each reagent together in an individual container. Good. In the latter case, each reagent is dispensed into a predetermined measurement container at the time of use. When reagents are provided for each sample measurement, the container containing each reagent may be integrally formed as a cartridge, and each reagent may be stored in a different section of the cartridge. If the first and second antibodies are included in the kit as lyophilized products, a buffer as described above suitable for dissolving them may be further included in the kit. Other containers for the assays that contain these antibodies or that may be included in the kit do not interact with the antibodies and antigens and are made of materials that do not interfere with the reactions used in the assays, such as enzymatic reactions and chemiluminescent reactions. Any material may be used as long as it is made. If necessary, the surface may be provided with a pre-treatment so as not to cause such an interaction. Such processing methods are well known to those skilled in the art. This kit is usually accompanied by an instruction manual.

[0026]

EXAMPLES Example 1: Materials and measuring method (1) Preparation of anti-human C-peptide antibody-bound particles 1 mg of anti-human C-peptide antibody was added to 2.5 ml of 50 mM phosphate buffer p
It was dissolved in H3.0 to obtain a sensitizing solution. Next, the magnetic particles (ferrite-coated latex particles, particle size 2 μm) subjected to ultrasonic cleaning treatment with 50 mM phosphate buffer pH 3.0
m, manufactured by Nippon Paint Co., Ltd.), 50 mg of the sensitizing solution was added, and the mixture was thoroughly mixed. After the end, attract the particles by magnetic force from the outside of the reaction vessel,
The reaction solution was removed by suction, and washed with 50 mM female buffer pH 5.5. The sensitized magnetic particles were suspended again in 50 mM female buffer pH 5.5, and 5 mg / ml of 1-ethyl-3- (3-dimethylaminopropyl) was added.
Carbodiimide hydrochloride (Nacalai Tesque) aqueous solution
After adding 1 ml, the mixture was rotated at 25 ° C. for 30 minutes. The particles were again magnetically attracted and washed, suspended in 50 mM Tris buffer pH 7.2 containing 0.1% sodium azide, and stored at low temperature until use. Antibody-bound particles are 50 mM fresh containing BSA, 150 mM sodium chloride and 0.1% sodium azide.
It was suspended in 0.03% (w / v) with M Tris buffer pH 7.2 and used for measurement as a particle liquid. 9 for N-recognition antibody for sensitization
Using 101 (Medix Biochemical), 9103 (Medix Biochemical) or CPT-3-F11 (Dako Japan) as an antibody that recognizes the C-terminal region from the center of the C-peptide molecule PEP-001 (manufactured by Dako Japan) was used.

(2) Alkaline Phosphatase-Labeled Anti-Human C-Peptide Antibody The anti-human C-peptide antibody described in (1) and alkaline phosphatase (manufactured by Oriental Yeast Co., Ltd.) were prepared by the method of Yoshitake et al., J Biochem. . 1982,92 (5), 141
3-1424) to give an enzyme-labeled antibody. The enzyme-labeled antibody was diluted to 0.2 μg / ml with a 50 mM female buffer (pH 6.8) containing 1% BSA, 150 mM sodium chloride, 0.3 mM zinc chloride and 0.1% sodium azide to obtain a labeled antibody solution.

(3) Standard Solution The standard solution was prepared by diluting human C-peptide (manufactured in-house) to an arbitrary concentration with Lumipulse sample diluent (manufactured by Fujirebio Inc .; hereinafter, referred to as sample diluent). .

