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. Author manuscript; available in PMC: 2014 Oct 27.
Published in final edited form as: Anal Biochem. 2012 Aug 21;430(2):141–150. doi: 10.1016/j.ab.2012.08.005

Figure 6. Specific detection of mAbs A-c and B-b using an indirect capture ELISA.

Figure 6

Top panels (A and B): Cartoon of the indirect capture ELISA.

Incubation: A fixed amount of mAb specific domain and increasing amounts of mAbs are mixed in individual wells and incubated together until binding reaches equilibrium. Amount of mAb bound is a function of the mAb solution KD. Capture: Each reaction mixture is then transferred to a new well on the capture plate which has been coated with the domain specific mAb for capturing of the domain. Domains bound by a mAb binding the same epitope are not captured and are washed away. Detection: Captured domains are detected with anti-SV5 antibody binding the C-terminal SV5 epitope tag followed by anti-mouse Ab-HRP (A) A-LC-HN mut domain binds mAb A-c with high affinity and mAb E-c with low affinity in solution at a non-overlapping epitope. At the mAb concentrations studied, the affinity of mAb E-c for the A-LC-HN domain is too low for binding to occur during the incubation stage. On the capture plate coated with mAb A-c, only free A-LC-HN mut not bound by mAb E-c is captured, allowing quantitation of the mAb E-c concentration. (B). The B-LC-HN-b domain binds both mAb B-b and mAb E-c with overlapping epitopes, and with a 500-fold higher affinity for mAb B-b than to mAb E-c. At the mAb concentrations studied, the affinity of mAb E-c for the B-LC-HN-b domain is too low for binding to occur during the incubation stage. On the capture plate coated with mAb B-b, the free B-LC-HN-b is captured, while B-LC-HN-b bound by mAb B-b in solution is washed away, allowing quantitation of the mAb E-c concentration. HRP = horse radish peroxidase.

Bottom panel (C). Binding specificity of direct vs. indirect capture ELISAs. C1. Direct ELISA with a plate coated with domain A-LC-HN mut shows binding of mAb E-c at higher mAb concentrations than required for mAb A-c. C2 Indirect capture ELISA showed specificity of domain A-LC-HN-mut for only mAb A-c. mAb A-c was coated on the plate as capture antibody, A-LC-HN-mut was incubated with either mAb A-c, or a mixture of eight mAbs (mAbA-a + mAbA-b + XOMA 3B + XOMA 3E) or a mixture of nine mAbs (XOMA 3AB + XOMA 3B + XOMA 3E). C3. Direct ELISA with a plate coated with domain B-LC-HN-b cannot distinguish between mAb B-b and mAb E-c. C4. Indirect capture ELISAs showing specificity of domains B-LC-HN-b for only mAb B-b. mAb B-b was coated on the plate as capture antibody, B-LC-HN-b was incubated with mAb B-b, a mixture of eight mAbs (XOMA 3 AB and mAb B-a, -b and XOMA 3E) or a mixture of nine mAbs (XOMA 3AB + XOMA 3B + XOMA 3E).