Development of time-resolved immunofluorometric assays for the detection of house dust mite-allergic IgE in human sera

doi:10.4236/health.2010.211190

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Vol.2, No.11, 1280-1286 (2010)

doi:10.4236/health.2010.211190

Health

Openly accessible at

Development of time-resolved immunofluorometric

assays for the detection of house dust mite-allergic

IgE in human sera

Ratchanoo Phiphatchaipaisarn1, Jundee Rabablert1*, Kornkarn Bramarapravati2,

Duangthep Thongdee1, Nares Wongpitoon3, Worawan Durongpisitkul3, Nat Malainual4

1Department of Biology, Faculty of Science, Silpakorn University, Nakorn Pathom, Thailand; *Corresponding Author: jundee@su.ac.th;

2Department of Preclinical Science, Faculty of Medicine, Thammasat University, Patumthani, Thailand;

3Department of Pediatric, Police General Hospital, Bangkok, Thailand;

4Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.

Received 6 September 2010; revised 15 September 2010; accepted 16 September 2010.

ABSTRACT

Dermatophagoides farinae and D. pteronyssi-

nus are the prevalent house dust mites (HDM) in

tropical countries and are associated with aller-

gic diseases. This investigation developed a time-

resolved immunofluorometric assay (TR-IFMA)

for the first time to detect specific IgE antibody

in patients with skin prick test positive to HDM

but no detectable IgE by other means. Levels of

IgE to natural and recombinant HDM allergens

were measured by TR-IFMA in 50 HDM-allergic

patients and 19 healthy participants compared

to sandwich enzyme-linked immunosorbent as-

say (ELISA). A recombinant allergen, rDerf2,

showed a 14 kDa band corresponding to broad

range proteins of natural HDM.TR-IFMA showed

sensitivity lower than 0.35 kUA/l. TR-IFMA em-

ploying three HDM antigens showed good cor-

relations with sandwich ELISA at R2 0.93-0.96.

TR-IFMA detected HDM IgE in 62, 62, 25 percent

of allergic patient serum sample compared to 28,

32, and 22 percent detected by ELISA result

using three HDM allergen. TR-IFMA also detec-

ted 26.3, 31.6, and 5.3 percent positive samples

from 19 healthy participants while ELISA showed

0, 5.3, and 0 percents IgE positive samples. The

use of rDerf2 as an HDM allergen for the assay

was verified with no statistically different from

other HDM allergens. TR-IFMA showed lower

detection limit than ELISA and yielded higher

sensitivity for serum of people with allergic sym-

ptoms with no detectable HDM IgE. It is antici-

pated that TR-IFMA for HDM-specific IgE detec-

tion will play an important role in future diagno-

sis of HDM allergy in clinical laboratories and for

different research purposes.

Keywords: House Dust Mites; Time-Resolved

Immunofluorometric Assay; Allergy; IgE

1. INTRODUCTION

House-dust mites (HDMs) represent one of the most

important allergen sources for the development of aller-

gic diseases worldwide, particularly asthma and allergic

rhinitis [1]. Dermatophagoides farinae (Df, originally

known as American HDM) and Dermatophagoides pte-

ronyssinus (Dp, European HDM) are the predominant

species in tropical and subtropical climates [2]. More

than 80% of allergic patients are sensitized to mite aller-

gens [3,4].

Positive skin prick tests (SPT) or serum IgE antibod-

ies to HDMs are demonstrable in allergic patients. How-

ever, if patients are allergic to histamine, skin prick test

could lead to complicated immunoreaction harmful to

the patients [5]. Enzyme-linked immunosorbent assay

(ELISA) is the method used to quantify serum HDM-IgE

in vitro in laboratories [6]. However, a more sensitive in

vitro test is needed for people who have dust mite aller-

gic clinical diagnosis but shown undetectable HDM-IgE

in their serum.

