Pharmacology & Pharmacy, 2010, 1, 27-32
10.4236/pp.2010.11004 Published Online July 2010 (http://www.SciRP.org/journal/pp)
27
The Porcine Pulmonary Surfactant Protein A
(pSP-A) Immunogenicity Evaluation in the
Murine Model
Sandra de Cássia Dias1,3, Fernanda Lucio dos Santos2, Dirce Sakauchi2, Dmitri Iourtov2, Isaias Raw3,
Flavia Saldanha Kubrusly3
1Engenharia de Bioprocessos-Campus Alto do Paraopeba, Universidade Federal de São João Del-Rei, Minas Gerais, Brazil; 2Divisão
Bioindustrial, Instituto Butantan, Sao Paulo, Brazil; 3Centro de Biotecnologia, Instituto Butantan, Sao Paulo, Brazil.
Received June 8th, 2010; accepted July 9th, 2010.
ABSTRACT
This paper investigated the porcine surfactant protein A (pSP-A) immunogenicity in murine model. Many elegant stud-
ies about SP-A therapeutic applications are available however specific studies about its exogenous immunogenicity
were not easily assumed. Therefore, we investigated the immunogenicity of this porcine protein in mice. The mice re-
ceived pSP-A sub cutaneously on days 0 and 7. The animals were observed during 90 days and the blood was collected
on days 30, 60 and 90 for assessment the immunogenic potential of pSP-A. Some animals showed circulating antibodies
above the screening cut point, which was calculated based on control mice sera signals. However, those antibodies
were considered false positive rea d-outs by the performed competitive in hibition assay. Also no neutralizing an tibodies
were detected able to avoid the porcin e protein ability to promote lipid aggregatio n. So far in this mode l, porcine sur-
factant protei n -A could be considered not imm u no genic.
Keywords: Immunogenicit y , Porcine Surfactant Protein A, Anti-Drug Antibody, Murine Model
1. Introduction
Alveolar type II cells produce surfactant protein A, SP-A.
This protein belongs to a group of soluble humoral pat-
tern recognition receptors, called collectins, which mod-
ulate the immune response to microorganisms [1]. The
primary unit of a collectin contains an amino-terminal
collagen like domain and a carboxyl terminal lectin or
carbohydrate recognition domain (CRD) united by a
more hydrophobic neck. SP-A is assembled as hexamers
of trimers. This super structure can bind, agglutinate,
opsonize and neutralize many different pathogens and
can also modulate the uptake of these microorganisms by
phagocytic cells as well as the inflammatory and the
adaptive immune responses. Recent data have also high-
lighted their involvement in clearance of apoptotic cells,
hypersensitivity and a number of lung diseases [2-4].
Because of its ability to protect from infection by a
wide variety of microorganisms and its capacity to regu-
late the inflammatory response SP-A might be used as a
paradigm to develop drugs to prevent or treat lung infec-
tions [3]. Despite of a huge number of reports deals with
the role of SP-A for pulmonary pathology and its proba-
bly use in lung disease treatment, very few reports men-
tioned the immunogenicity of SP-A after exogenous ad-
ministration [5]. The SP-A high molecular mass, oli-
gomeric structures and glycosylation can contribute for
immunogenicity development, but theses properties are
important to SP-A activity in vivo and should be main-
tained for therapeutics applications of this molecule.
Regulatory discussions about immunogenicity of the-
rapeutic proteins represent today a central issue of bio-
pharmaceuticals both by developers and by regulators
cause an unwanted immunogenicity may lead to a loss of
product efficacy besides severe side effects. These ef-
fects could develop more deleterious consequences to the
patient (allergy, anaphylaxis, serum sickness, neutraliza-
tion of the drug or native protein) [6,7].
Since we have been studying porcine lung as a raw
material for the development of potential lung disease
medicines, we purified porcine SP-A (pSP-A) as by-
product of lung surfactant production. The reduced
number of reports about immunogenicity of exogenous
SP-A administration prompted us to investigate the im-

The Porcine Pulmonary Surfactant Protein A (pSP-A) Immunogenicity Evaluation in the Murine Model 28
munogenicity of pSP-A in the murine model.
