Interference-suppressed pharmacokinetic immunoassay
Herein is reported a method for the determination of a bispecific antibody in a serum-containing sample comprising the steps of first incubating a solid phase to which a capture antibody specifically binding to the bispecific antibody has been immobilized with the sample to form an immobilized capture antibody-bispecific antibody-complex, thereafter incubating the solid phase with a replacement antibody that competes with the capture antibody for binding to the bispecific antibody and thereby forming a solution comprising a replacement antibody-bispecific antibody-complex, and determining in the solution the replacement antibody-bispecific antibody-complex.
1 . A method for the determination of a bispecific antibody in a serum-containing sample comprising the following steps:
a) incubating a solid phase with the serum-containing sample,
wherein the serum-containing sample comprises the bispecific antibody and a monospecific antibody,
wherein the bispecific antibody comprises a first binding site that binds to a first antigen and a second binding site that binds to a second antigen, and wherein the monospecific antibody comprises the second binding site,
wherein the solid phase comprises a capture antibody immobilized thereon,
wherein the capture antibody specifically binds the first binding site to form an immobilized capture antibody-bispecific antibody-complex, and wherein the monospecific antibody nonspecifically binds the solid phase,
b) optionally washing the solid phase whereby the capture antibody-bispecific antibody-complex remains bound to the solid phase,
c) incubating the solid phase with a solution comprising a replacement antibody that competes with the capture antibody for binding to the first binding site of the bispecific antibody, thereby forming a replacement antibody-bispecific antibody-complex in the solution, wherein the monospecific antibody remains bound to the solid phase, and
d) determining the replacement antibody-bispecific antibody-complex in the solution of step c) and thereby determining the bispecific antibody in a serum-containing sample.
2 . The method according to claim 1 , wherein the replacement antibody is labelled and the determining of the replacement antibody-bispecific antibody-complex is by detecting said label.
3 . The method according to claim 1 , wherein the replacement antibody is the capture antibody.
4 . The method according to claim 1 , wherein the solid phase in step c) is incubated with the replacement antibody at a concentration of 6.67 nM or more.
5 . The method according to claim 1 , wherein the bispecific antibody is an anti-CD3/CD20 bispecific antibody.
6 . The method according to claim 5 , wherein the bispecific anti-CD3/CD20 antibody is RG6026.
7 . The method according to claim 1 , wherein the monospecific antibody is an anti-CD20 antibody.
8 . The method according to claim 7 , wherein the anti-CD20 antibody is obinutuzumab.
9 . The method according to claim 1 , wherein the determining in step d) comprises (i) transferring the solution comprising the soluble replacement antibody-bispecific antibody-complex to a second solid phase comprising a second capture antibody, wherein the second capture antibody specifically binds to the second antigen binding site of the bispecific antibody, to form an immobilized second capture antibody-bispecific antibody-complex, and (ii) detecting the immobilized second capture antibody-bispecific antibody-complex.