Techniques for predicting, detecting and reducing aspecific protein interference in assays involving immunoglobulin single variable domains
This invention provides, and in certain specific but non-limiting aspects relates to: assays that can be used to predict whether a given ISV will be subject to protein interference as described herein and/or give rise to an (aspecific) signal in such an assay (such as for example in an ADA immunoassay). Such predictive assays could for example be used to test whether a given ISV could have a tendency to give rise to such protein interference and/or such a signal; to select ISV's that are not or less prone to such protein interference or to giving such a signal; as an assay or test that can be used to test whether certain modification(s) to an ISV will (fully or partially) reduce its tendency to give rise to such interference or such a signal; and/or as an assay or test that can be used to guide modification or improvement of an ISV so as to reduce its tendency to give rise to such protein interference or signal; methods for modifying and/or improving ISV's to as to remove or reduce their tendency to give rise to such protein interference or such a signal; modifications that can be introduced into an ISV that remove or reduce its tendency to give rise to such protein interference or such a signal; ISV's that have been specifically selected (for example, using the assay(s) described herein) to have no or low(er)/reduced tendency to give rise to such protein interference or such a signal; modified and/or improved ISV's that have no or a low(er)/reduced tendency to give rise to such protein interference or such a signal.
1 . An immunoassay method comprising:
(i) contacting a first immunoglobulin single variable domain (ISV), or a polypeptide comprising the first ISV at the C-terminal end of the polypeptide, with an antibody that binds to the C-terminal end of a second ISV, wherein the C-terminal end of the second ISV terminates in the sequence VTVSS (SEQ ID NO: 33), wherein the antibody is present in a human sample; and
(ii) determining whether the first ISV, or the polypeptide comprising the first ISV, is bound by the antibody.
2 . The immunoassay method of claim 1 , wherein: the first ISV is a VHH or a VH; and the second ISV is a VHH or a VH.
3 . The immunoassay method of claim 1 , wherein the first ISV is at the C-terminal end of a polypeptide that comprises the first ISV.
4 . The immunoassay method of claim 1 , wherein the second ISV comprises a proline at Kabat position 14 .
5 . The immunoassay method of claim 1 , wherein the antibody is a polyclonal antibody.
6 . An immunoassay method comprising:
(i) contacting an immunoglobulin single variable domain (ISV), or a polypeptide comprising the ISV, with a monoclonal IgG antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and a light chain comprising the amino acid sequence of SEQ ID NO: 36; and
(ii) determining whether the ISV, or the polypeptide comprising the ISV, is bound by the antibody.
7 . The immunoassay method of claim 6 , wherein the ISV is a VHH or a VH.
8 . The immunoassay method of claim 6 , wherein the ISV is at the C-terminal end of a polypeptide that comprises the ISV.
9 . An immunoassay method comprising:
(i) identifying an antibody that binds to the C-terminal end of a second immunoglobulin single variable domain (ISV), wherein the C-terminal end of the second ISV terminates in the sequence VTVSS (SEQ ID NO: 33), and wherein the antibody has been isolated and/or purified from a human sample;
ii) contacting a first ISV, or a polypeptide comprising the first ISV at the C-terminal end of the polypeptide, with the antibody; and
(iii) determining whether the first ISV, or the polypeptide comprising the first ISV, is bound by the antibody.
10 . The immunoassay method of claim 9 , wherein the second ISV comprises a proline at Kabat position 14 .
11 . The immunoassay method of claim 9 , wherein the antibody is a monoclonal antibody.
12 . The immunoassay method of claim 9 , wherein: the first ISV is a VHH or a VH;
and the second ISV is a VHH or a VH.
13 . An immunoassay method for predicting protein interference or risk thereof comprising:
(i) contacting a first immunoglobulin single variable domain (ISV) having a C-terminal region, or a polypeptide comprising the first ISV at its C-terminal end, with an antibody present in a human sample that binds the C-terminal region of a second ISV, or a polypeptide comprising the second ISV at its C-terminal end; and
(ii) predicting protein interference or the risk thereof by determining whether the antibody has increased or decreased binding to the first ISV, or the polypeptide comprising the first ISV, relative to the binding of the antibody to the second ISV, or the polypeptide comprising the second ISV, wherein the C-terminal region of the first ISV and the C-terminal region of the second ISV differ.
14 . The immunoassay method of claim 9 , wherein the first ISV is at the C-terminal end of a polypeptide that comprises the first ISV.
15 . The immunoassay method of claim 13 , wherein: the first ISV is a VHH or a VH;
and the second ISV is a VHH or a VH.
16 . The immunoassay method of claim 13 , wherein the first ISV is at the C-terminal end of a polypeptide that comprises the first ISV.
17 . An immunoassay method for predicting protein interference or risk thereof comprising:
(i) identifying an antibody that binds to the C-terminal region of an immunoglobulin single variable domain (ISV), and wherein the antibody has been isolated and/or purified from a human sample;
(ii) contacting a first immunoglobulin single variable domain (ISV) having a C-terminal region, or a polypeptide comprising the first ISV at its C-terminal end, with the antibody; and
(iii) predicting protein interference or the risk thereof by determining whether the antibody has increased or decreased binding to the first ISV, or the polypeptide comprising the first ISV, relative to the binding of the antibody to a second ISV, or a polypeptide comprising the second ISV, wherein the C-terminal region of the first ISV and the C-terminal region of the second ISV differ.
18 . The immunoassay method of claim 17 , wherein: the first ISV is a VHH or a VH;
and the second ISV is a VHH or a VH.
19 . The immunoassay method of claim 17 , wherein the first ISV is at the C-terminal end of a polypeptide that comprises the first ISV.