Immunopharmacology and Immunotoxicology, 2012; 34(4): 695–701

© 2012 Informa Healthcare USA, Inc.

ISSN 0892-3973 print/ISSN 1532-2513 online
DOI: 10.3109/08923973.2011.653645

RESEARCH ARTICLE

Cross reaction between proteins from Larrea divaricata

Cav. (jarilla) and cellular and extracellular proteins of
Pseudomonas aeruginosa
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C. Sasso1, M. A. Mattar de Anaya1, R. Davicino1, R. Martino1, Y. Casali2, S. Correa3, and B. Micalizzi1,*

Microbiology Unit, Department of Biochemistry and Biological Sciences, Faculty of Chemistry and Biochemistry and
1

Pharmacy, National University of San Luis, San Luis, Argentina, 2Bromatology, Test and Evaluation of Drugs Unit,
Department of Pharmacy, Faculty of Chemistry, Biochemistry and Pharmacy, National University of San Luis, San Luis,
Argentina, and 3Immunology, CIBICI (CONICET) Department of Clinical Biochemistry, Faculty of Chemical Sciences,
National University de Córdoba, University Campus, Córdoba, Argentina

Abstract
Larrea divaricata is widely used in folk medicine to treat different pathologies, but little is known about its immunological
properties. Pseudomonas aeruginosa is an opportunistic pathogen which causes several intrahospitalary infections.
We aimed to assess the immunological relation between proteins from a crude extract of L. divaricata Cav. (JPCE)
For personal use only.

and cellular and extracellular proteins (EP) of P. aeruginosa, as well as to establish the cross reactivity between
proteins of both species using a mouse anti-JPCE serum. Protein profiles of JPCE and P. aeruginosa were analyzed
by SDS-PAGE. The percentage of similarity of protein bands between these two species was 43–57%. However, JPCE
proteins were immunogenic. The reactivity of mouse anti-JPCE antibodies against different fractions was studied by
western blot. The anti-JPCE serum detected several antigenic bands on different bacterial proteins. Several common
immunoreactive bands were detected (27–100%) when bacterial proteins were incubated with anti-JPCE serum
(heterologous reaction) and anti-bacterial proteins serum (homologous reaction). By enzyme-linked immunosorbant
assay (ELISA) assays, high titers of anti-JPCE against different types of cellular bacterial fractions were observed
(1/1280–1/2080). Our data clearly demonstrate that antibodies elicited with L. divaricata crude extract are able to
cross-react with cellular and EP of P. aeruginosa. These findings could be relevant in the development of alternatives
therapies for patients suffering intrahospitalary opportunistic infections with P. aeruginosa.
Keywords:  Larrea divaricata, Pseudomonas aeruginosa, cross reaction, ELISA, antigen similarity

Introduction been determined. In JPCE are present mainly proteins

Larrea divaricata Cav. (jarilla) is a plant that belongs
to the Zygophyllaceae family. This species is the most
widespread of the entire genre, from Mexico to Chile and
Argentina.(1) This plant is widely used in folk medicine
for the treatment of microbial infections, wounds, rheu-
matism, gastric disturbances, and tumors. In this study,
we used proteins from a partially purified crude aqueous
extract of jarilla (JPCE), which chemical nature has not
of molecular weights greater than 20 kDa. Experimental
studies have demonstrated fungotoxic and antimicro-
bial activity of L. divaricata extracts,(2–4) antitumoral
effects,(5–7) immunostimulatory effects,(8,9) and cross reac-
tion between proteins of L. divaricata and proteins of
Gram-negative bacteria(10)
Pseudomonas aeruginosa (P. aeruginosa) is a Gram-
negative, aerobic, rod-shaped bacterium with unipolar

*Present address: Microbiology Unit, Department of Biochemistry and Biological Sciences, Faculty of Chemistry and Biochemistry and
Pharmacy, National University of San Luis, San Luis, Argentina.
Address for Correspondence:  Blas Micalizzi, Microbiology Unit, Department of Biochemistry and Biological Sciences,
Faculty of Chemistry and Biochemistry and Pharmacy, National University of San Luis, Chacabuco and Pedernera, 5700, San Luis,
Argentina. Tel: +54 (2652)-520300. Int: 242. Fax: +54 (2652) 520300. E-mail: blasmi@unsl.edu.ar
(Received 26 October 2011; revised 21 December 2011; accepted 24 December 2011)

