Detection of serum IgG autoantibodies to FcεRIα by ELISA in patients with chronic spontaneous urticaria.

BACKGROUND: Mast cells are a key effector cell in the pathogenesis of chronic spontaneous urticaria (CSU) and activated by circulating FcεRI-specific IgG as well as IgE. This study evaluated the prevalence of circulating autoantibodies to FcεRIα in the sera of CSU patients.
METHODS: Eighty-eight patients with CSU and 76 healthy controls (HCs) were enrolled. To detect circulating autoantibodies (IgG/IgA/IgM) to FcεRIα, ELISA was done using YH35324 (as a solid phase antigen), and its binding specificity was confirmed by the ELISA inhibition test. The antibody levels were presented by the ratio of YH35324-preincubated to mock-preincubated absorbance values. Clinical and autoimmune parameters, including atopy, urticaria activity score (UAS), serum total/free IgE levels, serum antinuclear antibody (ANA) and autologous serum skin test (ASST) results, were assessed. The autoimmune group was defined if CSU patients had positive results to ASST and/or ANA.
RESULTS: The ratio of serum IgG to FcεRIα was significantly lower in CSU patients than in HCs (P<0.05), while no differences were noted in serum levels of IgG to recombinant FcεRIα or IgA/IgM autoantibodies. The autoimmune CSU group had significantly lower ratios of IgG/IgA (not IgM) autoantibodies to FcεRIα than the nonautoimmune CSU group (P<0.05 for each). No significant associations were found between sex, age, atopy, urticaria duration, UAS, or serum total/free IgE levels according to the presence of IgG/IgA/IgM antibodies.
CONCLUSIONS: This study confirmed the presence of IgG to FcεRIα in the sera of CSU patients, especially those with the autoimmune phenotype.

Introduction

Chronic spontaneous urticaria (CSU) is defined as the occurrence of wheals, angioedema, or both for more than 6 weeks due to unknown causes [1]. The prevalence rate of CSU was reported at 0.5%-5% in the general population and has been increasing worldwide along with industrialization [2, 3].

Mast cell is a key effector cell, and when activated by various triggers, it releases mediators and cytokines like histamine. Recent guidelines recommend the measurement of serum autoantibody, IgG to thyroid peroxidase (TPO), which is related to not only thyroid diseases but also autoimmune diseases, to evaluate autoimmunity in patients with CSU [1, 4].

Although IgE-mediated mechanism is a major one to activate mast cells, recent studies demonstrate autoimmune-mediated mechanisms in patients with CSU, especially those with more severe phenotypes [5-7]. A type IIb hypersensitivity as an autoantibody-mediated mechanism was first reported in CSU [8]. A-positive result to the autologous serum skin test (ASST) was related to the presence of autoantibodies in CSU [9]. However, the prevalence or role of each autoantibody in CSU has not been fully understood [10, 11]. Among autoantibodies, IgG to FcεRIα has been reported to be a major autoantibody for activating mast cells in CSU patients; however, a recent report has demonstrated a higher prevalence of IgM to FcεRIα (than IgA and IgG antibodies to FcεRIα) in the sera of CSU patients [11].

Regarding the detection method of IgG autoantibody to FcεRIα, several different methods were reported; a standardized method is still unavailable. We could measure serum free IgE using YH35324 (capturing the FcεRIα part of free IgE) in the sera of asthmatics [12] and hypothesized that this YH35324 could capture the FcεRIα binding site of FcεRIα-specific antibodies. Then, we extended ELISA to detect circulating IgG/IgA/IgM autoantibodies to FcεRIα in the sera of CSU patients compared to controls. In the present study, we aimed to evaluate the prevalence of serum IgG/IgA/IgM autoantibodies to FcεRIα in association with clinical/autoimmune parameters in patients with CSU.

Methods

Study subjects

The present study enrolled 88 CSU patients and 76 healthy controls (HCs) from the Department of Allergy and Clinical Immunology, Ajou University Hospital (Suwon, South Korea). Patients who had urticaria symptoms for over 6 weeks without any inducible causes were diagnosed as having CSU. HCs had no history of allergic diseases like urticaria or autoimmune disease.

CSU patients were over 18 years old and had never been exposed to IgE-related biologics. They were allowed to use control medications including antihistamines, except steroids, for 4 weeks prior to the study, and their sera were collected at the diagnosis and stored at −80°C until estimation. The levels of serum IgG/IgA/IgM autoantibodies to FcεRIα were measured by ELISA with applying YH35324 and compared between CSU patients and HCs. All participants provided written informed consent and agreed to voluntarily participate in the study. This study was approved by the Institutional Review Board of Ajou University Hospital (AJIRB-BMR-SMP-20-435).

The demographic and clinical characteristics of the study subjects were obtained by the investigators. The severity of CSU including disease duration, current medication requirements, and the urticaria activity score (UAS-15) were analyzed. The UAS-15 included the degree of pruritus and the number/size/distribution/duration of wheal over the preceding week at the initial visit. Patients were classified according to the severity of symptoms as follows: 0, symptom-free; 1 to 5, mild urticaria; 6 to 10, moderate urticaria; and 11 to 15, severe urticaria. Concomitant allergic disease and atopic status were documented.

Skin prick tests (SPT) were performed with 55 common aeroallergens (Bencard, Brentford, UK), which included Dermatophagoides pteronyssinus, D. farinae, histamine, and a saline negative control. A positive reaction was defined as a wheal of more than 3 mm in diameter or wheal size larger than a positive control (histamine) after 15 minutes. If SPT was not possible, specific IgE antibodies to clinically relevant allergens, including house dust mite (HDM)-specific IgE, were measured. Atopy was defined when SPT showed a positive results or serum allergen specific IgE was elevated (≥0.35 IU/mL) to at least 1 common environmental allergen. The levels of serum total and specific IgE were measured by ImmunoCAPs (ThermoFisher Scientific, Waltham, MA, USA). Serum free IgE level was measured by ELISA as previously described [12].

Evaluation of autoimmunity in CSU

Routine laboratory measurements were taken and included complete blood count, liver enzymes, thyroid stimulating hormone (TSH), and the antinuclear antibody (ANA) screening test. In the cases of abnormal TSH results, thyroid autoantibodies (anti-TPO or anti-TG) were checked. The autologous serum skin test (ASST) was performed to evaluate autoimmune characteristics in CSU patients. The subjects who underwent the ASST had an intradermal injection of 50 μL of their own sera into their arm. After 30 minutes, the patient’s wheal and flare responses were compared with the responses to normal saline and histamine as controls. An induced wheal larger than 1.5 mm was considered a positive result [13].

ELISA for the measurement of serum autoantibodies

The levels of serum autoantibodies to FcεRIα were measured using ELISA by using YH35324 (Yuhan, Seoul, South Korea). This novel YH35324 is based on a human IgG4 Fc domain combined with a human FcεRIα extracellular domain on its Fab chain. As a FcεRIα domain, we speculated that YH35324 could have not only high affinity for serum free IgE but also circulating IgG autoantibodies to FcεRIα. Based on its affinity, we developed a new ELISA using this YH35324 for the measurement of IgG/IgA/IgM autoantibodies to FcεRIα. In brief, the YH35324 in bicarbonate buffer (pH 9.6) was coated at 1μg/mL onto each well (a solid phase) overnight at 4°C. After washing 3 times with 0.05% Tween 20 containing phosphate-buffered saline (PBST), the plates were blocked with buffer (10% FBS containing PBST) for 1 hour.

Sera from CSU and HCs were pre-incubated for 1 hour with 5 μg/mL YH35324 or mock (10% FBS containing PBST). Then, 2 kinds of sera (pre-incubated with YH35324 or mock at 1:10 dilution) were loaden onto antigen-coated wells at room temperature for 2.5 hours and washed 5 times with PBST. After washing, each well was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature and washed 5 times with PBST.

For the measurement of IgG autoantibodies to FcεRIα, purified serum by using the Gel-IgG-Spin purification kit (ThermoFisher Scientific, Rockford, IL, USA) was incubated, and HRP-conjugated anti-IgG antibody (1:2000, BD Biosciences, Franklin Lakes, NJ, USA) was used as a second antibody, followed by 2 hours of incubation. In the case of IgA and IgM autoantibodies to FcεRIα, sera purification was not applied. HRP-conjugated anti-human IgA and anti-human IgM (1:3000 for IgA, 1:2000 for IgM, SouthernBiotech, Birmingham, AL, USA) were used as second antibodies and incubated for 1.5 hours.

