Vol. 13, No. 6 Printed in U.S.A.

INFECTION AND IMMUNITY, June 1976, p. 1733-1742

Copyright C 1976 American Society for Microbiology

Identification and Quantitation of Capsular Antigen in Capsulated and Noncapsulated Strains of Haemophilus influenzae Type b by Crossed-Immunoelectrophoresis FRANCIS L. A. BUCKMIRE Department of Microbiology, The Medical College of Wisconsin, Milwaukee, Wisconsin 53233

Received for publication 26 January 1976

Sonicated preparations of capsulated Haemophilus influenzae type b, two of its spontaneous mutants, one containing patches of capsules (class I variant) and the other noncapsulated (class II variant), and a noncapsulated strain of H. influenzae type d were analyzed by crossed-immunoelectrophoresis using unadsorbed antiserum to capsulated H. influenzae type b. Twenty common antigens were present in all four cultures. Two type b-specific antigens were also identified in the three H. influenzae type b cultures when antiserum adsorbed with H. influenzae type d sonicates (AdR) was used. One of these, the type b capsular antigen, cross-reacted with an antigen ofBacillus pumilus. Further adsorption of AdR with the B. pumilus sonicate reduced, but did not eliminate, the antibodies to the type b capsular antigen, although all antibodies to B. pumilus were removed. Sonication sheared the type b capsular antigen, resulting in an increase in its electrophoretic mobility in agarose gel. The capsular antigen from all three H. influenzae type b cultures had the same electrophoretic characteristics. Reproducible quantitation of sheared and unsheared capsular antigen was demonstrated by rocket immunoelectrophoresis. As little as 2.5 ng of polyribophosphate pentose was identified and measured. Capsulated H. influenzae type b contained 78 ng of polyribophosphate pentose per gg of cell protein; class I contained 20 ng and class II contained 4 ng. The small amount of type b capsular antigen present in the class II variant may account for its lack of detection in this organism before now.

Haemophilus influenzae, the causative viewed by obliquely transmitted light. The agent of human infections, such as meningitis class I variant produces very rough-surfaced, and otitis media, may be isolated from patients noniridescent (R) colonies, and the class II vareither as capsulated or as noncapsulated orga- iant produces smooth, noniridescent (S) colonisms (12, 15, 34). The capsulated bacteria are nies. Capsulation, and hence the existence of capmore highly pathogenic (21). Of the six serological types of capsulated H. influenzae, type b is sular antigen, has generally been demonthe most frequent cause of life-threatening dis- strated by the Quellung reaction (2, 26), by eases (3, 28, 34). Whether the noncapsulated immunofluorescence using fluorescein-tagged, strains found in clinical material are derived type-specific antibody (9, 12, 27), or other imfrom capsulated strains is not known, but in munological techniques, such as double immusupport of this possibility is the fact that, in in nodiffusion (Ouchterlony), that allow soluble vitro experiments, capsulated bacteria sponta- capsular antigen to diffuse away from intact neously mutate to noncapsulated cells (12). The cells through a permeable matrix (usually pattern of the "dissociation" is generally from agar) and interact with specific capsular antithe normal, fully capsulated coccobacilli to (i) bodies (14). Data obtained with H. influenzae genetically unstable filamentous bacilli con- type b (HITh) using the above techniques have taining patches of capsules (class I variant) and led to the general conclusions that (i) antisera (ii) short bacilli, which are noncapsulated obtained from rabbits immunized with forma(class II variant) (13, 28). Several authors have linized, capsulated HITh contain highly specific used different terminologies to describe the dis- type b capsular antibodies (4, 6, 32); (ii) several tinctive colonial morphology produced by these gram-positive and -negative bacteria contain forms (13, 23). The capsulated cells produce antigens that cross-react with the type b capsumucoid (M) colonies that are iridescent when lar antigen, the cross-reacting antigen being 1733

