IMMUNOASSAY FOR MEASURING C-PEPTIDE

Abstract

The present invention relates to a method for detecting and in particular for measuring C-peptide. Specifically, the present invention relates to a displacement assay for quantitative determination of C-peptide in a sample.

Claims

1. A method for detecting C-peptide comprising: i) providing an antibody-ligand-complex, wherein the antibody is an antibody directed against C-peptide and wherein the ligand has a lower affinity to the anti-C-peptide antibody than wild type C-peptide; ii) contacting the antibody-ligand-complex with a sample to be analyzed; and iii) displacing the ligand from the antibody-ligand-complex by C-peptide analyte, thereby determining the presence or concentration of C-peptide analyte within the sample.

2. The method according to claim 1, wherein the ligand is a C-peptide variant, in particular a C-peptide variant wherein one of the amino acids no. 12 (L), no. 21 (L), no. 22 (Q) and/or no. 23 (P) is substituted and comprising optionally up to 25 further substitutions and/or deletions.

3. The method according to claim 1, wherein the ligand is a C-peptide variant, wherein the amino acid no. 21 (L) is substituted by T, I, F, C, Q, V or S, in particular by I, and/or wherein the amino acid no. 22 (Q) is substituted by S, A, N or M, in particular by A or S, and/or wherein the amino acid no. 23 (P) is substituted by A, V or S, in particular by A, and/or wherein the amino acid no. 12 (L) is substituted by C or P, in particular by C.

4. The method according to claim 1, wherein the ligand is a C-peptide variant, wherein 1 to 20 amino acids are deleted at the C-terminus and/or at the N-terminus.

5. The method according to claim 1, wherein the ligand is a variant C-peptide selected from TABLE-US-00004 Mut2: AA10-25; P(23)to A (SEQ ID NO: 2) N-VELGGGPGAGSLQALA-C Mut3: AA10-25; L(21)to I (SEQ ID NO: 3) N-VELGGGPGAGSIQPLA-C Mut5: AA10-25; Q(22)to S (SEQ ID NO: 4) N-VELGGGPGAGSLSPLA-C Mut6: AA10-25; Q(22)to A (SEQ ID NO: 5) N-VELGGGPGAGSLAPLA-C Mut18: AA8-17; L(12)to C (SEQ ID NO: 6) N-GQVECGGGPG-C

6. The method according to claim 1, wherein the ligand comprises a label and/or a linker.

7. The method according to claim 1, wherein the ligand comprises a label selected from biotin, optical labels, electrochemical labels, enzyme labels and/or mass label.

8. The method according to claim 1 being a displacement assay comprising the steps a) providing a solid phase, b) immobilizing an antibody directed against C-peptide on the solid phase, c) contacting the antibody with a ligand to provide an antibody-ligand-complex, d) contacting the antibody-ligand-complex with a sample to be analyzed, e) displacement of the ligand from the antibody-ligand-complex by C-peptide analyte, and f) determining the amount of displaced ligand.

9. The method according to claim 1, being a displacement assay comprising the steps a) providing a solid phase, b) immobilizing a ligand on the solid phase, c) contacting the ligand with an anti-C-peptide antibody to provide an antibody-ligand-complex, d) contacting the antibody-ligand-complex with a sample to be analyzed, e) displacement of the ligand from the antibody-ligand-complex by C-peptide analyte and f) determining the amount of displaced ligand.

10. A test kit for performing the method of claim 1 comprising a) optionally a solid phase, b) an anti-C-peptide antibody, and c) a ligand having a lower affinity to the anti-C-peptide antibody then wild type C-peptide.

11. The test kit according to claim 10, wherein the anti-C-peptide antibody and/or the ligand comprise a label.

12. The test kit according to claim 10 comprising an antibody-ligand-complex immobilized to a solid phase.

13. A C-peptide variant, wherein one of the amino acids no. 12 (L), no. 21 (L), no. 22 (Q) or no. 23 (P) of SEQ ID NO: 1 is substituted.

