Polyclonal IgY Antibody Development

Aviva is one of the most experienced IgY antibody companies in the world, providing complete antibody production services from gene sequences to antibody purification. Aviva's antibody production service is a one-stop full service: Understanding our client's specific needs, producing and delivering the products according to schedule, and providing technical support post-delivery. We respect our clients' confidential information, and guarantee that all intellectual property remains with the client.

Here is our standard protocol for IgY custom antibody development:

Also, learn more about our ISO13485 compliant antibody development, validation, and production.

Polyclonal IgG Antibody Development

Aviva has more than 15 years of rabbit polyclonal IgG production experience providing complete antibody production services from gene sequences to antibody purification. Aviva's antibody production service is a one-stop full service: Understanding our client's specific needs, producing and delivering the products according to schedule, and providing technical support post-delivery. We respect our clients' confidential information, and guarantee that all intellectual property remains with the client.

There are a variety of protocols and schedules that can be used for antibody production. Each protocol is designed to meet the timeline, budget, quality, and specifications of the client.

Here is our standard protocol for IgG custom antibody development:

Also, learn more about our ISO13485 compliant antibody development, validation, and production.

Phospho-Specific Antibody Development

The key to generating phospho-specific antibodies is to design specific and immunogenic antigens. Aviva has successfully produced phospho-specific antibodies that are specific to the phosphorylation sites of interest to research scientists. Aviva's phospho-specific antibody development service includes the following steps:

Antibody Design and Peptide Synthesis:

The common antigen for a phospho-specific antibody is a synthetic peptide carrying one or two phospho residues, tyrosine, threonine, or serine. Clients provide information about the desired phospho residue and its flanking sequence, then Aviva will design the phospho-peptide. Two peptides will be synthesized, a phospho-peptide and a non-phospho-peptide.

Antibody Generation:

Antibody production will use Aviva's expertise in producing polyclonal antibodies. Antibodies from egg yolk (or IgG in serum) will be prepared and further purified.

Phospho-Specific Purification:

Immunization with a phosphopeptide may generate two different types of antibodies:
a. Phospho-specific antibodies.
b. Pan-specific antibodies against the non-phosphoryl polypeptide.

Two antigen-affinity columns will be used to purify the phospho-specific antibodies and pan-specific antibodies. The first column to be used is the non-phosphopeptide affinity column. Antibodies recognizing the non-phospho residues of the peptide will bind to the column and be eluted as pan-specific antibodies. The flow-through fraction contains non-specific antibodies and antibodies that are specific to the phospho-residue. The collected flow-through fraction will then be applied to the second column, the phospho-peptide column. Antibodies recognizing the phospho-residue will bind to the column and then be eluted as phospho-specific antibodies. Non-specific antibodies will be removed as flow-through.

Antibody Characterization

Initial ELISA characterization will test the degree of cross reactivity of the two products toward each substrate. In general, there should be less than 0.1% binding of non-phospho peptide compared to the targeted phosphorylated peptide. Subsequent testing can involve WB, IHC, ICC, etc, for analyses of the antibodies produced, using available cell lines or samples provided by client. Aviva will collaborate with the client to design immunogen sequences and develop the specific project protocol. Each project delivers phospho-specific and pan-specific antibodies, antigen samples, pre-immune serum and total antibody.

Aviva will collaborate with the client to design immunogen sequences and develop the specific project protocol. Each project delivers phospho-specific and pan-specific antibodies, antigen samples, pre-immune serum and total antibody.

IgY Monoclonal Antibody Development

Aviva Systems Biology has many years of experience developing scFv chicken IgY antibodies. Chicken IgY scFv fragments combine the advantages of chicken IgY polyclonal antibodies, monoclonal antibodies, and scFv recombinant antibodies.

Recombinant scFv single-chain antibodies are gaining popularity in the therapeutic and diagnostic industries. Advantages of scFv antibodies include:

• Higher specificity
• Alterable affinity after clone selection by random mutagenesis
• Highly scalable manufacturing - fragments are expressed in bacteria and purified using regular protein tags
• Lower cross-reactivity due to the absence of invariable regions
• Consistency and reproducibility

Our general process for scFV antibody development is as follows:

Antigen is prepared using bioinformatics analysis and our proprietary fusion domains and vectors. Chickens are immunized and polyclonal antibodies are analyzed to indicate immune response of the host. Once the chicken with the best immune response is identified, its B-cells are isolated, and total RNA is prepared. Total RNA is later used for cDNA library generation and subsequent phage library preparation. Positive clones are selected and scFv fragments are expressed in E. coli.

Process Summary

• Immunization and Purification of Polyclonal IgY
• IgY Fragment coding cDNA preparation
• Phage library construction
• Phage library screening
• Antibody expression in E. coli
• Antibody characterization

IgG Monoclonal Antibody Development

Aviva Systems Biology has over 15 years of experience in development and production of monoclonal antibodies. Our experienced scientific team will work to design a protocol that will meet your needs for research, product development, or Clinical or IVD use.

Standard Hybridoma Development Description

I. Immunization

• Purchase of 5 Balb/C mice or 3 Fisher rats
• Pre-immunization test bleed for negative baseline
• Immunizations and test bleeds
• Antibody titer of test bleeds by ELISA
• One or two mice or rats with the best titers are selected for fusion
• Pre-spleen collection boost before fusing
• Typically 70 – 90 days depending on antigen and immune response

II. Fusion and Selection

• Lymphocytes from 2 mice or one rat are pooled and fused.
• Fused cells are grown on HAT selective single-step cloning media.
• Clones are transferred to 96-well plates and expanded.
• 10 - 14 days

III. Screening and Expansion of Hybridoma Clones

• Primary screening of hybridoma tissue culture supernatants by indirect ELISA.
• Positive clones are further tested by ELISA.
• Positive clones are isotyped and subcloned to identify stable expressing clones.
• Further evaluation by the client if needed (ship supertantant to client).
• Report to client
• 14 - 21 Days

IV. Subcloning and Cryopreservation

• Selection of 1 to 5 clones in conjunction with client.
• Cryopreservation of at least 5 vials per selected clone.
• Storage of vials for 3 months at no charge.
• Long term storage available (optional).
• Report to client
• 14 - 21 days

V. Antibody Production and Purification (Optional)

• Aviva offers antibody production and purification services to meet the needs of research, diagnostic development, or for use in FDA or CE medical devices