Monoclonal antibodies (mAbs) have been frequently

used in allergy research. Their uses include the quantifi-

cation of environmental allergen [7], allergens purifica-

tion [8], and crystal structure [9]. Specificity for unique

epitopes and unlimited in vitro production capability are

the advantage of allergen-specific mAbs.

The purpose of this study was to development a time-

resolved immunofluorometric assay (TR-IFMA) for the

measurement of HDM-specific IgE. The TRF is charac-

terized by lower detection limits and greater specificity,

R. Phiphatchaipaisarn et al. / HEALTH 2 (2010) 1280-1286

1281

reproducibility and practicability [10]. Two group II HDM

allergens and a group two recombinant vector were used

for validation of the assay.

2. METERIALS AND METHODS

2.1. Reagents

Commercial allergens were purchased from Allertech

(ALK, UK). DELFIA® Wash, DELFIA® Assay Buffer,

DELFIA® EU-labeled Streptavidin and DELFIA® En-

hancement Solution were purchased from Perkin-Elmer

Life Sciences (Wallac Oy, Turku, Finland). Monoclonal

anti-group 2 antibodies (mAb 1D8 clone, Indoor biotec-

nologies Ltd, Manchester, UK). Biotinylated rabbit-anti

mouse IgG solution and Biotinylated-labeled Mouse

anti-human IgE antibody purchased from SouthernBio-

tech (Birmingham, USA). BCIP/NBT 1 Component Sub-

strate and ABTS® Peroxidase 1 Component substrate So-

lution were purchased from KPL (MD, USA). C96 Mi-

croWell® plates were purchased from NuncTM (Roskilde,

Denmark). The instrument used for measurement - DE-

LFIA 1420 Automatic Immunoassay System - is the pro-

duct of Perkin-Elmer Life Sciences (Wallac Oy, Turku,

Finland).

2.2. HDM Extracts and Recombinants Mite

Allergens

Total HDM extracts of D. farinae (Df) and D. ptero-

nyssinus (Dp) were prepared from Spent Mite medium

from Siriraj Dust Mite Center, Bangkok, Thailand [7].

The extracts were stored at –20℃.

Recombinant rDerf2 allergen was constructed with

ligated cDNA in pPICZ vector. It was a kind gift from

Assist. Prof. Dr. Surapon Piboonpocanun, the Institute of

Molecular Biology and Genetics, Mahidol University,

Thailand. The positive transformants were subjected to

expression in Pichia pasroris [11,12]. The supernatants

were stored at -20℃ until used.

Proteins were separated by SDS-PAGE followed by

transferring the fractionated proteins to a nitrocellulose

membrane using method as described by the manufac-

turer with a wet blotting apparatus (Bio-Rad, USA). The

blotted membrane was blocked overnight at 4℃. The

membrane was further incubated with Monoclonal anti-

group 2 antibodies, followed by Biotinylated rabbit-anti

mouse IgG solution (1:5000) and BCIP/NBT 1 Compo-

nent Substrate.

2.3. Immunological Analysis

2.3.1. Enzyme-Linked Immuosorbent Assay

(ELISA)

Specific IgE to Df, rDerf2 and Dp were analyzed by

the ImmunoCAP™ system by using the cut-off limit 0.35

kilo units of aprotinin-specific antibodies per litre (kUA/l),

as recommended by the manufacturer. They were ana-

lyzed by sandwich ELISA (will be referred to as ELISA

thereafter) as previously described [13]. Wells were coated

with 1D8 clone mAb and let incubated over-night. Then

1 ng/well allergens in PBS containing 1% BSA were add-

ed. The wells were further incubated with serum (1:10),

followed by biotinylated-labeled Mouse anti-human IgE

antibody (1:1000), streptavidin-peroxidase and ABTS®

Peroxidase 1 Component substrate. The result was meas-

ured in a DELFIA 1420 Automatic Immunoassay System.

