Purification of Murine Monoclonal Antibodies

Abstract

Hybridoma technology has made possible the production of highly specific, homogeneous antibodies with predefined binding characteristics, which can be produced in large amounts, from immortal cell lines. They probably represent the immunochemist's ideal as reagents, and monoclonal antibodies (MoAbs) are invaluable for the investigation and assay of the entire antigen spectrum. However, it must be remembered that, although MoAbs can be exquisitely specific, they are far from pure unless specific procedures are used for their isolation. If produced as ascitic fluid, they contain proteins derived from the host animal, whereas in vitro production as cell culture supernatant produces MoAbs contaminated with tissue culture additives, nonimmunoglobulin secretion products, and material derived from dead disrupted cells.

Monoclonal Antibodies

“…The ESVP 1 MAb preparation contained little non-antibody material (Figure 4.1A) but it was prepared from ascitic fluid by sodium sulphate precipitation (Section 4.5.1 .1)and would therefore have contained a low concentration of endogenous mouse IgG (Galfr& and Milstein, 1981;Baines et al, 1990). The solubilities of some of the mouse IgG contaminants may have been decreased by biotinylation (Wadsley and Watt, 1987), thus causing the observed non-specific binding.…”

“…The radioactivity of each fraction is expressed as a percentage of the total applied to the column. (Baines et al, 1990).…”

“…Spent culture supernatant, on the other hand, has lower concentrations of non-MAb proteins but typically contains only 5-50 fig MAb/ml and so needs to be concentrated to allow for efficient MAb labelling. MAbs were therefore partially purified from ascitic fluid by sodium sulphate precipitation (Tijssen, 1985), and concentrated from hybridoma culture supernatant by PEG 8000 precipitation (Baines et al, 1990) or Protein A-Sepharose affinity chromatography (Ey et al, 1978). for IgG MAb precipitation from hybridoma culture supernatant because Neoh et al (1986) had reported that maximal precipitation occurred within the range 5-10% (w/v) PEG 8000, at a concentration that was characteristic for each MAb, although this information was obtained with ascitic fluid.…”

“…PEG precipitation was reported to be a rapid procedure, requiring only a short equilibration period of up to 1 hour before centrifugation (Ingham, 1984;Neoh et al, 1986;Baines et al, 1990 (Table 4.17), although this material was very impure because 30% of total serum proteins were reported to precipitate in 14% (w/v) PEG 8000 (Cripps et al, 1983 (Figure 4.3B). ESVP 2 was produced by bulk culture in FCS medium, and although FCS is usually considered to be antibody-free it often contains a low concentration of bovine IgG ( Figure 4.3B).…”