We recently described a new method for preparing. b and c and for making a polyvalent. conjugate specific for the S.

Transcription

1 INFECTION AND IMMUNITY, Apr. 1976, p Copyright 1976 American Society for Microbiology Vol. 13, No. 4 Printed in U.SA. Diethylaminoethyl-Cellulose-Bacterial Cell Immunoadsorbent Columns: Preparation of Serotype-Specific Globulin and Immunofluorescent Conjugates for Streptococcus mutans Serotypes a and d ROGER M. McKINNEY* AND LEROY THACKER Center for Disease Control, Atlanta, Georgia Received for publication 3 December 1975 Diethylaminoethyl (DEAE)-cellulose was used as a support material for preparing bacterial cell columns. Pretreatment of the bacterial cells with formalin was essential in obtaining satisfactory adherence of the cells to DEAE-cellulose. Cross-reacting antibodies were removed from antibody preparations against strains of Streptococcus mutans serotypes a and d by adsorption on appropriate bacterial cell columns. S. mutans serotype d was further divided into two subtypes on the basis of immunofluorescent staining with conjugates of immunospecifically adsorbed immunoglobulin G. The DEAE-cellulose-bacterial cell columns were regenerated after use by desorbing the cross-reacting antibodies with low-ph buffer and were used repeatedly over an 18-month period with no detectable loss in effectiveness. The etiological role of Streptococcus mutans in experimentally induced dental caries has been demonstrated in laboratory animals (1, 12, 13, 20, 27), and evidence of an association between S. mutans and dental caries in humans has been accumulated (6, 9-11, 15, 21, 23) Ḃratthall (2) demonstrated the existence of five antigenically different serotypes of S. mutans: a, b, c, d, and e. Recently, Perch et al. (28) proposed the addition of two new serotypes, f and g. A number of studies have related the composition of antigens in the cell walls of S. mutans to Bratthall's serological classification (4, 16, 18, 22, 26, 27, 33). Further study is needed on the incidence ofs. mutans in dental plaque from various populations and on the relative frequency with which the different serotypes occur. Currently, immunofluorescence (IF) techniques appear to be the only practical means of identifying S. mutans at the serotype level directly in dental plaque samples. For this identification, it is most desirable to have serotype-specific IF reagents that will not stain other morphologically similar organisms commonly found in the oral cavity. IF has been used to identify S. mutans (3, 14, 19), and serotype specificity has been obtained by batch-wise adsorption of the conjugates or sera with cells of the cross-reacting serotypes (3). Although batch adsorption is practicable for small amounts of serum, it is 1161 unsatisfactory for large-scale production of specific antisera. Various methods have been advocated for retaining bacterial cells in a column for applications involving affinity chromatography (7, 25, 32, 35). We recently described a new method for preparing bacterial cell columns for affinity chromatography in which diethylaminoethyl (DEAE)-cellulose is used as the support material. This method was used for removing crossreacting antibodies in the preparation of serotype-specific conjugates for S. mutans serotypes b and c and for making a polyvalent conjugate specific for the S. mutans species (24). In the present study, DEAE-cellulose-bacterial cell columns were used in the preparation of serotype-specific conjugates for S. mutans serotypes a and d. MATERIALS AND METHODS Reagents and buffers. DEAE-Sephadex A-50 was obtained from Pharmacia Fine Chemicals, Inc., Piscataway, N.J. DEAE-cellulose (N,N-diethylaminoethyl ether, 1 meq/g) was from Eastman Kodak Co., Rochester, N.Y. The ph 7.4 borate saline (BS) was prepared as 0.05 M boric acid containing 0.85% NaCl. The ph was adjusted to 7.4 with concentrated NaOH, and the conductivity was adjusted to 15,000,umhos/cm at room temperature by adding water or granular NaCl. Conductivity was measured with a Beckman conductivity bridge model RC 16B2 (Beckman Instruments, Inc., Cedar Grove, N.J.). The ph 8.0 BS was prepared as 0.05 M boric acid containing

2 1162 McKINNEY AND THACKER 0.85% NaCl and was adjusted to ph 8.0 with concentrated NaOH. Phosphate-saline, ph 2.3, was prepared as 0.05 M NaH2PO4 in 1% NaCl and adjusted to ph 2.3 with concentrated HCl. BS-bovine serum albumin (BS-BSA) diluent was 0.5% crystalline BSA (Miles Laboratories, Inc., Kankakee, Ill.) in ph 8.0 BS with 0.1% NaN3. The ph was readjusted to 8.0 after BSA and NaN3 were added. Source of bacterial strains. S. mutans strains AHT, JC1, JC3, PK1, B3, B7, OMZ 70, B2, B14, and AT10 were obtained from Douglas Bratthall, University of Goteborg, Goteborg, Sweden. Strains E49, HS-1, HS-6, OMZ 61, JC2, SL1-R, K1-R, B1.3, and OMZ 176 were from Arnold Bleiweis, University of Florida, Gainesville. Strains 3720, FA-1, BHT, NCTC-10449, GS5, SL-1, , OMZ 65, OIHI, LM7, and V-100 were from L. Ariel Thomson, National Institute of Dental Research, Bethesda, Md. Richard Facklam, Center for Disease Control, Atlanta, Ga., provided the following strains: S. salivarius