(4) Measuring Method A fully automatic chemiluminescence immunoassay system Lumipulse Forte (manufactured by Fujirebio Co., Ltd .; hereinafter referred to as Lumipulse Forte) was used as a measuring instrument, and the measurement was carried out by a one-step method and a two-step method. The measurement was performed according to the following procedure. 4-1) Measurement by one-step method As a reaction container for measurement, an immunoreaction cartridge dedicated to a fully automatic chemiluminescence immunoassay system Lumipulse was used. Particle liquid 50μ in the immune reaction tank of the immune reaction cartridge
(1) 120 μl of the labeled antibody solution was injected into the reagent dispensing tank, sealed with an aluminum seal to seal the reagent, and arranged in a dedicated cassette for setting a cartridge. Lumipulse washing solution composed of 10 mM Tris and 0.1% polyoxyethylene (10) octylphenyl ether (manufactured by Fujirebio Co., Ltd .; hereinafter, referred to as washing solution), Lumipulse substrate solution containing chemiluminescent substrate AMPPD as a main component (Fujirebio Co., Ltd.) (Hereinafter, referred to as a substrate solution), a cassette was set at a predetermined mounting position of Lumipulse Forte, and a series of operations and measurements were performed by a computer program attached to the measuring instrument. Details are shown below.

The standard solution or the sample to be measured is set in a dedicated sample rack. The sample rack is set at the sample rack installation location, and a measurement operation start command on the program is input. The measurement proceeds in accordance with instructions such as the measurement method, the number of samples to be measured, the amount of reagent dispensed, and the measurement order specified by the program. First, the sample rack moves to the sample dispensing position in the apparatus at the start of measurement. At the same time, the immune reaction cartridge is taken out of the dedicated cassette and moved to a linear reaction line maintained at 37 ° C. In the middle of the reaction line, there is a magnet for attracting the reaction solution, dispensing the washing solution, attracting the particles by magnetic force, a mixer for stirring the reaction solution, and a port for dispensing the labeled antibody solution and the substrate solution. Each operation is executed according to.

A sampling chip mounted on the tip of a sample dispensing arm aspirates 20 μl of a sample to be measured, and then moves to a reagent dispensing tank in an immunoreaction cartridge to aspirate 50 μl of labeled antibody. The chip moves to the immune reaction cartridge immune reaction tank, and discharges the sample to be measured and the reagent into the particle liquid. Subsequently, suction and discharge are repeated with the same chip to mix the liquid. The reagents are incubated in the bath for 16 minutes at 37 ° C. During that time, 30
It moves sequentially on the immune reaction line every second and proceeds to the washing section. In front of the washing section, magnets are installed on both sides of the line to attract particles. Next, the reagent suction nozzle moves to the reaction tank, sucks the residual liquid in the tank, and completely separates the particles and the reaction liquid.

The particles separated from the reaction liquid are separated from the magnet, while the cleaning liquid is injected from the cleaning liquid injection nozzle. After the particles are sufficiently mixed with the washing liquid by stirring, they are attracted again by magnetic force and separated from the washing liquid. A series of operations of attracting particles by magnetic force, removing residual liquid, injecting a cleaning solution, stirring, and attracting particles is repeated seven times, and the washing operation is completed.
After completion of the washing step, 200 μl of the substrate solution is dispensed, mixed well with the particles, and reacted on the reaction line at 37 ° C. for another 4 minutes and 30 seconds. The cartridge moves to the counting section and measures the amount of light emission for 30 seconds. The measured value of the sample to be measured was calculated based on a standard curve created from the luminescence of the standard antigen.

4-2) Two-Step Method An immunoreaction cartridge dedicated to Lumipulse, a fully automatic chemiluminescence immunoassay system, was used as a reaction vessel for measurement. Particle liquid 50μ in the immune reaction tank of the immune reaction cartridge
(1) 120 μl of the labeled antibody solution was injected into the reagent dispensing tank, sealed with an aluminum seal to seal the reagent, and arranged in a dedicated cassette for setting a cartridge. The washing solution, the substrate solution, and the cassette were set at predetermined mounting positions of Lumipulse Forte, and a series of operations and measurements were performed by a computer program attached to the measuring device. Details are shown below.