695
 696  C. Sasso et al.
motility. As it rarely causes infections in immunocom-
petent patients, it is considered an opportunistic patho-
gen. It is a major health problem in hospitals, especially
when dealing with immunocompromised patients
(i.e. transplanted, patients with cancer, severe burns,
or AIDS).(11) This situation is exacerbated by the diffi-
culty of treating infections with P. aeruginosa because
of its high natural resistance to several antibiotics
and disinfectants.(12,13) P. aeruginosa produces several
extracellular products which play a major role in acute
infection caused by this bacterium, for example, exo-
toxin A, exoenzyme S, phospholipase C, rhamnolipid,
elastase, and alkaline protease(14,15) Despite the high
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Monash University on 07/04/13
number of patients that may develop infections with P.
aeruginosa, there is no a vaccine currently available in
the market.(16,17)
The aim of our work was to assess the immunological
relation between proteins of L. divaricata Cav. and cel-
lular and extracellular proteins (EP) of P. aeruginosa, as
well as to establish the cross reactivity between proteins
of both species.
Materials and methods
Plant material
Leaves and little tender branches of L. divaricata Cav.
were collected in Nogolí, San Luis, Argentina, on March,
For personal use only.
2009. The plant was identified in the herbarium of the
National University of San Luis, where is placed the
voucher of the specimen number: UNSL No. 467.
Crude extract
The crude extract was obtained as following: Immediately
after collection, 15 g of leaves and tender branches fresh
in 100 mL of PBS, pH 7.4 (15% w/v) at 4°C during 24 h,
then it was grounded in a mortar, and the aqueous frac-
tion was filtered by using a filter paper (Whatman N° 40).
This crude extract was used to obtain partially purified
concentrated proteins.
Obtention of purified proteins of crude extract
The crude extract was filtered through a 0.45 µm mem-
brane and sterilized by a 0.22 µm membrane. Proteins
were concentrated and partially purified by using mem-
brane concentrators (Centriplus Amicon) with a 10 kDa
cutoff.(18)
Bacterial strain and inocula
The reference strain of P. aeruginosa ATCC 27853 was used,
obtained from the American Type Culture Collection
(MD, USA), and kindly provided by Prof. Olga Aliendres
of Centorbi (Laboratory of Microbiology UNSL, San Luis,
Argentina). P. aeruginosa was isolated on Mac Conkey
agar, at 37°C for 24 h; 1 mL of a bacterial suspension (in
distilled water) with turbidity comparable to 0.5 pattern
of the McFarland scale (1–2 × 108 CFU/mL) was added
to 150 mL of protein free Eagle medium and incubated at
37°C with constant stirring for 24 h.
 Immunopharmacology and Immunotoxicology
Cellular and EP of P. aeruginosa
Cellular fractions obtained as described by Michiels
et  al. soluble cellular proteins (SCP), total membrane
proteins (TMP), and soluble cytoplasmic and periplas-
mic proteins (CP) were obtained. Cells were separated
from the culture by centrifugation 10 min at 8000g. The
cells were washed and adjusted to OD600 = 1 and then
sonicated for 4 min at 20 kHz in sonication buffer (10 mM
Tris-HCl, 5 mM MgCl2 pH 8). For the obtention of SCP,
the sonicated was centrifugated 20 min at 1000g and the
supernatant used as antigen. For TMP, the proteins were
obtained by centrifugation of the SCP, 60 min at 40.000g.
The pellet was resuspended in PBS and used as antigen.
The CP were obtained from the supernatant of TMP by
adding three volumes of acetone and incubated 1 h at
4°C. Then centrifugated 10 min at 8000g, the pellet was
dry and resuspended in PBS.(19)
EP were obtained as described by Daza et al. The bac-
teria were collected from the culture by centrifugation
10 min at 27.138g, 4°C. The supernatant was filtered by
0.22 µm membrane and dialyzed with distilled water 24 h
at 4°C. Later, the EP were lyophilized and resuspended in
2 mL of saline solution (8.5%).(11)
The protein content was determined by Lowry’s
method(20) employing a standard of 0.2 mg/mL
albumin.
Animals
Rockland mice, 6–8 weeks old of both sexes (18–21 g)
were employed. Animals were housed and cared for at
the Animal Resource Facilities, Faculty of Chemistry,
Biochemistry, and Pharmacy, National University of San
Luis, in accordance with institutional guidelines.
Active immunization
Animals were immunized twice subcutaneously with
a 3-week interval, with 0.2 mL of 0.3 mg/mL JPCE or
0.1 mg/mL of SCP, TMP, CP, and EP in AlPO4 (1:1) as
adjuvant. Fifteen days after the second dose, serum
from retro-orbital and submandibular blood was
obtained.
SDS-PAGE and western blot
JPCE, SCP, TMP, CP, and EP were identified by using
SDS-PAGE.(21) Protein samples were electrophoresed (20
μl/lane) through a 12% separating polyacrylamide gel
using the discontinuous buffer system of Laemmli.(22)
The protein samples were boiled for 4 min in sample buf-
fer (6.25 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 5%
β-mercaptoethanol and 0.025% bromophenolblue). Gels
were run on a Miniprotean vertical slab electrophoresis
cell (Bio-Rad Laboratories, Richmond, CA, USA) and
protein bands visualized by Coomassie blue and silver
staining for proteins. Their molecular masses (Mm) were
identified by comparison with patterns whose Mm are
known (kDa): 66 (bovine serum albumin), 45 (egg albu-
min), 29 (carbonic anhydrase), and 20 (trypsin inhibitor)
(Pharmacia).