Finally, the plates were incubated with tetramethylbenzidine (TMB) substrate (BD Biosciences) for 20 minutes, followed by stopping the reaction with 2N H2SO4. The absorbance values were read using an ELISA reader (BioTek, Winooski, VT, USA) at 450 and 570 nm, and the subtracted values were analyzed. All samples were measured 3 times repeatedly, and mean values were analyzed after removing outliers. The coefficient of variation in the reproducibility of ELISA for IgG to FcεRIα was 13.7%. The serum levels of IgG to recombinant FcεRIα were measured simultaneously and compared between patients with CSU and HCs according to the same protocol for the measurement of IgG to FcεRIα except that each well was coated with recombinant soluble FcεRIα (MyBioSource, San Diego, CA, USA) as a solid phase antigen and recombinant soluble FcεRIα was used as an inhibitor instead YH35324.

The levels of IgG/IgA/IgM autoantibody were presented as the ratio of YH35324 preincubated to mock-preincubated values. Reduced levels after the pre-incubation was calculated, and lower ratios represent higher levels of circulating autoantibodies [11].

ELISA inhibition test

Competitive ELISA inhibition tests were performed to confirm the binding specificity of YH35324. Sera from patients with CSU were incubated overnight at 4°C, with increasing amounts (0–1000 μg/mL protein concentration) of YH35324 before use. The pretreated sample was incubated with the YH35324-coated microtiter plate, and ELISA was carried out as previously described. D. farinae was used as a nonspecific allergen. The inhibition rate was calculated by the formula: inhibition rate (%) = 100 –(absorbance with inhibitor/absorbance without inhibitor) x100.

Statistical analysis

To evaluate the association among 3 autoantibodies and clinical parameters, parametric and non-parametric continuous variables were compared using Student’s t test and the Mann-Whitney test. Categorical values were compared Pearson’s Chi-squared test and Fisher’s exact test. Parametric and non-parametric variables were compared among over 2 groups using ANOVA and the Kruskal-Wallis test. If standard deviations were not equal in parametric variables, Welch’s ANOVA test was used. Bonferroni and Dunnett were applied as post hoc tests. Correlation coefficients were calculated by the Pearson method. Cutoff values for the prevalence of circulation autoantibodies to FcεRIα were determined at the 5% percentile ratio of non-autoimmune CSU. All statistical analyses were performed, and graphs were created using R 4.1.0 (R Core Team, 2021), GraphPad Prism Version 9.2.0 for Mac (GraphPad Software, San Diego, CA, USA). Statistical significance was set at P less than 0.05 for all tests.

Results

Clinical characteristics of the study subjects

Table 1 shows the baseline characteristics of patients enrolled compared to HCs. Patients with CSU was younger than HCs (median 37.5 [IQR 25.0–48.0] vs. median 41.0 [IQR 34.0–52.0], P = 0.01). No differences were found in sex or atopy prevalence. The mean UAS was 9.2±4.3; positive rates of ASST and ANA were 22.7% and 18.2%, respectively. Both serum total and free IgE levels were significantly higher in CSU patients than in HCs, with significant differences (P<0.001 for each). Two patients with CSU had hyperthyroidism and 1 patient with CSU had hypothyroidism. Eight patients with abnormal results of TSH or a history of thyroid disease were positive to at least one thyroid autoantibody (anti-TPO or anti-TG). Patients with positive thyroid autoantibodies did not have any autoantibodies to FcεRIα. S1 Fig showed the correlation between IgG to YH35324 and IgG to recombinant FcεRI. (Pearson r = 0.41, P<0.0001).

Table 1

Clinical characteristics of the study subjects in the cohort.

Variable	CSU	Healthy controls	P value
	(n = 88)	(n = 77)
Female, n (%)	54 (61.4%)	39 (50.6%)	0.22
Age (years)	37.5 [25.0;48.0]	41.0 [34.0;52.0]	0.01
Atopy, n (%)	54 (61.4%)	36 (50.0%)	0.20
UAS-15	9.2±4.3	NA
ASST positive, n (%)	20 (22.7%)	NA
ANA positive, n (%)	16 (18.2%)	NA
Serum total IgE (kU/L)	135.5 [64.5;312.5]	50.7 [20.4;110.5]	<0.001
Serum free IgE (ng/mL)	252.9 [99.5;639.8]	105.9 [23.4;273.5]	<0.001

Non-parametric values are presented as median [IQR] and parametric values or mean ± SD. Categorical values are shown as number (percentage). P values were evaluated by the t test or the Mann-Whitney test. CSU, chronic spontaneous urticaria; HCs, healthy controls; UAS, urticaria activity score; ASST, autologous serum skin test; ANA, antinuclear antibody, NA: not available

The prevalence of serum autoantibodies (IgG/IgA/IgM) to FcεRIα in CSU patients

Fig 1 shows the comparison of serum IgG/IgA/IgM levels to FcεRIα between patients with CSU and HCs. The ratio of IgG to FcεRIα was significantly lower in patients with CSU than in HCs, while no differences were noted in the ratio of IgG to recombinant FcεRIα or IgA/IgM autoantibodies to FcεRIα. The ELISA inhibition test confirmed binding specificity by showing a dose-dependently increasing inhibition rate (%) with serial addition of the YH35324 (0–1000 μg/mL) in the sera of 1 patient with CSU (S2 Fig).

Fig 1

Comparison of serum IgG, IgA, and IgM autoantibodies to FcεRIα between patients with chronic spontaneous urticaria (CSU) and healthy controls (HCs).

The results were presented by the IgG ratio of YH35324-pretreated to mock-treated values (A), the IgG ratio of rhFc-pretreated to mock-treated value (B), the IgA ratio of YH35324-pretreated to mock-treated value (C) and the IgM ratio of YH35324-pretreated to mock-treated value (D). Horizontal lines and dotted lines show median values and cutoff values of each autoantibody to FcεRIα respectively. P values were evaluated by Mann-Whitney test. rhFc, recombinant human Fc; NS, no statistical significance.

Table 2 summarizes comparisons of clinical parameters with the results of serum IgG/IgA/IgM to FcεRIα. The cutoff value for the positive predictive value of IgG/IgA/IgM to FcεRIα was determined from the results of non-autoimmune CSU and shown as dotted lines (0.65, 0.72, and 0.71, respectively, Figs 1 and 2). The prevalence of serum IgG/IgA/IgM to FcεRIα in CSU were 8.1, 5.8, and 3.4% respectively. No significant differences were noted in demographic findings, clinical parameters (sex, age, atopic status, disease duration, UAS, and serum total/free IgE), or comorbid conditions (allergic rhinitis and asthma) according to the presence of serum IgG/IgA/IgM to FcεRIα. In addition, no differences were noted in peripheral eosinophil or basophil counts between the groups with positive and negative to IgG/IgA/IgM autoantibody. The levels of HDM-specific IgE were higher in the IgG-positive group than in the IgG-negative group (median values of D. pteronyssinus-specific IgE, 18.1 [IQR 1.1–24.9] kU/L vs. 0.1 [IQR 0.1–1.8] kU/L, P<0.05; the median values of D. farinae-specific IgE, 33.9 [IQR 1.3–45.7] kU/L vs. 0.3 [IQR 0.1–2.5] kU/L, P<0.05), while no significant associations were found with atopic status or serum IgE levels between the 2 groups. No differences were found in the results of IgA/IgM autoantibodies as shown in Table 2.

Table 2

Clinical characteristics of the patients with chronic spontaneous urticaria (CSU) according to the results of serum autoantibodies to FcεRIα.

