Imaging Assays
Axworthy teaches methods that relate to delivery of diagnostic agents (abstract). In one embodiment, a chemical conjugate or fusion protein with streptavidin (first member of a binding pair) is administered to a recipient for whom diagnosis is desired. The conjugate localizes to the target site. As a consequence of the rapid target site uptake, a clearing agent is then administered. The clearing agent serves to rapidly diminish the serum level of the conjugate. Next, biotin labeled with an imaging radionuclide such as Tc-99m (radioactively labeled compound conjugated to a second member of a binding pair) is administered. Biotin directs the localization of the administered moiety to the previously localized conjugate. Consequently, imagining of the target sites proceeds with minimal exposure of non-target sites to radioactivity which offers the advantages of speed, facilitating imaging of difficult target sites (¶77).
Axworthy further teaches that galactose is the protoypical clearing agent (¶117) and that galactose-based clearing agents also include galactosylated, biotinylated proteins to remove circulating streptavidin-targeting moiety conjugates (¶126).
Internalization Assays
Antibodies:
Gerritsen (US12443918) teaches a method for selecting an antibody on the basis of its ability to be internalized into a cell by providing antibodies which bind to the same antigen on a cell and a detection toll such as a Fab fragment with a detectable label such as CypHer5 that does not mediate internalization itself, incubating the antibodies, detection tool and cells having the antigen and detecting fluorescence.
Marks (US2002/0182643) teaches methods of identifying ligands such as antibodies which are internalized into a cell by providing antibodies to be tested for internalization which bind the same antigen and contacting eukaryotic cells expressing the antigen with the ligands. Marks also teaches providing a reporter that provides a detectable signal which is noncovalently coupled to the ligand. In one embodiment, the ligand is an antibody with an epitope tage that is recognized by anotehr binding partner such as a secondary antibody which can provide a detectable label.
Maruyama (WO2006055371) teaches providng antiboides which bind the same antigen to be tested for internalization, eukaryotic cells expressing the antigen, and secondary antibodies with a detectable lable such as Cyper5E
Determining the level of a drug in a patient’s sample:
Size Eclusion Chromatography (SEC):
Singh (US 8,574,855) discloses methods for detecting the presence or level of a drug (e.g., anti-TNFalpha) in a sample by contacting labeled TNFalpha with the sample haivng the drug (e.g., anti-TNFalpha) to form a labeled complex (immuno-complex or conjugate) and then subjecting the complex to size exclusion chromatography to separate the labeled complex from the free labeled TNF-alpha. The method can be modifed for detecting the presence or level of an autoantibody to an anti-TNFa drug by contacting a labeled anti-TNFalpha drug with the same suspected of having the autantibody to the anti-TNFalpha to form a labled complex with the autoantibody and subjecting the labeled complex to SEC.
–as part of competition based assays:
Singh (US 15/603,137, published as US 2017/0328923; see also (Singh, US Patent Application 16/536,777, published as US 2020/0088749); see also US 10422807)) also discloses a method for determining the presence or level of a biologic such as a drug (antibody) in a sample that includes contacting the sample with an unlabeled soluble antigen such as a soluble fragment of a cell surface molecule (e.g., alpha4 integrin or beta 7 integrin or alpha4beta7 polypeptide), then contacting this sample with a labeled form of the biologic/drug to form a labeled complex between the antigen and the labeled biologic, followed by subjecting the unlabeled and labeled complexes to size exclusion chromatography to separate the unlabeled and labeled complexes from free labeled biologic/drug to detect an amount of the free labeled biologic/drug and then comparing the amount of the free labeled biologic/drug detected to a standard curvie of known amounts of the biologic/drug thereby determining the presence or level of the biologic/drug in the sample. The principle behind such an indirect assay is that the amount of patient drug determines how much unlabeled antigen remains free to bind to a labeled version of the drug. By tracking changes in the area of the free (unbound) labeled drug, one can determine how much pateint drug is present. In a particular example, a sample such as serum containing a therapeutic drug such as vedolizumab (anti-alpha4beta7 integrin monoclonal antibody) is incubated with a fixed amount of antigen to the drug such as soluble alpha4beta7. Then, a fixed amount of labeled drug (vedoizumab couple to Alexa Fuor 488) is added. The amount of therapeutic drug in the sample determines how much antigen remains free and available to bind the labelled drug. This, in turn determines how much labeled drug is free. Since the peak area of the free labeled drug is proportional to the amount of therapeutic drug in the sample, one can quantify the amount of therapetuic drug by interpolation against a standard curve containing known amounts of the drug. In other words, the presence or level of the unlabeled biogic (vedolizumab) relies on detecting the amount of free (unbound) labeled biologic (vedolizumab) in the sample after unlabeled antigen and labeled biologic (vedolizumab) are added sequentially to teh sample. In a separte embodiment patient serum is combined with rIL12p40 to allow the therapeutic UTK to bind with rIL12p40. Subseqeuntly, fluorescently labed UTK competed with unlabled UTK in pateint sera for binding to tis target, rIL12p40, followed by separation on HPLC size exclusion chromatography. For the anti-UTK assay, standard curves were created by incubated normal human serum containing known amoutns of rabbit anti-UTK antibodies with fluorescently labeled UTK; bound and free UTK were then separated by SEC-HPLC.
Detecting Presence/level of autoantibodies:
Sing (WO 2011/056590) disloses a method for detecting the pressence or level of an anti-TNFalpha drug in a sample which includes contacting labeled TNFalpha with a sample suspected of having an anti-TNFalpha drug to form a labeled complex (i.e., immuno-complex or conjugate) with the anti-TNFalpha drug, subjecting the labeled complex to size exclusion chromatography to separate the labeled complex (e.g., from free labeled TNFalpha) and detecting the labeled complex thereby detecting the presence or level of the anti-TNFalpha drug. Such detection is important becasue adminsitration of TNFalpha inhibitors can induce an immune resposne to the drug and lead to the production of autantibodies.