# Immunochemical Characterization ofAcacia Pollen Allergens and Evaluation of Cross-Reactivity Pattern with the Common Allergenic Pollens

Authors: Mohammad-Hosein Shamsbiranvand; Ali Khodadadi; Mohammad-Ali Assarehzadegan; Seyed Hamid Borsi; Akram Amini
Journal: Journal of Allergy
 (2014)

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## Abstract

Pollen from theAcacia has been reported as an important source of pollinosis in tropical and subtropical regions of the world. The aim of this study was to characterize the IgE binding protein of Acacia farnesiana pollen extract and evaluate cross-reactivity with the most allergenic pollens. In this study, pollen extract was fractionated by SDS-PAGE and the allergenic profile was determined by IgE-immunoblotting and specific ELISA using forty-two Acacia allergic patients. Potential cross-reactivity among Acacia and selected allergenic plants was evaluated with ELISA and immunoblotting inhibition experiments. There were several resolved protein fractions on SDS-PAGE which ranged from 12 to 85 kDa. Several allergenic protein bands with molecular weights approximately between 12 and 85 kDa were recognized by IgE-specific antibodies from Acacia allergic patients in the immunoblot assay. The inhibition by the Prosopis juliflora pollen extract was more than those by other pollen extracts. Moreover, the wheal diameters generated by the Acacia pollen extract were highly correlated with those of P. juliflora pollen extracts. The findings suggest that several proteins such as 15, 23, 45, and 50 kDa proteins could be used as diagnostic and therapeutic reagents for patients allergic to A. farnesiana and P. juliflora.


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## Body

## 1. Introduction

Acacia farnesiana (Vachellia farnesiana), a member of the Fabaceae family, is common throughout tropical and subtropical regions of Asia, Africa, Australia, and America with hot and humid climates, where it is planted as a shade and/or ornamental tree or for binding sand [1, 2]. Pollens from the Fabaceae family have been reported as an important source of pollinosis in the United States, European countries, and Asia [1–3]. Moreover, the inhalation ofA. farnesiana pollen is one of the main causes of respiratory allergic diseases in semiarid countries such as Iran, Saudi Arabia, and the United Arab Emirates, where the frequency of sensitization ranges from 25% to 48% [2, 4–7].Howlett et al. reported a high level of cross-reactivity betweenAcacia (wattle) andLolium perenne(rye grass) pollen using the radioallergosorbent test (RAST) [8]. The results showed that IgE molecules which bound toAcacia pollen proteins also bound toL. perenne pollen extracts. In spite of a high rate of sensitization toAcacia pollen in Iran and neighboring countries, to our knowledge, there are few studies about the characterization ofA. farnesiana pollen extract, and cross-reactivity of this plant with the five most allergenic pollens in arid and semiarid areas (Prosopis juliflora,Salsola kali,Amaranthus retroflexus, Chenopodium album, andKochia scoparia) is not well documented [2, 5, 9, 10].The recognition of allergenic components of pollens is essential for component-resolved diagnosis, the design of patient-specific immunotherapy, and the explanation of sensitization mechanisms to various allergens [11, 12]. In this study, we evaluate proteins ofAcacia pollen extract which are specifically reactive to the immunoglobulin E (IgE) of pollen-allergic patients and the IgE cross-reactivity amongA. farnesianaand the selected plants usingin vivo andin vitro assessments.


## 2. Materials and Methods

### 2.1. Preparation of Extract

Polleniferous materials were collected fromA. farnesiana’s flowers during February–May throughout Ahvaz city, a tropical region in southwest Iran with a tropical climate and a population of more than 1.4 million [2].Collection and processing of pollen materials was done carefully by trained pollen collectors. Pollen grains were separated by passing the dried materials through different sieves (100, 200, and 300 meshes) successively. The final fine powder was subjected to a purity check for pollen content using a microscope. Pollen materials with more than 95% pollen and less than 5% floral parts of the same plant were taken for antigen extraction.Pollen materials were defatted using repeated changes of diethyl ether. Pollen was extracted as described previously [10]. In brief, two grams of pollen was mixed with 10 mL phosphate-buffered saline (PBS) 0.01 M (pH 7.4) by continuous stirring for 18 h at 4°C. The extract was centrifuged at 16,000 g, filtered through a 0.22 μm membrane under sterile conditions, and dialyzed against 10 mM phosphate buffer. The extract was then freeze-dried. The protein content of the extract was measured by Bradford’s method [13].


### 2.2. Patients and Skin Prick Test

Forty-two respiratory allergic patients were enrolled in this study presented to the Immunology and Allergy Department of Ahvaz Jundishapur University of Medical Sciences. The patients were asked to complete a detailed questionnaire. They were considered as having a history of allergy if they reported at least one eye, nasal, or respiratory symptom to common allergens such as house dust, domestic animals, food, or pollen. The patients were also evaluated by a clinical examination and a skin prick test (SPT) with common aeroallergens. Ten healthy subjects who presented negative SPTs and no specific IgE to theA. farnesianapollen extract were assigned as negative controls. The human ethics committee of the institute approved the study protocol with informed written consent from each patient.Skin prick tests were performed by an experienced nurse under physician’s supervision. In this test,A. farnesianaand the most allergenic pollen extracts (Prosopis juliflora, Salsola kali, Amaranthus retroflexus, Chenopodium album, andKochia scoparia) were put on the patients’ inner forearms and irritation of the epidermis was caused by prick method. The result was observed after 15 minutes. Next, the mean diameter of wheal reaction in every patient was measured and compared with negative (glycerol saline) and positive (histamine, 10 mg/mL) controls. Patients with a wheal diameter >3 mm were considered positive compared to negative and positive controls and were asked to donate a serum sample. Serum samples of patients were stored at −20°C before use.