2.3.2. Time-Resolved Immunoflurometric

Assay (TR-IFMA)

Specific IgE to all three allergens were analyzed by

the ImmunoCAP™ system by using the cut-off limit

0.35 kUA/l as recommended by the manufacturer. Df,

recombinant Derf2 and Dp were analyzed by TR-IFMA

[13]. Wells were coated with mAb and incubated over-

night. After incubation, 1 ng/well allergens in PBS con-

taining 1 % BSA were added. The wells were further

incubated with serum (1:10), followed by biotinylated-

labeled Mouse anti-human IgE antibody (1:1000), DEL-

FIA® EU-labeled Streptavidin and DELFIA® enhance-

ment solution. The result was measured in a DELFIA

1420 Automatic Immunoassay System.

2.4. Patients and Collection of Samples

Fifty rhinitis patients with/without intermittent or per-

sistent, mild-to-moderate asthma with positive result to

Skin Prick Test (SPT) were selected for this investiga-

tion by Dr. Nares Wongpitoon or Dr. Voravan Durong-

pisitkul of Department of Pediatric, Police General Hos-

pital, Bangkok, Thailand. As the control group, 19 heal-

thy subjects with no history of allergic diseases were in-

cluded. The study was approved by the Ethics committee

of the Police general Hospital and written informed con-

sent was obtained from all participants.

Five milliliter of venous blood was taken from each

participant. The blood was centrifuged within 6 hr of co-

llection at 400 g for 30 min. The serum was then collected

and stored frozen at –20℃.

In order to quantify specific IgE levels, serum from

three highest HDM-sensitized individuals as analyzed by

ELISA and TR-IFMA were analyzed by the Immuno-

CAP™ system at the laboratory of Faculty of Medicine,

Mahidol University, Bangkok, Thailand. The IgE levels

in several dilutions of the reference serum were analyzed.

Fifth calibrator points in two-fold dilutions ranging

0.22-3.49, 0.22-3.49 and 0.28-4.55 kUA/l of Df, rDerf2

and Dp specific IgE respectively were made for use in

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calibrating curve of both ELISA and TR-IFMA.

2.5. Statistical Analysis

Levels of specific IgE to HDM allergens in ELISA

and TR-IFMA were obtained from Version 13 (Chicago,

IL, USA).

3. RESULTS

3.1. HDM-Allergen Characteristics

HDM-allergen characteristics were determined by

SDS-PAGE and Immunoblot using monoclonal antibody,

as shown in Figure 1. Result from SDS-PAGE showed

the MW of Df and Dp ranged from 10 to 72 kDa, while

the molecular weight of rDerf2 was approximately 14 kDa.

Immunoblot also confirmed rDerf2 molecular weight at

14 kDa.

3.2. Calibration Curve and Detection Limit

A typical standard curve for HDM-specific IgE using

ELISA and TR-IFMA is shown in Figure 2. The sensi-

tivity (± 2 standard deviations) of the assay, as calculated

from X replicates of the zero standards, was about <

0.35 kUA/l. The calibration curve was linear.

3.3. Subject Characteristics

Specific antibodies to HDM allergens were examined in

a total of 69 subjects. Subjects were screened by SPT

using commercial Der f, Der p, CR, Grass, Dog, Cat,

Wheat, Shrimp, Feather, M1 and M2. The demographic

clinical characteristics of the subjects were shown in Ta-

ble 1.

(a) (b)

Figure 1. The protein components of HDM allergen were de-

termined by SDS-PAGE (a); Lane 1: Df; Lane 2: Dp; Lane 3:

Molecular mass standards (in kDa); Lane 4: rDerf2; Lane 5:

pPICZ. (b) Immunoblot Lane 1: Molecular mass standards (in

kDa); Lane 2: rDer f 02; Lane 3: pPICZ.