strains HHT, SS-2, NCTC 8618, CDC 262, and CDC 908; S. sanguis strains CDC 910, CDC 911, CDC 912, CDC 983, and JC43; S. uberis strains CDC 1001, CDC 842, CDC 843, and CDC 845; and S. mitis strains CDC 50-70, CDC 429, JC67, and JC74. Preparation of antigens and immunization schedule. Todd-Hewitt broth was dialyzed through cellulose tubing to exclude high-molecular-weight and possibly antigenic components of the media. This dialysate, with 3% glucose added, was used to grow the S. mutans type a strain 3720 and type d strain SL-1 for use as immunogens. The cells were killed by adding 1 ml of formalin per 100 ml of broth and allowing them to stand for 2 h at room temperature. The cultures were centrifuged, the cells were resuspended to a density of 40 international opacity units/ml (36) with 0.85% saline containing 0.5% formalin, and the adjusted antigens were stored at 4 C. Young New Zealand white rabbits (about 3 kg) were immunized intravenously with the antigen preparations according to the following schedule: days 1 and 2, 0.2 ml; day 3, 0.5 ml; day 4, 1.0 ml; and day 5, 2.0 ml. Boosters of 2.0 ml were given on days 30 and 31 and were repeated weekly on each of 2 successive days for 6 weeks. After the initial rest period, 50 to 60 ml of blood was taken weekly from the medial artery of the ear just before the first booster injection for the week was given. Sera of each serotype whose whole-serum conjugates gave homologous IF staining titers of 160 or higher were combined to give a pool of 3720 type a antiserum and a pool of SL- 1 type d antiserum. These two pools of immune serum were used in all of the column immunoadsorption studies. Separation and measurement of IgG. Immunoglobulin G (IgG) was separated from immune serum by chromatography on DEAE-Sephadex A-50 as previously described (24). The IgG fraction was concentrated to approximately 20 mg/ml, using an Amicon model 202 stirred cell with a PM 10 ultrafiltration membrane (Amicon Corp., Lexington, Mass.). The concentrated IgG was centrifuged to clarify it, and the protein content was determined by absorbance at 280 nm (A280) with a Beckman model 25 spectrophotometer (Beckman Instruments, Inc., Cedar Grove, N.J.), assuming E I %m = 14. Conjugate preparation. Whole serum and IgG were labeled with fluorescein isothiocyanate (FITC) as described previously (24). Whole-serum conjugates were prepared from serum obtained from each rabbit before initial immunization and also from serum taken at intervals during the course of immunization. Fluorescein/protein (F/P) ratios of IgG-FITC conjugates were determined from A ratios (A494/A280), which were then related to F/P ratios as determined by the biuret analysis for protein and A494 for FITC (17). F/P ratios of IgG conjugates were within the range of 14 to 20 /pg/mg. INFECT. IMMUN. Electrophoretic analyses. The Beckman microzone equipment was used for performing cellulose acetate strip electrophoresis analyses with fraction 1 immunoglobulin from the DEAE-Sephadex column (17). For immunoelectrophoresis (IE) studies, whole-cell extracts ofs. mutans strains 3720 and SL- 1 were prepared according to the method of Rantz and Randall (29). Extracts were dialyzed against water, lyophilized, and reconstituted to a concentration of 15 mg/ml. Unadsorbed and adsorbed preparations of anti-3720 and anti-sl-1 IgG were used at concentrations of 10 mg/ml. The method described by Campbell et al. (5) using borate buffers, ph 8.4 and ionic strength 0.1,u, was used for IE. CdSO4, %, was incorporated in the agar for increased sensitivity (8). IF staining. Serial twofold dilutions were made of conjugates with BS-BSA diluent for IF staining. Microscope slide smears were stained and examined by methods that were previously described (24). Staining titers were expressed as the dilution factor of the highest dilution of conjugate to give 4+ or very bright staining. Titers for whole-serum conjugates were expressed in relation to whole serum as undilute. Titers for IgG-FITC conjugates were expressed in relation to 10 mg of IgG per ml of conjugate as undilute. Preparation of DEAE-cellulose-bacterial cell columns and immunoadsorption procedures. The columns were 115 cm in length by 26 or 30 mm inside diameter, depending on the amount of material to be packed. The strains of bacterial cells and the volumes of packed cells of each strain in the various columns were chosen on the basis of the heterologous IF staining titers of the control conjugates (conjugates of immune IgG before any column immunoadsorptions). Bacterial cells were killed by adding formalin to the broth culture to give a 1% concentration of formalin or approximately 0.4% formaldehyde, after which the cells were allowed to stand for 2 h at room temperature before harvesting. A minimum of 2 g of DEAE-cellulose per ml of packed cells was found to be necessary to immobilize the cells and produce a column with a satisfactory flow rate. The DEAE-cellulose-bacterial cell columns were prepared as described previously (24). The four immunoadsorption columns used in the present study were a non-mutans oral streptococci column, an S. mutans type d (B13) column, an S.

