Carrier Protein Activation and Conjugation Data for Immunogen Preparation

Experiments with KLH variants and other immunogens to validate solubility, maleimide activation consistency, peptide conjugation efficiency and immunogenicity

by Brian D. Wolf, Ph.D.; Douglas A. Hayworth, Ph.D. - 06/05/13

In 2009, we updated and streamlined the structure of our immunogenic carrier protein products and conjugation kits. In doing so, we performed a number of experiments designed to validate and demonstrate the quality, properties, differences and uses of these reagents. In this article, we summarize the results of those experiments.

Thermo Scientific Imject Carrier Proteins are preparations of immunogenic proteins for conjugation to antigens (haptens) of various kinds to facilitate preparation of immunogens that can be used for immunization leading to antibody production.

The current offering of Imject Products includes the following proteins and varieties:

• Mariculture Keyhole Limpet Hemocyanin (mcKLH)
  • Protein and EDC Conjugation Kit
  • Maleimide-activated Protein and Kit (including a PEGylated version of KLH)
• Blue Carrier™ Protein
  • Protein and EDC Conjugation Kit
  • Maleimide-activated Protein
• Bovine Serum Albumin (BSA)
  • Protein and EDC Conjugation Kit
  • Maleimide-activated Protein and Kit
  • Cationized BSA (cBSA) Protein
• Ovalbumin (OVA)

Imject mcKLH is native keyhole limpet hemocyanin (KLH). The “mc” designation denotes the fact that that all Imject KLH products are purified from KLH that is obtained from sustainable maricultures of limpets rather than from wild-harvested limpets.

Blue Carrier™ Protein is a purified preparation of Concholepas concholepas hemocyanin (CCH). The large protein exhibits most of the same immunogenic properties as the popular carrier protein, keyhole limpet hemocyanin (KLH).

Typically, unmodified carrier proteins are conjugated to peptides or other haptens via carboxyl-to-amine crosslinking with EDC (Part No. 22980). Peptides containing terminal cysteines (sulfhydryl groups) are conjugated to carrier proteins using maleimide crosslinking chemistry, using carrier proteins that have been pre-activated to contain maleimide groups.

Carrier protein solubility

Solubility is an important consideration with regard to the use of carrier proteins. KLH is the most popular and highly immunogenic carrier protein, but it is also notorious for having poor solubility. This can make it difficult to prepare for crosslinking reactions and to recover conjugate in subsequent clean-up steps.

Our data indicate that the various forms of Imject mcKLH and related Blue Carrier Protein products have more than sufficient solubility for use in immunogen preparation and subsequent immunization protocols (Figure 1). Maleimide activation decreases the solubility of a given protein type, but the effect on KLH is offset by modification with polyethylene glycol (PEG). Blue Carrier Protein is more soluble than mcKLH, especially when maleimide-activated forms are compared.

Levels and quality of maleimide activation

Maleimide activation of carrier proteins makes it possible to conjugate sulfhydryl-containing haptens. For a number of reasons, this is an especially effective and popular strategy for conjugating cysteine-terminated peptide haptens. Maleimide activation can be accomplished using Thermo Scientific Pierce Sulfo-SMCC crosslinker (or similar reagent), but it is difficult to obtain and validate consistently high levels of activation.

Imject Maleimide-Activated Carrier Proteins are prepared using a rigorous, carefully optimized manufacturing procedure, and each lot is tested to confirm that a high level of activation was achieved. We use a cysteine-coupling assay to determine the moles of maleimide per mole of carrier protein. Briefly, maleimide-activated carrier proteins were reacted with a dilution series of L-cysteine and then the amounts of noncoupled cysteine were measured in a microplate assay using Thermo Scientific Ellman’s Reagent, also known as DTNB (Part No. 22582). See METHODS section for additional details.

There are several ways to visualize the results of the maleimide-activation assay. Figure 2 displays example results of the cysteine assay, graphed as micromoles of maleimide per gram of carrier protein. Because there are significant differences in molecular weights of tested carrier proteins, this presentation is more relevant to practical uses than the mole-to-mole comparison (i.e., conjugation protocols are alike in specifying a given milligram amount of carrier protein for reaction regardless of its molecular weight).

