Preparative electroelution of specific protein antigens from MicoplasmaPneumoniae: use in an enazyme-linked immunosorbent assay (Ellisa)

© ELSEVIER Paris 1988

Ann. Inst. Pasteur/MicrobioL 1988, 139, 589-603

PREPARATIVE ELECTROELUTION OF SPECIFIC PROTEIN ANTIGENS FROM MFCOPLASMA PNEUMONIAE: USE IN AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) G. Papierok (~), C. Derives (2), A. Daunizeau (l), P. Wattr~ (3) and J.-C. Derieux (2) O) Service de Microbiologie, Insdtut Pasteur, 59019 Lille Cedex (France), (2) Labora~'oire de Microbiolog:.e, Universit,4 des Sciences et Techniques, 59655 Villene.ive d'Ascq (France), and (3) Service de BacMriologie-Virologic B du CHU, 59019 Lille Cedex

SUMMARY A rapid, simple and preparative method is described for the recovery of the seven k,,ighest molecular weight proteins (HMWP) from Mycoplasma pneumoniae membrane. The yield of proteins obtained was approximately 90 %. The method involved the separation of M. pneumoniae proteins by sodium dodecyl sulphate polyacrylamide gel electropboresis (SDS-PAGE), followed by electroelution of HMWP. These eluted antigens were u~e~ '~n an ELISA to measure IgG antibodies in sera from 9 blood donors and 9 patients with M. pneumoniae infection. The specificity of M, pneum~niae H M W P was examined by competition ELIS~ and immunoblotting with different mycoplasma species encountered in the respiratory tract. KEY-WORDS: Protein, Mycoptasma pneurnoniae, Membrane; SDS-PAGE, Electroelution, ELISA, Immunobiotting. INTRODUCTION Mycoplasma pneumon&e, a human pathogen responsible for many respiratory tract infections [3, 7] is a slow glowing organism which requires appropriate culture medium. Therefore, biological diagnosis of the infections

Submitted November 12~ 1987, accepte5 September 27, 1988.

G. PAPIEROK A N D COLL.

590

is essentially based on detection of specific antibodies in patient's serum by various methods [4, 5, 26, 27]. The most commonly used is the complement fixation test (CFT), which employs a hapten antigenically related to glycolipids of different origins [1, 17, 20, 22]. More specific and sensitive techniques such as the enzyme-linked immunosorbent assay (ELISA) [6, 8], which detects antibodies against whole organisms or protein antigens, have expanded recently but have not yet replaced C~T for several reasons. The crude protein antigens obtained by Tween ether treatment of whole M. pneumoniae [24] gave better results in comparison with CFT, but presented minor cross-reactivity in common with bacterial components [25]. The recent dot ELISA using the 168-Kd protein (designated P 0 of M. pneumoniae as antigen [13] also gave promising results, but the extraction of antigen was time-consuming and required special costly equipment; furthermore, the preparation and use of dot ELISA is more complicated than indirect ELISA. Therefore, we decided to extract protein antigens through simple electroelution using the same electrophoretic systems used in the separation step. Various reasons led to this choice in the recovery of the 7 highest molecular weight proteins (HMWP) of M. pneumoniae membrane: the facility of their localization at the top of the separation gel; the fact that several have been demonstrated to be major immunogens [21] ; and the fact that ~hey arc implicated in pathogenicity due to their ability to attach to respiratory epithelial cells [I1, 18]. In th e present study, we were able to isolate milligram quantities of protein antigens of M. pneumoniae membrane with a yield of apr~roximately 90 %. A simple, rapid immunoenzymatic assay using the HMW~ as antigens was compared to the CFT in a serolegical study of 18 human sera. ~y competitive ELISA and immunoblotting, we also compared the H M W P to aw'2'-~ of different mycoplasma species encountered in the respiratery tract.

MATERIALS AND METHODS 1) Mycoplasm~ strains and culture conditions.

All strains of mycoplasmas used in the current investigation were a gift from Mine C. Bonissol (Laboratoire des Mycopiasmes, Institut Pasteur, Paris).

BSA CFT DTT ELISA HMWP MSE

= = = = -:

b odice serum a l b u m i n . c o m p l e m e n t fi:~ation test. dithiothreitol. enzyme-linked immunosorbent assay. high m o l e c u l a r weight protein. Measurin g Scientific E q u i p m e n t .

