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Protocols

Production of large quantities of monoclonal antibodies

In principle there are two ways to achieve this:

  1. Slow adaptation
    • Culture the hybridomas in normal medium containing FCS (5-10%). Since FCS contains minute amounts of IgG (depending on the batch but usually 100µg/ml) this can interfere with subsequent labeling of the antibody.
    • Decrease the percentage of FCS slowly from 10 to 8; 6; 4; 2; 1 %. This can take several weeks but depends on the hybridoma.
    • Collect the cells from the 1% FCS culture and suspend them in PFHM (protein free hybridoma medium). Cells sometimes go through crises but eventually start growing in this medium.
    • Collect the quantity needed. Check for the concentration: it should be approximately the same as in FCS cultures (Usually 1-50 µg/ml).
  2. Fast method
    • Expand the cells in normal medium with 10% FCS.
    • Collect the cells and suspend them in PFHM. Cells will look awkward and most of them will die soon but will produce antibody.
    • Use the FCS culture to expand the cells and collect cells for seeding in PFHM. Concentration of antibody is usually lower than in the FCS culture but method is really fast.

Processing the supernatant containing the antibodies:

The easiest way is to perform an ammonium sulfate precipitation to downsize the solution from liters to milliliters. Most antibodies will precipitate at 50% ammonium sulfate. The amount needed is easily obtained from the internet (http://www.encorbio.com/protocols/AM-SO4.htm). Slowly add ammonium sulfate while stirring the supernatant. Avoid local high concentrations of ammonium sulfate because it will precipitate unwanted proteins. After overnight stirring at the final concentration collect the solution and spin down at 10,000g for 30-45 minutes. Decant the supernatant and dissolve the pellet in PBS. Dialyze at least overnight against PBS. A precipitate will appear (probably cell debris). Spin down the solution at ≥10,000g for 30 minutes. The supernatant contains rather pure antibody. An estimate of the concentration can be obtained by measuring the absorbance at 280 nm. 1 mg/ml IgG has an A280 of 1.4. Be aware that this is dependent on the Trp content, which can vary from monoclonal to monoclonal.

Further purification can be performed with protein G beads using standard technology.

An alternative purification can be done with T-gel (see: http://www.piercenet.com/Products/Browse.cfm?fldID=01010326).