The standard solution or the sample to be measured is set in a dedicated sample rack. The sample rack is set at the sample rack installation location, and a measurement operation start command on the program is input. The measurement proceeds in accordance with instructions such as the measurement method, the number of samples to be measured, the amount of reagent dispensed, and the measurement order specified by the program. First, the sample rack moves to the sample dispensing position in the apparatus at the start of measurement. At the same time, the immune reaction cartridge is taken out of the dedicated cassette and moved to a linear reaction line maintained at 37 ° C. In the middle of the reaction line, there is a magnet for attracting the reaction solution, dispensing the washing solution, attracting the particles by magnetic force, a mixer for stirring the reaction solution, and a port for dispensing the labeled antibody solution and the substrate solution. Each operation is executed according to. The sampling tip attached to the tip of the sample dispensing arm is
Dispense μl, discharge 20 μl of it, and mix well with the particle liquid by vortexing. The reagents are incubated in the bath for 8 minutes at 37 ° C. During that time, the immune reaction cartridge moves sequentially on the immune reaction line every 30 seconds and proceeds to the washing section.

After the washing step is completed, the labeled antibody solution dispensing nozzle aspirates 50 μl of the labeled antibody solution from the reagent dispensing tank and injects it into the immune reaction tank. The particles and the labeled antibody are sufficiently mixed by vortexing, and an immunoreaction is performed for 8 minutes. During that time, the immune reaction cartridge moves sequentially on the immune reaction line every 30 seconds and proceeds to the washing section. After the washing step is completed, the substrate solution
Dispense 200 μl, mix well with the particles, and react on the reaction line at 37 ° C. for an additional 4 minutes and 30 seconds. The cartridge moves to the counting section and measures the amount of light emission for 30 seconds. The measured value of the sample to be measured was calculated based on a standard curve created from the luminescence of the standard antigen.

Example 2: C-peptide responsiveness and detection sensitivity The combination of the same antibody is excluded from the antibody-bound particles prepared from the four types of antibodies described in Example 1 (1) and the labeled antibody.
A total of 24 immunoassays were performed using the 1-step method and the 2-step method. 2S / N for the results obtained in each case
Are shown in Tables 1 and 2. As described above, 2S / N is the concentration of an antigen that produces a signal twice as high as the signal (noise signal) at the antigen-free condition, that is, at an antigen concentration of 0 ng / ml. This was used as an index for comparing the responsiveness of the samples.

[Table 1] Table 1.2 S / N sensitivity (1 step method) -: Not performed, ND: Low response or below detection sensitivity

[0037]

[Table 2] Table 2.2 S / N sensitivity (2-step method) -: Not performed, ND: Low response or below detection sensitivity

Example 3 Reactivity to Modified C-Peptide In a system established using an antibody having a high degree of discrimination of the C-peptide molecule terminal, it is expected that the response to proinsulin and split-type proinsulin will decrease. You. Therefore, a polypeptide in which two molecules of Arg are added to the N-terminus and Lys and Arg are added to the C-terminus of the C-peptide by the same combination and procedure as in Example 1 (hereinafter simply referred to as modified C-peptide) Was measured. Modified C-peptide (manufactured by Sigma) was adjusted to 5 ng / ml using a sample diluent and measured. The cross-reactivity was determined as a percentage of the apparent concentration measured as C-peptide divided by the actual concentration of modified C-peptide in the sample, and the results are shown in Tables 3 and 4.

[0039]

[Table 3] Cross-reactivity of modified C-peptide (one-step method) Unit:%-: Not implemented

[0040]

[Table 4] Cross-reactivity of modified C-peptide (two-step method) Unit:%-: Not implemented

As shown in Tables 3 and 4, in the combination using 9101 which is an antibody recognizing the N-terminal region, the cross-reactivity of the modified C-peptide is less than 0.5% to 9.3%. Clearly identified.

