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Gene Therapy (1999) 6, 637–642
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Inhibition of adenovirus-mediated gene transfer by
bronchoalveolar lavage fluid
A Bastian and B Bewig
First Department of Internal Medicine, Christian-Albrechts-University of Kiel, Schittenhelmstra␤e 12, 24105 Kiel, Germany
Human and animal trials with recombinant adenovirus
have been discouraging, since the level of recombinant
gene expression was low. Nonspecific and specific
immune response mediated by, for example, macrophages, T cells and immunoglobulins may prevent infection or cause death of infected cells. We analyzed the
effect of bronchoalveolar lavage fluid (BAL) on the
efficiency of adenoviral infection in vitro. A total of 26 BAL
samples of randomly selected patients was examined.
Adenovirus-mediated gene transfer was quantified using
AdCMV.Null, a recombinant adenovirus, in a modified titer
assay based on immunocytochemical detection of infected
293 cells. In addition, the concentration of anti-adenovirustype 5 IgA, IgG and IgM antibodies in BAL was determined
by ELISA. 53.8% of the BAL samples (14 out of 26)
reduced adenoviral infectivity by at least 50% (factor of
inhibition ⭓2). All BAL samples effecting a reduction in
adenoviral infectivity contained detectable amounts of antiadenovirus-type 5-IgA antibodies. However, the correlation
between the concentration of IgA antibody and the strength
of inhibition was weak (r = 0.336). Even high levels of antiadenovirus-type 5-IgM, IgG or IgA antibodies did not influence adenoviral infectivity consistently. This observation
indicates that BAL contains (a) anti-adenovirus-type 5 antibodies which are not directed against adenoviral epitopes
responsible for the viral adherence and uptake process;
and/or (b) inhibitors of viral infectivity different from antibodies.
Keywords: BAL; adenovirus; gene transfer; immunoglobulin
Introduction
Gene therapy may become a powerful tool for the treatment of inherited or acquired genetic diseases.1–4 A key
problem is the generation of efficient gene transfer vectors and strategies. Success of adenovirus-mediated gene
transfer is limited by the development of host immune
response resulting in diminished or lacking gene
expression. Two phases of elimination have been
described. Introduction of recombinant adenovirus into
mammalian airways stimulates destructive cellular
responses, leading to the replacement of the airway with
non-transgene-containing cells. This process is thought to
be responsible for the late phase of elimination.5–7 Modification of the adenovirus with inactivation of the E2a
gene in addition to E1 sequences was associated with
longer gene expression and less inflammation in vivo,
indicating possible strategies of circumvention.8,9 However, the recently observed early elimination of virus has
major implications, since 90% of vector DNA is eliminated within 24 h following in vivo administration.10
Macrophages are known to be involved in this process. In
addition, antibody-mediated immune response, primed
during a prior wild-type adenovirus infection, is likely to
reduce the initial efficiency of gene transfer. Depending
on the route of administration, special local features may
have implications on the efficiency of adenoviral gene
transfer. For the treatment of pulmonary manifestations
Correspondence: B Bewig
Received 24 March 1998; accepted 4 November 1998
of genetic diseases, application via the airways seems
promising, since this route provides direct and specific
access to epithelial cells of the bronchi. However, adenovirus administered via the airways has to cross the epithelial lining fluid barrier before reaching the target cell.
No qualitative or quantitative data are available with
regard to how adenovirus infection is influenced by noncellular substances in the bronchoalveolar fluid (BAL).
We examined the effect of BAL from unselected patients
on the infectivity of recombinant adenovirus in vitro
using a modified neutralization assay. Results from these
experiments were analyzed in relation to a quantitative
assessment of specific immunoglobulin A, M and G titers
in BAL directed specifically against recombinant adenovirus type 5, which is frequently used in gene transfer
experiments.
Results
Effect of BAL fluid on adenoviral infection of cells in
vitro
To evaluate the effect of BAL fluid on the infectivity of
recombinant adenovirus in vitro, a modified neutralization test was used. 293 Cells were incubated with normal saline as control or with BAL samples obtained from
different patients. The cells were then infected with
recombinant adenovirus at a dose of 0.001 p.f.u. per cell.
Infected cells were detected immunocytochemically and
counted. The number of infected cells after incubation
with BAL samples was compared to controls (Figure 1).