Variables (n = 86)	IgG to FcεRIα		IgA to FcεRIα		IgM to FcεRIα
	Negative	Positive	Negative	Positive	Negative	Positive
	(n = 79, 91.8%)	(n = 7, 8.1%)	(n = 81, 94.1%)	(n = 5, 5.8%)	(n = 83, 96.5%)	(n = 3, 3.4%)
Female, n (%)	50 (63.3%)	3 (42.9%)	49 (60.5%)	4 (80.0%)	51 (61.4%)	2 (66.7%)
Age (years)	37.0 [25.0;48.5]	44.0 [38.0;47.0]	38.0 [27.0;48.0]	22.0 [22.0;42.0]	38.0 [25.0;48.0]	47.0 [41.0;51.5]
Angioedema, n (%)	38 (48.1%)	3 (42.9%)	39 (48.1%)	2 (40.0%)	40 (48.2%)	1 (33.3%)
UAS-15	10.0 [6.0;13.0]	7.0 [5.5;11.5]	10.0 [6.0;13.0]	9.0 [2.0;12.0]	10.0 [6.0;13.0]	15.0 [9.0;15.0]
Duration (months)	10.0 [2.5;27.0]	7.0 [4.5;8.0]	8.0 [2.5;24.0]	10.0 [3.0;12.0]	9.0 [3.0;24.0]	1.0 [0.6;3.0]
ASST positive, n (%)	17 (21.5%)	3 (42.9%)	19 (23.5%)	1 (20.0%)	20 (24.1%)	0 (0.0%)
ANA positive, n (%)	13 (16.5%)	3 (42.9%)	15 (18.5%)	1 (20.0%)	15 (18.1%)	1 (33.3%)
Serum total IgE (kU/L)	132.0 [59.5;288.0]	396.0 [179.5;538.0]	139.0 [61.0;294.0]	182.0 [93.0;520.0]	143.0 [65.5;312.5]	68.0 [37.0;296.5]
Serum free IgE (ng/mL)	254.8 [100.7;555.3]	788.1 [129.1;797.8]	254.8 [99.3;611.8]	311.4 [250.9;781.1]	266.7 [100.7;639.8]	163.2 [83.3;478.3]
Atopy, n (%)	46 (58.2%)	7 (100.0%)	48 (59.3%)	5 (100.0%)	52 (62.7%)	1 (33.3%)
Der p IgE (kU/L)	0.1 [0.1;1.8] *	18.1 [1.1;24.9] *	0.2 [0.1;2.4]	0.5 [0.2;2.0]	0.2 [0.1;2.0]	0.2 [0.1;3.4]
Der f IgE (kU/L)	0.3 [0.1;2.5] *	33.9 [1.3;45.7] *	0.4 [0.1;4.3]	1.4 [0.2;1.8]	0.4 [0.1;3.9]	0.2 [0.2;3.0]
Eosinophil counts (cells/μL)	93.5 [57.6;161.8]	62.4 [44.9;155.1]	93.5 [57.6;162.8]	65.0 [36.0;156.0]	93.5 [55.2;168.9]	70.4 [35.2;96.0]
Basophil counts (cells/μL)	32.0 [22.8;43.0]	39.0 [33.1;54.8]	32.4 [22.8;44.0]	39.6 [32.5;42.7]	33.0 [23.7;43.6]	22.8 [14.6;47.4]

Non-parametric values are shown as median [IQR] and categorical values are shown as number (percentage).

P values were evaluated by Pearson’s Chi-squared test or the Mann-Whitney test.

*P<0.05, between patients with negative results and those with positive results to IgG to FcεRIα.

UAS, urticaria activity score; ASST, autologous serum skin test; ANA, antinuclear antibody; Der p, Dermatophagoides pteronyssinus; Der f, Dermatophagoides farinae

Fig 2

Comparisons of serum IgG/IgA/IgM levels to FcεRIα between the autoimmune and nonautoimmune groups.

The results were presented by the IgG ratio of YH35324-pretreated to mock-treated value (A), the IgA ratio of YH35324-pretreated to mock-treated value (B) and the IgM ratio of YH35324-pretreated to mock-treated value (C). The autoimmune group was defined if CSU patients have a positive result to ASST or ANA test. Horizontal lines and dotted lines show median values and cutoff values of each autoantibody FcεRIα respectively. P value was evaluated by Mann-Whitney test. ASST, autologous serum skin test; ANA, antinuclear antibody; NS, no statistical significance.

Comparison of IgG to FcεRIα levels with the results of ASST and ANA

When defined if CSU patients had at least 1 positive result to ASST or ANA, the autoimmune CSU group had significantly lower ratio of IgG/IgA to FcεRIα than the non-autoimmune CSU group (the median values of IgG to FcεRIα, 0.73 [IQR 0.68–0.81] vs. 0.78 [IQR 0.72–0.89], P<0.05; the median values of IgA to FcεRIα, 0.93 [IQR 0.87–1.01] vs. 0.99 [IQR 0.93–1.05])(P<0.05, Fig 2). IgM to FcεRIα showed no difference between the 2 groups. Comparison of serum autoantibodies between the ASST-positive and ASST-negative groups showed no significant difference (S3 Fig).

Discussion

The present study demonstrates the presence of circulating IgG antibody to FcεRIα by the home-made ELISA using purified IgG sera and the YH35324 (capturing the FcεRIα portion of specific IgG to FcεRIα) as the solid phase antigen. The IgG-ELISA inhibition test demonstrated the binding specificity with serial additions of YH35324 (up to 88.85% inhibition). In addition, higher levels of serum IgG/IgA to FcεRIα were noted in patients with CSU with autoimmune phenotypes, while serum IgG to recombinant FcεRIα were not detectable in the same serum set of the study subjects. These findings suggest that our ELISA is a reproducible and reliable method to detect serum IgG level to FcεRIα in the sera of various allergic and immunologic diseases, although further replication studies are needed.

For CSU, the autoimmune mechanism plays a major role in activating mast cells [14, 15]. CSU patients with type IIb autoimmunity have serum autoantibodies to FcεRIα or IgE on the surface of mast cells [11, 16]. From the first study on IgG to FcεRIα in patients with CSU to the latest study evaluating the prevalence of IgG, IgA and IgM to FcεRIα in those with CSU, autoimmune CSU has long been studied [11, 17]. However, there are no standardized method to measure serum IgG/IgA/IgM autoantibodies to FcεRIα, and the reference values for determining autoimmunity are not determined.

To define type IIb autoimmune CSU, 3 diagnostic criteria have been recommended: (1) positive ASST, (2) positive basophil histamine release assay and/or basophil activation test, and (3) positive IgG autoantibodies against FcεRIα and/or IgE [4, 7]. CSU patients with positive ASST results present more severe symptoms, including wheals, itching, and systemic manifestations, than those with negative ASST results [7]. However, no difference was noted in this study. The positive rate of the ASST was 22.7% in the present study, which is lower than those of previous studies (34% to 70%) [18, 19]. Autoimmune thyroiditis was reported to be related to the persistent positive result of ASST in patients with CSU independent of urticaria symptoms, questioning the role of ASST in the CSU patients with autoimmune thyroiditis [20]. Further long-term follow-up studies are needed to evaluate its clinical relevance (effects on drug responses or long-term clinical outcomes) in the management of CSU.

The ASST has been increasingly performed and revealed that it is associated with IgG antibodies against the FcεRIα or IgE. The positive reaction in the ASST was reported to confirm the presence of autoantibodies in CSU [9]. A previous study showed higher serum IgG to FcεRIα estimated by immunoblot assay in CSU patients, especially in those with a positive ASST, although further replication studies are not validated [10]. These findings suggest that IgG to FcεRIα may be involved in autoantibody-mediated mechanism of CSU. However, the present study showed no significant difference in IgG to FcεRIα between the ASST-positive and ASST-negative group (S3 Fig). The discordance between previous studies and ours in the same ethnicity may be attributed to: (1) differences in the positive rates of ASST found in the study subjects and (2) the different detection methods applied (immune-dot assay vs. ELISA). The sensitivity and specificity between these 2 different assays cannot be comparable [21].

Regarding autoimmune parameters in CSU, ANA has commonly been reported to be present in CSU patients, especially those associated with the autoimmunity type IIb [22]. The ANA-positive group is known to be associated with poor responses to antihistamines or omalizumab [23, 24]. The positive ANA rate in CSU patients were 18.2% in the present study, which is consistent with the positive rates (15%-29%) of previous studies [10, 23, 25]. However, no differences were found in UAS or other clinical parameters between the ANA-positive and ANA-negative groups, similar to a previous study [24].

To evaluate autoimmunity in CSU, basophil autoreactivity functional assay is needed, but it is difficult to apply in every patient in the aspects of technical difficulties, cost, and low availability in the real-world practice [26]. Therefore, basophil histamine release assay and/or basophil activation test were not widely performed in real-world study or practice.

The prevalence of IgG autoantibody to FcεRIα was reported from 4% to 64% in CSU patients [27]. For IgM and IgA autoantibodies, there has been a study reporting higher prevalence (60% and 57%, respectively) in CSU patients [11]. The present study showed a higher prevalence of IgG to FcεRIα in CSU patients than in HCs, but no differences were noted in the prevalence of IgM or IgA autoantibodies to FcεRIα between CSU patients and controls. In addition, when they were compared according to autoimmunity, the autoimmune group had a higher prevalence of IgG to FcεRIα than the nonautoimmune group, although the prevalence of IgG to FcεRIα was 8.1%, which were lower than those of previous studies [11]. The wide range of the prevalence of autoantibodies in CSU has not yet been clearly explained. There are a few explanations of these differences. First, the measurement methods were different among the studies, where western blot analysis, ELISA (with using different antigens/antibodies) or both assays were applied [27]. Further studies are required to confirm the results. Secondly, the discrepancies in autoantibody prevalence may have been attributed to differences in underlying causes and regional differences [1]. Even using the same measurement method, the prevalence rates were variable [10]. Thirdly, the cutoff criteria were different whether they were derived from HCs or other control subjects [10, 11]. The number of the study subjects having high levels of serum autoantibodies to FcεRIα was very low, and some of HCs had this autoantibody without any evidence of autoimmune disease or CSU. Therefore, the cutoff values from HCs may have failed to differentiate the autoimmune CSU group from the non-autoimmune CSU group. It is also supported by the fact that the prevalence of disease-related autoantibodies in the disease-free subgroup from the European cohort of autoantibody- related disease was found to be 23.6% [28].

