crude mite extract, Dermatophagoides farinae. Int. Arch. Allergy. Immunol. 1991; 96: 199-205. 7 Yasueda H, Mita H, Yui Y, Shida T. Isolation and characterization.
Allergology International (1996) 45: 195-198
Original
Article
Anti-Dermatophagoides in allergic
farinae
type
Matsunobu
Suko,2 Hirokazu
Department of Otolaryngology, School of Medicine, Showa University Therapy, Faculty of Medicine, University of Tokyo, Tokyo, Japan
ABSTRACT
Sera from 27 patients with mite-sensitive allergic rhinitis, without atopic dermatitis and bronchial asthma, were examined for anti-Der f I and anti-Der f II IgE antibody contents by enzyme-linked immunosorbent assay (ELISA).Anti-Der f I anti-Der
f II IgE
32.68±0.88ng/mL
antibody
levels
was
IgE antibody
patients.
in these
In comparison
and
while
that the
the
pattern
as in that
inverse
was
over
between
of patients the
case
in
anti-
anti-Der
f
study the pres-
serum
with allergic
and The
the
of a previous
ratio
in patients
14.78±1.34
respectively.
predominant
with the results
indicates
II IgE antibodies
the same
were
(mean±SEM),
Der f II IgE antibody
ent study
II IgE antibodies
rhinitis
Harumi Suzaki,1 Go Matsuzaki,2 and Yasuya Nomura1
and
I and
anti-Der
rhinitis
indicated
with bronchial
asthma,
patients
with
f I
atopic
dermatitis.
These results indicate that immunological features and major allergen molecules could be different in different atopic diseases. At present it is not clear where this difference comes from, but the route of immunological sensitization (via respiratory tract vs via skin) might result in the difference. Key words: Der f I, Der Ill, IgE antibody, major allergen, mite.
Okudaira2
and 2Department
of Medicine
and Physical
The purification of mite allergen has been attempted by many investigators. At present, there is some general consent that the major allergens purified from both species may be grouped into two main groups of mite allergen, one group with a molecular weight of about 25kDa and another with a molecular weight of about 15kDa. According to the new allergen nomenclature system,5 the major allergens for the house dust mites are to be designated as Der p I and Der f I for the large molecular-weight allergens, while the low molecular-weight allergens are called Der p II and Der f II, all isolated from either of the species. Anti-mite IgE antibodies have been routinely measured in clinical practice for the management of mite-sensitive atopic patients, however the allergen preparations used for the assay were crude extracts of mite body or mite culture. This limitation prevented detailed analysis of anti-mite IgE antibodies in different atopic diseases. Recent progress in mite allergen research enabled us to analyze the anti-mite IgE antibodies directed to different allergen molecules in different atopic diseases. However, there have been few reports on anti-Der f I and antiDer f II IgE antibodies in patients with allergic rhinitis. In this study, anti-Der f I and anti-Der f II IgE antibody contents were examined in patients with allergic rhinitis.
METHODS INTRODUCTION
Subjects
Allergy to house dust is an important cause for atopic diseases such as allergic rhinitis, bronchial asthma and atopic dermatitis in many countries. It has been well established that two different species of mite, Dermatophagoides pteronyssinus and Dermatophagoides farinae, are the major sources of allergen in house
Twenty-seven patients with perennial mite-sensitive allergic rhinitis, without bronchial asthma and atopic dermatitis, were subjected to the study. The group was composed of 18 males and 9 females aged between 15 and 55 years (mean age 31.1).
dust.1-4 Preparation Correspondence: Dr Harumi Suzaki, Department of Otolaryngology, Showa University,1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan. Received 16 February 1996. Accepted for publication 17 September 1996.
of crude
mite extract
of D. farinae
Dermatophagoides farinae was defatted with ether, 20mL of phosphate-buffered saline (2mmol/L, pH 7.4) was added to 1g of
defatted
mite and the
mixture
was
rotated
for
48h
at
4℃.
196
H SUZAKI ET AL.
Preparation
of Der f I and
II
Isolation of Der f I was attempted by a method described previously6 and isolation of Der f II by the method reported by Yasueda et al.,7 with slight modifications. The columna such as CM-cellulose (CM-52, Whatman, Springfield Mill, Kent, UK) and Sephadex G-75 (Pharmacia, Uppsala, Sweden) were used when the isolation of Der f IIwas carried out. These were different from those of Yasueda et al. Enzyme-linked immunosorbent I and II IgE antibodies
assay
for anti-Der
f
For anti-Der f I and II IgE antibody measurements, micro-titer plates (Immulon 2; Dynatech Laboratories, Chantilly,VA, USA) were
incubated
μg/mL
with
of Derf II
100μL/well
in 0.1mol/L
of
10μg/mL
corbonate
Der
buffer
(pH
f I
9.6)
or
50
overnight
in the cold in a humidified box. After three washes with phosphate-buffered were
blocked
Diluted
saline/Tween, with
100μL
100μL
serum
protein-binding
of
Block
samples
Ace
(1/2
sites of the wells
(Yukijirusi,
dilution
for
to the wells. After incubation
for 1 h at room
serum samples
and washed
μL of
were removed
1/3000-diluted
biotin-labeled
Tokyo, Japan). IgE)
were
added
temperature,
three
Fig. 1 Anti-Der f I and II IgE antibody levels in the sera of patients with allergic rhinitis.
the
times. Then 100
affinity-purified
goat
antihu-
man IgE fragment (Tago, Burlingame, CA, USA)was added to each well and incubated at room temperature for 1h. Afterthree washes,
100μL
of
1/4000-diluted
peroxidase-avidin
D
(Vector
Laboratories, Burlingame, CA, USA)was added to each well and incubated further for 1h. The wells were then washed three times with
phosphate-buffered
saline and
100μL
of
o-phenylenedi-
amine dihydrochlorine (Sigma Chemicals, St Louis, MO, USA) substrate
solution
was
added,
followed
by the
addition
of
100μL
of 4N sulfuric acid to terminate the reaction. Absorbance at 490 nm was read by an automatic ELISAreader (model MTP-32; Corona Electric, Ibaragi, Japan). A preparation of atopic serum which contained 526ng/mL of anti-Der f I and 139ng/mL of antiDer f II IgE antibodies was employed as a standard. Absolute amount of IgEantibody was calculated on the intensityof coloring reaction by plotting on the standard curve prepared using a known amount of PS IgEmyeloma protein. Statistical
analysis
Data were analyzed by applying paired sample t-test. P values
