JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1992, p. 2047-2053

Vol. 30, No. 8

0095-1137/92/082047-07$02.00/0 Copyright X 1992, American Society for Microbiology

Production and Characterization of Monoclonal Antibodies to Type 8 Lipooligosaccharide of Neisseria meningitidis XIN-XING GU, CHAO-MING TSAI,* AND ARTHUR B. KARPAS

Center for Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, Maryland 20892 Received 30 January 1992/Accepted 14 May 1992

Eight monoclonal antibodies (MAbs) to lipooligosaccharides (LOSs) of Neisseria meningitidis were produced by immunizing mice with purified LOS from group A meningococcal strain Al. The specificities of the MAbs were examined by enzyme-linked immunosorbent assay (ELISA), immunodot assay, and ELISA inhibition by using the homologous Al LOS, 12 immunotype LOSs of N. meningitidis (Li through L12), and LOSs or lipopolysaccharides from other gram-negative bacteria. Two of the MAbs, 4385G7 (immunoglobulin G2b [IgG2b]) and 4387A5 (IgG2a), had the strongest reactivities with the homologous Al LOS, moderate reactivities with the M978 (L8) LOS, but no reactivity with other LOSs. The other six MAbs (4 IgM and 2 IgG3) reacted with the Al LOS and with several or many of the 12 LOSs. ELISA inhibition at 50%o showed that the inhibitory activities of the LOSs from strains Al and BB431 (a group B strain) to the specific MAb 4387A5 were about 10 to 20 times greater than that of the M978 (U) LOS. When compared with MAb 2-1-L8 (U) by Western blot (immunoblot) analysis and ELISA inhibition, the two specific MAbs recognized a different epitope in the 3.6-kDa LOSs of strains Al and BB431. We propose that the new epitope is L8a, since the MAbs also reacted with the M978 (U) LOS. The expression of the U8a epitope in the Al LOS requires a few monosaccharide residues in its oligosaccharide moiety, and the fatty acid residues in its lipid A moiety also play a role. In a whole-cell ELISA, the two specific MAbs bound specifically to the homologous strain Al and the US prototype strain M978 but not to any other LOS prototype strains. These results suggest that the two specific MAbs can be used for LOS typing of N. meningitidis. MATERIALS AND METHODS Bacterial strains. N. meningitidis group A strain Al (also called 2E, LOS type 8) (21, 26) is one of the strains recommended by the World Health Organization for the production of group A capsular polysaccharide vaccine. It was isolated from a spinal fluid in Germany in 1964 (14, 21). Twelve LOS prototype strains of N. meningitidis were from the collections of the Walter Reed Army Institute of Research and our laboratory (Table 1); most of them have been characterized previously (21, 26, 42, 45, 46). N. meningitidis group B strain BB431 is a recent disease isolate (12). Preparation of purified LOSs. LOSs of N. meningitidis were prepared from outer membrane vesicles by gel filtration as described previously (16). Briefly, N. meningitidis was grown in tryptic soy broth, and during growth the organism released a large amount of outer membrane vesicles into the growth medium. Outer membrane vesicles in the culture supernatant were isolated and treated with 2% sodium deoxycholate, and the dissociated LOS was finally purified on a Sephacryl S-300 column. Production of hybridomas. Female BALB/c mice were inoculated subcutaneously three times in 1 week with 8 ,ug of purified LOS from strain Al in 0.2 ml of normal saline per dose. The mice were rested for 1 month, and they were again inoculated intraperitoneally three times in 1 week with 4 ,ug of LOS per dose. After resting for 3 months, the mice were given a final intravenous dose of 107 CFU organisms that were killed with 0.5% formalin 3 days prior to removal of their spleens. During the immunization period, mouse sera were obtained and tested for antibody activity by enzymelinked immunosorbent assay (ELISA) by using strain Al LOS as a coating antigen. Spleens were recovered from two immunized mice, and 1.53 x 10' spleen cells were combined with 0.76 x 108

Neisseria meningitidis strains can be classified into serothe basis of their capsular polysaccharides, into serotypes on the basis of their outer membrane proteins, and into immunotypes on the basis of their lipooligosaccharides (LOSs) (10, 11, 13, 26, 28, 42). By using passive hemagglutination and solid-phase radioimmunoassay inhibition of rabbit polyclonal antibodies, Zollinger and Mandrell (26, 45, 46) identified 11 LOS types (Li through Lii) in N. meningitidis. Later, LOS types 12 and 13 were identified (2, 44). Types Li through L8 are mainly found in serogroups B and C (26), while types L9 through L13 are primarily associated with serogroup A (2, 46). Several monoclonal antibodies (MAbs) to LOS immunotypes L8 through Lii of N. meningitidis have been reported (8, 21, 36, 37). These MAbs are assumed to be type specific, but some reactivities with other types were observed (20, 21). The MAbs described above and other MAbs have been used as tools for LOS typing and for characterizing pathogenic strains ofNeisseria species (3, 9, 15, 21, 24, 25, 35, 36). Additional specific MAbs are needed for LOS typing of N. meningitidis strains because of the diversity of the LOS antigens (6, 21, 39, 42), the presence of multiple LOS types on the same strain (13, 29, 45, 46), and the presence of strains with untyped LOSs (12, 21, 33, 46). We produced two MAbs which are specific for the L8a epitope on the LOS of N. meningitidis Al and BB431 (BB431 is a previously untyped group B strain). This epitope can be distinguished from the 13 known LOS antigens; therefore, the specific MAbs can be used for defining the new epitope of LOS in N. meningitidis. groups on

*

Corresponding author. 2047

2048

GU ET AL.