There have been several studies showing that the presence of IgG autoantibodies against FcεRIα is linked to more severe symptoms, a poor response to conventional antihistamine treatment, and a slower response to anti-IgE treatment [11, 29, 30]. IgM to FcεRIα was reported to have a correlation with peripheral basopenia and eosinopenia [11]. IgA to FcεRIα was related to present or past gastrointestinal-or mucosal-associated infections [11]. The present study showed no significant associations between IgG to FcεRIα and clinical/severity parameters. Recent studies suggest that, along with mast cells, eosinophils, basophils and other cells are involved in CSU [31]. Both lesional or non-lesional skin of CSU patients showed a mixed infiltrating cells such as eosinophils and basophils [1, 31–33]. Recruitment of eosinophils into the skin can be mediated by the interactions between mast cells and various mediators. Eosinophil granule proteins from activated eosinophils cause persistent mast cell activation, and IgG-anti-FcεRII/CD23 leads to eosinophil secretion via the low-affinity IgE receptor FcεRII/CD23 on eosinophils and histamine release from basophils, suggesting the role of interactions between mast cells and basophils/eosinophils in the pathogenesis of CSU. When peripheral eosinophil and basophil counts were compared according to the presence of serum IgG to FcεRIα, no differences were found in the present study, indicating that their immune cells are not associated with IgG autoantibody to FcεRIα in CSU.

There are 2 limitations in this study. One is that the operational definition of autoimmune CSU without basophil functional assay may be insufficient to reflect real autoimmunity. Basophil functional assays are the only currently available method to evaluate functional autoantibodies, however they are difficult to conduct in outpatient clinics due to technical difficulties and costs. Both tests for ANA and ASST are poor surrogate markers for a risk of false positivity and a possibility of the co-existence of atopic status [13, 34]. Nonetheless, they are potential alternatives to evaluate autoimmunity which is clinically relevant in CSU patients [13, 23, 35]. The other is that we could not find any clinical significance according to the presence of IgG to FcεRIα as well as IgA/IgM autoantibodies to FcεRIα, which may have been attributed to a lower number of positive responders. Further replication studies in a larger cohort of CSU patients are needed to validate these findings.

In conclusion, we detected higher IgG autoantibody levels to FcεRIα by the ELISA with applying the YH35324 in the sera of CSU patients compared to HCs, which were higher in the autoimmune group, suggesting a possible involvement of circulating autoantibodies against FcεRIα in the autoimmune mechanism of CSU.

Correlation between log transformed IgG to YH35324 and log transformed IgG to recombinant FcεRI before pretreatment analyzed by Pearson’s correlation.

(TIF)

Click here for additional data file.

Competitive ELISA inhibition tests with serial additions of YH35324 and D. farina extracts (200μg/mL).

ELISA, enzyme-linked immunosorbent assay; D. farinae; Dermatophagoides farinae.

(TIF)

Click here for additional data file.

Comparison of serum IgG/IgA/IgM levels to FcεRIα between the ASST-positive and ASST-negative group.

The results were presented by the IgG ratio of YH35324-pretreated to mock-treated value (A), the IgA ratio of YH35324-pretreated to mock-treated value (B) and the IgM ratio of YH35324-pretreated to mock-treated value (C). ASST, autologous serum skin test; NS, no statistical significance.

(TIF)

Click here for additional data file.

4 Jul 2022

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Additional Editor Comments :

July 4, 2022

Dear Dr. Hae-Sim Park

Ref: PONE-D-22-12472

Title: Detection of serum IgG autoantibodies to FcεRIα by ELISA in patients with chronic spontaneous urticaria

Journal: PLOS ONE

Thank you for your choosing us for your publication medium. Your study on the autoantibodies to the IgE receptor is interesting in our readers. However, there are some demerits in the discrete evidences and setting of experimental observation.

We have completed the review process of your manuscript. As you can read the attached comments, there are several contriversial issues in your study, causing for the 6 independent reviews, which are not general.

One reviewer is very positive, but the other criticisms are so important for your research to justify.

I believe that the comments can help you to revise your MS.

I hope you can easily revise it.

Thank you

Sincerely,

Cheorl-Ho Kim Prof

Sungkyunkwan Univ, Biol Science Dept.

Editor of Plos One

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: No

Reviewer #4: Partly

Reviewer #5: No

Reviewer #6: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: I Don't Know

Reviewer #5: Yes

Reviewer #6: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: Yes

Reviewer #6: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

Reviewer #6: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The objective of this study was to investigate the prevalence of circulating autoantibodies to FcεRIα in association with clinical/autoimmune parameters in CSU patients. In the era of tailored medicine, endotyping of CSU is strengthened in order to provide better treatment option for CSU patients. Authors performed important study and demonstrated the presence of serum autoantibodies to FcεRIα using their own reliable ELISA methods which were much higher in the autoimmune CSU group. Further studies are needed to validate this methods and results in the large number of CSU patients cohort.

Reviewer #2: Jang and co-workers investigate an ELISA method to detect IgG autoantibodies to the high affinity IgE receptor in patients with chronic spontaneous urticaria by using a mAb as solid phase antigen. Inhibition assays are used to assess the specificity of the method. They report that IgG to Fc�  RI are present in sera of CSU patients, especially in those with an autoimmune phenotype.

GENERAL COMMENTS

A standardized in-vitro method able to detect Fc�  RI autoantibodies has been sought for decades with little success. The present attempt is quite interesting, but the study shows a series of shortcomings that need to be addressed in order to increase its quality.

SPECIFIC POINTS

Line 85: Which allergens were tested?

Line 98: This is a mistake! In most cases CSU patients with thyroid autoantibodies don’t show any defect in thyroid function. Thus, testing TPO and TG auto-antibodies only in the presence of abnormal TSH levels leads to an underestimate of autoimmune study subjects.

Lines 166-7: The higher total IgE levels in CSU patients suggest autoallergy rather than IgG-mediated autoimmunity (see Schmetzer, at al. J Allergy Clin Immunol. 2018 Sep;142(3):876-882.)

Lines 168-171. See previous comment to line 98.

Line 227: Regarding the prevalence of ASST see Asero et al Clin Exp Allergy. 2001 Jul;31(7):1105-10.

Line 229: See Fusari et al. Allergy. 2005 Feb;60(2):256-8.

Lines 235-6: In most previous studies dealing with ASST and HRA, there has always been a marked discrepancy between the prevalence of these two markers, with ASST+ patients that were invariably much more frequent that HRA+ patients

Lines 251-2: The functional basophil activation tests maybe are not widely performed but remain the only reliable method to check whether the autoantibodies to the high affinity IgE receptor or to IgE are functionally active (see also Ref 17)

Lines 269-70: this is evidence against the specificity of the detection of these autoantibodies in the absence of a functional testing.

Lines 293-305: Evidence of an association between HDM and CSU is dated and poor. Atopic status as a whole prevails in auto-allergic CSU patients (type I after Gell-Cooms). See Schmetzer et al. suggested above.

Line 308: “Basophil functional assays… ”. These are missing but remain the only way to show that IgG autoantibodies are functional. Of autoimmunity

Line 309: ANA and ASST are poor surrogate markers. See also Asero et al. Eur Ann Allergy Clin Immunol. 2021 Dec 14. doi: 10.23822/

MINOR POINTS

Line 39: Why CU and not CSU?

Line 48: Reference 17 is also appropriate at this point

Line 56: a standardized method is still unavailable.

Line 114: Sera? Maybe the authors mean the YH35324 bound to the solid phase…

Line 117: dilution) were loaden onto…

Line 223: and/or IgE (delete anti- and antibodies)

Line 233: delete using.

Lines 242-3: Please delete the sentence in brackets.

Line 282: Suggestion: “Recent studies suggest that, along with mast cells, eosinophils, basophils and other cell types are involved…”

Reviewer #3: This report seeks to evaluate auto-antibodies specific for FcER1 that could be causally-linked to CSU. The approach uses a novel fusion protein in which an extracellular domain of FcER1alpha is linked to an IgG4 Fc domain. I have several concerns with the report. I found it challenging to understand the methodologies, which could relate to language, but also due to the way it is presented. Some of my concerns:

1) The main ELISA appears to actually be an inhibition assay. Why is it not described as such (noting that a separate section describes an inhibition ELISA with dose-response curve)? Why is there no data using a direct ELISA - it would likely be easier to interpret and more compelling than the current presentation? How was the inhibition concentration of 5 ug/mL chosen for the main assays, when in the dose-response study this same concentration exhibited very little inhibition?

2) Why the IgG purification in line 120, but not in the main assay? This seems like comparing apples and oranges between the approaches

3) How good are the performance characteristics of the selected cut-offs for distinguishing between CSU and HC?

