J Clin Pathol 1990;43:685-690

Evaluation of urinary antigen ELISA fordiagnosing Legionella pneumophila serogroup 1

infection

R J Birtles, T G Harrison, D Samuel, A G Taylor

AbstractThe enzyme linked immunosorbentassay (ELISA) described was developedto detect a soluble antigen in the urine ofpatients with Legionnaires' diseasecaused by Legionella pneumophilaserogroup 1 (L.pn 1). The assay wasevaluated and showed good specificity(100%) and intra-assay reproducibility.Antigen was detected in the urine of 93(77%) of 120 patients, overall, and in 86%of patients from whom a specimenobtained within seven days of onset ofillness was available. On all but one

occasion the first urine sample takenfrom a patient for whom a positiveELISA result was obtained, was itselfpositive. In one case antigen was notdetected at four days but was present on

the fifth day after onset of symptoms. Intwo patients urinary antigen was detec-table as early as two days after onset ofsymptoms. In another the antigen per-sisted for at least 60 days. More than halfthe patients, however, had stoppedproducing detectable antigen within 14days of onset of symptoms. It istherefore important that where Legion-naires' disease is suspected urine iscollected as early as possible in thecourse of the disease.

PHLS LegionellaReference Unit,Division ofMicrobiologicalReagents and QualityControl, CentralPublic HealthLaboratory, 61Colindale Avenue,London NW9 5HT

R J BirtlesT G HarrisonD SamuelA G TaylorCorrespondence to:Dr R J BirtlesAccepted for publication15 February 1990

At present the laboratory diagnosis of Legion-naires' disease in the United Kingdom isusually made either by culture of Legionellapneumophila from clinical material or byestimation of a patient's specific antibody res-

ponse to the bacterium using the indirectfluorescence antibody test (IFAT) or therapid microagglutination test (RMAT). Al-though a rapid diagnosis can often be estab-lished using either of these tests, the diagnosiscan sometimes be delayed.The diagnosis of Legionnaires' disease by

the detection of legionella antigens in patients'urine using an ELISA was first described byTilton,' and several further assays have beenreported.2' It is clear that the ability of suchassays to detect antigens early in the course ofthe illness makes them potentially useful diag-nostic tools. To date, however, no assay hasbeen thoroughly evaluated and none is widelyused in the United Kingdom. In this paper wedescribe an ELISA for the recognition of an

L.pn 1 urinary antigen, using a fluorescein-isothiocyanate (FITC)-anti FITC system toamplify the signal. The ELISA was evaluated

with respect to specificity, sensitivity, andreproducibility and the results were comparedwith those obtained using established diag-nostic methods.

MethodsHyperimmune rabbit antisera raised againstthe Philadelphia-i strain of L.pn 1 (NCTC11192) were produced as described by Har-rison and Taylor.5 The immunoglobulin Gportion of the immune serum was purified byaffinity chromatography on a protein A-Sepharose column (Pharmacia, Sweden) usingstandard methods. The concentration ofprotein in the purified preparation was

estimated by measuring optical density (OD)at 280 nm.A monoclonal antibody (LKC2b) was

raised against the Knoxville-i strain of L.pn 1

(NCTC 11286). The strain was grown on

buffered charcoal yeast extract agar (BCYE) at37°C for 48 hours. Cells were then removedinto 0-15M phosphate-buffered saline, pH 7 2(PBS), containing 2% formalin, andincubated at 37°C overnight to kill the bac-teria. The suspension was diluted to an OD at605 nm equivalent to 1 2 and mixed at a ratioof 1:1 with Freund's incomplete adjuvant.Balb/c mice were inoculated intraperitoneallyon five occasions with 100 gl of this prepara-

tion at fortnightly intervals. Spleens were

removed three days after the final inoculation.Spleen cells from the immunised mice were

fused with mouse myeloma cell line, JK-Ag8653, using standard methods.67 Culturesupernatants from the hybridomas were

