Buccutite™ Conjugation Kits: Quick and Easy Antibody Labeling

Crosslinkers for Bioconjugation

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Crosslinkers are a family of compounds that can be used to join together two or more macromolecules, such as proteins. The modification of proteins through crosslinking techniques provides analysis and insight into complex protein interactions as well as interaction domains. Crosslinking techniques are also an important pre-requisite to affinity purification of proteins and critical to immunological research. To select the best crosslinking method for an experiment, it is imperative to understand the functional and compositional structure of proteins that make them favorable targets for modification.

Complex protein structures are comprised of small amino acid building blocks linked together to form polypeptide chains. These amino acid building blocks contain many basic functional groups, such as amines and thiols, that can be targeted for chemical modification. This targeting mechanism provides the foundation for the development and production of a variety of synthetic crosslinkers.

The most critical feature of a crosslinker is its reactive ends which target specific functional groups on proteins. The most commonly used reactive group, NHS esters, is synthesized into homobifunctional crosslinkers for use in protein conjugations. These crosslinkers serve as powerful tools in protein analysis and detection techniques. Unfortunately, certain disadvantages and adverse side reactions such as self-polymerization are commonly associated with these crosslinkers. To combat this, Buccutite™ crosslinking technology was developed to provide a robust and efficient alternative for protein conjugations.

Functional Groups and Their Respective Reagents

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The most common and simplest functional group targeted for biomolecular modification is primary amines (-NH2) located on the N-terminus of polypeptides and the side chains of lysine residues. Because these primary amines are typically nucleophilic and positively charged, they have an outward facing conformation in physiological conditions. This makes them readily accessible and favorable targets for crosslinkers without denaturing the protein structure. Of the vast synthetic chemical groups targeting primary amines, N-hydroxysuccinimide esters (NHS esters) have been used with the greatest success.



NHS esters are reactive groups formed by EDC activation of carboxylate moieties in alkaline conditions. Activated NHS ester reagents react with primary amines on target proteins forming stable amide bonds.

Disadvantages of NHS Esters

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Despite their simplicity and ease of use, homobifunctional NHS ester crosslinkers have various drawbacks, specifically in protein-protein conjugation. These include self-polymerization, hydrolysis and purification.

First, homobifunctional NHS esters have a disadvantage in site specific protein-protein conjugation techniques because of its inability to differentiate between amine groups on the target protein from those on the label protein. This results in an uncontrollable tendency to self-polymerize proteins. For example, the use of homobifunctional crosslinkers when preparing antibody-HRP conjugations is not ideal because of the unfavorable antibody-antibody conjugations that may occur.

To mitigate this problem, AAT Bioquest has developed a convenient ReadiUse™ Preactivated HRP-NHS Ester (Cat# 11025) that contains the mono-NHS ester of HRP (Figure 2 for mechanism). By pre-activating HRP with one NHS ester, the rate of successful protein-HRP conjugation increases while the potential for self-polymerization of the target protein decreases. Our pre-activated HRP-NHS ester is ideal for labeling antibodies with HRP enzymes in a simple and efficient manner for use in ELISA and other immunoassay applications.



Aside from self-polymerization, hydrolysis of NHS esters and amide bonds is another undesirable drawback associated with NHS ester crosslinking. Hydrolysis competes with the primary amine reaction as the pH of the system increases resulting in less efficient crosslinking of protein targets. Given that optimal conjugation systems occur at pH 8-10, it is clear that this could pose an issue. A possible remedy to minimize the effects of hydrolysis and maximize the amine modification is to use a high concentration of target protein, but this can prove costly for researches.

In addition, after conjugation, purification steps must be taken to remove excess unreactive reagents and proteins when using homobifunctional NHS ester crosslinkers. These final steps present additional complications. First, purification lowers the yield of modified protein conjugates due to the residual loss of modified proteins within purification columns. Secondly, these additional steps prolong the duration it takes to successfully complete conjugation.