3.4. Comparison of TR-IFMA and ELISA

Number of positive samples from ELISA and TR-

IFMA in allergic patient and healthy participant serum as

seen using Df, rDerf2 and Dp allergen against group II

mAb is shown in Table 2. TR-IFMA detected HDM IgE

in higher percentage of allergic patient serum sample

when tested using Df, rDerf2 and Dp allergen (62, 62, 25

percent) compared to ELISA result (28, 32, and 22 per-

cent respectively). In 19 healthy subjects, ELISA de-

tected HDM IgE in 0, 5.3, and 0 percents samples tested

by the three HDM allergens while TR-IFMA found 26.3,

31.6, and 5.3 percent positive samples respectively.

TR-IFMA was compared to ELISA by parallel testing

of 50 serum specimens from rhinitis patients utilizing Df,

rDerf2 and Dp allergen respectively. Among allergic pa-

tients very good overall correlation was found (Figure

3). The linear regression coefficient values calculated

from the positive specimens varied from 0.93-0.96.

Table 1. Demographic and characteristics of the study indivi-

duals.

Groups

Characteristics allergic

individuals (A)

non-allergic

individuals (B)

Number of subjects (n) 50 19

Age (years, mean ± SD) 39.52 ± 9.93 25.21 ± 6.9

Gender (male/female) 24/26 4/15

Clinical diagnosis (N, %) 50, 100 % N/A

Catagorized by allergen (%) N/A

Der f 100 %

Der p 100 %

CR 20 %

Grass 8 %

Dog 4 %

Cat 8 %

Wheat 0 %

Shrimp 0 %

Feather 0 %

M1 12 %

M2 12 %

Der p, Dermatophagoides pteronyssinus; Der f, Dermatophagoides farinae;

CR, American Cockroach; Grass, Bermuda grass pollen; Dog, Dog epithe-

lium; Cat, Cat pelt; Feather, feather of Chicken, Duck, and Goose; M1,

Penicillium notatum, Aspergillus niger, Drechslera, Cladosprium spharos-

permum, Alternaria; M2, Curvularia, Fusarium, Aureobasidium, Mucor,

hizopus R

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(a) (b) (c)

(d) (e) (f)

Figure 2. Standard curve for sandwiched ELISA for (A) Df, (B) rDerf2, (C) Dp. Standard curve for TR-IFMA for (D) Df,

(E) rDerf2, (F) Dp. Each point on the standard curve is the mean of 3 duplicate measurements.

Table 2. Number of positive samples from ELISA and TR-

IFMA in allergic patient and healthy participant serum as seen

using Df, rDerf2 and Dp allergen against group II mAb.

Openly accessible at

Number of positive samples: n (frequency %)

Allergen tested Allergic patients (A)

Total n = 50

Healthy participants (B)

Total n = 19

ELISA 14 (28) 0 (0)

TR-IFMA 311 (62) 5 (26.3)

rDerf2

ELISA 16 (32) 1 (5.3)

TR-IFMA 312 (62) 6 (31.6)

ELISA 11 (22) 0 (0)

TR-IFMA 253 (50) 1 (5.3)

1, 2 and 3correspond to the same data in Table 3.

TR-IFMA positive cases from serum of allergic pa-

tients tested with all three HDM allergen were separated

into two groups, namely those from ELISA positive and

ELISA negative samples. Number of samples, frequency

and median of each group is shown according to specific

allergen in Table 3. Median, standard deviation, 25 and

75 percent values are presented in Figure 4. Among TR-

IFMA positive samples, 54.84, 48.39, and 56 percents of

Df, rDerf2, and Dp were identified as ELISA negative

samples.

Concentrations of HDM IgE TR-IFMA from all three

HDM allergens testing (allergic patients and healthy par-

ticipants) were subjected to test for several related sam-

ple using Friedman test. At N =69, p value equals 0.093.

The use of any of the three HDM allergens does not give

a statistically significant result in TR-IFMA technique.