1734

BUCKMIRE

ribitol phosphate (6, 10, 25, 32); and (iii) class II HITb does not contain capsular antigen. Leidy et al. have reported, however, that class II HITh populations treated with deoxyribonucleic acid from a capsulated type a culture have given rise to transformants containing both the a and b capsules (20). Thus, class II cells may have the potential to produce a type b capsule, although they do not produce it. Recently, a high-resolution and very sensitive immunochemical technique, crossed-immunoelectrophoresis (CIE), has been developed by Laurell to more clearly resolve mixtures of antigens (18). This technique utilizes the fact that different antigens have different electrophoretic mobilities and the observation of Ressler (29) that antigens can be forced by an electrical current into an agar matrix containing homologous antiserum to produce a number of narrow precipitation curves with a common base. Although this method was developed and has been primarily used for the analysis and semiquantitation of normal and abnormal serum antigens, it has proven extremely useful in resolving the maze of antigens present in certain microbial preparations, for example, mycobacteria (30), Candida albicans (8), and mycoplasma (17). Laurell, using the above principles, further developed a method (rocket immunoelectrophoresis) for quantitation of antigens (19). Different concentrations of antigen electrophoresed from different wells of the same size into an agarose/antibody gel exhibited a linear or near-linear relationship between the heights of the rocket-shaped precipitin peaks and the antigen concentration. In this communication, analyses of the antigens from the three classes of HITh, capsulated and class I and II variants, are presented. The antiserum used was prepared against capsulated type b strain "Rab." The results show (i) that all three HITh cultures produce the capsular antigen but in vastly different amounts and (ii) that CIE and rocket immunoelectrophoresis can be used to detect and quantitate very small quantities of type b capsular antigens in a system containing multiple haemophilus antigens and antibodies.

INFECT. IMMUN.

cated by the Quellung reaction with HITb type bspecific antiserum (Hyland) or by an immunofluorescence procedure with fluorescein-conjugated type b-specific antiserum (12). A class II spontaneous variant was similarly selected from a 12-h plate culture of the class I variant (12). H. influenzae strain Rd (HIRd), a noncapsulated variant from a type d isolate (5), was obtained from J. Bendler, Department of Microbiology, Medical College of Wisconsin. Bacillus pumilus strain 172 was obtained from the American Type Culture Collection. Stock cultures prepared from 6-h-old plate cultures were suspended in Trypticase soy broth (Baltimore Biological Laboratories, Inc.) containing 5% glycerol and 0.08% Noble agar (Difco) and stored at -85 C. Growth conditions. All H. influenzae strains were routinely cultivated on plates of solid medium incubated at 37 C in candle jars. The medium was prepared by supplementing autoclaved Trypticase soy agar (Baltimore Biological Laboratories, Inc.) with nicotinamide adenine dinucleotide (nicotinamide adenine dinucleotide-oxidized, P-L Biochemicals, Milwaukee, Wis.), recrystallized hemin (Nutritional Biochemicals Corp., Cleveland, Ohio) (16), and 0.1% a-D-glucose anhydrous (Calbiochem, La Jolla, Calif.), at final concentrations of 10 ,ug/ml, 10 ,ug/ml, and 0.1%, respectively. Supplements were individually sterilized by filtration through 0.20- Am pore size cellulose triacetate membrane filters (Gelman Instrument Co., Ann Arbor, Mich.). Media were stored at 4 C and were not used if refrigerated for longer than 14 days. The B. pumilus was cultured on Trypticase soy agar plates at 37 C. Preparation of cell suspensions and sonicates. Cell suspensions were prepared by suspending cells scraped from 9-h plate cultures in 10 mM tris(hydromethyl)aminomethane(Tris)-2.2 mM CaCl2 buffer, pH 8.0. All suspensions were adjusted to an optical density of 6 to 7 on a Spectronic 20 spectrophotometer at a wavelength of 600 nm. Onemilliliter aliquots of the suspensions were placed in an ice bath and sonicated to complete breakage using a Heat Systems Sonifier cell disruptor. Cell breakage was monitored by phase-contrast microscopy. These sonicates, which contained between 2.5 and 3.2 mg of protein per ml, were used as antigen preparations. Preparation of SDS lysates. Sodium dodecyl sulfate (SDS) lysates were prepared by adding SDS to a final concentration of 0.25% to cell suspensions prepared as above and heating the mixture at 50 C for 10 min. This procedure resulted in total clearance of the suspension. Preparation of HITb washings. A capsulated HITb cell suspension prepared as above was vorMATERIALS AND METHODS texed vigorously and then centrifuged at 48,200 x g Bacteria. The capsulated strain of HITb (Sim's for 10 min at 4 C. The supernatant was decanted and isolate) used in this study was generously provided used as the "Tb" wash antigen preparation. by B. W. Catlin of the Department of Microbiology, Preparation of crude capsular antigen. Crude Medical College of Wisconsin, Milwaukee. It was capsular antigen (polyribophosphate [PRP]) was obisolated from the cerebrospinal fluid of a child with tained from capsulated HITb cells by the method of meningitis. A class I spontaneous variant was se- Rodrigues et al. (31). Cells from plate cultures inculected from a 12-h plate culture of the capsulated bated for 9 h were suspended in chilled 1 mM sodium parent by its distinctive colonial and cellular mor- phosphate-150 mM sodium chloride buffer, pH 7.2 phology (12). Cultures ofboth strains were authenti- (phosphate-buffered saline), centrifuged, and resus-

