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Antibodies against the common polysaccharide and protein protective antigens of Pseudomonas aeruginosa in normal blood donors
FEMS MicrobiologyImmunology76 (1991) 321-326 © 1991 Federation of European Microbiological Societies 0920-8534/91/$03.50 Published by Elsevier
321
FEMSIM 00178
Antibodies against the common polysaccharide and protein protective antigens of Pseudomonas aeruginosa in normal blood donors T a t j a n a A. M a k a r e n k o , Lilija S. E d v a b n a j a , I r i n a A b r a m o v a a n d E.S. S t a n i s l a v s k y Mechnikoc Research Institute for Vaccine and Sera, Academy of Medical Sciences, Moscow, U.S.S.R.
(Received 10 January 1991) (Revised 10 June 1991) (Accepted 25 June 1991) Key words: Pseudomonas aeruginosa; Antibodies; C o m m o n polysaccharide antigen; Protein antigen
1. S U M M A R Y Screening of normal plasma obtained from 172 blood donors from the Helsinki area and from 46 blood donors from the Moscow area was performed in order to reveal 'natural' antibodies to the common polysaccharide (rhamnan) and protein antigens of P. aeruginosa. Antibodies were detected by ELISA. A m o n g blood donors from the Helsinki area high titres of antibodies to the protein antigens were detected in 42 active blood donors (24.4%) and very high titres in nine (5.3%) highly-active blood donors, whereas in the Moscow area in 15 (34.9%) and in one case (2.3%), respectively. Antibodies to the common polysaccharide antigen were determined in the Helsinki area in 23 active blood donors (13.4%) and in one (0.5%)
Correspondence to: E.S. Stanislavsky, I.I. Mechnikov Central Research Institute of Vaccines and Sera, Ministry of Public Health of the U.S.S.R., Mechnikov per. 5a, Moscow, U.S.S.R.
highly active blood donor, whereas in the Moscow area in four active blood donors (8.6%). The plasma contained both polysaccharide and anti-protein antibodies. The level of antibodies to the polysaccharide antigen was lower than the level of antibodies to the protein antigens. There was no statistically significant difference between the corresponding values of blood donor groups from the Helsinki and Moscow areas.
2. I N T R O D U C T I O N Pseudomonas aeruginosa is a typical nosocomial pathogen which causes purulent complications after surgery (12.2%), in medicine (9.5%), gynaecology (2.9), obstetrical hospital categories (1.1%), in paediatric (8.0%) and newborn services (2.0%) [1]. P. aeruginosa carriage is wide-spread, and 'natural' anti-Pseudomonas antibodies can be detected among healthy people [2].
322 A commercial preparation of ' n o r m a l ' gammaglobulin, obtained from pooled blood from healthy persons, which contained antibodies against slime and O-antigens of P. aeruginosa, protects mice against P. aeruginosa experimental infection. It has been found that the level of these antibodies varies greatly from batch to batch
[3]. P. aeruginosa also contains other antigens, including protein protective antigens, localized in the outer m e m b r a n e of the cell wall [4,5]. These antigens were used to prepare a P. aeruginosa vaccine (PV) which protected mice against experimental P. aeruginosa infection [6] and stimulated the formation of specific antibodies in 9 4 - 9 7 % of vaccinated volunteers. Moreover, in 45.6% of these volunteers the antibody titres reached high levels [7]. Recently, a common polysaccharide antigen (CPA) in P. aeruginosa has been described, namely rhamnan [8,9] which might be used for the identification of P. aeruginosa species. Rhamnan has been found in the lipopolysaccharide of P. aeruginosa as well as in phytopathogenic P. cerasi. Thus an immunological link was observed between these species [10]. Based on the above data, this study was carried out to determine the level of 'natural' antibodies to CPA and to protein, and protective antigens of the outer m e m b r a n e of P. aeruginosa in the plasma of blood donors. Such a study may assist in the selection of normal human plasma with high level of antibodies to the nosocomial pathogen, P. aeruginosa.
3. M A T E R I A L S A N D M E T H O D S
3.1. Antigens Two types of antigen were used: (1) P. aeruginosa vaccine (PV) was prepared from purified, protein protective antigens of the outer membrane as previously described by Stanislavsky et al. [6], and (2) LPS of P. cerasi, isolated from cells by the phenol-water method and purified by Cetavlon [10]. The P. cerasi strain was obtained from the D.K. Zabolotny Institute of Microbiol-
ogy and Virology, Academy of Sciences (Kiev, U.S.S.R.) and cultivated as described earlier [10].
