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Noll, M., and Kornberg, R. D. (1977), J . Mol. Biol. 109, 39 3-404. O h , A. L., Carlson, R. D., Wright, E. B., and Olins, D. E. (1976), Nucleic Acids Res. 3, 3271-3291. Oudet, P., Gross-Bellard, M., and Chambon, P. (1975), Cell 4, 281-300. Reeves, R.(1976), Science 194, 529-532. Richards, B. M., and Pardon, J. F. (1 970), Exp. Cell. Res. 62, 184- 196. Roark, D. E., Geoghegan, T. E., and Keller, G. H. (1974), Biochem. Biophys. Res. Commun. 59, 542-547. Sahasrabuddhe, C. G., and Van Holde, K. E. (1973), J . Biol. Chem. 249, 152-156. Shaw, B. R., Herman, T. M., Kovacic, R. T., Beaudreau, G. S . , and Van Holde, K. E. (1976), Proc. Natl. Acad. Sci. U.S.A.73, 505-509. Sollner-Webb, B., Camerini-Otero, R., and Felsenfeld, G. (1976), Cell 9, 179-193. Sollner-Webb, B., and Felsenfeld, G. (1975), Biochemistry 14, 2915-2920. Spadafora, C., Bellard, M., Compton, J. L., and Chambon, P. ( 1 976), FEBS Lett. 69, 28 1-285. Sperling, R., and Bustin, M. (1975), Biochemistry 14,
3322-3331. Thomas, J. O., and Kornberg, R. D. (1975), Proc. Natl. Acad. Sci. U.S.A.72, 2626-2630. Thomas, J. O., and Thompson, R . J. (1977), Cell 10. 633640. Van Holde, K. E., Shaw, B. R., Lohr, D., Herman, T. M., and Kovacic, R. T. (1975), Proc. FEBS Meet., l o t h , 57-72. Van Holde, K. E., and Weischet, W. 0.(1 977), Biopolymers (in press). Varshavsky, A. J., Bakayev, V. V., and Georgiev, G. P. (1 976), Nucleic Acids Res. 3, 477-492. Weintraub, H., and Groudine, J. (1976), Science 193, 848856. Weintraub, H., Palter, K., and Van Lente, F. ( 1 975), Cell 6, 85-1 10. Weintraub, H., and Van Lente, F. (1974), Proc. Natl. Acad. Sci. U.S.A.71, 4249-4253. Weintraub, H., Worcel, A,, and Alberts, B. (1976), Cell 9, 409-41 7. Woodcock, C. F. L. (1977), Science 195, 1350-1352. Wooley, J. C., Pardon, J. F., Richards, B. M., Worcester, D. L. and Campbell, A. M. ( 1 977), Fed. Proc., Fed. Am. SOC. Exp. Biol. 36, 810.
Isoelectric Focus Analysis of Rat Anti-phosphocholine Antibodies? Vivian Braciale,T Georges Der Balian, and Joseph M. Davie*
ABSTRACT: Anti-phosphocholine (PC) antibodies in sera from
four strains of rats were examined before and after immunization with either Streptococcus pneumoniae R36A, which contains PC as a cell wall component, or with PC-coupled keyhole limpet hemocyanin (PC-KLH). PC-specific protein was purified from pooled immune sera and shown by a combination of isoelectric focus (IEF) in acrylamide and crossed immunoelectrophoresis, as well as by molecular weight determination in NaDodS04-acrylamide, to be immunoglobulin. An additional, small molecular weight, nonimmunoglobulin
D e t a i l e d studies of antibodies with similar antigen binding specificities have revealed striking structural similarities. In particular, the analysis of mouse anti-phosphocholine (PC)' + From the Department of Microbiology and Immunology, Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, Missouri 631 10. Received April 15, 1977; revised manuscript received August IO, 1977. This work was supported by U S . Public Health Service Grant No. Ai-11635, National Science Foundation Grant No PCM76-09719, and by the following companies: Brown & Williamson Tobacco Corporation; Larus and Brother Co., Inc.; Liggett & Myers, Inc.; Lorillard, A Division of Loews Theatres, Inc.; Philip Morris, Inc.; R. J. Reynolds Tobacco Co.; United States Tobacco Co.; and Tobacco Associates, Inc. *Supported by US. Public Health Service Training Grant No. CA091 18. Present address: John Curtin School of Medical Research, Australian National University, Canberra, A C T . 2601, Australia. I Abbreviations used are: PC, phosphocholine; KLH, keyhole limpet hemocyanin; IEF, isoelectric focusing; PBS, phosphate-buffered saline; BGG, bovine y-globulin; BSA, bovine serum albumin; CFA and IFA, complete and incomplete Freund's adjuvant, respectively; PnC, C polysaccharide.
