Mycoplasma pneumoniae protein involved in the antibody response in human infection

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1 J Clin Pthol 1984;37: Myoplsm pneumonie protein involved in the ntibody response in humn infetion SUVI M RAISANEN,* JUKKA I SUNI,* ANTTI VAHERIt From the *Muniipl Bteriologil Lbortory, Auror Hospitl, Helsinki, nd the tdeprtment of Virology, University ofhelsinki, Helsinki, Finlnd SUMMARY Antigen from purified Myoplsm pneumonie orgnisms treted with Tween-80- ether ws used in solid phse enzyme immunossy nd ompred with the onventionl lipid ontining omplement fixtion ntigen for mesuring ntibodies in ser from ptients with septi or bteril meningitis or with pprent M pneumonie infetion. In immunoblotting of the enzyme immunossy ntigen, enzyme immunossy positive ser deteted polypeptide t Mr = } , while enzyme immunossy negtive ser whether positive or negtive in the omplement fixtion test did not. These results indite tht the enzyme immunossy ntigen ontining the high moleulr weight polypeptide n be used to mesure M pneumonie ntibodies more speifilly thn the onventionl lipid ontining omplement fixtion ntigen. Considerble inreses in the titres of omplement fixing ntibodies retive with the glyolipid ontining frtion of Myoplsm pneumonie hve been reported in mny kinds of infetions nd illnesses with or without other evidene of M pneumonie infetion.'-3 Exmples of suh illnesses re the infetions nd disorders of the entrl nervous system.4-" Some of these ntibody responses my be due to ross reting ntibodies stimulted by vnous onditions.'2 Suh ntibodies often develop during ute pnretitis.'3 14 On the other hnd, ntibodies ginst brin, lung, nd liver tissue develop during M pneumonie infetions's nd injetions of myoplsms other thn M pneumonie n use entrl nervous system symptoms in nimls.'"-8 Our previous studies with enzyme immunossy nd Tween-80-ether treted M pneumonie ntigen,'4 insted of the onventionl lipid ontining omplement fixtion ntigen, supported, in onjuntion with linil evidene, the view tht some of the rises in omplement fixtion ntibody titres my be due to flse positive retions ginst the glyolipid moieties of the ntigen. It seemed possible tht more purified M pneumonie protein ntigen ould eliminte these retions nd mke the serologil dignosis of M pneumonie infetion more relible. To lrify the possible role of M pneumonie in entrl nervous system infetions, we exmined Aepted for publition 22 June 1984 pired ser from ptients with meningitis nd meningoenephlitis with fourfold or greter rise in omplement fixtion ntibody titre to the M pneumonie lipid ntigen. Using the enzyme immunossy with protein ontining ntigen for M pneumonie,'4 ser from ptients with bteril meningitis did not show serologil response, wheres ser from ptients with M pneumonie respirtory infetion showed signifint inreses in ntibody titre. The ltter ser reted in immunoblotting with defined high moleulr weight polypeptide seprted by sodium dodeyl sulphte polyrylmide gel eletrophoresis (SDS-PAGE) nd ws trnsferble to nitroellulose pper. Mteril nd methods SERUM SPECIMENS These inluded pired ser from 11 ptients with septi meningitis nd meningoenephlitis nd four ptients with bteril meningitis, ll with fourfold or greter inreses in ntibody titre in the M pneumonie omplement fixtion test. Pired ser from five ptients with pneumoni showing fourfold or greter rises in the onentrtion of ntibody ginst M pneumonie in the omplement fixtion test, nd thus with probble M pneumonie infetion, were used s positive ontrols. The negtive ontrol group onsisted of pired ser from 12 ptients with pneumoni due to uses other thn M 1129

2 1130 pneumonie nd single ser from four one yer old helthy hildren, ll with low ntibody titres in the M pneumonie omplement fixtion test. ANTIGENS Myoplsm pneumonie enzyme immunossy ntigen ws prepred nd produed by Orion Dignosti (Espoo, Finlnd) ording to our speifition.'4 Stndrd M pneumonie omplement fixtion ntigen (Orion Dignosti), whih onsisted of lipid omponents extrted with hloroform nd methnol from ultivted purified M pneumonie orgnisms,'9 ws used in the omplement fixtion test. ENZYME IMMUNOASSAY The ntigen ws diluted in sodium rbontesodium birbonte buffer, ph 9-6. Serum nd onjugte dilutions were mde in phosphte buffered sline (PBS) supplemented with 0-2% Tween 20 nd 10% sheep or newborn lf serum. Hevy hin speifi, lkline phosphtse onjugted ntibodies ginst humn IgG nd IgM were used s onjugtes (Orion Dignosti). The solid phse enzyme immunossy proedure ws s desribed elsewhere.'4 All ser were diluted 1/100 for the ssy. SDS PAGE AND IMMUNOBLOTTING For immunoblotting both enzyme immunossy nd omplement fixtion ntigens