Polyclonal Antibody Development

Spring Valley offers two protocols for maximizing antibody production in terms of levels of antibody and volume of serum. These protocols represent a basic approach that can be extended and enhanced using the available options listed below or your custom study design.

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Standard Protocol

The standard protocol outlined below can be used with almost any antigen, usually of unknown or weak immunogenicity.

Day	Procedure
0	Pre-immunization test bleed
0	Primary immunization
21	Immunogen boost
42	Immunogen boost
52	Standard bleed
(ELISA available)
63	Immunogen boost
73	Standard bleed
(ELISA available)

Fast Protocol

The fast protocol can be used with almost any antigen, usually of known or strong immunogenicity.

Day	Procedure
0	Pre-immunization test bleed
0	Primary immunization
21	Immunogen boost
31	Standard bleed
(ELISA available)
42	Immunogen boost
52	Standard bleed
(ELISA available)

Protocol Options

The following options are available for tailoring the protocols to produce the antibodies you need: screening animals (test bleed) for existing antibodies prior to placement on a study, serum filtration through 0.45 micron filter, ELISA as screening/assessment tools, serum antibody purification, or your customized study parameters. In addition to these options, any protocol may be extended to obtain a larger volume of serum or to possibly increase antibody titers.

Standard Monthly Extension Protocol

Extention Day	Procedure
1	Immunogen boost
12	Standard bleed
27	Standard bleed (or exsanguination)

Antigen Preparation

Spring Valley can accept antigen for immunization in any form including, but not limited to, in solution, as a precipitate, as a cell suspension, or as a band in acrylamide gel. Freund’s Complete Adjuvant is used for the initial injection and Freund’s Incomplete Adjuvant is used for subsequent injections. Other adjuvants are available. The recommended minimum antigen dose per animal is 100 µg protein or 250 µg peptide. Minimum antigen requirements are listed below for several activities.

Minimum Antigen Requirements:

ELISA (up to 6 serum samples)	20 ug
Fast protocol (total per animal)	750 ug
Standard protocol (total per animal, or 5 mice)	1 mg
Protocol extension (total per animal)	250 ug

Antibody Production in Many Animal Species

Antibody production can be conducted in a variety of animals. Although the rabbit is, in most instances, the ideal animal for polyclonal antibody development, mice, rats, guinea pigs, ferrets, sheep, and goats are also available. The table below indicates expected serum volume per species per protocol.

Approximate serum volumes (ml) per bleed for multiple species

	Rabbit	Rat	Guinea pig & Ferret	Mouse	Sheep & Goat
Test bleed	5	0.25	0.5	0.2	15
Fast protocol
Day 0 pre-bleed	5	0.25	0.5	0.2	50
Day 31 standard bleed	15	0.5	1	0.2	400
Day 52 standard bleed	15	0.5	1	0.2	400
Standard protocol
Day 0 pre-bleed	5	0.25	0.5	0.2	50
Day 52 standard bleed	15	0.5	1	0.2	400
Day 73 standard bleed	15	0.5	1	0.2	400
Protocol extension
Day 12 standard bleed	15	0.5	1	0.2	400
Day 27 standard bleed	15	0.5	1	0.2	400
or
Day 27 exsanguination	60	5	6	0.5	1500*
Exsanguination	60	5	6	0.5	1500*
Plasmapheresis	-	-	-	-	1500
* Plasmapheresis performed in lieu of exsanguinations.

Due to the inherent variability between immunogens and animals, Spring Valley does not guarantee the quality or quantity of the polyclonal antibody.

Peptide Synthesis and Conjugation
Spring Valley protein chemists can analyze your protein sequence to predict potentially immunogenic peptide sequences. We strongly recommend you use this service unless you have already done so. There is no fee for this service if you have your peptide synthesized with us. Peptides are synthesized to greater than 85% purity by HPLC analysis. Other purity levels, analytical methods and modifications are available upon request. Conjugation of peptides to KLH is standard, but other carriers (ie. BSA, OVA) are available.