4. DISCUSSIONS

It has been reported that D. farinae which homolo-

gous with D. pteronyssinus is composed of broad range

of 10 to 72 kDa. In the present study, Immunoblotting

analysis of rDer f 2 using a specific mAb showed one

band of 14 kDa (Figure 1), agreeing with previous re-

ports and indicating that rDer f 2 corresponded to D.

farinae [14,15]. It is known that D. farinae usually coex-

its with D. pteronyssinus. As a result the assay measur-

ing IgE binding to Derf2 would not distinguish D. fari-

nae sensitization with cross reactivity [16].

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(a) (b) (c)

Figure 3. Correlation between levels of specific IgE against Df (a), rDerf2 (b) and Dp (c) measured by ELISA and TR-IFMA.

Figure 4. TR-IFMA positive cases from serum of allergic pa-

tients tested with all three HDM allergens. Median, standard

deviation, 25 and 75 percent values are presented. HDM aller-

gens Df: 1) ELISA negative 2) ELISA positive rDerf2: 3)

ELISA negative 4) ELISA positive Dp: 5) ELISA negative 6)

ELISA positive.

4.1. Levels of Specific IgE Reactivity to HDM

Allergens: Lower Detection Limit and

Increase Testing Sensitivity

The IgE reactivity profile for the sera from allergic

patients (n = 50) and healthy participants (n = 19) was

determined by three different allergens, namely Df, rDer

f 2 and Dp measured by TR-IFMA compared with

ELISA.

As shown in Table 2, the efficacy of recombinant an-

tigen (rDerf 2) and natural antigen (Df or Dp) were ex-

amined in allergic patients and non-allergic individuals

by both TR-IFMA and ELISA. In allergic patients, we

found that only 28, 32, and 22 percents result was ob-

tained by ELISA, while 62, 62, and 50 percent of patient

serum reacted with Df, rDerf2, and Dp respectively us-

ing TR-IFMA. The increase in rate of HDM IgE detec-

tion was significant at 34, 30 and 28 percents for all

three allergen respectively. This is the first indication

Table 3. TR-IFMA positive cases in serum from allergic pa-

tients categorized by ELISA result in Df, rDerf2 and Dp test-

ing.

TR-IFMA positive cases in

symptomatic patients

HDM allergens ELISA positive ELISA negative

Df: total 31 samples1

n (frequency %) 14 (45.16) 17 (54.84)

Median (kUA/l) 13.37 1.81*

rDerf2: total 31 samples2

n (frequency %) 16 (51.61) 15 (48.39)

Median (kUA/l) 16.67 2.74*

Dp: total 25 samples3

n (frequency %) 11 (44) 14 (56)

Median (kUA/l) 37.19 1.66*

Mann-Whitney Rank Sum Test; p < 0.001*

suggesting that TR-IFMA of all three HDM allergens

show higher sensitivity for serum of people with allergic

symptoms with no detectable HDM IgE. In addition,

when ELISA showed 0, 5.3, and 0 percent positive sam-

ples in healthy participant serum, TR-IFMA detected

26.3, 31.6, and 5.3 percent positive samples. That is the

26.3, 21.3 and 5.3 percent higher detection rate across

the board.

In 50 HDM-SPT positive allergic subjects, 48 per-

cents of this group showed cross-reactive IgE binding to

all three allergens measured by TR-IFMA, while only 22

percent showed cross-reactive IgE binding to all three

allergens measured by ELISA. Additionally, cross-reac-

tive IgE to two allergens was also observed by both TR-

IFMA and ELISA. TR-IFMA showed 10, 2 and 0 per-

cent cross-reactivity between Df/rDerf2, rDerf2/Dp and

Df/Dp, while ELISA result showed 2, 0, and 0 percent

respectively. In healthy participants there was no cross-

reactivity report over all three antigens. Only 15.8 and 5.3

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percent cross-reactivity were reported for Df/rDerf2 and

rDerf2/Dp. These numbers confirms TR-IFMA sensitivity

over ELISA, and suggests that rDerf2 is a slightly better

antigen as seen from cross-reactivity data.