of M pneumonie were used. The proteins were seprted by SDS 10% polyrylmide slb gel eletrophoresis prepred ording to Lemmli.20 After seprtion, the proteins were trnsferred eletrophoretilly to nitroellulose sheets nd immunoblotted ording to the proedure by Towbin et!2' modified s desribed elsewhere.22 The trnsfer ws heked by the reversible heprin toluidin blue stining proedure. For immunologil detetion of the trnsferred proteins, the sheets were sequentilly exposed to the ptient ser, diluted 1/50 in PBS supplemented with 0*2% Tween 20 nd 2% bovine serum lbumin, then to '251-lbelled stphylool protein A, nd finlly to utordiogrphy. Results CORRELATION BETWEEN ENZYME IMMUNOASSAY AND COMPLEMENT FIXATION TITRES The results of the enzyme immunossy were first obtined s bsorbne vlues (A405). A modertely positive onvlesent phse serum of the positive ontrol group ws hosen s the positive referene serum. The men of the negtive ontrol group results ws lulted in both the IgG nd IgM test._ 0 E 4Ė N w 4 o~~~~~44 Risnen, Suni, Vheri Fig. 1 IgM ntibody response ginst the Tween-ether treted ntigen in the enzyme immunossy test in vrious groups ofptients with dignosti rises or high titres of omplement fixtion ntibodies ginst M pneumonie. Helthy ontrols inlude ptierns with pneumoni with low omplement fition ntibody titres ginst M pneumonie. Vlues for the ute (1) nd onvlesent phse (2) re shown. nd used s the negtive referene. Using these referene vlues the results were expressed s enzyme immunossy units (EIU). x 100 EIU M2-Ml where x = result of the test, S = bsorbne vlue of the smple, Ml = men bsorbne of the negtive ontrol group,4,nd M2 = bsorbne of the positive referene serum. None of the ser from the four ptients with bteril meningitis (three used by Hemophilus influenze nd one by Neisserimeningitidis), showed pronouned inreses in eitb*t the IgM or the IG ntibody onentrtions in enzyme immunossy despite signifint inreses in omplement fixtion ntibody titres (Figs. 1 nd 2). Most ptients with septi meningitis nd enephlitis lso hd low onentrtions of ntibodies ginst the enzyme immunossy ntigen, gin despite the fourfold or greter rise in the omplement fixtion test. Two ptients with enephlitis like neurologil, symp-

3 Antigeni myoplsm protein vb "I E N w Fig. 2 IgG ntibody response ginst the enzyme immunossy ntigen in the ptient groups offig. 1. toms hd signifint onentrtions of ntibodies in enzyme immunossy s well. Both ptients hd respirtory infetions ssoited with neurologil symptoms: one hed&n ute respirtory infetion, negtive old gglutinin titres, nd lower-igg onentrtion ginst the enzyme immunossy ntigen in the onvlesent phse serum thn in the ute phse smple; the other hd rise in ntibody onentrtion in the enzyme immunossy nd n inrese in the old gglutinin titres nd developed pneumoni fter the entrl nervous system symptoms (Figs. 1 nd 2). Ser from ll the ptients with typil pneumoni nd positive omplement fixtion titres were lerly positive in the M pneumonie enzyme immunossy test s well (Figs. 1 nd 2). These ptients lso hd rised old gglutinin titres in their onvlesent phse ser. Ser from helthy one yer old hildren nd from ptients with pneumoni with no inrese in omplement fixtion ntibody titre did not respond in the enzyme immunossy test to the Tween 80- ether treted M pneumonie ntigen. IDENTIFICATION OF POLYPEPTIDE ANTIGEN SDS-PAGE showed over 10 protein bnds in the onentrted Tween 80-ether treted M 1131 pneumonie ntigen. Eletrophoresis of the omplement fixtion ntigen lso gve severl polypeptide bnds. When ptient ser, previously tested with both the enzyme immunossy nd the omplement fixtion test, were used in immunoblotting to identify the ntigeni polypeptides seprted by SDS-PAGE, one zone t Mr = ws repetedly deteted by enzyme immunossy positive ser (Fig. 3). No suh bnd ould be found in the omplement fixtion ntigen with ser positive both in the enzyme immunossy nd the omplement fixtion test. Also, enzyme immunossy negtive ser, whether negtive or positive in the omplement fixtion test, filed to detet this bnd in either type of ntigen. The ute phse ptient ser whih hd low response in the M pneumonie enzyme immunossy test often filed to show the Mr = ntigen or gve fint retion; onvlesent phse ser were ble to detet the zone lerly. Ailllw, z 0_: o.h -30 Fig. 3 Immunoblotting of M pneumonie polypeptides seprted by SDS-PAGE nd trnsferred to nitroellulose sheets, with known enzyme immunossy positive onvlesent phse serum of ptient with pneumoni (I nd 2) nd serum of helthy one yer old hild with no enzyme immunossy or omplement fixtion ntibodies ginst M pneumonie (3 nd 4). Both ser were diluted 1150 nd 1/100. Arrow indites the Mr = position.