The limitations, complications and targeting variability of homobifunctional NHS esters sparked the development of Buccutite™ crosslinking technology. This refined crosslinking technology addresses many problems associated with NHS ester crosslinkers while greatly improving upon the mechanisms and range of applications in which it can be used.

Buccutite™ Crosslinking Technology

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Buccutite™ crosslinking technology provides a simplistic and efficient approach to conjugate proteins with another macromolecule such as an antibody or an enzyme. Buccutite™ crosslinking technology utilizes two exclusive linkers, Buccutite™ FOL, SE and Buccutite™ MTA, SE. The unique properties of these Buccutite™ crosslinkers allow them to function separately as two halves of a single homobifunctional crosslinker. Each Buccutite™ crosslinker is engineered to have an amine-reactive group on one end which specifically targets primary amines on the desired protein. The other end contains our proprietary Buccutite™-reactive group, which has a high degree of affinity for only binding its respective Buccutite™ MTA/FOL crosslinker counterpart. When each is present, the two Buccutite™-protein conjugates covalently link together at their Buccutite™-reactive site, "like buckling a seat belt," to successfully create a protein-protein conjugate.

Sample Protocol

Utilizing a sequential conjugation technique, one can perform protein modifications with Buccutite™ crosslinking technology in three simple steps:

1. Run Protein 1-Buccutite™ MTA reaction by adding Protein 1 solution directly into the vial of lyophilized Buccutite™ MTA, mix and remove free MTA, SE by desalting.
2. Run Protein 2-Buccutite™ FOL reaction by adding Protein 2 solution directly into the vial of lyophilized Buccutite™ FOL, mix and remove free FOL, SE by desalting.
3. Crosslinking Protein 1-Buccutite™ MTA & Protein 2-Buccutite™ FOL conjugates is initiated by mixing the two conjugates together under relatively neutral conditions.

Advantages of Buccutite™ Crosslinking Technology

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Buccutite™ crosslinking technology improves upon amine-reactive conjugation techniques, creating a more efficient and robust two-step conjugation system. Individual preparation of each protein with its respective Buccutite™ crosslinker provides more control over the conjugation process while reducing the possibility of adverse side reactions. Since Buccutite™ crosslinkers are exclusively amine reactive, the use of catalysts and reducing reagents are no longer needed.

Homobifunctional NHS ester crosslinkers randomly target amine groups on proteins, allowing for self-polymerization and making it difficult for site-specific conjugation. The unique reactive property of each Buccutite™ crosslinker provides a more site specific conjugation technique. Each Buccutite™ crosslinker has one amine-reactive end for conjugation to a target protein and one Buccutite™ MTA/FOL-reactive end that binds only to its linker counterpart for successful protein-protein conjugation. Replacing one amine-reactive end with a Buccutite™ MTA/FOL reactive-end eliminates the self-polymerization experienced when using homobifunctional NHS ester, yielding highly stable macromolecular conjugates.

Buccutite™ technology, such as ReadiLink™ antibody labeling kits, provides a more refined approach to protein conjugations by eliminating the purification steps. Buccutite™ crosslinkers eliminate these purification steps because of the high degree of affinity they have to link together proteins only at their Buccutite™ MTA/FOL reactive site. This prevents any unreactive Buccutite™ crosslinkers from self-polymerizing proteins and interfering with the immunoassay of the desired protein-protein conjugation. This distinct feature of Buccutite™ crosslinkers proves useful in sandwich ELISA immunoassays where substrates are captured and detected between two layers of antibodies. Treatment of enzyme-linked secondary antibodies with Buccutite™ limits enzyme-antibody conjugation specifically to the secondary antibody and not to the capture antibody coating the microplate well.

Extensive testing and quality control have also demonstrated that Buccutite™ technology significantly improves conjugation yield over traditional labeling methods. For example the yield of modified proteins increases from 30%, when using NHS ester crosslinkers, to 50-60% when using Buccutite™ crosslinking technology. This makes Buccutite™ a fast and convenient alternative for protein conjugation, particularly when sample protein quantities are limited.