MW PAS PBS PMSF SDS-PAGE

= m o l e c u l a r weight. = pe ri odi c acid Schiff. = p h o s p h a t e - b u f f e r e d saline, = phenylmethylsulphony!fluodde. = s o d i u m dode e yl sulph~ae-polya c r y l a m i d e gel eleetrophoresis.

MYCOPLASMA

PNEUMONIAE

PROTEIN SPECIFICITY

591

a) M. pneumoniae strain FH was grown and harvestec2 as described previously [24]. The mycoplasma cells were iva~hed 5 times with phosphate-buffered saline pH 7.2 (PBS: 10 mM sodium phosphate 130 mM NaCi). They were then suspended in 20 ml of PBS and crushed using an MSE (Measuring Scientific Equipment, UK) ultrasonic di~;integrator (150 W, 14 ~m wavelength) for 1 min in a cold bath. The suspension was fractionated according to the differential centrifugation method of Hollingdale and Lemcke [9] with slight modification. After centrifugation at 5,000 g for 5 rain, the resuspended pellet was treated again as above. The supernatants were pooled and centrifuged at 40,000 g for 45 rain. The final pellet was washed 4 times with PBS and designated as crude membrane. b) M. orale (strain ATCC 23714), M'. salivarium (strain ATCC 23064) and M. hominis (strain ATCC 23114) were grown as described previously [2]. The crude membrane fractions were prepared according to the method described above. 2) Recovery of M. pneumoniae H M W P by electroelufion.

M. pneumoniae crude membrane proteins were fractionated for 2 h 30 rain by sodium dodecyl sulphate polyacrylamide gel ele~.trophoresis (SDS-PAGE) according to Laemmli [19]. About 70 mg of protein were layered on 11 tubes (inside diameter of 16 mm) of a GT tube gel unit (Hoofer Scientific Instruments, San Francisco) with 13 % separating gel and 4 % stacking gel. The H M W P were localizeO by electrophoresis of 60 ~tg of prestained protein molecular weight standards (Bethesda Research Laboratories, USA) in the 12th tube. After electrophoresis, the gel regions localized above the ~-galactosidase (116-Kd) were cut and fragmented over 11 elution tubes (fig. 1). Fhe region of gel containing the prestained ~3-galactosidase was cut and fragmented over the 12th elution tube. Electrophoresis was performed at 200 mA constant for 6 h with a Laemmli electrode buffer mixed with 1 mM dithiothreitol (DTT) and 0.1 mM phenylmethylsulphonylfiuoride (PMSF). Progress was easily monitored by coelectroelution of the prestained ~3-galactosidase. The buffer containing eluted proteins in the 11 dialysis bags was removed and stored at - 4 0 ° C . The recovered M. pneurnoniae H M W P were then identified by comparative SDS-PAGE with crude membrane proteins using a 0.75-mm slab gel consisting of a 4 0i0 stacking and an 8 % separating gel. Molecular masses of protein patterns were determined with molecular weight markers: myosin (205Kd), i?-galactosidase ( l l 6 K d ) , phosphorylase b (97.4 Kd), bovine albumin (66 Kd), egg albumin (45 Kd) and carbonic anhydrase (29 Kd) (Sigma Chemical Company, Saint Louis). Furthermore, we researched the eventual presence of glycoproteins by the periodic acid Schiff (PAS) staining procedure of Zaccharius e~t al. [30]° 3) CFT m~d EL!SA test. a) A micromethod described earlier by Sever [26] was used for the CFT. The complement fixation antigen was a glycolipid (Behring Institute, West GermanyL b) Flat-bottomed microtitration plates (Serocluster EIA plate, Costar, Cambridge, MA) were coated with 100 gl of duted H M W P at room temperature overnight ; the coating buffer consisted of 0. i M carbonate-bicarbonate buffer (pH 9°6). Serum and conjugate dilutions were made in PBS supplemented with 0.05 % Tween 20 and 1 % BSA. Rabbit anti-human IgG seram conjugated to alkaline phosphatase was used at 1/80 (lot n ° 410.941 B, Behring Institute, West Germany). A!l serum samples were tested at working dilution and distributed in duplicate in the weils. The enzyme immunoassay procedure was described elsewhere [81. Absorbance values were measured at 405 nm by an < (Diagnostics Pasteur, Marnes-ia-Coquette, France). Sera which gave an absorbance value equivalent to 0.30 (twice the background level) or greater were regarded as positive.