VOL. 13, 1976

H. INFLUENZAE TYPE b CAPSULAR ANTIGEN

pended in fresh phosphate-buffered saline containing 0.5% formaldehyde. This suspension was then stirred for 24 h at 37 C and recentrifuged and the supernatant was collected and concentrated under vacuum at 37 C on a flash evaporator (Buchler Instruments, Fort Lee, N.J.). The concentrate was ethanol precipitated and centrifuged, and the pellet, redissolved in glass-distilled water, was stored at -20 C. This material (PRP) contained 265 ,g of protein and 250 ug of pentose per ml. Protein determination. Protein was determined by the method of Lowry et al. (22), using lysozyme (Nutritional Biochemicals Corp.) as the standard. Optical densities were read in a Beckman DU spectrophotometer at a wavelength of 750 nm. Pentose determination. Pentose was determined by the Bial orcinol reaction of Meybaum (24) as modified by Albaum and Umbreit (1), using reagent-grade D-ribose (P-L Biochemicals, Inc., Milwaukee, Wis.) as the standard. The reaction mixtures were heated for 45 min in a boiling-water bath, and optical densities were read at 670 nm in a Beckman DU spectrophotometer. Orcinol monohydrate (Aldrich Chemical Co., Inc., Milwaukee, Wis.) was twice recrystallized in chloroform (J. T. Baker Chemical Co., Phillipsburg, N.J.). CIE. CIE was performed by essentially the procedure described by Roberts et al. (30) on 8- by 10-cm glass slides. One percent agarose, A grade (Calbiochem, San Diego, Calif.), dissolved in 73 mM Tris22 mM sodium barbital-0.47 mM CaCl2-0.2 mM NaN3 (IEP) buffer, pH 8.6, was used. Wicks were made from Whatman 3 MM filter paper. Ten microliters of the sample (antigen preparation) and 2 ,u1 of bromothymol blue (tracking dye) were routinely used. First-dimensional electrophoresis was carried out in the IEP buffer at 17.5 mA/slide at 5 C until the tracking dye had traveled a distance of approximately 4.5 cm from the antigen well. The agarose/ antibody gel used for the second-dimensional electrophoresis contained 0.4 ml of antiserum in 10 ml of 1% agarose prepared as above. The second-dimensional electrophoresis was carried out at 7 mA/slide for 20 to 22 h at 5 C. Upon completion, the slides were rinsed twice with 100 mM NaCl and twice with glass-distilled water. Excess moisture and protein were removed from the gels by pressing the slides between heavy glass plates lined with paper towelling after each rinse. The gels were then thoroughly dried and stained with 1% Coomassie brilliant blue dissolved in a 9:2 mixture of 95% ethanol to glacial acetic acid; destaining was carried out in the ethanol-acetic acid mixture without the Coomassie blue. Quantitative rocket immunoelectrophoresis. Rocket immunoelectrophoresis was performed by the procedure of Laurell (19) on 8- by 10-cm slides. The agarose/antibody gel contained 0.5 ml of the antiserum mixed with 12.5 ml of 1% agarose dissolved in the IEP buffer. Ten microliters of antigen was applied to each of seven evenly spaced antigen wells cut across the 8-cm edge of the slides. Electrophoretic current of 17.5 mA/slide was applied during application of the samples and for an additional 6 h afterward at 5 C. After electrophoresis the slides

