Vol. 30, No. 5JOURNAL OF CLINICAL MICROBIOLOGY, May 1992, p. 1198-12040095-1137/92/051198-07$02.00/0Copyright © 1992, American Society for Microbiology

Enzyme Immunoassay for Detection of Immunoglobulin M (IgM)and IgG Antibodies to Mycoplasma pneumoniae

S0REN A. ULDUM,* J0RGEN S. JENSEN, JAN S0NDERGARD-ANDERSEN,t AND KLAUS LINDMycoplasma Laboratory, Neisseria Department, Statens Seruminstitut,

Artillerivej 5, DK-2300 Copenhagen S, Denmark

Received 15 October 1991/Accepted 28 January 1992

An enzyme immunoassay (ETA) for detection of immunoglobulin M (IgM) and IgG antibodies to Mycoplasmapneumoniae was developed. The EIA was evaluated on the basis of results in the M. pneumoniae complementfixation (MPCF) test and the cold agglutinin test. Serum samples from 430 patients with respiratory infectionsof known or unknown etiology, from 91 healthy children and adults and from 20 patients with rheumatoidfactor, were investigated. By the criteria chosen for positive diagnostic EIA values, we found that the combinedmeasurement of specific IgM and IgG gave a specificity of 99.7% and a sensitivity of 97.8%. If only IgMantibodies were measured, the specificity was 100% and the sensitivity was 88%. For IgG alone the specificitywas 99.7%, but the sensitivity was only 46% because of the high EIA cutoff value chosen for IgG. We foundno false positives among serum samples from patients with non-M. pneumoniae respiratory infection of knownetiology, and there were no false IgM positives due to rheumatoid factor. In some cases the IgM EIA resultsbecame positive earlier in the course of illness than the MPCF titer. While children and teenagers respondedpredominantly with IgM antibodies, patients older than 40 years often had an IgG response only (56% ofcases), probably because of reinfection. We conclude that this ETA is a good alternative to the combined MPCFand cold agglutinin tests in the diagnosis of M. pneumoniae infection.

Mycoplasma pneumoniae is a common cause of lowerrespiratory tract disease, especially in children and youngadults. The diagnosis is in most cases confirmed by serology.M. pneumoniae is difficult to culture because of its fastidiousnature, and growth is too slow for diagnostic use. New rapidmethods for detection of the organism by immunologicaltechniques (8, 18), gene probe (8, 17), and polymerase chainreaction (13) are promising but need further evaluation fordiagnostic use.The M. pneumoniae complement fixation (MPCF) test

based on a glycolipid antigen has been the most commonlyused serological test since it was described by Kenny andGrayston in 1965 (14). The test has been evaluated and wasfound to be both sensitive and specific (15). Although itsspecificity has been questioned in patients with pancreatitis(19) and other extrapulmonary manifestations (16, 27), show-ing titer rises to low or moderate antibody levels, theseconditions do not generally motivate the clinician to have anMPCF test made.The cold agglutinin (CA) test has been retained for many

years as a supplement to the MPCF test. CAs develop inmore than half of the patients with M. pneumoniae infection(22), and being immunoglobulin M (IgM) antibodies, they arethe first to be detected and the first to disappear again (6).However, CA may be produced by patients without M.pneumoniae infection (24).

Several enzyme immunoassays (EIA) for the serologicaldiagnosis of M. pneumoniae infection have been publishedin the last 10 years. They include assays either for thedetermination of specific IgG only (30), for both IgG and IgM(7, 11, 12, 25, 31), and assays for IgM only (,u-capture) (9,34). However, most of them are not evaluated with respect

* Corresponding author.t Present address: Novo-Nordisk A/S, Immunkemisk Afdeling,

DK-2820 Gentofte, Denmark.

to predictive diagnostic values in a routine diagnostic set-ting. Some lack sufficient sensitivity and/or specificity, andinterpretation of the results is often hampered by patients'preexisting M. pneumoniae antibodies (7, 11, 25, 30).

In this report we present an evaluation of an EIA for thedetection of specific IgM and IgG to M. pneumoniae basedon a comparison with results from the MPCF test combinedwith the CA test. The predictive values of a diagnosticpositive and a negative EIA in different epidemiologicalsituations are discussed.

MATERIALS AND METHODS

Antigen preparation for EIA. M. pneumoniae Mac wasgrown at 37°C in 800-ml Nunclon plastic flasks (Nunc,Roskilde, Denmark) in 150 ml of Hayflick's medium (23)with 0.05 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer. When a dense layer of coloniesappeared and the color was turning orange, the medium wasremoved. Plast-adherent mycoplasmas were washed threetimes with 0.9% (wt/vol) NaCl with penicillin G (500 U/ml)and harvested by shaking the flasks with sterile glass beads.The organisms were then washed twice by suspension in0.9% NaCl with penicillin and centrifugation at 39,000 x gfor 10 min at 6°C. The pellets were suspended in 0.5 ml ofsaline per flask with penicillin and stored at -80°C.

Aliquots of 300 pul of the organisms were thawed, and 150,ul of 0.9% (wt/vol) NaCl and 50 ,ul of 10% (wt/vol) sodiumdodecyl sulfate (SDS) (lot L-4509; Sigma Chemical Co., St.Louis, Mo.) were added per 300 ,ul, giving a final SDSconcentration of 1% (wt/vol). The suspension was mixedwell and incubated at room temperature for 30 min. The celllysate was centrifuged at 30,000 x g at 15°C for 10 min. Thesupernatant, containing about 10 mg of protein per ml asestimated by the method of Lowry (24a), was stored at-80°C until use as antigen in the EIA.Conjugates. Horseradish peroxidase-conjugated rabbit

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anti-human IgM (code P215) and anti-human IgG (code P214)were both from DAKO, Copenhagen, Denmark.EIA procedure. After optimization with regard to antigen

dilution, serum dilution, enzyme-substrate reaction time,and type of microtiter plAte, the following procedure fordetermining IgM and IgG antibodies to M. pneumoniae wasfound to be the most appropriate. Each well except all outerwells of the microtiter plates (PolySorp; Nunc, Roskilde,Denmark) was coated with 100 RI of antigen diluted 1:1,000in 0.05 M carbonate buffer (pH 9.6) and incubated at 4°Covernight or longer, covered with parafilm (American Na-tional Can, Greenwich, Conn.). Under these conditions theplates could be stored for at least 2 weeks. The plates werewashed twice with phosphate-buffered saline (PBS; pH 7.4)with 0.05% (vol/vol) Tween 20 (article 822184; E. Merck AG,Darmstadt, Germany) (PBS-T), and then postcoated with120 ,ul of a 5% (wt/vol) solution of skim bovine milk powder(Irma A/S, R0dovre, Denmark) in PBS-T (PBS-T-M) at roomtemperature for 30 min. The wells were washed as describedabove, and 90 [lI of PBS-T-M was added to each well. Tenmicroliters of the serum to be tested, diluted 1:50 in PBS-T-M, was then added to each of two wells in two plates,giving a final serum dilution of 1:500. The plates werecovered with parafilm and incubated for 1.5 h at 37°C on ashaking table and washed four times in PBS-T, and then 100ptl of the two conjugates diluted 1:2,000 in PBS-T-M wasadded to the relevant plates. The plates were incubated for 1h as described above and washed three times in PBS-T andonce in deionized water. The wells were then filled withcitrate buffer (pH 5), incubated for 2 min, and emptied, and100 ,ul of substrate solution (0.38 mg of 1,2-phenylenedi-amine-dihydrochloride [78440; Fluka AG, Buchs, Switzer-land], 50 ml of citrate buffer, pH 5, and 20 pul of 30% H202)was added to each well. After incubation for 6 min, thereaction was stopped by adding 150 ,ul of 1 N H2SO4 to eachwell. The A490 was read with a photometer (ImmunoReaderNJ 2000; InterMed, Tokyo, Japan). The blank was a wellcoated with antigen and incubated with the conjugates only.

Reference sera and calculation of EIA values. Five refer-ence serum samples were included in duplicate in eachexperiment. Each of the five serum samples were given bothan IgM and an IgG EIA reference value, calculated as themean optical density (OD) value of 10 experiments multi-plied by 100. The EIA reference (ref.) values were asfollows: ref. 1, IgM, 1.8, and IgG, 1.7; ref. 2, IgM, 27, andIgG, 24; ref. 3, IgM, 63, and IgG, 71; ref. 4, IgM, 93, andIgG, 106; ref. 5, IgM, 144, and IgG, 146. The OD values foreach of the five reference serum samples (mean of two wells)were used to construct a calibration curve (OD values versustheir respective EIA reference values). The line of best fitwas used to determine the EIA values of each serum sampletested (mean of two wells). The linear regression and the testresults (EIA values) were calculated by a computer programmade in SuperCalc 5.0 (Computer Associates International,Inc.). Sera with values above 180 were given the value >180because OD values above 1.8 were not linear. For validationof the experiment the correlation coefficient (r2) of the lineshould be >0.95, and the calculated values for ref. 1 shouldbe <6, and those for ref. 5 should be >120.

Reproducibility. Interassay reproducibility of the EIA wasestimated by calculating the mean EIA value, the standarddeviation, and the coefficient of variation for 24 different testruns for the five reference serum samples, four controlserum samples from patients with MPCF titers of .64, andtwo control pools of sera, namely, pool E (sera from children

c 5 years old with MPCF titers of <16) and pool A (serafrom patients with MPCF titers of 32 to 128).MPCF test. The MPCF test was performed in principle by

the method of Kenny and Grayston (14, 20) but was slightlymodified and performed in a microtiter system (21). Briefly,a glycolipid antigen was extracted from plast-adherent my-coplasma cells with chloroform-methanol, and the dry pow-der was dissolved in 96% ethanol and stabilized with 5%bovine serum albumin (BSA) in veronal-buffered saline (pH7.6) (VBS). This CF antigen was stored until use at -80°C inaliquots of 0.6 ml.

Sera were prediluted 1:8 in PBS and heat inactivated at56°C; twofold dilutions to 1:4,096 were performed in vol-umes of 50 pul of VBS with 0.5% BSA in microtiter plates(MikroWell U-plate; Nunc). To all dilutions, 50 ,u1 of VBSwith 0.5% BSA containing 1.5 U of antigen and 2 U of guineapig complement was added. After incubation at 37°C for 1 h,50 pI of a 1.5% suspension of sensitized sheep erythrocyteswas added; for sensitization 2 U of rabbit antiserum to sheeperythrocytes (mainly IgM class antibody) was used. Afteranother 1 h of incubation at 37°C, the antibody titers wereread as the reciprocal of the highest serum dilution giving a60% hemolysis. Each experiment included two pools ofpositive human sera with MPCF titers of 128 and 2,560,respectively, and a negative serum.We considered the MPCF test as diagnostic of a current or

recent M. pneumoniae infection if the titer rises at leastfourfold to >64 between two consecutively collected serumsamples or if single or standing titers were >512; single orstanding titers from 64 to 256 were considered presumptiveof a current or past infection.CA test. CA titration was performed as previously de-

scribed (20). In brief, sera were diluted by twofold dilutionsin 0.4 ml of VBS (pH 7.6) from 1:16 to 1:2,048. A 2.5%suspension (25 pul) of washed human group OI erythrocyteswas added to each tube. The tubes were shaken and thenincubated overnight at 4°C. After a light shake, the tubeswere observed against an illuminated frosted green glassplate, and the titer was read as the reciprocal of the highestserum dilution giving visible agglutination. A titer of 264was considered positive. Although nonspecific, a positiveCA titer together with a positive MPCF titer supports thediagnosis of a current M. pneumoniae infection.

Cultivation of M. pneumoniae from patients. Cotton-tipswabs were taken from the naso-oropharynx and immedi-ately immersed into a vial with 3 ml of mycoplasma broth(SP4 medium) (29). The vial was transported within 24 h tothe laboratory, where two agar plates were streaked, andtwo vials with broth were inoculated with 0.1 ml each; thetwo media were the classical Hayflick's medium and SP4medium. After incubation of the agar plates at 37°C in amoist atmosphere of 5% CO2 and 8% 02, growth of M.pneumoniae was identified by the disk growth inhibition test(5).Groups of human serum samples. Sera from a total of 541

patients and healthy persons were included. The patientswere separated into two categories as follows: one of pa-tients with respiratory tract infections and positive MPCFtest results, divided into three groups, and the other ofpatients and healthy persons with MPCF negative sera,divided into four groups. The categories and groups aredescribed below.