14. The C-peptide variant of claim 13, wherein the amino acid no. 21 (L) is substituted by T, I, F, C, Q V or S, in particular by I, and/or wherein the amino acid no. 22 (Q) is substituted by S, A, N or M, in particular by A or S, and/or wherein the amino acid no. 23 (P) is substituted by A, V or S, in particular by A, and/or wherein the amino acid no. 12 (L) is substituted by C or P, in particular by C.

15. The C-peptide variant of claim 13, wherein the amino acid no. 21 (L) is substituted by T, F, C, V or S, and/or wherein the amino acid no. 22 (Q) is substituted by S, A, N or M, in particular by A or S, and/or wherein the amino acid no. 23 (P) is substituted by A, V or S, in particular by A, and/or wherein the amino acid no. 12 (L) is substituted by C or P, in particular by C.

Description

[0088] The invention is further illustrated by the enclosed Figures and the following Examples.

[0089] FIG. 1 shows a schematic scheme of a displacement assay, wherein an antibody is immobilized to a solid phase to which immobilized antibody a labeled ligand is coupled. This labeled ligand is displaced by the analyte resulting in release of labeled ligand, which can be measured by a detector.

[0090] FIG. 2 shows a schematic scheme of a displacement assay, wherein a ligand is immobilized to a solid phase to which ligand an antibody is coupled. By adding a sample comprising analyte, the ligand is displaced from the antibody-ligand-complex. The ligand remains immobilized on the solid phase and an analyte-antibody-complex is released from the surface.

[0091] FIG. 3 shows QCM-data (Quartz Crystal Microbalance data) of a stepwise performance of a displacement immunoassay. A negative frequency shift shows increase of mass attached to the surface (in this case an Au-surface). Ligand used: mut6-C-peptide.

[0092] FIG. 4 shows a further QCM-measurement of an displacement immunoassay. Ligand used: mut3-C-peptide.

[0093] FIG. 5 shows a further QCM-measurement of an displacement immunoassay. Ligand used: mut2-C-peptide.

[0094] FIG. 6 shows a further QCM-measurement of an displacement immunoassay. Ligand used: mut5-C-peptide.

[0095] FIG. 7 shows a continuous C-peptide measurement.

[0096] FIG. 8 shows a QCM-measurement, wherein a PEP001 antibody was immobilized on an Au-surface.

[0097] FIG. 9 shows a displacement immunoassay, wherein a ligand is coupled to the solid surface using a linker.

[0098] FIG. 10 shows a further QCM-measurement of an displacement immunoassay. Antibody used: CPT-3-F11, ligand used: mut18-C-peptide.

EXAMPLES

Example 1: Displacement Immunoassay for Detecting C-Peptide Using Mut6-C-Peptide

[0099] An Au-substrate is covered with a self-assembled monolayer (SAM). This self-assembled monolayer protects the surface from unspecific adsorption of peptides or proteins. Part of the SAM molecules is coupled with biotin, which is exposed on the surface. To this surface the biotin binding protein streptavidin is applied and bound by the biotin moieties. Since streptavidin has a plurality of binding sides for biotin, a ligand labeled with biotin can be bound to the streptavidin. Finally, anti-C-peptide antibody PEP001 is added, which binds to the ligand forming an immobilized antibody-ligand-complex, which is schematically depicted in FIG. 2. The stepwise build-up of an immobilized antibody-ligand-complex on the Au-surface can be monitored using a Quartz Crystal Microbalance (QCM). A negative frequency shift shows increase of mass on the Au-surface. Referring to FIG. 3, attachment of streptavidin and Mut6-C-peptide results in a frequency shift of about −20 Hz. The binding of antibody PEP001 to the ligand Mut6-C-peptide results in a frequency shift of about >−50 Hz.

[0100] Addition of a sample containing C-peptide results in displacement of the ligand by wild type C-peptide and release of a C-peptide-antibody-complex as schematically shown in FIG. 2. In FIG. 3 release of the antibody-C-peptide-complex results in an observable positive frequency shift.

[0101] A quantitative determination of the amount of C-peptide within a sample can be achieved by calibrating the assay using solutions having known C-peptide concentrations. The amount of frequency shift observed for a sample indicates the quantitative amount of C-peptide being present within that sample.

