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IBGRL RESEARCH PRODUCTS |  |
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IBGRL RESEARCH PRODUCTS | |
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Introduction
The IBGRL was set up in 1946 to centralise production of blood grouping reagents and provide a reference centre for the newly formed National Blood Transfusion Service. Until 1984, all blood grouping reagents were prepared by the labour intensive process of selecting, absorbing and pooling human sera donated by blood donors. In the early 1980’s, hybridoma technology was emerging, and the possibility of producing monoclonal blood typing reagents was being explored. A monoclonal development laboratory was set up at IBGRL in 1982. The laboratory also financed the development of human monoclonal anti-D at Babraham, Cambridge and UKTS, Bristol. At this time the Immunochemistry Section of the SW Regional Transfusion Centre (SWRTC) was exploring the possibility of developing monoclonal antibodies to blood group active structures on red cells. The immunochemistry department joined IBGRL and the SWRTC is now known as NBS (National Blood Service) Midlands and South West Zone, Bristol Centre. In 1986 the activities of IBGRL were split. The production of major blood group reagents was undertaken by our sister laboratory, the BioProducts Laboratory at Elstree, in a newly created Diagnostics Division (BPL (D)). Reference, research and development work remained at IBGRL. In 1988 a concentrated program of reagent development was undertaken by staff from IBGRL, BPL(D) and SWRTC to produce a range of major blood grouping reagents based on selected monoclonal cells lines developed by SWRTC. These reagents were successfully launched by BPL(D) in April 1990. BPL stopped production of blood grouping reagents in 1996. The cell lines were licensed to Bioscot and Excel Biotech. Many other monoclonal cell lines were produced by the SWRTC and IBGRL (both internally and from externally funded R & D). These antibodies were not exploited and marketed by BPL(D), as they did not have potential as major blood grouping reagents. During further characterisation and studies, the activities of some of these antibodies were studied by various International Workshops. As a result of the successful participation and performance of some of the antibodies, many requests were received from research workers for supplies of these antibodies. Most requests were fulfilled, but at IBGRL’s expense. The cost factor together with a lack of a carefully controlled quality and standardisation program led IBGRL during 1990 to undertake an extensive program of developing these popular cell lines for routine production and supply of antibody to other research workers.
Establishment and Maintenance of Cell Lines The aim of the work undertaken on the cell lines is to ensure that the cells are stable in continuous culture, can be cultured to high cell concentrations on a routine basis and will produce consistent amounts of active specific antibody. All cell lines are first tested for mycoplasma infection. Mycoplasma is a common contaminant of cultured cell lines, and, if present, can adversely affect the growth rate and antibody secretion rate of the cells and can also make it difficult to expand cell cultures into large growth vessels. If a mycoplasma infected cell line is grown in a laboratory, it is very difficult to avoid cross contamination into previously uninfected cultures. All new cell lines are kept in "quarantine" (in a separate incubator, and not handled at the same time as other lines) until they have been tested and shown to be free of mycoplasma. A DNA hybridisation method was used for mycoplasma detection (Gen-Probe1) until 1996 when a PCR (polymerase chain reaction) based method was used. If a cell line is mycoplasma positive, it undergoes a regime of treatment, developed in the laboratory, with the antibiotic ciprofloxacin. This is usually effective in eliminating mycoplasma from cell cultures. Treatment continues until the culture gives a negative result with the mycoplasma test. Once a treated line is mycoplasma test negative, normal work continues on the line, but the line is retested at later stages of growth, since mycoplasma infection can sometimes recur. Stability of cell lines is monitored by limiting dilution cloning analysis2. All lines are cloned at least three times after primary fusion, and cloning continues until all clones isolated are secreting specific antibody. At the final cloning stage, several clones are compared for (1) the growth rate, (2) growth of cells in larger culture vessels, (3) the secreted antibody potency (measured by serological titration, or intensity of staining immunoblots) and (4) antibody concentration measured by double antibody sandwich class specific ELISA3. The best clones are then selected - i.e., those showing optimal growth characteristics, together with a high level of secretion of specific antibody that has the highest potency for the lowest antibody concentration. Comparison of antibody concentration and potency is important if the avidity of the secreted antibody is to be maintained and/or improved. We have observed significant variation in antibody avidity between different sister clones4. The specificity and immunoglobulin class and subclass of the antibody secreted by the best clone are carefully confirmed. This clone is then expanded in culture to provide sufficient cells to freeze a master cell bank stored indefinitely in liquid nitrogen. One vial from each freezing is thawed to check the viability of the frozen cells.
