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IgM antibodies to P1 cytoadhesin of mycoplasma pneumoniae are part of the natural antibody repertoire expressed early in life
Immunology Letters 63 (1998) 59 – 62
Rapid Note
IgM antibodies to P1 cytoadhesin of mycoplasma pneumoniae are part of the natural antibody repertoire expressed early in life Fatma Ben-Aissa-Fennira a, Amel Ben Ammar-El Gaaied b, Annie Bouguerra a, Koussay Dellagi c,* a
Departement of Hematology, Faculte´ de Me´decine de Tunis, Tunis, Tunisia Laboratory of Immunology, Faculte´ des Sciences de Tunis, Tunis, Tunisia c Laboratory of Immunology, Institut Pasteur de Tunis, 13 Place Pasteur, 1002 Tunis, Tunisia b
Received 7 April 1998; accepted 14 April 1998
Keywords: Mycoplasma pneumoniae; P1 cytoadhesin; IgM antibodies; Natural antibodies
Mycoplasma pneumoniae (MP) is a procaryote which causes primary atypical pneumonia in humans [1]. This infection is most commonly reported in children and young adults [2]. Tissue colonization requires cytoadherence of the microorganism to respiratory epithelial cells [3]. A 180-kd molecular weight, trypsin sensitive, surface protein designated P1, is likely to be the major adhesin of MP [4,5]. The involvement of this P1 adhesin is supported by the demonstration that monoclonal [6] and monospecific [7] anti-P1 antibodies inhibit the MP attachment to respiratory epithelium. Moreover, protein P1 elicits a strong immune response in humans and experimental animals infected with MP [8,9] indicating that adhesin P1 is the major immunogen. The cytoadhesin P1 gene was cloned and protein sequence deduced [10]. An immunogenic epitope of 13 aminoacids length (Pep13P1) involved in microorganism cytoadherence has been identified [11,12]. P1 protein [9] as well as short synthetic peptides deduced from the P1 amino acid sequence [13] (including Pep13P1 peptide) could be used as defined specific antigens in mycoplasmal serology. We report here that IgM antibodies to Pep13P1 cytoadhesin can be detected by ELISA in all newborn and young infants serum samples and are most likely ascribed to the natural
* Corresponding author. Tel.: + 216 1 789608; fax: + 216 1 791833; e-mail: [email protected] 0165-2478/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0165-2478(98)00053-4
antibody reactivity detected early in life. Serum samples were obtained from four groups of individuals: (a) ten healthy adult donors; (b) ten healthy children aged 7 years; (c) 20 young infants aged 11–17 months; and (d) seven newborns after full term delivery. In the latter case, cord blood was collected by venopuncture of placental umbilical vein, after section of the umbilical cord. One serum sample was also obtained from a 6-year-old child, (patient LK), suffering acute primary atypical pneumoniae with high titer of cold agglutinins [14]. Polyclonal IgM were purified from pooled sera of each age group, by 40% ammonium sulfate precipitation, followed by Sepharose 6B chromatography. The first peak eluted from the column was then submitted to DEAE ion exchange chromatography to remove any contaminating IgG. IgM was eluted at 0.3 M phosphate buffer. Enzyme linked immunoassay (ELISA) was used to detect IgM antibodies to MP or Pep13P1 coated plates. Mycoplasma pneumoniae used in this study was the FHATCC No. 15531 strain which was grown in liquid medium at pH 7.8, containing (per liter) 21 g of PPLO broth, 10 g of tryptone and 5 g of yeast extract (all supplied from DIFCO, USA). The medium was supplemented by 20% v/v of fetal calf serum (Flow Laboratories V/C), 10 ml of glucose (50%; w/v) solution, 10 ml of ampicillin solution (66 mg/ml) and 15 ml phenol red (0.1% w/v solution). A synthetic peptide (Pep13P1) derived from
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F. Ben-Aissa-Fennira et al. / Immunology Letters 63 (1998) 59–62
Fig. 1. IgM antibodies binding curves of 20 sera from healthly infants, aged 11 – 17 months, to MP coated plates as revealed by ELISA. Results obtained with serum LK (from a patient with acute primary atypic pneumoniae) (b) and a serum sample from a normal adult (a) are also represented. Results are expressed as OD units.