J. CLIN. MICROBIOL. TABLE 1. Screening specificities of eight MAbs against N. meningitidis LOSs from 12 immunotype strains by ELISA

LOS from straina

126E 35E 6275 891 M981 M992 6155 M978 120M 7880 7889 7897 Al BB431

Presence of the following MAb and isotype": 4383D3 and

IgM

4362F4 and IgM

Li L2

+ ++

+ ++

B

12

L4 L5 L6 L7 L8 L9

+ + + ++ ++ ++ -

+

C B B

++ ++ + + ++ ++ ++ ++ ++ ++ -

Serogroup

C C

B B A A A A A B

Immunotype

L10 Lll L12

4361F3 and

++ ++

+ + ++ ++ ++

+ + ++ ++

1gM

4386B4 and IgM

++ ++ + ++

++ ++ ++ ++ ++ ++ -

4384H1 and IgG3

+ ++ + ++ ++ ++ ++ -

M7H4 and IgG3

+ -

4385G7 and IgG2b

4387A5 and IgG2a

-

-4-

-

-

-

+

--

+ +

+ ++ ++

++ ++ ++ -

+

++ ++

a Purified menincoccal LOSs from 12 LOS prototype strains (Li through L12) and strains Al and BB431. b + +, strongly positive; +, positive; +, weakly positive; -, negative.

nonsecreting Sp2/0-Agl4 myeloma cells. Fusions were performed by a method modified from that described previously (22, 32). Of the 1,152 original wells, 942 contained colonies, and most colonies produced antibodies. Eight wells that contained one or two colonies and with high antibody titers when measured by ELISA were selected. The selected hybridomas were cloned by limiting dilution two to three times after they were screened by ELISA. Selected clones were inoculated intraperitoneally into BALB/c mice primed with pristane. Antibodies from ascitic fluids were purified by using affinity columns (Pierce, Rockford, Ill.). Tissue culture fluids were also collected. ELISA. MAb responses to meningococcal LOSs were detected by an ELISA by using purified LOSs as coating antigens. LOS antigen (100 ,ul; 10 ,ug/ml) in phosphatebuffered saline (PBS; pH 7.4) was coated onto polystyrene microtiter plates (Immulon I plate; Dynatech Laboratories, Inc., Alexandria, Va.) overnight. A 3-h incubation was used for antibodies in mouse sera, hybridoma culture fluids, and ascites, each in serial dilutions. Alkaline phosphatase-conjugated rabbit anti-mouse immunoglobulin G (IgG) was added, and the solution was incubated for 3 h at room temperature. Saline containing 0.05% Tween 20 was used in five washings between steps. After the enzyme substrate was added, the reactions were read with a microplate autoreader (EL309; Bio-Tek Instruments). MgCl2 (10 mM) was added to all solutions in the ELISA to improve the test (18). Whole-cell ELISA (1) was also performed by using 12 LOS prototype strains and strain Al as coating antigens. MAbs in ascitic fluids were diluted to find their linear reactivities, and the optimal dilutions of MAbs 4385G7, 4387A5, and 2-1-L8 were 1:10,000, 1:20,000, and 1:2,000, respectively. Other steps were the same as those for the ELISA described above. MAb isotyping. Determination of immunoglobulin class and subclass was accomplished with an Immuno Select ELISA kit, which is a MAb-based isotyping system (GIBCO, BRL, Bethesda, Md.). The isotypes of hybridoma culture fluids were determined as described in the kit's instructions. Immunodot assay. The dot blot (immunodot) assay used in the study described here was based on the method of

Bio-Rad (Bio-Rad Laboratories, Richmond, Calif.), with some modifications. Briefly, 0.5 ,ug of purified LOS in 50 ,ul of 10 mM MgCl2 in PBS was dotted onto a nitrocellulose membrane under vacuum with Milliblot transfer equipment (Millipore Corporation, Bedford, Mass.). The dotted membrane was rinsed with 20% ethanol, cut, and dried at room temperature. The membranes were blocked for 1 h with 1% gelatin in Tris-buffered saline (20 mM Tris, 500 mM NaCl, [pH 7.5]) and were incubated with the culture fluids of eight monoclonal hybridomas at 4°C overnight. The membranes with LOS-antibody complexes were reacted with goat antimouse IgG or IgM (, chain) and horseradish peroxidase conjugate (Sigma Chemical Co., St. Louis, Mo.) for 4 h at room temperature. The reactions were visualized by using the purple insoluble product of the peroxidase from the substrate 4-chloro-1-naphthol (Sigma). ELISA inhibition test. The ELISA inhibition test used different purified LOSs to inhibit the reactions between MAbs and the coating LOS antigen of strain Al. First, MAbs 4385G7, 4387A5, and 2-1-L8 were assayed in serial dilutions by ELISA, and a curve based on the different MAb dilutions and their readings at A405 was drawn. The antibody dilution used for the inhibition test was at the middle of the linear portion of the dilution curve (A405, about 1.0). Second, 200 RI of MAbs was preincubated with 200 ,l of 1% bovine serum albumin in PBS containing 200 p,g of purified LOS (inhibitor) per ml or with only 1% bovine serum albumin in PBS (control) at 4°C overnight. For strains BB431, Al, M978, and 126E, the purified LOSs were serially threefold diluted (from 200 to 0.002 p,g/ml) and were incubated with MAb 4387A5 or 2-1-L8. Third, each 100 p,l of the incubated solutions (containing 10 p,g of purified LOS) was transferred to a microtiter plate coated with 10 p,g of purified LOS of strain Al per ml, and the subsequent reaction was detected as described above for the ELISA. Fourth, the percent inhibition was calculated as follows: (1 - inhibitors' mean A405/controls' mean A405) x 100. All the experiments contained at least quadruplicate non-LOS-incubated controls and triplicate LOS-incubated samples, and the experiments were repeated once. The variations in ELISA were ± 10%. The 50% inhibition value was determined as the amount of LOS inhibitor required for 50% inhibition.

