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Antibody-Drug Conjugate Assays

Bioassay Services for Antibody-Drug Conjugate (ADC) Characterization

We offer a full suite of bioassay services tailored for antibody-drug conjugates (ADCs), enabling precise binding characterization, internalization tracking, cytotoxicity evaluation, and serum & plasma stability analysis. Our high-throughput and customizable ADC solutions support lead selection, mechanism of action (MOA) evaluation, and comprehensive characterization for therapeutics development.

 

Our bioassays for antibody-drug conjugates (ADCs) provide:

  • Quantitative binding assessment using ELISA, SPR, and FACS
  • High-throughput internalization assays (pHrodo, acid quench, colocalization, and time-course imaging)
  • Cytotoxicity, apoptosis, and bystander killing assays to evaluate ADC potency
  • Serum/plasma stability assays for ADC integrity and drug-to-antibody ratio (DAR) assessment

Antibody-drug conjugate (ADC) bioassay services for ADC characterization.

Antibody-Drug Conjugate Assay Details:

Service Item Description Request A Quote 
Binding Assays

Protein-based (ELISA/SPR)

Cell-based (FACS)

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Cytotoxicity Assays CellTiter-Glo
Bystander Killing Assay​s

PI/AnnexinV staining (FACS)

Caspase 3/7 activation (Luminescence)
Internalization​ Assays Schedule a Free Consultation
Developability Evaluation​   Serum/plasma stability (Human/cyno/mouse)

Case Study #1: Customized ELISA and SPR Assays for Evaluating ADC Binding Before and After Conjugation

In this case study, customized ELISA and SPR binding assays for ADCs were developed to access binding during conjugation process, emphasizing the importance of re-evaluating ADC binding activity post-conjugation.

Case study on optimized ELISA and SPR assays for evaluating Antibody-Drug-Conjugate binding before and after conjugation, highlighting the importance of reassessing binding activity post-conjugation. 

Figure 1: (A) ELISA analysis showed that ADC1 exhibited a comparable EC50 to the naked antibody 1, but showed reduced maximum binding, suggesting that conjugation may affect binding efficacy. (B) SPR analysis demonstrated that ADC2 maintained comparable binding pre- and post-conjugation with naked antibody.

Case Study #2: Internalization Assays for High-Throughput Screening of Antibodies and Antibody-Drug Conjugates (ADCs)

We have developed multiple customized internalization assays to evaluate the internalization activities of antibodies and ADCs, including pHrodo, acid quench, colocalization, and time-course analysis. These assays provide robust tools for real-time ADC internalization monitoring with high-throughput screening capabilities.

Case study on customized internalization assays for evaluating antibodies and ADCs, including pHrodo, acid quench, colocalization, and time-course analysis for high-throughput screening and real-time monitoring.

 

Figure 1: (A) The pHrodo method demonstrated dose-dependent internalization in SK-BR-3 cells. (B) The acid quench method revealed stronger internalization of antibody 1 compared to antibody 2 after 2-4 hours. (C) The colocalization method showed antibody 1 transitioning from the plasma membrane to lysosomes. (D) The time-course method enabled real-time monitoring of ADC internalization over four days.

Case Study #3: mFab/hFab-MMAE and hFab-ZAP ADC Cytotoxicity Assays

This case study highlights the development of customized ADC cytotoxicity assays to assess the cell killing potency of naked antibodies prior to conjugation. The assays demonstrated strong consistency between the cytotoxic effects of naked antibodies and ADCs. Additionally, hFab-MMAE exhibited reduced non-specific killing compared to hFab-ZAP, making it a reliable tool for evaluating antibody potential as ADC candidates.

Case study on customized ADC cytotoxicity assays to assess cell-killing potency before conjugation, showing strong consistency and reduced non-specific killing with hFab-MMAE compared to hFab-ZAP.

Figure 1: (A) Cytotoxicity assay using anti-mouse Fc Fab-MMAE. (B) Cytotoxicity assay using anti-human Fc Fab-MMAE. (C) Cytotoxicity assay using anti-human Fc Fab-ZAP. (D) Cytotoxicity assay with the final ADC molecule. (E) Summary of cytotoxicity test results of antibodies and ADCs.