### 2.3. Enzyme-Linked Immunosorbent Assays (ELISAs)

Total serum IgE levels were measured by means of a commercially available ELISA kit according to the manufacturer’s instructions (Radim, Italy).To measure the levels of specific IgE toA. farnesianapollen in patients’ sera, an indirect ELISA was developed as described earlier [14]. Briefly, 0.2 μg ofA. farnesianapollen extract in 100 μL carbonate buffer (15 mM Na2CO3 and 35 mM NaHCO3, pH 9.6) was incubated at 4°C overnight per well of a 96-well microtiter plate (Nunc MaxiSorp, Denmark). Each well was then blocked for 1 h at 37°C with 150 μL of 2% BSA in PBS followed by incubation for 3 h with 100 μL of serum at room temperature with shaking. Each well was then incubated for 2 h at room temperature with 1 : 1000 dilution of biotinylated goat anti-human IgE antibody (Nordic Immunology Co., Netherlands) in 1% BSA. Each incubation step was followed by five washes with PBS-T (PBS containing 0.05% Tween 20). Wells were added by 100 μL of a 1 : 5000 dilution of horseradish peroxidase-conjugated streptavidin (Bio-Rad, USA). Following five washes, 100 μL of chromogenic substrate was added to each well and the plate was incubated for 15 min in the dark. The plate was read at 450 nm with an ELISA reader. Optical density (OD450) greater than four times the median values of the negative controls was considered to be positive.


### 2.4. Total Extract Preparation

Pollens fromP. juliflora (mesquite),S. kali,A. retroflexus,C. album, andK. scoparia were prepared from polleniferous materials and then their extracts were prepared as described above [10]. The protein content of each extract was then determined using Bradford’s method [13].


### 2.5. SDS-PAGE and IgE-Immunoblotting

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ofAcacia pollen extract (60 μg) was performed according to Laemmli [15] using 12.5% or 15% acrylamide separation gels under reducing and nonreducing conditions. Reducing and nonreducing sample buffers were the same except that the final reducing sample buffer contained 5% (vol/vol) 2-mercaptoethanol (2-ME). Separated protein bands from the electrophoresis ofA. farnesiana pollen extract were electrotransferred to polyvinylidene difluoride (PVDF) membranes (Immobilon P, Millipore Corp., Bedford, MA, US), as described earlier [10]. In brief, after washing and blocking, membranes were incubated with a 1/5 dilution of serum pool or individual sera from patients withA. farnesiana allergy or with control sera (1 : 5 dilutions). Biotinylated anti-human IgE (Nordic Immunology Co., Netherlands) (1 : 500 v/v in 1% BSA) was added to the blotted membrane strips and incubated for 2 h at room temperature. The unbound antibodies were removed from blots by washing with PBS and followed by incubation with 1 : 10000 v/v in BSA1% HRP-linked streptavidin (Sigma-Aldrich, USA) for 2 h at room temperature. The bound enzymatic activity of horseradish peroxidase was detected by high-sensitivity liquid diaminobenzidine (Liquid DAB+) chromogen (DAKO, Denmark).


### 2.6. ELISA Inhibition

ELISA inhibition assays were performed as described above, except that a pooled serum (1 : 2 v/v) fromA. farnesiana allergic patients (numbers 2, 7, 21, 24, and 36) was preincubated for one hour at room temperature with either 1000, 100, 10, 1, 0.1, or 0.01 μg of the selected pollen extract (includingP. juliflora, S. kali, A. retroflexus,C. album, andK. scoparia) as inhibitors or with BSA as a negative control. Percentage of inhibition was calculated using the following formula: (OD of sample without inhibitor − OD of sample with inhibitor/OD of sample without inhibitor) × 100.


### 2.7. IgE-Immunoblotting Inhibition

To study the cross-reactivity betweenA. farnesianapollen and selected allergenic plants, the IgE-immunoblot inhibition experiment was performed. Reducing SDS-PAGE resolvedA. farnesiana pollen proteins were transferred to PVDF membrane. After blocking, membrane strips were kept for 3 h at room temperature with a mix of 100 μL of pooled sera (1 : 5 v/v) (from patients 2, 7, 21, 24, and 36) which were preincubated with pollen extracts fromP. juliflora, S. kali, A. retroflexus,C. album, andK. scoparia, as well as BSA (as negative control).


