Enzyme Conjugation with Antibodies

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Enzyme conjugation with antibodies is a well-established and highly versatile technology widely used in immunoassays, bioimaging, biosensors, and emerging therapeutic applications. By covalently linking enzymes to antibodies or antibody fragments, highly sensitive detection reagents and targeted biocatalytic systems can be generated. Creative Enzymes provides comprehensive antibody–enzyme conjugation services, covering conjugate design, optimized conjugation chemistries, fusion protein strategies, purification, and in-depth characterization. Our services support applications ranging from routine ELISA reagent development to advanced antibody–enzyme conjugates (AECs) for targeted therapy and diagnostics, ensuring high bioactivity, defined conjugation ratios, and excellent batch-to-batch reproducibility.

Background: Scientific and Industrial Foundations of Antibody–Enzyme Conjugates

Antibody–enzyme conjugates represent one of the earliest and most successful examples of functional bioconjugation in life science research and diagnostics. Well-established practices of antibody–enzyme conjugates have already been widely applied in enzyme-linked immunosorbent assays (ELISAs), Western blotting, immunostaining, flow cytometry, and biosensor development. In these applications, enzymes serve as signal amplifiers, converting biochemical recognition events into measurable optical or electrochemical signals.

For ELISA and related immunoassays, there are two main types of enzymes that are commonly used to conjugate with secondary antibodies: horseradish peroxidase (HRP) and alkaline phosphatase (AP). HRP is favored for its high turnover rate, compatibility with a wide range of chromogenic and chemiluminescent substrates, and excellent sensitivity. AP, on the other hand, offers superior stability and is often preferred for long-term assays or multiplexed detection systems.

Beyond classical analytical applications, antibody–enzyme conjugates have recently attracted increasing attention as therapeutic agents, presenting precise targeting and catalytic activities that are not matched by traditional antibody–drug conjugates (ADCs). In contrast to ADCs, where each antibody delivers a finite payload of cytotoxic molecules, antibody–enzyme conjugates can catalytically activate prodrugs or detoxify harmful compounds at the target site, enabling signal or therapeutic amplification.

To achieve effective tissue penetration and biological performance, therapeutic antibody–enzyme products are often required to exhibit a defined antibody-to-enzyme ratio, frequently close to 1:1. In addition, site-specific conjugation or genetically encoded fusion strategies may be required to preserve antigen-binding affinity and enzymatic activity while minimizing heterogeneity.

Conjugation of enzymes to antibodies Figure 1. Conjugation of horseradish peroxidase (HRP) to antibody (IgG).

What We Offer: Comprehensive Antibody–Enzyme Conjugation Services

Creative Enzymes provides end-to-end solutions for the development and production of antibody–enzyme conjugates, tailored to diagnostic, imaging, biosensing, and therapeutic applications. Our services are designed to balance conjugation efficiency, structural control, and biological functionality.

Key offerings include:

Custom design of antibody–enzyme conjugates based on application requirements
Conjugation of enzymes to full-length antibodies or antibody fragments (Fab, scFv, Fc, etc.)
Development of controlled antibody/enzyme ratios, including near 1:1 conjugates
Chemical, enzymatic, and fusion protein–based conjugation strategies
High-purity conjugate production, free of unconjugated enzymes or antibodies
Advanced analytical characterization to confirm structure, activity, and consistency

Our multidisciplinary expertise enables us to support both standard reagent production and highly specialized development programs.

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Service Workflow

Workflow of enzyme conjugation with antibodies service

Our Advanced Technologies for Antibody–Enzyme Conjugation

Chemical Crosslinking Strategies for Antibody–Enzyme Conjugates

Antibody–enzyme conjugates are most commonly generated by inducing covalent bonds between the enzyme and antibody via chemical crosslinkers, enzyme-catalyzed reactions, or fusion protein technologies. Commonly used reagents for chemical crosslinking include:

Glutaraldehyde, which reacts with amine groups on lysine residues
Maleimide-based linkers, enabling thiol-specific conjugation
Aldehyde-functionalized reagents, often used in site-directed approaches

Enzymatic and Site-Controlled Conjugation

To improve conjugation homogeneity and preserve biological activity, enzymatic conjugation methods can be employed. These approaches enable more precise control over conjugation sites and reduce structural heterogeneity, which is particularly important for therapeutic applications.

Fusion Protein Strategies and Antibody–Enzyme Engineering

Fusion proteins represent an alternative strategy, especially in directed enzyme prodrug therapy (ADEPT) and targeted imaging. In this approach, antibody fragments and enzymes are genetically fused to form a single polypeptide or self-assembling complex.

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Why Choose Us

Extensive Expertise in Antibody and Enzyme Engineering

Decades of experience in enzyme modification and antibody technologies ensure robust and reliable conjugate design.

Multiple Conjugation Platforms Available

Chemical, enzymatic, and fusion protein strategies are offered to match diverse application needs.