2. Materials and Methods
2.1 Purification of Porcine Surfactant Protein
A (pSP-A)
The pSP-A was purified from a lung extract previously
clarified. This extract is a waste from the main produc-
tion. The short protocol involved an acid precipitation of
the rejected extract before affinity chromatography (pat-
ent pending). LPS free pSP-A was obtained after the en-
dotoxin removal by polymixin B-agarose (Pierce).
2.2 Gel Filtration Chromatography
Oligomerization was assessed by size exclusion chroma-
tography. The pSP-A was loaded onto Superdex 200 HR
column (10/30) (Ge Healthcare) in 0.1 M ammonium
acetate- acetic acid buffer (pH 8.0). The flow rate was
0.3 mL/min and the peaks were manually collected. The
chromatography was calibrated using the molecular mass
standards (Thyroglobulin-669 kDa, aldolase-158 kDa,
albumin-66 kDa, ovalbumin-43 kDa, chymotrypsinogen-
25 kDa).
2.3 Protein Assay
Protein concentration was determined using a bicin-
choninic acid protein assay Kit (BCA; Pierce) using bo-
vine serum albumin (BSA) as a standard.
2.4 Gel Electrophoresis
Purity of pSP-A were determined by 12.5% SDS-PAGE
under reducing (β-mercaptoethanol) and non-reducing
conditions. Relative molecular masses of pSP-A were
estimated using molecular mass standards run in parallel.
2.5 Western Blot
The pSP-A was resolved by SDS-PAGE (12.5%) under
reducing conditions and electroblotted from gel to the
polyvinylidene difluoride membrane. After blocking, the
membrane was incubated with rabbit anti-human SP-A
polyclonal antibody from patients with rheumatoid ar-
thritis or with mouse anti-human SP-A monoclonal anti-
body (HYB 238-04-S BIOPORTO) or with rabbit an-
ti-porcine SP-A polyclonal antibody.
2.6 Subcutaneous Administration of pSP-A
Forty female Swiss mice (20-22 g) were housed in cages
at room temperature (22 ± 1°C) and 12 h light-dark cycle.
The Instituto Butantan Committee for Research and
Animal Ethics approved the experimental protocol. Mice
were separated in two experimental groups: control and
treatment group. Treatment group-Twenty mice were
injected subcutaneously (sc) with 100 µl of pSP-A at
dose of 5 mg/kg on days 0 and 7. Control group-Twenty
mice were divided in two subgroups. Ten animals were
sc injected with 100 µl of saline on days 0 and 7 and the
other ten were not injected. All animals were bled
through the ophthalmic plexus on days 30, 60 and 90.
The serum was separated and individually stored at
–20°C.
2.7 Screening for Circulating Antibodies against
pSP-A
The presence of anti-pSP-A polyclonal antibody in mice
serum was investigated using an indirect ELISA assay.
Wells were coated with 100 ng of pSP-A in coating buf-
fer (carbonate-bicarbonate pH 9.6) overnight at 4°C fol-
lowed by blockage with 10% fetal calf serum in PBS-T.
Control or treated mice sera diluted 10, 100 and 1000
times were added to search circulating antibodies against
the protein. After that incubation, the second antibody
(goat anti-mouse IgG peroxidase-conjugate from Sigma)
was added. The optical density was read at 492 nm using a
microtiter plate spectrophotometer (Multiskam). The
screening cut point was calculated using the individual
control sera results in triplicate obtained from the three
bleedings and from the three used dilutions. ANOVA
estimated the mean and the standard deviation results to
be used in the parametric method: mean + 1.645SD [8].