Of 26 BAL samples, 14 demonstrated significant inhi-
Inhibition of adenovirus-mediated gene transfer by BAL
A Bastian and B Bewig
638
Table 1 Properties of samples from bronchoalveolar lavage
fluid
Factor of
inhibition
22.5
18.0
13.7
9.3
8.4
7.4
7.3
7.3
6.5
4.5
3.4
Figure 1 Effect of bronchoalveolar lavage samples on the infectivity of
recombinant adenovirus in vitro. 293 Cells were grown as monolayer in
96-well plates. After incubation with bronchoalveolar lavage sample
(20 ␮g protein) from unselected patients or normal saline as control, cells
were infected with a recombinant adenovirus AdCMV.Null at a multiplicity of infection of 0.001 p.f.u. per cell. Infected cells were stained
immunocytochemically utilizing an antibody specifically directed against
adenoviral proteins 48 h after infection and counted using light
microscopy. Points indicate percentage change of number of infected cells
for each individual patient when incubated with BAL as compared with
cells incubated with control solution.
2.4
2.3
2.0
1.9
1.9
1.8
1.6
1.5
1.2
1.0
1.0
1.0
1.0
0.9
0.8
Disease
sarcoidosis
healthy
sarcoidosis
sarcoidosis
sarcoidosis
tuberculosis
tuberculosis
carconoma
sarcoidosis
healthy
lymphocytic
pneumonitis
sarcoidosis
lymphoma
healthy
sarcoidosis
carcinoma
carcinoma
bronchitis
sarcoidosis
carcinoma
healthy
lymphoma
sarcoidosis
asthma
asthma
healthy
IgA-AK
quant
(ng/90 ␮g
protein)
Ad-IgG-AK Ad-IgM(extinction)
Ak
(extinction)
36.0
16.0
33.0
91.0
22.0
91.0
8.5
68.0
87.0
54.0
23.5
0.31
0.55
0.82
1.32
0.68
1.00
0.64
0.84
1.47
1.25
0.49
0.03
0.17
0.19
0.15
0.01
0.25
0.10
0.19
0.12
0.24
0.09
49.0
9.2
21.0
12.8
28.5
10.7
0.0
0.0
18.0
21.0
7.7
7.1
7.1
20.0
4.4
0.68
0.77
1.17
0.71
1.02
1.88
0.82
0.15
0.90
0.25
0.21
1.25
0.52
1.37
0.24
0.00
0.06
1.13
0.00
0.00
0.16
0.07
0.02
0.04
0.00
0.02
0.57
0.00
0.50
0.00
bition of adenoviral infection in 293 cells. The extent of
inhibition varied from factor 2 to 22.5, demonstrating a
reduction of infected cells to at least 50% in the presence
of BAL compared with noninfected cells. BAL samples
from three different patients each reduced the number of
infected cells to less than 10% compared with controls.
In 10 samples, BAL was found to have no or little
influence on infectivity of adenovirus (less than 50%
inhibition). Two BAL specimens were observed to
mediate a slight increase in adenovirus uptake into cells.
While three patients who suffered from sarcoidosis had
BAL samples with no significant effect on adenoviral
infectivity, four out of the five samples demonstrating the
most powerful inhibition of adenoviral transfer were
obtained from patients with sarcoidosis (Table 1).
to the inhibitory properties of the BAL specimens
(Figure 2). Most samples (20 out of 26) contained detectable amounts of antibodies against adenovirus type 5.
BAL samples with no effect on adenoviral infectivity contained varying titers of IgA antibodies (between 0 and
87 ng/20 ␮g protein). All samples but one inhibiting
adenovirus infection to less than 20% compared with
control (factor of inhibition less than 5) contained less
than 30 ng IgA/20 ␮g BAL protein, one of the samples
had 49 ng IgA/20 ␮g protein. There seems to be a correlation between inhibitory effect and concentration of IgA
up to a factor of inhibition of 9.3 and IgA of 91 ng/20 ␮g
protein. However, the most powerful inhibitory effects,
which clearly exceeded these samples, were obtained
with samples of medium range IgA titers as high as 36
ng IgA/20 ␮g protein. One sample demonstrating significant inhibition of infectivity contained only low IgA
anti adenovirus antibodies of 8.5 ng IgA/20 ␮g protein, a
range where other samples displayed no inhibitory effect.
Overall correlation was r = 0.336.