4) One of the main positive findings in the paper, namely Fig 1A, shows a difference between CSU and HC that is largely explained by several outliers in the HC group with high ratios. How are these values >1 explained? Are they biologically plausible or meaningful?

5) The discussion dominates the paper and is lengthy.

Overall, there may be a story here, but the methods and results as described and shown are hard to understand and raise concerns about validity and reproducibility.

Reviewer #4: The currently accepted definition for autoimmune urticaria is finding functional autoantibodies by basophil histamine release assay or basophil activation tests. ANA and ASST do not provide sufficient evidence. Positive thyroid antibodies with low total IgE are potential surrogate markers for patients with a positive positive BHRA. Functional anti-IgE autoantibodies have also been described in chronic spontaneous urticaria. They have not been assessed in this study. Immunoassays detect functional and non-functional antibodies. Earlier work by Dr Kaplan showed that the IgG subclass is important in determining functionality in the basophil histamine release assay. Why use UAS6 - the standard clinical scoring system is over 7 days? The methodology of the ELISA is difficult to follow. The clinical utility of this test is uncertain. Some of the references are misquote, such as 7 and 8.

Reviewer #5: The manuscript “Detection of serum IgG autoantibodies to FcεRIα by ELISA in patients with chronic spontaneous urticaria” reported the results of a study that investigated CSU patients using an innovative ELISA test to detect serum Ig to FcεRIα. The study is interesting, but there are some criticism in the methods and the results are not in line with literature.

In particular:

Methods, lines 90-91. Atopy was defined when SPT showed a positive results or serum allergen specific IgE was elevated (≥0.35 IU/mL) to at least 1 common environmental allergen. Why you didn’t considered also clinical symptoms? Considering only SPT or specific IgE, you cannot diagnosed atopic dermatitis.

Methods, lines 97-98. “In cases of abnormal TSH results, thyroid autoantibodies (anti-TPO or anti-TG) were checked”. Really, there are some patients with autoimmune thyroiditis and normal TSH. Also CSU guidelines recommended the measurement of serum autoantibody IgG to thyroid peroxidase. How you checked thyroid autoantibodies only in cases of abnormal TSH?

Results, lines 176-177. “The ratio of IgG to FcεRIα was significantly lower in patients with CSU than in HCs.” This result is singular. In fact, in literature the incidence of anti- FcεRIα autoantibodies is normally higher in CSU patients (Immunobiology. 2018 Dec;223(12):807-811 - Egypt J Immunol . 2020 Jan;27(1):141-155 - J Microbiol Immunol Infect. 2020 Feb;53(1):141-147 - J Immunol Res. 2022 Feb 25;2022:6863682).

Results, lines 198-199. “The autoimmune CSU group had significantly lower ratio of IgG/IgA to FcεRIα than the non-autoimmune CSU group”. Also, this result is singular. In fact, in literature the incidence of anti- FcεRIα autoantibodies is normally higher in CSU patients with autoimmunity (especially with positive ASST).

Discussion, lines 207-208. “Higher levels of serum IgG/IgA to FcεRIα were noted in patients with CSU with autoimmune phenotypes”. In results, you state “The autoimmune CSU group had significantly lower ratio of IgG/IgA to FcεRIα than the non-autoimmune CSU group”. How can you explain this difference?

Discussion, lines 232-234. “A previous study showed higher serum IgG to FcεRIα estimated by using immunoblot assay in CSU patients, especially in those with a positive ASST, although further replication studies are not validated”. Really, some other studies confirm higher serum IgG to FcεRIα in CSU patients, as above reported.

Discussion, lines 236-240. “The discordance between previous studies and ours in the same ethnicity may be attributed to … the different detection methods applied (immune-dot assay vs ELISA). The sensitivity and specificity between these 2 different assays cannot be comparable.” In this case, the lower serum IgG to FcεRIα that you have encountered can be attributed to: (1) lower sensitivity of the method that you used; (2) lower specificity of other methods, giving false positive results. Can you demonstrate this statement?

Discussion, lines 256-257. “The present study showed a higher prevalence of IgG to FcεRIα in CSU patients than in HCs”. In results, you state “The ratio of IgG to FcεRIα was significantly lower in patients with CSU than in HCs.” How can you explain this difference?

Discussion, lines 259-260. “The autoimmune group had a higher prevalence of IgG to FcεRIα than the nonautoimmune group”. In results, you state “The autoimmune CSU group had significantly lower ratio of IgG/IgA to FcεRIα than the non-autoimmune CSU group”. How can you explain this difference?

Reviewer #6: Authors have established an ELISA for IgG, IgM and IgA autoantibodies against FceRIa, using a recombinant chimeric molecule which consists of human IgG2 and FceRIa. Using this system, the authors studied the presence of IgG, IgA and IgM autoantibodies against FceRIa in sera of patients with chronic spontaneous urticaria (CSU) and healthy controls, in relation to autoimmune characteristics and other clinical backgrounds. They detected all IgG, IgA and IgM subclasses of autoantibodies against FceRI both in sera of patients with CSU and healthy controls, but the prevalence is higher in IgG subclass of patients with CSU, especially that in patients of autoimmune group, and IgA subclass in patients with CSU of autoimmune group, but not in IgM subclass. The reason for discrepancies between this report and previous reports of the autoantibodies against FceRIa remains unclear.

Major problems:

1. The presence of autoantibodies against FceRIa is commonly compared with reactions in autologous serum skin test (ASST) and basophil functional assay (histamine release test or basophil activation test) rather than the comprehensive autoimmune characteristics defined by the authors in this study, i.e. positive results to ASST and/or antinuclear antibody (ANA) (autoimmune group). The authors stated that they were not afford to do basophils functional assays. However, the reviewer argue that the authors should show relationships of the amounts of autoantibodies and results of ASST. Authors should also describe why they employed ASST in combination to ANA, which was not taken in their previous study of the autoantibodies using dot blot assay (ref 19).

2. Authors showed that the level of IgG autoantibodies against YH35324 in patients with CSU was higher than that of healthy control. Moreover, the level in patients in autoimmune group was higher than that of non-autoimmune group. How about the comparison between patients with CSU in non-autoimmune group and healthy control?

3. For the inhibition test in ELISA, authors used YH35324 as an inhibitor. I wonder why not recombinant FceRIa, which should reflect more specific binding of autoantibodies against FceRIa.

4. Figure 1. Cutoff values for autoantibodies in this study should be shown as dotted or broken lines.

5. Page 4, line 80-84. The way of evaluation by UAS6 should be described in more detail. Is it the sum of UAS in 6 days? If so, the worst score should be 36, but sever urticaria was defined as 11 to 15 in line 83.

6. Page 7, line 132-135. These descriptions read to me that YH35324 was used as an inhibitor. If authors intend to show the superiority of their ELISA using YH35324 to ELISA using recombinant soluble FceRIa, which is the method employed in reference 10, they should use recombinant FceRIa rather than YH35324. In any case, data of such comparison is more convincing for readers. Moreover, a figure of relationship of IgG binding to HY35324 and that to recombinant FceRIa in sera of individual donors should be shown as a supplementary material.

7. Page 10, line 211. A phrase, “as well as serum free IgE in the sera of various allergic” should be deleted. Data in this manuscript do not contain any information directly support this statement.

8. Page 11, line 235-236. “However, the present study showed no ….” As pointed above, data should be shown, even if there is no difference between ASST-positive and negative groups. Table 2 showed the number and rate of ASST positive patients in IgG autoantibodies positive and negative patients, but did not show the numbers of IgG autoantibodies positive and negative patients in ASST positive and negative patients.

9. Page 260, line 260. The prevalence should also be described in the Result section as well.

10. Page 13-15, Discussions in line 276 to the end, especially to 305 are too long and tedious. The level of anti-house dust mite IgE does not have much relevance to the study of autoantibodies taken in this study.

Minor problems]

1. Page 6, line 122. Is “IgG antibody” a mistake for “anti-IgG antibody”?

2. Page 11, line 215. “to FceRIa” should be “to FceRIa or IgE”.

**********

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Reviewer #2: No

Reviewer #3: No

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Reviewer #6: Yes: Michihiro Hide

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28 Jul 2022

Reviewer #1: The objective of this study was to investigate the prevalence of circulating autoantibodies to FcεRIα in association with clinical/autoimmune parameters in CSU patients. In the era of tailored medicine, endotyping of CSU is strengthened in order to provide better treatment option for CSU patients. Authors performed important study and demonstrated the presence of serum autoantibodies to FcεRIα using their own reliable ELISA methods which were much higher in the autoimmune CSU group. Further studies are needed to validate this methods and results in the large number of CSU patients cohort.

Our response: We thank the reviewer for the positive comment on this study. Indeed, this study highlighted the presence of autoantibodies to FcεRIα, and compared it between the autoimmune and non-autoimmune groups in patients with CSU, which should be further validated in larger cohorts of CU patients.

