- If I have any questions about antibody services, who do I contact?
- How do I order polyclonal antibodies?
- What is the difference between monoclonal and polyclonal antibodies?
- Which antibody services does metabion offer and what are the prices?
- What services are included with custom polyclonal antibody synthesis?
- Can metabion help with the design of an antigenic peptide?
- What type of antigen does metabion use?
- Should immunogenic peptides be coupled to a carrier?
- What is the likelihood, that antibodies generated against synthetic peptides will be able to efficiently recognize the intact protein?
- Which peptide purity degree is sufficient for immunization?
- Is it necessary to purify the specific antibodies out of the serum?
- How long does it take until I get a custom made antibody?
- What are the specifications of polyclonal antibodies and how is quality assured?
- How much serum will I get?
- Can I get the pre-immune sera before the start of the immunizations?
- What is required, when sending "your" protein for immunization?
- How should the serum be handled/stored?
- Standard protocol for immunization.
1. If I have any questions about antibody services, who do I contact?
If you cannot find an answer in our "FAQ-catalogue" just contact us.
The easiest way is to write an email to antibodies@mymetabion.com.
We will answer your question as soon as possible.
2. How do I order polyclonal antibodies?
- Via our homepage or
- Via email to antibodies@mymetabion.com. Please include customer number, shipping and billing address information, and sequence in 1 letter codes (see FAQ 11) of antigenic peptide.
If you need our advice for the design of an antigenic peptide or have any other question, please write an email to antibodies@mymetabion.com.
3. What is the difference between monoclonal and polyclonal antibodies?
Polyclonal antibodies are antibodies that are derived from different B-cell lines. They are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognising a different epitope. These antibodies are typically produced by immunization of a suitable mammal, such as a mouse, rabbit or goat. Larger mammals are often preferred as the amount of serum that can be collected is more. An antigen is injected into the mammal. This induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen. This IgG is purified from the mammal’s serum.
By contrast, monoclonal antibodies are derived from a single cell line.
Monoclonal antibodies (mAb or moAb) are antibodies that are identical because they are produced by one type of immune cell that are all clones of a single parent cell. Given (almost) any substance, it is possible to create monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry, molecular biology and medicine. When used as medications, the generic name ends in -mab (see "Nomenclature of monoclonal antibodies").
Monoclonal antibodies are produced by a single clone of hybridoma cells and are therefore single species of an antibody molecule (directed against one epitope).
4. Which antibody services does metabion offer and what are the prices?
- Polyclonal antibodies against a custom delivered protein
- Polyclonal antibodies against a recombinant protein made by us for you
- Polyclonal antibody against a synthetic peptide
- Monoclonal antibody
Click here for pricing information. For special orders please contact us.
5. What services are included with custom polyclonal antibody synthesis?
- Help with the design and the selection of the immunogenic peptide.
- Fast and efficient peptide synthesis.
- HPLC purification of the peptide and MALDI ToF mass spectrometric analysis.
- Peptide to carrier protein conjugation.
- Polyclonal antibodies from two rabbits according to our standard protocol.
- 40 ml of serum from each rabbit and 2 ml of preimmune serum (negative control) are supplied.
ELISA to verify titer and quality of the polyclonal antibodies are supplied together with the antibodies.
6. Can metabion help with the design of an antigenic peptide?
Yes! metabion will highlight the most antigenic regions of a protein, thus allowing us to choose the most immunogenic peptides. This help is provided at no additional charge, and it is based on a computer analysis of the protein sequence using algorithms that take into consideration the hydrophobicity and the knowledge of the tri-dimensional conformation of the different amino acid residues.
In some cases, it may be better to generate antibodies with a mixture of 2 or 3 different peptides whose sequence is derived from the same protein sequence. Such approach may increase the chance to obtain antibodies able to recognize the intact protein.
Just send the amino acid sequence to antibodies@mymetabion.com, and we try our best to propose some promising antigenic peptides. In general, we suggest to use peptides which consist of 11-18 amino acids.