Table 3 and Figure 4 revealed lower detection limits

and the sensitivity of TR-IFMA. All HDM allergens

provided significantly different median between ELISA

positive and negative samples in Mann-Whitney Rank

Sum Test with p < 0.001. Figure 4 shows that even

though ranges of all three ELISA negative groups show-

ed overlap with the ELISA positive ones, one hundred

percent of all three ELISA negative populations stayed

within 40 percentile of the corresponding ELISA posi-

tive population. TR-IFMA was able to showed real value

of some ELISA negative samples beautifully in this de-

monstration.

This investigation employed three different HDM al-

lergens, namely Df and Dp from natural sources, and a

recombinant rDerf2. Result from the test for several re-

lated sample using Friedman test showed that TR-IFMA

result using all three allergens was not significantly dif-

ferent from one another. Thus this investigation suggests

that rDerf2 can be used as a HDM allergen in TR-IFMA.

Since rDerf2 can be expressed in large quantity, this

should ensure a homogeneous batch supply of HDM al-

lergen for time to come.

Immunoassays based on time-resolved fluorometry

represents an attractive option that offers several advan-

tages over other traditional techniques, including very

high sensitivity, no use of radioactive reagents, stability

of the reagents, low background interference and a wide

test range [17, 18]. Additionally, the use of a specific mAb

as capture antibody in this study avoided over estimation

of allergens by reducing the cross-reactive epitopes rec-

ognition. The use of specific polyclonal serum avoided

the loss of detection produced by conformational changes

affecting one or more epitopes. Hence, a specific anti-

HDM polyclonal serum has been used as the secondary

antibody in this investigation, unlike most of other im-

munoassays described [19].

The time-resolved immunofluormetric assay develop-

ed in this investigation provides sensitive and highly

specific techniques for the determination of HDM-specific

IgE in human sera. Measurements achieved with TR-

IFMA and ELISA were highly correlated (R2 between

0.93-0.96). In regard to the limit of detection, the assay

allows the detection of HDM-specific IgE as low as <

0.35 kUA/l, which is well below limits of detection re-

ported by other techniques (Immunoblot, ELISA, RIA);

TR-IFMA can therefore be considered as the most sensi-

tive method for quantifying HDM-specific IgE. In addi-

tion, the assay ranges of TR-IFMA is the wider than

those of ELISA in our laboratory. Our data was consis-

tent with previous reports [20,21]. It is clearly seen that

TR-IFMA showed sensitivity to serum of allergic pa-

tients with symptom whose serum were formerly HDM

IgE negative using ELISA. The analysis of real samples

from HDM positive allergic patients compared to HDM

negative but positive to aeroallergen demonstrated that a

broad range of HDM-specific IgE could be detected with

the present techniques.

5. CONCLUSIONS

TR-IFMA was developed for HDM allergen IgE test-

ing and evaluated. Such improvements may be useful for

screening of HDM allergy and other aeroallergens. TR-

IFMA of all Df, rDerf2 and Dp showed lower detection

limit than ELISA and yielded higher sensitivity for se-

rum of people with allergic symptoms with no detectable

HDM IgE. Recombinant HDM allergen, rDerf2, was pro-

ven to be as good an allergen as its natural counterpart,

namely Df and Dp. It is anticipated that TR-IFMA for

HDM-specific IgE detection will play an important role

in future diagnosis of HDM allergy in clinical laborato-

ries and for different research purposes.

6. ACKNOWLEDGEMENTS

This study was supported by the TRF (MRG-WII 505S061), a par-

tial funding from Thailand Research Fund No. MRG-WII525S092 and

DIG5180004. The authors thank Associate Professor Dr. Darawan

Wanachiwanawin, Department of Parasitology, Faculty of Medicine

Siriraj Hospital, Mahidol University for laboratory facility, and Assis-

tant Professor Dr. Junya Pattaraarchachai, Faculty of Medicine, Tham-

masat University for statistic analysis.

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