## 2.1. Preparation of Extract

Polleniferous materials were collected fromA. farnesiana’s flowers during February–May throughout Ahvaz city, a tropical region in southwest Iran with a tropical climate and a population of more than 1.4 million [2].Collection and processing of pollen materials was done carefully by trained pollen collectors. Pollen grains were separated by passing the dried materials through different sieves (100, 200, and 300 meshes) successively. The final fine powder was subjected to a purity check for pollen content using a microscope. Pollen materials with more than 95% pollen and less than 5% floral parts of the same plant were taken for antigen extraction.Pollen materials were defatted using repeated changes of diethyl ether. Pollen was extracted as described previously [10]. In brief, two grams of pollen was mixed with 10 mL phosphate-buffered saline (PBS) 0.01 M (pH 7.4) by continuous stirring for 18 h at 4°C. The extract was centrifuged at 16,000 g, filtered through a 0.22 μm membrane under sterile conditions, and dialyzed against 10 mM phosphate buffer. The extract was then freeze-dried. The protein content of the extract was measured by Bradford’s method [13].


## 2.2. Patients and Skin Prick Test

Forty-two respiratory allergic patients were enrolled in this study presented to the Immunology and Allergy Department of Ahvaz Jundishapur University of Medical Sciences. The patients were asked to complete a detailed questionnaire. They were considered as having a history of allergy if they reported at least one eye, nasal, or respiratory symptom to common allergens such as house dust, domestic animals, food, or pollen. The patients were also evaluated by a clinical examination and a skin prick test (SPT) with common aeroallergens. Ten healthy subjects who presented negative SPTs and no specific IgE to theA. farnesianapollen extract were assigned as negative controls. The human ethics committee of the institute approved the study protocol with informed written consent from each patient.Skin prick tests were performed by an experienced nurse under physician’s supervision. In this test,A. farnesianaand the most allergenic pollen extracts (Prosopis juliflora, Salsola kali, Amaranthus retroflexus, Chenopodium album, andKochia scoparia) were put on the patients’ inner forearms and irritation of the epidermis was caused by prick method. The result was observed after 15 minutes. Next, the mean diameter of wheal reaction in every patient was measured and compared with negative (glycerol saline) and positive (histamine, 10 mg/mL) controls. Patients with a wheal diameter >3 mm were considered positive compared to negative and positive controls and were asked to donate a serum sample. Serum samples of patients were stored at −20°C before use.


## 2.3. Enzyme-Linked Immunosorbent Assays (ELISAs)

Total serum IgE levels were measured by means of a commercially available ELISA kit according to the manufacturer’s instructions (Radim, Italy).To measure the levels of specific IgE toA. farnesianapollen in patients’ sera, an indirect ELISA was developed as described earlier [14]. Briefly, 0.2 μg ofA. farnesianapollen extract in 100 μL carbonate buffer (15 mM Na2CO3 and 35 mM NaHCO3, pH 9.6) was incubated at 4°C overnight per well of a 96-well microtiter plate (Nunc MaxiSorp, Denmark). Each well was then blocked for 1 h at 37°C with 150 μL of 2% BSA in PBS followed by incubation for 3 h with 100 μL of serum at room temperature with shaking. Each well was then incubated for 2 h at room temperature with 1 : 1000 dilution of biotinylated goat anti-human IgE antibody (Nordic Immunology Co., Netherlands) in 1% BSA. Each incubation step was followed by five washes with PBS-T (PBS containing 0.05% Tween 20). Wells were added by 100 μL of a 1 : 5000 dilution of horseradish peroxidase-conjugated streptavidin (Bio-Rad, USA). Following five washes, 100 μL of chromogenic substrate was added to each well and the plate was incubated for 15 min in the dark. The plate was read at 450 nm with an ELISA reader. Optical density (OD450) greater than four times the median values of the negative controls was considered to be positive.


## 2.4. Total Extract Preparation

Pollens fromP. juliflora (mesquite),S. kali,A. retroflexus,C. album, andK. scoparia were prepared from polleniferous materials and then their extracts were prepared as described above [10]. The protein content of each extract was then determined using Bradford’s method [13].


## 2.5. SDS-PAGE and IgE-Immunoblotting

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ofAcacia pollen extract (60 μg) was performed according to Laemmli [15] using 12.5% or 15% acrylamide separation gels under reducing and nonreducing conditions. Reducing and nonreducing sample buffers were the same except that the final reducing sample buffer contained 5% (vol/vol) 2-mercaptoethanol (2-ME). Separated protein bands from the electrophoresis ofA. farnesiana pollen extract were electrotransferred to polyvinylidene difluoride (PVDF) membranes (Immobilon P, Millipore Corp., Bedford, MA, US), as described earlier [10]. In brief, after washing and blocking, membranes were incubated with a 1/5 dilution of serum pool or individual sera from patients withA. farnesiana allergy or with control sera (1 : 5 dilutions). Biotinylated anti-human IgE (Nordic Immunology Co., Netherlands) (1 : 500 v/v in 1% BSA) was added to the blotted membrane strips and incubated for 2 h at room temperature. The unbound antibodies were removed from blots by washing with PBS and followed by incubation with 1 : 10000 v/v in BSA1% HRP-linked streptavidin (Sigma-Aldrich, USA) for 2 h at room temperature. The bound enzymatic activity of horseradish peroxidase was detected by high-sensitivity liquid diaminobenzidine (Liquid DAB+) chromogen (DAKO, Denmark).