P1 cytoadhesin sequence was prepared by manual sequence at 99% of homogeneity and was a generous gift of Dr M. Bahraoui and Dr K. Mabrouk (Laboratoire de Biochimie, Faculte´ de Me´decine, Nord-Marseille). Pep13P1 sequence is Gly-Ile-Val-Arg-Thr-Pro-Leu-AlaGlu-Leu-Leu-Asp-Gly. Polystyrene microplates (NUNC) were coated with 50 mcl/well of whole MP resuspended in carbonate buffer pH 9.6 (50 mcg/ml protein content). After drying of the plates, MP were fixed 5 min in absolute ethanol and washed in phosphate buffered saline (PBS) 0.01 M pH 7.2. Alternatively the plates were coated with Pep13P1 in carbonate buffer at 1 mcg/well. The plates were saturated by 3% milk-PBS. Sera or human purified IgM, at adequate dilution, were added on coated plates and incubated overnight at 4°C. Binding of human anti-MP or anti Pep13P1 antibodies was revealed by mouse MAbs specific of human m chains followed by b-galactosidase labeled rabbit anti-mouse Ig (Amersham). Peripheral blood mononuclear cells (PBMC) were separated from heparinized cord blood by centrifugation over a Ficoll-hypaque gradient (Pharmacia). EBVtransformed lymphoblastoid cell lines were obtained by incubating 2 · 106 PBMC with 1 ml of supernatant from
the EBV-secreting marmoset cell-line B958, in RPMI 1640 containing 2 mM L-glutamine, 100 U/ml penicillin, streptomycin, 0.1 mg/ml of cyclosporin A and 20% fetal calf serum (FCS). When serum samples obtained from 20 heathly young infants aged 11–17 months were tested by ELISA for IgM antibodies to MP, we found that, unexpectedly, all sera showed significant reactivity with this antigen (Fig. 1). Serum LK, from a patient with acute primary MP infection, was used as positive control and also showed a strong reactivity (Fig. 1(b)). In contrast, a serum sample as well as purified IgM from a pool of sera from heathly adults, were only weakly reactive (Fig. 1(a)). Considering the very young age (B 1.5 years) of the healthy infants that were sampled, it seemed unlikely that the IgM reactivity with MP was indicative of a concomitant acute primary MP infection. We assumed that it rather indicates, that sera at this age group contained IgM cross reacting with some MP antigen(s). Since natural IgM antibodies are frequently polyreactive early in life [15], we investigated anti-MP/Pep13P1 IgM reactivity of newborn serum samples collected at full term normal deliveries. It is unlikely that newborns have been in contact with any infectious agent includ-
F. Ben-Aissa-Fennira et al. / Immunology Letters 63 (1998) 59–62
61
Fig. 2. IgM antibodies binding curves of seven human newborn sera (NS1 – NS7) to Pep13P1 (A) or MP (B) coated plates as revealed by ELISA. Sera are used at various dilutions (1/5–1/650). Results are expressed as OD units (abscisses are in logarithmic scale).
ing MP, and the expressed antibody repertoire at this age represents the natural, non-antigen driven, antibody repertoire. Interestingly, we found that newborn sera expressed constantly a strong reactivity with MP or P1 coated plates (Fig. 2(A and B)). This reactivity
was still significant at 1/650 dilution. This result was confirmed when IgM purified from a pool of newborn sera was used. Fifty percent binding was obtained with as low as 0.7 mcg/ml IgM. In contrast, polyclonal IgM purified from a pool of sera collected from healthy
Fig. 3. Binding curves of polyclonal IgM purified from pooled adult sera (IgM adult) or pooled sera from children aged 7 years (IgM children) or pooled cord blood sera (IgM newborn) to Pep13P1 coated plates as revealed by ELISA. Results are expressed as OD units (abscisses are in logarithmic scale).