4 1132 Disussion The enzyme immunossy test using protein ontining ntigen treted with Tween 80-ether seems to be more speifi for M pneumonie infetion thn the onventionl omplement fixtion test, whih is bsed on lipid ontining ntigen. In enzyme immunossy, the ser from ptients with bteril meningitis nd most ser from ptients with septi meningoenephlitis showed no signifint ntibody titres or dignosti rises in ntibody onentrtion. The smll inrese seen in some of the onvlesent phse ser my hve been due to minor ross retive ommon bteril omponents present in the enzyme immunossy ntigen. In ddition to its speifiity, ompred with the omplement fixtion test, enzyme immunossy is simple proedure nd it is possible to mesure both IgM nd IgG ntibody onentrtions seprtely, whih gives some informtion of the phse of the immune response. In some ses of septi meningitis nd enephlitis, M pneumonie my be the tul use of infetion, but definite onlusions on the ssoition between M pneumonie nd the entrl nervous system symptoms nnot yet be mde. The two ptients with enephlitis s the linil dignosis who hd high ntibody onentrtions in both the omplement fixtion nd enzyme immunossy test lso hd respirtory symptoms during the ourse of their illnesses, nd thus my hve experiened two diseses simultneously: respirtory M pneumonie infetion nd widespred entrl nervous system infetion. Moreover, one of these two ptients hd lowered enzyme immunossy IgG ntibody vlue in the onvlesent phse serum. Isoltion of M pneumonie from the respirtory trt would hve been most informtive, but ould not be done in this retrospetive study. Immunoblotting hs previously proved useful for deteting immunologilly tive omponents from otherwise unpurified mteril.2'23 In the present study we used Tween 80-ether treted frtion of M pneumonie orgnisms s ntigen in the enzyme immunossy test. This ntigen preprtion ws used in immunoblotting to define ntigeni polypeptides. Ser positive in the enzyme immunossy nd omplement fixtion test deteted one mjor polypeptide t Mr = , orresponding to the myoplsm surfe protein P1 onerned in the tthment of the orgnisms to respirtory epithelium desribed by Hu et l.2425 We did not detet this polypeptide when either omplement fixtion positive/enzyme immunossy negtive or omplement fixtion negtive/enzyme immunossy negtive ser were used. These results lso gree well with those mde with whole M pneumonie Risnen, Suni, Vheri orgnisms nd monolonl ntibodies.262" In these studies the min ntigeni entity of the M pneumonie orgnism is high moleulr weight protein. The enzyme immunossy ntigen ontining this ntigeni polypeptide ppers to be relible in the dignosis of M pneumonie infetion. We thnk Dr Antti Ponk for some of the ptient ser, Viivi Perttunen nd Pirjo Srjkivi for tehnil ssistne, nd Anne Brisk for seretril ssistne. This work ws supported by grnts from the Finnish Medil Soiety Duodeim nd the Finnish Medil Reserh Counil. Referenes Brunner H. Presott B, Greenberg H, Jmes WD, Horswood RL, Chnok RM. Unexpetedly high frequeny of ntibody to Myoplsm pneumonie in humn ser s mesured by sensitive tehniques. J Infet Dis 1977; 135: Ponk A. The ourrene nd linil piture of serologilly verified Myoplssm pneumonie infetions with emphsis on entrl nervous system, rdi nd joint mnifesttions. Ann Clin Res 1979; 1 I(suppl 24): Snds MJ Jr, Stz JE, Turner WE Jr, Soloff LA. Perirditis nd perimyorditis ssoited with tive Myoplsm pneumonie