1735

were processed in the same manner as for the CIE slides. Conventional immunoelectrophoresis. Slides were prepared in the same manner as for quantitative rocket immunoelectrophoresis but with 1% agarose not containing antiserum. Six evenly spaced antigen wells (3 mm in diameter) were cut along a 10-cm edge of the slide such that the well centers were 3 mm from the slide edge. Five microliters of sample (antigen) and 2 Al of bromothymol blue were placed in each of the wells. Electrophoresis was conducted using the IEP buffer (400 ml/slide) at 35 mA until the marker dye had migrated at least 40 mm from the well. After electrophoresis, antiserum (0.1 ml) was placed in each of five troughs cut equidistantly between the wells and parallel to the 8-cm edge of the slide. Precipitin curves were allowed to develop by placing the slides in a humidified chamber for 48 h (24 h at 25 C and 24 h at 4 C). Ouchterlony agarose-gel immunodiffusion. Plastic petri dishes (85 mm in diameter) containing 5 ml of solidified 1% agarose in phosphate-buffered saline were used. A center (antiserum) well, 3 mm in diameter, and six outer (antigen) wells, 6 mm in diameter, spaced 5 mm from the center well and equidistant from each other, were cut in the solidified agar. Ten microliters of antiserum and 50 ,ul of antigen were placed in the inner and outer wells, respectively, and precipitin lines were allowed to develop in a humidified chamber for about 40 h. Crude antiserum. Burro antiserum produced against capsulated HITb strain "Rab" was graciously provided by J. B. Robbins, National Institute of Child Health and Human Development, Bethesda, Md. (6). Before use, the antiserum was sterilized by passage through a 0.2-,um pore size cellulose triacetate membrane filter. This preparation had an agglutinin titer of 32 against capsulated HITb. Preparation of adsorbed antisera. A dense cell suspension (approximately 10 optical density units at 600 nm of 16-h-old HIRd suspended in 10 mM Tris2.2 mM CaCl2, pH 8.0) was sonicated to total breakage. One-tenth milliliter of the sonicated preparation was added to 20 ml of the crude antiserum; the mixture was incubated for about 16 h at 4 C and centrifuged at 12,000 x g for 15 min, and the supernatant (adsorbed antiserum) was saved. This procedure was repeated six times with a gradual increase to 1.2 ml in the amount of sonicate added on the final adsorption. The supernatant of this adsorption was called AdR antiserum. The AdR antiserum was further adsorbed in the same manner as above with sonicates of B. pumilus. This final adsorbed antiserum was called AdB antiserum.

RESULTS Detection of antigens released from cell suspensions and sonicates. As previously shown (12), whole cell suspensions of capsulated HITb examined by immunodiffusion against homologous antiserum gave a single precipitin line of complete identity with that formed to PRP. A similar line of complete iden-

1736

BUCKMIRE

tity, as well as other precipitin lines, was observed with class I HITb cell suspensions. No lines of complete identity were observed with class II HITb, HIRd, or B. pumilus cell suspensions. However, lines of partial identity were observed between cell suspensions of B. pumilus and capsulated HITb. When the cell suspensions were sonicated, on the other hand, not only were several precipitin lines observed in all H. influenzae samples, but the class II preparation then gave an apparent line of identity with PRP. Class II HITb is generally considered not to contain capsular antigen (PRP) (12). B. pumilus continued to show a line of partial identity with PRP and the other H. influenzae cultures. Resolution of the many precipitin lines observed in the sonicates by the immunodiffusion method was poor. Resolution and semiquantitation of the soluble antigens by conventional electrophoresis and CIE. To better evaluate the numbers and similarities of the antigens present in the HITb cultures, sonicates were further examined by conventional immunoelectrophoresis and CIE using the unadsorbed (crude) antiserum to capsulated HITb strain "Rab." By conventional immunoelectrophoresis, five precipitin lines were observed with the capsulated and class I and II HITb cultures. One of these antigens migrated toward the anode at a very similar rate to that of the tracking dye (bromothymol blue). This antigen was absent in the HIRd sonicate. A precipitating antigen with similar electrophoretic mobility was present in both the PRP and B. pumilus sonicates. Precipitin lines of partial identity between envelope preparations ofB. pumilus and type b capsular antigen from H. influenzae have previously been reported (7). Thus, the fast-moving antigen was considered the type b capsular antigen. With CIE, on the other hand, 21 antigen peaks were clearly resolved with the capsulated and class I and II sonicates and 20 with the HIRd sonicate (Fig. 2a and b). The antigen missing from the HIRd sonicate but present in the HITb cultures migrated at a similar rate to the tracking dye during first-dimensional electrophoresis. An antigen with a similar migration rate to the tracking dye, and which produced a precipitin peak with the antiserum, was also present in the sonicates of PRP and B. pumilus. It was the only antigen in these two sonicates to react with the antiserum. Thus, the fast-migrating antigen present in the HITb cultures was considered the type b capsular antigen and was designated P4. It was also the most conspicuous (very dense) of the precipitin peaks obtained with the sonicates (Fig. 1). To

INFECT. IMMUN.