3 VOL. 13, 1976 mutans type d (SL-1) column, and an S. mutans type a (AHT) column. The bacterial cell compositions and liters of broth used to grow the cells for the four columns are listed in Table 1. The non-mutans oral streptococci column (hereafter called the non-mutans column) was also used in the previous study (24). For immunoadsorption, the IgG fraction of immune serum obtained by DEAE-Sephadex A-50 chromatography was applied to the appropriate bacterial cell column. Monitoring of the columns, dissociation of the adsorbed cross-reacting or homologous antibodies, and reconditioning of the columns for repetitive use were performed as described previously (24). IgG fractions from the immunoadsorption col- TABLE 1. Composition of DEAE-cellulose-bacterial cell columns ofr cells cellu- Liters gake of Column Cells of cul- ced ture (ml) lose S. mutans Type d, B13 [ S. mutans Type d, SL S. mutans Type a, AHT Non-mu- S. salivarius tans oral (3)a strepto- S. sanguis (3)b 9 20 cocci S. uberis (2) 2 4 S. mitis (4)d 4 10 a Strains: Jablon's HHT, Gibbon's SS2, and NCTC b Strains: CDC 910 (ATCC 10556), CDC 911 (ATCC 10557), and JC 43. c Strains: CDC 842 and CDC d1 Strains: CDC (ATCC 9811), CDC 429, JC 74, and JC 67. IMMUNOADSORBENT COLUMNS 1163 umns were concentrated to 10 to 20 mg/ml for preparing conjugates or for further adsorption procedures. The control conjugates (conjugates of unadsorbed IgG) and conjugates of IgG that were processed on the various immunoadsorbent columns were evaluated by IF staining with all strains of S. mutans and non-mutans oral streptococci listed under "Source of bacterial strains." RESULTS Testing of antisera by IF staining. Wholeserum conjugates of the preimmune sera did not have appreciable IF staining titers against any of the S. mutans or non-mutans oral streptococcus strains that were observed to stain with the immune conjugates. Therefore the cross-reactions observed in IF staining with the immune conjugates must have been the result of immunizations. Satisfactory or 4+ staining of homologous cells at a 1:160 dilution (wholeserum conjugates) was usually obtained by the 5th or 6th week of immunization. Conjugates from the succeeding weekly bleedings showed an overall progressive increase in staining titers with prolonged immunization. IE. The results of IE of the antigen extracts of S. mutans strains 3720 and SL-1 against anti-3720 IgG before and after adsorption with the SL-1 and non-mutans columns are shown in Fig. 1. Absence of a precipitin line against the SL-1 extract after adsorption indicates removal of the cross-reacting antibody, which agrees with IF staining results. Likewise, adsorption of the anti-sl-1 IgG with the AHT and nonmutans columns removed the cross-reaction for 3720 (Fig. 2). A I [ : 3720 I' w I ~~SLI 1~3720 L 9 _l ~~~SLI FIG. 1. Immunoelectrophoretic patterns ofextracts ofs. mutans 3720 type a and SL-1 type d. (A) IgG in the trough was anti-3720 unadsorbed. (B) IgG in the trough was anti-3720 adsorbed with the SL-1 column and the non-mutans oral streptococcus column.

