ANTIBODIES - Production

Polyclonal Antibodies:

Biomedal’s Polyclonal Antibodies Production Service offers customised antibody production in rabbits utilizing proteins, peptides, small molecules, plasmics, etc. Moreover, in combination with our Peptides Production Services, we can synthesize any antigenic peptide necessary to generate the antibody of interest.

Polyclonal Antibody Production using a given sample in rabbit:

This service generates the quantity of polyclonal antibodies desired by the customer using an antigenic sample he or she has supplied.

Service Details:

- Peptide analysis for the selection of regions with greater antigenicity.

- Sample purification by HPLC.

- Sample conjugation with carrier KLH, BSA or Blue Carrier, througt N- or C-terminal sample residue.

- Two rabbit immunizations.

- Two serum extractions per rabbit during the immunization period.

- Providing the customer with 50 to 100 ml of serum per rabbit.

- Affinity purification of 2 ml of serum per rabbit.

Inmunization Protocol

The immunization protocol can be changed if the customer deems it necessary.

DAY	0	14	28	42	56	70	90
Inmunization	1º	2º		3º			4º
Sangrado	*Pre-inmune		*2-5 ml		2-5 ml		Final

(*) Optional

The purification includes:

- Purification by Protein G of 2 ml of serum.

- Concentration or dialysis of desired buffer (eg., 0,15 M PBS)

- Sterilization by filtration of the final product, with preservatives and stabilizers if they are required.

The customer receives:

- Between 50 to 100 ml of serum of each rabbit.

- Serum of each of the two extractions performed during the immunization routine.

- Purified antibody by protein G of two ml of the final extraction for each rabbit (1-5mg).

This service includes: Peptide analysis for the selection of regions with greater antigenicity. Sample purification by HPLC. Sample conjugation with carrier KLH, BSA o Blue Carrier, througt N- or C- terminal sample residue. Two rabbits inmunization. Two serum extraction per rabbit during inmunization period, providing the customer with 50 to 100 ml of serum per animal. Affinity purification of 2 ml of serum per rabbit.

Ref.	Product
SE-3452	Polyclonal Antibody Production using a given sample in rabbit

Polyclonal Antibody Production using peptide synthesis in rabbit:

This service includes the peptide synthesis protocol since the immunization is the same.

Service Details:

- Peptide analysis for the selection of regions with greater antigenicity.

- Peptide synthesis.

- Peptide purification by HPLC.

- Purity analysis by Mass Spectrometry MALDI.

- Sample conjugation with carrier KLH, BSA or Blue Carrier, through N- or C-terminal peptide residue.

- Delivery of the synthesized peptide that has not been conjugated.

- Two rabbit immunizations.

- Two serum extractions per rabbit during the immunization period.

- Between 50 to 100 ml of serum per rabbit.

- Affinity purification of two ml of serum per rabbit.

Immunization protocol:

The immunization protocol is the same as that of the previous service. It can be changed if the customer deems it necessary.

DAY	0	14	28	42	56	70	90
Inmunization	1º	2º		3º			4º
Sangrado	*Pre-inmune		*2-5 ml		2-5 ml		Final

(*) Optional

The purification includes:

- Purification by Protein G of 2 ml of serum.

- Concentration or dialysis of desired buffer (eg., 0,15 M PBS)

- Sterilization by filtration of the final product, with preservatives and stabilizers if they are required.

This service includes: Peptide analysis for the selection of regions with greater antigenicity. Peptide synthesis. Peptide purification by HPLC. Purity analysis by Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI). Sample conjugation with carrier KLH, BSA o Blue Carrier, througt N- or C-terminal peptide residue. Delivery of the synthesized peptide that has not been conjugated. Two rabbits inmunization. Two serum extractions per rabbit during inmunization period, providing the customer with 50 to 100 ml of serum per animal. Affinity purification of 2 ml of serum per rabbit.