Case Study #4: Bystander Killing Assays for Evaluating ADC Cytotoxicity Potency to TAA-Negative Cells

In this case study, customized bystander killing assays were developed to assess the cytotoxic potency of ADCs against TAA-negative cells for ADC characterization.

Case study on customized bystander killing assays to evaluate ADC cytotoxicity potency against TAA-negative cells.

Figure A: Bystander killing of the target ADC molecule. NCI-N87 (HER2+) cells and Raji.luc (HER2–) cells were co-cultured at 2500 cells/well. T-Dxd demonstrated a stronger bystander killing effect on Raji cells as the number of co-cultured NCI-N87 cells increased.

 

Case Study #5: Development of Customized Serum & Plasma Stability Assays for Antibody and ADC Characterization

In this case study, customized serum & plasma stability assays were optimized to evaluate the integrity and binding activity of antibodies and ADCs. These assays provided critical insights into ADC stability, including drug-to-antibody (DAR) analysis and the impact of serum & plasma incubation on antigen-binding activity.

Case study on customized serum and plasma stability assays to evaluate the integrity and binding activity of antibodies and ADCs, including DAR analysis and the impact of serum and plasma incubation on antigen-binding.

Figure 1: (A) The serum & plasma stability assay workflow. (B) ADCs maintained stable DAR values during 14-day incubation in rat plasma, as assessed by LC-MS. (C) Antigen-binding activity of human serum-treated ADC samples showed a slight decrease after 14 days, as measured by ELISA.

 

Speak with Our Expert!

Frequently Asked Questions for Antibody-Drug Conjugate Assays

Q: Have you worked with dual-payload ADCs, and what is your experience with them?

A: We have experience supporting dual-payload ADC projects. Many of the in vitro assays used for dual-payload ADCs, such as cytotoxicity and internalization assays, are similar to those used for single-payload ADCs. One potential difference is that dual-payload ADCs may show different toxicity patterns, so data interpretation may need to be assessed case by case.

Q: What is your high-content imaging capacity for internalization assays?

A: Our Shanghai site has two Operetta high-content imaging systems. These instruments support both 96-well and 384-well plate formats and provide enough capacity to screen hundreds of ADCs or antibody samples.

Q: What are the key differences between CTG, MTT, and WST cytotoxicity assays? When should each be used?

A: These assays differ mainly in their detection principles. CellTiter-Glo (CTG) measures cellular ATP levels and is generally considered a robust assay with a strong signal window. MTT and WST measure dehydrogenase activity in cells and generate a detectable product that can be read on a plate reader. Assay selection depends on the study objective and the biology of the molecule being evaluated.

Q: How does Operetta compare with Incucyte for internalization assays?

A: Operetta is well suited for high-throughput endpoint internalization assays, making it useful for early-stage screening of larger antibody or ADC panels. Incucyte is better suited for real-time kinetic studies, such as understanding the timing of internalization or when uptake reaches saturation. In general, Operetta is preferred for higher-throughput screening, while Incucyte is more appropriate for mechanistic or kinetic evaluation.

Q: How well do binding and internalization correlate?

A: Binding and internalization do not always correlate strongly. In some cases, antibodies with lower binding affinity can still show good internalization activity. Moderate binding may be sufficient for internalization, so strong binding alone is not always predictive of uptake performance.

Q: In internalization assays, is it useful to include antigen-negative or low-expression cells to demonstrate specificity?

A: Yes, antigen-negative or low-expression cells can serve as useful controls to help assess specificity. That said, internalization studies are typically performed using high antigen-expression cells, since these provide the clearest signal for evaluating uptake. Negative or low-expression cells are often included as supporting controls.

Q: If internalization is already measured, why is a binding assay still needed?

A: Binding assays are still valuable because they are typically simple, fast, and high throughput. In many workflows, binding is assessed first during early-stage screening to identify promising hits, and internalization is then evaluated in a second step.

Q: Is it important to assess Fc-mediated functions such as ADCC?

A: Fc-mediated functional assays can be important for certain ADCs, especially those in IgG1 format that may retain Fc effector function. In those cases, ADCC-related evaluations may be useful with binding and internalization studies to provide a more complete characterization profile.

Q: What are the main limitations of 2D cell-based assays for IND-enabling decisions?