MPCF-positive serum samples. (i) Group 1. At least twoconsecutive serum samples from each of 95 patients withrespiratory infections (mostly pneumonia), all showing a

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TABLE 1. Distribution of persons in the four MPCF-negativegroups with regard to IgM EIA values

No. (%) in following group:IgM EIAvalue 4 (n = 160) Sa (n = 42) 6 (n = 20) 7 (n = 91) T=t313)0-9 140 (88) 36 (86) 19 (95) 77 (85) 272 (87)10-19 20 (12) 6 (14) 1 (5) 14 (15) 41 (13)20-29 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

Total 160 (100) 42 (100) 20 (100) 91 (100) 313 (100)

a Values for convalescent-phase serum only.

>fourfold titer rise to at least 64 in the MPCF test; medianage, 21 years (range, 3 to 78 years).

(ii) Group 2. Single serum samples from 84 patients withMPCF titers of .512; median age, 22 years (range, 1 to 62years).

(iii) Group 3. Single serum samples from 49 patients withMPCF titers from 64 to 256; median age, 28 years (range, 1to 73 years).MPCF-negative serum samples. (i) Group 4. Single serum

samples from 160 patients with respiratory tract infection ofunknown etiology with MPCF titers of <16; median age, 50years (range, 1 to 90 years).

(ii) Group 5. Acute- and convalescent-phase serum sam-ples from 42 patients with respiratory infection showingsignificant rises in titer of antibodies to Chlamydia groupantigen (n = 6), Legionella sp. (n = 6), influenza A virus (n= 6), influenza B virus (n = 8), adenovirus (n = 7), andrespiratory syncytial virus (n = 9). All were with MPCFtiters of .32; median age, 46 years (range, 1 to 99 years).

(iii) Group 6. 20 single serum samples with differentconcentrations of rheumatoid factor (RF), 15 IgM RF-posi-tive sera and 5 IgA RF-positive sera, kindly supplied byMimi H0ier-Madsen, Department of Autoimmunology, Stat-ens Seruminstitut. All were with MPCF titers of s32;median age, 61 years (range, 30 to 90 years).

(iv) Group 7. Single serum samples from 91 healthy adultsand children with MPCF titers of .16; median age, 16 years(range, 1 to 90 years).

RESULTS

Criteria for a positive EIA test. IgM and IgG EIA valueswere chosen to give the best discrimination between groupsof patients with diagnostic positive MPCF test results(groups 1 and 2) and MPCF test-negative patients or healthypersons, as follows. (i) A significant increase in EIA valueswas defined either as a 2-fold or greater increase to 230 forIgM and/or to .40 for IgG or a 1.5-fold increase to .55 forIgM and/or IgG; they were all considered diagnostic of acurrent or recent M. pneumoniae infection, and these in-creases corresponded to at least four times the standarddeviation (see Table 7). (ii) A single IgM EIA value of 260and/or an IgG value of .100 was also considered diagnosticof a current or recent M. pneumoniae infection. (iii) A singleIgM EIA value of .30 and/or an IgG value of >60 wasconsidered presumptive of a current or past infection.

Specificity. The EIA results for MPCF-negative sera(groups 4 to 7) are presented as number and percentage ofpersons with IgM values in Table 1 and IgG values in Table2, stratified in various EIA ranges. None of the sera from thepatients with non-M. pneumoniae respiratory infections in

TABLE 2. Distribution of persons in the four MPCF-negativegroups with regard to IgG EIA values

No. (%) in following group:IgG EIAvalue 4 (n =160) 5 n=4)6n=207(n=9 Total5a(n42)6(n20) 7(n=91)(n =313)

0-910-1920-2930-3940-4950-5960-6970-100

>100

82 (51)51 (32)17 (11)4 (2.5)4 (2.5)1 (0.5)1 (0.5)

26 (62)15 (36)1 (2)

14 (70) 63 (69)5 (25) 21 (23)

6 (7)1 (1)

1 (5)

185 (59)92 (29)24 (8)5 (2)4 (1)1 (0.3)1 (0.3)0 (0)1 (0.3)

Total 160 (100) 42 (100) 20 (100) 91 (100) 313 (100)a Values for convalescent-phase serum only.

group 5 showed a significant increase in EIA value, indicat-ing absence of cross-reactivity between the causative micro-organisms and M. pneumoniae protein antigen. Table 1shows that sera from all the MPCF-negative groups had IgMEIA values of <20, giving a specificity of 100% for the IgMEIA.Table 2 demonstrates that some MPCF-negative sera had

higher EIA reaction levels for IgG than for IgM. One patientin group 4, a 71-year-old male with respiratory infection ofunknown etiology, had a positive IgG EIA value of 67 (IgMEIA = 0), which is not diagnostic. In group 6 anotherpatient, a 32-year-old male with a low positive IgM RF valueof 12 (the test is positive for values of >8 [10]) and a MPCFtiter of 32 had a diagnostic IgG EIA value of 103 (IgM EIA= 19). With one false diagnostic positive serum the specific-ity for measuring IgG was 99.7% (312/313 x 100). Forpatients with non-M. pneumoniae infection and healthyadults and children (groups 5 and 7), the specificity was100% with no IgG values of >40.

Sensitivity. EIA results for consecutive sera from patientswith respiratory infections and diagnostic MPCF titer rises(group 1) are shown in Table 3, and single serum sampleswith MPCF titers of .512 (group 2) are shown in Table 4.The results are grouped according to number and percentageof patients with positive EIA values and/or increases invalues and are stratified according to positive and negativeCA titers. It can be seen from Tables 3 and 4 that there is anassociation between CA-positive and IgM EIA-positive re-actions and between CA-negative and IgG-positive reac-tions, respectively. If groups 1 and 2 are calculated together,175 of 179 patient sera had diagnostic positive EIA values,giving a sensitivity of 97.8%. Among these, 18 (10%) wereexclusively IgG positive, 93 (53%) were exclusively IgMpositive, and 64 (37%) were both IgG and IgM positive. Ofthe patients, 156 (87%) had a diagnostic value by a singlecriterion: an IgM EIA value of .60. Among consecutive serain group 1, 63 (68%) of 92 EIA diagnostic positive patientsdeveloped significant increases in IgM and/or IgG values,and 4 paired sera were positive only by this criterion. Ofanother 4 patients in groups 1 and 2, there were three EIApresumptive positives and one negative. The latter was a12-year-old boy with pneumonia whose first serum hadMPCF and CA titers of < 16 and < 16, respectively, and IgMand IgG EIA values of 6 and 15, respectively, and whosesecond serum had MPCF and CA titers of 256 and <16,

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TABLE 3. Distribution of patients in group 1 with a diagnostic rise in MPCF titer

No. (%) positive for following EIA':

CA titer No. (%) of IgM IgG IgM and IgGpatients

30-59 . 60 Rise6 Total' 60-99 .100 Rise' Total' Total Diag"

.64 81 (85) 1 (1) 77 (95) 39 (48) 78 (96) 14 (17) 17 (21) 33 (40) 39 (48) 81 (100) 81 (100)c32 14 (15) 0 (0) 3 (21) 2 (14) 3 (21) 3 (21) 5 (36) 9 (64) 10 (71) 13 (93) 11 (79)

Total 95 (100) 1 (1) 80 (84) 41 (43) 81 (85) 17 (18) 22 (23) 42 (44) 49 (52) 94 (99) 92 (97)a The patients had positive EIAs according to the three criteria (EIA values of 30 to 59 or .60 for IgM or 60 to 99 or .100 for IgG or by significant increase

in EIA value). A CA titer of .64 is positive, and a CA titer of s32 is negative.A significant increase in EIA values (see text).

c Total number of patients positive by any of the three criteria.d Diag, number of patients EIA positive by the diagnostic criteria only.

respectively, and IgM and IgG EIA values of 8 and 38,respectively.

In group 1, 14 of 33 (42%) patients with an acute-phasenegative MPCF test were EIA positive, one for IgG and 13for IgM, including 7 with diagnostic values of .60. On theother hand, four patients with acute-phase MPCF positivetiters of 64 to 128 were EIA negative.The predictive diagnostic values (PV) (32) for the EIA

calculated for the above-mentioned groups of sera are asfollows: The PV for a positive EIA (PVpos) is 99.4%(175/176 x 100), and the PV for a negative EIA (PVneg) is98.7% (312/316 x 100). See also the Discussion for anestimate of the PVs when the EIA is used as a diagnosticroutine test in two different epidemiological situations.M. pneumoniae was isolated from 13 MPCF and CA

positive patients in groups 1 and 2. They had all diagnosticpositive EIA values.

Presumptive cases. Results for single serum sample from49 patients with presumptive positive MPCF titers of from 64to 256 (group 3) are shown in Table 5. Of 34 CA-positivepatients, 30 (88%) were EIA positive, including 27 (79%)who were diagnostic positive (mainly for IgM). Altogether ingroup 3, the EIA found 35 (71%) to be diagnostic positive, 5were presumptive positive, and 9 were negative.

Age-correlated ETA reactivity. Table 6 shows that withincreasing age of the MPCF-positive patients (groups 1, 2,and 3) the frequency of IgM reactivity decreased while thefrequency of IgG reactivity increased. CAs, which are IgMantibodies, were more common in patients younger than 40years than in older patients (Table 6).RF. None of the RF-positive sera showed positive IgM

EIA values (Table 1). To investigate further the possibilitythat IgM RF causes false-positive IgM reactions (7, 33), each

TABLE 4. Distribution of patients in group 2 withMPCF titers of .512

No. (%) No. (%) positive for following EIAa:

CA titer of pa- IgM IgG IgM and IgGtients

30-59 >60 60-99 .100 Total Diag"

264 78 (93) 4 (5) 73 (94) 8 (10) 33 (42) 78 (100) 77 (99)<32 6 (7) 0 (0) 3 (50) 1 (17) 4 (67) 6 (100) 6 (100)

Total 84 (100 4 (5) 76 (90) 9 (11) 37 (44) 84 (100) 83 (99)

of six strongly positive IgM RF (>100 IU/ml) and EIA-negative sera were mixed with equal parts of each of fourIgM EIA-negative sera (<30) of which two were IgG positive(from 90 to 140) and two were negative in the range of from30 to 59. When tested for EIA IgM and IgG reactivity, noneof the mixed sera in the final serum dilution of 1:250 had theiroriginal reactivity significantly changed (not shown).

Interassay reproducibility. An estimate of the reproducibil-ity is given in Table 7. The SD were generally higher for IgGthan for IgM and, as expected, slightly higher for the controlsera than for the reference sera. The CV values were ratherhigh for sera with low EIA values.

DISCUSSION

When using a diagnostic test the clinician must know itsreliability, expressed, for instance, as the diagnostic predic-tive value of a positive and a negative test result. For theestablishment of these values the investigator has to defineand use a group or population of patients with the disease inquestion and persons or patients without the disease. Inmany infectious diseases like those caused by M. pneumo-niae it is difficult to set up criteria for the "gold standard."Culture alone cannot be a gold standard because patientswith diagnostic titer rises may be culture negative (8). Facedwith this problem we chose to use the MPCF test as ourreference standard by defining diagnostic rises and levels ofantibodies to M. pneumoniae.We chose to measure IgM and IgG antibodies separately,

instead of using a mixed conjugate, because it gives moreinformation about the patient's immune status, and it is possi-ble to operate with different cutoff levels for IgM and IgG.

TABLE 5. Distribution of patients in group 3 withMPCF titers from 64 to 256

No. (%) No. (%) positive for following EIAa:

CA titer of pa- IgM IgG IgM and IgGtients

30-59 . 60 60-99 .100 Total Diag">64 34 (69) 2 (6) 25 (74) 1 (3) 2 (6) 30 (88) 27 (79).32 15 (31) 0 (0) 4 (27) 2 (13) 4 (27) 6 (67) 6 (53)

Total 49 (100) 2 (4) 29 (59) 3 (6) 6 (12) 40 (82) 35 (71)a See footnote a of Table 3 for descriptions of criteria.b Diag, number of patients diagnostic positive for an IgM value of .60

and/or an IgG value of .100.

a See footnote a of Table 3 for descriptions of criteria.b Diag, number of patients diagnostic positive for an IgM value of .60

and/or an IgG value of .100.

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TABLE 6. MPCF-positive patients in groups 1, 2, and 3 with lowand high EIA values and with positive CA titers

%' (no.) of patients with following result

Age (yr) patients IgM EIA value IgG EIA value titer

30-59 .60 30-59 .60) of .64

0-9 50 8 88 18 16 9210-19 51 2 90 25 25 8620-29 47 2 91 19 51 9130-39 50 2 78 16 58 8440-49 16 0 44 6 63 56

>50 14 0 36 21 71 64

Total 228 3 (7) 81(185) 20 (45) 41 (94) 85 (193)

It has been shown previously that healthy persons oftenhave elevated levels of specific M. pneumontiae IgG antibod-ies in their sera, probably because of past M. pnielurnoniiaeinfections (2, 7, 11, 25). This is one of the reasons why it isquestionable to base a diagnostic test for a current M.ptneuinoniae infection upon detection of specific IgG only(30). Our study confirmed that some control persons with a

negative MPCF test did have elevated levels of specific IgG(Table 2), which could be explained by a current M. pnel-moniae infection, but the evidence for this was insufficient.Therefore, the IgG EIA cutoff value had to be fixed at a

sufficiently high level to avoid false-positive results. If theEIA were to be based on detection of specific IgG only, thishigh cutoff value would have resulted in a low sensitivity.On the other hand, diagnosis of M. pnelnioniae infection

cannot be based on the demonstration of specific IgM only(2, 3, 11, 26, 28). We found that some, mainly older, patientsdid not produce IgM antibodies to M. pneumoniae at all butonly produced IgG antibodies, probably as a result ofreinfection; of 30 MPCF-positive patients older than 39 yearsof age, 56% had negative IgM EIA values of below 30 (Table6), and about 12% of all MPCF- and EIA-positive patientshad IgG responses only.

Other good reasons for measuring both IgM and IgG M.pneurnoniae-specific antibodies are (i) The level of IgGantibodies normally rises later than the IgM level, especiallyin children (2, 11, 26, 31), and (ii) some patients continue toproduce IgM predominantly during illness (2, 25). We foundthat 66% of MPCF-positive children younger than 10 yearshad negative IgG EIA values of <30 (Table 6).

In some cases the EIA detected positive levels of specificIgM earlier in the course of illness than the MPCF test. Of 33MPCF-negative acute-phase sera in group 1, 13 (39%) wereEIA positive for IgM, including 7 which were diagnosticpositive.Although the MPCF test and the EIA correlate well with

respect to sensitivity and specificity, individual MPCF titersand EIA values generally showed poor correlation (data notincluded), meaning that some sera with high EIA values hadonly moderately high MPCF titers and, more rarely, viceversa. Results from group 3 reflect this, showing that sera

from 35 of 49 patients (71%) with MPCF titers of 64 to 256had diagnostic positive EIA values.To investigate this further we tried to block some MPCF-

and EIA-positive sera with either the MPCF glycolipidantigen or with the EIA antigen before testing them in theEIA. The result was that all EIA reactions were significantlyreduced by the homologous ETA antigen, while only some

were blocked by the heterologous glycolipid CF antigen.

TABLE 7. Interassay reproducibility for the EIA"

Mean' SD CV'Serum

IgM IgG 1gM IgG IgM IgG

ref. 1 1.9 2.1 1.9 1.6 100.0 76.6ref. 2 27.0 23.4 2.5 3.1 9.2 13.3ref. 3 63.4 70.8 3.4 4.9 5.4 6.9ref. 4 93.3 104.2 3.2 6.4 3.5 6.2ref. 5 145.0 140.4 2.5 10.5 1.7 7.4Pool E 6.3 3.0) 2.2 1.7 34.9 56.7Pool A 39.2 81.4 4.2 6.0 10.8 7.48150 142.0 77.4 5.() 5.1 3.5 6.56948 5.3 145.4 2.5 12.5 47.2 8.617648 113.4 26.9 3.3 4.0 2.9 14.717981 9.8 10.5 3.4 2.2 34.7 20.9

" Reproducibility is on the basis of 24 tests, each inlcluding the fivereference sera and six control sera (two pools and four pallient sera).

" Mean (x) EIA values of 24 observations.CV, coefficient of variation.

Sera with both high MPCF titer and high EIA value had theirEIA reactivity almost totally blocked by the MPCF antigen,whereas sera with only moderate MPCF titers but high EIAvalues were only partly, if at all, blocked (data not included).This indicates that the two antigen preparations share someepitopes (carbohydrate?) and that the EIA antigen in addi-tion possesses epitopes (protein?) not prescnt in the MPCFglycolipid antigen. Further investigations are needed toassess whether the apparent better sensitivity of the EIAover that of the MPCF test proves correct.The combined MPCF and CA tests could be substituted by

this EIA, which has a specificity and sensitivity equivalent tothat of the MPCF test, and most MPCF- and CA-positivesera are IgM EIA diagnostic positive (from 74YG in group 3 to96%5 in group 1). Furthermore, it is an advantage to measurespecific M. pneulmt1oniae IgM over measuring the unspecificCA TgM, which may be produced also, although more rarely,by patients without M. pnleulnoniae infection.We are aware of the fact that our MPCF-positive groups

which were deemed to be true cases of M. pneulmotioaeinfection represent a highly artificial and selected populationwhich cannot be compared with the population met by theclinicians. The predictive diagnostic value of a positive EIAresult depends much on the prevalence of M. pneunoiniaeinfection in the population, and it will be lower than the onecalculated for this material (32). Examples from our experi-ence in a routine laboratory are given below for two periods,one with no epidemics with a low (2%,) prevalence, and oneincluding an epidemic with a high (10%7) prevalence of M.pneumnzoniae disease among patients with respiratory infec-tion whose sera were received for diagnosis by the MPCFtest. The specificity (99.7%) and sensitivity (97.8%) foundfor the EIA were used for the calculations; with a prevalenceof 2%, the PVneg would be 99.95% and the PVpos would be86.93%, giving a false-positive rate of 13.07%. With aprevalence of 10%, the PVneg would be 99.76% and thePVpos would be 97.31%, giving a false-positive rate of2.69%. It is obvious from these estimated values for PVposthat a diagnostic test for a disease with a low prevalencemust have a very high specificity.We have chosen a conventional direct EIA instead of a

l.-capture assay which has generally been preferred to avoidfalse-positive reactions caused by RF and to avoid false-negative IgM reactions caused by IgG antibodies occupyingthe epitopes of the solid-phase antigens (4). Apparently, we

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EIA FOR DETECTION OF M. PNEUMONIAE ANTIBODIES 1203

have no problems with RF, probably because of the highserum dilution of the sera (1:500), which also gave theadvantage that background reactions with the medium bind-ing microtiter plates (PolySorp) were avoided. When serawith high EIA IgG values (without IgM antibodies to M.pneumoniae) were mixed with sera that had IgM reactivityonly, some interference of the IgM assay by the IgG anti-bodies was observed. However, the reduction of the IgMreactivity was proportional to the IgG EIA value. Only serawith very high IgG value (>150) reduced the IgM reactionsignificantly (by 20 to 40%), thereby causing a risk offalse-negative IgM reactions. However, the EIA result sum-mary would still be positive because of the high IgG value(data not given).

Rabbit antisera raised against Mycoplasma genitalium (23)showed reactions with the M. pneurnoniae antigen used inthis EIA when an anti-rabbit immunoglobulin conjugate wasemployed, and MPCF-positive human sera showed reactionswith M. genitalium antigen preparcd in the same way as theM. pneumoniae antigen (unpublished observations). Obvi-ously, the EIA would not discriminate between humanantibodies to the two cross-reacting species (23). AlthoughM. genitaliumn has bcen isolated from throat swabs ofpatients (1), it is not yet known whether the organism is ahuman pathogen. In the search for possible M. genitaliuminfections, we performed immunoblotting with several EIA-positive sera against both M. ptnewunoniae and M. genital-iiwn; however, all positivc sera showed reactions with bothantigens; a few stronger reactions seen with M. genitaliumwere inconclusive. We have not considered the possibility ofM. genitaliumn infection in the evaluation of the specificity ofthe EIA.We believe that this EIA, which has a specificity of 99.7%,

a sensitivity of 97.8&% compared with the MPCF test, and arelative good reproducibility, is a realistic diagnostic alter-native to the MPCF test evcl if it is supplemented with theCA test.

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