G. P A P I E R O K A N D C O L L .

592

16 m m

Laemmli buffer with 1 mM DTT and O.I~M PMSF

!

" Fragmen~e~; g e l s l i c e s i n Laemmli b u f f e r w i t h ImMDTT, O. ImMPMSF and I0 % gIycerol

I0 mm

20 mm

I 1

.

.

SLacking gel

~ R u b b e r

band

R e c o v e r e d HMWP i n b u f f e r

Dialysis

membrane

FIG. 1. --Scheme of an elution tube for the recovery of M. pneumoniae HMWP from gel slices. The elution tube was constricted to an inner diameter of about 10 mm in order to avoid sliding gel during electroelution. The acrylamide concentration of the stacking gel was 4 % in 125 mM Tris HCl, pH 6.8, 0.1% SDS and 1 mM DTT. A dialysis tubing with an inside diameter of 16 mm (Union Carbide Film), initially boiled iv.,0. 1% SDS for 30 rain and washed thoroughly with distilled water, was fixed at the end of each elutica tube witil a rubber band.

4) Immunob|otting method and competitive ELISA. a) In orde~ to economize materials, i.e. eluted H M W P , series o f immunoblots were performed with proteins from crude membrane fractions. Samples (6 mg) of mycoplasma proteins were separated by SDS-PAGE using a 1.5-maJ slab gel consisting o f a 4 % stacking gel and an 8 % separating gel. After electrophoresis, the proteins were e!ectrophoretically transferred to nitrocellulose sheets (0.22-i~m pore size; Schleicher and Schiill Co, USA) by the method of Towbin et al. [29]. The immunoblot assay was performed as previously described [12]. The molecular mass markers were separated and transferred in the same way. b) In the competitive ELISA, we used soluble antigens extracted from mycoplasmas. The crude membrane fractions were crushed by the ultrasonic

MYCOPLASMA PNEUMONIAE PROTEIN SPECIFICITY

2

593

3 205 Kd

P2 ........

i16 97.4 66

45

29

FIG. 2. - - Analysis of the eluted HMWP of M. pneumoniae membrane by comparative SDS-PAGE. l) Eiuted HMWP ; 2) crude membrane of M. pneumoniae; 3) molecular weight markers (see text). The gel was stained with 0.25 % Coomassie brilliant blue R.

disintegrator for 5 rain in a cold bath. The suspensions were centrifuged at 100,000 g for 30 min and the supernatants were used as antigens. These soluble antigens and ~he crude membrane fractions showed similar patterns of proteins after SDS-PAGE analysis. To ELISA plates coated with M. Dneumoniqe H M W P , positive and control serum samples were added with various amounts of antigens of M. salivarium, M. orale, M. hominis and M. pneumoniae. Tile reaction was then performed like the ELISA test described above.

5) Serum samples, The serum samples that were used in the present s~udy were comprised of two groups. Group A consisted of 9 adult blood donors (Centre R6gional de Transfusion

594

G. P A P I E R O K

A

AND

COLL.

B

....

A

B

.~...P 2

m

a

b

FIG. 3. - - Comparison of eluted H M W P and of M. p n e u m o n i a e proteins by immunoblot-

ting with human sera. Antigens: lane A : proteins f r o m M. pneumoniae <; lane B : eluted H M W P . H u m a n sera: a: positive s e r u m ; b" control serum. A n t i s e r a were tested at 1/400 dilution. T h e goat a n t i - h u m a n IgG serum c o n j u g a t e d to peroxidase was used at 1/800 (lot n ° B12415/8032, Biosys, France).

M Y C O P L A S M A P N E U M O N I A E PROTEIN SPECIFICITY

595

Sanguine de Lille) between 20 and 35 years of age without clinical symptoms of respiratory disease. Group B included 9 sera from patients who had M. pneurnoniae disease confirmed by clinical symptoms and significant CFT titre (values of ~> 1/80).