## 2.6. ELISA Inhibition

ELISA inhibition assays were performed as described above, except that a pooled serum (1 : 2 v/v) fromA. farnesiana allergic patients (numbers 2, 7, 21, 24, and 36) was preincubated for one hour at room temperature with either 1000, 100, 10, 1, 0.1, or 0.01 μg of the selected pollen extract (includingP. juliflora, S. kali, A. retroflexus,C. album, andK. scoparia) as inhibitors or with BSA as a negative control. Percentage of inhibition was calculated using the following formula: (OD of sample without inhibitor − OD of sample with inhibitor/OD of sample without inhibitor) × 100.


## 2.7. IgE-Immunoblotting Inhibition

To study the cross-reactivity betweenA. farnesianapollen and selected allergenic plants, the IgE-immunoblot inhibition experiment was performed. Reducing SDS-PAGE resolvedA. farnesiana pollen proteins were transferred to PVDF membrane. After blocking, membrane strips were kept for 3 h at room temperature with a mix of 100 μL of pooled sera (1 : 5 v/v) (from patients 2, 7, 21, 24, and 36) which were preincubated with pollen extracts fromP. juliflora, S. kali, A. retroflexus,C. album, andK. scoparia, as well as BSA (as negative control).


## 3. Results

### 3.1. Patients

Forty-two patients, 20 males and 22 females (mean age,33.60±14.47 years; age range 10–70 years), were included in the study (Table 1). All patients suffered from respiratory allergies and seasonal rhinitis without asthma. The patients were all positive by skin prick test withA. farnesiana,P. juliflora, S. kali, A. retroflexus,C. album, andK. scoparia extracts (Table 1). A serum pool of 5 nonallergic subjects was used as a negative control.Table 1
Clinical characteristics, total and specific IgE values, and skin reactivity of allergic patients.
Samplenumber
Age(years)/sex1
Symptoms2
Acaciaspecific IgE3 (OD)
Total IgE (IU/mL)
Diameters (mm) of the papules obtained by prick test4
A. farnesiana
P. juliflora
S. kali
A. retroflexus
C. album
K. scoparia
Patients
1
34/M
L, N, E
0.9
181
7
5
8
9
10
9
2
41/M
N, E
2.33
453
8
9
10
30
11
11
3
39/F
L, N
2.71
156
5
7
16
11
10
9
4
14/F
L, N, E
1.11
165
7
9
7
5
14
11
5
12/M
L, N, E
1.10
123
5
6
4
3
2
0
6
10/M
L, N, E
1.19
313
6
5
10
10
9
8
7
29/F
L, N, E
1.89
345
7
8
8
7
11
5
8
43/F
N, E
1.98
234
6
7
10
12
11
12
9
21/M
N, E
2.07
381
7
9
10
10
12
11
10
26/F
N, E
0.89
225
5
7
10
9
10
11
11
26/F
L, N, E
1.02
468
5
5
10
7
12
8
12
23/F
L, N, E
0.85
265
6
8
13
9
10
10
13
30/M
N, E
1.63
167
7
7
10
7
8
9
14
21/F
L, N, E
1.50
275
7
9
10
10
11
12
15
70/M
L, N, E
1.97
187
6
6
12
11
15
10
16
43/F
N, E
1.85
212
8
7
12
8
11
10
17
32/F
L, N, E
1.94
250
6
8
10
11
13
15
18
46/F
L, N, E
1.52
261
5
7
10
7
9
10
19
34/F
N, E
1.42
301
5
6
5
5
4
0
20
13/M
N, E
2.01
463
8
9
12
5
8
6
21
22/M
N
2.21
280
9
8
8
8
6
7
22
17/F
L, N, E
2.02
302
8
10
8
7
5
5
23
61/M
N
1.04
186
5
6
9
10
9
10
24
66/M
L, N, E
2.10
293
10
12
9
8
6
7
25
10/M
L, N
1.83
220
8
7
4
4
5
0
26
33/M
L, N
1.55
214
7
7
10
10
9
9
27
40/F
L, N
1.65
122
5
5
10
9
11
9
28
53/F
N
1.87
200
7
9
8
8
9
10
29
46/M
N, E
1.91
192
7
6
10
9
10
10
30
43/M
L, N, E
2.15
284
7
7
8
7
9
5
31
19/F
L, N, E
1.92
187
8
7
9
8
9
6
32
36/F
L, N, E
1.89
156
7
8
15
23
25
15
33
19/F
L, N, E
1.29
360
7
10
10
9
10
10
34
37/F
N, E
1.93
267
7
8
12
12
11
11
35
32/M
N, E
1.87
228
7
9
8
9
10
10
36
51/M
N, E
2.11
358
7
8
14
13
11
12
37
28/F
L, N, E
1.60
294
6
8
12
11
11
10
38
29/M
L, N, E
1.96
304
6
7
18
16
18
14
39
44/M
N, E
2.02
418
6
8
10
8
9
9
40
40/M
N, E
2.12
315
8
9
13
18
16
17
41
38/F
N
2.09
480
7
8
8
9
9
6
42
40/F
N, E
1.72
257
6
7
8
7
9
5
Controls
44
28/F
—
0.09
32
0
0
0
0
0
0
45
45/F
—
0.05
12
0
0
0
0
0
0
46
32/M
—
0.06
88
0
0
0
0
0
0
47
38/M
—
0.10
65
0
0
0
0
0
0
48
22/M
—
0.05
22
0
0
0
0
0
0
1M: male; F: female.
2L: lungs symptoms (breathlessness, tight chest, cough, wheeze); N: nose symptoms (sneezing, runny, blocked); E: eyes    symptoms (itching, dry).
3Levels of specific IgE to A. retroflexus pollen extract by ELISA (optical density at 450 nm).
4The mean wheal diameters are displayed in mm. Histamine diphosphate (10 mg/mL) positive control; glycerin-negative control.