MAb TO MENINGOCOCCAL LOS

VOL. 30, 1992

Dephosphorylated or deacylated LOSs (43) from strain Al, oligosaccharide (OS) and lipid A from the Al LOS, and monosaccharides and disaccharides (Sigma) were also tested with MAb 4387A5 by the inhibition test described above. Western blot (immunoblot) assay. The Western blot procedure of Towbin et al. (38) was modified for meningococcal LOS antigens by using the Bio-Rad mini-gel electrophoresis and transfer system. Briefly, purified LOS (2 ,ug) was subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) in a 15% gel that was prepared by the method of Laemmli (23). The transfer from polyacrylamide gels onto nitrocellulose membranes was performed at 250 mA in Bio-Rad transfer buffer (pH 7.5) for 6 h. Other steps were as described above for the immunodot assay. Hybridoma culture fluids or ascites were used as the source of LOS-binding antibodies. Another duplicated gel was silver stained after SDS-PAGE (41). Isolation and analysis of OS. The OS was isolated from strain Al LOS by hydrolysis of the LOS in 1% acetic acid at 100°C for 2 h and was purified through a Bio-Gel P-4 column (1.6 by 90 cm) by using 25 mM ammonium acetate as an eluting buffer. A major OS peak measured by the phenolsulfuric acid method (7) was collected and lyophilized twice. The monosaccharide composition of the OS was determined as described previously (27). The OS was further analyzed by high-performance liquid chromatography (HPLC). A CarboPac-PAl anion-exchange column and pulsed amperometric detection (Dionex Corp., Sunnyvale, Calif.) (17) were used with an eluant of 1 mM NaOH and 125 mM sodium acetate, a flow rate of 1 ml/min, and a detector output of 300 nA.

RESULTS Screening and isotyping of MAbs. After the fusion with two BALB/c mouse spleen cells and mouse myeloma cells, eight MAbs against the LOS of N. meningitidis group A strain Al were selected from 942 fusion cell lines by screening with the homologous Al LOS in an ELISA. Immunoglobulin class and subclass analysis showed that four of the eight MAbs were IgM and that the others were IgG2a, IgG2b, and IgG3 (Table 1). All MAbs had kappa light chains. Characterization of MAbs with meningococcal LOSs from 12 LOS-typing strains by ELISA and immunodot assay. Eight selected MAbs were characterized by ELISA with the LOS antigens (Ll through L12) from 12 LOS prototype strains and strains Al and BB431 (Table 1). All eight MAbs reacted strongly with the strain Al LOS, but none of them reacted with the L6 LOS. Two MAbs, 4385G7 (IgG2b) and 4387A5 (IgG2a), reacted only with Al, BB431, and M978 LOSs but not with the other LOSs. The LOSs of strains Al and BB431 had higher reactivities than did that of M978. Six other MAbs reacted with several or many of the 12 LOS types. Similar results were also obtained by immunodot assay (Fig. 1). Two MAbs, 4385G7 and 4387A5, bound strongly to LOSs from strains Al and BB431 and moderately to the M978 LOS. Six other MAbs bound to 5 to 10 of the 14 LOSs listed in Fig. 1. ELISA inhibition by purified LOS from N. meningitidis and some other gram-negative bacteria. To further characterize the specificities of MAbs 4385G7 and 4387A5, inhibition with purified LOSs from meningococci was examined by ELISA. The two MAbs were strongly inhibited by strain Al and BB431 LOSs and moderately inhibited by strain M978 LOS, but they were not inhibited by other meningococcal LOSs at concentrations of up to 100 ,ug/ml. The LOSs of Neisseria

2049

1 2 3 4 5 6 7 8 9 10 11 12 13 14 A B D

EG F G H

S

*

s

*

C..

aS... 0

t0

0 f.

*

*4

* ...

