Omega-5 and Gamma Gliadin are the Major Allergens in Adult-Onset IgE-Mediated Wheat Allergy: Results from Thai Cohort with Oral Food Challenge

Figures & data

Table 1 Baseline Characteristics of the 15 Patients Enrolled in the Present Study and All 33 Patients in TAWAC Cohort

	Enrolled Patients in the Current Study (N=15)	All Patients in the TAWAC Cohort (N=33)	p-value
Gender
Male	2 (13.3)	9 (27.3)	0.46
Female	13 (86.7)	24 (72.7)
Age, mean ± SD, year	32.3 ± 11.6	32.6 ± 11.3	0.93
Age of onset, ± SD, year	29.9 ±10.3	29.7 ± 10.5	0.97
Wheat allergy phenotype ^a
WDEIA	11 (73.3)	23 (69.7)	1.00
WA	4 (26.7)	10 (30.3)	0.51
Duration of disease, year	1.0 (1.0–5) (min= 0.50, max= 8.0)	2 (1.00–4.00) (min= 0.25, max= 17)	0.857
Number of episode before recruitment	4(2–6)	4(2–6)	0.640
Anaphylaxis severity ^a			1.00
Grade 1	0 (0)	2 (6.1)
Grade 2	8(53.3)	16 (48.5)
Grade 3	7(46.7)	14 (42.4)
Grade 4	0 (0)	1 (3.0)
sIgE to wheat^b level, kUA/L	1.18 (0.58–3.03)	0.54 (0.28–1.53)	0.257
sIgE to ω-5 gliadin^b level, kUA/L	5.84 (1.03–9.48)	3.99 (1.28–9.06)	0.920
Total IgE (IU/mL), IU/mL	400 (68.5–1160)	211 (100–508)	0.430

Notes: Number of patients (% within group), mean±SD and median(P25-P75). ^a Anaphylaxis severity: Ring and Messmer: grade 1–4. ^b Allergen-specific IgE using solid-phase immunoassay, ImmunoCAP (Phadia, Uppsala, Sweden).

Abbreviations: IU/mL, international units per milliliter; kUA/L, kilounits of allergen-specific IgE per liter; nsLTP, non-specific lipid transfer protein; SD, standard deviation; sIgE, specific immunoglobulin E; TAWAC, Thai Adult-onset IgE-mediated Wheat Allergy Cohort; WA, conventional wheat allergy; WDEIA, wheat-dependent exercise-induced anaphylaxis.

Figure 1 Patient recruitment in the study.

Notes: Patient #8 refused oral wheat challenge, and patient#9 had a contraindication for oral wheat challenge (recent coronary heart disease).

Table 2 Clinical Characteristics of the Patients in the Allergen Study (n=15)

Patient	Sex/Age of Onset (Years)	Diagnosis After Wheat-Cofactor Challenge	Symptoms According to History	CAP^a sIgE Wheat (kUA/L)	CAP^a sIgE ω5-Gliadin (kUA/L)	CAP^a sIgE ns-LTP (kUA/L)	Total IgE (IU/mL)	Grass Pollen Sensitization^b
1	F/20	WA	U, A, D, GI	0.1	0.93	0.01	99.7	Ber
2	F/35	WA	U, A, D	1.42	7.44	0.12	3420	Ber
3	F/37	WA	U, A, D, BP	1.18	5.84	0.55	3340	Ber
4	F/18	WA	U, A, GI	35.4	0.23	0.14	1160	Ber
5	F/45	WDEIA	U, A, D, GI	3.03	24.2	0.04	1350	No
6	F/27	WDEIA	U, Syncope, BP	2.69	9.48	0.04	587	Ber, John
7	F/20	WDEIA	U, D	1.06	6.15	0.05	353	No
8^c	F/33	Probable WDEIA	U, A, D, Syncope	0.68	7.85	0.12	545	No
9^c	F/37	Probable WDEIA	U, A, D, Syncope, GI	0.58	1.28	0.04	53.9	No
10	F/19	WDEIA	U, D	1.24	1.03	0.08	195	Ber
11	F/18	WDEIA	U, A, D	10.9	43.8	0.08	640	Ber, John
12	M/30	WDEIA	U, Syncope	0.09	2.75	0	59.6	No
13	F/21	WDEIA	U, D, GI	0.22	1.84	0	24.6	No
14	F/40	WDEIA	U, D, Syncope, BP	1.09	15.2	0.01	400	No
15	M/48	WDEIA	U, D, Syncope	0.06	0.07	0	68.5	No