4 1164 McKINNEY AND THACKER INFECT. IMMUN. SLi1 A L.' ----m 3720 SLi 3720 FIG. 2. Immunoelectrophoretic patterns ofextracts ofs. mutans SL-1 type d and 3720 type a. (A) IgG in the trough was anti-sl-1 unadsorbed. (B) IgG in the trough was anti-sl-1 adsorbed with the AHT type a column and the non-mutans oral streptococcus column. Nonspecific adsorption of IgG by the DEAE-cellulose-bacterial cell columns. To measure the completeness of IgG recovery in the absence of any immunoadsorption, 100-mg samples of nonimmune rabbit IgG were applied to all four columns shown in Table 1. The columns were developed with ph 7.4 BS, and the eluted protein was quantitatively collected and determined by absorbance measurements at 280 nm. Essentially, the 100-mg samples of nonimmune rabbit IgG applied to the immunoadsorption columns were completely recovered, which indicates that nonimmune retention of IgG by the DEAE-cellulose-bacterial cell columns is negligible. S. mutans 3720 a antiserum and immune IgG. DEAE-Sephadex chromatography of 50 ml of whole serum yielded 837 mg of IgG. This fraction was shown to be gamma globulin by cellulose acetate strip electrophoresis and was only IgG by IE. Samples of the IgG were applied to bacterial cell columns as shown in Fig. 3. The results of the various adsorption procedures with respect to homologous and heterologous cell IF staining titers observed with conjugates of the IgG fractions are shown in Table 2. The control conjugate (conjugate of unadsorbed IgG) had a high titer for all strains of the a serotype that were tested (4+ at a 1:128 dilution). Cross-reactions of high titer were also observed for the d strains and most of the S. salivarius strains. Cross-reactions with other S. mutans serotypes and non-mutans oral streptococci that were tested were either negligible or could be eliminated by conjugate dilution. The titers obtained after each independent or sequential adsorption procedure in relation to the control conjugate titers (Table 2) were as follows. (i) Adsorption on the B13 d column removed only those antibodies cross-reacting with B13 and OMZ 176, which are both d strains, but did not appreciably affect the titer for the remaining d strains. Thus the d serotype was divided into two subtypes on the basis of this adsorption. Adsorption on the B13 column also essentially removed the antibodies that cross-reacted with the S. salivarius strains. (ii) Adsorption on the SL-1 d column essentially removed the antibodies that cross-reacted with all of the d strains. This adsorption also removed antibodies that cross-reacted with the S. salivarius strains and was the most practical procedure in terms of time invested and recovery of antibody that was specific for S. mutans serotype a. (iii) Adsorption on the non-mutans oral streptococcus column removed the cross-reactions with all of the non-mutans oral streptococcus strains that were tested. Staining titers for the d strains were appreciably reduced but not eliminated by this adsorption. (iv) The conjugate of the product obtained by sequential adsorption on the non-mutans oral streptococcus column and the SL-1 column was specific for S. mutans serotype a and was similar to the product obtained by adsorption on the SL-1 column only. IF staining titers for the conjugate of the cross-reacting antibody desorbed from the SL-1 column are shown in the last column in Table 2. All of the cross-reactions that were observed

5 VOL. 13, 1976 IMMUNOADSORBENT COLUMNS ml whole serum DEAE Sephadex A mg IgG 415 mg IgG 100nmg IgG 250 mg IgG \ pnonmutans oral 1> B 13 "d" column SL-1 "d" column ~)strep.columnsl1d"clm 55 mg cross-reacting antibody r ecovered by desorption 350 mg IgG 56 mg IgG 175 mg IgG 250 mg IgG K SL-1 "d" column 200 mg IgG FIG. 3. Scheme of column immunoadsorptions performed with anti-s. mutans 3720 type a IgG. TABLE 2. Immunofluorescence staining titers ofconjugates of unadsorbed and of adsorbed serotype a (3720) immune IgG Adsorbed on DEAE-cellulose-bacterial cell column(s) Control origi- Nnm- Non-mu- Cross-reacting Seronal IgG before Non-mu- tans oral ~~antibody de- Organism type ad rtion (4+ 3B13 (type SL-1 (type tans oral tans or sorbed from titer)a d) only d) only strepto- cocci, then (4- +ctiter (4+ titer) (4+ titer) cocci only SL-1 (4+ titer) (4+ titer) (4+ titer) S. mutans NIDR 3720b a SL-1c d _d B13 and OMZ d S. salivarius HHTI a 4+ immunofluorescent staining (brilliant fluorescence). All dilutions are expressed in relation to 10 mg of IgG per ml of conjugate as undilute. b Representative strain of S. mutans serotype a. Other type a strains that stained similarly: AHT, E49, HS-1, HS-6, and OMZ 61. c Representative strain of subtype ofs. mutans serotype d. Other d strains that stained similarly: 6715:15, SLl-R, Kl-R, OMZ 65, CDC 1083, and OIHI. d Negative (no fluorescence). e Representative strain of S. salivarius. Other S. salivarius strains that stained similarly: SS-2, NCTC 8618, and CDC 262.

