- Polyclonal Antibodies
- Peptides
- Monoclonal Antibodies
For further information do not hesitate to contact us.
1. Polyclonal Antibodies
What animal should I choose
to raise polyclonal antibodies?
What is a SPF animal?
When should I use chicken antibodies?
How many egg yolks does a standard immunisation protocol yield?
How should I supply my protein?
What buffers are compatible with immunizations?
How should I ship my antigen?
What is the use of the pre-immune serum?
Can Covalab help with designing the peptide?
How long should my peptide be?
What purity of peptide is best for antibody production?
How is the peptide conjugated to the carrier protein?
What is a carrier protein?
Which carrier protein should I use?
Does Covalab prepare phosphospecific antibodies?
Is there a need for sodium azide in my serum?
Why are my tests bleeds red?
How should I store the serum?
What does the antibody titre mean?
How do I interpret my ELISA results?
Does Covalab guarantee a minimum titre?
When is a continuation of the antisera project advisable?
For how long can a project be extended?
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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.
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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.
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.
Chickens typically lay an egg a day. The egg collection starts at the first bleeding date. Consequently the yield is approximately 30 eggs.
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How should I supply my protein?
The protein can be supplied lyophilised, in solution, included in gel slices or attached to sepharose beads.
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.
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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.
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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.
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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
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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.
- >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
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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.
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.
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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).
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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.
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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.
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.
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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.
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.
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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.
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.
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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.
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.
2. Peptides
What purity do I require for my application?
What is the minimum and maximum sequence length Covalab can synthesise?
Will my peptide be difficult to synthesise?
How long will it take to synthesise my peptide?
Which amino acid should I use to conjugate my peptide?
Which carrier proteins can I use?
How should I store my peptide?
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The purity required depends on the application. Peptides of >50% (and >70%) purity are recommended for screening purposes and polyclonal antibody production. Peptides with more than >80% purity can be used for qualitative biochemical applications, e.g. enzyme-substrate studies or production of affinity resins. For all quantitative enzymology and biological assays we recommend >95% pure peptides.
We routinely synthesise peptides between 6 and 50 amino acids long. Sequences with less than 6 residues may be difficult to cleave from the resin and purify. Peptides longer than 40 amino acids are often more difficult to synthesise with high purity.
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Will my peptide be difficult to synthesise?
Several factors can affect the synthesis and purification of a peptide including its amino acid composition, peptide length, hydrophobic stretches and successive amino acids.
Highly hydrophobic peptides including residues such as Leu, Val, Ile, Try, Phen and Met may be difficult to purify and to solubilise in aqueous solutions. We recommend that there should be one charged residue for every five amino acids.
High purity peptides with multiple Cys, Met or Try residues can also be difficult to obtain as these residues are prone to oxidation/hydrogen bonding. Their production may be delayed due to the need for repeating the synthesis and/or purification.
Several serine and/or aspartic acid residues in a row may result in a product of lower purity. Multiple prolines in the sequence may undergo cis-trans isomerisation which may result in lower apparent purity. For peptides with a N-terminal Asparagine or Glutamine, we recommend the removal addition or substitution of another amino acid in order to obtain a higher quality product.
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How long will it take to synthesise my peptide?
The turn-around time depends on the quantity and purity required of the peptide. Most peptides (up to 80% purity, 5-25mg) are synthesised within 2-3 weeks. Delays can occur for longer peptides (>25mer) and difficult sequences, for example very hydrophobic sequences at high purity (>95%).
Which amino acid should I use to conjugate my peptide?
Peptides which will be used for antiserum production (usually 12-18 amino acids) require conjugation to a carrier protein as the molecular weight of the peptide is generally too small to elicit an immune response. When raising anti-peptide antibodies that should recognise the native protein, the amino acid used for coupling the peptide to the carrier protein is very important and should correspond to the natural representation of the peptide in the protein.
N-terminal peptides should be coupled through the C-terminal amino acid and C-terminal sequences through the N-terminal end. Internal peptides can be coupled through either end, and preferably at the less immunogenic side of the sequence.
Amidation of the C-terminus and acetylation of the N-terminus is recommended for internal peptides to avoid the introduction of non-natural charges (positive charge at the N-terminus or negative charge at the C-terminus). For example, the C-terminus of an internal peptide (conjugated at its N-terminus) should be amidated as this functional group (CONH2) mimics best the neutral amine bond between two amino acids.
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Which carrier proteins can I use?
The best known carrier proteins are keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA) and bovine thyroglobulin (THY). KLH is the most commonly selected carrier due to its high immunogenicity and generally will not be used as a “blocking” reagent in experimental assays.
The peptides are supplied lyophilised and should be stored at –20°C. For further information please consult our recommendations storage.
3. Monoclonal Antibodies
What is the difference between monoclonal and polyclonal antibodies?
Which animals are used by Covalab to raise monoclonal antibodies?
What type of antigens can be used and how can it be suplied?
How pure should the antigen be?
Should my antigen be coupled to a carrier protein?
My expressed protein has a tag, do I need to remove the tag for the procedure?
How is the immune response determined?
What is a hybridoma?
Can Covalab freeze and store the hybridomas?
Can Covalab supply large quantities of antibodies?
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Monoclonal antibodies are identical. They recognise only one specific epitope and have a defined specificity for the antigen. Polyclonal antibodies recognise independent epitopes on the antigen. Monoclonal antibodies give reproducible results as the hybridoma parent line or final clone can be stored and recloned at a later stage to produce more monoclonal antibodies.
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Which animals are used by Covalab to raise monoclonal antibodies?
Covalab uses mice as host animals. Routinely, 4 mice are immunised for each project.
Proteins and haptens (e.g. peptides) which are either lyophilised, in solution or included in polyacrylamide gel slices are suitable for immunisation. For the screening procedure, the antigen must be provided lyophilised or in solution. Solutions containing (>6M) urea, guanidine hydrochloride, and acetic acid should be avoided. Please contact us if other antigen preparations, buffer conditions and protein concentrations are envisaged.
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How pure should the antigen be?
The protein should be as pure as possible, especially for the screening procedure. Peptides should be >70% pure.
Peptides or haptens are not immunogenic enough and need therefore to be conjugated to a carrier protein such as KLH, OVA or BSA.
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My expressed protein has a tag, do I need to remove the tag for the procedure?
For the immunisation the tag does not need to be removed. For the screening procedure however the protein without the tag is required to identify suitable hybridomas.
The supernatants of hybridoma cells are screened by specific ELISA tests. The screening method is developed for each individual project.
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What is a hybridoma?
Hybridomas come from the fusion of spleen cells from the immunised mouse with immortal myeloma cells. Hybridomas have therefore the capacity to grow in cell culture and to secrete epitope-specific antibodies.
Monoclonal antibody production can be performed in vivo or in vitro, please ask for further information. |
