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Antibodies specialized programs
Antibodies against Post Translational Modifications
Eurogentec has acquired a strong expertise in producing antibodies against PTMs (Post Translational Modifications). We can target classical modifications such as phosphorylation, acetylation, and methylation as well as more complex modifications with high steric hindrance like Ubiquitinylation or SUMOylation.
PTM program step by step
1. Antigen Design
Design and synthesis of 2 peptides (1 non-PTM + 1 harboring the PTM)
2. Antigen coupling
Coupling of the PTM peptide to a carrier
3. Immunization
Immunization of the hosts using the PTM peptide following the Speedy 28-day or a classical 87-day protocol.
Various epitopes
Please note that peptides used as immunogens are typically 13 to 16 amino acid long. Knowing that the size of an epitope may be as short as 5 to 6 amino acids, one peptide may contain several epitopes, each targeted by a specific antibody.
Antibodies mixture
The immune system of a host immunized with a PTM peptide will raise antibodies specifically targeting that antigen, i.e. the PTM-harboring peptide. Some epitopes will likely be located within the peptide sequence outside the PTM, while others will encompass the PTM. As a consequence, the host serum will contain a complex mixture of polyclonal antibodies, some recognizing the PTM (in blue), some others recognizing the non-modified peptide part (in yellow) These peptide-specific antibodies will be mixed up with antibodies targeting the protein carrier, strongly immunogenic, as well as unrelated antibodies.
4. Analysis of the immune response
The serum of the immunized host(s) is analyzed for the presence of anti-PTM antibodies by ELISA using the non-PTM and PTM peptides.
5. Purification
To specifically sort out the PTM-specific antibodies, a double affinity purification is performed. The serum is first passed through a column containing the PTM-peptide (in blue): non peptide-related antibodies are collected in the flow through (‘FT1’), and all antibodies specifically binding to the peptide are subsequently eluted (‘Peak 1’).
The ‘Peak 1’ sample is then passed through a column containing the non-PTM peptide. The PTM-specific antibodies are collected in the flow through (‘FT2’), while the peptide-specific, but not PTM-specific antibodies (in yellow) are eluted (‘Peak 2’).
This double purification step will allow obtaining polyclonal antibodies specific to the PTM, but a side-consequence is that the titer is lower, compared to a one-step classical purification.
6. ELISA
ELISA testing of the purified pAb (FT2 and Peak2) against the non-PTM and PTM peptides
7. Deliverables
Both FT2 and Peak2 are delivered to the customer, together with any remaining peptide (PTM or non-modified)
PTM program overview
| Speedy Anti-PTM* | Classical Anti-PTM* | |
|---|---|---|
|
Eurogentec designs and synthesises two peptides : one modified with the PTM and one unmodified
|
Eurogentec designs and synthesises two peptides : one modified with the PTM and one unmodified
|
|
| Peptide synthesis | 1 unmodified peptide | 1 unmodified peptide |
| >70% purity | >70% purity | |
| 10-13 aa | 10-13 aa | |
| 15-20 mg | 15-20 mg | |
| 1 modified peptide (PTM) | 1 modified peptide (PTM) | |
| >70% purity | >70% purity | |
| 10-13 aa | 10-13 aa | |
| 15-20 mg | 15-20 mg | |
| Coupling to carrierb | 5 mg of modified peptide | 5 mg of modified peptide |
| Coupled to | Coupled to | |
| KLH | KLH | |
| BSA | BSA | |
| OVA | OVA | |
| THY | THY | |
| or MAP carrier | or MAP carrier | |
| Available hosts (Number of host immunized animals) | Rabbit (2) | Rabbit (2) |
| Rat (6) | Rat (6) | |
| Guinea-pig (6) | Guinea-pig (6) | |
| ELISA Guaranteec | (O) | (O) |
| Affinity purification | Double purification of 50 mL serum | Double purification of 50 mL serum |
| QC validation | ELISA and SDS-PAGE | ELISA and SDS-PAGE |
| Delivery | Purified antibody | Purified antibody |
| Remaining serum | Remaining serum | |
| Remaining peptidesd | Remaining peptides |
a.The dates of injection and bleed in these schedules may be subject to slight changes depending on the program specifications
b. Please note that the carrier choice may significantly impact the immunization result, as for example KLH is usually much more antigenic than BSA, leading to much higher Ab titers.
c. The guarantee only applies for peptides when approved by Eurogentec
d.There is no a minimum amount of remaining peptide guaranteed after Coupling, Elisa tests and purification. If a specific quantity of unconjugated peptide is requested, it will be provided as separated aliquote for additional fee.
*Post Translational Modification
(O) optional