Immunoassays and methods of detecting and measuring intact fibroblast growth factor 23, and c-terminal and n-terminal fragments thereof

An immunoassay for detecting intact FGF-23 protein molecules and N-terminal fragments thereof, and further identifying the relative amounts of said intact FGF-23 and said N-terminal fragments, said immunoassay comprising:

• a N-terminal capture antibody, said N-terminal capture antibody having an affinity for and specifically binding to an antigenic region falling within amino acids 25-179 of FGF-23 (SEQ ID No.:
• 3);
• a N-terminal tracer antibody having a label conjugated therewith, said N-terminal tracer antibody having an affinity for and specifically binding to an antigenic sequence falling within amino acids 25-179 of FGF-23 (SEQ ID NO.:
• 3), said specificity of said N-terminal tracer antibody being dissimilar to that of said N-terminal capture antibody such that in use, said N-terminal capture antibody and said N-terminal tracer antibody are capable of sequentially and simultaneously binding to the respective antigenic regions to which each respective antibody specifically binds;
• a C-terminal tracer antibody having a label conjugated therewith, said C-terminal tracer antibody having an affinity for and specifically binding to an antigenic region formed within amino acids 180-251 of FGF-23 (SEQ ID NO.:
• 4), said C-terminal tracer antibody being able to bind to said antigenic region sequentially or simultaneously in combination with said N-terminal tracer antibody and said N-terminal capture antibody.

An immunoassay method for detecting intact FGF-23 molecules and C-terminal fragments thereof and further determining the relative amounts of said intact FGF-23 molecules and C-terminal fragments thereof in a liquid sample suspected of containing both intact FGF-23 molecules and C-terminal fragments thereof, said immunoassay method comprising the steps of:

• a) contacting said liquid sample with C-terminal capture antibodies, said C-terminal capture antibodies having a specificity for an antigenic region falling within amino acids 180-251 of FGF-23 (SEQ ID NO.:
• 4) such that substantially all intact FGF-23 molecules and substantially all C-terminal fragments having amino acids 180-251 (SEQ ID NO.:
• 4) are bound thereto;
• b) contacting said intact FGF-23 molecules and C-terminal fragments bound to said C-terminal capture antibodies in step a) with C-terminal tracer antibodies having a label conjugated thereto, each said C-terminal tracer antibody being specific for an antigenic sequence falling within amino acids 180-251 of FGF-23 (SEQ ID NO.:
• 4), said specificity of said C-terminal tracer antibody being dissimilar to said specificity of said C-terminal capture antibodies such that in use, a C-terminal capture antibody and a C-terminal tracer antibody simultaneously bind to an intact FGF-23 molecule and/or a C-terminal fragment;
• c) measuring said signal generated by said label conjugated to said C-terminal tracer antibodies bound in step b), said label conjugated to said C-terminal tracer antibodies providing a signal indicative of the degree of binding of said C-terminal tracer antibodies to both said intact FGF-23 molecules and said C-terminal fragments;
• d) contacting said bound intact FGF-23 molecules and C-terminal fragments thereof in step b) with an N-terminal tracer antibodies having a label conjugated thereto, said N-terminal tracer antibodies having an affinity for and specifically binding to an antigenic region formed within amino acids 25-179 of FGF-23 (SEQ ID NO.:
• 3), each N-terminal tracer antibody being able to bind to said antigenic region sequentially or simultaneously in combination with said C-terminal tracer antibody and said C-terminal capture antibody, said label conjugated to said N-terminal tracer antibody producing a signal corresponding to only the intact FGF-23 molecules bound by said C-terminal capture antibody;
• and
• e) measuring the signal produced by said labels of said N-terminal tracer antibodies of step d), said signal being indicative of only the intact FGF-23 molecules present in said liquid sample.

