Methods for detecting antibodies by surface plasmon resonance
Inventors
Kelemen, Mary Katherine Morr • Holmquist, Brett
Assignees
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Publication Number
US-12105083-B2
Publication Date
2024-10-01
Expiration Date
Abstract
The present disclosure relates to a method for detection of an antibody or antibody fragment in a biological sample from a subject. In some embodiments, the methods comprise immobilizing a first binding agent on a surface plasmon resonance (SPR) biosensor; adding a ligand that binds to the first binding agent under conditions such that a complex of the ligand and the first binding agent is formed; adding an aliquot of the biological sample under conditions such that the antibody and/or antibody fragment binds to the ligand that is complexed to the first binding agent; and detecting the presence of the antibody and/or antibody fragment as a change in signal obtained from SPR. In some embodiments, the antibody is an antibody therapeutic such as certolizumab pegol. Also disclosed are systems and kits for detecting an antibody in a biological sample from a subject using SPR.
Core Innovation
The invention provides a method for detection of an antibody or antibody fragment in a biological sample from a subject using a surface plasmon resonance (SPR) biosensor. The method includes immobilizing a first binding agent on an SPR biosensor surface, adding a ligand that binds to the first binding agent under conditions that form a complex of the ligand and the first binding agent, and then adding an aliquot of the biological sample under conditions such that the antibody or antibody fragment binds to the ligand that is complexed to the first binding agent.
After the antibody or antibody fragment binds to the ligand complex, the method includes adding a secondary antibody or a second binding agent that recognizes a moiety on the antibody or antibody fragment. The antibody or antibody fragment is detected as a change in signal obtained from SPR.
In specified embodiments, the therapeutic is certolizumab pegol, and the ligand corresponds to TNFα, with recognition of a PEG moiety by the secondary antibody or second binding agent. Example descriptions further refer to detection of antibody therapeutics in human serum or plasma, including sensor construction, immobilization layers for ligand attachment, and quantitative behavior such as lower limit of quantitation and upper limit of quantitation for certolizumab.
Claims Coverage
The independent claim is a complete SPR-based detection workflow with five core inventive features. These features are the immobilized first binding agent, ligand–first binding agent complex formation, binding of a therapeutic antibody or antibody fragment from a human-treated subject, secondary recognition of a moiety on the captured antibody, and detection as an SPR signal change.
Immobilizing a first binding agent on an SPR biosensor surface
Immobilizing a first binding agent on a surface plasmon resonance (SPR) biosensor.
Forming a ligand–first binding agent complex
Adding a ligand that binds to the first binding agent under conditions such that a complex of the ligand and the first binding agent is formed.
Binding therapeutic antibody or antibody fragment to the complexed ligand
Adding an aliquot of the biological sample under conditions such that the antibody or antibody fragment binds to the ligand that is complexed to the first binding agent, wherein the antibody or antibody fragment is an antibody therapeutic, and wherein the biological sample is from a human being treated with the antibody therapeutic.
Secondary binding agent recognizing a moiety on the antibody or antibody fragment
Adding a secondary antibody or a second binding agent that recognizes a moiety on the antibody or antibody fragment.
Detecting antibody or antibody fragment as an SPR signal change
Detecting the antibody or antibody fragment as a change in signal obtained from SPR.
PEG moiety recognition
The method of claim 1 is wherein the moiety on the antibody or antibody fragment is polyethylene glycol (PEG).
TNFα ligand selection
The method of claim 1 is further defined such that the ligand is TNFα.
Quantitation limits for certolizumab
The method is wherein a lower limit of quantitation (LLOQ) and an upper limit of quantitation (ULOQ) for certolizumab are 1.0 μg/mL and 90 μg/mL, respectively.
Ligand immobilization density threshold
The method further includes immobilizing a ligand at a density of at least 1,000 Response Units (RU).
Sample dilution constraint
The method of claim 1 includes diluting the biological sample by at least 2-fold.
Claim coverage focuses on SPR detection of therapeutic antibodies in human-treated subjects by immobilizing a first binding agent, forming a ligand–first binding agent complex, capturing the therapeutic antibody or antibody fragment, adding a secondary antibody or second binding agent that recognizes a moiety on the captured antibody, and detecting an SPR signal change. Dependent refinements further specify PEG moiety recognition, selection of TNFα as the ligand, and quantitative and assay constraints including ligand immobilization density, certolizumab LLOQ/ULOQ ranges, and sample dilution.
Stated Advantages
Enables detection of an antibody or antibody fragment as a change in signal obtained from SPR.
Provides quantified certolizumab measurement with specified lower limit of quantitation (LLOQ) and upper limit of quantitation (ULOQ).
Demonstrates accuracy and imprecision in example measurements.
Provides selectivity and specificity in the presence of interferents including adalimumab, golimumab, infliximab, ENBREL, and rituximab.
Documented Applications
Detection of antibody or antibody fragments, including antibody therapeutic certolizumab pegol, in human biological samples obtained from a human being treated with the antibody therapeutic.
Use in human serum or plasma matrices for SPR-based detection with example evaluation against interferents including adalimumab, golimumab, infliximab, ENBREL, and rituximab.
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