Vol. 15, No. 6JOURNAL OF CLINICAL MICROBIOLOGY, June 1982, p. 1077-10840095-1137/82/061077-08$02.00/0

Characterization of Antibody Responses in Legionellosis withan Immunofluorometric Assay

CHARLOTTE M. BLACK,'* LEO PINE,2 CHARLES B. REIMER,1 ROBERT F. BENSON,2 AND

THOMAS W. WELLS1

Immunology1 and Biological Products Divisions,2 Centers for Disease Control, Atlanta, Georgia 30333

Received 24 August 1981/Accepted 22 February 1982

A solid-phase immunofluorometric assay was used to qualitatively characterizeand precisely measure human immunoglobulin class-specific antibody responses

in legionellosis. Stable antigen preparations consisted of cells grown at 25°C thatwere killed, fixed with Formalin vapors, washed, and lyophilized. Working-curvematerial consisted of dilutions of selected convalescent sera. Linear regressions oflogit transformations of relative fluorescence intensities versus the logarithm ofthe relative concentrations of sera were determined to give immunoglobulin class-specific antibody levels from uninfected and infected individuals. Each fluores-cence intensity obtained with immunoglobulin class-specific antibody was con-

verted to a multiple of the median fluorescence intensity obtained with sera fromuninfected individuals. A presumptive-positive acute-phase legionellosis serum

was defined for each immunoglobulin class by a multiple of the normal medianfluorescence intensity that was greater than the multiple of the normal medianfrom approximately 97% of the uninfected population.

Immunofluorometric assay (IFMA) is becom-ing increasingly important as a means for pre-cisely measuring ligand binding (2, 3, 15, 16). Wehave developed and used a number of IFMAs tomeasure alpha-fetoprotein levels in maternal se-rum in antenatal screening tests for neural tubedefects (14), alpha-fetoprotein levels in patientswith primary or metastatic teratocarcinoma (13),and rheumatoid factors (Reimer et al., Int.Congr. Immunol. 4th, Paris, abstr. no. 19.3.09,1980); to quantitate the specificity and potencyof fluorescein isothiocyanate (FITC)-conjugatedantibody reagents (12); to detect mouse hybrid-oma monoclonal antibodies to viral (11) andrickettsial antigens (C. M. Black, T. Tzianabos,L. F. Roumillat, J. E. McDade, and C. B.Reimer, Abstr. Annu. Meet. Am. Soc. Micro-biol. 1982, E35, p. 66); and to measure theimmunoglobulin class-specific antibody re-sponses of mice infected with influenza virus(Gonchoroff et al., Fed. Proc. 40:4740, 1981). Inthis study, we have developed a solid-phaseIFMA to precisely measure the immunoglobulinclass-specific antibody responses to bacterialinfections, using legionellosis in humans as amodel.

In addition to direct fluorescent-antibody andculture techniques, Legionnaires disease is com-monly diagnosed retrospectively by the indirectfluorescent-antibody (IFA) test. Although thereactants are essentially the same in the IFA andthe IFMA, the latter test has several advantages.The IFMA is a precise quantitative test that

removes the subjectivity of estimating low-levelfluorescence in the presence of backgroundstaining of a slightly different hue from compo-nents concomitantly fixed to the slide with theorganism. The IFMA eliminates the need to useegg yolk sac membrane suspension as an absor-bent and adhesive agent. It automatically aver-ages the fluorescence from a large number ofindividual organisms, thereby eliminating thenecessity to examine several microscopic fieldsto ensure observational reliability or to searchfor isolated fluorescence in a presumably nega-tive slide. Although most human infections arestill caused by serogroup 1, Legionella pneu-mophila, the continuing discovery of multipleserogroups (4, 9, 10) and new species (1, 7, 8)makes the IFA an increasingly impractical meth-od for routine use when the serogroup is notpreviously known. Many serotypes or species orboth can be measured by IFMA at the same timein the same tube with a single dilution of apatient's serum. By using a semiautomated dilu-tion procedure and automated processing of thefluorescence readout data, hundreds of samplescan be processed in a day by a relatively un-skilled technician. The cost of the instrumenta-tion is about one-fourth of the cost of a fluores-cent-antibody microscope.

MATERIALS AND METHODS

Antigen preparation. Cells of L. pneumophila,strains Philadelphia-1 (serotype 1) and Togus-1 (sero-type 2), were grown on Feeley-Gorman (6) agar plates

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1078 BLACK ET AL.

at 37°C, washed aseptically from the plate surface, andplaced in distilled water and adjusted to approximately9 mg (dry weight) per ml. These suspensions weredispensed into small vials and stored at -70°C. Toprepare the lyophilized antigen, 0.1-ml volumes of thethawed suspensions were spread over the surfaces of20-ml charcoal yeast extract agar plates (5). The plateswere incubated at 25°C for 9 to 10 days in glass candlejars; in this period the cells had reached the latelogarithmic or very early stationary phase of growth.The lids of the petri dishes were then raised on oneside and held in place with masking tape. A fresh 37%formaldehyde solution (reagent grade, with 15% meth-anol as preservative; Mallinckrodt, Inc.) was pouredinto the bottom of the chamber to a depth of about 0.5inch (1.27 cm), and the chamber with the open petridishes was resealed and incubated for 43 to 48 h at25°C. The cells were removed from the agar surfaceand washed three times in distilled water. One millili-ter of a standard cell suspension adjusted to an opticaldensity reading of 1.0 at 660 nm in a 1-cm light path(Gilford Instrument Laboratories, Inc.) was lyophi-lized in 0.01 M phosphate-buffered saline (PBS), pH8.0, containing 0.25% bovine serum albumin. For usein the IFMA, the cells were reconstituted with 1.0 mlof distilled water and 23 ml of 0.01 M PBS-0.25%bovine serum albumin-0.1% sodium azide to give afinal volume of 24 ml of cell suspension.Serum samples. Normal serum samples were ob-

tained from paid, fasted, healthy blood donors fromthe Atlanta, Ga., area or from the Centers for DiseaseControl (CDC) serum banks. Pathological sampleswere obtained in-house from previous studies or sup-plied by investigators outside CDC (see Acknowledg-ments).

Selected positive convalescent sera were used asworking-curve material for each immunoglobulin mea-sured. Sera that showed a linear logit-log dose-re-sponse curve over a range of at least three logs ofserum dilution and one log of fluorescence intensitywere chosen for working-curve materials. To avoid theaccumulated imprecision and bias inherent in makingserial dilutions, single dilutions of the original serawere accurately made in 0.01 M PBS-0.25% bovineserum albumin-0.1% sodium azide with an automaticdiluter to give seven working-curve samples, each ofwhich was threefold different in analyte concentrationfrom an adjacent working-curve sample. The dilutedworking-curve sera were divided into samples in smallamounts and frozen so that a fresh set of dilutionscould be thawed for each assay and repeated freezingand thawing of the same sample would not be neces-sary. Quality control samples consisted of positivecontrol serum (CDC group 1-positive control lot num-ber 79-0411) and normal sera that were stored frozen,as described above, until used.

FITC-conjugated antisera. All conjugates were pro-duced in-house by FITC dialysis labeling of DEAEcellulose-purified immunoglobulin G (IgG) fractionsfrom immunized goats. Antisera were made classspecific by absorption with soluble or insoluble heter-ologous immunoglobulins or both. The immunoglob-ulin class specificity and potency of the conjugatedantisera were evaluated by the IFMA of Phillips et al.(12), wherein stable antigen standards consisting ofhighly purified immunoglobulins covalently bound topolyaminostyrene beads are used to quantify the ho-

mologous and heterologous potencies of the conju-gates. The conjugates were titrated by using 3.75 x 108IgG-, IgM-, and IgA-coated beads per ml. The back-ground fluorescence was determined with solid-phasebeads coated with IgG from the same animal species asthat from which the conjugate was derived. The work-ing dilution was that dilution which corresponded tothe fluorescence intensity of one-half of the plateaufluorescence brightness for homologous staining ob-tained when beads were saturated with antigen andconjugated antibody.

Assay procedure. Functional complement compo-nents in all samples were inactivated by pretreatmentof the sera at 56°C for 30 min. Each patient's serum orquality control specimen was analyzed in duplicate bymixing 0.1 ml of a sample with 0.1 ml of a cellsuspension in disposable, borosilicate round-bottomedtest tubes. Working-curve materials were always ana-lyzed in triplicate. Equal volumes of the serum sam-ples and the cell suspensions were always deliveredwith the same air-displacement pipette (Clay Adams,Parsippany, N.J.) that had been shown to give animprecision of 1.7% coefficient of variation of thedelivered volumes. All working-curve or serum dilu-tions were made with a liquid displacement dilutingmachine (LKB 2075; Broma, Sweden) in a mannerresulting in less than 3% mean accumulated inaccura-cy (bias) at any dilution.Although the first incubation of cells with each

patient's serum was complete in 30 min, a 1-h incuba-tion period at 37°C was used to make close control ofincubation time unnecessary. After this incubation,the tubes were transferred to multitube centrifugecarriers. Two milliliters of 0.01 M PBS-0.25% bovineserum albumin-0.1% sodium azide was added, and theincubation mixture was centrifuged at 3,000 rpm (2,100x g) for 15 min at 20°C. The supernatant fluids weredecanted by intertwining latex rubber tubing amongthe tubes so that the entire carrier of tubes could bedecanted in one motion. This wash procedure wasrepeated two more times. After the third wash, 50 ,u1 ofthe appropriate conjugate dilution was added to thecells. After incubation at 37°C for 1 h, the cells werewashed three times as described above, except thebuffer used was 0.01 M PBS containing 0.15 mg ofTween 20 per liter. The cells were maintained in thesame tube throughout the assay, and the washed pelletwas suspended in 1.0 ml of PBS-Tween 20 buffer tomeasure the fluorometric response. The fluorescenceintensity for a given sample remained constant afterthree washes, indicating that all of the unbound conju-gate had been removed and that there was no signifi-cant loss of organisms or antibody with additionalwashes.A Gilson Spectra/Glow Filter Fluorometer (Gilson

Medical Electronics, Middleton, Wis.) equipped witha quartz-halogen lamp and a 100 mV digital displayunit was used for all fluorometric measurements. Forexcitation, two interference filters were used in tan-dem; each filter transmitted at least 50% of the inci-dent blue-green light at 492 nm and less than 1% of theincident light below 484 nm or above 496 nm. A 520-nm barrier filter (Corning no. 3-69) was used to isolatethe apple-green fluorescence. Fluorometer stabilitywas monitored with a standard solution of fluoresceindiacetate in 0.1 N NaOH, which was also used tocalibrate the fluorescence output.

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IMFA FOR ANTIBODY RESPONSE IN LEGIONELLOSIS 1079

AO Patient Ao Patient Ba Patient C

.2U.

.5

.1I

10-i

._

,/0.,oc0'7

10-4 10-3

qelative Concentration (O/D)

10-i 10-4 10-3 10.2

Relative Concentration (O/D)

FIG. 1. Analysis of the potency of and parallelismamong convalescent sera from different patients. (A)Typical S-shaped response of relative fluorescenceintensity versus patient serum concentration; (B) cor-responding logit-log transformations of the data plot-ted in (A).

Data analysis. The data were processed by using a

least-squares linear regression of the logit of fluores-cence intensity versus the logarithm of the relativeconcentration of sera. The logit of the fluorescenceintensity (y) was calculated as: logit y = log [(y - yo)lymax]/[1 - (y - yo)lymax], where yo and ymax are

mathematically derived constants which optimally lin-earize the data; yo physically represents the fluores-cence intensity value expected when a tube containsall of the system components except human serum,

and ymax represents the fluorescence intensity ex-

pected with a saturating dose of antibody and FITC-labeled conjugate. All unknowns and quality controlsamples were read from the working curve and ex-

pressed as a fraction of the working-curve serum.

Antibody distribution curves for the normal popula-tion were established with sera from approximately100 uninfected individuals. The normal populationmean, standard deviation, and median were calculatedfrom these data. All patient antibody levels were

expressed as multiples of the normal population medi-an.

RESULTS AND DISCUSSION

Figure 1A shows typical dose-response dataobtained for working-curve material. The S-shaped curves indicate that the fluorescence athigh antibody concentrations approached a hori-zontal plateau (ymax) where cells were saturatedwith antibody and FITC-labeled conjugate; atvery low antibody concentrations, the curves

-approach a second plateau (yo) of backgroundfluorescence as read directly from the digitaldisplay unit. Figure 1B shows the correspondinglogit-log transformation offluorescence intensityversus the log concentration of sera and demon-strates that the logit of the fluorescence is a

linear function of the logarithm of the antibodyconcentration between dilutions of 10-2 and10-5. The mean ± one standard deviation, cal-culated from triplicate points, is indicated on thelogit-log plot. The results shown in these figuresalso demonstrated the log concentration-re-sponse parallelism that exists between sera fromdifferent patients and the working curve, so thatthe former can be considered mathematically as

a simple dilution of the latter.

TABLE 1. Immunoglobulin class specificity of conjugated antisera"Reiative fluorescence intensity of antisera to:

Ig(GAM) IgG IgM IgA

IgG 0.371 0.566 bIgM 0.348 0.415IgA 0.351 0.363Goat IgG

aThe molar fluorescein-to-protein ratios were 4.1, 4.0, 4.9, and 3.6 for Ig(GAM), IgG, IgM, and IgA,respectively.

b , Heterospecific reactivity was not above the level of background staining obtained with normal goat IgGcovalently bound to polyaminostyrene beads.

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1080 BLACK ET AL.

45 Ig(GAM) Serogroup 1

4035302520

5

45 - IgG Serogroup 1

40 -

35 -

30 -

25 -

20-

15 -

105~

45 IgA Serogroup 140

a 35

15105

IaM SroarouD 145- .,, ,._.40 -

353025-

201510

5-

45 - Ig(GAM) Serogroup40 -

35 -

30 -

25 -

20 -

1510

usou us us us

N _ * -Z td 4

p 1+2

uo us us usous u un u us

No. of Standard Multiples ofDeviations the Median

FIG. 2. Normal population distribution curves ofimmunoglobulin response to serogroup 1 only and a

mixture of serogroups 1 and 2. The left column offigures shows the arithmetic plot of standard devi-ations from the normal population mean. The rightcolumn of figures shows the logarithmic plot of MOMfrom the normal population median.

Table 1 shows the molar fluorescein-to-pro-tein ratios, potency, and specificity of the conju-gated antisera used as determined by our IFMAfor evaluating conjugates (12). All of the antiseraused were raised in goats; hence, normal goatIgG covalently bound to the polyaminostyrenebead was used to measure baseline fluores-cence. The polyvalent antiserum, when used at a

1/50 dilution, recognized approximately equallyall three classes of immunoglobulins studied.The class-specific antisera used showed no het-erologous staining above background levels atthe dilutions used for the assay, which were 1/50for anti-IgG, 1/30 for anti-IgA, and 1/100 for anti-IgM.Normal population distribution curves for

each immunoglobulin assayed are shown in Fig.2. Distributions are shown plotted both arith-metically (in standard deviation units) and loga-rithmically (as multiples of the normal median[MOM]). Subsequently, we examined antibodylevels with polyspecific antisera to the humanimmunoglobulins Ig(GAM) to L. pneumophila(Philadelphia-1) of sick patients from variousparts of the nation who clinically were suspectedof having legionellosis but who gave no serologi-cal (by either IFA or IFMA) or cultural evidenceof this disease (data not shown). By IFMA, themedian Ig(GAM) antibody concentration of 223sera (including both S, and S2 bleeds) from thesenon-legionellosis, sick controls was 0.9 times themedian value obtained from 96 healthy patientcontrols. The approximate 97 percentile cutoffwas 1.5 times the median for healthy controlsand 1.6 times the median for non-legionellosis,sick controls. These distributions in (IFMA-measured) antibody levels of healthy and non-legionellosis, sick controls were not markedlydifferent. However, one should be cautious be-cause of the possibility that individuals with pastLegionella infections may have persistent anti-body. The within-run coefficient of variation forIg(GAM) for the entire normal population was28% (n = 56). The coefficient of variation for theentire normal populations for IgG, IgA, and IgMwas 56, 37, and 43%, respectively. The within-run coefficient of variation for Ig(GAM) in theregion between 2 and 3 standard deviationsabove the normal population mean was 14%, avalue which was derived from duplicate determi-nations of all pathological samples (n = 10)occurring in this range. The within-run coeffi-cient of variation of the method decreased to 7%near the center of the working-curve dose re-sponse. Too few samples occurred for the indi-vidual immunoglobulins between + 2 and + 3standard deviations to determine reliably theimprecision in this region.The approximate 97.5 percentile of normals

(+2 standard deviation) was chosen as a cutoffvalue for pathological sera, assuming an approx-imate Gaussian arithmetic distribution. Howev-er, the median value used as a central measurefor a small sample is less affected by an occa-sional spurious observation than is the mean;therefore, MOMs were chosen to define anti-body levels. This approach (use of MOM toexpress the concentration of an analyte) proved

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IMFA FOR ANTIBODY RESPONSE IN LEGIONELLOSIS 1081

TABLE 2. IFMA antibody responses by immunoglobulin class to Legionella serogroup 1 and a mixture ofserogroup 1 and 2 organisms

MOM antibody potency for following serogroupa:Specimen 1 land2

Ig(GAM) IgG IgA IgM Ig(GAM)123456789101112131415161718192021222324252627282930313233343536373839

35.261.67

17.532.521.237.22

14.665.781.765.830.92

45.232.211.222.56

53.1718.777.885.894.113.98

18.364.975.151.052.54

27.145.92

86.3928.351.701.596.171.751.931.201.330.830.68

39.661.524.491.901.832.833.734.441.872.042.039.782.542.263.77

19.577.106.533.624.116.515.914.521.481.082.92

18.273.91

34.857.472.162.837.323.242.440.680.960.681.40

4.971.949.931.471.238.332.053.092.052.871.58

179.271.341.781.20

11.4212.5810.183.455.683.09

10.913.344.620.753.312.058.175.33

45.543.341.943.452.083.780.451.700.450.15

5.971.129.752.410.915.11

11.412.380.486.160.35

10.101.990.530.85

16.2413.592.743.415.952.43

11.073.443.440.651.744.382.44

42.615.180.410.784.840.651.040.881.39

-0.050.41

23.801.52

13.151.971.635.19

10.553.902.044.771.24

34.021.541.091.89

46.6015.655.634.217.334.04

15.493.052.712.761.80

13.803.93

53.8516.5215.111.133.982.231.150.650.530.600.66

a The cutoff MOM values were 1.38, 1.58, 2.16, and 1.99 for Ig(GAM), IgG, IgA, and IgM, respectively, ofserogroup 1 and 1.58 for Ig(GAM) of serogroups 1 and 2. Boldface numbers represent positive reactions.

particularly advantageous for the assay of mid-pregnancy maternal serum alpha-fetoprotein forthe antenatal detection of fetal neural tube de-fects (17). For results expressed in MOM units,the approximate 97 percentile or pathologicalcutoff levels used were 1.38 for Ig(GAM), 1.58for IgG, 2.16 for IgA, and 1.99 for IgM whentested with Philadelphia-1 alone. The MOM val-ue for Ig(GAM) is 1.58 when tested with Phila-delphia-1 and Togus-1 combined. When pairedacute and convalescent sera were tested, a posi-tive seroconversion is defined as any S2-to-Slratio greater than 1.60.The results for the first 39 specimens are

expressed in Table 2 as MOM antibody potency.Specimens 1 through 39 were single, putativeconvalescent sera from patients with antibodiesto various serogroups of Legionella. Specimen24 was the CDC serogroup 1-positive control,and specimen 25 was the CDC serogroup 2-positive control; both of these reference re-agents were convalescent sera that were predi-luted in normal human serum to give the desiredlow IFA-positive control serum titers. Speci-mens 31 and 34 were from patients with sero-group 2 infections, specimen 32 was from apatient with a serogroup 3 infection, and speci-men 33 was from a patient with a serogroup 4

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1082 BLACK ET AL. J. CLIN. MICROBIOL.

TABLE 3. Paired acute and convalescent IFMA antibody responses by immunoglobulin class to Legionellaserogroup 1 and a mixture of serogroup 1 and 2 organisms

MOM antibody potency for following serogroupa:

Patient Days after 1first bleed 1_1 and 2

Ig(GAM) IgG IgA IgM Ig(GAM)

40 0 4.53 2.09 4.62 1.50 2.15

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

1115

0

17

0

1552

0

20

0

23

0

10

0

33

0

19

0

14

0

518

0

29

0

25

0

6

0

28

0

43

0

33

0

24

0

14

0

12

88.8392.19

1.2423.87

1.162.531.06

1.0125.16

0.782.09

0.7310.79

0.971.35

1.223.42

1.429.04

1.437.975.15

0.802.53

2.5246.33

1.1331.45

0.901.67

1.531.23

1.204.26

2.2119.66

1.4415.85

2.195.06

7.156.61

0.722.69

0.680.900.72

0.723.96

1.431.37

0.593.63

1.180.69

0.931.44

1.573.43

2.492.132.05

1.101.44

1.458.38

0.690.81

0.560.75

1.350.89

1.081.51

b

149.99213.63

0.628.82

1.233.100.75

0.7521.26

0.750.96

0.374.98

0.860.99

1.231.81

2.0513.61

0.9919.2712.96

0.990.99

1.8119.83

0.5058.00

0.450.64

1.471.23

0.99

2.17

46.4942.75

0.5315.46

0.261.171.80

0.5316.29

0.351.46

0.356.62

0.411.58

0.693.03

1.044.44

0.654.792.80

0.532.24

1.1712.79

0.9510.20

0.480.70

-0.050.41

0.412.03

117.5994.59

0.727.20

0.641.490.74

0.6714.03

0.451.58

0.457.27

0.841.21

0.932.15

0.935.77

0.944.583.31

0.741.51

1.1147.17

0.6119.77

0.710.74

0.800.67

0.871.92

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IMFA FOR ANTIBODY RESPONSE IN LEGIONELLOSIS 1083

TABLE 3-ContinuedMOM antibody potency for following serogroupa:

Patient Days after 1 1 and 2first bleed _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

Ig(GAM) IgG IgA IgM Ig(GAM)

59 0 4.3812 29.62 -

60 0 1.6773 5.50

61 0 - - -

15 17.58

62 0 2.21 - -18 5.59

63 0 26.09 -15 72.81

64 0 2.229 3.97

65 0 1.4825 -

a The cutoff MOM values were 1.38, 1.58, 2.16, and 1.99 for Ig(GAM), IgG, IgA, and IgM, respectively, ofserogroup 1 and 1.58 for Ig(GAM) of serogroups 1 and 2. Boldface numbers represent positive reactions.

b , Not tested.

infection. All other specimens in this groupshowed a single high IFA titer, were culture- ordirect fluorescent-antibody-positive for sero-group 1, or both. Specimens 36, 37, 38, and 39were all negative in the IFMA. These sera frompostmortem, culture-positive patients were re-ported to be normal by IFA.Sample 5 appeared to be associated with sero-

group 2, as indicated by the response when bothPhiladelphia-1 and Togus-1 cells were used incombination; this specimen was also positivewith the IgG-specific antisera. Specimens 11 and14 were positive only with the IgG-specificantisera; specimens 31, 32, and 34 were frompatients with infections from other serogroupsand also showed a relatively greater responsewhen tested with the IgG-specific antisera. Inview of some of the quantitative differencesobserved with the polyvalent Ig(GAM)- andIgG-specific antisera, further investigation couldbe directed to the possibility that IgG subclass orallotypic immunoglobulin subsets respond pref-erentially to common determinants shared bysome of the Legionella serogroups. Specimen 33was from a patient with serogroup 4 infectionand showed a strong cross-reactivity among allthe immunoglobulin classes; this result indicateda broader immunoglobulin response to somecommon determinants.Table 3 lists IFMA antibody responses for

paired acute and convalescent sera. Pairs 40

through 43 were reported as having positiveserology by the IFA; these sera were also shownto convert by the IFMA. Patient 42 respondedonly with IgA production, emphasizing the ne-cessity to use a polyvalent antiserum that recog-nizes all three immunoglobulin classes[Ig(GAM)]. Patients 44 through 49 were reportedas having positive culture and positive serologyby IFA. Patient 46 showed no seroconversion bythe IFMA or by IFA when retested here. Pa-tients 50 through 55 were reported as havingpositive culture but negative serology by IFA.The IFMA showed a positive seroconversion infive of these six persons. Patients 56 through 65were diagnosed by a state lab and confirmed byCDC to show IFA seroconversion to serogroup1. The IFMA demonstrated a positive serocon-version on all of these patients when repeatbleedings were tested. In addition, the IFMAshowed a positive result in 56% of the acute-stage sera, in contrast to 8% by IFA, althoughthe IFA results were obtained by different labo-ratories at different times.A linear regression performed on Sl, S2

Ig(GAM) antibody levels measured by IFMA on49 paired sera that were positive by IFA serolo-gy (data not shown) indicated an average in-crease of Legionella-specific antibody of ap-proximately 15% per day (5-day antibodydoubling time) at 2 weeks after onset of illness,which often corresponds to the first week of

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1084 BLACK ET AL.

hospitalization. This would indicate that a 1.6-fold S2-to-S1 ratio conversion (greater than threetimes the assay standard error) would occureach half-week during this acute phase-earlyconvalescence period. These results suggest aneed for a definitive study of the potential of thisIFMA for the early diagnosis of legionellosis.

ACKNOWLEDGMENTS

We thank John P. Anhalt, Mayo Clinic, Rochester, Minn.;Paul H. Edelstein, Veterans Administration, Los Angeles,Calif.; Raymond A. Pelletier, State of Vermont Department ofHealth, Burlington, Vt.; Washington Winn, University ofVermont, Burlington, Vt.; Mary L. Fried, Tucson, Ariz.; andHazel Wilkinson, Centers for Disease Control, Atlanta, Ga.,for generously supplying pathological serum samples.

LITERATURE CITED

1. Brenner, D. J., A. G. Steigerwalt, G. W. Gorman, R. E.Weaver, J. C. Feeley, L. G. Cordes, H. W. Wilkinson, C.Patton, B. M. Thompson, and K. R. Lewalien Sasseville.1980. Legionella bozemanii species nova and Legionelladumoffii species nova. Classification of two additionalspecies of Legionella associated with human pneumonia.Curr. Microbiol. 4:111-116.

2. Burgett, M. W., S. H. Fairfield, and J. F. Monthony.1977. A solid-phase fluorescence immunoassay for thequantitation of the C4 component of human complement.J. Immunol. Methods 16:211-219.3.

3. Deelder, A. M., and J. S. Ploem. 1974. An immunofluores-cence reaction for Schistosoma mansoni using the definedantigen substrate spheres (DASS) system. J. Immunol.Methods 4:239-251.

4. England, A. C., III, R. M. McKinney, P. Skaley, andG. W. Gorman. 1980. A fifth serogroup of Legionellapneumophila. Ann. Intern. Med. 93:58-59.

5. Feeley, J. C., R. J. Gibson, G. W. Gorman, N. C. Lang-ford, J. K. Rasheed, D. C. Mackel, and W. B. Baine. 1979.Charcoal yeast extract (CYE) agar: a primary isolationmedium for Legionella pneumophila. J. Clin. Microbiol.10:436-441.

6. Feeley, J. C., G. W. Gorman, R. E. Weaver, D. C.Mackel, and W. W. Smith. 1978. Primary isolation mediafor Legionnaires diseases bacterium. J. Clin. Microbiol.8:320-325.

7. Hebert, G. A., A. G. Steigerwalt, and D. J. Brenner. 1980.Legionella micdadii species nova: classification of a thirdspecies of Legionella associated with human pneumonia.Curr. Microbiol. 3:255-257.

8. McKinney, R. M., R. K. Porschen, P. H. Edelstein, M. L.Bissett, P. 0. Harris, S. P. Bondell, A. G. Steigerwalt,R. E. Weaver, M. E. Ein, D. S. Lindquist, R. S. Kops, andD. J. Brenner. 1981. Legionella longbeachae sp. nov.,another etiologic agent of human pneumonia. Ann. Intern.Med. 94:739-743.

9. McKinney, R. M., B. M. Thompson, P. P. Harris, L.Thacher, K. R. Lewallen, H. W. Wilkinson, G. A. Hebert,and C. W. Moss. 1979. Recognition of a new serogroup ofLegionnaires' disease bacterium. J. Clin. Microbiol.9:103-107.

10. McKinney, R. M., H. W. Wilkinson, H. M. Sommers,B. J. Fikes, K. R. Sasseville, M. M. Yungbluth, and J. S.Wolf. 1980. Legionella pneumophila serogroup six: isola-tion from cases of legionellosis, identification by immuno-fluorescence staining, and immunological response toinfection. J. Clin. Microbiol. 12:395-401.

11. Phillips, D. J., A. P. Kendal, R. G. Webster, P. M. Feo-rino, and C. B. Reimer. 1980. Detection of monoclonalinfluenza antibodies synthesized in culture by hybridomacells with a solid-phase indirect immunofluorometric as-say. J. Virol. Methods 1:275-283.

12. Phillips, D. J., C. B. Reimer, T. W. Wells, and C. M.Black. 1980. Quantitative characterization of specificityand potency of conjugated antibody with solid-phaseantigen bead standards. J. Immunol. Methods 34:315-327.

13. Reimer, C. B., and L. H. Einhorn. 1979. Quantitativeimmunofluorometric assay (IFA) of serum alpha-fetopro-tein (AFP) in patients with primary or metastatic terato-carcinomas, p. 41-44. In R. B. Herberman (ed.), NationalCancer Institute compendium of assays for immunodiag-nosis of human cancer, vol. I of developments in cancerresearch. Elsevier/North Holland Biomedical Press, Am-sterdam.

14. Reimer, C. B., D. Phillips, C. M. Black, and T. W. Wells.1978. Standardization of ligand binding assays for alphafetoprotein, p. 189-200. In W. Knapp, K. Houlbar, and G.Wick (ed.), Immuno-fluorescence and related stainingtechniques. Elsevier/North Holland Biomedical Press,Amsterdam.

15. Soini, E., and J. Hemmila. 1979. Fluoroimmunoassay:present status and key problems. Clin. Chem. 25:353-361.

16. Sundeen, J. T., and S. K. Randall. 1979. A quantitativeassay for low levels of IgM by solid-phase immunofluores-cence. J. Immunol. Methods 26:229-244.

17. Wald, N. J., H. Cuckle, D. J. H. Brock, R. Peto, P. E.Polani, and F. P. Woodford. 1977. Maternal serum alpha-fetoprotein measurement in antenatal screening for anen-cephely and spina bifida in early pregnancy. Report of theUK collaborative study on alpha-fetoprotein in relation toneural tube defects. Lancet i:1323-1332.

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