pneumonia from the Pediatric Clinic of the University of Padua. Serological Methods

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1 THE YALE JOURNAL OF BIOLOGY AND MEDICINE 56 (1983), Seodiagnosis of M. pneumoniae Infections by Enzyme- Linked Immunosobent Assay (ELISA) FRANCO BUSOLO, M.D., AND GIOVANNI ANTONIO MELONI, M.D. Institute of Micobiology, Univesity of Padua Medical School, Padua, Italy Received Januay 4, 1983 The enzyme-linked immunosobent assay (ELISA) has been used fo detection of antibodies against Mycoplasma pneumoniae. The esults of ELISA and its sensitivity compaed with othe seological methods, such as complement fixation (CF), metabolic inhibition (Ml), mycoplasmacidal test (MC), and adioimmunopecipitation (RIP) ae epoted. ELISA and MC showed geate sensitivity than CF and MI, while RIP showed seum tite two- to 16-fold highe. ELISA was specific as detemined using othe human mycoplasma. A simplified method based on the detemination of ELISA antibody end-point tite by a single seum dilution has been poposed. ELISA pesented seveal advantages: sensitivity, apidity, and low cost and, if adequately standadized, could become a eliable method fo the seodiagnosis of M. pneumoniae infection. The enzyme-linked immunosobent assay (ELISA), intoduced by Engvall and Pelmann fo quantitative detemination of antibodies [1], has found wide application in the diagnosis of bacteial [2,3], vial [4,5], and paasitic [6] diseases. ELISA has also been poposed fo its simplicity, sensitivity, and specificity fo the seological diagnosis of M. pneumoniae infection [7,8]. The application of ELISA fo detecting anti-m. pneumoniae antibodies, and its sensitivity as compaed with othe methods, such as complement fixation (CF), metabolic inhibition (MI), mycoplasmacidal test (MC), and adioimmunopecipitation (RIP), ae epoted hee. MATERIALS AND METHODS Mycoplasma M. pneumoniae (stain 10119) was kindly povided by the National Collection of Type Cultue, Colindale, London, England. The cultue conditions have been peviously descibed [9]. Human Sea Human sea wee obtained fom outpatients with suspected pimay atypical pneumonia fom the Pediatic Clinic of the Univesity of Padua. Seological Methods The sea wee examined with complement fixation (CF), metabolic inhibition (MI), mycoplasmacidal (MC), and adioimmunopecipitation (RIP) methods as descibed in a pevious pape [9]. 517 Addess epint equests to: F. Busolo, M.D., Institute of Micobiology, Univesity of Padua, Via A. Gabelli 63, Padova, Italy Copyight 1983 by The Yale Jounal of Biology and Medicine, Inc. All ights of epoduction in any fom eseved.

