Zbl. Bakt. Hyg., I. Abt. Orig. A 255, 91-95 (1983)

Monoclonal Antibodies to Legionella pneumophila Serogroup 1:

Possible Applications in Diagnostic Tests and Epidemiologic

Studies

R. M. McKINNEY, L. THACKER, D. E. WELLS, M. C. WONG, W. J. JONES,and W. F. BIBB

Centers for Disease Control, Atlanta, Georgia 30333, USA

Abstract

Monoclonal antibodies were produced against two strains of Legionella pneumophilaserogroup 1. A panel of nine monoclonal antibodies were selectedfortheir unique specifici­ties observed in indirect fluorescent antibody tests with 130 strains of L. pneumophilaserogroup 1. One monoclonal antibody was reactive with an antigen possessed by 128 of130 strains. Two major subgroups were identified and 13 different antigen patterns wereobserved among the 130 serogroup 1 strains.

Keywords: Monoclonal antibodies, Legionella pneumopbila, Legionella pneumophila sero­group 1, fluorescent antibody tests, hybridomas.

Introduction

Fluorescent antibody testing is a useful means of detecting legionellae in clinicalspecimens from the respiratory tract (Cherry et al., 1978; Broome et al., 1979).Tests such as radioimmunoassay (Kohler et al., 1981) and enzyme-linked immuno­sorbent assays (Tilton, 1979; Berdal, Farshy and Feeley, 1979; Sathapatayauongset al., 1982), show much promise as rapid diagnostic tests for soluble antigens ofLegionella pneumophila in urine. The success of these tests is highly dependent onthe availability of potent and specific antibodies directed against the appropriateantigens of the Legionella organisms. With the development of hybridoma technol­ogy (Kohler and Milstein, 1975, 1976) it is now feasible to produce and selecthybridoma cell lines that secrete antibodies of the desired specificity. These anti­bodies can be produced in unlimited quantities and used individually for detectionof specific antigens, or combined for maximum specificity and broad spectrumrecognition of a particular serogroup or species. 1. pneumophila serogroup 1 waschosen for our initial studies because of the high incidence of involvement of thisserogroup in legionellosis.

92 R.M.McKinney, L.Thacker, D.E.Wells, M.C.Wong, W.J.Jones, and W.F.Bibb

Materials and Methods

Preparation of Monoclonal Antibodies

BALBlc mice were obtained from Jackson Laboratory, Bar Harbor, Maine (U.S.A.).L. pneumopbila strain Knoxville1 was used for one set of experiments and strain Oldawas used for a second set of experiments. Cells were harvested after 48 h growth at 37 DCon charcoal-yeast extract agar. Mice were immunized intraperitoneally on day 1 withapproximately 2 x 105 cells in 1 ml of formalin-saline (0.5% formalin, 0.85% NaCI)emulsified with an equal volume of complete Freund's adjuvant. On day 30 each mousewas given an intravenous injection of 2 x 105 cells in formalin-saline. The fusion procedurewas essentially that of Claflin and Williams (1978).

Results and Discussion

Hybridoma Cell Lines, Monoclonal Antibodies, and IFA Reactivity Patterns

Approximately 1000 clones that produced monoclonal antibody to L. pneumo­phila serogroup 1 strains were obtained from the fusion experiments. Nine mono­clonal antibodies were selected for unique IFA reactivity patterns in tests withL. pneumopbila serogroup 1 strains. These nine monoclonal antibodies and thecorresponding cell surface antigens with which they are reactive are designated bythe Arabic numerals 1-9 for discussion throughout this report. The antibodiesdesignated as 1,2, and 3, were obtained from fusions of spleens of mice immunizedwith Knoxville 1 cells. The remainder of the unique monoclonal antibodies de­signated as 4, 5, 6, 7, 8, and 9, were from the Olda experiments. The frequencyof the corresponding antigens found among 130 randomly selected strains of L.pneumopbila serogroup 1 is shown in Table 1.

Table 1. Frequencyof Presence of Specific AntigensAmong 130Randomly Selected Strainsof L. pneumophila Serogroup 1

Antigen Number1

Positive by IFA Test1

1282

963

154

705

646

467

228

239

41

1 Antigen Number is the number designation of the corresponding antibody.

The IFA reactivity patterns of the nine monoclonal antibodies with 130 L. pneu­mophila serogroup 1 strains and the number of strains demonstrating each typeof reactivity pattern are shown in Table 2.