FIG. 1. Immunodot assay of MAbs to purified meningococcal LOSs. LOSs and MAbs are those presented in Table 1. Columns 1 through 12, LOSs Li through L12, respectively; columns 13 and 14, strain Al and BB431 LOSs, respectively. Strips A through H, the eight MAbs presented in Table 1 in sequence (4361F1 to 4387A5); strip I, MAb 2-1-L8.

gonorrhoeae JM31R, Bordetella pertussis 114, Haemophilus influenzae Rab, and Acinetobacter calcoaceticus NCTC 10305 and smooth lipopolysaccharides of Escherichia coli Olll:B4, K235, and 026:B6, Salmonella typhimurium (Sigma), Salmonella minnesota (Sigma), and Brucella abortus 1119 were noninhibitory. The OS but not lipid A of the strain Al LOS showed strong inhibition, indicating that the specific MAbs recognize an epitope at the OS but not at the lipid A of the LOS. Comparison of differences in specificity among MAbs 4385G7, 4387A5, and 2-1-L8. The LOS of strain Al was reported to belong to LOS type L8 (21, 26). The specificities of MAbs 4385G7 and 4387A5 were compared with that of L8-specific MAb 2-1-L8 (36) to determine whether the former two MAbs were also against the same L8 epitope. ELISA and the immunodot assay showed that MAbs 4385G7, 4387A5, and 2-1-L8 all bound to Al, BB431, and M978 LOSs (L8), but with different reactivities. In addition to the three LOSs, MAb 2-1-L8 also bound to the strain 126E LOS (Ll, L8) (Fig. 1, row I). The relative reactivities of the LOSs of the four strains described above (Al, BB431, M978, and 126E) with MAbs 4387A5 and 2-1-L8 were further examined by ELISA inhibition. For MAb 4387A5 (Fig. 2A), strain BB431 and Al LOSs had the highest inhibition; this was followed by the inhibition by M978 (L8) LOS; 126E LOS showed no inhibition when 10 ,ug of the LOS was used. The 50% inhibitory activities of strain Al and BB431 LOSs to the MAb was about 10 to 20 times higher than that of strain M978 (L8) LOS (Table 2). For MAb 2-1-L8 (Fig. 2B), the strain M978 (L8) and Al LOSs had the highest inhibitions; this was followed by 126E LOS, and the lowest inhibition was with BB431 LOS. While the 50% inhibition values of BB431 LOS was 0.4 p.g for 4387A5 MAb, 10 ,ug of the LOS still could not reach 50% inhibition for MAb 2-1-L8 (Table 2). In contrast, the 50% inhibition value of strain 126E LOS was 0.3 ,g for 2-1-L8, but there was no inhibition at 10 ,ug for MAb 4387A5 (Table 2). We also examined the two MAbs with a LOS from strain M25 (L8), a mutant of strain 44/76 (31). The reactivities of strain M25 LOS were similar to those of strain M978 LOS (L8). Figure 3 is an immunoblot of MAbs 4387A5 and 2-1-L8 with the four LOSs from the strains described above (126E, Al, M978, and BB431). The results were similar to those obtained by ELISA inhibition. The specificity of the other

2050

GU ET AL.

J. CLIN. MICROBIOL.

A

Inhibition (%) 100i

A

80

0.-

ta a*

Os

60

40

B

20

4,01

MO.-

Itio

i'm

LOS (ug)

C

1

.001

.004

.013

.04

.12

.37

1.1

10

3.3

LOS (ug) FIG. 2. Inhibition of MAb 4387A5 (A) and 2-1-L8 (B) binding to meningococcal Al LOS by four LOSs (inhibitors) from strains BB431 ( ), Al (--- ), M978 (. ), and 126E (- - -) in ELISA. The four LOSs were diluted threefold in series, with dilutions ranging from 10 to 0.001 p,g. The percent inhibition was calculated as follows: (1 - inhibitors' mean A405controls' mean

A405) x 100.

MAb (4385G7) was similar to that of MAb 4387A5 (data not shown). The results presented above indicated that MAbs 4385G7, 4387A5, and 2-1-L8 all bound to a single 3.6-kDa band of the Al LOS in silver-stained SDS-polyacrylamide gels, but they recognized two different epitopes. We propose that the new epitope for MAbs 4385G7 and 4387A5 is L8a, since the L8 epitope has been used for MAb 2-1-L8 (21, 36). Analysis of OS isolated from the strain Al LOS. The strain Al LOS showed a single band on SDS-PAGE but bound MAbs that recognize two different epitopes, as described TABLE 2. Inhibition of two different MAbs by four N. meningitidis LOSs MAb

4387A5 2-1-L8

Amt (,ug) of LOS required for 50% inhibition ofa:

BB431

Al

M978

126E

0.4 >10

0.9 0.03

8.0 0.02

>10 0.3

a The 50% inhibition was based on an inhibition ELISA (see Fig. 2) in which purified LOSs from strains BB431, Al, M978, and 126E were used as the inhibitors to MAbs 4387A5 and 2-1-L8.

