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RD Biotech / Molecular biology / Biologie MoléculaireRD Biotech / Services analytiques / Analytical servicesRD Biotech / Anticorps monoclonaux (mAb) / Monoclonal antibodiesRD Biotech / ELISARD Biotech / Culture Cellulaire / Cell cultureRD Biotech / Production de cellules / Cell production

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RD-BIOTECH

3 rue Henri Baigue

F-25000 Besançon

+33(0)3 81 53 88 37

info@rd-biotech.com

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Monoclonal antibodies

PRODUCTION OF MONOCLONAL ANTIBODIES (murine, humanized, chimeric, rat)

For several years, RD-Biotech has developed a strong expertise in the in vitro production of monoclonal antibodies. Production in ascites, although still allowed in France, is recognised as painful for the animals and we have replaced it with an entirely in vitro cell culture method.
RD-Biotech offers this alternative technology at an equivalent cost to the old ascitic method but with higher reproducibility and quality.

customers can send their clones to RD-Biotech (by flask or cryovials) for production. You do not have to specify the nature of the clone, which guarantees confidentiality.

The different steps in the in vitro antibody procedure are as follows:
The customer provides the clone accompanied by a signed technical agreement that specifies the culture medium, the nature and the isotype of the antibody. The hybridomas can then be adapted to an appropriate media (bovine IgG-depleted serum, serum-free medium etc...).

Before production of large quantities of monoclonal antibodies (around 100 mg), RD-Biotech offers a small scale pilot assessment.

Following the feasibility test, the customer receives a report on the pilot study and an accurate quote for the production phase.Customers may receive an aliquot for their own validation before ordering large scale in vitro production..
RD-Biotech‘s scale up ranges from 1 liter to over 250 liters of supernatant which yields a few milligrams to several grams of antibody.

-Production follow-up:
Using assay kits for mouse IgG, rat IgG and human IgG , developed and marketed by RD-Biotech, we are able monitor antibody production to estimate quickly the amount of supernatant needed to meet the target amount.

- Purification (immunoglobulins G, M or A):
The antibody supernatant can be concentrated up to 15 times by tangential ultra-filtration. Purification of the monoclonal antibody is then performed by affinity chromatography on Protein A or Protein G columns according to the isotype. In the case of immunoglobulin M, the purification is performed by ion exchange chromatography.

Optionally, the antibody can be purified in endotoxin free conditions using special buffers and non-pyrogenic vessels after the affinity chromatography step, the purity of the antibody can also be improved by a ion exchange chromatography step, which further reduces endotoxin levels.Monoclonal antibodies produced by RD-Biotech’s “Endofree” protocol have less than 1 EU / mg of antibody.

Monoclonal antibodies produced in vitro can be delivered to the customer after concentration and equilibration in the buffer of choice.

- Quality Control:
Monoclonal antibodies are supplied with a quality control report including an electrophoresis (SDS-PAGE) profile and total protein quantification. When needed, a LAL test that determines the level of endotoxins, is performed. The LAL test (Limulus Amebocyte Lysate) is carried out using a kit according to the protocol prescribed by the supplier. If requested, additional HPLC (SEC) analysis is available.

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DEVELOPMENT OF MURINE HYBRIDOMAS

RD-Biotech can support all stages of your monoclonal antibody projects.

--The customer provides: the protein of interest (native or recombinant), or a peptide sequence on a computer file.

- RD-Biotech then immunizes between 5 and 10 mice depending on the project.

- The immune response is monitored by ELISA titration or Western-blotting.

- Cell fusion is only done after customer validation of the titration results.

- Screening: Several types of screening are available, ELISA, Western-blotting, flow cytometry or other methods.
After the first screening, positive hybridomas are paased then screened again, amplified and a back up is made.

- The positive hybridomas are then cloned by two rounds of limiting dilution.

- Positive clones are banked as soon as possible.

- Development and production of polyclonal antibodies
RD-Biotech prepares polyclonal antibodies from different types of immunogens (purified protein, peptide, cell lysate...).
The most popular species is the rabbit, but we can also produce chicken antibodies (IgY).

