USE OF FUSION PROTEINS TO IMPROVE THE AVAILABILITY OF ANTIGENIC PEPTIDE EPITOPES IN IMMUNOASSAYS
Methods, techniques, systems and compositions to reduce or prevent the aggregation and/or epitope masking of control/calibration peptides. The methods described herein may be particularly helpful for calibration and quantifying target proteins from immunoassays such as solid-phase enzyme immunoassays (EIAs).
1 . A method of quantifying a target protein from a patient sample using a synthetic calibration protein having increased solubility, the method comprising:
performing an immunoassay on the patient sample using an antibody to the target protein;
generating a plurality of calibration solutions comprising different concentrations of a fusion protein of a synthetic target protein and Glutathione S-transferase (GST) in a detergent solution; and
using the plurality of calibration solutions to calibrate the immunoassay and provide a concentration of the target protein, wherein the different concentrations of the calibration solutions correspond to molar concentrations of the fusion protein without value-assigning the concentrations to determine the concentration of the target protein.
2 . The method of claim 1 , further comprising using the plurality of calibration solutions to make a calibration curve.
3 . The method of claim 1 , wherein the immunoassay is a solid-phase enzyme immunoassay (EIA).
4 . The method of claim 1 , wherein the patient sample is a blood sample.
5 . The method of claim 1 , wherein the target protein is NT-proBNP.
6 . The method of claim 1 , wherein the target protein is β Amyloid peptide (AB42).
7 . The method of claim 1 , wherein the target protein is MR-proANP.
8 . The method of claim 1 , wherein the fusion protein comprises a His6 Tag (SEQ ID NO: 3).
9 . The method of claim 1 , wherein the fusion protein comprises a TEV cleavage site between the target protein and the GST.
10 . The method of claim 1 , wherein the fusion protein comprises the GST fused to the C-terminus of the target protein.
11 . The method of claim 1 , wherein the fusion protein comprises the GST fused to the N-terminus of the target protein.
12 . The method of claim 1 , wherein the detergent solution comprises one or more of: an anionic surfactant, a nonionic surfactant, a zwitterionic detergent, and a chaotropic agent.
13 . The method of claim 1 , wherein the detergent solution comprises one or more of: Sodium dodecyl sulfate (SDS), Triton X-100, Tergitol-type NP-40 (NP-40), octylphenoxypolyethoxyethanol (IGEPAL CA-630), 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and Guanidine Hydrochloride.
14 . A method of quantifying a target protein from a patient sample using a synthetic calibration NT-proBNP having increased solubility, the method comprising:
performing an immunoassay on the patient sample using an antibody to NT-proBNP;
generating a plurality of calibration solutions comprising different concentrations of a fusion protein of a synthetic NT-proBNP and Glutathione S-transferase (GST) in a detergent solution; and
using the plurality of calibration solutions to calibrate the immunoassay and provide a concentration of the NT-proBNP from the immunoassay, wherein the different concentrations of the calibration solutions correspond to molar concentrations of the fusion protein without value-assigning the concentrations to determine the concentration of the target protein.