2 518 BUSOLO AND MELONI Pepaation of Antigen-Coated Well fo ELISA Linbo-type micotite plates with flat-bottomed wells (Flow Laboatoies) wee used. Peliminay titations wee made to detemine the optimal concentations of M. pneumoniae. Mycoplasmas wee eithe gown diectly in micotite wells o altenatively in flasks and afte thee washings with phosphate buffe solution (ph 7.2) 50 1l of suspension (A660=0.48) containing 25 jg of potein adsobed onto micotite wells and fixed in 10 pecent fomalin. A moe detailed method is epoted elsewhee [8]. Pepaation of Conjugated Antibodies Goat anti-human immunoglobulin G, M, and A combined (IgG-IgM-IgA), IgM and IgA antisea (Hyland, Costa Mesa, CA) wee puified by gel filtation in Sephacyl S-200 supefine (Phamacia, Uppsala, Sweden). Factions wee collected and the antibodies concentated to 5 mg/ml and then they wee coupled with alkaline phosphatase (Sigma type VII; specific activity: 1,000 U/mg of potein) by "one-step" pocedue as descibed by Engvall and Pelmann [1]. Conjugated antibodies at 4 C, unde steile conditions, wee stoed fo almost six months without any appeciable loss in enzymatic o immunological activity. We have found that fo longe stoage they wee best peseved in 1 ml aliquots at -80 C. By using a woking solution of 1:200 in PBS-Tween a hunded patients' sea wee examined with a millilite of concentated solution. ELISA ELISA was pefomed as peviously descibed [8] with some modifications. The adsobed antigen was washed with PBS containing 0.05 pecent Tween 20, which pevented non-specific antibody adsoption to the plastic. Micotite plates with 50 t1l of diffeent seum dilutions (in PBS-Tween), stating at 1:16, wee incubated fo one hou at 37 C. At the end of the incubation peiod, the plates wee washed twice with PBS-Tween. Fifty Al of conjugated antibody solution, diluted 1:200 in PBS- Tween, wee added to each well and the plates wee incubated fo one hou at 37 C. Afte thee washings, 200 itl of p-nitophenyl phosphate (1 mg/ml) solution in 0.05 M sodium cabonate buffe (ph 9.8) was added, and the plates wee maintained fo one hou at 37 C. The eaction was stopped with 50 A1 of 3N NaOH. Absobance at 405 nm was measued by a photomete instument (Titetek, Multiskan, Flow Laboatoies). Usually, ten patients' sea wee tested fo each micotite plate, along with one negative and one positive efeence seum. The seum tite was expessed by the highest dilution giving absobance values geate than 0.1. RESULTS AND DISCUSSION Figue 1 shows seum tites obtained by ELISA as compaed with CF, MI, MC, and RIP. ELISA tites wee in the ange 1:16 to 1:2,048 and 43 wee highe than those obtained with MI. Futhemoe, 7/80 (8.75 pecent) MI-negative sea wee positive when examined by ELISA, indicating a geate sensitivity and giving the possibility of detecting low antibody levels in the ealy stages of M. pneumoniae infection. Statistical analysis showed that the values obtained with ELISA coelated well with those obtained by the MI method (p < 0.01). The CF method fo detecting M. pneumoniae antibodies was of fa lowe sensitivity. Thee of 65 sea tested which had a CF tite of 1:16 had a makedly highe tite in ELISA. MC and ELISA had simila sensitivities since most of the values wee distibuted in the pox-

3 ELISA FOR M. PNEUMONIAE 519 Ml Bowa CF 65 sa i ". 256 p p - /- 64 * 6 U.,* U * - 0. P i 0~~~~~ _, 68ts -" S * u. XELISA / a B ELISA U MC...s 71 RIP. 54se O2048"' U 16384'os * P of. e. _* S 256-* * : _ 64 EIS ffi ^ 16.c ELISA antibody tite (ecipocal) FIG. 1. Compaison between the sensitivity of ELISA and othe seological methods. These compaisons "two by two" wee caied out using diffeent sea. In some instances, the same seum was assayed by moe than two methods. Each closed cicle epesents one patient's seum. imity of the baseline of the scattegam. ELISA did not each the degee of sensitivity of RIP, which showed seum tites two- to 16-fold highe (up to 1:16,000). A compaative evaluation on the sensitivities of CF, MI, MC, and RIP was also epoted in a pevious pape [9]. The specificity of ELISA fo seological diagnosis of M. pneumoniae infection was assessed using eight positive patients' sea and othe human mycoplasmas. In these expeiments we used stains fom the intenational collection such as M. fementans NCTC 10117, M. hominis PG-21, M. oale I NIH (CH 19299), M. oale 2 NIH (NH 20247), M. salivaium PG-20, and U. uealyticum T-960. Of the eight sea taken at andom fom the seoteca, fou had low tite (1:64) and fou had highe tites (1:512). Only one out of 1:64 tite seum coss-eacted with M. fementans. This may be attibuted to the fact that the most impotant antigen of M. pneumoniae is a glycolipid and the majo complement-fixing antigen of M. fementans is also found in the lipid factions and contains glycolipids and phospholipids [10]. This coss-eactivity did not limit the diagnostic value of the ELISA, since M. fementans is not pathogenic fo humans, and we have not found a significant ise in the antibody tite. In ou study, we have demonstated that a foufold ise in tite to M. pneumoniae occued in only 50 pecent of patients. Pobably this is due to the insidious develop-

