CUSTOM ANTIBODY SERVICE (CABS)
Scientific Supervisor: Mª Pilar Marco
The antibodies production platform, CAbS (Custom Antibody Service), is a CSIC service located at the Institute of Advanced Chemistry of Catalonia (group NB4D) that belongs to the Biomedical Network Research Center in Bioengineering, Biomaterials and Nanomedicine (CIBER -BBN).
CAbS is part of NANBIOSIS (infrastructure of production and characterization of nanomaterials, biomaterials and biomedical systems). NANBIOSIS is one of the 29 ICTS (Scientific Technical Scientific Infrastructures) recently recognized by the Ministry of Economy and Competitiveness of Spain.
The platform has a cell culture laboratory, with the equipment necessary for the collection, selection, growth and storage of hybridomas (the antibody-producing cells). Likewise, the service has laboratories equipped for the synthesis of immunogens and for the characterization of the antibodies produced.
CAbS offers its services for the production of monoclonal and polyclonal antibodies to groups of the CSIC and CIBER-BBN, as well as to other research groups belonging to public or private institutions and companies.
The service adapts to the needs of each user and can produce antibodies against proteins, peptides, organic molecules or other antigens, by using standardized or personalized protocols, having plenty of experience on the design phase of the immunogen, essential for modulating selectivity and affinity of the antibody. Each service request is examined by a Scientific Committee of the NB4D group (IQAC), which elaborates a feasibility report, prior to the acceptance of the project. Moreover, the user is permanently informed about the evolution of the project.
In addition, It should be highlighted that CAbS service is one of the few laboratories that offers the counseling and assistance service regarding the preparation of immunogens and the production of antibodies for low molecular weight molecules, such as pigments or hormons.
Contact: cabs@iqac.csic.es
Pilar Marco
Scientific Supervisor
pilar.marco@iqac.csic.es
Nuria Pascual
Technical Supervisor
nuria.pascual@iqac.csic.es
David Santos
Specialist Technician
david.santos@iqac.csic.es
Hapten synthesis
The haptens are molecules of small molecular weight, too small to induce an immune response and must be conjugated to a carrier molecule, generally a protein. Some haptens require modification before they can be coupled successfully to the carrier molecule. Therefore each hapten is unique and requires various strategies for its functionalisation which start from the simple modification of a portion of the existing hapten to the complete synthesis of a new modified hapten.
The hapten synthesis service includes:
- Personalised design and development of the synthesis strategies and conjugation of the hapten which are adapted to the needs of the client. Selection of the adequate principal structures for the preparation of the antigen
- Hapten synthesis
- Modification of the hapten and derivation
- Conjugation of the haptens to HCH, KLH, BSA and other proteins or enzymes
Preparation of bioconjugates and molecular probes
The process of bioconjugation of biomolecules through covalent binding includes the union of small molecules to proteins such as BSA, HCH or KLH or the conjugation of protein-protein conjugates as well as the conjugation of antibodies to enzymes. Also, the bioconjugation with oligosaccharides, carbon nanotubes, synthetic polymers etc. can be performed.
The bioconjugation service includes various stages:
- Personalised design and development of the conjugation strategies that adapt to the client.Viability studies of the Project. Obtaining monofunctional or bifunctional spacers, modification of the biomolecules and evaluation of the chemistry of the conjugation.
- Development of the bioconjugation process.
- Purification and chemical characterisation of the obtained bioconjugates.
Antibody purification
The purification of the antibodies includes the isolation of the antibody from the serum (polyclonal antibodies) or culture supernatant from hybridoma cell lines (monoclonal antibodies). The necessary level of purification will depend on the application that is expected for the antibody. The services offers previous advise on the different possibilities for purification according to the needs of the user.
The methods of purification that we offer vary from crude methods (precipitation of the proteins from the sample including any antibody present) to general purification (purification by affinity of certain classes of antibodies without taking account of the specificity to the antigen) to specific (purification by affinity of the antibodies which join specifically to a definite antigen).
- Ammonium sulphate precipitation
- Protein A, protein G affinity purification
- Specific antigen affinity purification
Immunoassay development
The service offers the development of immunoassays for the detection and quantification of substances or analytes of interest (contaminants, pathogens, allergens, biomarkers etc.) in different samples (biological, food etc.) with the objective of diagnosing or guaranteeing food safety and environmental safety.