Inhibition of adenoviral infectivity and detection of IgA
antibodies against adenovirus in BAL
All samples examined in the infectivity assay were analyzed for the presence of antibodies specifically directed
against recombinant adenovirus type 5, the same adenovirus used in the infectivity assay. IgA antibody content
in BAL was determined using ELISA technique. Results
were expressed as immunoglobulin content relative to
the protein concentration of the BAL samples and related
Inhibition of adenoviral infectivity and detection of IgG
antibodies against adenovirus in BAL
In order to evaluate the relation between IgG specific for
adenovirus type 5 and the effect of BAL on adenoviral
infection of cells, the specific IgG level in BAL was
determined by ELISA and results were correlated with
the results from the infectivity assay. There was a wide
range of anti-adenovirus IgG antibody concentrations in
BAL samples having no effect on the extent of adenoviral
Inhibition of adenovirus-mediated gene transfer by BAL
A Bastian and B Bewig
639
Figure 2 Concentration of IgA-specific against adenovirus type 5 in
relation to the factor of inhibition of adenoviral infection in concentrated
BAL. BAL from unselected patients was rendered cell-free by centrifugation. Protein content was adjusted to 1 ␮g/␮l. IgA concentration was
determined using ELISA technique in a sandwich with an adenovirus type
5 covered titer plate, BAL sample and anti-human IgA-specific antibody
and a standard curve using IgA. Factor of inhibition was evaluated in an
infectivity assay using 293-cells incubated with BAL sample (20 ␮g
protein) or normal saline as control. Cells were infected with
AdCMV.Null at a multiplicity of infection of 0.001 p.f.u. per cell. Infected
cells were stained immunocytochemically utilizing an antibody specifically
directed against adenoviral proteins 48 h after infection and using light
microscopy. The factor of inhibition was defined as the ratio of the number
of infected cells in the control to the number of infected cells in the BAL
sample. Each symbol represents the mean of two values.
Figure 3 Concentration of IgG specific against adenovirus type 5 in
relation to the factor of inhibition of adenoviral infection in concentrated
BAL. Analysis was performed as described for Figure 2, but IgG concentration was determined using ELISA technique in a sandwich with an
adenovirus type 5 covered titer plate, BAL sample and anti-human IgG
specific antibody. Data are presented as extinction read at 405 nm in the
ELISA assay compared with the factor of inhibition. Each symbol
represents the mean of two values.
infection (Figure 3). The samples with the highest and the
lowest IgG titer had both similar effects on adenoviral
infectivity. Mid-range inhibition of infectivity was associated with mid-range IgG titers. The BAL sample which
inhibited infection most strongly, contained low IgG antibody titers comparable to others with no inhibiting
potential.
Inhibition of adenoviral infectivity and detection of IgM
antibodies against adenovirus in BAL
Evaluation of BAL samples for the presence of specific
anti-adenovirus type 5-IgM antibody titer by ELISA technique revealed low level, but detectable amounts of antibodies in most of the patients. Results were expressed in
relation to the extent of inhibition the corresponding BAL
sample exhibited in the infectivity assay. The overall level
of IgM directed against adenovirus type 5 in BAL was
low (Figure 4). There were only three samples demonstrating increased IgM levels clearly different from the
rest. Analysis showed no correlation between concentration of specific anti-adenovirus type 5-IgM and the
influence of the BAL sample on the infectivity of the
adenovirus in vitro. Even the patient with the highest
antibody titer against adenovirus type 5 did not display
relevant change in the infectivity assay if cells were pre-
Figure 4 Concentration of IgM specific against adenovirus type 5 in
relation to the factor of inhibition of adenoviral infection in concentrated
BAL. Analysis was performed as described for Figure 2, but IgM concentration was determined using ELISA technique in a sandwich with an
adenovirus type 5 covered titer plate, BAL sample and anti-human IgM
specific antibody. Results are presented as extinction read at 405 nm in
the ELISA assay compared with the factor of inhibition. Each symbol
represents the mean of two values.
Inhibition of adenovirus-mediated gene transfer by BAL
A Bastian and B Bewig
640
incubated with patient’s BAL. The patient with a BAL
which most strongly inhibited adenoviral infection, had
low antibody titers in the range of other patients with no
inhibition. BAL samples from all three patients
with significant anti-adenovirus IgM antibodies had no
potential to inhibit adenoviral infectivity.