3.2. Blood donors Blood donors from the Helsinki area included 172 healthy persons, mean age 36.5 years (range 18-62), 71 female and 101 male, who were regular blood donors at the Finnish Red Cross Blood Transfusion Centre. Blood donors from the Moscow area included 46 healthy male subjects, mean age 31.2 years (range 25-40), who were previously carefully checked in the All-Union Haematologic Centre [12]. 3.3. ELISA Antibodies were determined by E L I S A [11] in N U N C ImmunoPlates I with PV (0.1 ~ g of protein) and LPS (0.1 ~ g dry weight) of P. cerasi. All reactions were performed at room temperature. Plates were incubated at 18 h with PV or LPS in 0.5 M carbonate buffer (pH 9.0). The plates were washed with 0.15 M phosphate buffer (pH 7.2) and incubated for 3.5 h with test or reference plasmas diluted with the same buffer. After washing, rabbit anti-human IgG conjugated with horseradish peroxidase (Labsystems Oy, Helsinki, Finland) was added and incubated for 18 h when the plates were washed. Substrate solution containing 0.4% O-phenylenediamine (Sigma, U.S.A.) in 0.5 M citrate buffer (pH 6.0) and 0.42% hydrogen peroxide was added. The reaction was stopped after 0.5 h by the addition of 3 N sulphuric acid. Results were read in a Titertek Multiskan (Labsystems) at 450 nm. Two types of reference plasma were used. Reference 1, used in the E L I S A with PV, was the plasma of a volunteer who was vaccinated with PV and in whom the highest level of anti-PV-antibodies was detected. Volunteers were immunized with PV by subcutaneous injection in a subscapular area. The injections were repeated thrice at intervals of 7 days: approximately 0.6, 1.2, and 2.4 mg of protein per injection, respectively [7]. The activity of plasma with PV antigen was assessed as follows: in optimal dilution 1:3200 absorbance at 450 nm of Reference 1 showed plate variation between 0.9 and 1.3; the mean
323
absorbance was 1.0. The test plasma were considered to be active (active donor) when the absorbance values were equal or exceeded 50% of the reference absorbance. When the absorbance >_ 1.0 the plasma was considered to be highly active (highly active donor). In E L I S A with LPS a plasma of a normal blood donor with the highest level of 'natural' LPS antibodies (Reference 2) was used. The activity of the LPS reaction was lower than the activity of the PV reaction, therefore the optimal dilution used was 1 : 1600. The absorbance at 450 nm of Reference 2 varied from 0.5 to 0.8 with a mean of 0.7. The plasma under investigation in the LPS reaction was considered to be active when the corresponding absorbance was in the range 0.7-0.9 and highly-active when it was > 1.0. 3.4. Statistical methods Significance of the difference between groups was assessed by Van der Varden [13].
4. R E S U L T S Table 1 summarizes the results of the normal plasma study with the PV antigen. The plasma of 42 (24.4%) out of 172 blood donors from the Helsinki area contained high levels of PV-antibodies, at a dilution of 1:3200 (active donors) and 9 (5.3%) contained very high PV-antibody titres with absorbance > 1.0 at the same dilution (highly active donors). When plasma of 43 blood donors of the Moscow area were studied in PV-antigen test 15 (34.4%) had high levels of PV-antibodies (active donors) and in one donor (2.3%) there was a very high level of antibodies (A450 nm was 1.1 at dilution of 1:3200; highly active donor). There was no significant statistical difference between the number of active or highly active donors in both blood donor groups. Table 2 illustrates the results of the test with another antigen, the LPS of P. cerasi. In the
Table 1 Results of the screening of donors plasma by ELISA with PV-antigen Area
Total
Negative
Helsinki
172 (100%) 43 (100%)
121 (72.3%) 27 (63.8%)
Moscow
Number of donors:
Difference in % between Helsinki and Moscow:
active a
highly-active b
active ~
highly-active b
42 (24.4%) 15 (34.9%)
9 (5.3%) 1 (2.3%)
2`2 = 2.1 P>0.1
A~2 = 0.6 P>0.3
Active, absorbance at 450 nm 0.5-0.9; b highly-active, absorbance at 450 nm _> 1. The plasma samples were diluted 1:3200.