CAbS offers personalised development of immunoassays that begins with advice and counsel from the service’s scientific committee. We can work with the users own antibodies or develop monoclonal or polyclonal antibodies. The antibodies and immunoreagents that make up the immunoassay can be conjugated to different types of markers such as fluorophores, nanoparticles, enzymes… At the same time, other necessary immunoreagents can be synthesised for the development of the assay.
The development of the immunoassay includes different stages:
- Design of the immunoassay format, sample matrix, the sensibility and the working range. Identification of necessary reagents and their conditions to obtain optimal results. Preliminary definition of the characteristics of the assay.
- Development Phase. Establishing assay parameters, for example the detection limit and working range. Evaluation of the effects of the matrix on the assay. Reproducibility study.
- Assay validation. Evaluation of the stability and robustness of the reagents. Measurement of inter and intra-assay reproducibility, precision and specificity. Validation with blind samples. Assay platforms: ELISA, microarrays with different formats based on the detection of individual analytes or multiplexed assays.
Antibody production
The service, which adapts to needs of each client, can produce antibodies against proteins, peptides, organic molecules and other antigens, through standardised or personalised protocols and by special emphasis on the design phase of the immunogens, which is essential to modulate the selectivity and affinity of the antibody. It is also one of the few laboratories which offer advice and assistence in relation to the preparation of immunogens and the production of antibodies for low molecular weight molecules such as pigments, hormones or anabolic steroids as well as peptides.
The immunogen can be provided by the user or can be designed and synthesized or bionconjugated by the same antibody production service.
The protocols and the care of animals are performed according to the specifications of the Real Decreto 53/2013 about protection of the animals used for experimentation and other scientific activities, both for monoclonal and polyclonal antibody production.
Monoclonals Antibody Production:
The Monoclonal Antibody Production service is based on Balb/c mice and includes the following phases:
-PRELIMINARY: Discussion of the details of the project (screening system, selection criteria, special requirements), planning and work preparation.
-IMMUNISATION, CELLULAR FUSION, SELECTION AND SCREENING: Immunisation of at least 4 mice (Balb/c), cell culturing of myeloma cells of the P3x63Ag8.653 cell line, cellular fusion, selection and screening.
-CLONING: Cloning, screening and cultivation of positive hybridomas. The user will receive, if they wish, the supernatant of the necessary culture for each positive clone to test it using their own methods.
-CRYOPRESERVATION: Three vials will be cryopreserved for each stable positive clone. The cryovials and 10 mL of supernatant of the culture of each positive clone will be sent to the client.
-CHARACTERISATION OF THE SELECTED CLONES: The Ig isotype will be determined and detection of mycoplasm test will be performed. The corresponding report will be given.
Production of polyclonal antibodies:
The service has a standard immunisation protocol that includes monthly innoculations with Freund adjuvant during a period of 4 months. This protocol adapts to the needs of the required antiserums, keeping in mind the characteristics of the immunogen and the required characteristics of the antibody.
To determine the progress of the immune response, a bleed is taken after 10 days after the second immunization to obtain antiserum and the titer is determined through ELISA. Among the controls is included obtaining preimmune serum from the animals obtained from the blood previously to the first injection. At the end of the process, the exsanguinations of the animal is made using a surgical process under anaesthesia. Normally 50-70 mL of hyperimmune serum is obtained per animal.
Adicionales services:
- Isotyping of obtained monoclonal antibodies
- Conjugation of the antibodies or antigens
- Storage of hybridomas in liquid N2
- Growth of hybridomas and production of monoclonal antibodies at a small or medium scale
Nitrogen Tank Cryomemo (Cryopal)
The Cryomemo device is a nitrogen tank equipped with an electronic kit consisting of level and temperature indicators, and a control and configuration interface. It is used to control and regulate the nitrogen levels and temperature of cryogenic tanks (GT 40, Asperge, Espace and RCB) using a sensor and solenoid valves that control nitrogen intake and blowdown. It is also used to manage alarms.
Custom Antibody Service (CAbS) is incorporating this new piece of equipment as a result of its participation in the project FICTS1420-27, selected by the MINECO for co-financing by the FEDER Program in ICTS 2014-2020.