Embodiment 4 FIG . Cross-Reactivity with Proinsulin Proinsulin was measured as a sample in the same combination and procedure as in Example 1 to determine cross-reactivity. Each standard curve was created according to the following method. Human C-peptide solutions were prepared in which the human C-peptide concentrations were adjusted to 0, 0.8, 8, and 20 ng / ml, and used as standard solutions. This standard solution
Using 20 μl, immunoassay was carried out according to the method described in Example 1. FIG. 8 shows standard curves of the one-step method and the two-step method using the 9101 antibody as the labeling antibody and the CPT3F11 antibody as the second antibody.

Human proinsulin was diluted with a sample diluent to a concentration of 50, 200 or 400 ng / ml. Using these solutions as samples, immunoassay was performed according to the method described in Example 1. For example, the chemiluminescence counts of samples at concentrations of 50, 200, and 400 ng / ml are determined using the 9101 antibody as the labeling antibody and the CP as the second antibody.
It was measured using the T3F11 antibody. As a result, the results of the one-step method were 4139, 51,337 and 118, 126, respectively, and the results of the two-step method were 4401, 42,493 and 99,434, respectively. This concentration was calculated by reading the count from the standard curve. Proinsulin used as a sample was human C-
The concentration corresponding to human C-peptide was calculated from the molecular weight ratio to the peptide. For example, the molecular weight of proinsulin is 949
4. Since that of human C-peptide is 3019, a 50 ng / ml proinsulin sample corresponded to a human C-peptide concentration of 15.9 ng / ml. Divide the measured value calculated from the standard curve by the concentration equivalent to human C-peptide of proinsulin used for measurement,
The percentage was determined and defined as the cross-reaction rate. Tables 5 and 6 show the results.

[0044]

[Table 5] Cross-reactivity with human proinsulin (1
Step method) Unit:%-: Not implemented

[0045]

[Table 6] Cross-reactivity with human proinsulin (2
Step method) Unit:%-: Not implemented

As shown in Tables 5 and 6, when 9101 is used as either an antibody-bound particle or a labeled antibody, the value fluctuates depending on the combination, but the cross-reaction rate is up to 0.4%.
Down to As shown in Tables 5 and 6, the cross-reaction rate was generally lower in the two-step method than in the one-step method.

Example 5 Using Synthetic Human C-Peptide Fragment
As a means for confirming the antigen recognition site of the anti-human C-peptide antibody reactivity confirmation antibody, an ELISA method was employed. An ELISA plate on which human C-peptide was immobilized was prepared and performed according to the following procedure. (1) Preparation of human C-peptide antigen-sensitized plate Human C-peptide was dissolved in 0.1 M phosphate buffer (pH 7.0).
It was adjusted to a concentration of 10 μg / ml. Then, the solution of each of the above concentrations was added to a microwell module (Nunc) per well.
Each 0.1 ml was dispensed and left at 4 ° C. overnight. Next day in well C-
After discarding the peptide solution, wash well with Lumipulse washing solution, fill the wells with 0.3 ml of 50 mM Tris buffer (pH 7.0) containing 2% BSA and 0.1% sodium azide, and 37 ° C.
And stored at 4 ° C. until use.

(2) Confirmation test for inhibition of immobilized human C-peptide binding 1% BSA, 1 mM MgCl ₂ and 0.
0 to 10 μg / ml using 50 mM female buffer containing 1% NaN ₃
Was prepared and 0.1 ml was dispensed into each well. Subsequently, it was adjusted to 0 to 1.0 μg / ml using the same buffer.
0.1 ml of the labeled antibody solution of 9101 or CPT3F11 was dispensed into each well. Shake the plate gently and mix the solution at 25 ° C.
For 1 hour. After completion of the reaction, the wells were thoroughly washed with a Lumipulse washing solution, 0.1 ml of a 1.0 M diethanolamine buffer solution (pH 10.0) containing 10 mM 4 nitrophenyl phosphate and 1 mM MgCl ₂ was added to each well, and incubated at 25 ° C. for 30 minutes. The absorption at a wavelength of 405 nm was measured. From the obtained absorbance, the binding rate of the labeled antibody to the solid phase was determined (Table 7). The binding rate is a numerical value representing the change in the amount of labeled antibody bound to the solid phase antigen that occurred with the amount of antigen coexisting during the reaction, and the absorbance obtained under the antigen addition condition of 0.1 to 10 μg / ml was 0 μg. Percentage divided by the absorbance obtained under the / ml addition condition. Sufficient color was obtained on the plate on which the C-peptide was immobilized, and the binding of each labeled antibody was confirmed. Furthermore, in the wells to which human C-peptide was added together with the labeled antibody solution, the binding rate decreased in an addition concentration-dependent manner,
It was confirmed that the addition of human C-peptide inhibited binding to immobilized C-peptide.

