Bridged Nucleic Acids (BNA)
Oligonucleotide-Antibody conjugates have been used in numerous applications from
diagnostics to therapeutics. Researchers and assay developers worldwide continue to design new oligonucleotide-antibody conjugates for novel usages for siRNA-antibody delivery in therapeutic applications, vaccines adjuvant, or as pre-targeting cancer therapeutics.Oligonucleotide-antibody conjugates have been used in immuno-PCR reaction as a sensitive
method for protein detection and quantification.
Bio-Synthesis offers oligonucleotide synthesis and oligonucleotide-antibody conjugation by
combining our expertise in the field and over 30 years of a successful track record. We routinely assist clients in creating antibody-oligo/nucleic acid hybrid molecules using various cross-linking chemistries.
All antibody oligonucleotide conjugates are analyze by SDS-PAGE with less than 10% of unconjugated antibody. Nearly all antibodies are recovered when starting at 2-5 mg/ml in a proper buffer, .
Antibody molecules possess a number of functional groups suitable for modification
or conjugation purposes. Crosslinking oligonucleotide-antibody can be achieved through lysine
ϵ-amine and N-terminal α-amine groups. Carboxylate groups also may be used to couple with another molecule using the C-terminal end as well as aspartic aid and glutamic acid residues. Although amine and carboxylate groups are as plentiful in antibodies as they are in most proteins, the distribution of these functional groups is nearly uniform on the surface of antibodies. For this reason, if some of the modified or conjugated residues are located on the antigen binding sites, the method may produce partially active or inactive oligonucleotide-antibody conjugates that may not bind to the antigen.In such cases, an alternative conjugation method using a thiol reactive group by selectively cleaving an antibody with a reducing agent to create two half-antibody molecules, or using smaller antibody fragment such as Fab'. Conjugation done using hinge area-SH
groups will orient the attached oligonucleotide away from the antigen binding regions,thus preventing blockage of these sites and preserving activity.
The second alternative method of site-directed conjugation of antibody molecules
takes place at carbohydrate chains, typically attached to the CH2 domain within the
Fc region. Upon periodate oxidation, an aldehyde group can be introduced to the antibody,
which will allow it to react with an amine modified oligonucleotide.
Antibody is reduced at the hinge region to yield two half-antibody molecules, which then react with maleimide-activated oligos.
The price varies based on the project specifications. Our service includes materials and labor for conjugation only! Price does not include the cost of biopolymer synthesis
or purchase from a commercial vendor. If deemed necessary, biopolymer modification to introduce additional functional groups, extra linkers, or spacers will be an additional cost. Please contact us for a quote.
Up to 3 mgs of starting material and 1-2 mgs of antibody for initial pilot conjugationprior production, is required for a thorough optimization, and method development
of purification and analysis. We have been successful with as little as 200 micrograms in some cases. We provide a few test runs for our customers to test.The test conjugates are agreed upon within one week. Bio-Synthesis will also provide a scale up production. After conjugation, a standard desalting, or purification, a small percent of heterogeneous products containing single or multi-site conjugate per molecule.
Depending on project specification, a pool of heterogeneous products in a small percentage may exist.
Oligo synthesis and modification is manufactured under a strict quality control process.Analytical HPLC and MS analyses are performed in every development cycle. After activating amine modified-oligos with NHS ester-maleimide, followed by gel filtration to remove excess crosslinking reagents, reduction of the antibody is followed by the conjugation of the antibody to maleimide-activated. Final target conjugates must first be isolated
from excess or unreacted reagent. In many cases, simple dialysis removes unreacted reagent from the reaction solution (if the protein/antibody is significantly larger(>3-fold) than the modifying or coupling reagent). Additional purification such as stirred cell filtration, tangential flow filtration (TFF), or gel filtration chromatography may also be used to either remove excess reagent or to isolate and characterize the cross-linked product. Reagents that are similar in size or larger than the antibody (mostly protein and other biological molecules) may require other purification techniques such as affinity chromatography, ion-exchange chromatography,and hydrophobic interaction chromatography.
Cross-linked target molecules may then be further characterized by biochemical or
biophysical techniques for an additional fee. Once the product has been purified,it may be subject to many different types of studies including spectroscopic (MALDI-TOF,ESI, LC-MS Fluorescence), electrophoresis, immunochemical biochemical, and/or enzymatical analysis. QC (quality control) and QA (quality assurance) procedures are also followed independently to offer you double guarantee for the highest quality possible of every delivered conjugate. Moreover, our dedicated technical account managers will guide your project through every step of the process and constantly keep you informed of the latest project progress.
We can chemically link the intended components of a conjugated molecule, however, there exists the possibility that the binding sites/active sites of the protein can be altered/modified (partially or completely) independent of the stoichiometry used. Sometimes this loss of activity is caused by physically blocking the antigen binding sites during conjugation or by conformational changes in the complement-determining regions. Some proteins/antibodies are just too labile to undergo chemical modification reactions, regardless of the coupling methods used.
Bio-Synthesis can only guarantee the structure of our conjugates but not the suitability to specific biological applications.
For us to better understand your customized project, please complete our Bioconjugation Service Questionnaire. The more our chemists understand your project needs, the more accurate feedback we will be able to provide you. Provide us with your project details will enable to us to recommend the best reagents to use for your project. The most useful and readily available tools for bioconjugation projects are cross-linking reagents. A large number of cross-linkers, also known as bifunctional reagents, have been developed. There are several ways to classify the cross-linkers, such as the type of reactive group, hydrophobicity or hydrophilicity, and the length of the spacer between reactive groups. Other factors to consider are whether the two reactive groups are the same or different (for example, heterobifunctional or homobifunctional reagents), whether the spacer is cleavable, and whether the reagents are membrane permeable or impermeable. The most accessible and abundant reactive groups in proteins are the ϵ-amino groups of lysine. Therefore, a large number of the most common cross-linkers are amino selective reagents, such as imidoesters, sulfo-N-hydroxysuccinimide esters, and N-hydroxysuccinimide esters. Due to the high reactivity of the thiol group with N-ethylmaleimide, iodoacetate and a-halocarbonyl compounds, new cross-linkers have been developed that contain maleimide and a-carbonyl moieties. Usually, N-alkylmaleimides aremore stable than their N-aryl counterparts.
In addition to the reactive groups on the cross-linkers, a wide variety of connectors and spacer arms have also been developed. The nature and length of the spacer arm play an important role in the functionality. Longer spacer arms are generally more effective when coupling large proteins or those with sterically protected reactive side-chains. Other important considerations are the hydrophobicity, hydrophilicity, and the conformational flexibility. Long aliphatic chains generally fold on themselves when in an aqueous environment, which makes the actual distance spanned by such linker arms less than expected. Instead, spacers that contain more rigid structures (for example, aromatic groups or cycloalkanes) should be used. These structures, however, tend to be very hydrophobic which could significantly decrease the solubility of the modified molecules or even modify some of their properties. In such cases, it is recommended to choose a spacer that contains an alkylether (PEO) chain. Bio-Synthesis offers several cross-linkers with PEO chains, such as thiol-binding homobifunctional reagents, heterobifunctional based, and their derivatives.
Within 3-5 days upon receiving your project scope, we will provide you an appropriate quotation. An order can be placed with PO (Purchase Order) or major credit cards ( ). Your credit card will be billed under Bio-Synthesis, Inc.