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CELL BANKS

We have the facility to make and store Working and Mastercell banks

RD-Biotech realises the importance of safeguarding customers valuable hybridomas.
The customer provides us with the hybridoma culture or a cryovial.
The cells are amplified and, if necessary, productivity assessed with a FastELISA kit .

100% viable cells are frozen in exponential growth phase.
The cryovials are stored at -80 ° C then stored in liquid nitrogen before shipment to the customer.

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PURIFICATION OF IMMUNOGLOBULINS: IgG, IgM, IgA, IgY

The customer provides us with either serum, plasma, culture supernatant, or even egg yolk.

Using FastELISA kits dosing of mouse IgG, rat IgG or human IgG,RD-Biotech can quickly check the levels of immunoglobulin G in the sample and estimate how much it will be possible to purify.

RD-Biotech produces top quality antibodies with a purity from 80 to 99%.
Quality control: RD-Biotech antibodies come supplied with a quality control report including electrophoretic analysis of total protein (SDS-PAGE).
The purified antibodies are then shipped to the customer.

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ANTIBODY COUPLING AND OTHER MODIFICATIONS

RD-Biotech carries out fragmentation monoclonal or polyclonal antibodies (pepsin, papain...) and / or the coupling of antibodies to different markers:

- Peroxidase labeled (HRP)
- Labeling with alkaline phosphatase (ALP)
- Biotin labeleling
- Labeling with a fluorescent marker (FITC fluorescein, Cyanine, Alexa fluor...)
- Coupling to Luminex type beads (Luminex, Estapor...)

The couplings are performed using robust protocols routinely used by our teams.
After modification of the antibody and quality control, the sample is sent to the customer with the quality control report.

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DEVELOPMENT OF IMMUNOASSAYS

ELISA, Western-blotting, Flow Cytometry etc.

RD-Biotech lends its expertise and immunoassay platform to the development of a series of Immunological detection methods:

-We develop analytical methods according to the customer's specifications.
-Analysis of samples by methods approved by the customer.

For various applications:
-Animal and human in vitro diagnosis
-Quality control
-Research
-Clinical Studies

-ELISA: Method development
RD-Biotech produces ELISA based protocols based on customer specification.
RD-Biotech carries out an initial feasibility study to determine optimal parameters for the study such as: antibody choice, selection of raw materials, development and optimisation of each step (e.g coating micro-plates, staining, etc…)

-TEST 1: Simple indirect sandwich test: using extracted or purified protein coated wells stained using a polyclonal rabbit antibody, or a commercial peroxidase coupled antibody appropriate to the species under test.
-TEST 2: Immuno-competition assay: wells coated with specific antibody, incubated and then stained with a mixture of peroxidase-labeled protein and unlabeled purified protein.
-TEST-3: Indirect or direct double sandwich test (depending on the availability of specific antibody): wells are coated with the antibody followed by incubation with either a second antibody or the purified protein. Staining can be either direct if the second antibody is conjugated or indirectly with a third species specific conjugated antibody.
Different parameters are measured for optimising each strategy (eg. coating of micro-wells, sample incubation times and staining).
Data processing: All absorbance measurements are analysed by BPL and saved as Excel files; Results are also recorded in a bench notebook and copies are available to the customer upon request.

After this initial stage, RD-Biotech is able to identify the best methodology to use, specifying optimal operating conditions. The report provides a set of dose performance curves done under various conditions (eg.test sample volume, number and duration of incubation, enzyme conjugate) and exact details of all protocols performed.

The customer can then decide whether to initiate the transferable development phase of the robust ELISA technique we have developed.


- RD-Biotech: Analytical Services for ELISA, Western-blotting, flow cytometry, …
RD-Biotech samples analysis is performed using commercial kits or with a customer validated protocol. A raw data report is then sent to the customer.

Types of analysis:

- Titration of immune responses by ELISA
- Immune response caracterization by ELISA or by a Western-blotting (multi-track system, multiscreen)
- Determination of cytokine profiles from different types of samples (plasma, culture supernatant, biological fluid, etc…) by ELISA
- Cell sorting by flow cytometry

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ADDITIONAL SERVICES

Our sales team plus our scientific and technical experts are at your disposal to offer you what's right for your application!

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