### 3.2. Skin Prick Test

Mean diameters of positive wheal sizes wereA. farnesiana: 6.69±1.17 mm,P. juliflora: 7.57±1.50 mm,S. kali: 9.95±2.81 mm,A. retroflexus: 9.74±4.79 mm,C. album: 10.19±3.79 mm, andK. scoparia: 8.9±3.70 mm. Positive correlation coefficients were attained between theA. farnesiana andP. juliflora(r=0.64, P<0.01),A. farnesiana andS. kali (r=0.07, P=0.6),A. farnesiana andA. retroflexus(r=0.1, P=0.4), A. farnesiana andC. album (r=0.06, P=0.6), andA. farnesiana andK. scoparia (r=0.007, P=0.9) pollen extracts for wheal diameter on the SPT (ANOVA procedure).


### 3.3. Protein and Allergenic Profile

The protein composition ofA. farnesiana extract was analyzed by Coomassie Brilliant Blue staining (Figure 1(a)). The reducing SDS-PAGE separation of the pollen extract showed several resolved protein bands in theA. farnesiana extract with molecular weights in the range of approximately 12 to 85 kDa. The most prominent bands had MWs of approximately 50, 66, and 85 kDa. Other predominant bands were identified at 12, 15, 20, 23, 28, 37, and 45 kDa. Nonreducing SDS-PAGE ofAcacia pollen extract showed eight prominent protein bands with estimated molecular weights between 15 and 66 kDa (Figure 1(a)).Figure 1
(a) Coomassie Brilliant Blue stained SDS-PAGE of the crude extract ofA. farnesiana pollen in reducing and nonreducing conditions on 12.5% acrylamide gel. Lane MW, molecular weight marker (Fermentas, Lithuania); R, reducing condition; NR, nonreducing condition. (b) Immunoblotting ofAcacia pollen extract (with reducing SDS-PAGE). Each strip was first blotted withAcaciapollen extract. All strips were then incubated with allergic patients’ sera and detected for IgE reactive protein bands. Lane MW, low molecular weight. NC, negative control.IgE reactivity of the separated protein bands from the electrophoresis ofAcacia pollen extract was determined via immunoblotting assays. Specific IgE binding fractions probed with sera from all forty-two allergic patients are shown in Figure 1(b). The results showed several IgE reactive bands ranging from about 12 to 85 kDa. Figure 1(b) shows the apparent MW of each protein fraction and the prevalence of each one among all forty-two allergic patients. The most frequent IgE reactive bands among the patients’ sera were approximately 66 and 85 kDa. However, there were other IgE reactive protein bands among patients’ sera with molecular weights 15, 20, 23, 28, 39, 45, and 50 kDa (Figure 1(b)). No band was detected when a negative control serum pool was assayed.


### 3.4. Specific IgE Levels and Inhibition ELISA

The sera of forty-two allergic patients were evaluated for specific IgE binding to totalAcacia pollen extract. All allergic patients had detectable specific IgE levels to the total extract ofAcacia pollen (Table 1). These results are consistent with those obtained in the IgE-immunoblotting assays using the pollen extract ofAcacia (Figure 1(b)). To investigate cross-reactivity and allergenic potency amongAcacia andP. juliflora, S. kali, A. retroflexus, C. album,andK. scoparia, ELISA inhibition with their extracts was done (Figure 2). Almost complete inhibition was attained with 100 μg/mL ofAcacia pollen extract as positive control. Preincubation of pooled serum with high concentration (1000 μg/mL) ofP. juliflora revealed significant inhibition of IgE binding to allergenic proteins ofA. farnesiana pollen extract (68%) (Figure 2).Figure 2
Inhibition of IgE binding toAcacia pollen extract by ELISA using pollen extract from the most allergenic plants, mesquite,S. kali, A. retroflexus,C. album, andK. scoparia. Control experiments were performed with BSA.


### 3.5. Immunoblot Inhibition Assays

In order to evaluate the IgE cross-reactivity betweenAcaciaand five other allergenic pollen extracts, an immunoblot inhibition was carried out with theAcaciapollen extract as the solid phase. As shown in Figure 3, IgE reactivity to most of the allergenic proteins ofAcaciapollen was inhibited when theAcaciapollen extract was used as an inhibitor (positive control Lane 2). Complete inhibition of IgE binding to 15, 23, 45, and 50 kDa components ofAcacia extract occurred when the sera were preincubated withP. juliflora extract at 70 μg/mL. Meanwhile, the three bands at about 28, 66, and 85 kDa were partially inhibited. However, whenS. kali, A. retroflexus, C. album,andK. scoparia pollen extracts were used as inhibitors, the 20, 39, 45, and 50 kDa bands were completely inhibited, whereas the 15, 23, 28, 66, and 85 kDa were not inhibited (Figure 3).Figure 3
Immunoblotting inhibition assays. Lane MW, molecular weight marker (Fermentas, Lithuania). Lane 1,Acacia protein strip incubated with pooled serum without inhibitor (negative control). Lane 2,Acacia protein strip incubated with pooled serum containing 70 μg ofAcacia pollen extract as inhibitor (positive control). Lane 3,Acacia protein strip incubated with pooled serum containing 70 μgP. juliflora as inhibitor. Lane 4,Acacia protein strip incubated with pooled serum containing 70 μgS. kali as inhibitor. Lane 5,Acacia protein strip incubated with pooled serum containing 70 μgA. retroflexus as inhibitor. Lane 6,Acacia protein strip incubated with pooled serum containing 70 μgC. album as inhibitor. Lane 7,Acacia protein strip incubated with pooled serum containing 70 μgK. scoparia as inhibitor.