screened against the Knoxville-I strain byimmunofluorescence.5 Hybridoma culturesproducing antibodies which showed goodfluorescence were cloned by limiting dilution.Ascitic fluid was obtained by reintroducingone of the stable antibody secreting clones(LKC2b) into Balb/c mice treated with pris-tane.The antibody isotype of LKC2b was deter-

mined by immunofluorescence using class andsubclass specific conjugates (Tago, Burlin-game, California, USA). The serogroup andsubgroup specificity of the monoclonalantibody was determined by immunofluores-cence, firstly with each of the 14 serogroups ofL pneumophila then with the 10 L.pn 1 sub-groups.8LKC2b monoclonal antibody was purified

by a modification of the euglobin precipitationmethod described previously.9 Briefly, theascitic fluid was clarified by centrifugation,

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then 20 volumes of ice cold 2% boric acidwere slowly added to one volume of asciticfluid with stirring on ice for 30 minutes. Theeuglobin precipitate was recovered after cen-

trifugation at 600 x g for 20 minutes andresuspended in 2 ml of 0- 1M carbonate buffer,pH 9 2, containing 0-5M NaCl.

ANTI-L.PN 1 FITC CONJUGATEFITC labelled antibody was prepared usingmethods similar to those described by Samuelet al for protein-FITC conjugates.'0 Briefly,FITC (5 mg/ml in dry absolute ethanol) was

added at a molar ratio of 20:1 to a stirredpreparation of LKC2b IgM, diluted in 0 1Mcarbonate buffer, containing 0 5M NaCl (pH9 2) in the dark at room temperature. Thereaction was allowed to continue for a further60 minutes, then the LKC2b-FITC was

separated from free FITC by gel filtrationthrough Sephadex G25 using a PD 10 column(Pharmacia, Sweden) preblocked with 1%bovine serum albumin, and equilibrated withPBS. Fractions containing conjugate were

pooled and stored in PBS containing 30% (v/v) glycerol and 0-08% azide.

The production and characterisation of theanti-FITC monoclonal antibody (2F3. 1) usedin this study has been described previously.'0A horseradish peroxidase (EC1.11.1.7) con-

jugate of this antibody was prepared by theperiodate method."

URINARY ANTIGEN ELISA

Flat bottomed microtitre plates (Nunc-Immuno Plate 1) were coated with captureantibody by adding 200 gl of purified rabbitIgG (10 ig/ml) in PBS (pH 7-2) containing0-08% (w/v) sodium azide to each well. Plateswere sealed and incubated at 200C for 48hours before being stored at 40C until use.

Before use the wells were washed four timeswith PBS containing 0.1% (v/v) Tween 20(PBS-Tween). Urine to be tested was heatedin a boiling waterbath for 10 minutes, cooled,then centrifuged in a bench centrifuge at10 000 x g to remove any precipitate. Urine(100 [d) was added to each well and the plateincubated at 37°C for 60 minutes, thenwashed four times in PBS-Tween. LKC2b-FITC (90 gl), diluted 1/800 in PBS-Tweencontaining 5% horse serum (HS), was thenadded to each well and the plate was

incubated as above. After further washing 85gl of the anti-FITC horseradish peroxidase

conjugate, diluted 1/3000 in PBS-Tween-HS,was added to each well followed by an incu-bation and washing step as above.

Substrate was prepared by diluting a 10mg/ml stock solution of 3,3', 5,5' tetramethyl-benzidine in dimethyl-sulphoxide to 100 gg/ml in O.1M citrate-acetate buffer (pH 6-0)containing 0 006% (v/v) hydrogen peroxide.To each well was added 100 gl to detectbound anti-FITC horseradish peroxidase andafter a final 30 minute incubation the reactionwas stopped with 50 gl of 2M sulphuricacid. The intensity of the subsequent yellowcolour produced was measured spectro-photometrically at 450 nm wavelength (OD450)using a plate-reader blanked on a coated wellcontaining only substrate.Three control preparations were used in

each assay. These consisted of two positivecontrol urine specimens, A and B, collectedfrom patients with confirmed Legionnaires'disease, and a negative control urine sample,C, collected from an apparently healthy mem-ber of staff. For a dose-response study urinesamples from controls A and B were dilutedwith distilled water to concentrations rangingbetween 10% and 85% (v/v) of the neat urine,then each concentration was tested in quadru-plicate in the ELISA.

