Title:  Immunoassay for measuring human C-peptide and kit therefor

United States Patent:  6,534,281

Issued:  March 18, 2003

Inventors:  Kitajima; Sachiko (Tokyo, JP); Kurano; Yoshihiro (Tokyo, JP); Nakatsubo; Kaoru (Tokyo, JP); Nishizono; Isao (Tokyo, JP)

Assignee:  Fujirebio Inc. (Tokyo, JP)

Appl. No.:  878380

Filed:  June 12, 2001

An immunoassay for selectively measuring human C-peptide as well as a kit therefor is disclosed. In the method, human C-peptide contained in a sample, a first anti-human C-peptide antibody, and a second anti-human C-peptide antibody which is immobilized on a solid support are reacted to form an immune complex among these three components. The formed immune complex is separated from the non-reacted antibodies and sample; and then the separated immune complex is quantified. The first antibody recognizes an epitope existing in the region from 1st to 16th amino acid residue from the N-terminal of the human C-peptide, and the second antibody recognizes an epitope existing in the region from 1st to 16th amino acid residue from the N-terminal of human C-peptide; with the proviso that the first and second antibodies do not recognize the same epitope so that they can simultaneously bind to said human C-peptide.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a sandwich immunoassay for measuring human C-peptide wherein first and second anti-human C-peptide antibodies which recognize different epitopes are used.

The first anti-human C-peptide antibody used in the immunoassay according to the present invention is one which recognizes an epitope existing in the region from 1st to 10th amino acid from the N-terminal of human C-peptide (The 10th amino acid residue, for example, from the N-terminal is hereinafter indicated as "10a.a." for convenience. The positions of amino acids will hereinafter be indicated in the same manner. Further, for example, the region from 1st to 10th amino acid residue from the N-terminal of human C-peptide is herein after indicated as "1-10a.a." for convenience. The regions in C-peptide will hereinafter be indicated in the same manner). The first antibody preferably recognizes an epitope existing in 1-8a.a., more preferably an epitope existing in 1-8a.a. including at least a part of 1-3a.a. The term "recognize an epitope" means that the antibody undergoes antigen-antibody reaction with the epitope thereby binding to the epitope. The first antibody preferably does not substantially recognize human C-peptide having Arg-Arg-attached to the N-terminal thereof

(residues 31-63 of SEQ ID NO:1) (hereinafter referred to as "Arg-Arg'C-peptide"). The term "does not substantially recognize" means that the cross-reactivity of the first antibody to Arg-Arg-C-peptide is not more than 10% when the concentration of Arg-Arg-C-peptide is 5 ng/ml. The cross-reactivity is preferably not more than 5%, more prefereably more than 1%. The cross-reactivity of the first antibody to Arg-Arg-C-peptide is determined by first preparing a calibration curve by the method of the present invention in a conventional manner using different known concentrations of human C-peptide solutions as samples, and measuring the apparent concentration of Arg-Arg-C-peptide solution whose actual concentration is known (5 nl/ml). The cross-reactivity of the immunoassay to Arg-Arg-C-peptide is the percentage of the apparent concentration measured based on the calibration curve to the actual concentration of Arg-Arg-C-peptide, the the proviso that the actual concentration is compensated so as to compensate the difference in molecular weights between C-peptide and Arg-Arg-C-peptide by multiplying the actual concentration by the ratio of molecular weights (see Example 4 below). It should be noted that the cross-reactivity of the antibody to Arg-Arg-C-peptide can be measured by using Arg-Arg-C-peptide to which Lys-Arg is attached at the C-terminal (i.e., "modified C-peptide" used in Example 3 below, residues 31-65 of SEQ ID NO:1) because the first antibody recognizes an epitope existing in 1-10a.a. region and so the precise structure of the C-terminal of the peptide does not inflkuence on the binding of the first antibody to the peptide. The first antibody is preferably a monoclonal antibody in view of reproducibility and specificity. Antigen-binding fragments such as Fab fragment and F(ab')₂ fragment of the first antibody may also be used in place of the first antibody. The first antibody of the antigen-binding fragment thereof is not immobilized, but used in free state.