## 3.1. Patients

Forty-two patients, 20 males and 22 females (mean age,33.60±14.47 years; age range 10–70 years), were included in the study (Table 1). All patients suffered from respiratory allergies and seasonal rhinitis without asthma. The patients were all positive by skin prick test withA. farnesiana,P. juliflora, S. kali, A. retroflexus,C. album, andK. scoparia extracts (Table 1). A serum pool of 5 nonallergic subjects was used as a negative control.Table 1
Clinical characteristics, total and specific IgE values, and skin reactivity of allergic patients.
Samplenumber
Age(years)/sex1
Symptoms2
Acaciaspecific IgE3 (OD)
Total IgE (IU/mL)
Diameters (mm) of the papules obtained by prick test4
A. farnesiana
P. juliflora
S. kali
A. retroflexus
C. album
K. scoparia
Patients
1
34/M
L, N, E
0.9
181
7
5
8
9
10
9
2
41/M
N, E
2.33
453
8
9
10
30
11
11
3
39/F
L, N
2.71
156
5
7
16
11
10
9
4
14/F
L, N, E
1.11
165
7
9
7
5
14
11
5
12/M
L, N, E
1.10
123
5
6
4
3
2
0
6
10/M
L, N, E
1.19
313
6
5
10
10
9
8
7
29/F
L, N, E
1.89
345
7
8
8
7
11
5
8
43/F
N, E
1.98
234
6
7
10
12
11
12
9
21/M
N, E
2.07
381
7
9
10
10
12
11
10
26/F
N, E
0.89
225
5
7
10
9
10
11
11
26/F
L, N, E
1.02
468
5
5
10
7
12
8
12
23/F
L, N, E
0.85
265
6
8
13
9
10
10
13
30/M
N, E
1.63
167
7
7
10
7
8
9
14
21/F
L, N, E
1.50
275
7
9
10
10
11
12
15
70/M
L, N, E
1.97
187
6
6
12
11
15
10
16
43/F
N, E
1.85
212
8
7
12
8
11
10
17
32/F
L, N, E
1.94
250
6
8
10
11
13
15
18
46/F
L, N, E
1.52
261
5
7
10
7
9
10
19
34/F
N, E
1.42
301
5
6
5
5
4
0
20
13/M
N, E
2.01
463
8
9
12
5
8
6
21
22/M
N
2.21
280
9
8
8
8
6
7
22
17/F
L, N, E
2.02
302
8
10
8
7
5
5
23
61/M
N
1.04
186
5
6
9
10
9
10
24
66/M
L, N, E
2.10
293
10
12
9
8
6
7
25
10/M
L, N
1.83
220
8
7
4
4
5
0
26
33/M
L, N
1.55
214
7
7
10
10
9
9
27
40/F
L, N
1.65
122
5
5
10
9
11
9
28
53/F
N
1.87
200
7
9
8
8
9
10
29
46/M
N, E
1.91
192
7
6
10
9
10
10
30
43/M
L, N, E
2.15
284
7
7
8
7
9
5
31
19/F
L, N, E
1.92
187
8
7
9
8
9
6
32
36/F
L, N, E
1.89
156
7
8
15
23
25
15
33
19/F
L, N, E
1.29
360
7
10
10
9
10
10
34
37/F
N, E
1.93
267
7
8
12
12
11
11
35
32/M
N, E
1.87
228
7
9
8
9
10
10
36
51/M
N, E
2.11
358
7
8
14
13
11
12
37
28/F
L, N, E
1.60
294
6
8
12
11
11
10
38
29/M
L, N, E
1.96
304
6
7
18
16
18
14
39
44/M
N, E
2.02
418
6
8
10
8
9
9
40
40/M
N, E
2.12
315
8
9
13
18
16
17
41
38/F
N
2.09
480
7
8
8
9
9
6
42
40/F
N, E
1.72
257
6
7
8
7
9
5
Controls
44
28/F
—
0.09
32
0
0
0
0
0
0
45
45/F
—
0.05
12
0
0
0
0
0
0
46
32/M
—
0.06
88
0
0
0
0
0
0
47
38/M
—
0.10
65
0
0
0
0
0
0
48
22/M
—
0.05
22
0
0
0
0
0
0
1M: male; F: female.
2L: lungs symptoms (breathlessness, tight chest, cough, wheeze); N: nose symptoms (sneezing, runny, blocked); E: eyes    symptoms (itching, dry).
3Levels of specific IgE to A. retroflexus pollen extract by ELISA (optical density at 450 nm).
4The mean wheal diameters are displayed in mm. Histamine diphosphate (10 mg/mL) positive control; glycerin-negative control.