Antigen 1 appears to be an excellent marker for the L. pneumophila serogroup1, since it is present in 128 of the 130 strains (Table 1). On the basis of reactivitywith the panel of monoclonal antibodies, these 128 strains may be divided into twodistinct subgroups designated as subgroup la and subgroup Ib (Table 2). Strainsin subgroup la possess both antigens 1 and 2 and may possess additional antigensas indicated by IFA reactivity with other monoclonal antibodies in the panel. Strains

Monoclonal Antibodies to Legionella pneumophila Serogroup 1 93

Table 2. Indirect Fluorescent Antibody Reactivity Patterns of Nine Monoclonal Antibodieswith 130 1. pneumophila Serogroup 1 Strains

Representative Strain Monoclonal Antibody Number'1 2 3 4 5 6 7 8 9

Subgroup laKnoxville 1 (14) 2 +2 + + _4

Los Angeles 2 (34) + +Birmingham 1 (2) + + + W5 WPhiladelphia 1 (12) + + W +Allentown 1 (16) + + + + WAdelaide 1 (6) + + + W W +SK-267 (11) + + + + + +Subgroup lbOlda (21) + + + + + + +Togus 2 (10) + + +San Francisco 1 (1) + + + + +BL 434 GIE3 (1) + + + + + +D5041 (1) W + + + + +Darby 1 (1) + + + + +

, Number designation given to the monoclonal antibody and hybridoma cell line thatproduced it.

2 Number of strains among a total of 130 with this reactivity pattern.3 Bright fluorescence.4 Negligible fluorescence.5 Weakly positive reaction visible in some tests and absent in others.

in subgroup lb possess antigen 1 but not antigen 2, and may possess any combina­tion of the remaining antigens as indicated by IFA reactivity with the panel ofmonoclonal antibodies.

Our study provided some indication of the scope of antigenic diversity withinL. pneumophila serogroup 1. As shown in Table 2, 13 different antigen combina­tions among the 130 serogroup 1 strains were revealed by selective reactivity withthe panel of nine monoclonal antibodies. The choice of strains Knoxville 1 andOlda for immunizing the mice used for hybridorna experiments was based on ab­sorption studies of rabbit antisera which demonstrated that there were antigenicdifferences between these strains. However it appears certain that additional mono­clonal antibodies with different specificities than those reported here could be pro­duced if other strains of serogroup 1 were used as immunogens.

The limited number of environmental isolates that were tested in this study doesnot allow a meaningful interpretation as to geographic distribution of strains withparticular antigen combinations. However, these results do indicate the potentialof monoclonal antibodies for typing isolates and identifying a common source ofinfection in nosocomial situations and epidemics. Some isolates from commonsources and their antigen patterns are shown in Table 3.

All of the eight serogroup 1 clinical isolates obtained from the Wadsworth VAHospital that were tested were of the subgroup la, antigen combination (1, 2),indicating a probable common source of infection. The three clinical isolates from

94 R.M.McKinney, L.Thacker, D.E. Wells, M.e. Wong, W.].]ones, and W.F.Bibb

Table 3. Antigen Combinations Observed for Clinical Isolates of L. pneumopbila Sero­group 1 from Specific Hospitals or Cities

Strains

Los Angeles 2, 3, 4, 6, 20, 21,22, 23

Miami Beach1 (1978)Miami Beach2 (1981)Miami 1 (1981)Philadelphia 1, 2, 3, 4Philadelphia 5SK267, SK279, SK294SK289, SK291, SK301, SK302

Source

Wadsworth VA HospitalMt, Sinai HospitalMt, Sinai HospitalMiami VA HospitalPhiladelphia epidemic (1976)Childrens' Hospital (1980)Pontiac, Mi.'Pontiac, Mi.2

Antigen Combination

la (1,2)1a (1, 2, 3)la (1,2,3)la (1,2,3)1a (1,2,5)la (1,2)1a (1, 2, 4, 5, 6, 9)la (1,2,4,5,6,9)

i Strains isolated from guinea pigs exposed to aerosols of evaporative condenser waterfrom the health department building in Pontiac, Mi., in studiesof the Pontiac feverepidemicin 1968 (Kaufmann et al., 1981).

2 Strains isolated from guinea pigs exposed to unfiltered air in the health departmentbuilding in Pontiac, Mi., in studies of the Pontiac fever epidemicin 1968 (Kaufmann et al.,1981).