F. Ben-Aissa-Fennira et al. / Immunology Letters 63 (1998) 59–62
62
adults was weakly reactive and did not reach 12.5% OD values of newborn IgM, at 10 mcg/ml. IgM purified from a pool of sera collected from children aged 7 years gave intermediate results (Fig. 3). In order to formally exclude the possibility that neonate IgM could have been contaminated by maternal antibodies, we established EBV derived B lymphoblastoid cell lines from PBMC of six newborns. All culture supernatants, tested after full establishment of the cell lines, showed significant IgM reactivity with MP/Pep13P1 coated plates (OD range: 0.576–0.917). Our results show that anti-P1 reactivity of newborn and infants IgM is likely an expression of the natural antibody repertoire generated independently of specific antigenic stimulation. P1 (or at least the Pep13P1 peptide) therefore belongs to the panel of self and non-self antigens recognized by the primitive B cell repertoire. It has been largely demonstrated, both in mice and humans, that natural antibodies are: (i) mainly of IgM class; (ii) early expressed in life; (iii) frequently polyreactive; and (iv) react with both self and non-self antigen including bacterial antigen (for review: [13]). Antigens recognized by natural antibodies are also frequently recognized by human monoclonal IgMs from patients with benign gammapathies or lymphoproliferative diseases. These MIgM were usefull reagents to characterize the structural basis of the broad antibody reactivity of natural antibodies and have demonstrated that they make preferential use of germ line, frequently unmutated, VH and VL genes. Interestingly, we have found that 4 out of 20 human monoclonal IgMs from patients benign gammapathies or Waldenstro¨m macroglobulinemia expressed anti-P1 reactivity (Fennira et al., manuscript in preparation) supporting the data obtained with newborn sera. Finally and considering the natural reactivity of neonate IgM to P1, ELISA tests for IgM antibodies based on whole MP or P1 protein coated plates should be cautiously interpreted when used as a diagnostic tool of mycoplasma infection in newborns or young infants.
Acknowledgements The authors are grateful to M. Bahraoui and K.
.
Mabrouk for providing Pep13P1 synthetic peptide and to Denise Dellagi for providing MP antigens. References [1] S. Razin, in: B.K. Ghosh (Ed.), Organization of Procaryotic Cell Membranes, vol. 1, CRC Press, Boca Raton, FL, 1981, pp. 165 – 250. [2] H.M. Foy, Infections caused by Mycoplasma pneumoniae and possible carrier state in different populations of patients, Clin. Infect. Dis. 17 (Suppl. 1) (1993) 37 – 46. [3] F. Gorski, W. Bredt, Studies on adherence-mechanism of Mycoplasma pneumoniae, FEMS Microbiol. Lett. 1 (1977) 265–267. [4] P.C. Hu, A.M. Collier, J.B. Baseman, Surface parasitism by M. pneumoniae of respiratory epithelium, J. Exp. Med. 145 (1977) 1328 – 1343. [5] E.J. Hansen, R.M. Wilson, W.A. Clyde Jr., J.B. Baseman, Characterization of hemadsorption negative mutants of Mycoplasma pneumoniae, Infect. Immun. 32 (1980) 127 – 136. [6] P.C. Hu, R.M. Cole, Y.S. Huang, J.A. Graham, D.E. Gardner, A.M. Collier, W.A. Clyde Jr., Mycoplasma pneumoniae infection: role of a surface protein in the attachment organelle, Science 216 (1982) 313 – 315. [7] D.C. Krause, J.B. Baseman, Inhibition of Mycoplasma pneumoniae hemadsorption and adherence to respiratory epithelium by antibodies to a membrane protein associated with hemadsorption and virulence, Infect. Immun. 35 (1983) 809 – 813. [8] P.C. Hu, Y.S. Huang, J.A. Graham, D.E. Gardner, Identification of immunogens of Mycoplasma pneumoniae by protein blotting, Biochem. Biophys. Res. Commun. 