## 3.2. Skin Prick Test

Mean diameters of positive wheal sizes wereA. farnesiana: 6.69±1.17 mm,P. juliflora: 7.57±1.50 mm,S. kali: 9.95±2.81 mm,A. retroflexus: 9.74±4.79 mm,C. album: 10.19±3.79 mm, andK. scoparia: 8.9±3.70 mm. Positive correlation coefficients were attained between theA. farnesiana andP. juliflora(r=0.64, P<0.01),A. farnesiana andS. kali (r=0.07, P=0.6),A. farnesiana andA. retroflexus(r=0.1, P=0.4), A. farnesiana andC. album (r=0.06, P=0.6), andA. farnesiana andK. scoparia (r=0.007, P=0.9) pollen extracts for wheal diameter on the SPT (ANOVA procedure).


## 3.3. Protein and Allergenic Profile

The protein composition ofA. farnesiana extract was analyzed by Coomassie Brilliant Blue staining (Figure 1(a)). The reducing SDS-PAGE separation of the pollen extract showed several resolved protein bands in theA. farnesiana extract with molecular weights in the range of approximately 12 to 85 kDa. The most prominent bands had MWs of approximately 50, 66, and 85 kDa. Other predominant bands were identified at 12, 15, 20, 23, 28, 37, and 45 kDa. Nonreducing SDS-PAGE ofAcacia pollen extract showed eight prominent protein bands with estimated molecular weights between 15 and 66 kDa (Figure 1(a)).Figure 1
(a) Coomassie Brilliant Blue stained SDS-PAGE of the crude extract ofA. farnesiana pollen in reducing and nonreducing conditions on 12.5% acrylamide gel. Lane MW, molecular weight marker (Fermentas, Lithuania); R, reducing condition; NR, nonreducing condition. (b) Immunoblotting ofAcacia pollen extract (with reducing SDS-PAGE). Each strip was first blotted withAcaciapollen extract. All strips were then incubated with allergic patients’ sera and detected for IgE reactive protein bands. Lane MW, low molecular weight. NC, negative control.IgE reactivity of the separated protein bands from the electrophoresis ofAcacia pollen extract was determined via immunoblotting assays. Specific IgE binding fractions probed with sera from all forty-two allergic patients are shown in Figure 1(b). The results showed several IgE reactive bands ranging from about 12 to 85 kDa. Figure 1(b) shows the apparent MW of each protein fraction and the prevalence of each one among all forty-two allergic patients. The most frequent IgE reactive bands among the patients’ sera were approximately 66 and 85 kDa. However, there were other IgE reactive protein bands among patients’ sera with molecular weights 15, 20, 23, 28, 39, 45, and 50 kDa (Figure 1(b)). No band was detected when a negative control serum pool was assayed.


## 3.4. Specific IgE Levels and Inhibition ELISA

The sera of forty-two allergic patients were evaluated for specific IgE binding to totalAcacia pollen extract. All allergic patients had detectable specific IgE levels to the total extract ofAcacia pollen (Table 1). These results are consistent with those obtained in the IgE-immunoblotting assays using the pollen extract ofAcacia (Figure 1(b)). To investigate cross-reactivity and allergenic potency amongAcacia andP. juliflora, S. kali, A. retroflexus, C. album,andK. scoparia, ELISA inhibition with their extracts was done (Figure 2). Almost complete inhibition was attained with 100 μg/mL ofAcacia pollen extract as positive control. Preincubation of pooled serum with high concentration (1000 μg/mL) ofP. juliflora revealed significant inhibition of IgE binding to allergenic proteins ofA. farnesiana pollen extract (68%) (Figure 2).Figure 2
Inhibition of IgE binding toAcacia pollen extract by ELISA using pollen extract from the most allergenic plants, mesquite,S. kali, A. retroflexus,C. album, andK. scoparia. Control experiments were performed with BSA.


## 3.5. Immunoblot Inhibition Assays

In order to evaluate the IgE cross-reactivity betweenAcaciaand five other allergenic pollen extracts, an immunoblot inhibition was carried out with theAcaciapollen extract as the solid phase. As shown in Figure 3, IgE reactivity to most of the allergenic proteins ofAcaciapollen was inhibited when theAcaciapollen extract was used as an inhibitor (positive control Lane 2). Complete inhibition of IgE binding to 15, 23, 45, and 50 kDa components ofAcacia extract occurred when the sera were preincubated withP. juliflora extract at 70 μg/mL. Meanwhile, the three bands at about 28, 66, and 85 kDa were partially inhibited. However, whenS. kali, A. retroflexus, C. album,andK. scoparia pollen extracts were used as inhibitors, the 20, 39, 45, and 50 kDa bands were completely inhibited, whereas the 15, 23, 28, 66, and 85 kDa were not inhibited (Figure 3).Figure 3
Immunoblotting inhibition assays. Lane MW, molecular weight marker (Fermentas, Lithuania). Lane 1,Acacia protein strip incubated with pooled serum without inhibitor (negative control). Lane 2,Acacia protein strip incubated with pooled serum containing 70 μg ofAcacia pollen extract as inhibitor (positive control). Lane 3,Acacia protein strip incubated with pooled serum containing 70 μgP. juliflora as inhibitor. Lane 4,Acacia protein strip incubated with pooled serum containing 70 μgS. kali as inhibitor. Lane 5,Acacia protein strip incubated with pooled serum containing 70 μgA. retroflexus as inhibitor. Lane 6,Acacia protein strip incubated with pooled serum containing 70 μgC. album as inhibitor. Lane 7,Acacia protein strip incubated with pooled serum containing 70 μgK. scoparia as inhibitor.