FIG. 1. CIE of sonicated capsulated HITb suspensions in 10 mM Tris-2.2 mM CaCl2 buffer. Ten microliters containing 32 ,ug ofprotein was used as the antigen. The antibody source was unadsorbed (crude) antiserum to capsulated HITb strain Rab.

aid in further comparative analysis of the antigens three other prominent precipitin peaks were labeled P,, P., and P:, (Fig. 2a and b). Table 1 is a comparative analysis of the data obtained by CIE from all the haemophilus cultures (see Fig. 1, 2a and b). Whereas the areas (per microgram of cell protein) under peaks P1, P2, and P3 were remarkably similar for all four H. influenzae cultures, that of P4 (the type b antigen peak) was largest in the capsulated strain, intermediate in the class I, minimal in the class II HITb cultures, and absent from the HIRd culture. The data (Table 1) strongly indicated that the type b capsular antigen was present in all three HITb cultures including the class II variant, a mutant generally considered devoid of this antigen. To explore this possibility further, the crude antiserum was appropriately adsorbed and used to examine the antigen preparations for the presence of the capsular antigen. When the crude antiserum was adsorbed with class II HITb sonicates, antibodies to all haemophilus antigens were removed from the antiserum; that is, no precipitin peaks were obtained with any of the H. influenzae cultures or with PRP and B. pumilus sonicates. However, when the crude antiserum was repeatedly adsorbed with the HIRd sonicate, the antiserum preparation obtained (AdR) gave the same three precipitin peaks (Pu, P3, P4) with all three HITh sonicates but only one of these peaks (P3) with the HIRd sonicate (Fig. 2c). These results indicate that there are two antigens (P, and P4) specific for H. influenzae type b. Of these, one (P4) corresponded to the capsular antigen in its electrophoretic mobility and by the fact that it was the only peak formed when the PRP and B. pumi-

VOL. 13, 1976

H. INFLUENZAE TYPE b CAPSULAR ANTIGEN CAP-21

a

b

Rd- 20

P1,P2 PP4 I

F2P1

I

" I

I

P3

,,I II

.

C

I

~~CAP-3

FIG. 2. Schematic drawings of the precipitin peaks resolved by CIE of sonicated H. influenzae (HI) suspensions in 10 mM Tris-2.2 mM CaCl, buffer versus antiserum to capsulated HITb strain Rab. (a) Represents the precipitin peaks obtained for capsulated HITb (see Fig. 1) versus the unadlsorbed antiserum. Similar patterns were obtained for class I and II HITb. (b) Represents the precipitin peaks obtained for HIRd versuls the unadsorbed antiserum. (c) Represents the precipitin peaks obtained for capsulated

HITb versus antiserum adsorbed with an HIRd sonicate Similar patterns were obtained for class I and II HITb. Peaks P,, P., P:,, P,, and P,, are explained in the text and in the legend to Table 1.

lus sonicates were reacted with the AdR antiserum. The other peak (P,,) appeared in approximately the same position as P,, but since it was not present in the HIRd sonicate it was considered different form P,. P, was an unadsorbed common

haemophilus antigen.

The adsorbed antiserum (AdR) was then adsorbed repeatedly with B. pumilus sonicate to remove the antibodies that cross-react with B.pumilus. This double-adsorbed antiserum (AdB) removed the single precipitin peak previously obtained when the B. pumilus sonicate was reacted with the AdR antiserum. Reaction of the PRP and all the haemophilus sonicates with the AdB antiserum, however, gave identical peaks to those previously obtained with the AdR antiserum (Fig. 2c). There was also an increase in the area, per microgram of cell protein, of P, (the capsular peak) in all of the HllTb sonicates. Concomitant with the increase in

1737

area of P4 was a proportionate decrease in the titer of the adsorbed antiserum, AdB, to capsulated HITb cells (Table 1). Effect of preparative procedures on the migration rate of the type b antigen. An antigen in the class II HITb variant, which cross-reacts with the antibodies to the type b antigen from the capsulated and class I HITb strains, could be "released" as a result of the procedure used to procure the antigen. Figure 3 shows that sonication, the method used for disrupting cells, greatly altered the migration rate of the type b antigen during first-dimensional electrophoresis. Antigen preparations, obtained either by the method of Rodrigues et al. (31) or from washings of the capsulated HITb cells, which were then sonicated, migrated at approximately the same rate as the tracking dye, whereas the preparations that were not sonicated migrated at a much slower rate and gave a broad peak, which spanned the entire length of the gel (from the antigen well to the tracking dye) (Fig. 3a and b). The increased migration rate of the type b antigen after sonication was presumably caused by the shearing of the polysaccharide molecule by sonication. It was still possible, however, that antigens possessing similar determinants but of different electrophoretic mobility to the type b antigen (PRP) obtained from the capsulated strain could be present in the other HITb cultures. To test this possibility, antigen preparations obtained by SDS lysis, a method not likely to shear highmolecular-weight capsular polysaccharide, were examined by CIE against unadsorbed and AdR antisera. All three HITb cultures gave a profile for the type b antigen similar to that obtained with the nonsonicated PRP and Th washings both in migration rate and in staining quality. These similarities in appearance and migration rate of the unsheared capsular antigen released from all three HITb cultures suggest that the antigen may be the same. Figure 3c also shows that the rate of migration of all the noncapsular antigens in the SDS lysates (seen only in samples run against crude antiserum) increased to approximately 0.9 relative to the tracking dye. These noncapsular antigens, also seen with the HIRd SDS lysate, appeared as parallel peaks with a common base. Quantitation of the type b capsular antigen by rocket immunoelectrophoresis. There is only one published report that implicates class II HlTb cultures as having the genetic capability of producing capsular antigen (20), and in no case has the capsular antigen been positively identified in class II cultures. It was, therefore,

1738

BUCKMIRE

INFECT. IMMUN.