[0049]

[Table 7] Table 7. Confirmation test of inhibition of immobilized human C-peptide binding

[0050]

[Table 8] Table 7 (continued)

(3) Inhibition test on binding of immobilized human C-peptide by human C-peptide fragment 1 The plate used in (1) was prepared by adding 1% BSA, 1 mM MgCl ₂ and 0.
Seven human C- peptide fragments using 50mM female buffer containing 1% NaN ₃ (the homemade) diluted prepared in 0~1mg / ml concentration, aliquoted 0.1ml min to each well. Human C- used
The amino acid sequence of the peptide fragment is N-1: 2 residues + 1 to 8 (a sequence obtained by adding 2 amino acid residues Arg-Arg to the human C-peptide N-terminal), N-2: 1 to 8 and N-3 : 3-8, N-4: 5-12, N-
5: 2nd to 8th, N-6: 3rd to 10th, N-7: 4th to 10th (Fig. 1), C-1: 24th to 31st + 2 residues (human C-peptide C
Sequence with two amino acid residues Lys-Arg added at the end), C-2: 2
Fragments corresponding to Nos. 4 to 31, C-3: Nos. 24 to 29 (FIG. 2). The positions of these peptide fragments in the human C-peptide are shown in FIG. 1 and FIG. Then, using the same buffer
9101, CPT3F11, PEP-001 or 910 adjusted to 0.1 μg / ml
0.1 ml of the labeled antibody solution of No. 3 was dispensed to each well. Thereafter, the same operation as in (2) was performed, and the absorption at a wavelength of 405 nm was measured. The higher the binding ability of the antibody to each fragment, the lower the absorption at 405 nm of the same peptide fragment. The measurement results are shown in FIGS. When the 9101-labeled antibody solution was used, the absorption was reduced by the addition of N-2, N-3 and N-6 (FIG. 4A). Since these are sequences containing the N-terminal 3rd to 8th amino acids, 9101 is considered to recognize the sequence presented by this fragment.

(4) Binding inhibition test of immobilized human C-peptide by human C-peptide fragment 2 The plate used in (1) was prepared by adding 1% BSA, 1 mM MgCl ₂ and 0.
Seven human C- peptide fragments using 50mM female buffer containing 1% NaN ₃ (the homemade) diluted prepared in 0~1mg / ml concentration, aliquoted 0.1ml min to each well. The amino acid sequence of the human C-peptide fragment is N-4: 5-12, N-8: 7-14, N-
9: 9-16, N-10: 7-13, N-11: 7-12, N-12: 8
Nos. 14 to 14, N-13: Nos. 9 to 14, and N-14: Nos. 10 to 14 (FIG. 1). The positions of these peptide fragments in the human C-peptide are shown in FIG. 1 and FIG. Subsequently, 0.1 ml of a CPT3F11-labeled antibody solution adjusted to 0.1 μg / ml using the same buffer solution was dispensed into each well in an amount of 0.1 ml. Thereafter, the same operation as in (2) was performed, and the absorption at a wavelength of 405 nm was measured. The higher the binding ability of the antibody to each fragment, the more identical the peptide fragment
The absorption at 405 nm decreases. The measurement results are shown in FIG. Of each fragment of N-8 to N-14 consisting of a sequence of 5 or more consecutive residues, the terminal residue has N-11 consisting of either No. 10 or No. 12
In N-14, the absorption did not decrease, and in other cases, it decreased significantly. From this, the recognition site of CPT3F11 is at least 9
The antibody was found to recognize the 5th consecutive 5th residue and a sequence different from 9101.

