Genosphere Biotechnologies offers this unique guarantee to our customers for anti-peptide antibodies as well as for anti-protein antibodies services provided sample requirements are fulfilled.
Should you have any questions or requests, please do not hesitate to contact us.
- Polyclonal antibodies production from rabbits, guinea pigs and chicken
- Standard offers: immunization of 2 animals
- Standard protocol: 4 immunizations, 70 days
- Custom protocols, more animals, host combination, antigen combinations
- Any antigens, or haptens: antibiotics, small organic compounds, proteins, peptides, etc.
- Anti-peptide packages: synthesis, purification & KLH conjugation included
- Standard peptide carrier conjugation via a (C- or N-) terminal cys
- Unconjugated peptide delivered with purity levels of 70 to 90%
- HPLC analytical trace and mass spectrum documentation
- Antigenic peptide design
- ELISA titer for all projects
- A guaranteed good titer for every anti-peptide antiserum we deliver
- Pre-immune antibodies for all projects
- Anti-protein package: WESTERN blot included
- Affordable monospecific antibodies libraries for antibodies arrays
- Affordable guaranteed phospho-specific antibodies
- Antigen expression and/or purification for immunization
- Personalized service
Should you require a service that is not listed or believe your needs are not covered here, please do not hesitate to contact us, we will do our best to match your research requirements at a very affordable price.
Genosphere biotechnologies offers full custom anti-peptide antibodies services at a competitive price. Provide your peptide sequence, select your species, and we’ll produce a high titer antisera within 14 weeks. We can help you with design and choice of your peptide sequence. Drawing on many years of industrial experience in producing all kinds of peptides, Genosphere Biotechnologies manufactures peptides and protein conjugates (KLH, BSA, OVA…) with purities of 70-90%.
While many factors will influence overall antibodies quality, controlled peptide synthesis is important for achieving good specificity and refined sequence discrimination (Figure 1).
Standard package offer: rabbits & guinea pigs
The standard anti-peptide service package includes:
- Synthesis, purification & KLH conjugation of your peptide
- Immunization of 2 animals for:
- Rabbits: 100 ml of ELISA titered antiserum
- Guinea pigs: 20 ml of ELISA titered antiserum
- Pre-immune control serum
- Full report with ELISA analysis data
- 5 to 10 mg of unconjugated peptide (with HPLC & MS data, purity 70 to 90%)
Alternatively, we can use your own program or yet design a specific protocol to meet your requirements such as different time periods collection, more animals, or several species per project. All projects may be extended beyond initial termination date for additional antiserum collection.
Peptide-carrier protein conjugation
Synthetic peptides are too small to induce a good immune response for the purpose of antibodies production in animals. As a result, they are rarely used as antigens. Instead, peptides are covalently linked to a larger protein and the resulting conjugate is used as immunogen. It is critical for further successful immunization that the peptide-hapten to protein-carrier molar ratio be very high, and that all epitopes on the peptides be properly oriented in order to induce high titer specific immune response. To that end, peptide quality and well-controlled conjugation chemistry are key to a successful immunization project.
Our standard offer includes the following peptide conjugation conditions:
- Protein carrier: KLH (Keyhole Limpet Hemocyanin). It is a high molecular weight protein isolated from a marine mollusc species with very potent antigenicity when submitted to the immune systems of mammals or birds. In addition, it has a high number of free amino groups available for hapten coupling.
- Coupling: via a terminal cys (N- or C-terminal) on peptides using sulfhydryl chemistry and amino groups on KLH. This approach using two different coupling chemistries, in a two-steps reaction with isolation of intermediate products permits directed and controlled orientations of the peptides onto the globular KLH carrier.
- Figure 1. Three 15 aa peptides were synthesized, purified, conjugated to KLH, and the resulting immunogens were used independently to raise polyclonal antibodies in rabbits. Sequences differed by 8 aa alternatively. ELISA cross-analysis using 1:10 000 dilutions was performed as shown. Results indicate a complete discrimination of the produced antisera against other sequences with ELISA responses similar to that of pre-immune sera.
- Click on figure to enlarge
- Figure 2. Every antibodies project undergoes stringent quality control procedures including ELISA titration against pre-immune sera as negative control, peptide analytical HPLC, and mass spectrometry.
- Click on figure to enlarge
Peptide should be at least 10 residues long and include a terminal cys for conjugation purposes.
However, in some cases, antibodies to shorter sequences are required, and we have developed a special efficient protocol for smaller chains peptides (5-10 aa). In addition, some projects call for the coupling of the carrier protein directly to the N-terminal amine of the peptide. Please contact us with your project details.