Table 2 Results of the screening of donors plasma by ELISA with LPS of P. cerasi Area
Helsinki Moscow
Total
172 (100%) 43 (100%)
Number of donors:
Difference in % between Helsinki and Moscow:
active a
highly-active b
active a
highly-active b
23 (13.4%) 4 (8.6%)
1 (0.5%) 0
X 2 = 0.73 P > 0.3
X 2 = 0.27 P > 0.5
a Active, absorbance at 450 nm 0.7-0.9; b highly-active, absorbance at 450 nm _> 1. The plasma samples were diluted 1 : 1600.
324 Helsinki group there were 23 (13.4%) active donors and one highly active donor (0.5%), whereas in the Moscow group there were 4 active donors (8.6%).
5. D I S C U S S I O N The results of the present study suggest that among healthy persons there are subjects with a high or very high level of 'natural' antibodies to the common polysaccharide antigen (CPA) or protein protective antigens of the outer membrane of P. aeruginosa. The suggested criterion for the evaluation of the activity of normal plasma by an E L I S A provides an easy screening method of more than 200 blood donor plasma specimens in order to select active donors with specific anti-P, aeruginosa antibodies. Earlier [3] ' n o r m a l ' gamma-globulin manufactured by Biomed (Petrovo-Dalnee, Moscow, U.S.S.R.) was examined. In some batches of pooled IgG high titres of antibodies to O-antigens and to P. aeruginosa slime antigens were detected. These preparations protected mice against P. aeruginosa experimental infection. When clinical trials of such pooled IgG of gamma-globulin were performed, a therapeutic effect was found when infants younger than two years with severe forms of P. aeruginosa infection were treated [14]. Results of the present study show that normal blood donor plasma containing high or very high levels of anti-P, aeruginosa antibodies (so-called selected plasma) can probably be used for the treatment of P. aeruginosa infections in humans. The other use of selected plasma is the production of anti-Pseudomonas immunoglobulin for intravenous administration. A question arises: what is more u s e f u l - - i m m u n i z a t i o n of donors with PV in order to obtain immune plasma or selection of normal donor plasma based on the P. aeruginosa antibody level? According to previous data [7] immunization of volunteers with PV stimulates the formation of specific antibodies in 9 4 - 9 7 % and approximately in half of them a high level of antibodies was
revealed. High levels of antibodies against P.
aeruginosa are seen in approximately 25-35% of normal donors (active donors) and very high levels in approximately 2 - 5 % (highly active donors). It is known that so-called 'natural' antibodies against the nosocomial pathogen, P. aeruginosa, can be detected in blood of healthy people [2]. The formation of such antibodies is induced by an asymptomatic carriage of P. aeruginosa or earlier infection in anamnesis. We called these antibodies as 'natural', to distinguish them from the antibodies produced by immunization. It is worth mentioning that high or very high levels of 'natural' antibodies to PV (i.e. to protein protective antigens), revealed approximately in one third of non-immunized donors, almost coincides with the high level of corresponding antibodies in approximately 50% of immunized donors. We consider that both methods of preparation of anti-P, aeruginosa plasma should be used: immunization and selection. Both types of anti-P. aeruginosa plasma might find a broad application in practical health care for the treatment of severe forms of P. aeruginosa infection, especially P. aeruginosa sepsis.
ACKNOWLEDGEMENTS We are grateful to Dr. G u n n a r Myllyla from the Finnish Red Cross Blood Transfusion Centre for 172 normal blood donor plasmas, for the discussion of the present paper, and for his valuable comments. We wish to acknowledge Dr. D.E.S. Stewart-Tull from Glasgow University for his valuable comments and revision of the paper.