Ref.	Product
SE-3453	Polyclonal Antibody Production using Peptide Synthesis in rabbit

Other animals

Ref.	Product
SE-4822	Policlonal antibody production using a given sample in guinea pig
SE-4826	Policlonal antibody production using peptide synthesis in guinea pig
SE-4823	Policlonal antibody production using a given sample in goat
SE-4827	Policlonal antibody production using peptide synthesis in goat
SE-4824	Policlonal antibody production using a given sample in rat
SE-4828	Policlonal antibody production using peptide synthesis in rat
SE-4825	Policlonal antibody production using a given sample in chicken
SE-4829	Policlonal antibody production using peptide synthesis in chicken
SE-5014	Policlonal antibody production using a given sample in hen
SE-5136	Policlonal antibody production using peptide synthesis in hen

Monoclonal Antibodies:

Biomedal’s Standard Monoclonal Antibody Production Service is based on the Balb-c mouse line. The service includes the next phases:

1. PRELIMINARY: Discussion about details of the project (screening system, selection criteria, special requests), planning and work preparation.

2. INMUNIZATION, CELLULAR FUSION. SELECTION AND SCREENING: Immunization at least of 2 mice (Balb - C), culture of SP2 line myeloma cell, cellular fusion, selection and screening.

3. CLONING: Cloning, screening and culture of positive hybridomas. The customer will receive the supernatant of the culture necessary by each positive clone for verification under his or her own system.

4. CRYOPRESERVATION: Three vials of each stable positive clone will by cryopreserved. The cryopreservation vials and 10 ml of the supernatant of the culture of each positive clone will be sent to the client. Also, we will store another two criopreservation vials in our installations until the customer is satisfied with what has been received (for a minimal additional cost).

5. CHARACTERIZATION OF SELECT CLONES: The Ia isotope will be determined and a microplasma detection test performed. The corresponding certificate will be issued.

6. THE CUSTOMER WILL PAY ONLY IN CASE OF SUCCESS: From the third phase it will be known if clones have been obtained that are positive against the immunogen. The customer will pay only if the third phase is achieved satisfactorily. In this case, he or she will pay 50% of the total cost of the service. The second part of the payment will be made upon delivery of the product.

7. RIGHTS OF THE CLIENT: The client acquires the ownership of all positive clones and will receive complete documentation of all the work carried out during the production of the monoclonal antibody.

8. OTHER SERVICES: We offer large scale production service for the chosen antibody, as well as its purification and conjugation with different fluorochromes and enzymes.

Monoclonal Antibody Production Protocol:

PHASE I: Inmunization

The period of immunization can be changed between 30 to 90 days according to requirements. The customer will provide the protein. If it is necessary for Biomedal to synthesise the protein, an additional fee will be applied.

PHASE II: Fusion, screening and selection

This phase includes the generation of a primary culture, selection in HAT medium and screening. The fusion of spleen cells with myeloma of the SP2/0 cellular line will be handled according to our standard system. All the primaries cultures with growth and Ig production will be tested by the customer.

PHASE III: Cloning and screening

All the positive clones will be used for future cloning. Cloning will be carried out using the dilution limit method. After each cloning a screening of the positives will be carried out. The customer receives 1 ml of supernatant of each clone with growth for its test.

PHASE IV: Second cloning, screening, stabilisation and cryopreservation

In this phase steps are taken to obtain pure clones, including that tested by the client. At the end we obtain three cryoprervation vials and 10 ml of culture for each positive clone. Biomedal will store two cryopreservation vials of each clone for some months that will only be used with the client’s authorisation.

PHASE V: Characterisation of selection clones

Selected clones are sent previously tested for:

- Determination of the Ig subclass.

- Microplasma.

We provide a certificate with the results.

Conditions and Warranty

Biomedal guarantees a minimum of three positive clones producing the lg against the immunogen used.

The clone will be tested by the customer.

PHASE VI: (optional)

Purification:

The purification type varies according to the requests and needs of the customer:

- Precipitate with ammonium sulfate.

- Purification with Protein A or G.

- Purification by affinity with antigen.

- Purification by filtration gel.

Conjugation:

The antibody produced will be conjugated with fluorochromes or enzymes.

Ref.	Product
SE-3446	Monoclonal antibody conjugation with FITC service
SE-3447	Monoclonal antibody conjugation with R-PE service
SE-3448	Monoclonal antibody conjugation with APC service
SE-3449	Monoclonal antibody conjugation with HRP service
SE-3451	Production using hybridomas
SE-3454	Hybridoma Cryopreservation