6 1166 McKINNEY AND THACKER 50 ml whole serum Y 461 mg IgG 435 mg IgG f mg IgG 293 mg IgG DEAE-Sephadex AO50 AHT a" column 72 mg cross-reacting antibody recovered by desorption K ) nonmutans oral strep. column 240mg IgG 100 mg IgG 100 mg IgG j SL- l "d" column 18 mg purified antibody recovered by desorption 58 mg IgG (all antibodv for S. mutans removed) 65mg IgG B 13 "d" column FIG. 4. Scheme of column immunoadsorptions performed with anti-s. mutans SL-1 type d IgG. with the control conjugate were apparent in IF staining with the conjugate of this desorbed antibody. S. mutans SL-1 d antiserum and immune IgG. DEAE-Sephadex chromatography of 50 ml of whole serum yielded 461 mg of IgG. Samples of the IgG were applied to bacterial cell columns (Fig. 4). Homologous and heterologous IF staining titers for conjugates of the control and samples from the various columns are shown in Table 3. The control conjugate gave a titer of 128 with all of the d strains and cross-staining of all of the a strains (4+ at a 1:16 dilution). High titers were also observed for all of the S. salivarius strains. The results obtained after each independent or sequential adsorption procedure in relation to the control conjugate titers (Table 3) were as follows. (i) Adsorption on the AHT type a column removed the antibody cross-reacting with the a strains without appreciably affecting the staining titer for the d strains or the S. salivarius strains. INPFECT. IMMUN. (ii) Sequential adsorption on the AHT type a column and the non-mutans column completely removed the staining of the a strains and the non-mutans strains without appreciably reducing the staining titer for the d strains. (iii) Sequential adsorption on the AHT a column, non-mutans column, and B13 d column resulted in a division of the d serotype. Staining was completely eliminated for the d strains B13. OMZ 176, and OIHI, but the staining titer for the remaining d strains was 16. (iv) Sequential adsorption on the AHT a column, non-mutans column, and SL-1 d column completely removed the staining of all type d and type a S. mutans as well as the S. salivarius strains (not shown in Table 3). The antibody-conjugate titers of the crossreacting antibody that was desorbed and recovered from the AHT a column are also shown in Table 3. The conjugate of this cross-reacting antibody had a considerably higher IF staining titer than the control conjugate (256 versus 16) for all of the type a strains and a titer of 128 for all of the type d strains except B13, OMZ 176, and OIHI, which were 4+ at a maximum 1:16 dilution. Titers for the non-mutans strains were similar to those obtained with the control conjugate. The antibody conjugate of specifically purified SL-1 antibody (Table 3, last column) desorbed from the SL-1 d column was specific for the d strains and showed a higher staining titer (512 or 256) than did the control conjugate. Stability of bacterial cell immunoadsorption columns. The immunoadsorption columns have been used for as long as 18 months with as many as 13 runs being made on a single column with no detectable loss in capacity for antibody adsorption or in flow rate. All four columns performed satisfactorily when last used. DISCUSSION Although use of a pure IgG fraction of immune serum is not essential to the successful application of bacterial cell column immunoadsorption methods in which DEAE-cellulose is a component, it is much easier to monitor the antibody-containing fractions and to quantitatively account for the protein applied to the column if pure IgG is used. If serum fractions other than pure IgG are applied to the column, ion exchange fractionation may occur in addition to the antigen-antibody interactions. Further, ion exchange fractionation would be expected to occur if FITC conjugates of IgG were applied to the column. There was general homogeneity among the S. mutans type a strains as indicated by IF staining titers with the control conjugates as