4 520 A4os BUSOLO AND MELONI FIG. 2. Coelation between A 405 value at 1:32 seum dilution and ELISA end-point antibody tite. The 0.3 egession line and the standad eos wee I I 0.7- X/ 1y=0.092 x calculated on 110 ELISA-positive sea, using anti-igg-igm- II IgA conjugated an- ELISA end-point antibody tite (ecipocal) tibodies. ment of the disease and the acute-phase sea aleady show high antibody levels when they ae tested with ELISA. In ode to educe the cost of the assay and to examine moe sea in each micotite plate, we tied to find a elationship between the absobance value at the vaious sea dilutions and the seum end-point tite. In this way, an absobance value detemined on a single seum dilution (1:32, in ou case) might be intepeted quantitatively to give the anti-m. pneumoniae end-point tite of that seum. One hunded and ten ELISA-positive sea wee tested, using anti IgG-IgM-IgA conjugated antibodies. The egession line with the coesponding standad eos is shown in Fig. 2. To test the validity of this simplified method, an additional 48 sea wee examined in ELISA at a single seum dilution. Only 10 pecent of these sea showed a discepancy in end-point tite detemination. Theefoe we suggest that the simplified method might be the test of choice fo epidemiological studies whee a positivity o negativity can be immediately ascetained. A question aises on the kind of antibodies measued by the diffeent seological methods. Late seum samples contain a pedominance of IgG antibodies which ae moe effective in the MI [11], while in CF only IgM and some IgG subclasses ae involved. On the othe hand, the geate sensitivity of RIP and ELISA might depend on the ability of these methods to measue antibodies belonging to any molecula class and eacting not only with glycolipid antigen, but also with othe antigens of the plasma membane. In addition, ELISA can be class-specific. Ninety pecent of patients with M. pneumoniae infection had IgM while a lowe pecentage had IgA. We have found that IgM antibodies may pesist long afte one yea following the onset of the disease. This finding was in ageement with the hypothesis that silent infections occu quite commonly [12] and that M. pneumoniae disease might esult fom einfection of patients peviously sensitized to the oganism [13]. In this field, ELISA pesents the advantages of apidity, low cost, and avoidance of the poblems which might be caused in the MI and MC by the pesence of antibiotic in the seum. Futhemoe, ELISA, if adequately standadized by its adaptability to automation, could become a eliable method fo the seodiagnosis of M. pneumoniae infection.

5 ELISA FOR M. PNEUMONIAE 521 REFERENCES 1. Engvall E, Pelmann P: Enzyme-linked immunosobent assay (ELISA). Il1. Quantitation of specific antibodies by enzyme labeled anti-immunoglobulin in antigen-coated tubes. J. linmunol 109: , Russell H, Facklam RR, Edwads IR: Enzyme-linked immunosobent assay fo steptococcal M potein antibodies. J Clin Micobiol 3: , Lewis VJ, Thacke WL, Mitchell SH: Enzyme-linked immunosobent assay fo chlamydial antibodies. J Clin Micobiol 6: , Foghani B, Schmidt NJ: Antigen equiements, sensitivity and specificity of enzyme immunoassays fo measles and ubella vial antibodies. J Clin Micobiol 9: , Volle A, Bidwell DE: Enzyme immunoassays fo antibodies in measles, cytomegalovius infections and afte ubella vaccination. B J Exp Pathol 57: , Volle A, Batlett A, Bidwell DE: Enzyme immunoassays fo paasitic diseases. Tans R Soc Top Med Hyg 70:98-106, Raisanen SM, Suni J, Leinikki P: Seological diagnosis of Mycoplasma pneumoniae infection by enzyme immunoassay. J Clin Pathol 33: , Busolo F, Tonin E, Conventi L: Enzyme-linked immunosobent assay fo detection of Mycoplasma pneumoniae antibodies. J Clin Micobiol 12:69-73, Busolo F, Zozenon M, Piacentini 1: Compaative evaluation of some methods fo detection of Mycoplasma pneumoniae antibodies. Boll Ist Sieote Milanese 58: , Kenny GE: Antigens of the Mycoplasmatales and Chlamydiae. The antigens. Edited by M Sela. New Yok, Academic Pess, 1975, pp Clyde WA J: Mycoplasma pneumoniae infections of man. In Human and Animal Mycoplasmas. Vol II. Edited by JG Tully, RF Whitcomb. New Yok, Academic Pess, 1979, pp Fenald GW, Collie AM, Clyde WA J: Respiatoy tact infection due to Mycoplasma pneumoniae in infants and childen. Pediatics 55: , Bunne H, Hoswood RL, Chanock RM: Moe sensitive methods fo detection of antibody to Mycoplasma pneumoniae. J Infec Dis 127:52-55, 1973