2 3 4

FIG. 3. SDS-PAGE and immunoblots of purified meningococcal LOSs from strains 126E, Al, M978, and BB431 (lanes 1 through 4, respectively). (A) Silver-stained gel. (B and C) Immunoblots of the corresponding LOSs from panel A with MAbs 4387A5 and 2-1-L8. The arrowheads on the left indicate the locations of the 3.6-kDa band of the strain Al LOS.

above. Analysis of the OS from strain Al was performed to determine whether the OS is a single component, since MAbs 4385G7 and 4387A5 were inhibited by the OS but not the lipid A of the Al LOS and the OS moiety in meningococcal LOS is usually responsible for the specificities of LOS types (19). After mild acid hydrolysis of the strain Al LOS to remove lipid A, the OS showed a major and a few minor OS peaks by Bio-Gel P-4 chromatography. The major OS peak was further analyzed by HPLC, as shown in Fig. 4. The OS contained at least four components (Fig. 4A), and the major peak 2 could be further separated into peaks 1 and 2 with a different eluant (Fig. 4B). These results showed that strain Al OS contains a mixture of OSs, indicating the presence of more than one LOS type in the LOS. Preliminary characterization of the L8a epitope. Monosaccharide composition analysis of the Al OS purified by Bio-Gel P-4 chromatography revealed that it contained galactose, glucose, glucosamine, heptose, and 2-keto-3-deoxyoctulosonic acid in a molar ratio of about 1.6:2:1:2:1. Inhibition of MAb 4387A5 by sugars and LOS antigens in an ELISA was used to examine the chemical nature of the L8a epitope (Table 3). The Al OS had 50 and 93% inhibition at 1 and 10 mM, respectively. In contrast, the possible terminal monosaccharides lactose and phosphoethanolamine, on the basis of the structure of meningococcal LOS (34, 40), had no inhibition at 1 mM or had only 10 to 20% nonspecific inhibitions at 50 mM. These results indicate that expression of the L8a epitope requires a few monosaccharide residues in the OS. When the OS was coupled to tetanus toxoid, the inhibitory ability of the OS was enhanced about 20 times because of the polyvalent nature of the OS-protein conjugate (5).

MAb TO MENINGOCOCCAL LOS

VOL. 30, 1992

2051

2

B

A

2

I

5

5

FIG. 4. Analysis of strain Al OS by HPLC on a Dionex CarboPac-PAl column. The strain Al OS (2.5 p.g) was eluted with 4 mM NaOH-125 mM sodium acetate (A) or 1 mM NaOH-125 mM sodium acetate (B). The retention times of OS peaks changed with different eluants. The retention times (in minutes) of the peaks were as follows: (A) peak 2, 12.0; peak 3, 15.0; peak 4, 17.0; and peak 5, 18.9; (B) peak 1, 9.9; peak 2, 10.6; peak 3, 14.0; peak 4, 14.6; peak 5, 16.9. The two minor peaks around the arrows were observed when only buffer was injected into the column.

As expected, the Al LOS is a very potent inhibitor since the LOS exists as micelles and is a polyvalent antigen (Table 3). Modification of lipid A in the LOS by partial deacylation, but not dephosphorylation, reduced the reactivity of the LOS 10-fold. By comparison, deacetylation of the OS did not reduce its inhibitory activity (data not shown). These results suggest that the fatty acid residues in the lipid A of the LOS also play a role in the expression of the L8a epitope

in the LOS, as has been observed by Yamasaki et al. (43) in gonococcal LOS. LOS typing of N. meningitidis by MAbs by whole-cell ELISA. Table 4 shows the reactivities of 12 LOSs from prototype strains of N. meningitidis with MAbs 4385G7, 4387A5, and 2-1-L8 by whole-cell ELISA. The L8a MAbs 4385G7 and 4387A5 bound well to Al and M978 whole bacteria and were more specific than MAb 2-1-L8. Some strains of E. coli and other gram-negative bacteria showed no reactivity (data not shown).

TABLE 3. Inhibition of MAb 4387A5 (L8a) by LOS and OS from strain Al and other sugars by ELISA Concn (mM)a at 50% inhibitione

Inhibitors

LOSs Native .........................................

Dephosphorylated ......................................... Deacylated .........................................

0.0028 0.0027 0.028

Sugars OS ........................................ 1 Lactose ......................................... >50 (12) N-Acetyllactosamine ....................................... >50 (12) Galactose ................. >50 (16) ........................ Glucose ......................................... >50 (15) N-acetylglucosamine ...................................... >50 (19) 2-Keto-3-deoxyoctulosonic acid ......................... >50 (19) Phosphorylethanolamine .................................. >50 (16) Conjugate, OS-tetanus toxoidc.

0.057c a The concentrations of Al LOS and OS were based on molecular weights of 3,600 and 1,400, respectively. bValues in parentheses are percent inhibition at 50 mM.

pg

The OS-tetanus toxoid conjugate contained 200 of OS and 1,100 pg of protein per ml (16a) and the concentration of the conjugate was based on the Al OS. c

TABLE 4. Reactivities of MAbs to N. meningitidis LOS in whole-cell ELISA Strain'

126E 35E 6275 89I M981 M992 6155 M978 120M 7880 7889 7897

Al

4385G7

Reactivityb (A40) with MAb: 4387A5

2-1-L8

0 0 0 0 0 0 0 0.51 0.04 0 0 0 0.73

0 0 0 0 0 0 0 0.70 0.04 0.04 0.03 0.03 1.08

0.2 0.03 0.15 0.03 0.03 0.03 0.12 0.65 0.14 0.11 0.11 0.12 0.78

a Twelve meningococcal LOS prototype strains and Al strain as described in Table 1. Whole cells (about 1.5 x 109) were used as coating antigens. b The reactivities are given averages of triplicate A405 determinations in whole-cell ELISA.

2052

GU ET AL.