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Growth and Harvesting of Cell Lines All antibodies are grown in in vitro cell culture conditions. Because we have optimised conditions for producing antibodies at high levels there is no need for in vivo ascitic fluids to be produced. When antibody is required, one vial of frozen cells is taken from the master cell bank and rapidly expanded in culture to the required final volume. If only small volumes of supernatant are required, the cells are grown in static flasks. If greater than 500 ml is required, the cells are grown in suspension spinner vessels. If highly concentrated antibody at levels of 50-1000 mg are required, then the cell line is grown in hollow fibre culture. |
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This allows concentrations of approximately 1 mg/ml (depending on the concentration of the antibody output in static flasks) to be harvested. Volumes vary but on average 30 ml at 1 mg/ml may be harvested every week i.e. 30 mg/week. This varies for each cell line and the conditions for growth must be optimised for each cell line. For growth of cell cultures using hollow fibre technology, dissolved oxygen, pH, temperature and glucose/glutamine levels can be carefully controlled, giving optimum growth conditions for high levels of antibody secretion. The Tecnomouse (Integra Biosciences, Letchworth, UK) has been successfully used to produce concentrated amounts of antibody. The advantage with using the Tecnomouse is that 5 different cell lines can be grown at the same time on the same hollow fibre unit. Other hollow fibre systems have also been used at IBGRL.
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Growth and production of antibody is monitored daily by cell counts and ELISA. Following the peak of antibody production, cultures are harvested by centrifugation followed by sterile filtration of supernatant. Each batch of supernatant is assessed for specificity, potency and antibody concentration. Supernatant is then aseptically distributed into aliquots at a range of different volumes, and stored at -25°C until required. The activity of the frozen material is monitored after three months and thereafter every six months.
Purification of Antibodies Antibodies can be supplied as purified immunoglobulin on request, although limited antibodies are available from stock. IgG antibodies are purified by affinity chromatography on a protein-G Sepharose column in an FPLC system. IgM antibodies are purified by affinity chromatography (using an anti-mouse IgM column) followed by size exclusion chromatography on Sephacryl S-300. Purity of antibodies is checked by non-reducing SDS polyacrylamide gel electrophoresis, ELISA and serological methods. Purified antibodies are stored at high concentration in aliquots at -80°C.
Conjugated Antibodies Purified antibodies conjugated to either FITC or R-phycoerythrin (R-PE) have been made available since 1994.
Range of Antibody Specifications and Applications Originally in 1991, sixteen different monoclonal antibodies were available. In 1999, 37 different monoclonal antibodies are available. In 1997-1998, we took over the routine production of cell lines that had originally been made at the Birmingham Transfusion Centre and have made some of the antibodies available to customers within the National Blood Authority, at cost. The antibodies available cover a wide range of research interests. The anti-DAF (CD55) and CD59 antibodies are of interest to workers studying the regulation of complement activity in health and disease. Anti-LFA-3 (CD58) and CD44 antibodies are useful in studies of the mechanisms of antigen specific immune recognition and lymphocyte migration. CD55, CD59 and CD58 antibodies identify three well characterised glycosylphosphatidylinositol (GPI) linked proteins which are deficient from the peripheral blood cells of patients with paroxysmal noctural haemoglobinuria (PNH). The popularity of these antibodies is, in part, a result of their usefulness for the analysis of PNH cells by flow cytometry. The anti-M and anti-N antibodies have been used by Langlois5 in a flow cytometric assay to determine the proportions of red cells not bearing M or N antigens and thus the significance of raised numbers of cells with the M-N-phenotype in disease, particularly in cancer. They have shown that this assay is a good predicator of mutagenesis and it is used by various countries for environmental and radio/chemo-therapy monitoring. Glycophorins A and C are interesting cell membrane sialoglycoprotein markers. Glycophorin C has a wide tissue distribution, and is known to interact with cytoskeletal elements. The anti-Wrb antibody causes a reduction of red cell membrane deformability and inhibits invasion by malarial parasites. The Tn antigen is a cryptantigen, and its expression in some tissues has been associated with oncogenesis. Other blood group antibodies are also available. The anti-A, anti-B and anti-Rh(D) antibodies are of use to workers studying the structure and distribution of these blood group antigens. These antibodies are IgG and are useful for work involving flow cytometry or tissue staining but have not been selected for haemagglutination. Those cell lines producing IgM antibodies that are good blood grouping reagents have been licensed to Bioscot and Excel Biotech. BRAD 3 is a human monoclonal anti-D that reacts as an indirect agglutinin with all Rh D positive red cells tested except those of the rare DVI or Rohar types (pattern 136). By flow cytometry, FITC-BRAD 3 can be used to quantitate accurately the numbers of Rh D positive cells in a mixture of Rh D positive and negative cells, and thereby estimate the size of feto-maternal bleeds7,8. BRAD 3 can also discriminate weak D in feto-maternal bleeds where the site/cell numbers are above 1000 RhD sites9. FITC conjugated BRAD 3 has also been used in conjunction with R-phycoerythrin conjugated BRIC 256 (anti-Glycophorin A) in a dual labelling flow cytometry method for feto-maternal hemorrhage (FMH) quantitation10.
Most of the antibodies we have available have been submitted to either one or several of the following workshops.
Marketing and Presentation Marketing and sales of the antibodies was originally handled for IBGRL by BPL commercial department Elstree, but now all are processed and fulfilled by IBGRL in Bristol where antibody stocks are held. Any technical queries on the performance and use if the antibodies must be referred to IBGRL. A data sheet has been prepared for each cell line, providing the customer with information concerning the origin of the cell line and the specificity and known uses of the antibody, together with information about the antigen recognised and its distribution, including published references. Each shipment of antibody is accompanied by a package insert giving information on the antibody concentration of that particular batch, together with the class, specificity and cell culture conditions.
We accept orders by post or fax and orders are usually despatched within forty eight hours of receipt, although purified antibodies and conjugates may take up to two weeks if they are not held in stock. Orders are taken from both the UK and overseas. Address orders to: Admin Office International Blood Group Reference Laboratory NHSBT North Bristol Park Filton Bristol BS34 7QG Tel: +44 (0) 117 921 7500 FAX: +44 (0) 117 912 5796
UK - Delivery within the UK is by first class post and is free of charge. Overseas - To ensure rapid delivery we ship the goods by a door to door courier service (e.g. FedEx) which is an extra charge. If an import licence is required for importation from the UK into your country please send a copy of it with your order. We will include it with the goods to speed up customs clearance. Delivery will take 2-4 days, customs clearance permitting.
Useful Contact Numbers IBGRL Fax : +44 (0)117 912 5796 IBGRL Office for placing orders : + 44 (0) 117 921 7500 IBGRL Technical and stock level enquiries: + 44 (0) 117 921 7560 Rosey Mushens IBGRL Research Division Manager : + 44 (0) 117 921 7471 Marion Scott IBGRL E-mail: enquiries.ibgrl@nbs.nhs.uk
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Produced for  by the Intranet Design TeamThis page was last updated on 03-May-11. |
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