Example 2: Displacement Immunoassay for C-Peptide Using Mut3-C-Peptide, Mut2-C-Peptide or Mut5-C-Peptide

[0102] An immunoassay as described in Example 1 was provided except that Mut3-C-peptide, Mut2-C-peptide or Mut5-C-peptide were used as ligand.

[0103] As can be seen in FIG. 4, the stepwise build-up of a streptavidin/Mut3-C-peptide/PEP001 assembly results in a mass build-up on the surface which can be seen as negative frequency shift in the QCM-data. Addition of a sample containing WT-C-peptide (shortly after 2 hours and 15 minutes) results in displacement of the ligand Mut3-C-peptide by WT-C-peptide and in a considerable frequency increase. This frequency increase can directly be correlated to the amount of WT-C-peptide in the sample. FIG. 5 and FIG. 6 show similar results for Mut2-C-peptide and Mut5-C-peptide respectively.

Example 3: Continuous Immunoassay for Detection of C-Peptide

[0104] As described in Example 1, a SAM/biotin/streptavidin/biotin-ligand/antibody-complex is immobilized on an Au-surface. In this Example the ligand was C-peptide mutant B10-25_Mut3, the antibody was PEP001. The stepwise build-up monitored using QCM is shown in FIG. 7. Adding a sample containing 500 ng/ml WT-C-peptide results in release of antibody-WT-C-peptide-complex, which is observed in the QCM-data as positive frequency shift (addition of WT-C-peptide at about 4 hours).

[0105] It is then possible to regenerate the assay by again adding antibody PEP001 forming ligand/antibody-complex immobilized to the surface. Thus, it is possible to use the assay for a second and further measurement. Exemplary in FIG. 7, three consecutive measurements are shown.

Example 4: Displacement Immunoassay for C-Peptide Using an Immobilized Antibody

[0106] Antibody PEP001 was directly immobilized to an Au-surface as shown in FIG. 1. Unspecific protein binding sides were saturated with BSA (Bovine Serum Albumin). Then a WT-C-peptide covalently coupled with BSA was added to reveal build-up of an immobilized antibody-C-peptide-complex. The results are shown in FIG. 8. Due to the weight of BSA (70 kDa) the binding of C-peptide to the immobilized can be made visible.

Example 5: Displacement Immunoassay Using a Linker to Immobilize a Ligand to a Surface

[0107] Immobilization of a ligand to a surface can also be performed using a linker (instead of a self-assembling monolayer). A suitable linker is for example a cysteine linker. In this configuration immobilization of an antibody via an antibody-ligand-complex to the surface can be detected by measuring impedance. A ligand having the sequence N-CGSGSGSGSVELGGGPGAGSLAPLA-C(SEQ ID NO:7) was directly immobilized on an Au-surface. Additionally, 6-ferrocenyl-1-hexanethiol was bound to the Au surface for enhanced impedance measurement. Unspecific protein binding to the surface was avoided by incubation with a polyethylene glycol (ML-PEG) and bovine serum albumin (BSA). The QCM-data for this assay are shown in FIG. 9. Addition of PEP001 antibody results in a considerable negative shift of the frequency and addition of WT-C-peptide results in release of the bound antibody.

Example 6: Displacement Immunoassay for C-Peptide Using Mut18-C-Peptide

[0108] An immunoassay as described in Example 1 was provided except that Mut18-C-peptide was used as ligand.

[0109] As can be seen in FIG. 10, the stepwise build-up of a streptavidin/Mut18-C-peptide assembly results in a mass build-up on the surface which can be seen as negative frequency shift in the QCM-data. Addition of the anti-C-peptide antibody CPT-3-F11 resulted in a frequency shift of almost −30 Hz. Addition of a sample containing WT-C-peptide (shortly after 2 hours and 15 minutes) results in displacement of the ligand Mut18-C-peptide by WT-C-peptide and in a considerable frequency increase. This frequency increase can directly be correlated to the amount of WT-C-peptide in the sample.