Enzyme Labeling of Antibodies

As a professional enzyme labeling service provider, BOC Sciences offers enzyme antibody conjugation services. We offer a wide range of enzymes for antibody labeling, including alkaline phosphatase, horseradish peroxidase, β-galactosidase, etc.

What is Enzyme Conjugated Antibody?

Enzyme conjugated antibodies are made by attaching an enzyme to a specific antibody by appropriate methods. The quality of enzyme-conjugated antibody mainly depends on the enzyme and antibody with good purity, high activity, and high affinity. Once prepared, enzyme-labeled antibodies can be used for a variety of applications including enzyme-linked immunosorbent assay, immunohistochemistry (IHC), protein blotting, and other immunoassays.

Fig.1 Scheme of an antibody modification with enzyme. (Falck et al., 2018)

Services for Enzyme Labeled Antibody

Alkaline Phosphatase (AP) Labeled Antibody

Alkaline phosphatase (AP) is a hydrolase enzyme often used in conjunction with antibodies for immunoassays. AP has a lower catalytic rate than HRP but is more stable and maintains linear reaction kinetics for longer periods.

Horseradish Peroxidase (HRP) Labeled Antibody

Horseradish Peroxidase (HRP) can be directly conjugated to an antibody or secondary antibody. In order to produce maximum specificity and a high signal-to-noise ratio, it is preferable to use direct primary antibody.

β-Galactosidase (β-gal) Labeled Antibody

β-Galactosidase (β-gal) has a high turnover rate and can be used with substrates that yield both soluble and insoluble products. β-Galactosidase antibody conjugates are an excellent choice for photometric and electrochemical enzyme multiplication assay techniques (EMAT). In addition, most tissues exhibit very low levels of endogenous enzyme activity.

Enzyme-linked Secondary Antibody

Selected enzymes are conjugated with secondary antibodies to form enzyme-secondary antibody complexes. The conjugated enzyme-secondary antibody complexes need to be validated and optimized to assess the catalytic activity of the complexes as well as to test specificity.

Enzyme Antibody Conjugation

BOC Sciences can maintain antibody activity by directing the location of the label so that it does not interfere with the antigen binding site. For some conjugates, it may be necessary to modify/purify the antibody or introduce functional groups and linkers to the enzyme or antibody.

• Aldehyde introduction via diol oxidation
• Thiol introduction through Traut's reagent
• Thiol introduction through SATA
• Disulfide bond reduction

For the conjugation of enzymes to antibodies, we typically use heterobifunctional cross-linkers.

• NHS ester-maleimide
• Reductive amination
• Oxime
• Dydrozone formation

Purification of Antibody Enzyme Conjugate

The final conjugate first needs to be separated from the excess or unreacted reagent by gel filtration or dialysis. Other purification techniques such as stirred cell filtration, tangential flow filtration (TFF), and gel filtration chromatography can also be used to remove excess reagents or to isolate and characterize the cross-linking product. For reagents that are similar in size to or larger than the antibody, other purification techniques such as affinity chromatography, ion exchange chromatography, and hydrophobic interaction chromatography must be used.

Quality Control of Antibody Enzyme Conjugate

After the purification of the product, many different types of analyses are required, including spectroscopy (MALDI-top, ESI, LC-MS fluorescence), electrophoresis, immunochemical biochemistry, and enzyme analysis. We also independently follow QC (Quality Control) and QA (Quality Assurance) procedures, providing you with the dual assurance that each delivered conjugate is of the highest possible quality.