Web: www.ImmunoK.com
Email: IKinfo@amsbio.com
IMMUNOKONTACT CUSTOM SERVICES:
Polyclonal Antibody Production
Basic Package
SERVICE
1
Peptide
synthesis
DESCRIPTION
TIME
 Custom FMOC Peptide synthesis up to 18 amino acids (20-30 mg).
Peptide with more than 18 amino acids will be charged pro rata. We can
provide assistance on designing peptide with respect to protein
homology, antigenicity, hydrophilicity, and synthetic suitability.
1.5 week
 HPLC purification if peptide purity is <90%.
2
Conjugation
 Conjugation of 4 mg peptide (using Cystein) to KLH.
0.5 week
 Conjugation to IKERSA (an unique 28 kDa recombinant protein from
Immunokontact, conjugation using Cysteine residue at the N-terminal of
peptide) for western blotting analysis and other functional studies.
TM
3
Immunization
and anti-sera
production

Conjugated peptides or recombinant proteins are used to immunize
rabbits. See next page for detail protocol. 
7-10 weeks
 Ten-week protocol to produce antibodies in 2 rabbits.
 Pre-immune and three immune bleeds (100-120 ml antiserum).
4
Test
 ELISA test until the titre reaches 1:4,000.
1 day
 Western blot analysis when ELISA reaches target titre.
5
Purification
 Protein G purification
1 week
 Antibodies are eluted using both pH11 and pH2.5 buffers and neutralized
immediately followed by dialysis in PBS

See Rabbit Polyclonal Antibody Production Protocol for detailed
description of steps 3-5.
6
10-13 weeks
Total
Additional Options
SERVICE
3
4
5
DESCRIPTION
TIME
Immunization
and anti-sera
production
 Add for each additional rabbit
Test
 Immunohistochemistry on tissues or cultured cells
1 week
 Immunoprecipitation on tissue or cell lysate
1 week
 Peptide affinity purification
1 week
Purification
 Extend protocol for each additional week
UNITED KINGDOM, INTERNATIONAL:
AMS BIOTECHNOLOGY (EUROPE) LTD.
SWITZERLAND
AMS BIOTECHNOLOGY (EUROPE) LTD.
63B MILTON PARK, ABINGDON OXON OX14 4RX
CENTRO NORD-SUD 2E, 6934 BIOGGIO LUGANO
TEL: +44 (0) 1235 828200
FAX: +44 (0) 1235 820482
TEL: +41 (0) 91 6045522
FAX: +41 (0) 91 6051785
DEUTSCHLAND:
AMS BIOTECHNOLOGY (EUROPE) LTD.
TEL: +49 (0) 69-779099
FAX: +49 (0) 69-13376880
Web: www.ImmunoK.com
Email: IKinfo@amsbio.com
RABBIT POLYCLONAL ANTIBODY PRODUCTION PROTOCOL
1
Subject
New Zealand White Rabbits.
2
Adjuvant
Complete Freund's Adjuvant (CFA) initially, followed by Incomplete Freund's
Adjuvant (IFA) for all subsequent injections.
3
Immunogen
Typically proteins and conjugated peptides. 50 to 100 µg immunogen is used for
each immunization.
4
Procedure
The immunogen is diluted to 0.5 ml with sterile saline and combined with 0.5 ml of
the appropriate adjuvant. The antigen and adjuvant are mixed thoroughly to form a
stable emulsion that is injected beneath the skin of the rabbit (subcutaneously) in
the area around the shoulders and intra-muscularly into the large muscle of the rear
legs. (That is about 1/4 of the antigen being used in each area). This provides
enhanced immune response from the sustained presentation of the immunogen.
5
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 2,500 RPM for
20 minutes.
6
Schedule
Week 0
7
Note
Bleed 10 ml (yields 5-8 ml pre–immune serum)
Immunize with 200 g/rabbit antigen in CFA.
Week 1
Immunize with 100 g/rabbit antigen in IFA.
Week 2
Immunize with 100 g/rabbit antigen in IFA.
Week 3
Immunize with 100 g/rabbit antigen in IFA.
Week 4
Immunize with 100 g/rabbit antigen in IFA.
Week 5
Test bleed 20-30 ml.
ELISA on appropriate cell/tissue.
Immunize with 100 g/rabbit antigen in IFA.
Week 6
1St Production bleed 20-30 ml if test positive.
Immunize with 100 g/rabbit antigen in IFA.
ELISA test again if previous tests were negative.
Week 7
2nd Production bleed 20-30 ml.
Immunize with 100 g/rabbit antigen in IFA.
ELISA test again if previous tests were negative.
The continuation of the project needs to be discussed if negative.
Week 8
3rd Production bleed 20-30 ml.
Immunize with 100 g/rabbit antigen in IFA.
Week 9
4th Production bleed 20-30 ml.
Immunize with 100 g/rabbit antigen in IFA.
Week 10
Terminating bleed 100-120 ml.
Protein G purification of pooled bleeds.
Average yield of purified Ig fraction is 100-150 mg.
Final ELISA and WB QC test.
The rabbits can be maintained on a ten-weeks cycle of immunizations and bleeds as
required by the project. The results of testing will determine whether the program
should be continued, modified, or terminated. Extra bleeds can be taken upon
customer’s requirement of extended project with extra charge.
UNITED KINGDOM, INTERNATIONAL:
AMS BIOTECHNOLOGY (EUROPE) LTD.
SWITZERLAND
AMS BIOTECHNOLOGY (EUROPE) LTD.
63B MILTON PARK, ABINGDON OXON OX14 4RX
CENTRO NORD-SUD 2E, 6934 BIOGGIO LUGANO
TEL: +44 (0) 1235 828200
FAX: +44 (0) 1235 820482
TEL: +41 (0) 91 6045522
FAX: +41 (0) 91 6051785
DEUTSCHLAND:
AMS BIOTECHNOLOGY (EUROPE) LTD.
TEL: +49 (0) 69-779099
FAX: +49 (0) 69-13376880