RESULTS 1) Preparation of HMWP from 114. pneumoniae through electroelution. The soluble extracts from M. pneumoniae crude membrane were loaded on a 13 °70 polyacrylamide gel. Following the migration of proteins, only the H M W P were electroeluted. The yield showed purity of about 90 %. Approximately 10 mg of M. pneumoniae H M W P in 4 ml of electroelution buffer were obtained in one step by electroelution from 70 mg of crude me~vbrane proteins. The crude m e m b r a n e fraction and the eluted H M W P exhibited the same proteins with higher molecular weights (fig. 2). After PAS staining of gel acrylamide, no glycoproteins was detected. The antigenicity of these eluted H M W P was confirmed by the recognition of antigens with corresponding antibodies in the immunoblot (fig. 3). The h u m a n serum antibody response to M. pneumoniae proteins was particularly significant with proteins P1 and P2.

2) H M W P - E L I S A . a) Determination o f working conditions for HMWP-ELISA. To determine the most suitable working dilution for H M W P antigens, the absorbance values of a positive h u m a n reference serum (CFT titre equivalent to 1/640) and a control serum (CFT titre equivalent to 1/10) diluted at 1/100 were measured (fig. 4a). The maximal value was reached with 1.5 ~g of I-IMWP antigens per well and this amount was chosen in subsequent tests. Sera used previously were tested in 2-fold dilution series, and the working dilution of serum was determined. Positive serum gave a falling curve and was markedly different from control serum (fig. 4b); the absorbance value remained significant when the dilution was 1/800. Therefore, all serum samples were diluted to 1/100 in duplicate. Furthermore, all experiments were conducted several times. b) Comparison o f HMWP-ELISA and CFT. There was no difference in the results obtained by CFT and ELISA using several patient sera (fig. 5). H u m a n sera with a CFT titre equivalent or superior to 1/80 showed an absorbance value superior to 0.50. One of the 9 sera from

G. P A P I E R O K A N D COLL.

596

absorbance

at 405 nm 1 patient serum

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0,5

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1.5

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l

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Determination of working conditions for HMWP-EL1SA.

Top : determination of optimal H M W P antigen concentration for indirect ELISA. Sera were diluted at 1/100; Bottom: determination of the dilution of serum.

MYCOPLASMA

absorbance a t 4 0 5 nm

PROTEIN

PNEUMONIAE

SPECIFICITY

597

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B

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0.5

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FIG.

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A

gro, p B

Total IgG antibody response against M. pneumoniae H MWP antigens in ,human sera.

G r o u p A : control group composed o f 9 sera from adult blood donors ; group B : group composed o f 9 sera from patients with M. pneumo~ffae disease confirmed by clinical symptoms and significant C F T titre o f >~ 1/80.

598

G° P A P i E R O K A N D COLL.

group A gave an absorbance value superior to 0.30; this result was confirmed by immunoblot.

3) Specificity of HMWP antigens. The specificity of HMWP antigens was determined by immunoblots and competitive ELISA. Proteins which had elicited a prominent immune response in patients with M. pneu;no;:iae disease (group B) were of high molecular masses, particularly proteins P1 and P2 (fig, 6 ) Or13 protein P: had corresponding antibodies in all sera. Furthermore, the intensity of the protein PI band on immunoblots correlated with the higher absorbance values in ELISA. Patients with M. pneumoniae disease showed a restrictive response to M. oraie, M. saiivatium arid ?v[. /tO?~iiii,~S, and the proteins of these three mycoplasma species recognized by the human sera were different from those of M. pneumoniae (fig. 6)° Competitive ELISA confirmed the res.tlts of immunoblots. Soluble antigens prepared from M. orale, M. saiivarium and M. hominis and added in three different amounts (1, 2 and 3 [zg per vcell) to human sera from group B did not affect the reaction between human serum igG antibodies and HMWP antigens coated on microtitrafion plates. I~owever, if 2 [zg of M. pneumoniae soluble antigens were added per well to human serum, the reaction between IgG antibodies and HMWP antigens was strongly affected and the absorbance value feI1 frem 1.10 to 0.35.