Embodiment 7 FIG . C-Peptide Immunoassay Kit A C-peptide immunoassay kit containing a container containing each of the reagents 1 to 5 having the following composition was constructed. 1. Antibody-bound particle solution: 50 mM Tris, 150 mM sodium chloride, 2% BSA, 0.1% sodium azide in buffer (p
H7.2) contains 0.03% (w / v) of anti-human C-peptide antibody-bound particles. 2. Labeled antibody solution: 50 mM female, 100 mM sodium chloride, 0.3
Buffer consisting of mM zinc chloride and 0.1% sodium azide (pH
6.8) with alkaline phosphatase-labeled anti-C-peptide antibody
Include at a concentration of 0.2 μg / ml. 3. Standard solution: 50 mM Tris, 150 mM sodium chloride, 2% B
SA, buffer solution consisting of 0.1% sodium azide (pH 7.2)
Including peptides. 4. Substrate solution: 50 mM ciethanolamine, 0.02% AMPPD,
Buffer consisting of 0.1% sodium azide (pH 10.0).

[0054]

According to the method of the present invention, human C-peptide can be detected with extremely high sensitivity and specificity. In particular, according to the present invention, it is possible to overcome the low reliability of the human C-peptide measurement system caused by the cross reaction with human proinsulin. In addition, the present invention can be easily carried out by using the kit of the present invention.

[0055]

[Sequence List] SEQUENCE LISTING <110> Fujirebio Inc. <120> A method for assaying human C-peptide <130> Y1I0547 <140> <141> <150> JP 2000-174691 <151> 2000-06-12 <160 > 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 86 <212> PRT <213> Homo sapiens <400> 1 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 2 <211> 31 <212> PRT <213> Homo sapiens <400> 2 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 1 5 10 15 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln 20 25 30

[Brief description of the drawings]

FIG. 1 is a diagram showing the position and length of a synthetic N-side region human C-peptide fragment in human C-peptide. The number of amino acid residues was numbered 1 at the N-terminal of the natural C-peptide fragment.

Fig. 2 Synthetic C-terminal region human C-peptide fragment of human C-
FIG. 3 is a diagram showing positions and lengths in a peptide. The number of amino acid residues was numbered 1 at the N-terminal of the natural C-peptide fragment.

FIG. 3 shows the results of a binding inhibition test between CPT3F11 and human C-peptide using a human C-peptide fragment.

[Fig. 4] 9101 and human C-peptide using human C-peptide fragment
3 shows the results of a binding inhibition test with a peptide.

FIG. 5 shows the results of a binding inhibition test between PEP-001 and human C-peptide using a human C-peptide fragment.

[Fig. 6] 9103 and human C-peptide using human C-peptide fragment.
3 shows the results of a binding inhibition test with a peptide.

FIG. 7 shows the results of a binding inhibition test between CPT3F11 and C-peptide using a human C-peptide fragment.

FIG. 8 illustrates the standard curves used in the one-step and two-step methods.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Kaoru Nakatsubo 2-62-5 Nihonbashi-Hamacho, Chuo-ku, Tokyo Inside Fujirebio Inc. (72) Isao Nishizono 2-62-Nihonbashi-Hamacho, Chuo-ku, Tokyo No. 5 Inside Fujirebio Corporation

Claims (19)