A: Although 2D assays do not fully capture in vivo biology, they remain highly important for ADC discovery. Commonly used commercial tumor cell lines are still valuable as tool cell lines for early pharmacology assessment, and 2D cell-killing assays continue to be an important part of in vitro evaluation packages.

Q: Is the bystander assay typically performed in 2D or 3D culture?

A: Bystander assays are typically performed in 2D culture systems. While 3D approaches may be possible, they are generally more difficult to optimize.

Q: How do you define high-, medium-, and low-antigen expression cell lines?

A: There is no universal cutoff that applies across all targets, and antigen expression levels are usually defined relative to the target of interest. We can use quantitative FACS to measure antigen density across candidate cell lines and then group them into relative high-, medium-, and low-expression categories for that specific target.

Q: How many conjugates can your high-throughput workflow support, and at what scale?

A: For assays such as cytotoxicity and binding, our high-throughput workflow can support hundreds of antibodies or ADCs in a single experiment, depending on study design and assay format.

Q: How do you determine the right DAR for an ADC candidate?

A: The optimal DAR is highly molecule dependent and can be difficult to define without supporting data. DAR influences both efficacy and toxicity, so the most suitable DAR generally needs to be evaluated together with in vivo safety window data.

Q: How do you select the endpoint for high-throughput internalization or cytotoxicity assays?

A: Endpoint selection is usually based on assay optimization studies. For cytotoxicity assays, different time points such as 72 hours, 96 hours, or longer may be evaluated to identify the time point that provides the best assay window and reproducibility. For internalization assays, real-time studies can first be performed to help select an appropriate endpoint for higher-throughput screening.

Q: How do you select cell line pairs for bystander assays? Should the two cell lines have similar proliferation rates?

A: Cell line selection for bystander assays requires more than just differences in antigen expression. Typically, one cell line should have high target expression and the other low target expression, but both should first be tested individually. It is important to confirm that the low-expression cell line is not intrinsically sensitive to the ADC on its own. In addition, both cell lines need to be compatible in shared culture conditions.

Q: Besides antigen expression, what other factors matter when selecting cell lines for ADC assays?

A: In addition to antigen expression level, cell lines should also be relevant to the clinical indication and ideally have published or historical data available. Cell lines that have been used previously in in vivo or in vitro studies are often preferred because they provide stronger context and comparability.

Q: Do you support autoimmune or inflammation-related cell-based models?

A: For certain popular autoimmune and inflammatory targets, relevant assays are available, using targets such as IL-13, IL-18, TL1A, and TSLP. Available approaches may include PBMC-based assays, ligand-dependent cytokine release studies, proliferation analysis, and other functional readouts in primary cell systems.

Q: Can you recommend high-, medium-, or low-antigen-expressing cell lines for ADC assays?

A: We work closely with clients to help identify suitable cell lines for ADC assays. This may include literature review, database analysis, and quantitative FACS assessment to evaluate target expression levels. The antigen density data has been generated through client-specific collaborations, so those data cannot be shared across projects.

Q: What are reasonable ratios of the two cell types in a bystander assay? Is 10:1 acceptable?

A: The optimal ratio depends on the specific cell lines, antigen expression levels, and payload release efficiency, so it should be determined case by case. As a starting point, ratios such as 3:1 and 1:1 are commonly evaluated. A 10:1 ratio may also be feasible in some cases, but in 96-well plate formats, high proportions of antigen-high cells may reduce the number of antigen-low cells and limit the assay window.

Q: How much can the payload affect antibody binding and internalization?

A: Payload can affect antibody binding and internalization in some cases. For example, certain hydrophobic payloads may alter antibody conformation, which can reduce binding activity or internalization efficiency. The extent of this impact depends on the antibody, payload chemistry, and overall ADC design.

Q: Can you share more details about dual-binding assays?

A: Dual-binding can be assessed using several methods, including SPR, ELISA, and FACS. In SPR or ELISA-based approaches, one antigen is typically immobilized on a chip or plate, followed by addition of the bispecific antibody and then the second antigen. In FACS-based methods, cells expressing one antigen are incubated with the bispecific antibody, and binding to the second labeled antigen is then evaluated by flow cytometry.

Q: How difficult is it to test ADCs using 3D cell culture or organoid models?

A: Testing ADCs in 3D culture systems is feasible. However, additional data are typically needed to establish how well these models correlate with in vivo outcomes.

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