For western blot assays, separated proteins were elec-
troblotted onto nitrocellulose membrane as previously
described.(10,23) Membranes were blocked and incubated
overnight with anti-JPCE, anti-SCP, anti-TMP, anti-CP,
and anti-EP sera. Immunodetection was performed
using Western Lightning Chemiluminescence Reagent kit
(PerkinElmer Life Sciences, Inc., Boston, USA), accord-
ing to the protocol provided by the manufacturer. The
bands were digitalized with a scanner (UMAX S-GE) with
Corel Photo-Paint X5 version 15.0.0.489 (C) 2010 Corel
Corporation and the relative intensity (RI) of individual
bands was analyzed. Each protein was normalized to
the estimated value of the background relative intensity
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Cross reaction between proteins  697
Results
Protein profiles
Protein profiles of JPCE and SCP, TMP, CP, and EP of P.
aeruginosa were analyzed by SDS-PAGE. A high num-
ber of bands in the JPCE (18 bands) were identified
(Figure 1A, Lane 2). The Mm of bands ranged from 20
to 176 kDa. However, a large number of bands were
observed in the profiles of the different fractions of P.
aeruginosa: SCP, 19 bands; TMP, 17 bands; CP, 18 bands
and EP, 20 bands (Figure 1A, Lanes 3–6).
We analyzed the percentages of similarity of protein
bands between the different fractions of P. aeruginosa

(membrane of nitrocellulose RI). Arbitrarily, a minimum and JPCE and several common bands were detected. We
of 20% of intensity was required to be considered as appre- found a 43% of similarity between JPCE and SCP, a 57%
ciable band.(10,18) between JPCE and TMP, a 56% between JPCE and CP and
a 47% of similarity between JPCE and EP.
Enzyme-linked immunosorbant assay
The enzyme-linked immunosorbant assay (ELISA) assay
was performed as described by Mattar et  al.(10) Wells of
ELISA plates (Costar, Cambridge, MA, USA) were coated
with 100 μl per well of JPCE, SCP, TMP, CP, and EP at con-
centrations of 50 μg/mL. Anti- JPCE, anti-SCP, anti-TMP,
anti-CP, and anti-EP sera were assayed. To determine
the titer of antibodies the mean absorbance plus two
standard deviations of a 1:50 normal mouse serum was
adopted as cutoff absorbance.
For personal use only.

ELISA inhibition
A qualitative ELISA test was carried out with the anti-JPCE
serum to check the cross reactivity between JPCE and
SCP, TMP, CP, and EP proteins. ELISA plates were coated
with JPCE and blocked. Inhibition experiments were
performed as previously.(10,24) A previously titrated mice
anti-JPCE polyclonal antiserum was preincubated for 1 h
at 37°C with equal volumes of different concentrations of
JPCE, SCP, TMP, CP, and EP proteins (4, 16, and 64 μg/mL).
After incubation, samples were centrifuged at 44700 g and
100 μl of each supernatant was transferred to wells coated
with JPCE and incubated for 1 h at 37°C. Anti- JPCE with-
out preincubation was used as positive control. Results
were expressed as % of inhibition compared with the posi-
tive control.(24)