Controlled Antibody-to-Enzyme Ratios

We specialize in achieving defined conjugation ratios, including therapeutic-grade 1:1 constructs.

High-Purity Products with Rigorous Purification

Advanced purification ensures conjugates are free of residual enzymes and antibodies.

Comprehensive Analytical Characterization

Mass spectrometry, activity assays, and structural analysis validate conjugate quality and consistency.

Scalable and Application-Oriented Solutions

Services support projects from early research to translational and industrial development.

Case Studies: Representative Applications of Antibody–Enzyme Conjugation

Case 1: Zipbodyzyme–A New Antibody-Enzyme Fusion Protein

A novel antibody–enzyme fusion protein, termed Zipbodyzyme, was successfully expressed in the cytoplasm of Escherichia coli. This bifunctional construct consists of a Fab antibody fragment fused to an enzyme and stabilized by a leucine zipper pair at the C-termini of the Fab heavy and light chains, enabling proper assembly in the bacterial cytoplasm. Zipbodyzymes incorporating an anti-E. coli O157 Fab and either luciferase or green fluorescent protein retained both antigen-binding capability and enzymatic or fluorescent activity. The fusion proteins were directly applicable in ELISA without secondary antibodies, demonstrating their potential to simplify immunoassays and expand antibody–enzyme conjugation applications.

Structure of Zipbody, Zipbody-Luc and Zipbody-GFP Figure 2. Modelled structures of Zipbody and Zipbodyzymes. Blue, heavy chain (Hc); cyan, light chain (Lc). In the Zipbody structure, leucine zipper (LZ) sequences were fused to the C-terminal end of both the Hc and the Lc of the Fab antibody to promote association of the two chains. For construction of Zipbody-enzyme complexes (i.e., Zipbodyzymes), luciferase (Luc) or green fluorescent protein (GFP) was genetically fused to the C-terminus of the Hc of Zipbody. (Mori et al., 2018)

Case 2: Antibody–Urease Immunoconjugate L-DOS47 for Targeted Cancer Therapy

This study describes the development and characterization of L-DOS47, a novel antibody–enzyme immunoconjugate targeting CEACAM6-expressing tumors. A single-domain antibody (AFAIKL2) was expressed in E. coli and site-selectively conjugated to highly purified urease using SIAB as a cross-linker under controlled pH conditions. The optimized process achieved high conjugation efficiency, yielding 8–11 antibodies per urease molecule with over 95% purity and minimal free enzyme. Comprehensive analytical characterization confirmed structural integrity and binding activity. L-DOS47 demonstrated specific cytotoxicity toward CEACAM6-positive cancer cell lines and is currently under phase I clinical evaluation for non-small cell lung cancer.

The antibody-urease immunoconjugate L-DOS47 is used for cancer therapy Figure 3. Production and characterization of a camelid single domain antibody–urease enzyme conjugate for the treatment of cancer. (Tian et al., 2015)

Frequently Asked Questions (FAQs): Antibody–Enzyme Conjugation Services

Q: Which enzymes are most commonly used for antibody conjugation?

A: Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most widely used enzymes for immunoassays. Other enzymes can be used depending on detection or therapeutic requirements.
Q: Can you conjugate enzymes to antibody fragments instead of full antibodies?

A: Yes. We routinely work with full-length antibodies as well as Fab, scFv, Fc, and other antibody fragments.
Q: How do you control the antibody-to-enzyme ratio?

A: Conjugation conditions, linker chemistry, and purification strategies are optimized to achieve defined ratios, including near 1:1 conjugates.
Q: Are site-specific conjugation options available?

A: Yes. Enzymatic methods and thiol-specific chemistries can be used for site-controlled conjugation to reduce heterogeneity.
Q: How are conjugates characterized?

A: Characterization may include enzymatic activity assays, binding assays, mass spectrometry, molecular weight analysis, and stability testing.
Q: Can your conjugates be used for therapeutic research?

A: Our antibody–enzyme conjugates are suitable for research and preclinical development. Clinical application depends on regulatory pathways and further validation.
Q: What scales can be supported?

A: We support small research-scale production as well as larger-scale manufacturing for diagnostic and industrial applications.

References:

Mori A, Ojima-Kato T, Kojima T, Nakano H. Zipbodyzyme: Development of new antibody-enzyme fusion proteins. Journal of Bioscience and Bioengineering. 2018;125(6):637-643. doi:10.1016/j.jbiosc.2017.12.021
Tian B, Wong WY, Hegmann E, Gaspar K, Kumar P, Chao H. Production and characterization of a camelid single domain antibody–urease enzyme conjugate for the treatment of cancer. Bioconjugate Chem. 2015;26(6):1144-1155. doi:10.1021/acs.bioconjchem.5b00237

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For research and industrial use only. Not intended for personal medicinal use. Certain food-grade products are suitable for formulation development in food and related applications.

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