DISCUSSION We have described a preparative method of extraction of Mo pneumoniae specific protein antigens for the detection of IgG antibodies by indirect ELISA. The recovered antigens contained 7 HMWP. Among them, proteins P1 and P2, with apparent molecular weights of 170 and 110 Kd respectively, were previously implicated in attachment of M. pneumoniae to host eells [18]. Furthermore, we showed by immunoblots that HMWP were the main antigenic proteins, as suggested by previous reports [12, 15]. Different molecular masses have been described for protein Pt, rauging from 160 [21] to 190 Kd [11]. The molecular weight uetermined in our experiments was 170 Kd and was the same as that described earlier by Jacobs etai. [12]. The advantages of our electroelution method were as follows. (1) Efficiency of recovery was good; indeed, the recovery of 10 mg of M. pneumoniae HMWP in one step would allow the coating of 66 microtitration plates at the rate of 1.5 ~.g per well. (2) No special equipment, chemicals or tubes are needed. Samples can be eluted using the same apparatus used for the initial separation of proteins on SDS gels. The unique difference between buffer

|a

e

A

A

FIG. 6, - - Immunoblots against mycop!asma proteins with human sera.

i

lb A

~t~

t

Antigens: lane 1, M. pneumoniae; lane 2, M. hominis; lane 3, M. orale; and lane 4, M. salivarium. Human sera: lanes la, 2, 3 and 4, sera from patients with M. pneumoniae infection (group B); lane lb, sera from adult blood donors (group A). The protein markers M (see text) and proteins of mycoplasmas A were stained by aurodye (Janssen Biotech) after transfer. Large arrow indicates lower limit of HMWP.

a

la

c3

© 7~ >.

Z

>

600

G. P A P I E R O K A N D COLL.

electrophoresis and buffer electroelution is the presence of an antiprotease PMSF and DTT during electroelution. DTT prevented the reoxidization of free SH groups, and thus the formation of dimers or oligomers; we noted a significant decrease in recovery when DTT was omitted. (3) The procedure was rapid and reproducible. Electrophoresis and electroelution can be run in 9-10 h. Another important condition for electroelution was the sample volume. The yield was markedly decreased when a 1/1 ratio for stacking gel and sample volume was not maintained. Our results confirmed the earlier observation in this regard [14]. The eluted proteins contained 0 . 1 % SDS; this detergent did not inhibit adsorption of HMWP on polystyrene wells, provided the antigens were diluted sufficiently [16]. Therefore, contrary to a previous report [13], we did not use the special dot ELISA, but opted for the classic indirect ELISA wt'~ich was faster and simpler. Sera from all patients with M. pneumoniae infection and positive CFT were clearly positive in HMWP-ELISA. Furthermore, the high absorbance values obtained in 3 sera from group B patients, which had only low CFT antibody levels, indicated the greater sensitivity of the ELISA technique. Serum from group A, which was positive by ELISA, showed a mild response to several HMWP by immunoblotting. This serum was obtained from a 23-year old patient showing pharyngitis for the last several months. We presumed that the pharyngitis was due to M. pneumoniae. The 8 other sera from the Same group presented ~ slight antibody level against protein Pl by immunoblotting. This level of pre-existing IgG anti-M, pneumoniae antibodies in sera from apparent healthy s-abjects was previously demonstrated by Jacobs et al. [13]. However, the presence of these antibodies does not protect the individuals [12], and in case of reinfection, IgG antibody levels increase. Thus, paired sera will be necessary for a serological diagnosis of M. pneumoniae disease. Protein antigens were demonstrated to be more specific than the glycolipids used in CFT [8, 25]. In previous data [13], the authors reported that the protein P1 did not cross-react with other common oral mycoplasmas. Our data confirm this result and demonstrate that there is no cross-reactivity between M. pneumoniae H M W P and the proteins of 3 other mycoplasma species: M. orale, M. salivarium and M. hominis. We did not investigate possible cross-reactivity between M. pneumoniae and M. genitalium in the present study for two reasons. (1) Bredt et al. [2] showed that antigenic cross-reactions between proteins of M. pneumoniae and M. genitalium did not play an essential role in the serological diagnosis of M. pneumoniae disease. (2) M. genitalium is not implicated in respiratory disease. Its pathogenic role is not well elucidated [23]. Furthermore, recent data [10] with a DNA

MYCOPLASMA PNEUMONIAE PROTEIN SPECIFICITY

601

probe do not support a substantial aetiological role for 34. genitalium in men with urethritis. In conclusion, the present study shows that H M W P antigens obtained through electroelution should permit development of a more sensitive, specific and reproducible ELISA. Since H M W P antigens contain protein P1 which is implicated in M. pneumoniae virulence, the ELISA test developed in this study appears to be a satisfactory way of confirming clinical diagnosis. Furthermore, monoclonal antibodies against H M W P antigens can be used in a rapid identification of M. vneurnoniae on sputum or throat washings at an early stage of the infection. Such experiments are now under way.

I~SUME I~,LECTROI~LUTION PRI~P~RATIVE D'ANTIGI~.NES PROTEIQUES SPI~CIFIQUES DE f:/[YCOPLASMA PNEUMONIAE: UTILISATION DANS UNE MI~THODE IMMUNOENZYMATIQUE

Nous d6crivons une m6thode s/imple, rapide et pr6parative pour l'obtertion des 7 prot6ines membranaire.~ e s plus lourdes de Mycopiasma pneumoniae, avec un rendement appror, imatif de 90 %. La s6paration des prot6ines se fait par 61ectrophorbse sur gel de polyacrylamide en pr6sence de sodium dod6cyl sulfate, suivie ae l'61ectro61ution des prot6ines Ies plus lourdes. Ces antig6nes 61u6s ont 6t6 utilis6s dans une technique immunoenzymatique pour mesurer les IgG s6riques de 9 sujets sains et de 9 patients pr6sentant des infections h M. pneumoniae. La sp6cificit6 des prot6ines les plus lourdes de M. pneumoniae a 6t6 6tudi6e par technique immunoenzymatique comp6titive et technique d'immunoblotting par comparaison avec celle des prot6ines de diff6rentes espbces de mycoplasmes du tractus respiratoire. MOTS-CLI~S: Mycoplasma pneumoniae, Prot6ine, Meml~rane; Electro61ution, SDS-PAGE, ELISA, Immunoblots.

REFERENCES

[1~ BIBERFELD,G., Antibodies to brain and other tissues in cases of Mycoplasma pneumoniae infection. Clin. exp. IramunoL, 1971, 8, 319-333. [2] BREDT,W., KLEINMANN,B. & JACOBS,E., Antibodies in the sera of Mycoplasma pneumoniae-i~afected patients against proteins of Mycoplasma genitalium and other mycoplasmas of man. ZbL Bakt. Hyg., 1987, A266, 32-42. [3] BROUGHTON,R.A., Infections due to Mycoplasma pneumoniae in childhood. Pediatr. infect. Dis., 1986, 5, 71-85.

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G. P A P I E R O K A N D C O L L .

['~] I]RUNNER, H. & CHAN. , R.M., A radioimmunoprecipitation test for detection of Mycoplas : pneurnoniae antibody. Proc. Soc. exp. Biol. (N.Y.), 1973, 143, 95-10 [5] BRUNNER,H., JAMES, aq.D., HORSWOOD,R.L. & CnANOCK, R.M., Measurement of Mycoplasma pneurnoniae mycoplasmacidal antibody in human serum. J. Immunol., 1972, 108, 1491-1498. [6] BusoLo, F., TONIN, ~. & CONVENTI, L., Enzyme-linked immunosorbent assay for detection o: ~ycoplasrna pneurnoniae antibodies. J. clin. Microbiol., 1980, 12, 69-77 [7] CASSEL, G.H. & C~LE, B.C., Mycoplasma as agent of human disease. New Engl. J. M 7 1981, 304, 80-89. [8] DUSSAIX, E., ~ A. • TOURNIER, P., Comparison of enzyme-linked immunosorbent ~ ,~ay(ELISA) and complement fixation test for detection of Mycoplasma pneumoniae antibodies. J. clin. Path., 1983, 36, 228-232. [9] HOLLINGDALE,M.R. & LEMCKE, R.M., The antigens ol Mycoplasrna horninis. J. Hyg. (Camb.), 1969, 67, 585-602. [111] HOOTON,T.M., ROBERTS,P.L., STAMM,W.E., ROBERTS,M.C., HOLMES,K.K. & KENNV, G.E., Prevalence of Mycoplasrna genitaliurn determined by DNA probe in men with urethritis. Lancet, 1988, I, 267-268. [11] Hu, P.C., COLE, R.M., HUANG, Y.S., GRAHAM, J.A., GARDNER, D.E., COLLIER, A.M. & CLYDE, W.A. Jr, Mycoplasma pneurnoniae infection: role of a surface protein in the attachment organelle. Science, 1982, 216, 313-315. [],]] JACOBS, E., BENNEWITZ, A. & BREDT, W., Reaction pattern of human antiMycoplasrna pneumoniae antibodies in enzyn-~e-linked in~J~a,~osorbent assays and immunoblotting. J. clin. Microbiol., 1986, 23, 517-522. [13] JACOBS,E., FUCHTE,K. & BREDT, W., A 168-kilodalton protein of Mycoplasrna pneurnoniae used as antigen in a dot enzyme-linked immunosorbent assay. Europ. J. clin. Microbiol., 1986, 5, 435-440. [14] JOVIN, T.M., Multiphasic zone electrophoresis. -- I. Steady-state movingboundary systems formed by different electrolyte combinations. Biochemistry, 1973, 12, 871-898. [15] KENNY, G.E. & CARTWRIGHT,F.D., Immunoblotting for determination of the antigenic specificities of antibodies to the Mycoplasrnatales. Israel. J. rned. Sci., 1984, 20, 908-911. [16] KENNV,G.E. & DUNSMOOR,C.L., Principles, problems and strategies in the use of antigenic mixtures for the enzyme-linked immunosorbent assay. J. clin. Microbiol., 1983, 17, 655-665. [17] KENN¥, G.E. & NEWTON, R.M., Close serological relationships between glycolipids of Mycoplasma pneumoniae and glycolipids of spinach. Ann. N. Y. Acad. Sci., 1973, 225, 54-61. [18] KRAUSE~D.C. 8~ BASEMAN,J.B., M. pneurnoniae proteins that selectively bind to host cells. Infect. Imrnun., 1982, 37, 382-386. [19] LAEMMLI,U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Long.), 1970, 22"/, 680-685. [20] LEINIKKI,P.O., PANTZAR,P. & TYKKA, H., Antibody response in patients with acute pancreatitis to Mycoplasrna pneumoniae. Scand. J. Gastroent., i973, 8, 631-635. [21] LEITH, D.K., TREVINO, L.B., TtJLLV, J.G., SENTERFIT, L.B. & BASEMAN,J.B., Host discrimination of Myeoplasrna pneurnoniae proteinaceous immunogens. J. exp. Med., 1983, 15"/, 502-519. [22] LIND, K., Immunological relationship betwe.zn Mycoplasrna pneurnoniae and streptococcus M.G. Acta. path. rnicrobiol, scand., 1968, 73, 237-244. [23] LIND, K. & KRISTENSEN, G.B., Significance of antibodies to Myeoplasma genitaliurn in salpingitis. Europ. J. clin. Microbiol., 1987, 6, 205-297. [24] RAISNNEN,S.M., SUN1, J. & LEINIKKI,P., Serological diagnosis of Mycoplasrna pneumoniae infection by enzyme immunoassay. J. clin. Path., 1980, 33, 836-840.

MYCOPLASMA PNEUMONIAE PROTEIN SPECIFICITY

603

[25] RAISANEN, S.M., SUNI5 J. • VAHERI, A., Mycoplasma pneumoniae protein involved in the antibody response in human infection. J. clin. Path., 1984, 37, 1129-1133. [26] SEVER,J.L., Application of microtechnique to viral serological investigations. J. Dnmunol., 1962, 88, 320-329. [27] SKAUG,K., ENG, J., ORSTAV[K,I. & HAUG,K.W., The diagnosis value of determination of IgM antibodies against Mycoplasma pneumoniae by the indirect immunofluorescent test. Acta path. microbiol, scand., 1976, 84, 170-176. [28] TAYL,~-RO~INSON, D., PURCet.L, R.M., WONG, D.G. & C~ANOCK, R.M., A colour test for measurement of antibody to certain mycoplasma species based vpon the inhibition of acid production. J. Hyg. (Camb.), 1966, 64, 91-104. [29] TOWBIn, H., StAEHLIN, T. & GORGON, J., Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. nat. Acad. Sci. (Wash.), 1979, 76, 4350-4354. [301 Z~CHARIUS,R.M., ZELL, E.L., MORRISON,J.H. & WOODLOCK,J.J., Glycoprorein staining following electrophoresis on acrylamide gels. Analyt. Biochem., 1969, 30, I48-152.