An immunoassay method for detecting intact FGF-23 molecules and N-terminal fragments thereof and further determining the relative amounts of said intact FGF-23 molecules and N-terminal fragments thereof in a liquid sample suspected of containing both intact FGF-23 molecules and N-terminal fragments thereof, said immunoassay method comprising the steps of:

• a) contacting said liquid sample with N-terminal capture antibodies, said N-terminal capture antibodies having a specificity for an antigenic region falling within amino acids 25-179 of FGF-23 (SEQ ID No.:
• 3) such that substantially all intact FGF-23 molecules and substantially all N-terminal fragments having amino acids 25-179 (SEQ ID NO.:
• 3) are bound thereto;
• b) contacting said intact FGF-23 molecules and N-terminal fragments bound to said N-terminal capture antibodies in step a) with N-terminal tracer antibodies having a label conjugated thereto, each said N-terminal tracer antibody being specific for an antigenic sequence falling within amino acids 25-179 of FGF-23(SEQ ID NO.:
• 3), said specificity of said N-terminal tracer antibody being dissimilar to said specificity of said N-terminal capture antibodies such that in use, an N-terminal capture antibody and an N-terminal tracer antibody are capable of simultaneously binding to an intact FGF-23 molecule or an N-terminal fragment;
• c) measuring said signal generated by said label conjugated to said N-terminal antibody bound in step b), said tracer conjugated to said N-terminal tracer antibody providing a signal indicative of the degree of binding of said N-terminal tracer antibody to both said intact FGF-23 molecules and said N-terminal fragments;
• d) contacting said bound intact FGF-23 molecules and N-terminal fragments thereof in step b) with C-terminal tracer antibodies having a label conjugated thereto, said C-terminal tracer antibodies having an affinity for and specifically binding to an antigenic region formed within amino acids 180-251 of FGF-23 (SEQ ID NO.:
• 4), each C-terminal tracer antibody being able to bind to said antigenic region sequentially or simultaneously in combination with said N-terminal tracer antibody and said N-terminal capture antibody, said label conjugated to said C-terminal tracer antibody producing a signal corresponding to only the intact FGF-23 molecules bound by said N-terminal capture antibody;
• and
• e) measuring the signal produced by said label of said C-terminal tracer antibody of step d), said signal being indicative of only the intact FGF-23 molecules present in said liquid sample.

An immunoassay method for detecting intact FGF-23 molecules and the N-terminal and C-terminal fragments thereof and further determining the relative amounts of said intact FGF-23 molecules and the N-terminal and C-terminal fragments thereof in a liquid sample suspected of containing both intact FGF-23 molecules and N-terminal and C-terminal fragments thereof, said immunoassay method comprising the steps of:

• a) contacting said liquid sample with a combination of N-terminal and C-terminal capture antibodies, said N-terminal and C-terminal capture antibodies being present in fixed proportion to one another, said C-terminal capture antibody having a specificity for an antigenic region falling within amino acids 180-251 of FGF-23 (SEQ ID No.:
• 4) such that intact FGF-23 molecules and C-terminal fragments having amino acids 180-251 (SEQ ID NO.:
• 4) can bind thereto, said N-terminal capture antibody having a specificity for an antigenic region falling within amino acids 25-179 (SEQ ID No.:
• 3) of FGF-23 such that intact FGF-23 molecules and N-terminal fragments having amino acids 25-179 (SEQ ID NO.:
• 3) can bind thereto;
• b) contacting said intact FGF-23 molecules and C-terminal fragments bound to said C-terminal capture antibodies in step a) with C-terminal tracer antibodies having a label conjugated thereto, said C-terminal tracer antibodies being specific for an antigenic sequence falling within amino acids 180-251 of FGF-23 (SEQ ID NO.:
• 4), said specificity of said C-terminal tracer antibodies being dissimilar to said specificity of said C-terminal capture antibodies such that in use, each said C-terminal capture antibody and said C-terminal tracer antibody can simultaneously bind to said intact FGF-23 molecules and said C-terminal fragments;
• c) contacting said intact FGF-23 molecules and N-terminal fragments bound to said N-terminal capture antibodies in step a) with a N-terminal tracer antibody having a label conjugated thereto, said N-terminal tracer antibodies being specific for an antigenic sequence falling within amino acids 25-179 of FGF-23 (SEQ ID NO.:
• 3), said specificity of said N-terminal tracer antibodies being dissimilar to said specificity of said N-terminal capture antibodies such that in use, each said N-terminal capture antibody and said N-terminal tracer antibody can simultaneously bind to said intact FGF-23 molecules and said N-terminal fragments;
• d) measuring said signal generated by said label conjugated to said C-terminal tracer antibody bound in step b), and comparing said signal to the proportional amounts of said N-terminal and C-terminal capture antibodies;
• and
• e) measuring said signal generated by said label conjugated to said N-terminal tracer antibody bound in step c), and comparing said signal to the proportional amounts of said N-terminal and C-terminal capture antibodies.