Data analysis
All the experiments were performed independently at
least two times. Statistical significance was determined
by one way analysis of variance and for further com-
parisons we used the Tukey test. For nonhomogeneous
variance we used the Student’s t-test. A p ≤ 0.05 was con-
sidered statistically significant. The similarity between
homologous and heterologous reactions on western
blot and the similarity of protein bands on SDS-PAGE
were expressed as the Dice correlation coefficient (SD),
as determined with the following equation: SD = 2nAB/
(nA + nB) × 100, where nAB is the number of matching
bands and nA and nB are the total numbers of bands in
profiles A and B.(25)
Figure 1.  SDS-PAGE. Protein profiles of proteins from partially
purified crude extract of jarilla (JPCE) and different fractions of
P. aeruginosa. Lane 1, Mm, molecular mass markers expressed in
kDa, Lane 2, JPCE; Lane 3, SCP, Lane 4, TMP; Lane 5, CP, and Lane
6, EP. The gels were stained with Coomassie brilliant blue (Lanes
1–5) and silver staining (Lane 6). B. Anti-JPCE IgG levels against
different antigens: SCP, TMP, CP, EP, and JPCE. *Significant
differences in levels of IgG anti-JPCE regarding the reaction
with pre-immune serum, C(-). *p < 0.05, ***p < 0.001.

© 2012 Informa Healthcare USA, Inc.

 698  C. Sasso et al.

Assessment of cross-reactivity anti-JPCE with cellular
and extracellular antigens of P. aeruginosa
The reactivity of mouse anti-JPCE antibodies against dif-
ferent fractions was studied by ELISA (Figure 1B). The
reactivity of anti-JPCE sera was significantly higher with
plant and bacterial fractions compared with unimmu-
nized controls. No significant differences in the reactivity
among the different fractions of P. aeruginosa (heterolo-
gous reaction) were observed. Conversely, the homolo-
gous response was significantly lower than that obtained
in the heterologous reaction (p < 0.01).
Assessment of anti-JPCE IgG titers
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with 4 µg/mL of protein. The inhibition observed with the
different antigens proved to be more than 50% compared
with the serum anti-JPCE without inhibition.
Immunoreactivity of sera obtained from JPCE and
cellular and extracellular fractions of P. aeruginosa
We evaluated the immunoreactivity of sera anti-JPCE,
anti-SCP, anti-TMP, anti-CP, and anti-EP against the dif-
ferent antigenic preparations by western blot. Results are
shown in Figure 4 and Table 2.
For the assay, we used a representative pool of sera with
a titer of 1/1600 against homologous and heterologous

Serial dilutions of sera from mice immunized with JPCE were

tested against JPCE, SCP, TMP, CP, and EP. No significant
differences in the IgG titers were obtained when anti-JPCE
was confronted with CP (1/254) and EP (1/730) compared
with JPCE (1/644) (Figure 2). The titers for SCP (1/2080) and
TMP (1/1280) were significantly higher than those obtained
when JPCE was used as antigen in the test (p < 0.05).

IgG levels in sera from mice immunized with

JPCE, SCP, TMP, and EP
Figure 3 shows IgG levels of anti-JPCE, anti-SCP, anti-TMP,
and anti-EP against SCP, TMP, EP, and JPCE, respectively.
Sera from pre-immune mice were used as negative con-
trols. The strongest reactivity was observed when SCP was
For personal use only.

