Should I choose mAb or pAb?
Monoclonal antibodies A monoclonal hybridoma cell line is first created by the fusion of antibody secreting immune cells from immunised mice, with immortal myeloma cells. The resultant hybridoma cell line then expresses a specific antibody when cultured in vitro. The antibodies may subsequently be purified from the cell culture supernatant, if required. 3-9 months, due to the time it takes to develop a successful hybridoma cell line with first screen data typically available at 3 months. Highly specific antibodies. mAbs recognise a single epitope on a single antigen as long as it is unique. A renewable resource due to the creation of the hybridoma cell line that permits unlimited production of mAbs. Low batch-to-batch variation due to the clonal population of cells and single antibody. Western blot, ELISA development, IP/IHC, affinity purification, flow cytometry, infectious disease characterisation, potential therapeutics, target validation and diagnostic tool.
Polyclonal antibodies Host animals are immunised with a predefined antigen, causing an immune response resulting in the secretion of antibodies by different B-lymphocytes. As each B-lymphocyte recognises a different epitope, this process results in a heterologous population of antibodies which can then be recovered directly from the host’s serum. Standard schedules are 77-98 days in rabbit. Broad spectrum recognition across the target due to the presence of a cocktail of antibodies that recognise multiple epitopes. pAbs provide more robust detection due to the presence of multiple epitopes. Batch-to-batch variation is likely, due to the inherent variability in the immune response of the host animals. Western blot, ELISA development, affinity purification, IP/IHC. Supply of antibody is limited by the available harvest and terminal bleeds.
Production Timescale Specificity Advantages Batch-to-batchvariability Applications
How much antigen do I need to supply? Typical (minimum) recommended antigen concentrations per immunisation are as follows:
Host species Mouse Rat Rabbit Sheep/Goat Alpaca/Llama
Deliverable: Average serum yield/animal 0.3 - 0.5 ml 2 - 3 ml 50-100 ml depending on breed 750 – 1,000 ml with harvest bleeds also available to increase yield 500 – 750 ml with harvest bleeds also available to increase yield
Minimum amount of antigen required TOTAL: 1,000 µg (1 mg for 4 animals) TOTAL: 1,000 µg (1 mg for 4 animals) 200 µg (0.2 mg) of antigen (0.4 mg/mL) for each of 6 immunisations. TOTAL: 1,200 µg (2,400 µg for 2 animals) 200 - 500 µg (0.2 - 0.5 mg) of antigen (0.4 - 1 mg/ml dependent on antigenicity) for each of 6 immunisations. TOTAL: 1,200 - 3000 µg 200 - 500 µg (0.2 - 0.5 mg) of antigen (0.4 - 1 mg/ml dependent on antigenicity) for each of 6 immunisations. TOTAL: 1,200 - 3000 µg
*Please note: Due to natural variations, exact volumes cannot be guaranteed
What is the purity of the custom antibodies? Various purification options offering different levels of purity are available and we would be happy to discuss these on a project-by-project basis with antigen affinity purification being the gold standard for polyclonal antisera.
How much purified antibody or antisera will I receive for custom antibodies? The concentration of purified antibody available at the end of the project will depend upon many factors, including the method of purification chosen. If a specific amount of purified antibody is required, this should be indicated at the commencement of the project to ensure that this is taken into account at the project design stage. Typically, working concentrations need to be determined empirically.
How should I store my antibody? Custom antibodies typically should be considered to have a shelf life of at least 1 year. However aliquots may be frozen at -20°C or below to increase the shelf life. Preservatives may be added, as required, in order to prevent microbial contamination.
What specifications are provided for antibodies? The following parameters will be reports on a Certificate of Analysis for each antibody:- Concentration- Label- Buffer- Preservative- Volume
What purification methods are available for custom antibodies? Purification of IgG is typically carried out by Protein A or Protein G affinity purification. However, APS offers a range of purification methods for monoclonal and polyclonal antibodies. The purification methods used are dependent upon the isotype of the antibody for culture. Other purification methods available include: - Ammonium Sulphate Precipitation- Dialysis & Ultrafiltration- Ion Exchange Chromatography- Size exclusion chromatography
What species are available for my antibody project? Mouse, rat, Rabbit, Goat, Sheep, Alpaca, Llama, Chicken (blood and eggs) and Emu (eggs).