Calibrator/control for simultaneous assay of proteins capable of complexing with one another
View Patent ↗Disclosed herein are compositions and methods comprising two or more proteins in which at least one of the proteins has been altered to reduce their mutual recognition and binding. Such compositions are useful as reference, calibrators or controls in methods and assays for determining the amount of one or more of the proteins that may be present in a sample of interest or in confirming the presence of one or more of the proteins in the sample. More particularly, it relates to compositions and methods comprising altered placental growth factor-1 (PlGF-1) and soluble fms-like tyrosine kinase (sFlt-1) and methods for determining the amount or confirming the presence of sFlt-1 and/or PlGF-1 in a sample of interest.
1. A method of calibrating or running controls in an assay for sFlt-1 and PlGF, the method comprising:
selecting as a calibrator or control a composition comprising known amounts of non-immobilized sFlt-1 and non-immobilized PlGF, wherein one or more amino acids or one or more non-amino acid groups of the non-immobilized sFlt-1 and/or the non-immobilized PlGF have been deleted, modified, or replaced with a different amino acid or non-amino acid group or groups thereby reducing or substantially eliminating formation of a non-covalent association complex between the non-immobilized sFlt-1 and the non-immobilized PlGF;
contacting the composition with a first immobilized receptor specific for sFlt-1 and a second immobilized receptor specific for PlGF, thereby forming a first complex of the non-immobilized sFlt-1 and the first immobilized receptor, and a second complex of the non-immobilized PlGF and the second immobilized receptor; and
detecting amounts of the first complex and the second complex, wherein amounts of the first complex and the second complex are compared to the known amounts of the non-immobilized sFlt-1 and the non-immobilized PlGF to calibrate or control the assay.
2. The method of claim 1 wherein the non-immobilized sFlt-1 is labeled with a first detectable label, and the non-immobilized PlGF is labeled with a second detectable label, and the detecting comprises detecting the first detectable label and the second detectable label.
3. The method of claim 1 wherein the detecting comprises contacting the first complex with a first labeled moiety and the second complex with a second labeled moiety, thereby forming a labeled first complex and a labeled second complex, and wherein the detecting comprises detecting the labeled first complex and the labeled second complex.
4. The method of claim 1 wherein the PlGF is PlGF-1.
5. The method of claim 4 wherein PlGF-1 comprises alanine in place of:
a) proline at position 25 of SEQ ID NO: 1, or
b) glutamine at position 27 of SEQ ID NO: 1, or
c) cysteine at position 60 of SEQ ID NO: 1, or
d) aspartate at position 72 of SEQ ID NO: 1 or
e) glutamate at position 73 of SEQ ID NO: 1, or
f) asparagine at position 84 of SEQ ID NO: 1, or
g) proline at position 98 of SEQ ID NO: 1, or
h) tyrosine at position 100 of SEQ ID NO:1, or
altered PlGF-1 having glycine in place of cysteine at position 70 of SEQ ID NO: 1, or any combination of the alanine replacements in a) to h) and the glycine replacement.
6. The method of claim 4 wherein PlGF-1 comprises alanine in place of aspartate at position 72 of SEQ ID NO: 1 and alanine in place of glutamate at position 73 of SEQ ID NO: 1.
7. The method of claim 4 wherein PlGF-1 comprises glycine in place of cysteine at position 70 of SEQ ID NO: 1, alanine in place of aspartate at position 72 of SEQ ID NO: 1 and alanine in place of glutamate at position 73 of SEQ ID NO: 1.