J. CLIN. MICROBIOL.

DISCUSSION

Other investigators have used whole bacteria as immunoin the production of anti-LOS hybridomas (3, 8, 36, 37). In that case, most of the hybridomas were against outer membrane proteins. We used purified LOS as an immunogen gens

without adjuvant, resulting in many anti-LOS MAbs but not anti-protein MAbs. Considering the LOS's endotoxic property and its lower antigenicity compared with those of proteins, we inoculated BALB/c mice with low doses of LOS by two routes: subcutaneous and intraperitoneal. All hybrid lines made antibodies which were found to be against LOS antigens. Of the 1,152 original wells, 942 contained colonies and 95% of them secreted antibodies to the strain Al LOS antigen. This suggests that the immunization method mostly triggers lymphocytes which recognize LOS as an antigen. The LOS of strain Al has previously been identified by Mandrell and Zollinger (26) as L8 by hemagglutination inhibition of rabbit antisera. Kim et al. (21) also showed the same result by using LOS MAb 2-1-L8, which was made to strain H355 (B:15:L8) by Zollinger and Mandrell (44, 47). Dudas and Apicella (8) have described MAbs 6B7 and 4C4, which were made by using strain Al bacteria. The two MAbs mainly bind to Lii LOS, and MAb 4C4 can also bind to L8 and L9 LOSs of some group A strains (21). In our study, several different MAbs were induced by the strain Al LOS (Table 1 and Fig. 1). All the results indicated that the strain Al LOS is a multitype LOS; and it can express at least the L8, L8a, and Lii epitopes. We used HPLC to analyze strain Al OS fractions that were purified by Bio-Gel P-4 column chromatography after mild acid hydrolysis of the Al LOS. The Al OS could be separated into at least four components by HPLC (Fig. 4). This may explain why strain Al LOS is a multitype LOS. However, the possibility that different epitopes reside in a single OS cannot be excluded. The L8a epitope reported here appears to be different from the L13 epitope, which resides in a higher molecular-weight LOS and does not react with many MAbs, including 2-1-L8, as reported by Achtman et al. (2). We also tested LOSs from N. meningitidis BB431 and a mutant of strain M986 by ELISA. The LOSs were indistinguishable from the strain Al LOS by SDS-PAGE; the strain Al LOS had a 3.6-kDa band. These two LOSs could be differentiated by the specific MAbs 4385G7 and 4387A5; the BB431 LOS was reactive, but the mutant M986 LOS was not. On the other hand, strain BB431 and Al LOSs could be distinguished by using MAb 4384H1, which did not bind with to the strain BB431 LOS (Table 1). Thus, we can use the L8a MAbs' for LOS typing and the other MAbs for further antigenic differentiation. Expression of different LOS types in N. meningitidis because of phase variation has been reported previously (4, 30). In the study described here, some prototype LOS strains not only expressed a specific LOS type but also expressed other types. For instance, strain 126E mainly expressed LOS type Li but it also contained small amounts of LOS type L8, which caused the reaction of MAb 2-1-L8 but not MAb 4387A5 in 'immunoblots (Fig. 3). We also observed a phase variation in strain M978 which showed two LOS bands by SDS-PAGE, but the intensities of the two bands varied under different growth conditions. Figure 3A presents the LOS pattern of strain M978, which mainly expressed an LOS type L8 major 3.6-kDa band. MAbs 4385G7 and 4387A5 did not bind to the other higher-molecular-weight band. Since MAbs 4385G7, 4387A5, and 2-1-L8