103 (1981) 1363. [9] E. Jacobs, A. Buchlotz, B. Kleinmann, W. Bredt, Use of adherence protein of Mycoplasma pneumoniae as antigen for enzyme linked immunosorbant assay (ELISA), Isr. J. Med. Sci. 23 (1987) 709 – 712. [10] M.J. Inamine, T.P. Denny, S. Loechel, M. Schaper, C.H. Huang, K.F. Bott, P.C. Hu, Nucleotide sequence of the P1 attachment protein gene of Mycoplasmas pneumoniae, Gene 64 (1988) 217– 219. [11] S.F. Dallo, C.J. Su, J.R. Horton, J.B. Baseman, Identification of P1 gene domain containing epitope(s) mediating Mycoplasma pneumoniae cytadherence, J. Exp. Med. 167 (1988) 718–723. [12] E. Jacobs, A. Pilatschek, B. Gerstenecker, K. Oberle, N. Bredt, Immunodominant epitopes of adhesin of Mycoplasma pneumoniae, J. Clin. Microbiol. 28 (1990) 1194 – 1197. [13] L. Hirschberg, T. Holme, A. Krook, Human antibody response to the major adhesin of Mycoplasma pneumoniae: Increase in titers against synthetic peptides in patients with pneumoniae, APMIS 99 (1991) 515 – 520. [14] F. Ben-Aı¨ssa-Fennira, A. Bouguerra, R. Hafsia, D. Dellagi, A. Ben Ammar-El Gaaied, K. Dellagi, Analyse immunochimique d’une IgM monoclonale humaine a` activite´ anticorps agglutinine froide au cours d’une infection a` Mycoplasma pneumoniae, Arch. Inst. Pasteur Tunis 73 (1/2) (1996) 3 – 8. [15] A.K. Stewart, C. Huang, A.A. Long, B.D. Stollar, R.S. Schwartz, VH-gene representation in autoantibodies reflects the normal human B cell repertoire, Immunol. Rev. 128 (1992) 101–122.
Rapid Note
IgM antibodies to P1 cytoadhesin of mycoplasma pneumoniae are part of the natural antibody repertoire expressed early in life Fatma Ben-Aissa-Fennira a, Amel Ben Ammar-El Gaaied b, Annie Bouguerra a, Koussay Dellagi c,* a
Departement of Hematology, Faculte´ de Me´decine de Tunis, Tunis, Tunisia Laboratory of Immunology, Faculte´ des Sciences de Tunis, Tunis, Tunisia c Laboratory of Immunology, Institut Pasteur de Tunis, 13 Place Pasteur, 1002 Tunis, Tunisia b
Received 7 April 1998; accepted 14 April 1998
Keywords: Mycoplasma pneumoniae; P1 cytoadhesin; IgM antibodies; Natural antibodies
Mycoplasma pneumoniae (MP) is a procaryote which causes primary atypical pneumonia in humans [1]. This infection is most commonly reported in children and young adults [2]. Tissue colonization requires cytoadherence of the microorganism to respiratory epithelial cells [3]. A 180-kd molecular weight, trypsin sensitive, surface protein designated P1, is likely to be the major adhesin of MP [4,5]. The involvement of this P1 adhesin is supported by the demonstration that monoclonal [6] and monospecific [7] anti-P1 antibodies inhibit the MP attachment to respiratory epithelium. Moreover, protein P1 elicits a strong immune response in humans and experimental animals infected with MP [8,9] indicating that adhesin P1 is the major immunogen. The cytoadhesin P1 gene was cloned and protein sequence deduced [10]. An immunogenic epitope of 13 aminoacids length (Pep13P1) involved in microorganism cytoadherence has been identified [11,12]. P1 protein [9] as well as short synthetic peptides deduced from the P1 amino acid sequence [13] (including Pep13P1 peptide) could be used as defined specific antigens in mycoplasmal serology. We report here that IgM antibodies to Pep13P1 cytoadhesin can be detected by ELISA in all newborn and young infants serum samples and are most likely ascribed to the natural
* Corresponding author. Tel.: + 216 1 789608; fax: + 216 1 791833; e-mail: [email protected] 0165-2478/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0165-2478(98)00053-4