2.8 Competitive Immunoassay to Confirm the
Positive Read-Outs against pSP-A
A competitive immunoassay was performed to dis- cri-
minate false positive to the actual positive read-outs. The
same indirect ELISA procedure described above was
used with exception that potential positive samples di-
luted 1:10 were previously incubated or not with pSP-A
25 μg/mL, overnight at 4°C. The assay was standardized
using the rabbit polyclonal anti-pSP-A produced with
Freund complete adjuvant (FCA) as the positive control
diluted 1:2500 versus different pSP-A concentrations to
built a calibration curve to establish the necessary
amount of pSP-A to assure the specific inhibition or the
specificity cut point. The chosen pSP-A concentration
exceeded forty times the necessary amount. The per-
centage of signal inhibition is the ratio of pSP-A inhib-
ited sample by uninhibited sample calculated by the for-
mula:
100 1
perce ntage of the signal inhibition
s
tudy dtug inhibited sample
unhibited sample


2.9 Screening for Neutralizing Antibodies (NAbs)
against pSP-A
The presence of NAbs was investigated in potential posi-
tive mice sera. The proposed assay verified the pSP-A
dependent of Ca+2 ability to aggregate phospholipids

The Porcine Pulmonary Surfactant Protein A (pSP-A) Immunogenicity Evaluation in the Murine Model 29
vesicles. The following reagents were used: Instituto
Butantan porcine lung surfactant solution (125 μg/mL)
and pSP-A solution (0.24 mg/mL), both in assay buffer
(5 mM Tris-HCl pH 7.4 plus 150 mM NaCl); and aque-
ous CaCl2 solution (2.42 mg/mL). The lipid:pSP-A
weight ratio was of 10:1 [9]. The assay was performed at
25°C in microplates. Phospholipids vesicles (200 μL)
were added in each well, and the turbidity was measured
at 405 nm and monitored at 1 min intervals over 10 min.
Afterward, 10 μL of controls and samples were added on
the respective wells, and the turbidity monitored. Finally,
Ca2+ (10 μL) was added to all and again monitored. For
better specification: pSp-A was the drug reference, the
positive control was the rabbit anti-pSP-A polyclonal
antibody made with adjuvant, the negative controls were
the assay buffer, non-treated mice sera and the rabbit
anti-bovine aprotinin polyclonal antibody and the sam-
ples were pSP-A treated mice sera of the second bleeding
(high and lower responders). All controls and samples
were diluting in a ratio of 1:9 in pSP-A solution. The
reference was prepared as 9 μL of the pSP-A solution
plus 1 μL of the assay buffer.
2.10 Statistical Analysis
Results are expressed as geometric means with the stan-
dard error of three independent observations. The data
were statistically analyzed by ANOVA using the Prisma
program (Graphpad Prism 5, San Diego, Califórnia). P <
0.05 was considered the level of statistical significance.
3. Results and Discussion
3.1 Characterization of pSP-A
The pSP-A was characterized in relation of its purity,
oligomerization and identity (Figures 1 and 2). From
those results it is important to highlight that the protein
shows oligomeric forms (mainly hexamers) able to in-
duce aggregation of phospholipid vesicles [10], one fun-
damental role of the protein in the intra-alveolar surfac-
tant phospholipid organization [9]. For us, one important
tool used to characterize its functionality.
After the subcutaneously injection of pSP-A in mice,
we tried to develop a strategy for the assessment of anti-
body immune responses against pSP-A. First we per-
formed a screening test for detecting the circulating an-
tibodies. The screening cut point was calculated based on
control mice sera response after each bleeding and for
each dilution and the obtained value was 0.20 ± 0.019.
The percentage of reactive samples was determined for
each bleeding and for each dilution and they are 75, 40
and 30 for the first, 90, 85 and 70 for the second and 60,
30 and 20 for the third bleeding (Figure 3). Five animals
(04, 08, 10, 13 and 18) were reactive in all bleeding and
dilutions (Figure 3).
abc
97
66
45
30
20
14
Figure 1. Characterization of pSP-A. Purity: 12.5% SDS-
PAGE (5 μg/lane) under reducing (a) and non-reducing (b)
conditions. (a) Lanes: 1-molecular markers. 2-pSP-A, (b)
Lane: 1-pSP-A. Oligomerization (c): pSP-A (2.0 mg/200 μl)
was loaded onto Superdex 200 column (flow rate-0.3 ml/min,
2 mm/min). I–Molecular mass standards (1-thyreoglobulin,
2-aldolase, 3-albumin, 4-ovalbumin, 5-chymotripsinogen).