TABLE 1. CIE analyses of the antigens present in sonicated bacteria which were detected by their reaction with antibodies in unadsorbed and adsorbed antisera to HITba Source of anti-

Antigen prepn

bodYb

No. of antigens resolved 21 21 21 20 1 3 3 3 1

Area (mm2) of precipitin peaks per ,g of cell proteinc Pu P1 P3 P2 P4 P 2.2 2.2 ++ 6.3 2.8 2.0 ++ 2.3 2.8 2.8 ++ 1.3 ++ 3.3 1.8 0 0 0 0 4.6 0 0 0.4 16.0 2.2 0 0 0.8 4.8 1.5 0 0 0.8 1.5 2.0 0 0 0.8 0 0 0.4 0 0 0.8 57.0 2.3 0 0 0.4 9.5 1.5 0 0 0.8 3.5 3.5

Capsulated HITb Crude Ab Class I HITb Crude Ab Class II HITb Crude Ab H. influenzae Rd Crude Ab B. pumilus Crude Ab Capsulated HITb AdR Class I HITb AdR Class II HITb AdR H. influenzae Rd AdR B. pumilus AdR Capsulated HITb AdB 3 Class I HITb AdB 3 3 Class II HITb AdB H. influenzae Rd AdB B. pumilus AdB 0 0 0 0 0 0 a The data summarized in this table are from Fig. 1 and 2. b Crude Ab refers to unadsorbed antiserum to capsulated HITb strain Rab; AdR to crude Ab after adsorption with a sonicate of H. influenzae Rd; and AdB to "AdR" after adsorption with a sonicate of B. pumilus. The titers were: crude Ab, 32; AdR, 16; AdB, 8. c Peaks P1 to P4 and Pu, are indicated in Fig. 2. + + indicates that a peak was present but was too small to be measured. The amount of the sonicate used ranged from 11 to 32 ,ug of protein.

b

a IL

0

"

Uh.I.. lo4mk

6.

il#

%f

f

%ib

d-

C

m Ag*.

sw

b

FIG. 3. CIE of sonicated, nonsonicated, and SDS lysates otfH. influenzae. The source of the antibodies was unadsorbed antiserum to capsulated HITb strain Rab. The antigen preparations used were: (a) sonicated "washings" from capsulated HITb; (b) "washings" from capsulated HITb that were not sonicated; (c) SDS lysate of capsulated HITb. Similar patterns were obtained for SDS lysates of class I and II HITb.

VOL. 13, 1976

H. INFLUENZAE TYPE b CAPSULAR ANTIGEN

of interest to know the quantitative relationship of the capsular antigen present in all three HITh cultures since this might, in part, explain why the antigen was not previously identified in class II cultures. Such quantitation may be accomplished by the rocket immunoelectrophoretic technique (19). Since the electrophoretic mobility of the capsular antigen was dependent on the method of preparation, it was first necessary to determine if a definite quantitative relationship existed between the fast-moving sonicated and the slower migrating, nonsonicated capsular antigen and between concentrations of the antigen and the heights of the precipitin peaks obtained in a given antiserum gel. Figure 4 shows that (i) a linear or near-

E E CD

LLI C-)

of

1739

linear relationship existed between concentrations of capsular antigen and the height of the precipitin peaks and (ii) the height of the precipitin peak for a given amount of capsular antigen was independent of antigen preparation, sonication versus nonsonication. The linear relationship held true for peak heights of 5 to 50 mm and had a standard error of 1 to 4%. The same linear relationship was maintained for both PRP and capsulated HITb sonicates as the antigen and unadsorbed and adsorbed (AdR and AdB) antisera (Fig. 4) as the antibody source. Decreasing the titer of the antiserum while keeping the antigen (PRP) concentration constant also resulted in an increase in the height of the precipitin peaks (Table 2). The increase in height was inversely proportional to the dilution of the antiserum. The density of the stained peaks also decreased with decreasing antibody titer. Table 2 also shows that significant peaks for the capsular antigen can be obtained by greatly diluting the antiserum. A peak height of 7 mm for 2.5 ng of PRP pentose was obtained with antiserum having a titer of 3.0. Quantitation of an antigen by the rocket immunoelectrophoretic technique is usually performed only when either a single antigen is used or the antiserum is monospecific. However, if an antiserum that contains multiple antibodies, only one of which is present in a high titer, is reacted with a preparation containing several homologous antigens, it should be possible, by suitably diluting the antiserum, to obtain a single precipitin peak for only the TABLE 2. Quantitative detection of PRP pentose by rocket immunoelectrophoresis in agarose gels containing low titers of antibodies to capsulated HITba