used as sensitizing antigen, high levels of IgG anti-SCP were

observed. Whereas the IgG levels were significantly lower
(p < 0.0001) for anti-JPCE. Interestingly, when JPCE were Figure 2.  Anti-JPCE IgG titers against JPCE and cellular and
employed as sensitizing antigen, levels of anti-SCP IgG extracellular antigenic fractions of P. aeruginosa: SCP, TMP, CP,
detected were higher compared with anti-JPCE (p < 0.05). and EP. The results are expressed as the mean of log titers anti-
When TMP were employed as sensitizing antigen, JPCE. *p < 0.05, **p < 0.01.
high levels of IgG anti-TMP were observed, whereas the
IgG levels were significantly lower (p < 0.0001) for anti-
JPCE. When JPCE were employed as sensitizing antigen,
IgG levels were observed with anti-TMP and anti-JPCE,
with the latter levels significantly lower (p < 0.0001).
When JPCE and EP were tested as sensitizing antigens,
no significant differences in the levels of IgG anti-JPCE
and anti-EP were observed.
The statistical analysis shows that the levels of IgG in
the sera of mice immunized with JPCE, SCP, TMP, and EP
were significantly higher than the reaction obtained with
sera from pre-immune mice (C(-)) compared the four
antigens tested (p < 0.05), except between the levels of
IgG anti-EP when JPCE were used as sensitizing antigen.
Figure 3.  IgG titers of anti-JPCE, anti-SCP, anti-TMP, and anti-EP
against SCP, TMP, EP, and JPCE as sensitizing antigens respectively.
ELISA inhibition test
Pre-immune sera, C(-) were tested with JPCE, SCP, TMP, and EP.
The cross reaction between JPCE proteins and cellular and *p < 0.05, ***p < 0.0001.
EP of P. aeruginosa was established by ELISA inhibition
assay using sera from animals immunized with JPCE. The
Table 1.  ELISA inhibition test using different antigens: JPCE,
titer of anti-JPCE was defined as the maximum dilution of SCP, TMP, CP, and EP with anti-JPCE antibodies.
the antiserum that produced 50% of maximum binding to JPCE SCP TMP CP EP
JPCE as sensitizing antigen. The binding of anti-JPCE to %I 80 80 65 60 70
the specific antigen JPCE was inhibited by preincubation The results are expressed as percentage of inhibition (% I)
with 4, 16, and 64 µg/mL of JPCE (+control), SCP, TMP, CP, compared with the absorbance of positive control (anti-JPCE
and EP. Table 1 shows only the percentages of inhibition serum not inhibited).

 Immunopharmacology and Immunotoxicology

 Cross reaction between proteins  699
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For personal use only.

Figure 4.  Western blot: Ag: JPCE, Ag: SCP, Ag: TMP, Ag: CP, and Ag: EP. Mm, molecular mass markers expressed in kDa; Anti-SCP, antibodies
anti-soluble cellular proteins; Anti-TMP, antibodies antitotal membrane proteins; Anti-CP, antibodies anticytoplasmic and periplasmic
proteins, Anti-EP, antibodies antiexoproducts, Anti-JPCE, antibodies antijarilla partially purified proteins of crude extract; C(-), pre-
immune serum. Arrows indicate common antigenic bands between the heterologous reaction using anti-JPCE serum and the homologous
reaction with anti-SCP, anti-TMP, anti-CP, and anti-EP, respectively.

antigens. For JPCE, 10 immunoreactive bands were detected,
whose Mm were 91, 75, 62, 57, 51, 37, 27, 23, 22, and 20 kDa,
whereas the anti-JPCE serum reacted with 14 bands in SCP,
8 bands in TMP, 4 bands in CP and 2 bands in EP.
However, the immunoreactive profiles of anti-SCP,
anti-TMP, anti-CP, and anti-EP against their homologous
antigens were analyzed. In SCP and TMP 12 immunore-
active bands were observed, in CP, 11 bands, and in EP
only 2 bands were detected.
The immunoreactive heterologous profiles with
anti-JPCE were compared with those homologous
profiles obtained with anti-SCP, anti-TMP, anti-CP,
and anti-EP. Several common immunoreactive bands
were detected in the different immunoreactive profiles
obtained: in SCP, 11 bands (SD = 84%); in TMP, 7 bands
(SD = 70%); in CP, 2 bands (SD = 27%); and in EP, 2 bands
(SD = 100%).
Discussion
Previous studies in our laboratory have reported the
cross reaction between proteins of L. divaricata Cav. and
proteins of Gram-negative bacteria.(10) In this work, we
present the cross reaction with Pseudomonas aeruginosa.
First, we carried out the SDS-PAGE technique, which has
been widely used in the analysis of plant and bacterial
proteins from species closely related.(26–31) The SDS-PAGE
revealed a high number of bands in JPCE, and the expres-
sion of them was comparable to protein profiles obtained
from cellular and EP of P. aeruginosa, demonstrating the
presence of common bands between the two species.(10)
When JPCE was used as immunogen generated titers
to 1/3200 (mean titer 1/600) by the ELISA technique and
the same showed highly immunogenic bands in western
blot. Conversely, we observed in the qualitative ELISA
assay, that the levels of IgG anti-JPCE reacted with cell
antigens and to a lesser extent, extracellular antigens.
These results could indicate that there are antigenic
determinants in JPCE that generate antibodies, and
they recognize epitopes that are distributed in a wide
variety of proteins of P. aeruginosa. The cross reactivity
of anti-JPCE against bacterial antigens was significantly
higher than that obtained in the homologous reaction.
These results demonstrate that the generation of cross-

© 2012 Informa Healthcare USA, Inc.