all bound to the 3.6-kDa band, further studies are needed to find a strain which expresses only the type L8a epitope. The better reactivity of the strain BB431 LOS than that of the strain Al LOS (Table 2) may suggest that more of the L8a epitope exists in the strain BB431 LOS. ACKNOWLEDGMENTS We thank Carl E. Frasch for supporting this project and providing us with strains from his collection. We also thank W. D. Zollinger for sending us some prototype strains and the MAb 2-1-L8 used in this study. REFERENCES 1. Abdillahi, H., and J. T. Poolman. 1987. Whole-cell ELISA for typing Neisseria meningitidis with monoclonal antibodies. FEMS Microbiol. Lett. 48:367-371. 2. Achtman, M., B. Kusecek, G. Morelli, K. Elickmann, J. Wang, B. Crowe, R. A. Wall, M. Hassan-King, P. S. Moore, and W. Zollinger. 1992. A comparison of the variable antigens expressed by clone IV-1 and subgroup III of Neissena meningitidis serogroup A. J. Infect. Dis. 165:53-68. 3. Apicella, M. A., K. M. Bennett, C. A. Hermerath, and D. E. Roberts. 1981. Monoclonal antibody analysis of lipooligosaccharide from Neisseria gonorrhoeae and Neisseria meningitidis. Infect. Immun. 34:751-756. 4. Apicella, M. A., M. Shero, G. A. Jarvis, J. M. Griffiss, R. E. Mandrell, and H. Schneider. 1987. Phenotypic variation in epitope expression of Neissena meningitidis lipooligosaccharide. Infect. Immun. 55:1755-1761. 5. Crothers, D. M., and H. Metzger. 1972. The influence of polyvalency on the binding properties of antibodies. Immunochemistry 9:341. 6. Crowe, B. A., B. Wall, B. Kusecek, B. Neumann, T. Olyhoek, H. Abdillahi, M. Hassan-King, B. M. Greenwood, J. T. Poolman, and M. Achtman. 1989. Clonal and variable properties of Neisseria meningitidis isolated from cases and carriers during and after an epidemic in The Gambia, West Africa. J. Infect. Dis. 159:686-700. 7. Dubois, M., H. Gillis, J. K. Hamilton, A. A. Rebers, and R. Smith. 1956. Colorimetric method for the determination of sugars and related substances. Anal. Biochem. 28:250-256. 8. Dudas, K. C., and M. A. Apicella. 1988. Selection and immunochemical analysis of lipooligosaccharide mutants of Neisseria gonorrhoeae. Infect. Immun. 56:499-504. 9. Estabrook, M. M., R. E. Mandrell, M. A. Apicella, and J. M. Griffiss. 1990. Measurement of the human immune response to meningococcal lipooligosaccharide antigens by using serum to inhibit monoclonal antibody binding to purified lipooligosaccharide. Infect. Immun. 58:2204-2213. 10. Frasch, C. E. 1979. Noncapsular surface antigens of Neisseria meningitidis. Semin. Infect. Dis. 2:304-337. 11. Frasch, C. E. 1983. Immunization against Neisseria meningitidis, p. 115-144. In C. S. F. Easmon and J. Jeljaszewicz (ed.), Medical microbiology, vol. 2. Academic Press, London. 12. Frasch, C. E., L. F. Mocca, and A. B. Karpas. 1988. Appearance of new strains associated with group B meningococcal disease and their use for rapid vaccine development, p. 97-104. In J. T. Poolman, C. Beuvery, and H. Zanen (ed.), Gonococci and meningococci. Martinus Nijhoff Publishers, Dordrecht, The Netherlands. 13. Frasch, C. E., W. D. Zollinger, and J. T. Poolman. 1985. Serotyping of Neisseria meningitidis and a proposed scheme for designation of serotypes. Rev. Infect. Dis. 7:504-510. 14. Gotschlich, E. C., T. Y. Liu, and M. S. Artenstein. 1969. Human immunity to the meningococcus. III. Preparation and immunochemical properties of the group A, group B, and group C meningococcal polysaccharides. J. Exp. Med. 129:1349-1365. 15. Griffiss, J. M., H. Schneider, R. E. Mandrell, R. Yamasaki, G. A. Jarvis, J. J. Kim, B. W. Gibson, R. Hamedeh, and M. A. Apicella. 1988. Lipooligosaccharides: the principal glycolipids of the neisserial outer membrane. Rev. Infect. Dis. 1O(Suppl.):

S287-S295.

VOL. 30, 1992 16. Gu, X. X., and C. M. Tsai. 1991. Purification of rough-type lipopolysaccharides of Neissena meningitidis from cells and outer membrane vesicles in spent media. Anal. Biochem. 196: 311-318. 16a.Gu, X. X., and C. M. Tsai. Unpublished data. 17. Hardy, M. R., R. R. Townsend, and Y. C. Lee. 1988. Monosaccharide analysis of glycoconjugates by anion exchange chromatography with pulsed amperometric detection. Anal. Biochem. 170:54-62. 18. Ito, J. I., Jr., A. C. Wunderlich, J. Lyons, C. E. Davis, D. G. Guiney, and A. I. Braude. 1980. Role of magnesium in the ELISA for lipopolysaccharides of rough E. coli strain J5 and Neissena gonorrhoeae. J. Infect. Dis. 142:532-537. 19. Jennings, H. J., M. Beurret, A. Gamian, and F. Michon. 1987. Structure and immunochemistry of meningococcal lipopolysaccharides. Antonie Leeuwenhoek 53:519-522. 20. Kim, J. J., R. E. Mandrell, and J. M. Griffiss. 1989. Neisseria lactamica and Neisseria meningitidis share lipooligosaccharide epitopes but lack common capsular and class 1, 2, and 3 protein epitopes. Infect. Immun. 57:602-608. 21. Kim, J. J., R. E. Mandrell, Z. Hu, M. A. J. Westerink, J. T. Poolman, and J. M. GrUiss. 1988. Electromorphic characterization and description of conserved epitopes of the lipooligosaccharides of group A Neisseria meningitidis. Infect. Immun. 56:2631-2638. 22. Kohler, G., and C. Milstein. 1975. Continuous culture of fused cells secreting antibody of predetermined specificity. Nature (London) 256:495-497. 23. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 277:680-685. 24. Mandrell, R. E., J. M. Griffiss, and B. A. Macher. 1988. Lipooligosaccharides (LOS) of Neisseria gonorrhoeae and Neisseria meningitidis have components that are immunochemically similar to precursors of human blood group antigens. J. Exp. Med. 168:107-126. 25. Mandrell, R. E., A. J. Lesse, J. V. Sugai, M. Shero, J. M. Griffiss, J. A. Cole, N. J. Parsons, H. Smith, S. A. Morse, and M. A. Apicella. 1990. In vitro and in vivo modification of Neisseria gonorrhoeae lipooligosaccharide epitope structure by sialylation. J. Exp. Med. 171:1649-1664. 26. Mandrell, R. E., and W. D. Zollinger. 1977. Lipopolysaccharide serotyping of Neisseria meningitidis by hemagglutination inhibition. Infect. Immun. 16:471-475. 27. Phillips, N. J., C. M. John, L. G. Reinders, and B. W. Gibson. 1990. Structural models for the cell surface lipooligosaccharides of Neisseria gonorrhoeae and Haemophilus influenzae. Biomed. Environ. Mass Spectrom. 19:731-745. 28. Poolman, J. T., C. T. P. Hopman, and H. C. Zanen. 1980. Immunochemical characterization of Neisseria meningitidis serotype antigens by immunodiffusion and SDS-polyacrylamide gel electrophoresis immunoperoxidase techniques and the distribution of serotypes among cases and carriers. J. Gen. Microbiol. 116:465-473. 29. Poolman, J. T., C. T. P. Hopman, and H. C. Zanen. 1982. Problems in the definition of meningococcal serotypes. FEMS Microbiol. Lett. 13:339-348. 30. Poolman, J. T., F. B. Wientjes, C. T. P. Hopman, and H. C. Zanen. 1985. Influence of the length of lipopolysaccharide molecules on the surface exposure of class 1 and class 2 outer membrane proteins of Neisseria meningitidis 2996 (B:2b:Pl.2), p. 562-570. In G. K. Schoolnik, G. F. Brooks, S. Falkow, C. E. Frasch, J. S. Knapp, J. A. McCutchan, and S. A. Morse (ed.), The pathogenic neisseria. Proceedings of the Fourth International Symposium, Asilomar, California, 21-25 October 1984. American Society for Microbiology, Washington, D.C.