REFERENCES [1] Hughes, J.M., Culver, D.H., White, J.W., Jarvis, W.R. et al. (1983) Nosocomial infection Surveillance, 1980-1982. In: U.S. Department of Health and Human Services/ Public Health Service Centers for Disease Control from the Morbility and Mortality Weekly Report Surveillance Summaries. November, Vol. 32, No 4SS. [2] Stanislavsky, E.S. and Kolker, I.I. (1978) Pseudomonas
325
[3]
[4]
[5]
[6]
[7]
[8]
infection (Review). All-Union Research Institute of Medical and Medico-Technical Information. Moscow, 102pp. Stanislavsky, E.S., Boultuci, L.G., Yushkova, N.A., Chekan, L.V., Zaidner, I.G., Koutomova, L.R., Mignivitsh, L.R. and Landsman, N.M. (1983) Immunite passive dans l'infection experimentale P. aeruginosa. Rev. Inst. Pasteur de Lyon, 3, 325-344. Stanislavsky, E.S., Edvabnaya, L.S., Bulk, V.F., Zhvanetskaya, M.I. and, Mashilova, G.M. (1986) Immunocbemical and immunological study of cell-wall proteins of Pseudomonas aeruginosa Acta Microbiol. Hungarica 33, 245-255. Specht, B.U. Von. Strigl, G., Ehret, W. and Brendel, W. (1987) Protective effect of an outer membrane vaccine against Pseudomonas aeruginosa infection. 'Infection' 15, 408-412. Stanislavsky, E.S., Edvabnaya, L.S, Bandman, O.A., Bulk, V.F., Zhvanetskaya, M.I. and Vargina, A.K. (1989) Experimental studies on the protective efficacy of a Pseudomonas aeruginosa vaccine. Vaccine 7, 562-566. Stanislavsky, E.S., Balayan, S.S., Sergienko, A.I., Makarenko, T.A., Edvabnaya, L.S., Krohina, M.A., Rusanov, V.M. (1991) Clinico-immunological trial of Pseudomonas aeruginosa vaccine. Vaccine 285 (in press). Sawada, Sh. and Takashi, K. (1985) A new common polysaccharide antigen of strains of Pseudomonas aerugi-
[9]
[10]
[11]
[12] [13] [14]
nosa detected with a monoclonal antibody. J. Infect. Dis. 152, 1290-2199. Kocharova, N.A., Knirel, Yu.A., Kochetkov, N.K. and Stanislavsky, E.S. (1988) Characterization of a rhamnan derived from preparations of Pseudomonas aeruginosa lipopolysaccharides. Biorgacheskaya Ckimia, 14, 701-703. Kocharova, N.A., Knirel, Yu. A., Romanowska, A., Romanovska, E., Makarenko, T.A., Edvabnaya, L.S., Stanislavsky, E.S., Zdorovenko, G.M. and Jakovleva, L.M. (1991) Detection of common polysaccharide antigen of P. aeruginosa with O-antiserum to P. cerasi. FEMS Microbiol./Immunol. 76, 69-74. Brodeur, B.R., Ashton, F.E. and Diena, B.B. (1976) Enzyme-linked immunosorbent assay for the detection of Neiseria gonorrhoeae specific antibodies. Can. J. Microbiol. 12, 113-121. Instructions for plasmapheresis application. Approved by the USSR Ministry of Health, 1972. Moscow, 1972, 26pp. Van der Varden, B.I. Mathematical statistics. Inostranaya Litertura. Publ. Hous. Moscow, 1060, 527pp. Bulay, P.I., Krassilnikov, A.P. Adarchenko, A.A., Jedvabnaja, L.S., Boltutsy, L.G. and Stanislavsky, E.S. (1986) Therapeutic effect of normal human gamma-globulin used for treatment of P. aeruginosa infection in infants. Ann. Immunol. Hung. 26, 629-637.
321
FEMSIM 00178
Antibodies against the common polysaccharide and protein protective antigens of Pseudomonas aeruginosa in normal blood donors T a t j a n a A. M a k a r e n k o , Lilija S. E d v a b n a j a , I r i n a A b r a m o v a a n d E.S. S t a n i s l a v s k y Mechnikoc Research Institute for Vaccine and Sera, Academy of Medical Sciences, Moscow, U.S.S.R.
(Received 10 January 1991) (Revised 10 June 1991) (Accepted 25 June 1991) Key words: Pseudomonas aeruginosa; Antibodies; C o m m o n polysaccharide antigen; Protein antigen
1. S U M M A R Y Screening of normal plasma obtained from 172 blood donors from the Helsinki area and from 46 blood donors from the Moscow area was performed in order to reveal 'natural' antibodies to the common polysaccharide (rhamnan) and protein antigens of P. aeruginosa. Antibodies were detected by ELISA. A m o n g blood donors from the Helsinki area high titres of antibodies to the protein antigens were detected in 42 active blood donors (24.4%) and very high titres in nine (5.3%) highly-active blood donors, whereas in the Moscow area in 15 (34.9%) and in one case (2.3%), respectively. Antibodies to the common polysaccharide antigen were determined in the Helsinki area in 23 active blood donors (13.4%) and in one (0.5%)
Correspondence to: E.S. Stanislavsky, I.I. Mechnikov Central Research Institute of Vaccines and Sera, Ministry of Public Health of the U.S.S.R., Mechnikov per. 5a, Moscow, U.S.S.R.
highly active blood donor, whereas in the Moscow area in four active blood donors (8.6%). The plasma contained both polysaccharide and anti-protein antibodies. The level of antibodies to the polysaccharide antigen was lower than the level of antibodies to the protein antigens. There was no statistically significant difference between the corresponding values of blood donor groups from the Helsinki and Moscow areas.