protein (PI = 7.1-7.3) was present in sera from normal and germ-free rats which had the ability to bind the C-carbohydrate of S.pneumoniae R36A, but without specificity for PC. The IEF profile of normal and immune sera showed marked sharing of bands of anti-PC antibody between individual rats as well as between strains. In addition, other anti-PC antibodies which focused between pH 8.5 and 9.5 were less regularly shared. The uniformity of IEF profile of the bulk of anti-PC antibodies in rats is most consistent with their being the products of germ line genes.
immunoglobulins by antigenic (Claflin and Davie, 1974a), functional (Claflin and Davie, 1974b), and structural means (Claflin and Rudikoff, 1977) has shown that all mice regardless of genetic background produce similar antibodies. This conservation of variable region structure is consistent with the existence of a germ line gene for anti-PC antibodies in mice. In addition, preliminary examination of the fine specificity of anti-PC antibodies raised in different species of rodents showed that each species developed distinctive binding patterns for PC and its analogues and that within a species, indistinguishable patterns were found in all individuals examined (Claflin and Davie, 1974b). These studies were based on the ability of soluble haptens to inhibit plaque formation by antibody secreting cells. The present paper extends these studies in the rat by examining the isoelectric focusing pattern of 7 s anti-PC antibodies in normal and immune rat serum. While minor differences in these spectra exist, extensive sharing of antibody patterns is seen regardless of the genetic background of the animal. BIOCHEMISTRY, VOL. 16, NO. 24, 1977
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quentially in two changes of 18% sodium sulfate (2-4 h), in 0.01% glutaraldehyde in 18% sodium sulfate ( 1 h), and in Animals. Young adult female Wistar/Furth (W/Fu) and 0.001% sodium borohydride in borate buffer (0.2 M borate, Fischer 344 rats were obtained from National Animal Labo0.15 M NaCI, pH 8.2) overnight. This procedure precipitates ratory Co., O'Fallon, Mo.; Lewis/maif (Lew) and Brown and cross-links immunoglobulins without affecting antigen Norway/maif (BN) rats were purchased from Microbiological binding capacity. The gel was then incubated with 30 pCi of Associates, Walkersville, Md.; axenic F344 weanling rats were ['*'I]PnC in 13 mL of 1% BSA for 24 h. After several seobtained from ARS/Sprague Dawley, Madison, Wis. quential washes in PBS, water, and 40% ethanol, the gel was Immunization. Animals were immunized intraperitoneally allowed to air dry. The dried gel was autoradiographed using (ip) with either 1 X IO8 heat-killed (56 O C , 30 min) StreptoKodak single-coated SB-54 x-ray film for 1 to 3 days. coccu.5 pneumoniae strain R36A (ATCC 27336) bacteria, In the case of immunoelectrophoresis in a second dimension, which contain PC as a cell wall component (Tomasz, 1967), a strip of the isoelectric focused gel (one well in width) was or with 250 p g of PC-coupled keyhole limpet hemocyanin removed and overlaid onto a horizontal 1% agarose (Sigma, (PC-KLH) in complete Freund's adjuvant (CFA) and bled at St. Louis, Mo.) gel plate in 0.025 M sodium barbital (pH 8.6) weekly intervals. PC-KLH, prepared as previously described containing goat anti-rat 7 s Ig (64 p L / m L agarose; Gateway (Claflin et al., 1974), contained 19 PC groups per 1 Os daltons lmmunosera Co., Cahokia, Ill.). Electrophoresis was then used of protein. The same dose of antigen and route were used for to move the focused proteins into the agarose gel. The direction secondary immunizations with either vaccine without adjuvant of electrophoresis was perpendicular to the long axis of the IEF or PC-KLH in incomplete Freund's adjuvant (IFA). strip in the plane of the agarose gel. The electrode running Measurement of Antibody Response. Antigen binding cabuffer was 0.1 M sodium barbital (pH 8.6). After electropacity was determined by a double isotope Farr assay phoresis for 30 min at 4 OC, 34 mA constant current (-IO (Gotschlich, 1971) using "NaCI as a volume marker and V/cm). the polyacrylamide strip was removed and the run '251-labeled C polysaccharide (PnC) from S. pneumoniae continued for an additional 5 h. K36A (Liu and Gotschlich, 1963). [I2'I]PnC was prepared Preparatice IEF and Sodium Dodecyl Sulfate Gel Elecby chloramine-T iodination (Greenwood et al., 1963) of t y trophoresis. About 8.7 mg of purified anti-PC antibodies raminyl PnC (Keck, 1972). Twenty microliters of serial which were dialyzed overnight against 2 M urea was fracthreefold