In some instances an alternative to protein carrier conjugation of the peptide hapten may be the use of multiple antigen peptides (MAP). MAP contains 8 copies of the peptide, synthesized directly on an 8-armed branched lysine core using a special resin. The resulting multimeric is a large macromolecule (10-15 kDa) which has a high molar ratio of peptide antigen to core molecule and does not require further conjugation to a carrier protein. Please note that MAP should not be used when the peptide sequence is derived from the C-terminus of the target protein.
Quality control
Our synthetic peptides are controlled by reversed-phase HPLC and mass spectrometry (Figure 2). Immune responses are monitored all along and immunization program adapted when necessary. Final ELISA is performed on your antisera and pre-immune sera for individual animals and we guarantee a minimum good titer for all your projects (Figure 2). A detailed ELISA report is supplied with every project and complete instruction and reagents used are described so you can reproduce the analyses.
Antigenic Peptide Design
When evaluating a protein sequence for antigenic determinants, it is critical to choose regions that are antigenic, hydrophilic, and with secondary structures that allows good solvent accessibility (Figure 3). Peptide chemical synthesis feasibility will guide the decision as well. One or several peptides are then synthesized and used as epitopes simultaneously or separately for immunisation. The antibodies thus generated should recognize the peptide sequence(s) within the parent protein. Using appropriate computer algorithms, we will analyze your protein sequence and we will suggest several peptide candidates for your antibodies project.
- Figure 3. Graph representation of the results generated by computer algorithm. The darken areas represent peptide stretches within the protein sequence that meet criteria for antigenicity.
- Click on figure to enlarge
- Fig 4. An equimolar mixture of 3 KLH-conjugates was used as immunogen in two rabbits. Following co-immunization protocol, antisera were collected and ELISA run. As shown antisera were positive against each individual peptide. The specific response was > 10-fold higher than that of pre-immune sera.
- Click on figure to enlarge
In some instances, in order to improve the chances of producing a successful antibodies pool, one may choose several target peptides within the same protein sequences and use them as antigen in a co-immunization protocol. Having several epitopes derived from a single protein will mathematically increase the chances of obtaining a pool of antibodies that will recognize the target protein. One will experience some background increase as well.
We will synthesize, purify and conjugate each peptide separately. Individual KLH conjugates are then mixed in equimolar ratios and used as immunogen. Individual antisera are titered by ELISA against each peptide separately.
- Figure 5. WESTERN blot analysis is performed for every project. Semi-purified protein antigen was provided as shown on the coomassie-stained polyacrylamide gel. We further purified the the 48 kDa protein of interest to > 90% purity and used it to raise polyclonal antibodies in guinea pig. The resulting antiserum was diluted 1/2000 for WESTERN blot analysis of the provided protein extract. The blot reveals a single band as expected.
- Click on figure to enlarge
Standard offer
This service includes the following features:
- Immunization of 2 animals with your antigen
- Rabbits: 100 ml of ELISA titered antiserum
- Guinea pigs: 20 ml of ELISA titered antiserum
- Pre-immune control serum
- ELISA data
- WESTERN blot analysis
Provided antigen samples
Antigens may be supplied in solution, lyophilized, as gel slice, inclusion bodies, insoluble aggregates, GST/His fusions, protein conjugates, etc. It should be noted that small proteins (<10 kDa), peptides, organic molecules need to be conjugated to a carrier protein in order to elicit a good immune response in animals.
Sample requirements
For protein antigens >10 kDa: 5 mg of protein should be provided for hen and rabbit antibodies and 2 mg for guinea pigs (solubilized or lyophilized). A minimum concentration of 1 mg/ml is required. Buffered NaCl solutions are preferred. Please dialyze out any urea or imidazole.
Quality control
Final ELISA are performed on your antisera and pre-immune sera for individual animals and we guarantee a minimum good titer for all your projects. A detailed ELISA report is supplied with every project and complete instruction and reagents used are described so you can reproduce the analyses. In addition, depending upon quality and quantity of antigen supplied, we control your antibodies by WESTERN blotting ensuring perfect specificity (Figure 5).
If you have a PCR fragment or a cDNA of your protein of interest we may be able to express it with a his6 tag into our bacterial expression system. Further purification on Ni-NTA columns yields immunization-grade antigen.
If the cDNA is not available, we may synthesize your gene of interest or a fragment of it (e.g. 300-450 bp corresponding to 100-150 aa on N-terminus).
Please contact us with your project details and we will design and quote the most efficient strategy for you.
In many instances, raising antibodies in chicken has proven essential. Where genetically distant species is required e.g. conserved mammalian proteins, Genosphere Biotechnologies will produce your antibodies in chicken. Antibodies are extracted and purified in high yields from egg yolks of immunized laying hens.