the Miami area, Miami Beach 1, Miami Beach 2, and Miami 1 were in subgroupla with antigen combination 1, 2, 3. Even though a time span of 3 years wasinvolved in these cases, in view of the relative infrequency of this antigen combina­tion, it is possible that tne source of infection was common. The strains Philadelphia1, 2, 3, and 4, were clinical isolates obtained from the Philadelphia epidemic of1976 (McDade et al., 1977). All were of the subgroup la, antigen pattern (1,2,5),indicating a probable common source of the etiologic agent in that epidemic. Phila­dlephia 5, which was not associated with that epidemic, belongs to subgroup la,antigenic pattern (1, 2). The SK isolates from the health department building inPontiac, Mi., (specimens collected in 1968) are divided into two groups in Table 3.The first group was isolated from lung tissue of guinea pigs exposed to aerosols ofevaporative condenser water and the second group was isolated from the lungtissue of guinea pigs that breathed unfiltered air in the building. Kaufmann et al.(1981) concluded that these serogroup 1 organisms found in the air of the healthdepartment building were disseminated from the evaporative condenser via the airconditioning system. The common antigen pattern identified for these two groupsof isolates further strengthens their conclusion. Indeed, the antigen pattern (1, 2, 5,6, 9) was found only for the SK series of isolates from that epidemic, and was notseen in any of the other 130 serogroup 1 isolates that were tested in this study.

The present limited study has in some measure shown the potential for use ofmonoclonal antibodies in epidemiologic studies. Another possible application of themonoclonal antibodies is in DFA tests for detecting Legionella cells in clinicalspecimens. It is clear from the reactivity spectrum (Table 2) that a combination ofmonoclonal antibodies 1, 2, and 4 should give a sufficiently broad coverage todetect all serogroup 1 antigenic variations that might be encountered in clinicalspecimens. Positive specimens could be further tested with the nine individual anti­bodies to determine antigen patterns when this information is desired. Monoclonal

Monoclonal Antibodies to Legionella pneumophila Serogroup 1 95

antibodies may also be used as capture antibodies in enzyme-linked immunosorbentassays (ELISA) or radioimmunoassays to detect antigenuria in legionellosis. In pre­liminary studies in our laboratory, antigens 1 and 2 were detected by ELISA inurine from a legionellosis patient infected with 1. pneumopbila serogroup 1 strainwith the (1, 2) antigen combination. Further studies are in progress to determinethe sensitivity and specificity of this test to detect antigenuria in patients withlegionellosis when monoclonal antibodies are employed.

References

Berdal, B.P., C. E. Farshy, and J. C. Feeley: Detection of Legionella pneumophila antigen inurine by enzyme-linked immunospecific assay. J. Clin. Microbiol. 9 (1979) 575-578

Broome, C. V., W. B. Cherry, W. C. Winn, and B.R. MacPherson: Rapid Diagnosis of Le­gionnaires disease by direct immunofluorescent staining. Ann. intern. Med. 90 (1979) 1-4

Cherry, W.B., B.Pittman, P.P.Harris, G.A.Hebert, B.M. Thomason, L. Thacker, and R.E.Weaver: Detection of Legionnaires' disease bacteria by direct immunofluorescent stain­ing. J. Clin Microbiol 8 (1978) 329-338

Claflin, L. and K. Williams: Mouse myeloma-spleen cell hybrids: Enhanced hybridizationfrequencies and rapid screening procedures. Curro Top. Microbial. Immuno!. 81 (1978)107-109

Kaufmann, A.F., .f. E. McDade, C.M.Patton, J. V.Bennett, P.Skaliy, J.C.Feeley, D.C.An­derson, M.E.Potter, V.F.Newhouse, M.B.Gregg, and P.S.Brachman: Pontiac fever:isolation of the etiologic agent (Legionella pneumophila) and demonstration of its modeof transmission. Amer. J.Epidem. 114 (1981) 337-347

Kohler, G. and C.Milstein: Continuous cultures of fused cells secreting antibody of pre­defined specificity. Nature 256 (1975) 495-497

Kohler, G. and C.Milstein: Derivation of specific antibody producing tissue culture andtumor cell lines by cell fusion. Europ. J. Immuno!. 6 (1976) 511-519

Kohler, R.B., S.E.Zimmerman, E. Wilson, S.D.Allen, P.H.Edelstein, L..f. Wheat, andA. White: Rapid radioimmunoassay diagnosis of Legionnaires' disease. Detection andpartial characterization of urinary antigen. Ann. intern. Med. 94 (1981) 601-605

McDade, J. E., C. C. Shepard, D. W. Fraser, T. F.Tsai, M. A. Redus, W.R. Dowdle, Labora­tory Investigation Team: Legionnaires' Disease: Isolation of a bacterium and demon­stration of its role in other respiratory diseases. New Eng!. J. Med. 297 (1977) 1197-1203

Sathapatayauongs, B., R. B. Kohler, L.]. Wheat, A. White, W. C. Wiml [r., ]. C. Girod, andP. H. Edelstein: Rapid diagnosis of Legionnaires' disease by urinary antigen detection.Comparison of ELISA and radioimmunoassay. Amer. J. Med. 72 (1982) 576-582

Tilton, R. c.: Legionnaires' disease detected by enzyme-linked immunosorbent assay. Ann.intern. Med. 90 (1979) 697-698