The first antibody may be obtained by a method essentially known in the art. For example, anti-human C-peptide antibodies, preferably monoclonal antibodies, are produced by a conventional method well-known in the art, and the obtained antibodies are then examined for their corresponding epitopes. The corresponding epitope of an antibody may be determined by binding inhibition assay using a series of fragments of human C-peptide as exemplified in Example 5 described below. If antigen-antibody reaction between the antibody to be tested and human C-peptide is concentration-dependently inhibited by addition of a known amount of a polypeptide fragment, the antibody to be tested recognizes an epitope existing in the added polypeptide fragment. Thus, by using a polypeptide fragment consisting of at least five consecutive amino acids in the region of 1-10a.a. of human C-peptide as a competitor in the binding inhibition assay, the antibody which can be used as the first antibody can be selected. From the anti-human C-peptide antibodies, those which recognize epitopes existing in the region from 1-10a.a. of human C-peptide are selected in this manner and may be used as the first antibody in the method of the present invention. It should be noted that it is not necessary to determine the epitope exactly, but the first antibody may be selected by determining whether the epitope recognized by the antibody exist in the region of 1-10a.a. The first antibody which does not recognize human C-peptide having Arg-Arg- attached to the N-terminal thereof may also be selected in the similar manner. That is, such an antibody may be selected by excluding those which recognize human C-peptide having Arg-Arg- attached to the N-terminal thereof in the competition immunoassay described above, using a polypeptide fragment including the N-terminal of human C-peptide, to which Arg-Arg- is attached, is used as the competitor. Alternatively, the first antibody may also be obtained by using as an immunogen a peptide fragment having at least five consecutive amino acid in the region of 1-10a.a. attached to a protein carrier such as KLH or BSA, and by selecting the antibodies which recognize the peptide fragment used as the immunogen.

Anti-human C-peptide monoclonal antibodies are commercially available. Among these commercially available monoclonal antibodies, those which recognize epitopes in 1-10a.a. of human C-peptide may conveniently be employed as the first antibody in the method of the present invention. Anti-human C-peptide monoclonal antibody 9101 commercially available from Medix Biochemica is an example of the commercially available monoclonal antibodies which can be used as the first antibody in the method of the present invention.

The antigen-binding fragments of the first antibody may be obtained by the methods well-known in the art. That is, the Fab fragment can be obtained by digesting the first antibody with papain and the F(ab')₂ fragment can be obtained by digesting the first antibody with pepsin.

The second antibody used in the sandwich immunoassay according to the present invention recognizes an epitope existing in 1-16a.a. of the human C-peptide. As shown in Examples below, by using an antibody which recognizes an epitope existing in 1-16a.a. of the human C-peptide, cross-reactivity to human proinsulin is much more decreased when compared to the cases where an antibody which recognizes an epitope existing in the C-terminal half of human C-peptide is used as the second antibody. This phenomenon was originally discovered by the present inventors. In view of reproducibility and specificity, the second antibody is also preferably a monoclonal antibody.

To carry out sandwich immunoassay, it is necessary that the second antibody recognize an epitope which is different from the epitope recognized by the first antibody, and that both the first and second antibodies can simultaneously bind to human C-peptide. If two antibodies can simultaneously bind to human C-peptide, it is regarded in the present invention that the two antibodies recognize different epitopes even if the region of the epitopes partly overlap each other.

Antigen-binding fragments such as Fab fragment and F(ab')₂ fragment of the first antibody may also be used in place of the second antibody. Such fragments may be obtained by the methods well-known in the art as mentioned above.

The second antibody may be obtained as follows: First, anti-human antibodies which recognize epitopes existing in 1-16a.a. of human C-peptide molecule are obtained. These antibodies may be obtained in the similar manner as described above for obtaining the first antibody, using a polypeptide fragment consisting of at least 5 consecutive amino acids in 1-16a.a. of human C-peptide as a competitor in the binding inhibition assay, or as an immunogen bound to a protein carrier. It should be noted that it is not necessary to determine the epitope exactly, but the second antibody may be selected by determining whether the epitope recognized by the antibody exist in the region of 1-16a.a. Then those antibodies which can bind to human C-peptide simultaneously with the first antibody are selected. This can be carried out by checking whether human C-peptide can be measured by a usual sandwich immunoassay using the first antibody and the antibody which is a candidate as the second antibody.

Among the commercially available monoclonal antibodies, those which recognize epitopes in 1-16a.a. of human C-peptide and which can bind to human C-peptide simultaneously with the first antibody may conveniently be employed as the second antibody in the method of the present invention. When the first antibody is anti-human C-peptide monoclonal antibody 9101 commercially available from Medix Biochemica, anti-human C-peptide monoclonal antibody 9103 commercially available from Medix Biochemica and anti-human C-peptide monoclonal antibody CPT-3-F11 commercially available from Dako may be employed as the second antibody.

Except that the above-described characteristic first and second antibodies are used, and that the second antibody is immobilized, the sandwich immunoassay according to the present invention may be carried out in the conventional way. In the following description (except for the Examples below), unless otherwise specified or unless otherwise apparent from the context, the term "antibody" means antibody or an antigen-binding fragment thereof.

The second antibody is immobilized on a solid support. As experimentally shown in the Examples below, the cross-reactivity of the immunoassay to human proinsulin is smaller in cases where the second antibody is immobilized and the first antibody is free than in the cases where the first antibody is immobilized and the second antibody is free. The solid support per se is well-known in the art and any solid supports conventionally used in immunoassays may be employed. Thus, the solid support may preferably be made of a polymer such as polystyrene, polyethylene, Sepharose (Pharmacia), cellulose or the like. Latex particles coated with ferrite, which have magnetism may also preferably be used as the solid support. The shape of the solid support is not important and preferably be one with which the immobilization of the antibody on the surface thereof may easily be carried out, and the immune complex may easily be separated from the reaction mixture and the non-reacted antibody after the immunological reactions. Thus, the solid support may preferably be in the form of magnetic particles. Alternatively, the inner wall of a well in a plastic microplate may also preferably be used as the solid support. In view of ease of handling, storage stability, ease of separation and the like, use of magnetic particles such as latex particles coated with ferrite is especially preferred.

To quantify the immune complex, it is preferred to label the first antibody which is not immobilized on the solid support. Labeled antibodies are well-known in the art, and any of the labels conventionally employed in immunoassays may be employed. Thus, as the label, radioactive isotopes, enzymes and fluorescent substances may preferably be employed. Examples of the radioactive isotopes include ¹²⁵ I, ¹³¹ I and the like. Examples of the enzymes include peroxidase, .beta.-galactosidase, alkaline phosphatase and the like. As the substrates for the enzyme reactions, o-nitrophenyl-.beta.-D-galactopyranoside, p-nitrophenyl phosphate, AMPPD (disodium 3-[4-methoxyspiro(1,2-dioxetane-3,2'-tricyclo[3,3,1,1³,7 ]decane)-4-yl]phenyl phosphate) and the like may be employed. As the fluorescent substance, fluorescein isothiocyanate (FITC) and the like may be employed. These labels may be quantified by the conventional methods well-known in the art, thereby the formed immune complex is quantified. In a preferred mode of the present invention, alkaline phosphatase-bound antibody is used as the enzyme-labeled antibody, and AMPPD which is a chemiluminescent substance is used as the substrate for the enzyme reaction.

As is well-known, labeling of the first antibody is not mandate to attain the quantification of the immune complex. The immune complex may also be quantified by reacting a labeled third antibody with the immune complex on the solid support, which third antibody recognizes the first antibody which is not immobilized.

Using the above-described first and second antibodies, the sandwich immunoassay may be carried out in a conventional way. Thus, the sandwich immunoassay may be carried out either by one-step method or by two-step method. In the one-step method, the first and second antibodies, and human C-peptide contained in the sample are simultaneously reacted. After washing, the immune complex bound to the solid support is quantified. This method has an advantage that the immunological reactions are carried out in one step, so that the operations are simple and the time needed for the immunoassay is short. In the two-step method, the human C-peptide contained in the sample is first reacted with the immobilized second antibody. After washing, the first antibody which is not immobilized is then reacted with the human C-peptide captured on the solid support through the second antibody. After washing, the immune complex bound to the solid support is quantified. This two-step method has an advantage that cross-reactivity to human proinsulin is lower than the one-step method.

The immunological reactions between one of the antibodies and human C-peptide in the sample may be carried out at a temperature and for a period of time under which immune complex between the antibody and human C-peptide is sufficiently formed. For example, the immunological reactions may be carried out at a temperature between room temperature to 37^o C. for not less than about 5 minutes, preferably for 7 to 30 minutes, or at 4^o C. overnight, as in the conventional sandwich immunoassays.

The buffer used for the immunological reactions or for dilution may be any buffers which are conventionally used in immunoassays. Examples of such buffers include PBS, 50 mM Tris buffer (pH7.2) containing or not containing bovine serum albumin (BSA) and 50 mM MES buffer (pH6.8). The buffer may contain about 0.1% of sodium azide in order to prevent growth of bacteria.

The sandwich immunoassay according to the present invention gives high detection sensitivity and low cross-reactivity to human proinsulin and split type proinsulin (i.e., proinsulin lacking 31-32a.a. or 64-65a.a. in proinsulin). The cross-reactivity of the sandwich immunoassay of the present invention to human proinsulin may preferably be not more than 10%, more preferably not more than 5%, still more preferably not more than 1% when the actual concentration of human proinsulin is 50 ng/ml. The cross-reactivity of the immunoassay to human proinsulin is determined by first preparing a calibration curve by the method of the present invention in a conventional manner using different known concentrations of human C-peptide solutions as samples, and measuring the apparent concentration of human proinsulin solution whose actual concentration is known (50 ng/ml). The cross-reactivity of the immunoassay to human proinsulin is the percentage of the apparent concentration measured by the method of the present invention based on the calibration curve to the actual concentration of the human proinsulin, with the proviso that the actual concentration is compensated so as to compensate the difference in molecular weights between human C-peptide and human proinsulin by multiplying the actual concentration by the ratio of molecular weights (see Example 4 below). The detection sensitivity of the sandwich immunoassay according to the present invention is preferably not more than 0.05 ng/ml, more preferably not more than 0.02 ng/ml. The detection sensitivity is herein defined as 2S/N, which means the concentration of human C-peptide which gives the signal twice the signal (noise signal) yielded when the concentration of human C-peptide is 0 ng/ml.

The sample which is subjected to the sandwich immunoassay according to the present invention is not restricted, and may be, for example, body fluids such as blood, serum, plasma and urine, as well as feces, in which C-peptide may be secreted.

The present invention also provides a kit for easily carrying out the sandwich immunoassay according to the present invention. The kit according to the present invention includes a vessel containing the first antibody and a vessel containing the second antibody. The kit may further comprise one or more appropriate buffers for immunological reactions or for dilution, or concentrates thereof, the substrate for enzyme reaction or solution thereof, human C-peptide as a standard sample or solution thereof, and a vessel for carrying out the immunoassay or for dilution. Preferred examples of the buffer for suspending the anti-human C-peptide-bound particles include 50 mM Tris buffer (pH7.2) containing BSA, 150 mM sodium chloride and 0.1% sodium azide. Preferred examples of the buffer for dissolving the labeled anti-human C-peptide include the same buffer just mentioned above which further contains 0.3 mM zinc chloride.

In the kit according to the present invention, second antibody is immobilized on the solid support, and the first antibody is labeled. The first antibody may be in the form of a solution in an appropriate buffer or may be in the frozen-dried state. The second antibody may be in the frozen-dried state together with the solid support, or may be in the form of a suspension when the solid support is particles. The reagents contained in the kit according to the present invention may be provided separately in the respective vessels, each vessel containing a reagent in an amount for carrying out one immunoassay for one sample. Alternatively, each reagent may be contained in a vessel in an amount for carrying out immunoassays for a plurality of samples. In this case, each reagent is dividedly used in each immunoassay. In cases where each vessel contains a reagent in an amount for carrying out one immunoassay for one sample, the vessels containing the different reagents may be in the form of compartments integrally formed in a cartridge. In cases where the first and/or second antibodies are contained in the kit in frozen-dried state, a buffer such as those mentioned above suited for dissolving the antibodies may be included in the kit. The vessels containing the antibodies and other vessels included in the kit may be made of any materials as long as they do not interact with the antibodies and they do not adversely affect the enzyme reactions, chemiluminescent reactions and the like. If necessary, the surfaces of the vessels may be treated so as to prevent such interactions. Such surface treatments are well-known in the art. A manual carrying the instructions for using the kit is usually attached to the kit.

Claim 1 of 19 Claims

We claim:

1. A method for measuring human C-peptide comprising the steps of:

(i) reacting human C-peptide contained in a sample, a first anti-human C-peptide antibody or an antigen-binding fragment thereof, and a second anti-human C-peptide antibody or an antigen-binding fragment thereof immobilized on a solid support to form an immune complex among three components;

(ii) separating the formed immune complex from non-reacted antibodies and/or antigen-binding fragments thereof, and sample, and

(iii) quantifying the separated immune complex;

said first antibody recognizing an epitope existing in the region from residues 1-10 of SEQ ID NO:2 from the N-terminal of said human C-peptide; said second antibody recognizing an epitope existing in the region from residues 1-16 of SEQ ID NO:2 from the N-terminal of said human C-peptide; and said first and second antibodies recognizing different epitopes so that they can simultaneously bind to said human C-peptide.

 

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