2. I N T R O D U C T I O N Pseudomonas aeruginosa is a typical nosocomial pathogen which causes purulent complications after surgery (12.2%), in medicine (9.5%), gynaecology (2.9), obstetrical hospital categories (1.1%), in paediatric (8.0%) and newborn services (2.0%) [1]. P. aeruginosa carriage is wide-spread, and 'natural' anti-Pseudomonas antibodies can be detected among healthy people [2].
322 A commercial preparation of ' n o r m a l ' gammaglobulin, obtained from pooled blood from healthy persons, which contained antibodies against slime and O-antigens of P. aeruginosa, protects mice against P. aeruginosa experimental infection. It has been found that the level of these antibodies varies greatly from batch to batch
[3]. P. aeruginosa also contains other antigens, including protein protective antigens, localized in the outer m e m b r a n e of the cell wall [4,5]. These antigens were used to prepare a P. aeruginosa vaccine (PV) which protected mice against experimental P. aeruginosa infection [6] and stimulated the formation of specific antibodies in 9 4 - 9 7 % of vaccinated volunteers. Moreover, in 45.6% of these volunteers the antibody titres reached high levels [7]. Recently, a common polysaccharide antigen (CPA) in P. aeruginosa has been described, namely rhamnan [8,9] which might be used for the identification of P. aeruginosa species. Rhamnan has been found in the lipopolysaccharide of P. aeruginosa as well as in phytopathogenic P. cerasi. Thus an immunological link was observed between these species [10]. Based on the above data, this study was carried out to determine the level of 'natural' antibodies to CPA and to protein, and protective antigens of the outer m e m b r a n e of P. aeruginosa in the plasma of blood donors. Such a study may assist in the selection of normal human plasma with high level of antibodies to the nosocomial pathogen, P. aeruginosa.
3. M A T E R I A L S A N D M E T H O D S
3.1. Antigens Two types of antigen were used: (1) P. aeruginosa vaccine (PV) was prepared from purified, protein protective antigens of the outer membrane as previously described by Stanislavsky et al. [6], and (2) LPS of P. cerasi, isolated from cells by the phenol-water method and purified by Cetavlon [10]. The P. cerasi strain was obtained from the D.K. Zabolotny Institute of Microbiol-
ogy and Virology, Academy of Sciences (Kiev, U.S.S.R.) and cultivated as described earlier [10].
3.2. Blood donors Blood donors from the Helsinki area included 172 healthy persons, mean age 36.5 years (range 18-62), 71 female and 101 male, who were regular blood donors at the Finnish Red Cross Blood Transfusion Centre. Blood donors from the Moscow area included 46 healthy male subjects, mean age 31.2 years (range 25-40), who were previously carefully checked in the All-Union Haematologic Centre [12]. 3.3. ELISA Antibodies were determined by E L I S A [11] in N U N C ImmunoPlates I with PV (0.1 ~ g of protein) and LPS (0.1 ~ g dry weight) of P. cerasi. All reactions were performed at room temperature. Plates were incubated at 18 h with PV or LPS in 0.5 M carbonate buffer (pH 9.0). The plates were washed with 0.15 M phosphate buffer (pH 7.2) and incubated for 3.5 h with test or reference plasmas diluted with the same buffer. After washing, rabbit anti-human IgG conjugated with horseradish peroxidase (Labsystems Oy, Helsinki, Finland) was added and incubated for 18 h when the plates were washed. Substrate solution containing 0.4% O-phenylenediamine (Sigma, U.S.A.) in 0.5 M citrate buffer (pH 6.0) and 0.42% hydrogen