Bioconjugation of Antibodies to Horseradish Peroxidase (HRP) Protocol

Introduction of Antibody Bioconjugation with HRP

Horseradish peroxidase (HRP) is a 40,000 Da enzyme that can catalyze the reaction of hydrogen peroxide with an organic electron- donating substrate to form a color, fluorescent, or chemiluminescent product upon oxidation. The use of antibody- HRP or streptavidin-HRP bioconjugates as part of an enhanced chemiluminescent (ECL) assay provides an extremely sensitive reporter for the detection of target antigen by ELISA and Western blotting applications.

The relatively small size of HRP facilitates access to antigenic sites or structures in the target antigen. Moreover, the enzyme remains stable and functional under multiple conditions that include chemical cross-linking, freeze drying, or prolonged storage at 4 °C.

HRP is a glycoprotein, and its polysaccharide chains are often used in cross-linking reactions to couple the enzyme to an antibody. Mild oxidation of sugars with sodium periodate generates reactive aldehyde groups that can be used for conjugation to amine-containing proteins. Reductive amination of oxidized HRP to antibodies in the presence of sodium cyanoborohydride is one simple method to prepare highly active antibody-HRP conjugates.

Another well-established method is the use of heterobifunctional reagents such as the water-soluble (10 mg/mL) sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1- carboxylate (sulfo-SMCC) and N-succinimidyl S-acetylthioacetate (SATA) to generate stable antibody-HRP conjugates. The sulfo-SMCC reagent contains an amine-reactive NHS ester on one end and a sulfhydryl-reactive maleimide group on the other end. In this type of reaction, the NHS ester of SMCC first creates sulfhydryl-reactive maleimide groups at the two HRP lysine residues, and cross-linking is achieved when mixed with sulfhydryl- containing antibody (SATA-modified) to create the final antibody-HRP conjugate. Maleimides are maleic acid imides derived from the reaction of maleic anhydride and ammonia or amine derivatives. The double bond of the maleimides can undergo alkylation when reacted with sulfhydryl groups to form stable thioether bonds. Maleimide reactions are specific for thiols at pH 6.5–7.5 but may also undergo hydrolysis to an open maleamic acid that is unreactive to sulfhydryls, a process that occurs faster at a higher pH.

Figure 1. Conjugation of SATA-modified antibody with sulfo-SMCC-activated HRP.

These heterobifunctional cross-linking reagents are used in controlled multistep protocols with great success to yield useful antibody-HRP conjugates. Sulfo-SMCC has a very stable maleimide functionality that affords the activation of either the HRP or antibody at amines reactive to the NHS ester end by limiting hydrolysis (see Note 1). This allows the resulting maleimide-activated intermediate to be purified from excess cross-linker, and other unwanted by-products, before mixing with the antibody and thereby exerting control over the extent of cross-linking while limiting unwanted polymerization of the conjugated protein.

The primary goal of antibody bioconjugation is the preparation of a stable antibody-enzyme conjugate that can serve as a target- directed reporter to detect or quantitate a specific antigen by immunoassay. The methods used to achieve this goal should yield an antibody that retains high antigen-binding affinity with robust enzymatic activity.

In this protocol, we describe the activation of HRP with sulfo- SMCC to create reactive maleimide groups for coupling to sulfhydryl groups introduced into antibodies by thiolation (see Note 2). The frequency of sulfhydryl occurrence in proteins is low when compared to amine or carboxylates, and this site limitation can restrict target antibody modification thereby increasing the probability that the antibody-HRP conjugate will retain antigen- binding activity.

SATA is a versatile thiolation reagent for introducing sulfhydryl groups into antibodies. The active NHS ester end of SATA reacts with amino groups in proteins to form stable amide linkage resulting in a protein with an acetylated sulfhydryl that can be stored as stock solutions without degradation. Reactivity of the SATA-modified stock requires deacetylation prior to cross-linking by simple exposure to excess hydroxylamine. The SATA thiolation process results in nearly random attachment over the surface of the antibody structure, and it has been shown that as many as six SATA per antibody can have minimal effect in antigen-binding affinity. SATA creates sulfhydryl target groups necessary to conjugate with maleimide-activated HRP (see Note 3 ).