antibody reactivity detected early in life. Serum samples were obtained from four groups of individuals: (a) ten healthy adult donors; (b) ten healthy children aged 7 years; (c) 20 young infants aged 11–17 months; and (d) seven newborns after full term delivery. In the latter case, cord blood was collected by venopuncture of placental umbilical vein, after section of the umbilical cord. One serum sample was also obtained from a 6-year-old child, (patient LK), suffering acute primary atypical pneumoniae with high titer of cold agglutinins [14]. Polyclonal IgM were purified from pooled sera of each age group, by 40% ammonium sulfate precipitation, followed by Sepharose 6B chromatography. The first peak eluted from the column was then submitted to DEAE ion exchange chromatography to remove any contaminating IgG. IgM was eluted at 0.3 M phosphate buffer. Enzyme linked immunoassay (ELISA) was used to detect IgM antibodies to MP or Pep13P1 coated plates. Mycoplasma pneumoniae used in this study was the FHATCC No. 15531 strain which was grown in liquid medium at pH 7.8, containing (per liter) 21 g of PPLO broth, 10 g of tryptone and 5 g of yeast extract (all supplied from DIFCO, USA). The medium was supplemented by 20% v/v of fetal calf serum (Flow Laboratories V/C), 10 ml of glucose (50%; w/v) solution, 10 ml of ampicillin solution (66 mg/ml) and 15 ml phenol red (0.1% w/v solution). A synthetic peptide (Pep13P1) derived from
60
F. Ben-Aissa-Fennira et al. / Immunology Letters 63 (1998) 59–62
Fig. 1. IgM antibodies binding curves of 20 sera from healthly infants, aged 11 – 17 months, to MP coated plates as revealed by ELISA. Results obtained with serum LK (from a patient with acute primary atypic pneumoniae) (b) and a serum sample from a normal adult (a) are also represented. Results are expressed as OD units.
P1 cytoadhesin sequence was prepared by manual sequence at 99% of homogeneity and was a generous gift of Dr M. Bahraoui and Dr K. Mabrouk (Laboratoire de Biochimie, Faculte´ de Me´decine, Nord-Marseille). Pep13P1 sequence is Gly-Ile-Val-Arg-Thr-Pro-Leu-AlaGlu-Leu-Leu-Asp-Gly. Polystyrene microplates (NUNC) were coated with 50 mcl/well of whole MP resuspended in carbonate buffer pH 9.6 (50 mcg/ml protein content). After drying of the plates, MP were fixed 5 min in absolute ethanol and washed in phosphate buffered saline (PBS) 0.01 M pH 7.2. Alternatively the plates were coated with Pep13P1 in carbonate buffer at 1 mcg/well. The plates were saturated by 3% milk-PBS. Sera or human purified IgM, at adequate dilution, were added on coated plates and incubated overnight at 4°C. Binding of human anti-MP or anti Pep13P1 antibodies was revealed by mouse MAbs specific of human m chains followed by b-galactosidase labeled rabbit anti-mouse Ig (Amersham). Peripheral blood mononuclear cells (PBMC) were separated from heparinized cord blood by centrifugation over a Ficoll-hypaque gradient (Pharmacia). EBVtransformed lymphoblastoid cell lines were obtained by incubating 2 · 106 PBMC with 1 ml of supernatant from
the EBV-secreting marmoset cell-line B958, in RPMI 1640 containing 2 mM L-glutamine, 100 U/ml penicillin, streptomycin, 0.1 mg/ml of cyclosporin A and 20% fetal calf serum (FCS). When serum samples obtained from 20 heathly young infants aged 11–17 months were tested by ELISA for IgM antibodies to MP, we found that, unexpectedly, all sera showed significant reactivity with this antigen (Fig. 1). Serum LK, from a patient with acute primary MP infection, was used as positive control and also showed a strong reactivity (Fig. 1(b)). In contrast, a serum sample as well as purified IgM from a pool of sera from heathly adults, were only weakly reactive (Fig. 1(a)). Considering the very young age (B 1.5 years) of the healthy infants that were sampled, it seemed unlikely that the IgM reactivity with MP was indicative of a concomitant acute primary MP infection. We assumed that it rather indicates, that sera at this age group contained IgM cross reacting with some MP antigen(s). Since natural IgM antibodies are frequently polyreactive early in life [15], we investigated anti-MP/Pep13P1 IgM reactivity of newborn serum samples collected at full term normal deliveries. It is unlikely that newborns have been in contact with any infectious agent includ-