PATIENT SPECIMENSUrine specimens from 120 confirmed cases ofLegionnaires' disease were available for evalua-tion of the diagnostic sensitivity of the ELISA.For inclusion in the evaluation Legionnaires'disease was confirmed using one ofthe diagnos-tic criteria outlined in table 1.Where possible, the following information

was also obtained: (1) date of onset of symp-

toms; (2) dates of collection of the urine sam-

ples; (3) dates of collection of serum samplesand the IFAT results obtained; (4) the sub-group ofL.pn 1 isolated; (5) date ofexposure toinfectious source (outbreak situations only).Urine samples from 80 patients diagnosed as

having an infection due to an agent other thanLegionella were available for evaluation ofthe specificity of the ELISA (table 2). Thespecimens were obtained from two sources.

(i) Forty seven urine samples (one from eachpatient) were kindly given by the BritishThoracic Society (BTS). These urines hadbeen collected from patients with pneumonia as

part of a prospective study organised by theBTS and the Public Health Laboratory Service

Table 1 Evidence of infection in 120 patients with confirmed Legionnaires' disease and sensitivity ofELISA in thesecases

Antigen* AntigentDiagnosis established by: Number of cases positive negative Sensitivity

Isolation of Legionella pneumophila serogroup 1 51 43 8 840Diagnostic serology$ 47 33 14 70°oRaised antibody titres and outbreak association 22 17 5 77%OTotal 120 93 27 77"h

*At least one urine sample taken from each patient was positive when tested in the L.pn 1 urinary ELISA.tNone of the patient's urine samples was positive when tested in the ELISA.Fourfold rise in IFAT titres to at least 64 or standing titre of 128.

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Legionella urinary antigen ELISA

Table 2 Details of infection and diagnosisfor 80 patients with non-L.pn I pneumonia of known aetiology

Source of Number ofAetiological agent specimen Method of diagnosis patients

Streptococcus pneumoniae BTS' Isolation from blood 10Isolation from sputum 14

EBTS2 Blood culture 1Sputum culture 9

LRU3 Evidence not stated4 1Mycoplasma pneumoniae BTS Serology' 11

LRU Serology 3Klebsiella pneumoniae LRU Evidence not stated 1Haemophilus influenzae LRU Evidence not stated 1Salmonella virchow6 LRU Isolation 1Streptococcus spp BTS Evidence not stated 1Aerobic Gram negative bacilli BTS Evidence not known 2Influenza A virus BTS Serology 3

LRU Serology 3Evidence not stated 2

Influenza B virus LRU Serology 1Varicella zoster virus BTS Evidence not stated 1Cytomegalovirus LRU Evidence not stated 1Coxiella burnetii BTS Serology 2Pneumocystis carinii LRU Direct immunofluorescence on sputum 8Chlamydia psittacii BTS Serology 3

LRU Evidence not stated 1

1 British Thoracic Society and Public Health Laboratory Service study.2 Patients with pneumonia excluded from the final BTS evaluation.3 Specimens sent to the PHLS Legionella Reference Unit.4 Diagnosis confirmed by submitting laboratory but evidence not stated.5 Fourfold rise in titres or high conversion titres.6 Patient had fever ofunknown origin and septicaemia.