Methods of Antibody Bioconjugation Protocol with HRP

Activation of HRP with Sulfo-SMCC

1. Dissolve HRP in 0.1 M sodium phosphate with 150 mM NaCl at pH 7.2 (PBS) to a concentration of 20 mg/mL (see Note 4).
2. Add 3 mg of sulfo-SMCC to the HRP solution and vortex to mix, allow reaction to incubate at room temperature for 15 min, and then add another 3 mg of sulfo-SMCC and incubate an additional 15 min (see Note 5).
3. Immediately purify the maleimide-activated HRP away for excess cross-linker and reaction by-products by gel filtration using a desalting column and PBS (Sephadex G25; 1/8th sample to bed volume) (see Note 6).
4. Collect 1 mL fractions and pool the first peak containing HRP (see Note 7).
5. Measure the protein concentration of the purified HRP and adjust to 10 mg/mL for the conjugation reaction. At this point, the maleimide-activated HRP should be used immediately for conjugation or freeze dry to preserve its maleimide activity.

Thiolation of Antibody

1. Freshly dissolve SATA in DMSO or DMF at 8 mg/mL.
2. Add 10–40 μL of SATA stock per 1 mg/mL of antibody in PB pH 6.5–7.5 (see Note 8).
3. React for 30 min at room temperature.
4. Purify SATA-modified antibody by gel filtration (Sephadex G25) using PBS pH 7.2 containing 1–10 mM EDTA (see Note 9).

Deacetylation of Sulfhydryl Groups on SATA- Modified Antibody

1. Freshly prepare 0.5 M hydroxylamine-HCL in PB (pH 7.2) with 10 mM EDTA.
2. Add 100 μL of hydroxylamine stock to each mL of SATA modified antibody to achieve a final concentration of hydroxylamine in the antibody solution to 50mM.
3. React for 2 h at room temperature.
4. Purify the deacetylated sulfhydryl-modified antibody by gel filtration (G25 Sephadex) using PBS with 1–10 mM EDTA pH 7.2 (see Note 10).

Bioconjugation of Sulfo-SMCC Maleimide- Activated HRP with SATAThiolated Antibody

1. Combine deacetylated sulfhydryl-modified antibody (SATA) with maleimide-activated HRP (sulfo-SMCC) using a ratio of4:1 excess HRP enzyme to antibody (see Note 11).
2. Allow the reaction to proceed at room temperature for 2 h or overnight at 4 °C.

Immunoaffinity Purification of antibody-HRP Conjugate by Liquid Chromatography

1. Prepare protein-G column by washing with 3–5× column volumes of protein-G binding buffer at a rate of 1 mL/min to remove preservative and exchange buffer and pH (see Note 12).
2. Dilute antibody-HRP conjugate with protein-G in binding buffer to adjust pH and pass over column 1–3× at a rate of 0.5–1 mL/min (see Note 13). Monitor protein absorbance at 280 nm using an in-line UV monitor (see Note 14).
3. Pass at least 5× column volume of protein-G binding buffer at 1 mL/min to wash beads of residual unbound protein and return to baseline 280 nm absorbance values.
4. Apply protein-G elution buffer (0.1 M glycine-HCL, pH 2–3) at a rate of 1 mL/min and begin collecting 1 mL fractions into tube containing 0.1 mL of neutralization buffer (1 M PB buffer, pH 8.0). Monitor protein absorbance at 280 nm UV to identify desorption of the IgG from the resin as an A280 protein peak and collect fractions until baseline protein measurement is achieved (see Note 15).
5. Determine protein concentration of each elution fraction by measuring absorbance at 280 nm or performing a protein assay such as Bradford or BCA.
6. Evaluate affinity-purified elution fractions for antibody and HRP activity by ELISA.
7. Pool peak fractions and dialyze against desired buffer. Centrifugal concentrators can be used to increase desired concentration of purified antibody-HRP. The addition of 10 % glycerol to the final antibody-HRP stock is useful for long-term storage (see Note 16).