Anti-peptide service offer:
- Synthesis, purification & KLH conjugation of your peptide
- Immunization of 2 hens
- Eggs collection, yolk isolation and IgY purification
- 1 g of ELISA titered IgY antibodies fraction
- Pre-immune purified IgY
- Full report with ELISA analysis data
- 5 to 10 mg of unconjugated peptide (with HPLC & MS data, purity 70 to 90%)
Anti-protein service offer (you provide the antigen):
- Immunization of 2 hens
- Eggs collection, yolk isolation and IgY purification
- 1 g of ELISA titered IgY antibodies fraction
- Pre-immune purified IgY
- Full report with ELISA analysis data
- WESTERN blot analysis
Pre-immune eggs are collected and control IgYs are purified. Following four immunizations over a 70-days period, six eggs per hen are collected, yolks are pooled and we isolate the IgY fraction (purity 90-95%). The final antibodies preparation is then titered by ELISA.
Protocols may be extended beyond the initial dispatching and more eggs would be collected.
In addition, the IgY fraction may be further purified by affinity chromatography using a peptide or antigen-column.
For large scale IgY production, multiple hen immunization, and timely supply over long periods, please contact us with your project details and we will design the most efficient strategy for you.
| Anti-Peptide | Anti-Protein (provided) |
Anti-Protein (with expression) |
Anti-Protein (with gene synthesis & expression) |
|
| We need: | Your sequence(s) | 5 mg of protein | cDNA | DNA sequence |
| Turn around time: | about 3.5 month | about 3 month | about 3.5 month | about 4 month |
Following polyclonal antibodies production as described above, you may wish to purify your antiserum by chromatography. Genosphere Biotechnologies offers two standard purification services.
Total IgG purification
Total IgG purification service is recommended for improving signal to noise ratio by eliminating all irrelevant serum proteins and macromolecules.
This service includes for 100 ml of antisera:
- Affinity chromatography of your antiserum
- Analysis of collected fractions
- ELISA
Purification of total IgG from the serum results in a highly pure (>90%) and stabilized polyclonal antibodies preparation.
Affinity purification
The second purification service is antigen affinity purification. This method is recommended when the antiserum reveals interfering background or when one antibodies sub-population is sought, or if further antibodies labeling is intended.
This service includes for 100 ml of antisera:
- Preparation of the affinity chromatographic support by covalently linking of your peptide
- Affinity chromatography of your antiserum
- Analysis of collected fractions
- ELISA
- Fig 6. (a) Affinity chromatography typical elution profile.
- Click on figure to enlarge
- Fig 6. (b) ELISA responses of the various fractions for a 15aa anti-peptide antisera.
- Click on figure to enlarge
- Fig 6. © SDS PAGE of a typical affinity-purified antibody.
- Click on figure to enlarge
- Fig 7. ELISA response of the phospho-specific antibodies against (a) the phosphorylated sequence and (b) the non-phosphorylated sequence.
- Click on figure to enlarge
This service allows the production of antibodies that will specifically recognize the phosphorylated state of your target protein. The approach includes the synthesis and purification of phosphorylated and non-phosphorylated peptides, immunisation, fabrication of two affinity columns for successive purification. Extensive ELISA on phospho-specific antibodies as well as on the fraction specific to the non-phosphorylated state is performed. We have successfully utilized this method with other post-translational modifications including acetylation and methylation.
Pricing and quotation
Genosphere Biotechnologies endeavors to offer cost-effective custom antibodies production that fit your needs. We offer discounts for larger volume orders. Contact us with your project details and we will work with you to select the most suitable service offer.
A quotation form is a available online for your convenience: Antibodies quote request
Ordering
An Online Order Form is available for your convenience.
- Anti-peptide Polyclonal Antibodies
Simply provide us with your sequence(s) of interest and we will start the procedures.
- Anti-protein polyclonal antibodies: sending your sample
Please send your sample clearly marked and wrapped with parafilm in a padded envelope. Include your Institutional Purchase Order and a letter describing your samples (mass amount, solution content, concentration if applicable).
Please provide a minimum of 5 mg of protein antigen for rabbits and hens antibodies and 2 mg for guinea pigs antibodies.
Please send it using express delivery courier or postal services to:
GENOSPHERE BIOTECHNOLOGIES
Antibodies Services
2 rue des Gravilliers
75003 PARIS
FRANCE
For a faster processing of your order and safer tracking of your samples, please advise our Customer Services of your sending a sample with courier airway bill number if available: info@genosphere-biotech.com.
Receipt of your samples is confirmed to you by e-mail and an approximate date of completion is released.
Product delivery
All antibodies are stored frozen prior to dispatch. The antibodies are shipped by express courier in insulated package with cold blocks and a complete report. Accompanied peptides are dispatched as a lyophilized powder.
Final note
Should your testing in your experimental system indicate that your anti-peptide antisera require further affinity purification, please contact us for special pricing offer. You may then send your antibodies back to us along with your peptide.