7. What type of antigen does metabion use?
metabion normally produces polyclonal antibodies against synthetic peptides, recombinant or natural proteins or other antigens as required by you. In case of synthetic peptides, the peptide shall be conjugated to a carrier protein which in general is either Ovalbumin or KLH.
The synthetic peptides can be provided/synthesized by metabion or supplied by you. We also offer recombinant protein synthesis services.
The conjugation of peptides with large proteins such as Ovalbumin or KLH is necessary in order to render the peptides immunogenic enough to provoke the desired immunological response which leads to the generation of specific antibodies (see FAQ 8). Of course, in case the antigen is already a protein, there is no need for conjugation to carriers.
As a general principle, we can start the immunization scheme with any antigen provided by the customer, knowing however, that the resulting antibodies will be the consequence of the antigen used. For instance, if the antigen is a mixture of different proteins, very likely we will generate different antibodies against the different proteins present in the mixture.
8. Should immunogenic peptides be coupled to a carrier?
Yes! Peptides which are used for immunizations (approximately 11-18 amino acids) are low in molecular weight.
Immune response will be enhanced if the peptide is coupled to a carrier protein (OVA, BSA or KLH).
Carrier proteins have a high molecular mass and a good antigenicity.
The conjugation of the peptide to the carrier is performed using a cross-linking molecule that will bridge the peptide sequence with the carrier protein. There are options for cross-linking.
- Gluterhaldeyde, reacts with all amino groups of the carrier and the peptide creating a very heterogeneous conjugate, where the peptide is bound in many different ways.
- The second one makes use of a bi-functional cross-linking agent which reacts with the amino groups on the carrier protein and with thiol groups (Cysteine residue) of the peptide. In this case, it is necessary to have a Cysteine residue in the amino acid sequence of the peptide and all peptide molecules coupled using this approach will display the same basic conformation after conjugation. In other words they will have a known and predictable orientation, leaving the majority of the molecule free to interact with the immune system.
In case of option 2), the peptide is coupled to the carrier via a single terminal cysteine:
- For a C-terminal peptide, add the Cys to the N-terminus so that the COOH is free like in the natural peptide.
- For an N-terminal peptide add the Cys to the C-terminus so that the NH2 is free like in the natural peptide.
For an internal peptide the Cys can be placed at either end. In addition we recommend to amidate the C-terminus if the Cys is attached to the N-terminus and to acetylate the N-terminus if the Cys is attached to the C-terminus.
9. What is the likelihood, that antibodies generated against synthetic peptides will be able to efficiently recognize the intact protein?
Please be aware that the peptide antigen provided by you or designed and synthesized by us is different from the proteins, as it only represents parts of it. While computational simulations for identification and selection of the most efficient immunogenic peptides show a very high success rate in terms of recognition of the native/intact protein, there is a 10-15% likelihood, that the antibodies generated through the use of synthetic peptides may not recognize the intact “original” protein from which the peptide(s) has/have been derived.
This could be observed particularly in immunoprecipitation or immunohistochemistry experiments.
10. Which peptide purity degree is sufficient for immunization?
A purity degree of 70-80% is usually sufficient.
11. Is it necessary to purify the specific antibodies out of the serum?
For certain applications such as immunohistochemistry, it may be necessary that the specific immunoglobulins are purified by affinity chromatography carried out by using a sepharose matrix to which the specific antigen (peptide or protein) is bound.
If you want to make sure that the background is very low, then we recommend an affinity purification of the serum. The antigenic peptide is bound to a column. Antibodies with low affinity are washed from the column while the ones with high affinity bind to the peptide. The high affinity antibodies are then eluted from the column.
If you are undecided about whether or not to opt for purification, we recommend that you decide later after having used the sera delivered to you at the end of the immunization protocol.