## 4. Discussion

Acacia is one of the major trees throughout arid and semiarid areas of Iran and neighboring countries along the Persian Gulf and the Sea of Oman [2, 16]. SDS-PAGE revealed several bands from theAcaciapollen extract with estimated MWs from 12 kDa to 85 kDa (Figure 1(a)). Among those bands, six IgE binding protein fractions with apparent MWs of 85, 66, 39, 45, 28, 23, and 15 kDa were detected from the blot (Figure 1(b)). Moreover, the results of SDS-PAGE showed relative similarity patterns of migration by protein components of the five selected pollens, mainly those of 15–85 kDa (data not shown). In previous studies the proteins with apparent MWs 39, 45, 66, and 85 kDa have been the most allergenic proteins in the selected allergenic weeds in this study [10, 17–19].In the current study, the immunoblot analysis with individual patient’s serum demonstrated diverse IgE reactivity with seven proteins of 15, 23, 28, 39, 45, 66, and 85 kDa as major IgE binding components. However, immunoblots showed that in the allergenic profile ofAcaciapollen extract the proteins with apparent MWs of 66 and 85 kDa are the major reactive proteins.The results of SDS-PAGE revealed that the overall pattern of migration ofAcacia pollen proteins changed under both reducing conditions. It was thus suggested that probable cysteine residues of the pollen proteins may be associated with interchain disulfide bonds. In previous studies, it was demonstrated that some proteins in pollen extract such as methionine synthase ofS. kali orA. retroflexus were partially degraded into two fragments with approximate MWs of 45 and 39 kDa [19, 20]. Taken together, these observations suggested that some proteins inAcacia pollen extract are susceptible to degradation as a result of proteolysis or exposure to a reducing condition. Moreover, it is possible that the number or size of the products of these proteins’ degradation depends on the conditions of pollen extract preparation and storage. Nevertheless, additional studies are required to elucidate the patterns of degradation and the number and the size of cleavage products.Cross-reactivity amongAcacia pollen components withLolium perenne pollen allergens has been described [8].Several studies reported that proteins with apparent MWs of 45 and 66 kDa are allergenic in the pollen extracts of mesquite [18] and the selected member of the Chenopodiaceae family [10, 19]. The results of immunoblotting inhibition revealed that the IgE binding reactivity of the allergenic proteins with 12, 20, 39, and 45 from theA. farnesianapollen extract was partially inhibited by all four pollen extracts and also partially withK. scopariapollen extract. As shown in Figure 3, IgE reactivity to most of the allergenic proteins ofA. farnesianapollen was inhibited when theP. juliflorapollen extract was used as an inhibitor. This indicates a significant IgE cross-reactivity between the two pollens and is also in line with the results of the SPTs (see below).The results of SPTs indicated highly significant correlations between the wheal diameters from theA. farnesianapollen extract and those fromP. juliflora pollen extracts (r=0.64, P<0.01). These results suggest an extensive IgE cross-reactivity among these pollens and are concurrent with the results of immunoblotting and inhibition experiments. These results collectively suggest that in these pollen extracts, protein components with MWs range 45 to 66 kDa may play a greater role in cross-reactivity compared to others. Earlier studies had also indicated one of the most allergenic proteins ofS. kali pollen, named Sal k 1, with a MW of 40–43 kDa [21, 22]. Sal k 1 displayed pectin methylesterase (PME) properties and is considered a major allergen ofS. kali pollen [14, 21, 22].In general, the knowledge of pollen cross-reactivity is crucial for diagnostics as well as formulation of immunotherapy vaccines. Cross-reactivity among pollens belonging to the same genus and/or different genera has been demonstrated earlier [23]. Some cross-reactive proteins have been genetically engineered and are found to have potential for use in immunotherapy [24].Another dominant IgE binding protein band with an estimated MW of 15 kDa was also detected by immunoblotting ofAcacia pollen extract (Figure 1(b)). Earlier, the allergens belonging to the profilin family with apparent MWs of 14 to 15 kDa were found fromS. kali(Sal k 4),A. retroflexus (Ama r 2), andC. album (Che a 2) pollens [25–27]. It may be that the 15 kDa- protein ofAcacia pollen is homologous with the 15-kDa IgE reactive band in these plants. However, further studies are required to prove the nature of this allergenic protein ofAcacia pollen.


## 5. Conclusion

In conclusion,A. farnesiana pollen is a potent allergen source with several IgE binding components. The observations altogether suggest a close allergenic relationship betweenAcacia and mesquite. Regarding the extensive cross-reactivity between these two trees and the abundance of them in tropical and subtropical regions, identification and production of the recombinant forms of common allergens of these pollens may lead to the exploration of new guidelines for diagnostic, therapeutic, and preventive purposes. Efforts are now underway to clone cDNAs encoding allergenic cross-reactive proteins fromAcacia pollen.



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*Source: 409056-2014-05-18.xml*