7 VOL. 13, 1976 TABLE 3. IMMUNOADSORBENT COLUMNS 1167 Immunofluorescence staining titers ofconjugates of unadsorbed and ofadsorbed serotype d (SL-1) immune IgG Adsorbed on DEAE-cellulose-bacterial Adsorbed on: column(s) AHT, type a; non-mutans Control be- AHT, type AHT,typeCros-reating a,then antibody de- oral strepto- Organism Sero- Seo fore oea-a ad- a, then hn a, the non-muantboycd-- sorbed from cci SL-1 L1 type sorption AHT, type non-mu- noral AHT t a type d. De- (4 + titer)a a tans oral tan to- AH', tytera sorbed from: (4+ titer) strepto- cocci, then (4+ tter) homologous cocci cocc, thpend cell column (4+ titer) (4terd (4+ titer) S. mutans SL lb d B13, OMZ d _d , and OIHI NIDR 3720c a S. salivarius HHTe a 4+ immunofluorescent staining (brilliant fluorescence). b Representative strain ofs. mutans serotype d. Other type d strains that stained similarly: , SLl- R, Kl-R, OMZ 65, and CDC c Representative strain of S. mutans serotype a. Other type a strains that stained similarly: AHT, E49, HS-1 HS-6, and OMZ 61. d Negative (no fluorescence). e Representative strain of S. salivarius. Other S. salivarius strains that stained similarly: SS-2, NCTC 8618, CDC 262, and CDC 908. well as with the conjugates of IgG through the various adsorption columns (Table 2). In their immunodiffusion studies, Mukasa and Slade (26) observed only one antigen site in cell wall extracts of HS6 type a cells that was common to both serotype a and serotype d strains; however, it is apparent from the present analysis by column immunoadsorption methods and IF staining that at least two antigens of the a serotype are shared with members of the d serotype. As seen from the data in Table 2, it was not possible to obtain serotype specificity with antisera against strain 3720 a by adsorbing with just the B13 d column. IF staining reactions of the 3720 type a crossreacting antibody desorbed from the SL-1 d column indicate the presence of common antigens among the S. mutans a and d strains and strains of S. salivarius and clearly show the feasibility of this method of isolating cross-reacting antibodies for the study of antigen-antibody interrelationships among closely related species and serotypes. The S. mutans d strains appear to be a more antigenically heterogeneous group than the a strains. Adsorption of SL-1 IgG on the B13 type d column removed all antibody to strains B13, OMZ 176, and OIHI (Table 3), thus separating these three strains as a subtype within the d serotype; these strains are deficient in at least one antigen that is present in the other d strains tested. The subtyping of B13 and OMZ 176 within the d serotype was also arrived at by the staining reactions of conjugates of sequentially adsorbed 3720 type a IgG, although OIHI, in this case, appeared to fit antigenically with the other five d strains. The conjugate of SL-1 cross-reacting antibody desorbed from the AHT type a column stained B13, OMZ 176, and OIHI to a much lower degree than the other d strains (Table 3), which indicates that these d strains have fewer antigens in common with the a serotype than do the other d strains that were examined. On the basis of these findings, one might speculate that B13 or OMZ 176 strains would be better immunogens than the other d strains listed in Table 3 for the preparation of d type antiserum in rabbits since less a-d crossreacting antibody would be expected. Perch et al. (28) observed the heterogeneity among the d strains and proposed a further breakdown into additional serotypes. Our findings, in general, appear to be consistent with theirs, with the exception of observations of the SL-1 strain. Perch et al. reported that SL-1 does not possess an antigen in common with the a serotype. In addition, Mukasa and Slade (26) failed to get a precipitin line with a hot-water extract of SL-1 against anti-hs 6 type a whole serum in immunodiffusion. Contrary to their findings, we find that the SL-1 strain reacts at a very high titer with conjugates of type a IgG

8 1168 McKINNEY AND THACKER (strain 3720) even after exhaustive adsorption on the B13 d column (Table 2). As further evidence of SL-1 having antigen in common with the a serotype, our unadsorbed or control conjugate of anti-sl-1 IgG had a high titer with all of the a strains (Table 3). The differences in observations regarding the SL-1 strain in these studies are probably due to the different strains of the a and d serotypes that were used as immunogens to prepare the antisera and to differences in the SL-1 cultures. Appreciable loss of antibody occurs in the preparation of pure antibody by adsorption and desorption on a homologous cell column when relatively small amounts of immune IgG are applied to the column. As seen in Fig. 4, for 100 mg of anti-sl-1 IgG applied to the SL-1 d column, 58 mg of IgG passed through. Since the portion that passed through had no IF titer, the total antibody of the IgG applied to the column must have been about 42 mg, and the 18 mg of pure antibody recovered represents about 43% of the antibody that was present in the 100-mg sample. Recoveries of purified antibody as high as 77% of the total antibody