Applications for Buccutite™ Crosslinking Technology

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Protein conjugates are extensively used in the purification and detection of complex biological samples. Commonly, antibodies are popular targets for bioconjugation with enzymes, biotin, and fluorescent labels for use in ELISA assay and flow cytometry. To facilitate the synthesis of protein-protein conjugates for use in immunoassays, AAT Bioquest provides a ReadiLink™ Rapid Protein Crosslinking Kit (Cat# 1315) for robust and efficient crosslinking. This kit comes with all the necessary components to effectively link two desired proteins. Included are both Buccutite™ MTA and FOL crosslinkers and the necessary reaction buffer needed to perform protein-protein conjugation. This method uses our distinct Buccutite™ crosslinking technology to synthesize protein-protein conjugates at high yields.

Enzyme-Linked Immunosorbent Assays (ELISA)

ELISA is a plate-based immunoassay technique that uses a solid-phase to detect antigen-antibody interactions in a biological sample. Antibodies are typically linked to an enzyme, such as horseradish peroxidase (HRP) via amine reactive groups, to amplify detectable colorimetric changes brought about by antibody-antigen interactions. Buccutite™ crosslinking technology provides an easy and robust alternative for antibody-HRP conjugations which, unlike traditional homobifunctional NHS esters, occur without the competing hydrolysis and self-polymerization side reactions. Using homobifunctional NHS esters for this protein-enzyme conjugation would require intermediate purification steps to remove unreactive NHS esters resulting in a low yield of antibody-enzyme conjugates. Replacing NHS esters with Buccutite™ crosslinkers allows for site specific conjugation of antibodies to enzymes without purification, boosting conjugation yields 50-60% modified proteins.



For convenience purposes, AAT Bioquest provides a ReadiLink™ Peroxidase Antibody Conjugation Kit (Cat# 5503) to facilitate an efficient way to conjugate antibodies to HRP. This kit provides HRP pre-activated with our proprietary linker, Buccutite™ FOL, ready for conjugation. Simply prepare the target antibody with the Buccutite™ MTA linker, and then mix the two Buccutite™ conjugated molecules together for protein-protein conjugation (Figure 4 for mechanism). It enables faster and quantitative conjugation of antibody to HRP with higher efficiencies and yields, ready for immediate use in ELISA assays.

Flow Cytometry

Another widely accepted application for protein modification is the antibody-fluorophore conjugates used in flow cytometry. This provides researchers with a powerful technique to analyze multiple parameters of individual cells within a heterogeneous sample, giving insight to cell characteristics such as size, structural complexity and phenotype. Common fluorescent probes used in these applications are phycobiliproteins because of the many advantages associated with them. Phycobiliproteins, such as allophycocyanin and r-phycoerythrin, are quench-resistance as a result of their protein backbone and exhibit long-wavelength fluorescence emissions. This makes them preferred fluorescent probes for applications that require high sensitivity or simultaneous multicolor detection.

Allophycocyanin (APC, Cat# 2554) and r-phycoerythrin (PE; Cat# 2558) are bright fluorescent proteins with high absorptivity and quantum efficiency that can easily be linked to antibodies for detection in immunoassays. Homobifunctional NHS esters are used to synthesize these antibody-fluorophore conjugates. However, using NHS esters is not the most efficient crosslinking technique for this type of protein conjugation. NHS esters random targeting of amine groups will self-polymerize antibodies lowering the yield of antibody-fluorophore conjugates. This can make it difficult for detection in flow cytometry. For this reason, AAT Bioquest offers a ReadiLink™ Rapid Protein Crosslinking Kit (Cat# 1315) to produce efficient and highly stable antibody-fluorophore conjugates for use in flow cytometry. This kit provides everything necessary to link antibodies and fluorophores together with a high conjugation yield for optimal results in immunoassay applications.

References

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