Reviewer #2: Jang and co-workers investigate an ELISA method to detect IgG autoantibodies to the high affinity IgE receptor in patients with chronic spontaneous urticaria by using a mAb as solid phase antigen. Inhibition assays are used to assess the specificity of the method. They report that IgG to Fc�  RI are present in sera of CSU patients, especially in those with an autoimmune phenotype.

GENERAL COMMENTS

A standardized in-vitro method able to detect Fc�  RI autoantibodies has been sought for decades with little success. The present attempt is quite interesting, but the study shows a series of shortcomings that need to be addressed in order to increase its quality.

SPECIFIC POINTS

Line 85: Which allergens were tested?

Our response: We evaluated the atopic status of enrolled patients using skin prick tests with common aeroallergens including Dermatophagoides pteronyssinus, D. farinae, cat, dog, cockroach, tree pollen mixture, grass pollen mixture, mugwort, ragweed, Humulus japonicus, Aspergillus, and Alternaria which are the most prevalent inhalant allergens in our environment.

Line 98: This is a mistake! In most cases CSU patients with thyroid autoantibodies don’t show any defect in thyroid function. Thus, testing TPO and TG auto-antibodies only in the presence of abnormal TSH levels leads to an underestimate of autoimmune study subjects.

Our response: We completely agree with your comments. In this study we used the laboratory test results retrospectively collected from the enrolled patients. In fact, the healthcare system of our country recommends that testing TPO and TG auto-antibodies would be preceded by the abnormal thyroid function test results. In this study, thyroid autoantibodies were measured only if the patients were indicated. Therefore, with this limitation, we did not use thyroid autoantibodies to define the autoimmune group of CSU.

Lines 166-7: The higher total IgE levels in CSU patients suggest autoallergy rather than IgG-mediated autoimmunity (see Schmetzer, at al. J Allergy Clin Immunol. 2018 Sep;142(3):876-882.)

Our response: Thank you for your comments. Indeed, higher levels of serum total IgE were reported to be more related to autoallergy in CSU than in type IIb CSU in previous studies. Consequently, higher total IgE levels in patients with CSU may result in the lower prevalence of autoantibodies to FcεRIα in this study. However, CSU patients having serum autoantibodies to FcεRIα had higher median value of serum total IgE levels than that of HCs (219.0 [68.0;525.0] vs. 46.1[22.0;97.9], P value 0.004) in this study. Therefore, higher total IgE levels in CSU patients does not always rule out the IgG-mediated autoimmunity in CSU patients in this cohort.

Lines 168-171. See previous comment to line 98.

Our response: Thank you for your comments. As we demonstrated, we evaluated thyroid autoimmunity in this study with the limitation of the healthcare system. Therefore, we evaluated thyroid autoimmunity in the patients if they were indicated.

Line 227: Regarding the prevalence of ASST see Asero et al Clin Exp Allergy. 2001 Jul;31(7):1105-10.

Our response: Thank you for your comments. We added the reference which you mentioned in the manuscript. (L 232)

Line 229: See Fusari et al. Allergy. 2005 Feb;60(2):256-8.

Our response: Thank you for your comments. We added the reference which you mentioned in the manuscript as following: Autoimmune thyroiditis was reported to be related to the persistent positive result of ASST in patients with CSU independent of urticaria symtpoms, questioning the role of ASST in the CSU patients with autoimmune thyroiditis. (L 233:234)

Lines 235-6: In most previous studies dealing with ASST and HRA, there has always been a marked discrepancy between the prevalence of these two markers, with ASST+ patients that were invariably much more frequent that HRA+ patients

Our response: Thank you for raising this point. This discrepancy may basically come from the methodological difference between these two methods. Each test could cover different spectrum of CSU with autoimmunity. A recent review about ASST in CSU demonstrated that almost all the patients with the positive result of basophil functional test including HRA were also ASST-positive, whereas a partial of ASST-positive patients were also basophil functional test positive. Circulating histaminergic factors in addition to autoantibodies were pointed as the potential reason for the auto-reactivity of ASST-positive patients. The positive result of ASST is a poor predictor of a positive basophil functional test, but ASST-negative patients could be excluded for the presence of functional circulating autoantibodies detectable by HRA based on high negative predictive value of ASST result.

Lines 251-2: The functional basophil activation tests maybe are not widely performed but remain the only reliable method to check whether the autoantibodies to the high affinity IgE receptor or to IgE are functionally active (see also Ref 17)

Our response: We entirely agree with your comments. It is the fact that basophil histamine release assay and basophil activation test are reliable methods to evaluate functional autoantibodies to FcεRIα in autoimmune CSU. However, the basophil function test must need considerable amount of samples, and it was not enough for the present study to evaluate the basophil function test considering the amount of stored patient samples. The present study aimed to evaluate the presence of serum autoantibodies to FcεRIα in CSU using YH35324. Further studies should be followed to validate the functional effect of these autoantibodies on basophils.

Lines 269-70: this is evidence against the specificity of the detection of these autoantibodies in the absence of a functional testing.

Our response: Thank you for your comments. We agree with your comments. However, in order to confirm its binding specificity, we did IgG-ELISA (competitive) inhibition test with serial additions of YH35324 as shown in Supplementary Figure 2. Significant inhibitions were noted with increasing doses of YH35324 preincubated in dose-dependent manners, while no significant inhibition was noted with addition of high dose of house dust mite allergen, indicating that circulating IgG autoantibodies detected in sera using our ELISA have binding specificity to FcεRIα, although we could not confirm their function whether they could release histamine from basophils or not.

Lines 293-305: Evidence of an association between HDM and CSU is dated and poor. Atopic status as a whole prevails in auto-allergic CSU patients (type I after Gell-Cooms). See Schmetzer et al. suggested above.

Our response: Thank you for your comments. We agree with your comments. Therefore, we have deleted the section describing the association between HDM and CSU as suggested.

Line 308: “Basophil functional assays… ”. These are missing but remain the only way to show that IgG autoantibodies are functional. Of autoimmunity

Our response: Thank you for your comments. We added as suggested. (L304:L305)

Line 309: ANA and ASST are poor surrogate markers. See also Asero et al. Eur Ann Allergy Clin Immunol. 2021 Dec 14. doi: 10.23822/

Our response: Thank you for your comments. We admitted that the absence of the basophil function assays is critical limitation of this study. However, as we mentioned, this kind of test is not yet appropriate for performing in outpatient clinic. It is easier to do ASST or ANA test for screening autoimmunity in CSU patients than doing basophil functional test considering high negative predictive value of ASST, ranging from 59% to 100%. We added the reference as suggested. (L 306:311)

MINOR POINTS

Line 39: Why CU and not CSU?

Our response: Thank you for your comments. We have changed from the word “CU” to CSU through the manuscript. (L39)

Line 48: Reference 17 is also appropriate at this point

Our response: We have modified as suggested. (L48)

Line 56: a standardized method is still unavailable.

Our response: We have modified as suggested. (L56)

Line 114: Sera? Maybe the authors mean the YH35324 bound to the solid phase…

Our response: Thank you for your comments. We have changed the word “sera” to plate. (L114)

Line 117: dilution) were loaden onto…

Our response: We have modified as suggested. (L117) …

Line 223: and/or IgE (delete anti- and antibodies)

Our response: We have modified as suggested. (L239) …

Line 233: delete using.

Our response: Thank you for your comments. We have removed using in the revised manuscript as suggested. (L241)

Lines 242-3: Please delete the sentence in brackets.

Our response: Thank you for your comments. We have deleted the sentence as suggested. (L 250)

Line 282: Suggestion: “Recent studies suggest that, along with mast cells, eosinophils, basophils and other cell types are involved…”

Our response: We have modified as suggested. (L289:L290)

Reviewer #3: This report seeks to evaluate auto-antibodies specific for FcER1 that could be causally-linked to CSU. The approach uses a novel fusion protein in which an extracellular domain of FcER1alpha is linked to an IgG4 Fc domain. I have several concerns with the report. I found it challenging to understand the methodologies, which could relate to language, but also due to the way it is presented. Some of my concerns:

1) The main ELISA appears to actually be an inhibition assay. Why is it not described as such (noting that a separate section describes an inhibition ELISA with dose-response curve)?

Our response: Thank you for your comments. We demonstrated the result of ELISA inhibition test with serial additions of YH35324 in the supplementary Figure 2.

Why is there no data using a direct ELISA - it would likely be easier to interpret and more compelling than the current presentation?

Our response: We already performed a direct ELISA with YH35324, but there was much background activity of non-specific IgG bindings as each serum have very high IgG level. Therefore, we compared the ratio of inhibition could be better for lowering the background signals of the measurement as previously reported by European investigators (Allergy 2020;75:3208 by Maurer M et al.)

