Phases  (Production Protocol)

Lead Time

I. Peptide Design and Synthesis
Custom Peptide synthesis up to 15 amino acids. Peptide with more than 15 amino acids will be charged for $15 per additional residue. We can provide assistance on designing peptide with respect to protein homology, antigenicity, hydrophilicity, and synthetic suitability with reasonable fee.

2 weeks

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
Conjugated peptides or recombinant proteins are used to immunize rabbits.
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 Time

13 weeks

     IV. Additional MAX Bleed
      •  MAX bleed 50 ml from each rabbit

1 week

     V. Extension (two additional booster injection, 20 day
         intervals 
      •  50 ml antiserum bleed after 50 days)

7 weeks 

     VI. ELISA Test
      •  Will conjugate peptide to BSA 
         ELISA test for final total bleed

1 week

     VII. Purification
      •  Peptide specific affinity purification
        ELISA data will be included for free

1 week

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 adjavant 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 
  needle gauge 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:

Genemed Synthesis offers phosphospecific antibody affinity purification which includes:

1. 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
2. 10 mg non-phosphospecific peptide (up to 15 amino acids) used for affinity depletion
3. Immunization of 2 rabbits
4. Phosphospecific and non-phosphospecific peptide ELISA titration of antibody for final bleeds
5. 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)