CellMosaic® has designed this personalized antibody-oligo conjugation kit to work with any antibody and an amine-modified oligo. The user supplies the amine-modified oligo (preferably a C6 amino modified oligo), which is readily available from many commercial oligo suppliers. Using the kit components, the customer converts the amine-oligo to a maleimide-oligo (Step 1A in Scheme 1). The customer also supplies their own antibody. Using the kit components, the customer converts some of the surface amines of the antibody to free thiol groups (Step 1B in Scheme 1), followed by reaction of the maleimide-modified oligo to generate the conjugates (Step 2 in Scheme 1). If there are any unreacted thiols on the antibody, they are subsequently capped (Step 3 in Scheme 1). The final conjugate is desalted and buffer exchanged with PBS buffer.
This kit provides materials to perform one (CM53405x1) or three (CM53405x3) antibody oligo conjugations. Scale of the reaction: 0.25−1 mg antibody (protein content).
Note to previous users: If you purchased this kit previously, the current one is an upgrade/improved kit with slightly different components from the first-generation kit, including:
- More precise loading (~2 oligos per antibody) for better performance;
- Increased recovery of the conjugate (~85%);
- No restriction of the oligo with high number of nucleobases;
- Able to accommodate limited antibody sample up to 0.25 mg.
Requirement for antibody:
- Amount: 0.25−1 mg IgG needed per reaction
- Preferably >90% pure by gel electrophoresis
Requirement for amine-oligo (≥5-mer):
- Amount: 4−15 nmol oligo content as measured by UV
- HPLC purified and lyophilized oligo with amino-oligo content ≥90% by C18 HPLC.
- Additional components of acid or other molecules containing primary or secondary amine groups and thiol should NOT be present
- ≥5-mer. (oligos longer than 60-mer may result in lower loading and high impurities)
Note: You can purchase HPLC purified C6 amino-modified oligo from standard oligo manufacturers up to 90-mer length for this kit preparation. Oligos tested at CellMosaic for this kit were from either Integrated DNA technologies (https://www.idtdna.com/) or TriLink Biotechnologies (https://www.trilinkbiotech.com/). We have not tested oligos made by other oligo manufacturers. It is highly recommended that customers analyze and quantify oligos prior to using them (Disclaimer: CellMosaic has no financial interest in IDT or TriLink).
Key Features of this Antibody-Oligo Conjugation Kit:
Offers a convenient way to prepare antibody−oligo conjugate with heterobifunctional crosslinking reagents
- Optimized precise loading with an average two oligos per antibody for high performance
Preparation can be done in a day (less than 6 h) with various break points
- ≥90% conjugate if the quality of the oligo is at par
All reagents and supplies included for preparation to purification
- Options to choose tailored services at CellMosaic after conjugation
- o You can choose to send your conjugates to CellMosaic for HPLC analysis of the sample or complete removal of trace oligo impurities and unreacted antibody.
Scheme 1: Synthetic route to antibody−oligo conjugate
Typical Kit Performance Data (LC analysis, CellMosaic)
Antibody information: A therapeutic antibody (human IgG1 subtype)
Oligo information: HPLC-purified 18mer oligo with 5’ NH2-C6 modification
Kit lot number: S331.S9.040721
Scale of the reaction: 1 mg of antibody
Specification of the final conjugates:
Calculated average DOL: 1.9 Unreacted antibodies: 9.3%
Unreacted oligo: ~0.5% Conjugate Purity: >90% (85% recovery)
Figure 1: Size-exclusion HPLC analysis of the antibody (Ab, red), oligo (blue), and purified antibody-oligo conjugates (green).