0.2 0.3 0.4 0.1 RELATIVE AMOUNT OF ANTIGEN FIG. 4. Normalized standard curves of the capsular antigen obtained by rocket immunoelectrophoresis. Two antigen preparations were used. Sonicates of capsulated HITb, 3.2 mg of protein per ml, and isolated type b capsular antigen, 250 ,ug of pentose per ml. The source of antibodies was either adsorbed or unadsorbed antiserum to capsulated HITb. The antiserum titer varied from 32 to 5 depending on the preparation used. Symbols: (0) Sonicated HITb versus HIRd adsorbed antiserum (AdR). Scale: 50 mm on ordinate = 10 mm in "rocket" height. (A) Sonicated HITb versus unadsorbed antiserum. Scale: 50 mm on ordinate = 7.5 mm in "rocket" height. (O) Sonicated HITb versus B. pumilus adsorbed AdR; also sonicated or nonsonicated PRP versus unadsorbed antiserum. Scale: 50 mm on ordinate = 25 mm in "rocket" height.

PRP pentose

(ng)

Peak heights" (mm) at antiserum titers of: 10.6

5.3

2.7

TH 51 313 35 56 156 21 43 78 48 28 39 13 32 20 20 9 19 13 10 7 10 5 7 5 2.5 a Antiserum to capsulated HITb was used as the antibody source. PRP refers to capsular antigen isolated from capsulated HITb by the method of Rodrigues et al. (31). The data are expressed as the amount of pentose present in PRP. b Average of at least six determinations. The standard error among determinations was less than 4%. TH, To high to read.

1740

INFECT. IMMUN.

BUCKMIRE

antigen for which there was a high antibody titer. When HITb sonicates (32 ,ug of protein) were reacted against the undiluted (crude) antiserum (titer of 32) by rocket immunoelectrophoresis, several rocket-shaped precipitin peaks were obtained. Dilution of the antiserum to a titer of 5 or less, however, gave a single precipitin peak. This peak was similar in shape and staining quality to the type b capsular peak obtained for PRP and HITh sonicates with the adsorbed antisera, AdR and AdB. Further verification that the single precipitin peak obtained by rocket immunoelectrophoresis was indeed the type b capsular peak was obtained by CIE, using capsulated HITb sonicate (32 ,tg of protein) as the antigen and the diluted antiserum (titer of 5) as the antibody source. A single precipitin peak, which corresponded to P4 in migration rate and in staining quality, was obtained. Relative amounts of PRP pentose present in HITb cultures. The amount of PRP pentose per microgram of cell protein present in the HITh cultures was determined by the rocket immunoelectrophoresis technique using crude antiserum (titer of 5). Our PRP preparation was used as the standard. Table 3 shows that the capsulated HITb contained 78 ng, class I contained 15 ng, and class II gave 4 ng of PRP pentose per ,tg of cell protein. Thus, the capsulated strain contained at least five times more capsular antigen than class I and 20 times more than class II.

DISCUSSION CIE gives better resolution of the antigens present in extracts of cell origin than either conventional immunoelectrophoresis or immunodiffusion. This is clearly demonstrated in the TABLE 3. Nanograms of PRP per microgram of cell protein in HITb cultures determined by rocket immunoelectrophoresis in agarose gels containing unadsorbed and AdR antiseraa ng/gg of pro-

Ratio of PRP teinb pentose 1 78 Capsulated HITb 0.19 14.7 Class I HITb 0.05 3.9 Class II HITb aPRP refers to capsular antigen isolated from capsulated HITb by the method of Rodrigues et al. (31). Unadsorbed antiserum to capsulated HITb strain Rab as well as antiserum adsorbed with a sonicate of H. influenzae strain Rd (AdR) were the antibody sources. Both antisera gave similar results. b PRP was used as the standard for computing the quantity of capsular antigen. The data are expressed as the amount of pentose present in PRP.