peroxide was added. The reaction was stopped after 0.5 h by the addition of 3 N sulphuric acid. Results were read in a Titertek Multiskan (Labsystems) at 450 nm. Two types of reference plasma were used. Reference 1, used in the E L I S A with PV, was the plasma of a volunteer who was vaccinated with PV and in whom the highest level of anti-PV-antibodies was detected. Volunteers were immunized with PV by subcutaneous injection in a subscapular area. The injections were repeated thrice at intervals of 7 days: approximately 0.6, 1.2, and 2.4 mg of protein per injection, respectively [7]. The activity of plasma with PV antigen was assessed as follows: in optimal dilution 1:3200 absorbance at 450 nm of Reference 1 showed plate variation between 0.9 and 1.3; the mean
323
absorbance was 1.0. The test plasma were considered to be active (active donor) when the absorbance values were equal or exceeded 50% of the reference absorbance. When the absorbance >_ 1.0 the plasma was considered to be highly active (highly active donor). In E L I S A with LPS a plasma of a normal blood donor with the highest level of 'natural' LPS antibodies (Reference 2) was used. The activity of the LPS reaction was lower than the activity of the PV reaction, therefore the optimal dilution used was 1 : 1600. The absorbance at 450 nm of Reference 2 varied from 0.5 to 0.8 with a mean of 0.7. The plasma under investigation in the LPS reaction was considered to be active when the corresponding absorbance was in the range 0.7-0.9 and highly-active when it was > 1.0. 3.4. Statistical methods Significance of the difference between groups was assessed by Van der Varden [13].
4. R E S U L T S Table 1 summarizes the results of the normal plasma study with the PV antigen. The plasma of 42 (24.4%) out of 172 blood donors from the Helsinki area contained high levels of PV-antibodies, at a dilution of 1:3200 (active donors) and 9 (5.3%) contained very high PV-antibody titres with absorbance > 1.0 at the same dilution (highly active donors). When plasma of 43 blood donors of the Moscow area were studied in PV-antigen test 15 (34.4%) had high levels of PV-antibodies (active donors) and in one donor (2.3%) there was a very high level of antibodies (A450 nm was 1.1 at dilution of 1:3200; highly active donor). There was no significant statistical difference between the number of active or highly active donors in both blood donor groups. Table 2 illustrates the results of the test with another antigen, the LPS of P. cerasi. In the
Table 1 Results of the screening of donors plasma by ELISA with PV-antigen Area
Total
Negative
Helsinki
172 (100%) 43 (100%)
121 (72.3%) 27 (63.8%)
Moscow
Number of donors:
Difference in % between Helsinki and Moscow:
active a
highly-active b
active ~
highly-active b
42 (24.4%) 15 (34.9%)
9 (5.3%) 1 (2.3%)
2`2 = 2.1 P>0.1
A~2 = 0.6 P>0.3
Active, absorbance at 450 nm 0.5-0.9; b highly-active, absorbance at 450 nm _> 1. The plasma samples were diluted 1:3200.
Table 2 Results of the screening of donors plasma by ELISA with LPS of P. cerasi Area
Helsinki Moscow
Total
172 (100%) 43 (100%)
Number of donors:
Difference in % between Helsinki and Moscow:
active a
highly-active b
active a
highly-active b
23 (13.4%) 4 (8.6%)
1 (0.5%) 0
X 2 = 0.73 P > 0.3
X 2 = 0.27 P > 0.5
a Active, absorbance at 450 nm 0.7-0.9; b highly-active, absorbance at 450 nm _> 1. The plasma samples were diluted 1 : 1600.
324 Helsinki group there were 23 (13.4%) active donors and one highly active donor (0.5%), whereas in the Moscow group there were 4 active donors (8.6%).