How was the inhibition concentration of 5 ug/mL chosen for the main assays, when in the dose-response study this same concentration exhibited very little inhibition?

Our response: Thank you for your comments. We did preliminary experiments to determine the optimal concentration of YH35324 to determine the presence of IgG autoantibody level compared to controls and found that 5 µg/mL is the optimal concentration to differentiate positive and negative groups in each serum.

2) Why the IgG purification in line 120, but not in the main assay? This seems like comparing apples and oranges between the approaches

Our response: Thank you for your comments. We applied IgG purification for the preparing measurement of IgG to FcεRIα to reduce non-specific binding to YH35324. In case of measurement of IgA and IgM, the levels of IgA and IgM were so low for using purification process.

3) How good are the performance characteristics of the selected cut-offs for distinguishing between CSU and HC?

Our response: The sensitivity and the specificity are 8.14% and 85.71%. The cut-off values for the presence of autoantibodies in patients with CSU were determined at the 5% percentile ratio of non-autoimmune CSU. Therefore, it is not appropriate using this cut-off values for distinguishing between CSU and HC. The sensitivity and the specificity for determining the autoimmune CSU and non-autoimmune CSU groups are 15.63% and 96.30% respectively.

4) One of the main positive findings in the paper, namely Fig 1A, shows a difference between CSU and HC that is largely explained by several outliers in the HC group with high ratios. How are these values >1 explained? Are they biologically plausible or meaningful?

Our response: We thank the reviewer for raising this point. We do also have been concerned about the value over 1. However, it has been concluded that they might be derived from non-specific binding effects in this assay.

5) The discussion dominates the paper and is lengthy.

Our response: Thank you for your comments. We have removed the part about relationship between HDM sensitization and CSU. The revised manuscript appears tidier.

Overall, there may be a story here, but the methods and results as described and shown are hard to understand and raise concerns about validity and reproducibility.

Our response: Thank you for your comments. We did our best to trim the manuscript as suggested.

Reviewer #4: The currently accepted definition for autoimmune urticaria is finding functional autoantibodies by basophil histamine release assay or basophil activation tests. ANA and ASST do not provide sufficient evidence. Positive thyroid antibodies with low total IgE are potential surrogate markers for patients with a positive positive BHRA.

Our response: Thank you for your comments. Indeed, the absence of the basophil functional test is critical limitation of this study. We aimed to validate new measurement of serum autoantibodies to FcεRIα in CSU patients. The currently accepted definition for type IIb autoimmune CSU needs the presence of IgG anti-FcεRIα which means a signal reduction by more than 50% and it is not clearly standardized. The ASST with a high negative predictive value ranging from 59% to 100% could be used to exclude the presence of functional circulating autoantibodies to FcεRIα in CSU patients. In this study, we used ANA and ASST result to define autoimmune CSU and non-autoimmune CSU for validating the new measurement of serum autoantibodies. In addition, CSU patients with positive thyroid antibodies did not have autoantibodies to FcεRIα independent of serum total IgE levels in our cohort.

Functional anti-IgE autoantibodies have also been described in chronic spontaneous urticaria. They have not been assessed in this study.

Our response: We focused on the measurement of autoantibodies to FcεRIα in CSU patients. However, we evaluated IgG to IgE autoantibodies in this cohort, which showed no significant difference. (Data were not shown)

Immunoassays detect functional and non-functional antibodies.

Our response: Thank you for your comments. We admitted the absence of evaluating functional antibodies was the limitation of this study.

Earlier work by Dr Kaplan showed that the IgG subclass is important in determining functionality in the basophil histamine release assay.

Our response: Unfortunately, we did not evaluate the IgG subclass of autoantibodies to FcεRIα.

Why use UAS6 - the standard clinical scoring system is over 7 days?

Our response: Thank you for your comments. We have replaced UAS with UAS-15 and removed the word “UAS6” to descript the scoring method more precisely. (L80)

The methodology of the ELISA is difficult to follow. The clinical utility of this test is uncertain.

Our response: The measurement of serum autoantibodies to FcεRI in CSU patients has not yet been standardized. We aimed to demonstrate the presence of serum autoantibodies using ELISA with YH35324 in our cohort and evaluated its functional relevance according to autoimmune status (based on the results of ASST and ANA). We used the same ELISA method published by Maurer M et al. (Allergy 2020;75:3208). Further replication studies are needed in other cohorts in other regions.

Some of the references are misquote, such as 7 and 8.

Our response: Thank you for your comments. We have modified the quotation of the references.

Reviewer #5: The manuscript “Detection of serum IgG autoantibodies to FcεRIα by ELISA in patients with chronic spontaneous urticaria” reported the results of a study that investigated CSU patients using an innovative ELISA test to detect serum Ig to FcεRIα. The study is interesting, but there are some criticism in the methods and the results are not in line with literature.

In particular:

Methods, lines 90-91. Atopy was defined when SPT showed a positive results or serum allergen specific IgE was elevated (≥0.35 IU/mL) to at least 1 common environmental allergen. Why you didn’t considered also clinical symptoms? Considering only SPT or specific IgE, you cannot diagnosed atopic dermatitis.

Our response: Thank you for your comments. In fact, the word “atopy” does not mean the disease as atopic dermatitis in this context. We aimed to define the atopic tendency of the enrolled patients as “atopy”. There is no need to consider clinical symptoms to define atopic tendency.

Methods, lines 97-98. “In cases of abnormal TSH results, thyroid autoantibodies (anti-TPO or anti-TG) were checked”. Really, there are some patients with autoimmune thyroiditis and normal TSH. Also CSU guidelines recommended the measurement of serum autoantibody IgG to thyroid peroxidase. How you checked thyroid autoantibodies only in cases of abnormal TSH?

Our response: Thank you for your comments. We could not screen the presence of serum autoantibodies to thyroglobulin (TG) or thyroid-specific peroxidase (TPO) antigens in all study subjects with CSU enrolled in this study. The measurement of autoantibodies related to thyroid disease is recommended to be followed by abnormal thyroid function test results (including high TSH level) following the health care system guideline in this country. Therefore, we limitedly evaluated thyroid autoantibodies.

Results, lines 176-177. “The ratio of IgG to FcεRIα was significantly lower in patients with CSU than in HCs.” This result is singular. In fact, in literature the incidence of anti- FcεRIα autoantibodies is normally higher in CSU patients (Immunobiology. 2018 Dec;223(12):807-811 - Egypt J Immunol . 2020 Jan;27(1):141-155 - J Microbiol Immunol Infect. 2020 Feb;53(1):141-147 - J Immunol Res. 2022 Feb 25;2022:6863682).

Our response: Thank you for your comments. In this study, the ratio was defined as YH35324 preincubated to mock-preincubated values. Theoretically, preincubated with YH35324 could diminish the concentration of autoantibodies to FcεRIα. Therefore, in this study, the lower ratio of IgG to FcεRIα indicates higher levels of anti-FcεRIα IgG which was described in the text and published by Maurer M et al. (Allergy 2020; 75:3208).

Results, lines 198-199. “The autoimmune CSU group had significantly lower ratio of IgG/IgA to FcεRIα than the non-autoimmune CSU group”. Also, this result is singular. In fact, in literature the incidence of anti- FcεRIα autoantibodies is normally higher in CSU patients with autoimmunity (especially with positive ASST). Discussion, lines 207-208. “Higher levels of serum IgG/IgA to FcεRIα were noted in patients with CSU with autoimmune phenotypes”. In results, you state “The autoimmune CSU group had significantly lower ratio of IgG/IgA to FcεRIα than the non-autoimmune CSU group”. How can you explain this difference?

Our response: Thank you for your comments. The lower ratio of YH35324 preincubated to mock-preincubated values demonstrated the higher concentration of autoantibodies measured by YH35324. This result is consistent with previous studies (Allergy 2020; 75:3208, and Figure 1 in the present study), therefore increased circulating autoantibody to FcεRIα is associated with the autoimmune phenotype of CU (based on results of ASST and ANA).

Discussion, lines 232-234. “A previous study showed higher serum IgG to FcεRIα estimated by using immunoblot assay in CSU patients, especially in those with a positive ASST, although further replication studies are not validated”. Really, some other studies confirm higher serum IgG to FcεRIα in CSU patients, as above reported.

Our response: Thank you for your comments. The lower ratio of IgG to FcεRIα indicate s higher level of circulating IgG to FcεRIα, therefore, the result of the present study is comparable with previous reports showing higher prevalence of serum autoantibodies, IgG to FcεRIα in CSU patients (Allergy 2020; 75:3208).

Discussion, lines 236-240. “The discordance between previous studies and ours in the same ethnicity may be attributed to … the different detection methods applied (immune-dot assay vs ELISA). The sensitivity and specificity between these 2 different assays cannot be comparable.” In this case, the lower serum IgG to FcεRIα that you have encountered can be attributed to: (1) lower sensitivity of the method that you used; (2) lower specificity of other methods, giving false positive results. Can you demonstrate this statement?