Discussion
Numerous applications revealed adenovirus as a powerful gene transfer vector in vitro. However, success of in
vivo gene therapy utilizing adenovirus as the vector was
limited by the transient expression of the transferred
gene. Several studies analyzing adenovirus-based gene
transfer to different organs in animals and humans
demonstrated progressive loss of recombinant gene
expression over days and weeks.2–5,11–14 The reason for
this phenomenon was in part the development of an
adaptive immune response with generation of antigenspecific cytotoxic T cells recognizing processed viral or
recombinant gene products on the surface of infected
cells. These cytotoxic T cells are thought to destroy
infected cells directly or to mediate apoptosis.5,6,14–17
Consequently, expression from the adenovirus vector
was markedly prolonged in nude mice lacking the T cellbased immune response.17 Strategies to circumvent T cellmediated host defense included modified viral vectors
with diminished expression of viral antigens or enhanced
expression of genes suppressing MHC presentation on
infected cells.18 Even immunosuppressive agents (eg
FK506) were used to prevent cellular immune response.19
Recent studies suggest a more rapid elimination of
adenovirus DNA from the target organ within the first
24 h.10 After systemic vector administration to mice, the
vast majority of vector genome was cleared in the liver.
Innate actions of the immune system like unspecific barrier functions, humoral or cell-mediated functions were
thought to be responsible. However, the precise location
(eg Kupffer cells) and mechanism of vector elimination
remains to be determined. Depending on the route
of administration, local effects possibly inhibiting
expression from adenoviral vectors are of certain interest.
Gene transfer into the lung, a possible target organ for
gene therapy in cystic fibrosis, can be achieved via the
airways. In this scenario systemic clearance is of no relevance. However, adenovirus may face a variety of cellular and non-cellular barriers within the epithelial lining
fluid before finally entering the epithelial cells. Alveolar
macrophages as part of the innate immune system and
T-lymphocytes may be involved in eliminating intruding
viruses. Our study analyzed the role of the non-cellular
fraction of the BAL in modulating the infectivity of
recombinant adenovirus. The data demonstrate that some
BAL samples contain components reducing the infectivity of recombinant adenovirus. Most BAL samples
with the property of reducing adenoviral infectivity contained detectable amounts of anti-adenovirus-type 5-IgA
antibodies. However, concentration of IgA antibody did
not correlate with the strength of inhibition.
Animal and human studies indicate the development
of humoral immune response after wild-type or recombinant adenoviral infection. In a mouse model, airway
infection with adenovirus type 5 produced neutralizing
antibodies in sera and BAL. Isotype analysis revealed that
in sera primarily IgG was found, whereas in BAL both
IgG and IgA were detectable. These antibodies were sufficient to prevent repeated gene transfer. The blocking
humoral responses depended on class II presentation of
viral proteins and activation of T-helper cells finally
resulting in the formation of neutralizing antibodies.14
Twenty-eight days after infection with recombinant adenovirus via airways, BAL samples from mice displayed
neutralizing activity. This neutralizing effect increased
parallel to the rise in IgA antibodies, but was independent of the presence of IgG antibodies.20 The limited
effect IgG from BAL has on inhibiting adenoviral infection as seen in our data was also demonstrated by Kaplan
et al.7 In his study, BAL from adenovirus-infected rhesus
monkeys retained neutralizing activity even after
depletion of IgG antibodies. While IgG does not contribute to inhibition of adenoviral infection, its role in
response to adenovirus remains unclear. IgG is mainly
found in the lower respiratory tract with no increase after
local nasal adenovirus application.21 Hypothesis was that
IgG might be produced in regional lymph nodes and
reach lung parenchyma by diffusion. Since IgG is known
to be involved in opsonization to facilitate phagocytosis,
it may be speculated that this process is not effective in
eliminating adenovirus from mucosal surfaces.
The effect of a single dose of adenovirus application
on systemic antibody titer remains controversial. Crystal
et al3 found no neutralizing antibodies in sera after
recombinant adenovirus-mediated gene transfer to the
human lung. The lack of systemic response may be contributed to by a low virus concentration and the fact that
recombinant adenovirus does not replicate. Data about
BAL findings were not reported. One year after a single
application of adenovirus to the nose of rhesus monkeys,
no antibody titer or neutralizing activity was detectable
in nasal washes.7 In mice, serum IgA raised to a low level
after a first administration of adenovirus vector to the
nose.21 Antibody titers increased after repeated intranasal
administration. Antibody titers found in this study were
significantly higher than those described before, differing
by one order of magnitude.12,22
The potential of antibodies in BAL or sera to neutralize
adenovirus is subgroup-specific, although each subgroup
demonstrates a low level of antigenic similarity. The
major antigenic proteins are hexon, penton and fiber. The
type-specific antigenic epitopes are located on fiber and
hexons, neutralizing antibodies are directed mainly
against hexons.23 Since epitopes on hexons cross-reacting
between the serotypes are directed mainly towards the
inside of the virion, infection with an adenovirus of one
serotype does not produce relevant immunity against
other types.24 Therefore, repeated application of adenovirus-mediated gene transfer using different serotypes
seems feasible. However, in human studies previous
infections with wild-type adenovirus may significantly
reduce efficiency of gene transfer due to preformed IgA
antibodies. In this regard previous adenovirus type 5 or
2 infections are of particular interest since most recombinant adenovirus vectors are derived from these types.