Figure 2. High levels of maleimide activation of Thermo Scientific Imject Carrier Proteins. Surface primary amines (i.e., lysine side chains) of Imject Carrier Proteins were maleimide activated by reaction with excess Thermo Scientific Pierce Sulfo-SMCC crosslinker. Activation levels (moles of maleimide per gram of carrier protein) were determined with a cysteine coupling assay. KLH and Blue Carrier Proteins are very large (approx. 8000kDa), enabling activation with 600 to 900 maleimide groups per protein molecule. BSA and ovalbumin are smaller (67kDa and 45kDa, respectively), allowing for activation with 5 to 20 maleimide groups per protein molecule.

A key practical feature of any useful carrier protein is its ability to efficiently conjugate with haptens, especially peptides of various kinds. Thus, we compared our various carrier protein products for their ability to conjugate two peptides having very different chemical properties (hydrophilic vs. hydrophobic).

We tested two fluor-labeled peptides:

• CaM Kinase II Substrate 281-291, 5-FAM labeled
  (5-FAM-MHRQETVDCLK-NH2, hydrophilic)
  Reconstituted in ultrapure water for use
• EMP17, FITC-LC labeled
  (FITC-LC-TYSCHFGPLTWVCKPQGG-OH, hydrophobic)
  Reconstituted in DMSO for use

For cysteine coupling, 0.3 to 0.4mg of peptide was combined with 1mg of maleimide-activated carrier protein in a total reaction volume of 300µL. For coupling with EDC, 0.8mg of peptide was combined with 1mg carrier protein and an appropriate amount of EDC crosslinker in a total reaction volume of 300µL. Completed conjugation reaction were then desalted to remove unconjugated peptides. Conjugation efficiency was determined by measuring the relative fluorescent units (RFU) of the conjugates using a compatible fluorescent plate reader. The RFU was compared to a standard of unconjugated peptides. Results were normalized to the mcKLH-peptide conjugates.

These results (Figure 3) demonstrate that immunogen preparation (among conditions within in each respective method, maleimide or EDC) yields similar conjugation efficiencies regardless of carrier protein or peptide hydrophilicity.

Hapten-carrier protein conjugate recovery

Typically, hapten-carrier conjugates must be filtered or otherwise “cleaned up” following crosslinking reactions to remove excess crosslinker and exchange buffer components before using the prepared immunogen for immunization. Usually this is accomplished by gel filtration, i.e., a desalting column. Traditionally, and in the former versions of our Imject Kits, gravity-flow desalting columns were used for this purpose. Our updated kits use much faster, simpler, and convenient centrifuge desalting columns: Thermo Scientific Zeba Spin Desalting Columns, 7K MWCO.

To illustrate the advantages of this newer desalting technology, we compared results obtained with samples processed using both traditional drip columns (Thermo Scientific Pierce Dextran Desalting Columns) and the newer Zeba Columns (Figure 4).

Conjugate immunogenicity and antibody production

Ultimately, the goal of immunogen preparation is to produce an immune response and concomitant production of hapten-specific antibodies by the immunized host animal. KLH is widely recognized as the benchmark or standard of immunogenicity: if another carrier protein or a variant of KLH elicits an immune response similar to KLH (or mcKLH), it is considered to be a very good immunogen.

To compare and illustrate the immunogenic quality of our Imject PEGylated Maleimide Activated mcKLH and Blue Carrier Protein, we performed parallel conjugation, immunization and antibody-titer experiments using these products and the corresponding mcKLH. Rabbits (n=2) were immunized with a synthetic peptide (QVPRRMIGTDAC) coupled to each carrier protein by three different methods (maleimide, glutaraldehyde, EDC). Collected sera (pre-immune and 35-day post-immunization) were screened by ELISA for peptide-specific antibody (Figures 5 and 6).

For a variety of reasons, KLH is the standard carrier protein of choice for immunogen preparation in antibody production procedures. Peptide-carrier conjugation is nearly always done by EDC crosslinking or via maleimide-activation. The data presented here demonstrate similarities and differences among varieties of KLH and related Imject carrier proteins. All varieties perform similarly in most respects, except solubility. Redesigned Imject Conjugation Kits use spin desalting columns and protocol improvements to improve conjugate recovery and reliability.