present have been obtained from an S. mutans homologous cell column of the c serotype by applying a much greater load of immune IgG to the column (24). Apparently the additional protein in the greater antibody load has a protective effect in the desorbing process and results in a greater percentage of recovery. Antibacterial IF conjugates of pure antibody, from which the crossreacting antibodies have been removed, are readily obtainable on a practical scale by the methods described. DEAE-cellulose-bacterial cell column immunoadsorption has major advantages over simple batch adsorption methods. The columns can be readily washed and equilibrated to the optimum ph and salt concentration for maximum antigen-antibody interaction. The use of columns allows monitoring with an ultraviolet absorptiometer and quantitative collection of protein fractions. The desorption and collection of the cross-reacting antibodies, or of homologous antibodies in the case of homologous cell columns, is probably much more quantitative by column than by batch methods. Finally, perhaps the greatest advantage of column immunoadsorption is that DEAE-cellulose-bacterial cell columns can be readily regenerated and used repeatedly over a long period of time with no detectable loss in effectiveness. The means by which the bacterial cells are bound to DEAE-cellulose was originally thought to be ionic (24), with extensive hydrogen bonding at low ph. However, work in this INFECT. IMMUN. laboratory has shown that pretreatment of bacterial cells with formalin is essential for satisfactory binding to DEAE-cellulose. The mechanism of adherence may be covalent bonding or a combination of ionic and covalent bonding. Since DEAE-cellulose tends to retain the IgM component of serum (30), this method of removing unwanted antibodies would not be satisfactory for preparing reagents for serological tests that are dependent on IgM antibody. With this exception, column immunoadsorption methods with formalin-treated bacterial cells mixed with DEAE-cellulose generally should be widely applicable for the systematic removal and collection of cross-reacting antibodies in classification studies and for the isolation of specific antibody fractions for various serological tests. This method should be an ideal tool for large-scale commercial preparation of highly specific immunofluorescent reagents and may also have potential for use in conducting processes of chemical synthesis that depend on enzymatic properties of bacterial cells. Svensson et al. (31) observed inhibition of precipitin reactions where certain polysaccharide antigens were involved, due to complexing of sugar residues by borate. Although we have not observed any inhibition due to the presence of borate in either column immunoadsorption procedures or IF staining with S. mutans, we have recently discontinued the use of borate in our systems. In subsequent column immunoadsorption work, we have substituted 0.01 M tris(hydroxymethyl)aminomethane-saline adjusted to ph 7.4 with HCl in place of the ph 7.4 BS. Tris(hydroxymethyl)aminomethane must be removed from the antibody preparation before labeling with FITC. ACKNOWLEDGMENTS We thank Richard R. Facklam, Center for Disease Control, for confirmatory biochemicals tests; L. Ariel Thomson, National Institute of Dental Research, for advice and assistance in obtaining cultures; and Bertie Pittman, G. Ann Hebert, and Patricia Harris, Center for Disease Control, for their assistance during the course of this research. This investigation was performed pursuant to Public Health Service Interagency Agreement no. Y01-DE with the National Institute of Dental Research. LITERATURE CITED 1. Bowen, W. H The induction of rampant dental caries in monkeys (Macaca irus). Caries Res. 3: Bratthall, D Demonstration of five serological groups of streptococcal strains resembling Streptococcus mutans. Odontol. Revy 21: Bratthall, D Immunofluorescent identification of Streptococcus mutans. Odontol. Revy 23: Burgess, J. E., and J. R. Edwards Chemical characterization of a cell wall antigen from Streptococcus mutans FAl. Infect. Immun. 8:

9 VOL. 13, Campbell, D. H., J. S. Garvey, N. E. Cremer, and D. H. Sussdorf Methods in immunology. W. A. Benjamin, Inc., New York. 6. Carlsson, J Presence of various types of nonhaemolytic streptococci in dental plaque and in other sites of the oral cavity in man. Odontol. Revy 18: Cope, J. B., J. J. Redys, E. W. Hibbard, and E. K. Borman Improved methods of adsorbing immunofluorescence reagents. Health Lab. Sci. 5: Crowle, A. J Immunodiffusion, p Academic Press Inc., New York. 9. De Stoppelaar, J. D., J. Van Houte, and 0. Backer- Dirks The relationship between extracellular polysaccharide-producing streptococci and smooth surface caries in 13-year-old children. Caries Res. 3: Duany, L. F., D. D. Zinner, and J. M. Jablon Identification of types of potentially cariogenic streptococci on human tooth surfaces by the fluorescent antibody technique. J. Dent. Res. 49: Edwardsson, S Characteristics of caries inducing human streptococci resembling Streptococcus mutans. Arch. Oral Biol. 13: Fitzgerald, R. J., and P. Keyes Demonstration of the etiologic role of streptococci in experimental caries in the hamster. J. Am. Dent. Assoc. 61: Gibbons, R. J., K. S. Berman, P. Knoettner, and B. Kapsimalis Dental caries and alveolar bone loss in gnotobiotic rats infected with capsule forming streptococci of human origin. Arch. Oral Biol. 11: Grenier, E. M., W. C. Eveland, and W. J. Loesche Identification of Streptococcus mutans serotypes in dental plaque by fluorescent antibody techniques. Arch. Oral Biol. 18: Guggenheim, B Streptococci of dental plaques. Caries Res. 2: Hardie, J. M., and G. H. Bowden Cell wall and serological studies on Streptococcus mutans. Caries Res. 8: Hebert, G. A., B. Pittman, R. M. McKinney, and W. B. Cherry The preparation and physicochemical characterization of fluorescent antibody reagents. U.S. Department of Health, Education, and Welfare, Washington, D.C. 18. Iacono, V. J., M. A. Taubman, D. J. Smith, and M. J. Levine Isolation and immunochemical characterization of the group-specific antigen of Streptococcus mutans Infect. Immun. 11: Jablon, J. M., and D. D. Zinner Differentiation of cariogenic streptococci by fluorescent antibody. J. Bacteriol. 92: Krasse, B Human streptococci and experimental caries in hamsters. Arch. Oral Biol. 11: Krasse, B., H. V. Jordan, I. Svenssen, and L. Trell The occurrence of certain "caries inducing" streptococci in human dental plaque material. Arch. Oral Biol. 13: Linzer, R., and H. D. Slade Purification and IMMUNOADSORBENT COLUMNS 1169 characterization of Streptococcus mutans group d cell wall polysaccharide antigen. Infect. Immun. 10: Littleton, N. W., S. Kakahashi, and R. J. Fitzgerald Recovery of specific "caries-inducing" streptococci from carious lesions in the teeth of children. Arch. Oral Biol. 15: McKinney, R. M., and L. Thacker Improvement in specificity of immunofluorescent reagents for identifying Streptococcus mutans by DEAE-cellulose-bacterial cell column immunosorption methods. J. Dent. Res. 55(Special Issue A):A50-A Morrison, R. B A new method for agglutinin adsorption. Lancet 2: Mukasa, H., and H. D. Slade Extraction, purification, and chemical and immunological properties of the Streptococcus mutans group "a" polysaccharide cell wall antigen. Infect. Immun. 8: Mukasa, H., and H. D. Slade Structure and immunological specificity of the Streptococcus mutans group b cell wall antigen. Infect. Immun. 7: Perch, B., E. Kjems, and T. Ravn Biochemical and serological properties of Streptococcus mutans from various human and animal sources. Acta Pathol. Microbiol. Scand. Sect. B 82: Rantz, J. A., and E. Randall Use of autoclaved extracts of hemolytic streptococci for serological grouping. Stanford Med. Bull. 13: Schultze, H. E., and J. F. Heremans Fractionation by adsorption and chromatography, p In H. E. Schultze and J. F. Heremans (ed.), Molecular biology of human proteins, vol. 1. Elsevier Publishing Co., New York. 31. Svensson, S., S. G. Hammarstrom, and E. A. Kabat The effect of borate on polysaccharide-protein and antigen-antibody reactions and its use for the purification and fractionation of cross-reacting antibodies. Immunochemistry 1: Thomson, R. O., P. D. Walker, and E. Harris Bacterial immunoadsorbent columns. Appl. Microbiol. 18: Van de Run, I., and A. S. Bleiweis Antigens of Streptococcus mutans. I. Characterization of a serotype-specific determinant from Streptococcus mutans. Infect. Immun. 7: Vaught, R. M., and A. S. Bleiweis Antigens of Streptococcus mutans. HI. Characterization of an antigen resembling a glycerol teichoic acid in walls of strain BHT. Infect. Immun. 9: Weetall, H. H Immunoabsorbent for the isolation of bacterial specific antibodies. J. Bacteriol. 93: World Health Organization W.H.O. Tech. Rep. Ser. no. 68, p World Health Organization, Geneva, Switzerland. 37. Zinner, D. D., J. M. Jablon, A. P. Aran, and M. S. Saslaw Experimental caries induced in animals by streptococci of human origin. Proc. Soc. Exp. Biol. Med. 118: