C-peptide specific assay procedure
The present invention relates to a specific C-peptide assay method which can eliminate all interference due to proinsulin and its intermediates, in particular des-31,32-proinsulin and/or des-64,65-proinsulin. It also relates to antibodies for carrying out this assay.
1. A method for assaying C-peptide in a sample suspected of containing C-peptide, intact proinsulin, des-31,32 proinsulin, and/or des-64,65-proinsulin comprising:
a) contacting the sample with at least a first anti-peptide antibody which specifically binds an epitope present entirely in the C-peptide and a second antibody which is selected from:
an anti-proinsulin antibody which is specific to intact proinsulin and des-31,32-proinsulin and recognizes an epitope located in an intact A-chain-C-peptide (AC) junction of proinsulin and des-31,32 proinsulin, said anti-proinsulin AC junction antibody epitope being either adjacent to or overlapping with the epitope of said at least one anti-C-peptide antibody, or
an anti-proinsulin antibody which is specific to intact proinsulin and des-64,65-proinsulin and recognizes an epitope located in an intact B-chain-C-peptide (BC) junction of proinsulin and des-64,65-proinsulin, said anti-proinsulin BC junction antibody epitope being either adjacent to or overlapping with the epitope of said at least one anti-C-peptide antibody, or
a mixture of said anti-proinsulin AC junction antibody and said anti-proinsulin BC junction antibody under conditions such that said at least one anti-C-peptide antibody binds to C-peptide in the sample, binding of the anti-proinsulin AC junction antibody to any proinsulin and/or des-31,32-proinsulin interferes with binding of said at least one anti-C-peptide antibody to intact proinsulin and des-31,32-proinsulin in the sample, and/or binding of the anti-proinsulin BC junction antibody to any proinsulin and/or des-64,65-proinsulin interferes with binding of said at least one anti-C-peptide antibody to intact proinsulin and des-64,65-proinsulin in the sample; and
b) detecting amount of C-peptide bound to said at least one anti-C-peptide antibody in said contacted sample as an assay of the amount of C-peptide in the sample.
2. The method according to claim 1 , wherein said epitope located in the AC junction of proinsulin and des-31,32-proinsulin includes a peptide sequence: Lys Arg.
3. The method according to claim 1 , wherein said epitope located in the AC junction of proinsulin and des-31,32-proinsulin includes a peptide sequence:
Leu Gln Lys Arg Gly Ile Val Glu (SEQ ID NO: 3).
4. The method according to claim 1 , wherein said epitope located in the AC junction of proinsulin and des-31,32-proinsulin includes a peptide sequence:
Leu Gln Lys Arg Gly Ile Val Glu Gln (SEQ ID NO: 4).
5. The method according to claim 1 , wherein said epitope located in the BC junction of proinsulin and des-64,65-proinsulin includes a peptide sequence: Arg Arg.
6. The method according to claim 1 , wherein the sequence of said epitope located in the BC junction of proinsulin and des-64,65-proinsulin is selected from the group consisting of:
Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val (SEQ ID NO: 5)
Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val (SEQ ID NO: 2); and
Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu (SEQ ID NO: 6).
7. The method according to claim 1 comprising:
a) contacting the sample with the anti-C-peptide antibody and an anti-proinsulin antibody which is specific to intact proinsulin and des-31,32-proinsulin and recognizes an epitope located in the AC junction of proinsulin and des-31,32-proinsulin, said proinsulin AC junction epitope either being adjacent or overlapping the epitope of said anti-C-peptide antibody, thus interfering with binding of the anti-C-peptide antibody to intact proinsulin and des-31,32-proinsulin and
b) detecting the amount of C-peptide bound to said anti-C-peptide antibody.
8. The method according to claim 1 comprising:
a) contacting the sample with the anti-C-peptide antibody and an anti-proinsulin antibody which is specific to intact proinsulin and des-64,65-proinsulin and recognizes an epitope located in the BC junction of proinsulin and des-64,65-proinsulin, said proinsulin BC junction epitope either being adjacent or overlapping the epitope of said anti-C-peptide antibody, thus interfering with binding of the anti-C-peptide antibody to intact proinsulin and des-64,65-proinsulin and
b) detecting the amount of C-peptide bound to said anti-C-peptide antibody.
9. The method according to claim 1 in which at least one of said antibodies is a monoclonal antibody.
10. The method according to claim 1 in which the sample is obtained from serum, plasma, or urine.
11. The method according to claim 1 , said method being a sandwich assay.
12. The method according to claim 1 , said method being a competitive radioimmunological assay.