Solubility tests

The different proteins were added at 200mg/mL to ultrapure water. Additional ultrapure water was added to the carrier protein solution to decrease the concentration by increments of 50mg/mL until the solution was clear with no insoluble protein material. At each water addition, the protein was allowed to dissolve for 10 minutes. Reported values are the highest concentrations at which the carrier proteins appeared fully dissolved.

Maleimide activation assay

We use a reliable cysteine coupling assay to determine the moles of maleimide per mole of carrier protein. Briefly, activated carrier proteins are dissolved in water at 10mg/mL and then diluted to 0.5mg/mL in Assay Buffer (0.1M sodium phosphate, 0.1M EDTA, pH 7.2). The diluted activated carrier proteins are transferred to a microplate (200µL/well). A dilution series of L-cysteine is prepared (1mg/mL to 0.25mg/mL) in Assay Buffer and 10µL of each dilution is added to a well containing protein solution. For a blank control, Assay Buffer is added to the test sample. The maleimide activated carrier protein is allowed to react with the cysteine for 2 hours at room temperature. Finally, the amount of noncoupled cysteine is determined using Thermo Scientific Ellman’s Reagent (5,5′-Dithio-bis-[2-nitrobenzoic acid]) (Part No. 22582). Samples are incubated for 15 minutes with 20µL of a 6.3mM Ellman’s Reagent prepared in Assay Buffer, after which the absorbance is measured at 405nm. The proportion (and absolute moles) of cysteine coupled is calculated by comparing the absorbance of the maleimide activated carrier samples to the absorbance of nonactivated carrier protein controls.

Peptide coupling efficiency

Fluor-labeled peptides (CaM Kinase II Substrate and EMP17) were from Anaspec.

For cysteine coupling, peptides were reconstituted at 10mg/mL, then 0.3 to 0.4mg of peptide was combined with 1mg of maleimide-activated carrier protein and conjugation buffer (Part No. 77164) for a total reaction volume of 300µL. For EMP17 peptide DMSO was added to a final concentration of 30% in the reaction and DTT added to a final molarity of 1mM. Reactions were incubated for 2 hours at room temperature. The conjugates were desalted to remove uncoupled peptide and EDTA.

For coupling with EDC, peptides were reconstituted at 20mg/mL. Carrier proteins were dissolved at 10mg/mL in ultrapure water. EDC was dissolved at 10mg/mL in ultrapure water immediately before addition. Then, 0.8mg peptide was combined with 1mg carrier protein and the volume adjusted to 300µL with EDC conjugation buffer. For EMP17 peptide, DMSO was added to a final concentration of 30%. Finally, 25µL of EDC solution was added and the reaction was incubated for 2 hours at room temperature. The reaction was then desalted into Imject Purification Buffer (Part No. 77159).

Desalting column tests

Tests were performed with Pierce Dextran Desalting Columns (Part No. 43230) and Zeba Spin Desalting Columns, 7K MWCO (Part No. 89889). Peptide conjugate samples (350µL) were desalted using columns equilibrated in Imject Purification Buffer (0.083M sodium phosphate, 0.9M sodium chloride, 0.1M sorbitol, pH 7.2). The Dextran Columns were equilibrated with 3 bed-volumes of buffer. The Zeba Columns were equilibrated by centrifugation with 3 x 1mL of buffer. Samples loaded on the Dextran Columns were desalted by gravity flow with 3 x 0.5mL aliquots of buffer; conjugates were recovered in about 1mL volume. Peptide-carrier conjugates were applied to the Zeba Spin Desalting columns, followed by a 50µL chase of buffer. The columns were centrifuged for 2 minutes at 1000 x g. Peptide-carrier conjugates were recovered in 350µL.

Conjugate immunogenicity

Rabbits (n=2) were immunized with a synthetic peptide (QVPRRMIGTDAC) coupled to each carrier protein by three different methods (maleimide, glutaraldehyde, EDC). Immunizations were performed using Thermo Scientific Pierce Custom Antibody Production Service (2-Rabbit, 70-day Protocol). Collected sera (pre-immune and 35-day post-immunization) were screened by ELISA for peptide-specific antibody. Samples were diluted and screened by an ELISA in which the immunizing peptide was coated directly onto the microplate. ABTS substrate (absorbance 405nm) was used for colorimetric detection of the HRP-conjugated secondary antibody.