Polyclonal Antibody Development

Immunization of various hosts (chickens, rabbits, alpacas, etc.) with the antigen of interest provokes an immune response that leads to the generation of antibodies recognizing a variety of different epitopes on the antigen. The antibody-containing fraction from the animal is then isolated and either used directly in the desired immunoassay or subjected to purification. In either case, the antibody preparation is ‘polyclonal’ (i.e. a mixture of different antibodies recognizing different epitopes on the antigen).

Advantages of this approach include:

• economical;
  
• large amounts of antibody produced;
• polyclonal nature of antibody preparation increases likelihood that antibodies will perform across a variety of sample types (native/denatured/fixed);
  
• polyclonality of antibody preparation results in binding to multiple epitopes on the same target molecule and consequently yields greater signal amplification than is realized by monoclonal antibodies.

Disadvantages of this approach include:

• greater likelihood of undesirable cross-reactivity (this can be reduced by immunizing with a peptide-based antigen targeting a defined epitope);
• continued production of antibody requires continued immunization of the host, as a result the binding characteristics of the antibodies obtained can change over time;
• no ability to isolate epitope-specific antibodies.

We can help you select the optimal host animal.

Some host animal options include Chicken, Rabbit, and Alpaca. Additional hosts are available as well. Consult our host selection guide to learn more about the particular advantages and limitations of each host animal, and get in touch with us for further guidance. Or, if you already know which host animal you would like to use, see below for processes.

Host Option 1: Chicken Polyclonal Antibody Development

Immunogen Selection (Variable timeline)

• Determination of optimal immunogen for your project.
• We can either use an immunogen supplied by you or, in consultation with you, we can design and synthesize a peptide immunogen that meets your criteria (species reactivity, degree of reactivity with related proteins etc.).
   

Immunization Schedule (10 weeks)

• Purified Immune IgY from two (2) chickens (approx. 800 mg of IgY per hen)
• Pre-Immune IgY from each chicken (~150mg of IgY per hen)
• Four (4) Immunizations per animal.
   

ELISA Report (Less than one week)

• Eggs are collected and IgY is analyzed by ELISA and compared to the pre-immune eggs to evaluate titer (recommended).
   

IgY Purification (1 week)

• Twelve (12) eggs are used to prepare purified IgY using our proprietary protocol yielding an IgY preparation that is >90% pure.
• The purified IgY prep can be used in most common applications (immunohistochemistry, immunocytochemistry, Western blotting etc.).
• To obtain only antigen-specific binders, the IgY prep can be further subjected to affinity purification (see below).
   

Affinity Purification (2 weeks)

• The IgY fraction is subjected to antigen-specific affinity purification yielding an antigen-specific antibody preparation that is >90% pure.

Host Option 2: Rabbit Polyclonal Antibody Development

Immunogen Selection (Variable timeline)

• Determination of optimal immunogen for your project.
• We can either use an immunogen supplied by you or, in consultation with you, we can design and synthesize a peptide immunogen that meets your criteria (species reactivity, degree of reactivity with related proteins etc.).
   

Immunization and Bleeds (8 weeks)

• Collection of 10 mL pre-immune bleed for use as control (~6 mL serum)
• 57 day immunization protocol- includes 5 immunizations and a test bleed taken after 3rd immunization.
• Single production bleed taken after 5th immunization (~15 mL serum)
• Serum can be used directly in most common assays (immunohistochemistry, immunocytochemistry, Western blotting etc.) or purified by either Protein A or affinity purification (see below).
   

ELISA Report (Less than one week)

• Test bleed analyzed by ELISA and compared to the pre-immune bleed to evaluate titer (recommended).
   

Protein A Purification (2 weeks)

• Production bleed is subjected to protein A purification yielding an IgG preparation that is >90% pure.
• The purified IgG prep can be used in most common applications (immunohistochemistry, immunocytochemistry, Western blotting etc.).
• To obtain only antigen-specific binders, the production bleed can alternatively be purified by affinity purification (below)
   

Affinity Purification (2 weeks)

• Production bleed is subjected to antigen-specific affinity purification yielding an antigen-specific antibody preparation that is >90% pure.

Host Option 3: Alpaca Polyclonal Antibody Development

Camelids produce an entirely different type of antibody that is devoid of light chains and is commonly referred to as a "heavy chain only antibody" (HCAb). The recombinant variable domains of HCAbs are known as nanobodies which possess unique properties such as small size that permits deep tissue penetration and crossing of the blood brain barrier.

Immunogen Selection (Variable timeline)

• Alpacas generally do not mount robust immune responses against small molecules or peptides.
• They have gained popularity due to the relative ease of cloning intact "single-domain" antibodies from their immune repertoire for use in phage display screening. Customers typically submit recombinant protein antigens for use as immunogens.
   

Immunization and Bleeds (8 weeks)

• Collection of 10 mL pre-immune bleed for use as control
• 63-day immunization protocol includes two test bleeds taken after second and fourth immunizations.
• Single production bleed taken after fifth immunization, up to ~250mL whole blood (can be shipped as whole blood or sent as serum).
   

ELISA Report (Less than one week)

• First and second test bleeds are analyzed by ELISA and compared to the pre-immune bleed to evaluate titer (recommended).