Our results do not support the hypothesis that inhibitory properties of BAL are mediated just by immunoglobulins, such as IgA. Inhibitory effects may be caused by
other components, such as proteases, proteins binding
adenovirus unspecifically, or molecules competing with
adenovirus for cellular binding sites. Possible candidates
are fibronectin and vitronectin which are both present in
Inhibition of adenovirus-mediated gene transfer by BAL
A Bastian and B Bewig
BAL and which inhibit uptake of adenovirus into cells.25,26
Like adenoviral penton base these molecules have RGD
motifs involved in adenovirus internalization.27 Diseases
associated with increased levels of inhibiting molecules
in BAL (eg fibronectin in interstitial lung diseases) may
therefore be less favorable for adenovirus-mediated gene
therapy. In our study population, four of the patients
whose BAL samples inhibited adenoviral infection most
powerfully had sarcoidosis. However, the total number
of patients was too small for this observation to be significant. Further studies will be necessary to detect
components other than immunoglobulins in BAL which
influence adenoviral infectivity. In this regard differences
in diseases and in treatment, eg the application of steroids to alter the composition of the protein profile in
BAL, may be important (eg effects on fibronectin or
vitronectin).
Although no in vitro scenario can simulate physiologic
phenomena in vivo, we suggest pretesting BAL fluid from
those patients who are to be treated for pulmonary diseases in gene therapy protocols using recombinant adenovirus. An assay for neutralizing activity may have significant implications for the treatment, since an inhibitory
factor of 10 means that the dose necessary to be applied
for equivalent effect needs to be increased 10-fold,
possibly resulting in enhanced toxic side-effects.
Materials and methods
Subjects
Twenty-six patients who underwent diagnostic bronchoscopy and bronchoalveolar lavage for sarcoidosis (9),
tuberculosis (2), carcinoma of the lung (4), non-Hodgkin
lymphoma (2), asthma (2), lymphocytic pneumonia (1),
bronchitis (1) or exclusion of pulmonary disease (5) were
randomly selected (Table 1).
Bronchoalveolar lavage (BAL)
Bronchoscopy was done using flexible fiberoptic bronchoscopes (Olympus, Hamburg, Germany). Patients
received atropine 0.5 mg and hydrocodon 7.5 mg subcutaneously half an hour before examination. Up to 10 ml
1% oxybuprocainhydrochloride (Novesine; Wander,
Nürnberg, Germany) or 2% lidocainehydrochloride
(Xylocain; Astra, Hamburg, Germany) were used as local
anesthesia. Supplemental oxygen was administered at a
rate of 2 to 6 l/min.
BAL was performed in the middle lobe bronchus or
the bronchial segment affected by radiological changes.
200 ml of 0.9% sodium chloride solution was applied in
20 ml fractions, each fraction was aspirated by gentle suction and pooled in polypropylene vessels for further
examination. Lavage fluid was filtered through sterile
gauze and centrifuged at 500 g (10 min, 4°C). 50–100 ml
of supernatant free of cells was applied to a centriprep
10 (Amicon, Beverly, MA, USA) and spun at 3000 g to a
final volume of 500–700 ␮l. After concentration, protein
content was determined and adjusted with phosphate
buffered saline (PBS) to a concentration of 1 ␮g
protein/␮l fluid.
Quantification of adenoviral infectivity
293 Cells, a cell line derived from human embryonic
nephroma, were seeded on 96-well culture dishes (Nunc,
Wiesbaden, Germany) and grown to a density of 1 × 105
per well in 100 ␮l of DMEM culture media (GIBCO BRL,
Grand Island, NY, USA) containing 10% fetal calf serum
(FCS), 100 U/l penicillin and 100 ␮g/ml streptomycin.
Cells were incubated with 20 ␮l BAL (corresponding
20 ␮g protein) or normal saline as control for 15 min.