II–pSP-A: 1-octadecamers, 2-hexamers, 3-monomers
ac b
28
56
212 1 2 1
Figure 2. Characterization of pSP-A. Identity: Western Blot
(10 μg protein/lane), Lanes: 1-hSP-A, 2-pSP-A. (a)–mouse
anti-human SP-A monoclonal antibody. (b)–rabbit anti-human
SP-A polyclonal antibody from patients with rheumatoid
arthritis and (c)–rabbit anti-porcine SP-A polyclonal anti-
body
Our second step of the program must discriminate
false positive read-outs from the actual ones using a
competitive immunoassay. The specificity cut point was
determined using the rabbit pSP-A polyclonal antibody
made with adjuvant as positive control when incubated
or not with different pSP-A concentrations. When 0.625
μg/mL pSP-A was added in the positive control, an inhi-
bition of 58.18% was obtained (Figure 4). According
results, the specificity cut point was defined as an inhibit-
tion equal to or greater than 50%. When 1:10 reactive
samples from the second bleeding were incubated with

The Porcine Pulmonary Surfactant Protein A (pSP-A) Immunogenicity Evaluation in the Murine Model 30
Figure 3. Humoral immune response to porcine SP-A administered subcutaneous rout. The animals were bled and their indi-
vidual serum used for detection of anti-pSP-A circulating antibodies. The screening cut point (SCP) (0.20 ± 0.019) was ob-
tained using the control group sera response
Figure 4. Competitive inhibition curve of rabbit anti-pSP-A
antibody made with adjuvant. The antibody was incubated
with different concentrations of pSP-A, and the inhibition
quantified by Elisa. The specificity binding cut point was
defined as inhibition equal to or greater than 50%
pSP-A 25 μg/mL (forty times greater to have drug ex-
cess), they showed different inhibitions, but they were
less than 50% for all of them (Figure 5). Thus, pSP-A
treated mice did not develop specific antibodies against
this protein.
Exogenous SP-A administration routes and treatment
regime are still unknown. In this study we used highly
immunogenic conditions, subcutaneous rout and two
doses on days 0 and 7 with physiologic protein concen-
tration. However, in the last bleeding we observed a de-
crease of the supposed anti-pSP-A antibodies. Theses
results appointed to an antibody transient production and
probably do not do have clinical relevance.
One last step was performed to conclude our valida-
tion program, we screened for NAbs in potential positive
samples. For that purpose we used the pSP-A ability to
Figure 5. Competitive inhibition assay. Mice second bleed-
ing individual serum (1:10) were incubated with pSP-A (25
μg/mL). The protein concentration was forty times greater
than used in the inhibition curve. Elisa quantified the inhi-
bition
aggregate phospholipid vesicles [11]. Ruano et al. (1996)
showed that after Ca2+ addition the light absorbance of
SP-A/lipid aggregates increases 20-25% [9]. We had
similar results (Figure 6). Moreover, any mice treated
sera did not affect the p-SP-A ability to phospholipids
aggregation, which means we did not detected NAbs
against p-SP-A (Figure 6). All reactive samples are false
positives.
Our pSP-A immunogenicity evaluation led us to con-
clude that at least for the murine model the porcine pro-
tein has no immunogenic potential.
Are those results enough to propose p-SP-A as one
safety and efficacy drug for human lung disease treatment?
Or we must consider the predictive value of animal mod-
els for the evaluation of immunogenicity in humans to be
usually low [8]. So, what to do?

The Porcine Pulmonary Surfactant Protein A (pSP-A) Immunogenicity Evaluation in the Murine Model 31
Figure 6. Assay for detect neutralizing antibodies (Nabs) in serum of mice treated with pSP-A. The Nabs must destroy pro-
tein ability to lipid aggregation. Samples and references were filled with 125 μg/mL phospholipids. After 10 min, buffer, sam-
ples, positive and negative controls were added in respective wells. Next, Ca2+ (1 mM final concentration) was added in all wells

The Porcine Pulmonary Surfactant Protein A (pSP-A) Immunogenicity Evaluation in the Murine Model 32
4. Acknowledgements
Thanks to Dr. RMF Piazza for kindly donation of the
reagents human SP-A and human SP-A antiserum.
Support: FAPESP, PRONEX, SADIA, Fundação Bu-
tantan.
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