In our in vitro services for Antibody-directed enzyme prodrug therapy (ADEPT), Creative Biolabs utilizes advanced biophysical techniques and high-throughput screening methods to accurately assess the binding affinity and specificity between antibodies and target enzymes. Our expert team has extensive experience in the ADEPT field, allowing us to provide customized experimental plans tailored to the specific needs of our clients. By thoroughly analyzing antibody activity, we assist clients in optimizing their ADEPT designs, ultimately enhancing the success rate of drug development.

Introduction

Evaluating the binding capacity of antibodies to their target antigens, as well as assessing the specificity of these antibodies towards target cells, is essential for ensuring the optimal effectiveness of ADEPT. Currently, Creative Biolabs offers a range of analytical services to evaluate the binding affinity and specificity of ADEPT components, providing scientific foundations for drug development and establishing a solid groundwork for clinical applications of ADEPT. By conducting quantitative analyses of the binding abilities between various antibodies and their corresponding enzymes or prodrugs, we can identify candidate antibodies with higher affinities, maximizing the accumulation of drugs in tumor cells while minimizing damage to normal cells. Additionally, through the assessment of antibody specificity, our team can select the most suitable antibodies, thereby enhancing precision in the treatment process.

Fig.1 The Affinity and Specificity Evaluation Analysis Strategies.^1,3

Services

Over the past decade, ADEPT has garnered significant research attention due to its ability to enhance the selectivity and efficacy of drugs within tumor cells. In the process of directing enzymes to tumor sites using specific antibodies, evaluating the affinity and specificity of these antibodies for their targets is important for drug design and optimization. As a result, Creative Biolabs has specifically developed a series of in vitro experimental services for ADEPT discovery.

• Antibody-Enzyme Complex Binding Affinity Assessment

Utilize Surface Plasmon Resonance (SPR) technology alongside stable isotope-labeled bioanalytical techniques to accurately measure the binding affinity of antibody-enzyme complexes.

Conduct affinity assessments under various conditions such as different pH levels and salt concentrations, assisting clients in identifying the optimal experimental parameters.

• Antibody Specificity Analysis

Perform specificity tests for the interaction of antibodies with target enzymes as well as non-target molecules using competitive binding assays and ELISA, to evaluate the selectivity of antibodies in diverse environments.

Examine antibody specificity in in vitro settings (cell culture media) to ensure their effectiveness and safety in practical applications.

• Dynamic Monitoring and Evaluation

Employ real-time monitoring technology to observe the kinetic changes during the binding process between antibodies and targets, providing data support for further optimization.

Offer analyses of binding kinetics models to enhance the understanding of drug action mechanisms.

• Binding Site Analysis

Use molecular docking and bioinformatics approaches to predict and analyze key binding sites of antibodies with target enzymes, thereby improving the efficiency of antibody design and selection.

• Competitive Binding Testing

By introducing various competing antigens into the reaction system, we can observe the impact on the antibody's binding to the specific target, allowing us to assess its specificity.

• Cross-Reactivity Detection

The antibody's binding ability is tested against multiple related or unrelated antigens to evaluate its cross-reactivity with non-target tissues.

Fig.2 The Detection of Binding Affinity and Specificity.^2,3

Case Study:

1. The objective of the Experiment

The purpose of this experiment is to evaluate the binding affinity and specificity of specific antibodies with prodrugs, providing essential data to support the subsequent development of ADEPT.

2. Experimental Procedures

1) Preparation of Antibodies and Enzymes

- Initially, the target antibodies and enzymes are extracted from source materials to ensure their authenticity and activity. We will purify the antibodies to eliminate impurities and analyze their purity using High-Performance Liquid Chromatography (HPLC).

2) Assessment of Binding Affinity

- Preparation of Solid Phase: Our lab uniformly immobilizes the purified antibodies onto the surface of a 96-well plate, maintaining the orientation and structural integrity of the antibodies.

- Competitive Binding Assay: Different concentrations of enzymes are added to the antibody-coated wells in a gradient manner. We set up blank and negative controls to observe the binding behavior of the enzymes at varying concentrations.

- Elution and Detection: After a specified binding period, unbound enzymes are removed using a wash buffer. We then quantify the amount of bound enzyme using an Enzyme-Linked Immunosorbent Assay (ELISA), comparing the binding amounts across different concentrations to calculate the binding affinity (Kd value).

3) Specificity Evaluation

- Control Experiments: To establish background binding, we introduce a non-specific enzyme with no binding affinity and perform the same binding experiments.

- Biological Reactivity Specificity Testing: Our scientists conduct binding experiments with other relevant molecules to ascertain the specificity of the antibodies across different targets, subsequently quantifying the specificity with a Selectivity Ratio.

3. Data Processing and Analysis

- We utilize non-linear regression methods, such as the Hill equation, to fit the binding data. Our data analysts will determine the Binding Affinity along with its confidence intervals, and use statistical software to conduct specificity analyses, ensuring the reliability and reproducibility of the results.

Advantages

• High-Throughput Screening: With the aid of automated systems, we can efficiently process a large number of samples in a short period, swiftly identifying the optimal antibody-enzyme combinations and enhancing our research and development efficiency.
• High Sensitivity Assessment: Using cutting-edge bioanalytical techniques, Creative Biolabs can accurately measure the binding affinity between antibodies and target enzymes. This high-sensitivity assessment is effective for identifying low-abundance binding patterns.
• Specificity Testing: Our platform is capable of conducting comprehensive evaluations of antibody specificity, ensuring accurate target recognition across various biological environments while minimizing interference from non-specific binding.
• Diverse Experimental Design: We provide customized experimental designs tailored to our client's specific needs, encompassing different types of antibodies and enzymes to ensure the validity and reliability of experimental outcomes.
• Enhancing Drug Development Efficiency: Through early assessment of binding affinity and specificity, our development team can promptly adjust research directions during the preclinical stage, significantly boosting the efficiency of ADEPT development.
• Reducing R&D Costs: Accurate assessments can effectively minimize the need for subsequent modifications and testing due to adverse non-specific binding, thereby lowering overall research and development expenses.
• Data Accuracy: By employing highly sensitive techniques such as SPR, our labs can obtain real-time, dynamic binding data, allowing for a more precise evaluation of affinity and specificity while minimizing potential errors.
• Comprehensive Results Interpretation: Beyond providing raw data, our staff delivers detailed analysis reports to our clients, which include potential improvement suggestions and recommendations for follow-up ADEPT experiments, helping them make informed decisions.

By integrating sensitive detection techniques with efficient experimental design, Creative Biolabs can deliver reliable assessments of ADEPT Binding Affinity and Specificity in a short timeframe. We can thoroughly assess the binding characteristics of ADEPT, ensuring a comprehensive validation of the results from multiple perspectives. Feel free to contact us anytime to discuss your research needs and to obtain more information and service quotes.

References

1. Makowski, Emily K., et al. "Co-optimization of therapeutic antibody affinity and specificity using machine learning models that generalize to novel mutational space." Nature communiCommunications2022): 3788.
2. Gu, Huajie, et al. "Graphene oxide-assisted non-immobilized SELEX of okdaic acid aptamer and the analytical application of aptasensor." Scientific Reports 6.1 (2016): 21665.
3. Distributed under Open Access license CC BY 4.0, without modification.

For Research Use Only | Not For Clinical Use