F. Ben-Aissa-Fennira et al. / Immunology Letters 63 (1998) 59–62
61
Fig. 2. IgM antibodies binding curves of seven human newborn sera (NS1 – NS7) to Pep13P1 (A) or MP (B) coated plates as revealed by ELISA. Sera are used at various dilutions (1/5–1/650). Results are expressed as OD units (abscisses are in logarithmic scale).
ing MP, and the expressed antibody repertoire at this age represents the natural, non-antigen driven, antibody repertoire. Interestingly, we found that newborn sera expressed constantly a strong reactivity with MP or P1 coated plates (Fig. 2(A and B)). This reactivity
was still significant at 1/650 dilution. This result was confirmed when IgM purified from a pool of newborn sera was used. Fifty percent binding was obtained with as low as 0.7 mcg/ml IgM. In contrast, polyclonal IgM purified from a pool of sera collected from healthy
Fig. 3. Binding curves of polyclonal IgM purified from pooled adult sera (IgM adult) or pooled sera from children aged 7 years (IgM children) or pooled cord blood sera (IgM newborn) to Pep13P1 coated plates as revealed by ELISA. Results are expressed as OD units (abscisses are in logarithmic scale).
F. Ben-Aissa-Fennira et al. / Immunology Letters 63 (1998) 59–62
62
adults was weakly reactive and did not reach 12.5% OD values of newborn IgM, at 10 mcg/ml. IgM purified from a pool of sera collected from children aged 7 years gave intermediate results (Fig. 3). In order to formally exclude the possibility that neonate IgM could have been contaminated by maternal antibodies, we established EBV derived B lymphoblastoid cell lines from PBMC of six newborns. All culture supernatants, tested after full establishment of the cell lines, showed significant IgM reactivity with MP/Pep13P1 coated plates (OD range: 0.576–0.917). Our results show that anti-P1 reactivity of newborn and infants IgM is likely an expression of the natural antibody repertoire generated independently of specific antigenic stimulation. P1 (or at least the Pep13P1 peptide) therefore belongs to the panel of self and non-self antigens recognized by the primitive B cell repertoire. It has been largely demonstrated, both in mice and humans, that natural antibodies are: (i) mainly of IgM class; (ii) early expressed in life; (iii) frequently polyreactive; and (iv) react with both self and non-self antigen including bacterial antigen (for review: [13]). Antigens recognized by natural antibodies are also frequently recognized by human monoclonal IgMs from patients with benign gammapathies or lymphoproliferative diseases. These MIgM were usefull reagents to characterize the structural basis of the broad antibody reactivity of natural antibodies and have demonstrated that they make preferential use of germ line, frequently unmutated, VH and VL genes. Interestingly, we have found that 4 out of 20 human monoclonal IgMs from patients benign gammapathies or Waldenstro¨m macroglobulinemia expressed anti-P1 reactivity (Fennira et al., manuscript in preparation) supporting the data obtained with newborn sera. Finally and considering the natural reactivity of neonate IgM to P1, ELISA tests for IgM antibodies based on whole MP or P1 protein coated plates should be cautiously interpreted when used as a diagnostic tool of mycoplasma infection in newborns or young infants.
