FRENQUENTLY ASKED QUESTIONS

What animal should I choose to raise polyclonal antibodies ?

The choice of animal is dependent on practical factors such as the source of the antigen, quantity of serum required for research, the titer of the antibody and the time required to generate antibodies. The rabbit is the most frequently used host for polyclonal antibody production. Covalab uses New Zealand White, Specific Pathogen Free (SPF) rabbits.

What is a SPF animal?

Specific Pathogen Free animals are bred uniquely for the production of antibodies. These animals are housed in a pathogen free environment ? all air, food, water entering the environment is guaranteed not to contain foreign organisms that could provoke an immune response. The sera of these animals have a very low background signal due to the restricted environment in which they live.

When should I use chicken antibodies?

Chickens are not mammals and therefore produce high-avidity antibodies to mammalian antigens. The production capacity of the chicken is very large. Further, the Fc region of the IgY is different to that of mammalian IgG?s hence a reduced background.

How many egg yolks does a standard immunisation protocol yield?

Chickens typically lay an egg a day. The egg collection starts at the first bleeding date. Consequently the yield is approximately 30 eggs.

How should I supply my protein?

The protein can be supplied lyophilised, in solution, included in gel slices or attached to sepharose beads. What buffers are compatible with immunizations? Most biologically related buffers are compatible with immunization, this includes PBS, Tris buffer, phosphate buffer, in moderate molarity. The antigen should be at a concentration of 1mg/ml or greater.

How should I ship my antigen?

Samples should be shipped on dry ice whenever possible. Peptides and SDS-PAGE gel slices can be sent at room temperature.

What is the use of the pre-immune serum?

Evaluation of pre-immune serum for a particular application permits the selection of the animal whose pre-immune serum gives the lowest background for the subsequent production of antibodies. The criterion for selection is no-signal when used in the procedure (eg no band Western Blot) due to the presence of previously generated antibodies. Pre-screening is particularly important when making antibodies against common bacterial, viral or common allergens. We recommend screening 5 to 10 animals and selecting the best for immunization.

Can Covalab help with the design of the peptides?

We can assist with the design of your peptide. Covalab uses a combination of prediction software and in-house expertise to select immunogenic peptide sequences. We select peptide sequences for immunisation based on a number of criteria. ? Antigenicity, Hydrophobicity and Accessibility ? Blast searches to confirm specificity of the proposed peptide candidates ? Alignment of sequences to identify specific antigenic peptides

How long should my peptide be?

Epitopes are generally 6-8 amino acids in length, by using a peptide of 16 amino acids in length there is the possibility of raising several different antibodies against the sequence used. Long peptides (>20?mers) are not recommended as these could contain secondary structures not found in the natural antigen.

What purity of peptide is best for antibody production?

* >50% and >70%: recommended for screening purposes and polyclonal antibody production * >80%: recommended for biochemical applications, e.g. qualitative enzyme-substrate studies or production of affinity resins * >95%: recommended for all quantitative enzymology and biological assays

How is the peptide conjugated to the carrier protein?

Peptides can be conjugated to a carrier protein via the N-terminus, C-terminus, an internal amino acid (not recommended) or an additional cysteine. Addition of a cysteine residue to the selected peptide sequence prior to coupling is a very common approach.

What is a carrier protein?

A carrier protein is a protein that is used to introduce the peptide to the immune system. As the molecular weight of the peptide is too small for it to elicit an immune response on its own, the peptide is conjugated to a protein to increase its apparent molecular weight. The host will then generate antibodies against the peptide and the carrier protein.

Which carrier protein should I use?

For best results a carrier protein should elicit a strong immune response. For this reason the carrier protein should be unrelated to the host species. Severals carrier proteins (BSA, OVA, THY) are available, but usually we used KLH (Keyhole limpet hemocyanin).

Does Covalab prepare phosphospecific antibodies?

Covalab produces phosphospecific antibodies and our method follows the procedure below: The phosphorylated peptide will be conjugated and used to immunise the host animals. Following the final bleed, phosphospecific antibodies will be selected by affinity purification. Two affinity columns will be used, the first one with non-phosphorylated peptide and the second one with the phosphorylated peptide. The serum will be passed through the first column and the flow ?through will be kept. The flow ?through will be purified on the second column in order to isolate those antibodies which recognise the phosphorylated peptide. ELISA tests will be performed to ensure that the antibodies recovered from the second column are phosphospecific.

Is there a need for sodium azide in my serum?

Sodium azide is a preservative which prevents bacterial growth. We recommend adding sodium azide to the serum for long-term storage. Please note that sodium azide interferes with some biological assays. Sodium azide can easily be removed by dialysis or buffer exchange.

Why are my tests bleeds red?

During collection and processing of the sera, haemolysis of red blood cells may occur which leads to the release of haemoglobin. The immunoreactivity of the serum will not be affected.

How should I store the serum?

For short term storage (up to one month) the serum can be kept at 4?C. For long-term storage we recommend aliquoting the serum in small vials and storing the aliquots at -20?C. Repeated freezing and thawing should be avoided.

What does the antibody titre mean?

The titre (or titer) of an antibody sample is a measure of the antibody concentration determined under a defined set of conditions. Serial dilutions of the antibody sample (test bleed or purified antibody) are allowed to react with a fixed amount of antigen. The antibody titre is defined as the lowest dilution to bind significantly to the antigen.

How do I interpret my ELISA results?

The results of the ELISA tests are reported on a graph. The antibody titre is defined as the reciprocal dilution giving an optical density (OD) reading at 450nm of 1. When the titre is >8000, the immunoreactivity of the sample is considered to be good. The pre-immune serum of the animal represents the negative control.

Can Covalab guarantee a minimum titre?

Due to the unpredictable nature of the immune response in host animals, we cannot guarantee the success of your antibody in your assays. We follow the project and help customers throughout the protocol and do everything possible to induce a significant immune response.

When is a continuation of the antisera project advisable?

If the response against your antigen is starting to increase at the end of the protocol, we recommend extending the project for another boost. Continuation of the project is also a good option if the titre is high and the customer requires more serum.

How long can a project be extended?

A project can be extended up to one year. This could be advantageous if the titre is satisfactory and large quantities of sera are required.