[Claims] (I) a method comprising: (i) a human C-peptide in a sample;
Forming an immune complex with the anti-human C-peptide antibody or its antigen-binding fragment thereof and the second anti-human C-peptide antibody or its antigen-binding fragment immobilized on a solid phase (Ii) separating the immune complex formed in (i) from unreacted antibodies and / or antigen-binding fragments thereof and a sample; and (iii) quantifying the immune complex. A method for measuring a C-peptide, wherein the first antibody recognizes an epitope present in a region consisting of amino acid residues 1 to 10 at the N-terminus of the human C-peptide molecule, and the second antibody Recognizing an epitope present in a region consisting of amino acid residues 1 to 16 at the N-terminus of said human C-peptide molecule, and wherein said first and second anti-human
C-peptide antibodies recognize different epitopes and
The method for measuring human C-peptide, wherein the method can simultaneously bind to C-peptide. 2. The method according to claim 1, wherein the first anti-human C-peptide antibody or the antigen-binding fragment thereof does not substantially recognize a human C-peptide having an Ng-terminal-added Arg-Arg-sequence. The method of claim 1. 3. The method of claim 3, wherein said solid phase is a magnetic particle. 4. The method according to claim 1, wherein (i) comprises the step of preparing the human C-peptide, the first anti-human C-peptide antibody or an antigen-binding fragment thereof, and the second anti-human C-peptide antibody in a sample. 2. The method according to claim 1, wherein the antigen-binding fragment is reacted with the antigen-binding fragment simultaneously. 5. The method according to claim 1, wherein (i) is performed in the sample first.
Reacting the C-peptide with the second anti-human C-peptide antibody or an antigen-binding fragment thereof immobilized to form an immune complex with the human C-peptide on the solid phase; The human C-peptide not bound to the solid phase is removed by washing, and then the first anti-human C-peptide antibody or an antigen-binding fragment thereof is reacted with the human C-peptide bound to the solid phase. 2. The method according to claim 1, wherein the reaction is a two-stage reaction. 6. The first antibody or the antigen-binding fragment thereof is labeled, and the unreacted labeled antibody or the antigen-binding fragment thereof which has not been reacted in (iii) is removed by washing. The method according to claim 1, wherein the bound label is quantified. 7. The method according to claim 1, wherein said first antibody and said second antibody are used. 8. The cross-reactivity with human proinsulin is 1
The method according to any one of claims 1 to 7, which is 0% or less. 9. The cross-reactivity with human proinsulin is 5
%. 10. The method according to claim 9, wherein the cross-reactivity with human proinsulin is 1% or less. 11. The detection sensitivity of human C-peptide is 0.05 ng / m.
The method according to any one of claims 1 to 7, characterized in that: 12. A (a) first container containing a labeled first anti-human C-peptide antibody or an antigen-binding fragment thereof, and (b) a second anti-human C-immobilized on a solid phase. -A second container containing a peptide antibody or an antigen-binding fragment thereof immobilized on a solid phase, wherein the first antibody is derived from the N-terminal amino acid residues 1 to 10 of the human C-peptide molecule. The second antibody recognizes an epitope present in a region consisting of amino acid residues 1 to 16 at the N-terminus of the human C-peptide molecule;
And a second anti-human C-peptide antibody that recognizes different epitopes and can simultaneously bind to the human C-peptide. 13. The method according to claim 1, wherein the first anti-human C-peptide antibody or the antigen-binding fragment thereof does not substantially recognize a human C-peptide having an Arg-Arg-sequence added to the N-terminus. 13. The kit according to claim 12. 14. The kit according to claim 12, wherein said first and second containers are stored in different compartments of an integrally molded cartridge. 15. The method according to claim 12, further comprising a first container containing the first anti-human C-peptide antibody and a second container containing the anti-second anti-human C-peptide antibody. Or 1
The kit according to the item. 16. Cross-reactivity with human proinsulin is 10% in an immunoassay performed using the kit.
The kit according to any one of claims 12 to 14, which is: 17. The kit according to claim 16, wherein cross-reactivity with human proinsulin is 5% or less in an immunoassay performed using the kit. 18. The kit according to claim 17, wherein the cross-reactivity with human proinsulin is 1% or less in an immunoassay performed using the kit. 19. The human C-peptide has a detection sensitivity of 0.05.
ng / ml or less.
The kit according to any one of claims 1 to 4.