infetion. Ann Intern Med 1977;86: Sterner G, Biberfeld G. Centrl nervous system omplitions of Myoplsm pneumonie infetion. Snd J Infet Dis 1969; 1: Goldsmith B, Mennonn J, Fortunto J, Dowling P, Cook S. Myoplsm ntibody in Guillin-Brre syndrome nd other neurologil disorders. Ann Neurol 1979;7: Steele JC, Thnsophon S, Gldstone RM, Fleming PC. Myoplsm pneumonie s determinnt of the Guillin-Brre syndrome. Lnet 1969;ii: Clyde WA Jr. Neurologil syndromes nd myoplsm infetions. Arh Neurol 1980;:37:65-6. Wrren P, Fishbein C. Msoli N, Rudolph J. Hodder DH. Poliomyelitis-like syndrome used by Myoplsm pneumonie. J Peditr 1978; 93: Burmn LG, Lofgren S. Reent pneumoni nd enephlitis due to Myoplsm pneumonie. Snd J Infet Dis 1979; 11: Lind K, Hoffmn H, Lrson SO, Jessen 0. Myoplsm pneumonie infetion ssoited with ffetion of the entrl nervous system. At Med Snd 1979;205: Koskiniemi ML, Vheri A. Aute enephlitis of virl origin. Snd J Infet Dis 1982; 3: Leinikki PO, Pntzr P, Tykk H. Antibody response in ptients with ute pnretitis to Vtyoplsm pneumonie. Snd J Gstroenterol 1973; 8: Leinikki PO, Pntzr P, Tykk H. Immunoglobulin M ntibody response ginst Myoplsm pneumonie lipid ntigen in ptients with ute pnretitis. J Clin Mirobiol 1978;8: Risnen SM, Suni JI. Leinikki PO. Serologil dignosis of Myoplsm pneumonie infetion by enzyme immunossy. J Clin Pthol 1980;33: Biberfeld G. Antibodies to brin nd other tissue in ses of Myoplsm pneumonie infetion. Clin Exp Immunol 1971;8: I Mnuelids EE, Lewis T. Olusion of brin pillries by endothelil swelling in myoplsm infetions. Pro Ntl Ad Si 1873;70:706-9.

5 Antigeni myoplsm protein 7 Thoms L, Aleu F, Bitensky MW, Dvidson M, Gesner B. Studies of PPLO infetion. II. The neurotoxin of Myoplsm neurolytium. J Exp Med 1966; 124: "Thoms L, Bitensky MW. Studies of PPLO infetion. IV. The neurotoxiity of intt Myoplsm nd their prodution of toxin in vivo nd in vitro. J Exp Med 1966; 124: Kenny GE, Gryston JT. Eton pleuropneumoni-like orgnism (Myoplsm pneumonie) omplement-fixing ntigen. J Immunol 1965; 95: Lemmli UK. Clevge of struturl proteins during the ssembly of the hed of bteriophge T4. Nture 1970;227: Towbin H, Stehelin T, Gordon J. Eletrophoreti trnsfer of proteins from polyrylmide gels to nitroellulose sheets proedure nd some pplitions. Pro Ntl Ad Si 1979; 76: Vrtio T, Zrdi L, Blz E, Towbin H, Vheri A. Monolonl ntibodies in nlysis of thepsin G-digested proteolyti frgments of humn plsm fibronetin. J Immunol Methods 1982;55: Burnette WN, 'Western blotting: eletrophoreti trnsfer of proteins from sodium dodeyl sulfte-polyrylmide gels to 1133 unmodified nitroellulose nd rdiogrphis detetion. Anl Biohem 1981;112: Hu PC, Collier AM, Bsemn JB. Surfe prsitism by Myoplsm pneumonie of respirtory epithelium. J Exp Med 1977; 145: Hu PC, Cole RM, Hyng YS, et l. Myoplsm pneurnonie infetion: Role of surfe protein in the tthment orgnelle. Siene 1982;216: b Feldner J, Gobel U, Bredt E. Myoplsm pneumonie dhesion lolized to tip struture by monolonl ntibody. Nture 1982; 298: Hu PC, Hung C-H, Collier AM, Clyde WA. Demonstrtion of ntibodies to Myoplsm pneumonie tthment protein in humn ser nd respirtory seretions. Infet Immun 1 983;41: Requests for reprints to: Dr Suvi M Risnen, Muniipl Bteriologil Lbortory, Auror Hospitl, Nordenskjoldinktu 20, SF Helsinki, Finlnd. J Clin Pthol: first published s /jp on 1 Otober Downloded from on 4 April 2019 by guest. Proteted by opyright.