(PHLS) in 1982-83.1' The aetiological agentwas known for each patient's infection. Urinesamples from a further 10 patients withlaboratory evidence of pneumococcal pneu-monia were also obtained from the BTS, butthese patients had been excluded from theirstudy.(ii) Twenty three urine samples (one from eachpatient) were submitted to the PHLS Legion-ella Reference Unit (LRU) from patients withpossible legionella infections, but a diagnosisother than Legionnaires' disease was sub-sequently established.Urine was also available from three patients

with Legionnaires' disease confirmed by cul-ture caused by non-Sgp 1 L pneumophila.These cases were shown to have infectioncaused by L pneumophila serogroups 3, 8, and12, and their urine samples were examined todetermine the serogroup specificity of theELISA.

REPRODUCIBILITYStatistical evaluation of within batch and be-tween batch reproducibility was carried out asdescribed by Strike.'4 Briefly, 10 urine samples(including negative, weakly positive, stronglypositive and control urines A, B, and C) wererandomly allocated to microtitre wells andtested in duplicate in the same microtitre plate.The mean and standard deviation of each pairof OD4so readings was determined for eachurine sample. The ELISA was then repeated afurther nine times over a period of days. Thesedata were then used for the calculation of thewithin batch standard deviation (Sw) and thebetween batch standard deviation (Sb).

Initially an Sw was calculated as describedabove but only using data for control urines A,B, and C. The SW was then updated after eachbatch of new results using the standard devia-tions of control urines A, B, and C from the 10most recent assays. Acceptance or rejection ofabatch of results was decided using the criteriaoutlined by Strike.'3

ResultsThe isotype of LKC2b monoclonal antibodywas determined to be IgM. When tested byimmunofluorescence against the 10 subgroupsof L.pn 1, LKC2b reacted with representativestrains from the "Philadelphia", "Allentown","Benidorm", "Knoxville" and "France" sub-groups of L.pn 1; these are the same subgroupsrecognised by 2 Mab" of the Internationalpanel.8A dose-response curve determined using

dilutions of control urine A is shown in thefigure. The coefficients of variation (CV) cal-culated from quadruplicate samples of eachdilution are shown in table 3." Also shown inthe figure is a dose-response curve for controlB; both curves seem to be parallel, althoughstatistical confirmation was not attempted.

REPRODUCIBILITYThe SW was found to be very low (0-051 ODunits within a possible range of values between0 0 and 2-0), allowing small differences in theconcentration of urinary antigen to be deter-mined with confidence. The Sb, however (cal-culated for each of the three control urinesamples) were found to be too high to allowantigen concentrations to be compared fromone plate to another (table 4). Results were

2-0

1 5

cfCtn1.0.

050

0.5-

0*

Control A0

/ ,-' Control B

ve, eel1-1-*--8-ao-.18

6 10 20 30 40 50 60 70 80 90 100Dilution of urine ('I. of neat)

Dose-response curvesfor control urines A and B.

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Birtles, Harrison, Samuel, Taylor

Table 3 Coefficient of variation (CV00) for dilutions of control A urine

Concentration of urine(0,, v/v of neat) Mean OD4,, Standard deviation CV%

100 1-94 008 4 185 171 009 5370 1-48 008 5-455 1-12 0 10 8940 078 009 11525 043 005 11-610 017 002 1180 005 0-01 140

therefore only compared when determinedwithin the same batch.

DISCRIMINATION BETWEEN POSITIVE ANDNEGATIVE RESULTSIf the OD450 (mean of duplicate estimation) ofthe urine being examined is greater than theOD450 of the negative control C + 2-77 S, thenthe urine was regarded as positive. In practicemost results could easily be determined by eye.

SPECIFICITYThe aetiological agents responsible for diseasein 80 patients known to have infection otherthan L.pn 1 pneumonia are shown in table 2.All the samples from these patients gavenegative results in the urinary antigen ELISAand the specificity was therefore calculated tobe 100%. In preliminary experiments a falsepositive reaction was obtained on one occasionwhen a urine specimen had not been boiled andclarified by centrifugation before being tested.Three patients had Legionnaires' disease

caused by L pneumophila of serogroups otherthan serogroup 1. These patients produced nodetectable antigen in their urine, suggesting theassay detects an antigen which is serogroupspecific.