MAb TO MENINGOCOCCAL LOS

2053

31. Rosenqvist, E., and C. E. Frasch. 1986. Development of Neisseria meningitidis serotype 15 protein/polysaccharide vaccine and evaluation in a mouse model. Antonie van Leeuwenhoek J. Microbiol. 52:260-262. 32. Rouse, D. A., S. L. Morris, A. B. Karpas, P. G. Probst, and A. D. Chaparas. 1990. Production, characterization, and species specificity of monoclonal antibodies to Mycobacterium avium complex protein antigens. Infect. Immun. 58:1445-1449. 33. Salih, M. A. M., D. Danielsson, A. Backman, D. A. Caugant, M. Achtman, and P. Olc6n. 1990. Characterization of epidemic and nonepidemic Neisseria meningitidis serogroup A strains from Sudan and Sweden. J. Clin. Microbiol. 28:1711-1719. 34. Schneider, H., J. M. Griffiss, J. W. Boslego, P. J. Hitchcock, K. M. Zahos, and M. A. Apicella. 1991. Expression of paragloboside-like lipooligosaccharides may be a necessary component of gonococcal pathogenesis in men. J. Exp. Med. 174:16011605. 35. Schneider, H., J. M. Griffiss, R. E. Mandrell, and G. A. Jarvis. 1985. Elaboration of a 3.6-kilodalton lipopolysaccharide, antibody against which is absent from human sera, is associated with serum resistance of Neisseria gonorrhoeae. Infect. Immun. 50:672-677. 36. Schneider, H., T. L. Hale, W. D. Zollinger, R. C. Seid, Jr., C. A. Hammack, and J. M. Griffiss. 1984. Heterogeneity of molecular size and antigenic expression within the lipopolysaccharides of individual strains of Neissena gonorrhoeae and Neissena meningitidis. Infect. Immun. 45:544-549. 37. Sugasawara, R. J., C. Prato, and J. E. Sippel. 1983. Monoclonal antibodies against Neisseria meningitidis lipopolysaccharide. Infect. Immun. 42:863-868. 38. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 39. Tsai, C. M., R. Boyidns, and C. E. Frasch. 1983. Heterogeneity and variation among Neisseria meningitidis lipopolysaccharides. J. Bacteriol. 155:498-504. 40. Tsai, C. M., and C. V. Civin. 1991. Eight lipooligosaccharides of Neisseria meningitidis react with a monoclonal antibody which binds lacto-N-neotetraose (GalI1-4GlcNAcP1-3GalI31-4Glc). Infect. Immun. 59:3604-3609. 41. Tsai, C. M., and C. E. Frasch. 1982. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119:115-119. 42. Tsai, C. M., L. F. Mocca, and C. E. Frasch. 1987. Immunotype epitopes of Neisseria meningitidis lipopolysaccharide types 1 through 8. Infect. Immun. 55:1652-1656. 43. Yamasaki, R., H. Schneider, J. M. Griffiss, and R. Mandrell. 1988. Epitope expression of gonococcal lipooligosaccharide (LOS). Importance of the lipoidal moiety for expression of an epitope that exists in the oligosaccharide moiety of LOS. Mol. Immunol. 25:799-809. 44. Zollinger, W. D. (Walter Reed Army Institute of Research). 1989. Personal communication. 45. Zollinger, W. D., and R. E. Mandrell. 1977. Outer membrane protein and lipopolysaccharide serotyping of Neissena meningitidis by inhibition of a solid phase radioimmunoassay. Infect. Immun. 18:424-433. 46. Zollinger, W. D., and R. E. Mandrell. 1980. Type-specific antigens of group A Neisseria meningitidis: lipopolysaccharide and heat-modifiable outer membrane proteins. Infect. Immun. 28:451-458. 47. Zollinger, W. D., and R. E. Mandrell. 1983. Importance of complement source in bactericidal activity of human antibody and murine monoclonal antibody to meningococcal group B polysaccharide. Infect. Immun. 40:257-264.