Notes: ^aAllergen-specific IgE using solid-phase immunoassay: ImmunoCAP (Phadia AB, Uppsala, Sweden). A positive test result is defined by a level of allergen-specific IgE ≥ 0.35 kUA/L ^b Demonstrated by skin prick test. Histamine (10 mg/mL) and normal saline were used as negative and positive controls. A positive test result is defined as a wheal diameter >3 mm larger than the negative control. ^c Did not undergo wheat-cofactor challenge due to refusing challenge (Patient#8) and severe reaction with cardiovascular comorbidity (Patient#9).

Abbreviations: Ber, bermuda grass; BP, hypotension; D, dyspnea; GI, gastrointestinal; IgE. immunoglobulin E; IU/mL, international units per milliliter; John, Johnson grass; kUA/L, kilounits of allergen-specific IgE per liter; nsLTP, non-specific lipid transfer protein; SD, standard deviation; SPT, skin prick test; sIgE, specific immunoglobulin E; U, urticarial; WA, conventional wheat allergy; WDEIA, wheat-dependent exercise-induced anaphylaxis.

Table 3 Identification of IgE-Bound Gliadin and Glutenin

Immunoblot	Molecular Mass (kDa)	Protein
Gliadin fraction
L	30–40	α/β-gliadin
M	40–59	γ-gliadin, peroxidase
M, H	45–70	ω-gliadin
Glutenin fraction
L, M	40–59	LMW glutenin, alpha-amylase/subtilisin inhibitor
H	70–100	HMW glutenin

Notes: L = proteins in MW range of <40 kDa; MW = Molecular weight; M = proteins in MW range of 40–59 kDa; H = proteins in MW range of >60 kDa.

Abbreviations; kDA, kilodaltons; LMW, low molecular weight; HMW, high molecular weight.

Figure 2 IgE immunoblotting of gliadin (A) and glutenin (B) extracts.

Notes: L = proteins in MW range of <40 kDa; M = proteins in MW range of 40–59 kDa; H = proteins in MW range of >60 kDa.

Abbreviations: WA, Wheat allergy group (patients 1–4); WDEIA, Wheat-dependent exercise-induced anaphylaxis group (patients 5–15); MW, molecular weight marker; kDa, kilodaltons.

Table 4 ELISA Results of γ-Gliadin and ω5-Gliadin Compared with ImmunoCAP Results

Patients	IgE Bound γ-Gliadin (Mean OD650 nm)	IgE Bound ω5-Gliadin (Mean OD650 nm)	SIgE ω5-Gliadin ImmunoCAP (kUA/L)	SIgE Wheat ImmunoCAP (kUA/L)
1	0.52	0.18^a	0.93	0.1
2	0.28	1.19	7.44	1.42
3	0.50	1.70	5.84	1.18
4	0.49	0.22	0.23	33.4
5	1.48	2.24	24.2	3.03
6	0.92	1.33	9.48	2.69
7	0.28	1.70	6.15	1.06
8	0.35	0.50	7.85	0.68
9	0.20	0.27	1.28	0.58
10	0.11^a	0.38	1.03	1.24
11	0.49	2.59	43.8	10.9
12	0.20	0.42	2.75	0.09
13	0.46	0.36	1.84	0.22
14	0.31	1.32	15.2	1.09
15	0.19^a	0.29	0.07	0.06

Note: ^a OD₆₅₀ nm level < 0.2 cut-off.

Abbreviations: mean OD _{650 nm}, the mean of optical densities at 650 nanometers; ELISA, enzyme-linked immunosorbent assay; kUA/L, kilounits of allergen-specific IgE per liter.

Figure 3 Direct binding ELISA. (A) γ-gliadin, (B) ω5-gliadin.

Notes: The dotted-line denotes a cut-off value. The median is shown for each group. The solid line denoted mean OD_{650 nm} level within the group. The serum of one wheat-dependent exercise-induced anaphylactic patient (patient #15) was excluded due to a low value of specific IgE to both wheat and ω5-gliadin.

Abbreviations: ELISA, enzyme-linked immunosorbent assay; OD, optical density; nm, nanometer.