5. D I S C U S S I O N The results of the present study suggest that among healthy persons there are subjects with a high or very high level of 'natural' antibodies to the common polysaccharide antigen (CPA) or protein protective antigens of the outer membrane of P. aeruginosa. The suggested criterion for the evaluation of the activity of normal plasma by an E L I S A provides an easy screening method of more than 200 blood donor plasma specimens in order to select active donors with specific anti-P, aeruginosa antibodies. Earlier [3] ' n o r m a l ' gamma-globulin manufactured by Biomed (Petrovo-Dalnee, Moscow, U.S.S.R.) was examined. In some batches of pooled IgG high titres of antibodies to O-antigens and to P. aeruginosa slime antigens were detected. These preparations protected mice against P. aeruginosa experimental infection. When clinical trials of such pooled IgG of gamma-globulin were performed, a therapeutic effect was found when infants younger than two years with severe forms of P. aeruginosa infection were treated [14]. Results of the present study show that normal blood donor plasma containing high or very high levels of anti-P, aeruginosa antibodies (so-called selected plasma) can probably be used for the treatment of P. aeruginosa infections in humans. The other use of selected plasma is the production of anti-Pseudomonas immunoglobulin for intravenous administration. A question arises: what is more u s e f u l - - i m m u n i z a t i o n of donors with PV in order to obtain immune plasma or selection of normal donor plasma based on the P. aeruginosa antibody level? According to previous data [7] immunization of volunteers with PV stimulates the formation of specific antibodies in 9 4 - 9 7 % and approximately in half of them a high level of antibodies was
revealed. High levels of antibodies against P.
aeruginosa are seen in approximately 25-35% of normal donors (active donors) and very high levels in approximately 2 - 5 % (highly active donors). It is known that so-called 'natural' antibodies against the nosocomial pathogen, P. aeruginosa, can be detected in blood of healthy people [2]. The formation of such antibodies is induced by an asymptomatic carriage of P. aeruginosa or earlier infection in anamnesis. We called these antibodies as 'natural', to distinguish them from the antibodies produced by immunization. It is worth mentioning that high or very high levels of 'natural' antibodies to PV (i.e. to protein protective antigens), revealed approximately in one third of non-immunized donors, almost coincides with the high level of corresponding antibodies in approximately 50% of immunized donors. We consider that both methods of preparation of anti-P, aeruginosa plasma should be used: immunization and selection. Both types of anti-P. aeruginosa plasma might find a broad application in practical health care for the treatment of severe forms of P. aeruginosa infection, especially P. aeruginosa sepsis.
ACKNOWLEDGEMENTS We are grateful to Dr. G u n n a r Myllyla from the Finnish Red Cross Blood Transfusion Centre for 172 normal blood donor plasmas, for the discussion of the present paper, and for his valuable comments. We wish to acknowledge Dr. D.E.S. Stewart-Tull from Glasgow University for his valuable comments and revision of the paper.
REFERENCES [1] Hughes, J.M., Culver, D.H., White, J.W., Jarvis, W.R. et al. (1983) Nosocomial infection Surveillance, 1980-1982. In: U.S. Department of Health and Human Services/ Public Health Service Centers for Disease Control from the Morbility and Mortality Weekly Report Surveillance Summaries. November, Vol. 32, No 4SS. [2] Stanislavsky, E.S. and Kolker, I.I. (1978) Pseudomonas
325
[3]
[4]
[5]
[6]
[7]
[8]
infection (Review). All-Union Research Institute of Medical and Medico-Technical Information. Moscow, 102pp. Stanislavsky, E.S., Boultuci, L.G., Yushkova, N.A., Chekan, L.V., Zaidner, I.G., Koutomova, L.R., Mignivitsh, L.R. and Landsman, N.M. (1983) Immunite passive dans l'infection experimentale P. aeruginosa. Rev. Inst. Pasteur de Lyon, 3, 325-344. Stanislavsky, E.S., Edvabnaya, L.S., Bulk, V.F., Zhvanetskaya, M.I. and, Mashilova, G.M. (1986) Immunocbemical and immunological study of cell-wall proteins of Pseudomonas aeruginosa Acta Microbiol. Hungarica 33, 245-255. Specht, B.U. Von. Strigl, G., Ehret, W. and Brendel, W. (1987) Protective effect of an outer membrane vaccine against Pseudomonas aeruginosa infection. 'Infection' 15, 408-412. Stanislavsky, E.S., Edvabnaya, L.S, Bandman, O.A., Bulk, V.F., Zhvanetskaya, M.I. and Vargina, A.K. (1989) Experimental studies on the protective efficacy of a Pseudomonas aeruginosa vaccine. Vaccine 7, 562-566. Stanislavsky, E.S., Balayan, S.S., Sergienko, A.I., Makarenko, T.A., Edvabnaya, L.S., Krohina, M.A., Rusanov, V.M. (1991) Clinico-immunological trial of Pseudomonas aeruginosa vaccine. Vaccine 285 (in press). Sawada, Sh. and Takashi, K. (1985) A new common polysaccharide antigen of strains of Pseudomonas aerugi-
[9]
[10]
[11]
[12] [13] [14]
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