dilutions of test sera was mixed with an equal volume tionated by liquid isoelectric focusing on the LKB 8101 ( 1 10 of diluent ( 1 % BSA, 0.01% BGG) containing 2 X IO4 cpm of mL) column (LKB, Chicago, Ill.). The liquid system was ??NaCI and 2 X I O 4 cpm of [12s1]PnC(sp act. 19 pCi/pg). stabilized against convection by means of a sucrose gradient lmniunoglobulins were precipitated by addition of 50 pL of (0-40%). Carrier ampholytes, in the pH 6-8 range, were added 85% saturated ammonium sulfate; the amount of [1?51]PnC to the sample at loading time (2% final concentration). The precipitated was measured in a y counter, and the dilution of focusing was run in 3 M urea at 2 or 15 W for 2 days at I O O C . serum needed to bind 33% of the ligand was determined At the end of the run I -mL fractions were collected and indi(AB('%{). vidually focused in an analytical gel and the similar fractions I n some cases, sera were separated by sucrose density ulwere pooled. The pools (fraction a tog) were concentrated on tracentrifugation into 7 s and 1 9 s fractions as described by Amicon filters (exclusion limit 25 000) and dialyzed against Perlmutter et al. ( I 977) prior to determination of ABCjj. PBS. Aliquots of the fractions were labeled with I2'I (GreenPurification of Anti-PC Antibodies. Rat sera were collected wood et al., 1963) and characterized by sodium dodecyl suland pooled 7 days following secondary immunization with 250 fate-polyacrylamide gel electrophoresis as described by F g of PC-KLH in saline. Seventeen milliliters of pooled imPerlmutter et al. ( 1 977). Reference proteins included lysomune serum was passed through 3 mL of PC-Sepharose. The zyme, bovine albumin, and bovine IgG. beads were washed extensively with phosphate-buffered saline Results (PBS, 0.056 M Na2HP04,0.016 M KH2P04,0.077 M NaCI, pH 7.4) until the absorbance a t 280 nm returned to baseline. Humoral Anti-PC Response. Rats were shown previously A IO-' M PC solution in PBS was then applied. The eluate (20 to respond to pneumococcal vaccine with the production of m L ) was concentrated on Amicon filters (exclusion limit direct (IgM) plaque-forming cells (Claflin and Davie, 1974b). 25 000) and dialyzed against PBS. The yield of antibody was The serum antibody response was measured in the present 500 pg/mL of serum, which corresponds well with values obstudies in response to either 1 X I O 8 pneumococci given ip in tained by precipitation of immune sera with PnC. Antibodies saline or to 250 p g of PC-KLH in C F A . Table I summarizes were also purified from normal serum by similar techthe PnC-binding capacity of normal and immune sera from niques. four strains of rats as measured by a Farr assay. It can be seen Isoelectric Focusing. Isoelectric focusing in vertical slab that all strains except Brown Norway have detectable PnCpolyacrylamide gels was performed as described by Briles and binding activity in normal serum. Immunization with either Davic ( 1 975). The gel was 5% acrylamide (Bio-Rad Laborapneumococcus or PC-KLH augments the binding capacity in tories, Richmond, Calif.), 2% ampholytes, pH 5 to 9.5 (LKB, all strains except Wistar which has high levels of antibody even Chicago, Ill.), and 3 M urea (Schwarz-Mann Ultra Pure, before immunization. To determine whether both 7 s and 1 9 s Orangeburg, N.Y.). The anode solution (upper trough) was antibodies were present, sera were fractionated by sucrose 1.5% phosphoric acid and the cathode solution (lower trough) density ultracentrifugation before assaying for antibody. was 2% monoethanolamine. Whole serum samples or purified Primary immune sera of the 3 strains tested had the bulk of the antibodies were focused for 15 h, 4 "C, 2 mA constant current, binding activity in the 7 s fractions. Surprisingly, even normal and then for an additional 0.5 h at 800 V. sera had substantial activity in the 7 s fractions. The specificity Iletection of Focused Antibody. Anti-PC antibodies in the of the PnC-binding activity from both normal and immune sera focused gels were detected by autoradiography (Briles and was directed to P C determinants since adsorption of the sera Davie, 1975) or by immunoelectrophoresis in a second diwith PC-Sepharose removed detectable PnC-binding activity. mension (Pierce et al., 1976). While differences exist between strains in the magnitude of the I n the case of autoradiography, the gel was incubated seresponse and in the antigenic preference, the significance of
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Vaccine immune Primary Secondary
Normal serum ABCx
Fischer Brown/Norway Wistar Lewis
%
1.7 (1.2)
