892

Electrophoresis 1990,11,892

A. Slavitkova

Short communications Alena SlaviEkova Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Praha

Electrophoresis of nonreduced IgM and IgG monoclonal antibodies on mixed agarose-polyacrylamide gels A gel matrix composed of 0.5 % agarose and 2.5 % polyacrylamide allows the parallel electrophoretic separation of nonreduced IgM and IgG monoclonal antibodies. The mobilities of IgM and IgG differ at pH 9.5, which enables apreliminary Ig class designation. It allows rapid detection of incomplete cloning ifboth IgM and IgG are present. A semiquantitative evaluation of nonreduced IgM in ascitic fluid is possible as well.

Besides monoclonal antibodies (MAbs) of IgG isotypes, some hybridoma clones secrete pentameric IgM isotypes. IgM polymeric molecules may partially or completely dissociate under reducing conditions. Consequently, for routine electrophoresis of MAbs, a gel matrix permitting the penetration of both IgG and nonreduced IgM molecules is desirable. Thin layers of agarose at corresponding concentrations (e.g. 0.8 %) or highly diluted polyacrylamide gels are not only difficult to handle but also have other inconvenient properties. Composite agarose-polyacrylamide gels, developed originally for separation of fragmented DNA and RNA 11, 21, are also applicable for electrophoresis of murine MAbs. Gels containing 2-2.5 % acrylamide and 0.5 % agarose are as easy to handle as more concentrated polyacrylamide gels. Under the conditions ofelectrophoresis in carbonate buffer at pH 9.5, as described by Hofejli and Hilgert [31,IgG, IgM and other proteins of mouse ascitic fluids are well distinguished by their mobilities.

Figure 1 . Electrophoresis of ascitic fluids in a mixed agarose-polyacrylamide gel in 0.05 M NaHCO,, pH 9.5. Lane (1) purified nonreduced monoclonal IgM; (2) and (3) ascitic fluids containing different monoclonal antibodies of the IgM class; (4) ascitic fluid containing antibodies of both IgCi and IgM class (before subsequent cloning): ( 5 ) and (6) ascitic fluids containing different monoclonal antibodies of IgG classes; (7) purified nonreduced monoclonal IgG. The gel (50 x 75 x 1 mm) was stained with Coomassie Brilliant Blue R-250. The positions of monoclonal IgM, IgG, mouse nonspecific immunoglobulin (Ig), transferrin (79and albumin (Al) and the polarity are indicated.

Mixed agarose-polyacrylamide gel electrophoresis was carried out in a 1 mm gel layer, containing 0.5 % w/v agarose, cellophane sheets. The mixed gels adhere well to the GelBond 2.5 % w/v acrylamide, 0.06 % w/v N,N’-methylenebis- films for agarose gels. acrylamine and 0.1 % ammonium persulfate in 0.05 M N a H C 0 3 (titrated to pH 9.5 with 2 M NaOH). Agarose (1 Yo) In the mixed agarose-polyacrylamide gels, nonreduced IgM of electrophoretical grade was dissolved in water with cons- MAbs migrate slower than their monomers and nonreduced tant stirring in a heated water bath ( 100°C) until the solution IgG MAbs. Mouse albumin, transferrin and nonspecific imwas free of air bubbles. In the meantime the same volume of munoglobulin are also well separated, as verified by the 5 % acrylamide with the respective N,N’-methylenebisacryl- purified proteins (not shown). Thus, the system is applicable amide and N,N,N’,N’-tetramethylethylenediaminein 0.1 M for the analysis of nonreduced ascitic fluids containing both NaHCO, was prepared and heated to 4OOC. After the cooling IgG and IgM MAbs (Fig. 1). Of 30 MAbs, the isotypes of of the agarose solution to the same temperature as the which were determined by specific antisera, 11 IgMs showed acrylamide solution, with care being taken to prevent the for- comparably slower mobilities than 19 IgGs. In conclusion, mation of lumps, both solutions were mixed, ammonium electrophoresis in mixed agarose-polyacrylamide gels is a persulfate was added and the solution was poured into an elec- useful method for preliminary screening of newly produced trophoretic cuvette. In larger vertical slab gels, a bottom layer MAbs and may reveal uncloned hybridoma lines, where both of 5 % polyacrylamide was poured first to prevent the IgM and IgG are secreted. The method also enables semicomposite gel from sliding out of the cuvette. Gels were stored quantitative determination of nonreduced IgM in an ascitic for several hours at room temperature and overnight at 4OC. fluid as described by HoiejSi and Hilgert for IgG MAbs using The running conditions were similar to those used in polyacrylamide gel electrophoresis [31. polyacrylamide gel electrophoresis, namely 0.05 M NaHCO,, pH 9.5, as the electrode buffer and avoltage of 10-20 V/cm in I am grateful to Dr. V. HoFejS;. and Dr. P. Dra’ber of this the gel. Electrophoresis was run until the zone of Institute for critical reading of the manuscript, and to Mr. Bromophenol Blue reached the bottom of the gel. The gels J. Picha f o r the photograph. were fixed 30min in 10 % w/v trichloroacetic acid. Usually the gels were stained with0.05 % Coomassie Brilliant Blue R-250 Received February 23, 1990 in 45 % ethanol and 10 % acetic acid, but immunofixation is also applicable. After washing with water, the gels were dried References from a 25 % ethanol - 3 % glycerol solution between I I1 Uriel, J . and Berges, J., in: Allen, R. C . and Maurer, H. R. (Eds.),

Correspondence: Dr. A. SlaviEkova, Institute of Molecular Genetics, Videiiska 1083, CS-142 20 Praha 4, Czechoslovakia Abbreviations: MAb, monoclonal antibody 0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

Electrophoresis and Isoelectric Focusing in Polyacrylamide Gel, de Gruyter, Berlin 1974, pp. 235-245. 121 Peacock, A.C. andDingman,C. W.,Biochemistry 1968,7,668-674. [31 HoiejSi, V. and Hilgert,I.,J.lmmunoZ. Methods 1986,86,103-105. 0 173-0835/90/0909-0892 %3.50+.25/0