|
Phases |
Lead Time |
|
I. Peptide synthesis
- Peptide synthesis up to 15 amino acids.
- Each phospho-residue (Phospho-Ser, Tyr or Thr)
|
2 week |
|
II. Conjugation
- Conjugation of 5 mg peptide (using Cysteine) to KLH.
- 10 mg free peptide will be provided for client
|
1 week |
|
III. Immunization and anti-sera production
- Ten-week protocol to produce antibodies in 2 rabbits.
- Pre-immune 5 ml/per rabbit and total final bleeds from two rabbits (100 ml antiserum) will be provided to client.
|
10 weeks |
|
Total Lead Time |
13 weeks |
Optional Services:
|
IV. Additional MAX Bleed
• MAX bleed 50 ml from each rabbit |
1 week |
|
V. Extension
(two additional booster injection, 20 days interval, 50 ml antiserum bleed after 50 days) |
7 weeks
|
|
VI. Test
ELISA test for final total bleed (including conjugating peptide to BSA) |
1 week
|
|
VII. Purification
- Synthesis the control peptide of the same sequence without phospho residue.
- Phospho-peptide antibody purification by 2-steps peptide affinity purification including phospho-peptide column and non-phospho-peptide column
- ELISA data will be included for free
|
1 week
|
Polyclonal Antibody Production Protocol
Protocol Definition:
|
Subject: |
|
New Zealand White Rabbits |
|
Adjuvant: |
|
Complete Freund's Adjuvant (CFA) initially, followed by incomplete
Freund's Adjuvant (IFA) for all subsequent injections. |
|
Immunogen: |
|
Typically, proteins and conjugated peptides. Amount of immunogen used
for each immunization is 0.5 mg /per rabbit /per injection. |
|
Procedure: |
|
The immunogen is diluted to one mL with sterile saline and combined
with one mL of the appropriate adjuvant. The antigen and adjuvant are
mixed thoroughly to form a stable emulsion which is injected
subcutaneously and provides enhanced immune response from the
sustained presence of the immunogen. |
|
Bleeds: |
|
Blood is collected from the central ear artery with a 19 gauge needle and
allowed to clot and retract at 37°C overnight. The clotted blood is then
refrigerated for 24 hours before the serum is decanted and clarified by
centrifugation at 2500 rpm for 20 minutes. |
|
Schedule: |
|
|
Day 1 |
Bleed 25 mL (yields 10 mL pre-immune serum). 1 st Immunize with antigen in CFA. |
|
Day 20 |
2 nd Immunize with antigen in IFA. |
|
Day 40 |
3 rd Immunize with antigen in IFA. |
|
Day 50 |
Test Bleed 10 mL for ELISA screen(internal quality control only, not provide to client). |
|
Day 60 |
4 th Immunize with antigen in IFA. |
|
Day 70 |
Final Total Bleed 50 mL from Each Rabbit. | |
Phosphospecific Antibody Affinity Purification:
Genemed Synthesis offers phosphospecific antibody affinity purification which includes:
- Phosphospecific peptide synthesis (up to 15 amino acids) -- 10 mg used for phosphospecific epitope affinity purification, 10 mg used for antibody production, and 10 mg delivered to client
- 10 mg non-phosphospecific peptide (up to 15 amino acids) used for affinity depletion
- Immunization of 2 rabbits
- Phosphospecific and non-phosphospecific peptide ELISA titration of antibody for final bleeds
- Pre-immune sample of serum (5 mL/each rabbit) and anti-serum (100 mL total) from two rabbits
- Affinity purification of serum
- Shipment of purified phosphospecific antibody which does not cross-react with
non-phosphospecific peptides (confirmed by ELISA)
|