Our response: The discordance between the previous study and the present study was that the present study showed no significant difference in IgG to FcεRIα between the ASST-positive and ASST-negative group even in the same ethnicity. The previous study compared nearly the same number of patients according to the ASST results (64 ASST-positive and 61 ASST-negative patients) compared to relatively small number of CSU patients having positive ASST results (n=20) in the present study. Considering various positive rates of ASST results, we could not compare sensitivity and specificity of these two different studies.

Discussion, lines 256-257. “The present study showed a higher prevalence of IgG to FcεRIα in CSU patients than in HCs”. In results, you state “The ratio of IgG to FcεRIα was significantly lower in patients with CSU than in HCs.” How can you explain this difference?

Our response: Thank you for your comments. As we mentioned, the lower ratio implied the higher concentration of autoantibodies FcεRIα in CSU patients. Therefore, the lower ratio of IgG to FcεRIα in CSU patients could be translated into a higher prevalence of IgG to FcεRIα in CSU than in HCs.

Discussion, lines 259-260. “The autoimmune group had a higher prevalence of IgG to FcεRIα than the nonautoimmune group”. In results, you state “The autoimmune CSU group had significantly lower ratio of IgG/IgA to FcεRIα than the non-autoimmune CSU group”. How can you explain this difference?

Our response: Thank you for your comments. The lower ratio of IgG to FcεRIα in the autoimmune CSU group (than in the non-autoimmune CSU group) could imply a higher concentration of IgG to FcεRIα, which can be translated into a higher prevalence of IgG to FcεRIα in the autoimmune CSU group.

Reviewer #6: Authors have established an ELISA for IgG, IgM and IgA autoantibodies against FceRIa, using a recombinant chimeric molecule which consists of human IgG2 and FceRIa. Using this system, the authors studied the presence of IgG, IgA and IgM autoantibodies against FceRIa in sera of patients with chronic spontaneous urticaria (CSU) and healthy controls, in relation to autoimmune characteristics and other clinical backgrounds. They detected all IgG, IgA and IgM subclasses of autoantibodies against FceRI both in sera of patients with CSU and healthy controls, but the prevalence is higher in IgG subclass of patients with CSU, especially that in patients of autoimmune group, and IgA subclass in patients with CSU of autoimmune group, but not in IgM subclass. The reason for discrepancies between this report and previous reports of the autoantibodies against FceRIa remains unclear.

Major problems:

1. The presence of autoantibodies against FceRIa is commonly compared with reactions in autologous serum skin test (ASST) and basophil functional assay (histamine release test or basophil activation test) rather than the comprehensive autoimmune characteristics defined by the authors in this study, i.e. positive results to ASST and/or antinuclear antibody (ANA) (autoimmune group). The authors stated that they were not afford to do basophils functional assays. However, the reviewer argue that the authors should show relationships of the amounts of autoantibodies and results of ASST. Authors should also describe why they employed ASST in combination to ANA, which was not taken in their previous study of the autoantibodies using dot blot assay (ref 19).

Our response: Thank you for your comments. Data were not shown, but we mentioned that the ratio of IgG to FcεRIα had no significant difference comparing between the ASST-positive and ASST-negative group. For this reason, we combined ANA result to ASST to define the non-autoimmune group in CSU who might have low prevalence of serum autoantibodies. The previous study sharing same ethnicity instead compared ASST-positive and ASST-negative patients with nearly same number of patients. Unfortunately, we could not evaluate functional effects of IgG to FcεRIα detected in this study.

2. Authors showed that the level of IgG autoantibodies against YH35324 in patients with CSU was higher than that of healthy control. Moreover, the level in patients in autoimmune group was higher than that of non-autoimmune group. How about the comparison between patients with CSU in non-autoimmune group and healthy control?

Our response: Thank you for your comments. Patients with CSU in the non-autoimmune group tended to show lower ratio of IgG autoantibody to FcεRIα than that of HCs, however, no statistical significance was noted. The ratio of IgA/IgM to FcεRIα showed no difference between the 2 groups.

3. For the inhibition test in ELISA, authors used YH35324 as an inhibitor. I wonder why not recombinant FceRIa, which should reflect more specific binding of autoantibodies against FceRIa.

Our response: We evaluated the ratio of IgG to FcεRIα using recombinant FcεRIα (as a solid phase antigen) and showed no significant differences between CSU patients and HCs, therefore we did not use recombinant FcεRIα as an inhibitor for ELISA inhibition test.

4. Figure 1. Cutoff values for autoantibodies in this study should be shown as dotted or broken lines.

Our response: Thank you for your comments. We added cutoff values of each autoantibody to FcεRIα as dotted lines in both Figure 1 and 2.

5. Page 4, line 80-84. The way of evaluation by UAS6 should be described in more detail. Is it the sum of UAS in 6 days? If so, the worst score should be 36, but sever urticaria was defined as 11 to 15 in line 83.

Our response: Thank you for your comments. We evaluated disease activity using the 15-point urticaria activity score (UAS-15). UAS-15 includes degree of pruritus and the number/size/distribution/duration of wheal during the preceding week. Each parameter is scored from 0 to 3 and the maximum UAS-15 with total five parameters can be 15, which were revised in the text.

6. Page 7, line 132-135. These descriptions read to me that YH35324 was used as an inhibitor. If authors intend to show the superiority of their ELISA using YH35324 to ELISA using recombinant soluble FceRIa, which is the method employed in reference 10, they should use recombinant FceRIa rather than YH35324. In any case, data of such comparison is more convincing for readers. Moreover, a figure of relationship of IgG binding to HY35324 and that to recombinant FceRIa in sera of individual donors should be shown as a supplementary material.

Our response: Thank you for your comments. We aimed to show compare the efficacy of ELISA with YH35324 and recombinant FcεRIα, and do ELISA under the same protocol, except only replacing YH35324 with recombinant FcεRIα. The recombinant FcεRIα was used as an inhibitor measuring IgG binding to recombinant FcεRIα. The manuscript has been edited as suggested. (L 136) And we added Supplementary Figure 1 showing the correlation between IgG binding to YH35324 and that to recombinant FcεRIα. The manuscript has been edited as suggested. (L 172:L173)

7. Page 10, line 211. A phrase, “as well as serum free IgE in the sera of various allergic” should be deleted. Data in this manuscript do not contain any information directly support this statement.

Our response: Thank you for your comments. The phrase has been deleted.

8. Page 11, line 235-236. “However, the present study showed no ….” As pointed above, data should be shown, even if there is no difference between ASST-positive and negative groups. Table 2 showed the number and rate of ASST positive patients in IgG autoantibodies positive and negative patients, but did not show the numbers of IgG autoantibodies positive and negative patients in ASST positive and negative patients.

Our response: Thank you for your comments. We attached the supplementary figure S3 comparing autoantibodies between ASST positive and negative groups in CSU. (L205:L207)

9. Page 260, line 260. The prevalence should also be described in the Result section as well.

Our response: Thank you for your comments. We additionally demonstrated the prevalence of autoantibodies to FcεRIα in patients with CSU in the Result section and Table 2.

(L187)

10. Page 13-15, Discussions in line 276 to the end, especially to 305 are too long and tedious. The level of anti-house dust mite IgE does not have much relevance to the study of autoantibodies taken in this study.

Our response: Thank you for your comments. We have removed the section describing the relationship between house dust mite and CSU in the text.

Minor problems]

1. Page 6, line 122. Is “IgG antibody” a mistake for “anti-IgG antibody”?

Our response: Thank you for your comments. We have changed as suggested. (L122)

2. Page 11, line 215. “to FceRIa” should be “to FceRIa or IgE”.

Our response: Thank you for your comments. We have changed as suggested. (L221)

Submitted filename: response_letter-26072022.docx

Click here for additional data file.

9 Aug 2022

Detection of serum IgG autoantibodies to FcεRIα by ELISA in patients with chronic spontaneous urticaria

PONE-D-22-12472R1

Dear Dr. Park,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Academic Editor

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Additional Editor Comments (optional):

August 9, 2022

Dear Dr Park,

Thanks for your submission to Plos One.

I have checked your revision and found it valuable for publication, although there are still controversial issues on the clinical tests or examinations.

As you know, the serum IgG autoantibodies to FcεRIα has been detected by other works before your submisison to our Plos One.

However, I recognized your work as a systemic validation from patients with chronic spontaneous urticaria.

Thank you

Sincerely

Cheorl-Ho Kim PhD

Academic Editor

Plos One

Reviewers' comments:

11 Aug 2022

PONE-D-22-12472R1

Detection of serum IgG autoantibodies to FcεRIα by ELISA in patients with chronic spontaneous urticaria

Dear Dr. Park:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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