 700  C. Sasso et al.

Table 2.  Immunoreactive bands of JPCE and cellular and extracellular expression of antigenic proteins that depends on the
antigens of P. aeruginosa. phases of growth.(34) Our results are showing that all
Pseudomonas aeruginosa antigens bacterial proteins, including both cellular and extracel-
SCP TMP CP EP JPCE lular, are reactive with anti-JPCE serum.
α-SCP α-JPCE α-TMP α-JPCE α-CP α-JPCE α-EP α-JPCE α-JPCE Previous studies in P. aeruginosa vaccines investigations
130
demonstrate a low response against specific bacterial anti-
100
gens (i.e. flagella, pilli).(35,36) Although it is well established
91 91 91
88 88
that optimal immunity to acute P. aeruginosa infection is
75 75 75 75 75
mediated by antibodies to the O antigen of the lipopolysac-
65 65 65 charide, it has been technically and conceptually challeng-
62 62 62 62 62 62 ing to produce an immunogenic and protective vaccine
59 59 based on this principle. Reasons include the serologic
57 57 57 57 57 57 57 diversity of these antigens, the poor immunogenicity of the
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51 51 51 51 51 51 purified O antigens, and the need to conjugate isolated O

48 48 antigens to protein carriers for maximal effects.(37) Studies
47 have been performed with outer membrane proteins and
43 EP, but to date, there is not a single candidate antigen that
39 39 39
can be identified as having a large advantage over other
37 37 37 37
bacterial antigens.(16,17,33) Interestingly, our work demon-
33 33 33
strates a good immunological response against those P.
31 31 31 31
29 29
aeruginosa antigens using proteins obtained from a jarilla
27 27
partially purified crude extract. In conclusion, our data
26 26 clearly demonstrate that antibodies elicited with L. divari-
25 cata crude extract are able to cross-react with cellular and
23 23 23 23 23 23 EP of P. aeruginosa. Therefore, our results highlight an
22 22 22 22 22 22 unknown feature of L. divaricata extract activity. Further
20 20 studies are under way to determine whether the anti-JPCE
For personal use only.

antibodies are protective. These findings could be relevant

reactive antibodies depends on the conformational sta-
bility and integrity of the immunogen.(32) We observed a
higher immunogenicity of bacterial cell antigens (SCP
and TMP), which can yield a wide variety of antibodies
capable of recognizing antigenic determinants present in
JPCE.(33)
The anti-JPCE serum detected 10 immunoreactive
bands in JPCE on the western blot technique. When the
serum was treated with cellular bacterial antigens (SCP
and TMP), a higher number of bands were observed.
These results show that the heterologous reaction is
higher than the homologous reaction with anti-JPCE,
possibly due to substantial differences in the nature
and amount proteins. As the generation of cross-re-
active antibodies depends on the conformational sta-
bility and integrity of the immunogen, the denatured
conditions of the SDS-PAGE could be precluding the
detection of certain conformational epitopes shared by
several proteins or subunits.(10) However, we observed
the presence of common and uncommon bands. The
immunoreactive heterologous profiles with anti-JPCE
were compared with those homologous profiles (anti-
SCP, anti-TMP, anti-CP, anti-EP) detecting large simi-
larity of immunoreactive bands: SCP (SD = 85%) and
TMP (SD = 64%) and lower similarity in CP (SD = 27%).
Although there was a 100% similarity with the pro-
files obtained with EP, only few bands were detected.
The low immunoreactivity of anti-EP serum with EP
of P. aeruginosa could be caused by the differential
in the development of alternatives therapies for patients
suffering intrahospitalary opportunistic infections with P.
aeruginosa.
Acknowledgments
This work was supported by funds of CyT project 9601
from the National University of San Luis, Argentina.
Roberto Davicino is a Assistant Researcher from
CONICET.
Declaration of interest
The authors declare no conflict of interest.
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