DIAGNOSTIC SENSITIVITYAt least one urine specimen was available fromeach of 120 patients with confirmed Legion-naires' disease. L.pn 1 urinary antigen wasdetected in 93 of 120 (77%) of these patients(table 1).The date of onset of symptoms was known

for 112 of 120 patients. Altogether, 202 urinesamples were collected from these patients atvarious dates during their illness. The diagnos-tic sensitivity of the assay decreased as the timeinterval between onset of symptoms and thedate ofurine collection increased (table 5). Thesensitivity was significantly higher (86%) whentesting urines which had been collected withinseven days of onset of symptoms than testingurines collected after this time (64%)(X2 = 3-90, 0-01 < p < 0 05). It is note-worthy that when antigen was detected in the

Table 4 Between assay standard deviation (Sb) ofcontrol urine samples

Control urine sample Mean OD4,S 5b value

Positive A 1-133 0-224Positive B 0-478 0-212Negative C 0095 0-058Possible range of OD450 values = 0-0-2-0

ELISA, with one exception, it was alwayspresent in the earliest available specimen.

Urine specimens giving a positive ELISAresult were obtained from patients withLegionnaires' disease confirmed by isolation ofthe following L.pn 1 subgroups: "Philadel-phia"; "Benidorm"; "Knoxville"; "France";and "Allentown". Urine from one patient withLegionnaires' disease confirmed by isolation ofL.pn 1 subgroup "Camperdown" contained nodetectable urinary antigen, but this sample wascollected 22 days after onset ofsymptoms whenthe sensitivity of the ELISA is reduced.

ONSET AND DURATION OF ANTIGEN EXCRETIONEighty seven patients with Legionnaires' dis-ease with known onset dates had at least oneurine sample collected which gave a positiveresult when tested in the ELISA. Urine sam-ples which contained detectable quantities ofurinary antigen were collected as early as twodays after onset of patient symptoms.

Fifteen of these 87 patients produced urinesamples at later dates which did not containdetectable antigen and these patients providedinformation about the duration of antigenexcretion (table 6). The first urine sampleswhich were negative when tested in the ELISAwere collected from these patients betweenseven and 90 days after onset of symptoms.Eight of the 15 patients were producing urinewhich contained no detectable antigen within14 days ofthe onset oftheir symptoms. Antigenwas still detected in urine of one of the 87patients 60 days after onset of symptoms.The onset of antigen excretion was also

related to the time after exposure to the sourceof infection. Detailed epidemiological inves-tigations showed a period of three days as theprobable period of exposure to L pneumophilain a recent outbreak. 14 The dates ofcollection of79 urine samples from 41 patients infected inthis outbreak were related to the earliest ofthese dates. The shortest interval betweenexposure and collection of urine was 10 days;urine taken at this time was available from twopatients and both specimens contained detect-able urinary antigen.

SEROLOGICAL RESPONSE OF PATIENTS EXCRETINGURINARY ANTIGENThe dates of collection and IFAT results wereknown for 53 serum samples from 22 patientswho were urinary antigen positive within sevendays of onset of symptoms (table 7). Only twoof22 of the samples showed diagnostic titres inIFAT within seven days of onset of symptoms.All sera taken between eight and 14 days after

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Legionella urinary antigen ELISA

Table 5 Sensitivity ofELISA in relation to time after onset ofsymptoms*

Number of days after onset ofsymptoms

01-07 08-14 15-21 22-28 > 28

Total number of urine samples 42 82 33 20 25Number of positive urine samples 36 57 23 14 8Sensitivity 86% 69% 70% 70% 32%

onset had positive titres, and 14 of 19 of thesehad diagnostic titres. Of the remaining serataken 15 days or more after onset of symptoms,10 of 12 gave diagnostic titres. Nineteen of the22 patients were diagnosed serologically. Theremaining three patients were diagnosed byisolation of L.pn 1 from their respiratory tract,but sera collected from all three early in theirillnesses showed positive rising titres. No con-valescent sera were availale from these patients.