Acknowledgements The authors are grateful to M. Bahraoui and K.
.
Mabrouk for providing Pep13P1 synthetic peptide and to Denise Dellagi for providing MP antigens. References [1] S. Razin, in: B.K. Ghosh (Ed.), Organization of Procaryotic Cell Membranes, vol. 1, CRC Press, Boca Raton, FL, 1981, pp. 165 – 250. [2] H.M. Foy, Infections caused by Mycoplasma pneumoniae and possible carrier state in different populations of patients, Clin. Infect. Dis. 17 (Suppl. 1) (1993) 37 – 46. [3] F. Gorski, W. Bredt, Studies on adherence-mechanism of Mycoplasma pneumoniae, FEMS Microbiol. Lett. 1 (1977) 265–267. [4] P.C. Hu, A.M. Collier, J.B. Baseman, Surface parasitism by M. pneumoniae of respiratory epithelium, J. Exp. Med. 145 (1977) 1328 – 1343. [5] E.J. Hansen, R.M. Wilson, W.A. Clyde Jr., J.B. Baseman, Characterization of hemadsorption negative mutants of Mycoplasma pneumoniae, Infect. Immun. 32 (1980) 127 – 136. [6] P.C. Hu, R.M. Cole, Y.S. Huang, J.A. Graham, D.E. Gardner, A.M. Collier, W.A. Clyde Jr., Mycoplasma pneumoniae infection: role of a surface protein in the attachment organelle, Science 216 (1982) 313 – 315. [7] D.C. Krause, J.B. Baseman, Inhibition of Mycoplasma pneumoniae hemadsorption and adherence to respiratory epithelium by antibodies to a membrane protein associated with hemadsorption and virulence, Infect. Immun. 35 (1983) 809 – 813. [8] P.C. Hu, Y.S. Huang, J.A. Graham, D.E. Gardner, Identification of immunogens of Mycoplasma pneumoniae by protein blotting, Biochem. Biophys. Res. Commun. 103 (1981) 1363. [9] E. Jacobs, A. Buchlotz, B. Kleinmann, W. Bredt, Use of adherence protein of Mycoplasma pneumoniae as antigen for enzyme linked immunosorbant assay (ELISA), Isr. J. Med. Sci. 23 (1987) 709 – 712. [10] M.J. Inamine, T.P. Denny, S. Loechel, M. Schaper, C.H. Huang, K.F. Bott, P.C. Hu, Nucleotide sequence of the P1 attachment protein gene of Mycoplasmas pneumoniae, Gene 64 (1988) 217– 219. [11] S.F. Dallo, C.J. Su, J.R. Horton, J.B. Baseman, Identification of P1 gene domain containing epitope(s) mediating Mycoplasma pneumoniae cytadherence, J. Exp. Med. 167 (1988) 718–723. [12] E. Jacobs, A. Pilatschek, B. Gerstenecker, K. Oberle, N. Bredt, Immunodominant epitopes of adhesin of Mycoplasma pneumoniae, J. Clin. Microbiol. 28 (1990) 1194 – 1197. [13] L. Hirschberg, T. Holme, A. Krook, Human antibody response to the major adhesin of Mycoplasma pneumoniae: Increase in titers against synthetic peptides in patients with pneumoniae, APMIS 99 (1991) 515 – 520. [14] F. Ben-Aı¨ssa-Fennira, A. Bouguerra, R. Hafsia, D. Dellagi, A. Ben Ammar-El Gaaied, K. Dellagi, Analyse immunochimique d’une IgM monoclonale humaine a` activite´ anticorps agglutinine froide au cours d’une infection a` Mycoplasma pneumoniae, Arch. Inst. Pasteur Tunis 73 (1/2) (1996) 3 – 8. [15] A.K. Stewart, C. Huang, A.A. Long, B.D. Stollar, R.S. Schwartz, VH-gene representation in autoantibodies reflects the normal human B cell repertoire, Immunol. Rev. 128 (1992) 101–122.