Recombinant adenovirus containing an empty expression
cassette (AdCMV.Null) was prepared as previously
described.28 10 ␮l of AdCMV.Null in serum-free DMEM
were applied to the 293 cells yielding a final concentration of 0.001 p.f.u. per cell. 1.5 h later complete
medium, containing bovine serum at a concentration of
10% was added. Incubation was continued for additional
48 h. Media were aspirated and cells were allowed to dry
in a laminar flow hood. Ice-cold methanol (100%) was
added followed by an incubation at −20°C for 15 min.
Methanol was aspirated and cells were washed twice
with PBS containing 1% BSA (PBS/BSA). The following
steps were performed at room temperature. A polyclonal
rabbit antibody directed against adenoviral antigens was
added to the cells (diluted 1:2000 in PBS/BSA, 50 ␮l per
well). After incubation for 1 h, cells were washed three
times in PBS/BSA. A goat anti-rabbit antibody labeled
with horseradish peroxidase was added (diluted 1:200 in
PBS/BSA, 50 ␮l per well) followed by another wash step
(three times PBS). Infected cells were detected using the
Pure Metal Enhanced Substrate Kit (Pierce, Rockford, IL,
USA) which was applied according to the instructions of
the manufacturer. After development of color, substrate
was replaced by 200 ␮l of PBS. All colored cell spots in
each well were counted microscopically. Each experiment
was done twice and as duplicate.
Protein assay
Protein concentrations were determined using the micro
BCA protein assay reagent kit (Pierce) following the
instructions of the manufacturer. The assay is based on
the biuret reaction and adapted to microtiter plate usage.
Briefly, 2 ␮l of protein standards plus 148 ␮l normal
saline, blanks or samples were added to the plate followed by the application of 150 ␮l of working solution
(containing sodium carbonate, sodium bicarbonate and
sodium tartate in NaOH, bicinchoninic acid in water and
cupric sulfate and pentahydrate in water). After mixing
and 120 min of incubation at 37°C absorbance was taken
at 540 nm. Protein concentration was determined using a
standard curve.
Detection of IgA IgM and IgG anti-adenovirus antibodies
Adenovirus stock solution was diluted 1:1000 in carbonate buffer (1.59 g Na2CO3 and 2.93 g NaHCO3 in 1 liter
H2O, pH adjusted to 9.6). 75 ␮l of this solution was
applied to each well of a 96-well plate (Nunc, Wiesbaden,
Germany) and incubated at 37°C in a humidified
chamber overnight. Plates were washed three times with
0.05% Tween in PBS (PBS-Tween). 75 ␮l of 2% BSA in
PBS were added to each well and incubated at room temperature for 3 h. After three washings with PBS-Tween
BAL samples containing 90 ␮g of protein in 100 ␮l NaCl
were added. Incubation was performed for 4 h at 37°C.
Washing was performed three times with PBS-Tween.
75 ␮l of a 1:4000 dilution of goat anti-IgA antibody labeled with horseradish peroxidase (DAKO, Hamburg,
Germany; No. PO216) were applied to the wells and
incubated for 1 h at 37°C. 10 ml of substrate (0.1 m NaAc,
641
Inhibition of adenovirus-mediated gene transfer by BAL
A Bastian and B Bewig
642
0.05 m NaH2PO4, adjusted to pH 4.2 with acetic acid,
0.5 mm 2,2′-azino-di-(3-ethylbenzthiazoline sulfonate)
(ABTS), Boehringer, Mannheim, Germany) were mixed
with 30 ␮l of 3% H2O2. 100 ␮l of this mixture were added
to each well and absorbance was taken at 405 nm on an
ELISA-plate reader. A standard curve was produced
using dilutions of samples obtained from a patient with
high concentrations of IgA.
A 1:4000 dilution of goat anti-IgG antibody labeled
with horseradish peroxidase (DAKO; No. PO214) and a
1:6000 dilution of goat anti-IgM antibody labeled with
horseradish peroxidase (DAKO; No. PO215) was used, to
determine IgG and IgM anti-adenovirus antibodies.
Statistical analysis
Factor of inhibition and content of immunoglobulin in
BAL were analyzed to determine the correlation (r).
Acknowledgements
We would like to thank RG Crystal, Division of Pulmonary and Critical Care Medicine, CUMC, New York, for
providing the adenoviral vector AdCMV.Null; E FalckPedersen, Department of Microbiology, CUMC, New
York, for providing the rabbit anti-adenovirus antibody;
and Sonja Rohweder for her excellent technical assistance.
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