DiscussionThe ELISA described here uses a monoclonalantibody to detect the captured antigen. Thisapproach has advantages over most previouslyreported L pneumophila urinary antigenELISAs which use polyclonal antisera.' 3 4Firstly, the monoclonal antibody can beprepared in large quantities which overcomesproblems of reproducibility encountered whenpreparing polyclonal antisera." Secondly,FITC conjugates of LKC2b are easilyprepared and do not give rise to problems ofnon-specific binding. The limitation of usingLKC2b is that it only reacts with the L.pn 1subgroups recognised by the "Mab 2"antibody of the international panel.8 Althoughthis specificity was only shown by immuno-fluorescence, it might be expected that it wouldalso apply to the ELISA. This is supported bythe observation that a urine sample from apatient infected with a strain of the L.pn 1subgroup "Camperdown" (not reactive withLKC2b by immunofluorescence) was negativewhen tested in the ELISA. Previous studiesindicate that over 95% of all cases of Legion-naires' disease are caused by L.pn 1,`6 of whichabout 90% are caused by strains belonging tosubgroups reactive with LKC2b."7 All out-breaks of community acquired Legionnaires'disease in the United Kingdom have also been

Table 6 Duration of detectable antigen in 15 confirmedcases of Legionnaires' disease

Last day* that a First day that apositive urine sample negative urine sample

Case number was taken was taken

1 3 72 5 73 5 134 6 115 8 96 8 107 9 118 11 129 13 9010 14 1711 15 3012 22 3913 27 2814 30 4515 31 32

*Number of days after onset of symptoms.

Table 7 Serological results obtainedfor 53 serumspecimensfrom 22 patients who produced detectableurinary antigen within seven days of onset

Days after onset ofsymptoms

Serology* 00-07 08-14 > 14

Negative 16 0 1Positive but not diagnostic 4 5 1Diagnostic 2 14 10Total (Sera/patients) 22/17 19/16 12/8

*Results as defined by Harrison and Taylor.21.

caused by L.pn 1 subgroups recognised byLKC2b (unpublished observations); thus theinsensitivity of the LKC2b to the non-serogroup 1 L pneumophila serogroups andpossibly some L.pn 1 subgroups is not a majordisadvantage.The use of the FITC-anti-FITC amplifica-

tion system in the ELISA has several potentialbenefits. This system has been shown to be avery sensitive method of detecting proteins inboth ELISA and dot blots and may be prefer-able to other enhancement systems because theconjugates are easy to prepare and the anti-FITC enzyme conjugate exhibits very lownon-specific binding.' 18A dose-response curve was shown for the

ELISA using positive controls A and B,indicating that quantification of the concentra-tion of antigen in a test urine is possible. In theform presented here the ELISA is a qualitativetest used to determine the presence or absenceof antigen, but proposed work on the nature ofthe antigen being detected and the timing andrelevance of its concentration may permitfuture quantification of the ELISA.The reproducibility of the ELISA was very

good where specimens were examined withinthe same batch. The comparison of OD4sovalues using statistical methods was essentialduring the evaluation of this assay, but inpractice for most urines tested in the ELISAthe difference between a positive and negativeresult was distinct enough to be determined byeye. The between batch reproducibility waspoor, prohibiting comparison of resultsobtained in different assay batches. Problemswith between batch reproducibility are thoughtto arise from the deterioration and variation ofconsumable reagents used in the ELISA,specifically horse serum and hydrogen perox-ide. This limitation is not relevant, however, asthe assay in its present form is designed foracute phase diagnosis of Legionnaires' diseasefrom single samples only, and there is nonecessity to compare different urine samplesover a series ofELISA batches.The specificity of the ELISA was calculated

to be 100% as no false positive results wereobtained using urine samples from patientswith non-L.pn 1 pneumonia. This estimatemust be treated with caution as urine samplesfrom only 80 patients were available forexamination and the time at which these urineswere collected, in relation to the patients'illnesses, was unknown. It is clear, however,that the ELISA is highly specific. As Legion-naires' disease accounts for only about2% ofall

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cases of pneumonia in the United Kingdom,'2this high specificity is very impn tant as even alow percentage of false positive results wouldgreatly reduce the predictive value of a positiveresult. 'The overall diagnostic sensitivity of the

ELISA was estimated to be about 77%, a figuresimilar to those reported for previously de-scribed ELISAs'5 and serological tests.20 Thesensitivity of the ELISA, however, was shownto be optimal (86%) during the first seven daysof illness. It is therefore clear that a urinesample should be examined from a suspectedcase as soon as possible after onset of symp-toms. The sensitivity of the ELISA decreasedto about 70% when the urine samples testedwere taken between the eighth and twentyeighth days of illness, and there was anothersignificant decline in sensitivity when testingurines taken at any later times. The role of theELISA in late or retrospective diagnosis ofLegionnaires' disease is therefore limited. Thisis in contrast to serology where high titres ofantibody often persist for a prolonged period.2'

In two patients urinary antigen becameundetectable within seven days of onset ofsymptoms, and eight of 15 patients had stoppedproducing detectable levels of antigen within14 days of onset. The duration of antigenexcretion, however, is clearly very variable andin the case of one patient remained positive forat least 60 days after onset of symptoms. Theseobservations are similar to those of Kohler eta1 4 who also showed that the duration ofurinary antigen excretion was extremely vari-able in patients with Legionnaires' disease,ranging from four to 327 days.

In only one instance did a patient whose firsturine sample was negative when tested in theELISA excrete detectable levels of antigen in asubsequent urine sample. Even in this caseantigen was detectable in urine taken on thefifth day after onset of the patient's symptoms.Thus if patients are not producing urine inwhich antigen can be detected within the firstweek of illness, they are probably unlikely to doso at all and "follow up" samples need not becollected. The validity of this conclusion willneed further investigation.The potential use of an ELISA to detect

antigen in the urine of patients during the acutephase of Legionnaires' disease has been discus-sed for some time."'' The results of this studyrealise this potential with a rapid, non-invasiveassay which has been evaluated usingspecimens from a large number of patients.The ELISA described is relatively simple toperform and results can be available withinfour hours of the sample being collected. Largenumbers of urines can be screened simultan-eously making the test particularly suitable foruse in an outbreak situation. Microtitre platescan be coated and stored for at least ninemonths before use without evidence ofdeterioration. The ability of the ELISA todetect antigen during the very acute stages of

infection (as soon as two days after onset ofsymptoms in some patients) make it a valuableaddition to existing diagnostic tests. Duringa recent outbreak of community acquiredLegionnaires' disease the urinary antigenELISA was the first laboratory assay to identifyL.pn 1 infection in 12 of 43 cases (unpublishedobservations).

We thank Mrs N Doshi for her help in the production of themonoclonal and polyclonal antibodies, the British ThoracicSociety for providing urine specimens, and the staff of theCommunicable Disease Surveillance Centre for epidemiologicaldata.

1 Tilton RC. Legionnaires' disease antigen detected byenzyme-linked immunosorbent assay. Ann Intern Med1979;90:697-8.

2 Bibb WF, Amow PM, Thacker L, McKinney RM. Detec-tion of soluble Legionella pneumophila antigens in serumand urine specimens by enzyme-linked immunosorbentassay with monoclonal and polyclonal antibodies. J ClinMicrobiol 1984;20:478-82.

3 Sathapatayavongs B, Kohler RB, Wheat LJ, et al. Rapiddiagnosis of Legionnaires' disease by urinary antigendetection. Am JMed 1982;72:576-82.

4 Kohler RB, Winn WC, Wheat LJ. Onset and duration ofurinary antigen excretion in Legionnaires' disease. J ClinMicrobiol 1984;20:605-7.

5 Harrison TG, Taylor AG. Identification of Legionellae byserological methods. In: Harrison TG, Taylor AG, eds. Alaboratory manual for Legionella. Chicester: J Wiley andSons Ltd, 1988:57-68.

6 Kohler G, Milstein C. Continuous cultures of fused cellssecreting antibody to predefined specificity. Nature1975;256:495-7.

7 Tedder RS, Yao JL, Anderson MS. The production ofmonoclonal antibodies to Rubella haemagglutinin andtheir use in antibody capture assays for Rubella specificIgM. JHyg 1982;88:335-50.

8 Joly JR, Mckinney RM, Tobin JC, Bibb WF, Watkins ID,Ramsay D. Development of a standardised subgroupingscheme for Legionella pneumophila serogroup 1 usingmonoclonal antibodies. J Clin Microbiol 1986;23:768-71.

9 MasseyeffR, Gombert J, Josselin J. Methode de preparationde la B2-macroglobuline du serum humain. Immuno-chemistry 1965;2:177-80.

10 Samuel D, Patt RJ, Abuknesha RA. A sensitive method ofdetecting proteins on dot and Western blots using mono-clonal antibody to FITC. J Immunol Methods 1988;107:217-24.

11 Wilson MB, Nakane PK. Recent developments in theperiodate method of conjugating horseradish peroxidase(HRPO) to antibodies. In: Knapp W, Holubar K, Wick G,eds. Immunofluorescence and related staining techniques.Amsterdam: Elsevier, 1978:215-24.

12 British Thoracic Society Research Committee. Community-acquired pneumonia in adults in British hospitals in 1982-1983: a survey of aetiology, mortality, prognostic factorsand outcome. Q J Med 1987;239:195-220.

13 Strike PW. Medical laboratory statistics. Bristol: JohnWright and Sons Ltd, 1981:73-95.

14 Westminster Action Committee. Broadcasting House Legion-naires' Disease. London: Department of EnvironmentalServices, City of Westminster, 1988:58-69.

15 Kohler RB, Sathapatayavongs B. Recent advances in thediagnosis of serogroup 1 Legionella pneumophilapneumonia by detection of urinary antigen. ZentralblBakteriol MikrobiolHyg 1983;255 (1 Orig A):102-7.

16 Dournon E, Mayaud C, Bure A, Desplaces N, Christol D.Epidemiological features of Legionnaires' disease in theParis area. Zentralbl Bakteriol Mikrobiol Hyg 1983;255 (1Orig A):76;83.

17 Dournon E, BibbWF, Rajagopalan P, Desplaces N, McKin-ney RM. Monoclonal antibody reactivity as a virulencemarker for LegioneHa pneumophila serogroup 1 strains. JInfect Dis 1988;157:496-501.

18 Harmer IJ, Samuel D. The FITC-anti-FITC system is asensitive alternative to biotin-streptavidin in ELISA. JImmunol Methods 1989;122:115-21.

19 Vecchio TJ. Predictive value of a single diagnostic test inunselected populations. NEnglJ Med 1966;274:1 171-3.

20 Harrison TG, Dournon E, Taylor AG. Evaluation ofsensitivity of two serological tests for diagnosingpneumonia caused by Legionella pneumophila serogroup1. J Clin Pathol 1987;40:77-R2.

21 Harrison TG, Taylor AG. Diagnosis of Legionnaires'disease by antibody levels. In: Harrison TG, Taylor AG,eds. A Laboratory manual for Legionella. Chichester: JWiley and Sons Ltd, 1988:115-9.

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