Organisms

H. influenzae system studied in this paper. The method allowed us to resolve 21 antigens, whereas the most antigens resolved by the other methods was 5 (by conventional immunoelectrophoresis). Comparison of the antigens obtained from three HITb cultures, capsulated and class I and II (noncapsulated), with those of a noncapsulated strain of HIRd and with the cross-reacting antigen from B. pumilus allowed us to positively identify the type b capsular antigen. Antiserum to a capsulated strain of HITh (Rab) gave only one antigen peak with sonicates ofB. pumilus. This cross-reacting antigen had a similar electrophoretic mobility to the fastest migrating of the HITh antigens (P4 in Fig. 2a). Argaman et al. (7) have demonstrated that the ribitol teichoic acid present inB. pumilus crossreacts with the type b capsular antigen, and that the type b capsular antigen does contain ribitol-5-phosphate in addition to ribose phosphate (7). Further corroborative evidence that the P4 peak was indeed the capsular antigen was (i) that it was absent from HIRd sonicates and (ii) that sonicates of washings and PRP isolated from capsulated HITh by the procedure of Rodrigues and co-workers (31) both gave single peaks, which had a similar electrophoretic mobility to P4. CIE with antiserum exhaustively adsorbed with HIRd sonicates also enabled us to identify another type-specific HITb antigen (designated as P,, in Fig. 2c). Our adsorption procedure removed the antibodies to all but one (P3) of the common haemophilus antigens (see Fig. 2). Further adsorption with the HIRd sonicates would probably have enabled us to obtain a type b-specific antiserum. Pr,, although being of a similar electrophoretic mobility to the common haemophilus antigen (P1), was unlikely P1 since it was not present in Rd sonicates. A type-specific haemophilus antigen other than the capsular antigen was suggested previously (23). This antigen was considered to be heat labile, since heated haemophilus cell suspensions gave lower agglutination titers than unheated preparations. The antigen was not identified, however. It is likely that the type bspecific noncapsular antigen (Pa) observed in all our HITb sonicates is the same as the postulated antigen. Heated antigen preparations would confirm this. Should this antigen (Pa) prove to be present only in type b strains, it would provide a means of identifying noncapsular antigen-bearing strains derived from HITh. All three strains of HITb, capsulated and class I and II variants, used in this study demonstrated the presence of the type b capsular

VOL. 13, 1976

H. INFLUENZAE TYPE b CAPSULAR ANTIGEN

antigen. Others (12, 33), using a variety of techniques, have demonstrated the presence of capsular antigen in the capsulated strain and the class I variant, but the class II variant has always been regarded as not possessing the antigen. Our experiments designed to verify that the class II variant does possess the capsular antigen assured us that the antigen was there, although in much lesser amounts than in either the capsulated strain or the class I variant. There was at least 20 times more capsular antigen in the capsulated culture and 4 times more in the class I variant. The relatively small amount of capsular antigen (4 ng/,ug of cell protein), coupled with the fact that the antigen was only released by methods that disrupt the cell envelope, may account for its lack of recognition before. The capsular antigen, like many polyanions such as deoxyribonucleic acid, may be sheared by sonication (11) and, like deoxyribonucleic acid molecules, the electrophoretic mobility of the sonicated capsular antigen in a nonrestrictive matrix (agarose) greatly increases. This increased electrophoretic mobility to a single sharp peak may indicate fragmentation of the molecule to a constant minimum size. Variation in the degree of sonication of the preparation would undoubtedly lead to heterogeneity in fragment distribution. The apparent nonshearing of the nonsonicated capsular antigen preparations, whether SDS lysed or vigorously mixed by vortexing, however, suggests that this substance is less susceptible to shearing than deoxyribonucleic acid. We obtained good quantitation of the capsular antigen by rocket immunoelectrophoresis. Accurate and reproducible results, as well as a near-linear relationship between peak heights and antigen concentrations, were dbtained for peak heights within the range of 5 to 50 mm. Similar quantitation was obtained with both sheared (by sonication) and unsheared capsular antigen despite their different electrophoretic mobilities. This was accomplished by electrophoresing for at least an additional one-third the time it takes the tracking dye to migrate to the anodic end of the agarose/antiserum gel. This method enabled us to measure as little as 2.5 ng of PRP pentose, the smallest amount attempted in the study. With appropriate dilution of antiserum, however, smaller amounts can undoubtedly be detected. Quantitation of the capsular antigen by CIE (accomplished by calculating the area under the antigen peak) was less reproducible and more time consuming than rocket immunoelectrophoresis. Only one sample can be done in the time it takes to do at

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least 14 samples by rocket immunoelectrophoresis. ACKNOWLEDGMENT I am grateful for the excellent technical assistance of Sharon Wilton.

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