In any case, don´t worry, we routinely offer purification and also provide the sepharose column carrying the antigen. Link zu Preisliste Antibody Purification
12. How long does it take until I get a custom made antibody?
Our carefully developed immunization protocols enable us to generate a good titer of antibodies in a reasonable time (7-8 weeks). We are well aware that in some cases, longer protocols may lead to higher titers of antibodies. However, our protocol is an ideal compromise between quality and time.
In general, you should plan with the following timelines:
- Polyclonal antibody:
About three months if we also synthesize the peptide for you. Depending on the ELISA results we do 4-5 rounds of immunizations. If the antigen is supplied by you, we need about 2 months for immunization, purification, QC and delivery. - Monoclonal antibody:
The development of a monoclonal antibody takes between 4-6 months.
We prefer to closely communicate your needs and the progress of the project with you. This includes but is not restricted to discuss the potential need for a prolongation or change of the immunization protocol in the event that the standard immunization scheme does not yield antibodies passing the established specifications.
As a side remark, we are also willing to start the immunization schedule with a protocol suggested by you. A specific quotation will be issued in this case.
13. What are the specifications of polyclonal antibodies and how is quality assured?
The antibodies pass QC specs if they are able to recognize the antigen used for immunization at a dilution of the serum not lower than 1:1,000. As negative control, we collect the pre-immune serum, which is used for internal QC. The pre-immune serum will be delivered to you together with the antibodies.
Serum collected from the animals is tested by ELISA. According to our procedures, the antigen used for immunization is coated on a microtiter plate and allowed to react with the specific serum. The reaction is followed by labelled secondary antibodies, which are able to recognize the specific immunoglobulins bound to the antigen. The intensity of the resulting label signals provides a quantitative measure of the antibodies. The use of pre-immune sera allows to establish the specificity of the antibodies.
In case of antibodies generated against synthetic peptides, the free un-conjugated peptide is coated on the microtiter plate. This way, we do not measure the immunoglobulins raised against the carrier protein (Ovalbumin or KLH).
Last but not least, we always use 2 animals since the immune response of a given animal may be different from that of a different animal. Moreover, the risk of losing one animal through sickness or death, which would prevent us from a successful completion of your order, is minimized of not completely covered.
14. How much serum will I get?
You will get 50 ml from each of the two immunized rabbits.
The serum contains 0.02% sodium azide as anti-bacterial preservative.
15. Can I get the pre-immune sera before the start of the immunizations?
Yes, this is important if the antigen is from bacterial, viral or yeast origin
16. What is required, when sending "your" protein for immunization?
We need at least 1.5-2mg of purified protein. The minimum protein concentration is 0,5 mg/ml.
The protein should be either lyophilized or dissolved in Tris or PBS buffer. A gel picture should give us an idea of the protein purity and its molecular weight.
17. How should the serum be handled/stored?
Prepare small aliquots and store them at -20 or -80°C.
Avoid repeated freeze and thaw cycles.
You can keep an aliquot at 4°C for several weeks.
18. Standard protocol for immunization.
| Step A, Day 0 | 5.0 ml of blood is drawn from the rabbit's ear to prepare about 2.0 ml of pre-immune serum utilizing as negative control. For each rabbit, 200 µg of antigen in PBS are emulsified with CFA and injected subcutaneously. |
| Step B, Day 21 | 200 µg of antigen in PBS are emulsified with CFA and injected subcutaneously into each rabbit. |
| Step C, Day 28 | 200 µg of antigen in PBS are emulsified with IFA and injected subcutaneously into each rabbit. |
| Step D, Day 35 | 1.0 ml of blood is drawn from the rabbit's ear to prepare about 0.5 ml of serum. This sample is tested for its positivity versus the pre-immune serum. A positive signal allows the proceed to Step E, a negative signal results in the process returning to Step B. |
| Step E, Day 37 | 100 µg of antigen in PBS are emulsified with CFA and injected subcutaneously into each rabbit. |
| Step F, Day 44 | 100 ml of blood are drawn from each rabbit. At least 40 ml of serum is then obtained which can then be utilized at a dilution from 1:1,000 to 1:10,000. |