Determination of ADC Concentration by Ligand-Binding Assays

  • Hsuan-Ping Chang
  • Dhaval K. ShahEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2078)


Total antibody, conjugated antibody or antibody-conjugated drug, and free drug are key analytes required to establish exposure–response relationships for ADCs. Therefore, bioanalytical strategies for ADCs include ligand-binding assays (LBA) and LC–MS/MS methods. Here we describe detailed methodology to develop a solid-phase-based enzyme-linked immunosorbent assay (ELISA), which is the most widely used LBA to quantify large-molecule components of ADC in biological matrices such as plasma, serum, tumor, or tissue homogenates. The approach presented here is designed to quantify total antibody concentrations in ADC containing samples, and can be easily adapted to quantify conjugated antibody concentrations.

Key words

Antibody–drug conjugate (ADC) Biodistribution Ligand-binding assay (LBA) Pharmacokinetics (PK) ELISA Sandwich ELISA 


  1. 1.
    Gorovits B et al (2013) Bioanalysis of antibody-drug conjugates: American Association of Pharmaceutical Scientists antibody-drug conjugate working group position paper. Bioanalysis 5(9):997–1006CrossRefGoogle Scholar
  2. 2.
    Khan MN, Findlay JWA (2009) Ligand-binding assays development, validation, and implementation in the drug development arena. Hoboken, NJ WileyGoogle Scholar
  3. 3.
    Khot A, Sharma S, Shah DK (2015) Integration of bioanalytical measurements using PK-PD modeling and simulation: implications for antibody-drug conjugate development. Bioanalysis 7(13):1633–1648CrossRefGoogle Scholar
  4. 4.
    Kaur S et al (2013) Bioanalytical assay strategies for the development of antibody-drug conjugate biotherapeutics. Bioanalysis 5(2):201–226CrossRefGoogle Scholar
  5. 5.
    Stephan JP, Kozak KR, Wong WL (2011) Challenges in developing bioanalytical assays for characterization of antibody-drug conjugates. Bioanalysis 3(6):677–700CrossRefGoogle Scholar
  6. 6.
    Dowell JA et al (2001) Pharmacokinetics of gemtuzumab ozogamicin, an antibody-targeted chemotherapy agent for the treatment of patients with acute myeloid leukemia in first relapse. J Clin Pharmacol 41(11):1206–1214CrossRefGoogle Scholar
  7. 7.
    Lewis Phillips GD et al (2008) Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res 68(22):9280–9290CrossRefGoogle Scholar
  8. 8.
    Pollack VA et al (2007) Treatment parameters modulating regression of human melanoma xenografts by an antibody-drug conjugate (CR011-vcMMAE) targeting GPNMB. Cancer Chemother Pharmacol 60(3):423–435CrossRefGoogle Scholar
  9. 9.
    Hwu P et al (2009) A phase I/II study of CR011-vcMMAE, an antibody-drug conjugate (ADC) targeting glycoprotein NMB (GPNMB) in patients (pts) with advanced melanoma. J Clin Oncol 27(15S):9032–9032Google Scholar
  10. 10.
    Sznol M et al (2009) Pharmacokinetics (PK) of CR011-vcMMAE, an antibody-drug conjugate (ADC), in a phase (Ph) I study of patients (pts) with advanced melanoma. J Clin Oncol 27(15S):9063–9063Google Scholar
  11. 11.
    Junutula JR et al (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol 26(8):925–932CrossRefGoogle Scholar
  12. 12.
    Singh AP, Shah DK (2017) Measurement and mathematical characterization of cell-level pharmacokinetics of antibody-drug conjugates: a case study with Trastuzumab-vc-MMAE. Drug Metab Dispos 45(11):1120–1132CrossRefGoogle Scholar
  13. 13.
    Singh AP et al (2019) A cell-level systems PK-PD model to characterize in vivo efficacy of ADCs. Pharmaceutics 11(2):pii: E98CrossRefGoogle Scholar
  14. 14.
    Stephan JP et al (2008) Anti-CD22-MCC-DM1 and MC-MMAF conjugates: impact of assay format on pharmacokinetic parameters determination. Bioconjug Chem 19(8):1673–1683CrossRefGoogle Scholar
  15. 15.
    Xie H et al (2004) Pharmacokinetics and biodistribution of the antitumor immunoconjugate, cantuzumab mertansine (huC242-DM1), and its two components in mice. J Pharmacol Exp Ther 308(3):1073–1082CrossRefGoogle Scholar
  16. 16.
    Kovtun YV et al (2006) Antibody-drug conjugates designed to eradicate tumors with homogeneous and heterogeneous expression of the target antigen. Cancer Res 66(6):3214–3221CrossRefGoogle Scholar
  17. 17.
    Advani A et al (2010) Safety, pharmacokinetics, and preliminary clinical activity of inotuzumab ozogamicin, a novel immunoconjugate for the treatment of B-cell non-Hodgkin's lymphoma: results of a phase I study. J Clin Oncol 28(12):2085–2093CrossRefGoogle Scholar
  18. 18.
    DiJoseph JF et al (2004) Antibody-targeted chemotherapy with CMC-544: a CD22-targeted immunoconjugate of calicheamicin for the treatment of B-lymphoid malignancies. Blood 103(5):1807–1814CrossRefGoogle Scholar
  19. 19.
    Alley SC et al (2008) Contribution of linker stability to the activities of anticancer immunoconjugates. Bioconjug Chem 19(3):759–765CrossRefGoogle Scholar
  20. 20.
    Tolcher AW et al (2003) Cantuzumab mertansine, a maytansinoid immunoconjugate directed to the CanAg antigen: a phase I, pharmacokinetic, and biologic correlative study. J Clin Oncol 21(2):211–222CrossRefGoogle Scholar
  21. 21.
    Sanderson RJ et al (2005) In vivo drug-linker stability of an anti-CD30 dipeptide-linked auristatin immunoconjugate. Clin Cancer Res 11(2 Pt 1):843–852PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical SciencesThe State University of New York, University at BuffaloBuffaloUSA

Personalised recommendations