Reliable diagnosis of insect venom allergy is based on the combination of patient’s history, skin testing (SPT) and laboratory tests for the detection of specific IgE (sIgE) [
The methodology for the measurement of sIgE has evolved during the recent years [
Principle of ALFA. A test cassette showing a positive test result is presented in (a) and the principle of the test in (b). The patient’s sample is transferred to the sample application point. Immediately afterwards the allergen solution of interest is applied. During incubation time of 20 min, the liquid is driven through the device by capillary flow. Allergen specific IgE of the sample binds specifically to the corresponding antigens of the allergen solution. The antigens are labeled and are retained at the test line (T) by a capture molecule. At the same time the sIgE bound to the allergen is bound by an antibody coupled to colored particles (conjugate). The intensity of the color reaction at the test line is proportional to the amount of immune complexes consisting of ligand tagged antigens, sIgE, and IgE specific conjugate. The signal intensity ranges from faintly pink (low titer of sIgE) to dark ruby (high titer of sIgE). Access conjugate, which is not bound at the test line, will form a dark ruby control line (C) after 20 min of incubation.
Sera of four groups were analyzed as follows. 40 patients with insect venom allergy to either bee ( 40 patients with insect venom allergy and double sensitization (serological) to bee and wasp venom, determined by ImmunoCAP, patient’s history and skin test. Mean age 42.25 y (SD 17.5 y). 20 (50%) males, 20 (50%) females. 4 (10%) Patients presented with anaphylactic reactions grade 1 (according to Ring and Messmer [ Atopic individuals (Sx1 positive, total IgE mean = 2986 kU/L, range 186–23813 kU/L, history of at least one atopic disease) without history of insect venom allergy ( Non atopic individuals without history of insect venom allergy (
Diagnosis of insect venom allergy was based on patient’s history (anaphylaxis due to bee or wasp sting [
In patients with insect venom allergy, skin prick tests with increasing concentrations of BV and WV (1, 10, 100
All sera in this study (
Spearman correlation,
When compared to the diagnosis (based on patients history, skin test, and sIgE detection by ImmunoCAP) comparative receiver operating characteristic analysis of groups A and B revealed area under the curve values for ALFA of 0.97 (BV) and 0.91 (WV).
In monosensitized patients (group A only), 12/12 BV allergic patients and 23/28 WV allergic patients were positive in ALFA using a cut-off value for ALFA of 10.0 RU, corresponding to a sensitivity of 100% for BV and 82% for WV. Similar results were obtained by ALLERG-O-LIQ. In ALLERG-O-LIQ 12/12 BV allergic patients and 24/28 WV allergic patients were found positive (Figure
Receiver operating characteristic of ALFA and ALLERGO-LIQ for the diagnosis of bee (a) and wasp (b) venom allergy in monosensitized patients (group A) and control groups (C and D). Curve with dots indicates results for ALFA and curve with squares for ALLERGO-LIQ. Diagnosis of insect venom allergy was based on patient’s history, skin testing, and detection of sIgE to bee or wasp venom by ImmunoCAP. Sensitivity/specificity for ALFA is 100%/83% (BV) and 82%/97% (WV) at a cut-off value of 10.0 RU and 100%/93% (BV) and 82%/93% (WV) for ALLERG-O-LIQ.
For groups A and B high agreement between ALFA, ALLERG-O-LIQ, and skin tests of up to 92% was observed (data not shown). Using atopic (without history of insect venom allergy—group C) and nonatopic (group D) sera as controls, the specificity of ALFA for the detection of sIgE to BV was calculated as 83% and for sIgE to WV as 97% (Figure
For insect venom allergic patients (groups A and B) the Spearman correlation coefficient for ALFA versus ImmunoCAP was 0.79 (
Spearman correlation diagram of ALFA versus ImmunoCAP for the detection of sIgE to bee venom. Quantitative agreement was found with a Spearman correlation coefficient of 0.72 (
Spearman correlation diagram of ALFA versus ImmunoCAP for the detection of sIgE to wasp venom. Quantitative agreement was found with a Spearman correlation coefficient of 0.82 (
These results demonstrate a good quantitative agreement for the detection of sIgE to BV and WV between ALFA and ImmunoCAP for sera of allergic patients (groups A and B) but poor quantitative agreement for the control groups, especially for BV (groups C and D). This seems to be caused by a surprisingly high number of positive ImmunoCAP results in the control groups, in particular in the group of atopic individuals (without history of insect venom allergy) with high total IgE-a finding that has been described before for different allergens [
Results for the detection of bee and wasp venom of ALFA and ImmunoCAP for group A (
ImmunoCAP | ALFA (cut-off 10 RU) | |||||||
Bee venom positive | Bee venom negative | Wasp venom positive | Wasp venom negative | Bee venom positive | Bee venom negative | Wasp venom positive | Wasp venom negative | |
Group A | 12 | 28 | 28 | 12 | 17 | 23 | 23 | 17 |
Group B | 36 | 4 | 39 | 1 | 27 | 13 | 30 | 10 |
Group C | 19 | 11 | 9 | 21 | 4 | 26 | 1 | 29 |
Group D | 3 | 27 | 4 | 26 | 6 | 24 | 1 | 29 |
Graphical illustration of the relation of total IgE (green diamond, determined by ImmunoCAP) and sIgE to BV (red) and WV (blue) for ImmunoCAP (a) and ALFA (b) for each study group.
Since sIgE to CCDs (cross-reactive carbohydrate determinants) can be responsible for major cross-reactivity of glycosylated allergens (e.g., of pollen, foods, insect venom, etc.), sIgE to CCDs could also account for the high rate of BV ImmunoCAP positives in the atopic negative controls (Group C). However analyzing CCD reactivity by ImmunoCAP showed that only less than 50% of the ImmunoCAP BV positive sera also displayed CCD reactivity (data not shown). This suggests that the high rate of BV positive results by ImmunoCAP in atopic patients (without history of insect venom allergy) with strongly elevated total IgE levels (mean = 2986 kU/L, range 186–23813 kU/L) cannot be explained by CCD reactivity but rather may reflect nonspecific IgE binding due to the solid phase architecture of the ImmunoCAP assay system.
Recently recombinant allergens have been introduced into the in vitro diagnostic of insect venom allergy. In particular for dissecting true double sensitization from cross-reactivity recombinant allergens seems to be of great relevance [
Detection of sIgE to bee and wasp venom by ALFA shows in the present study a good discrimination between sera of bee or wasp allergic patients and control sera with a comparable performance to the ALLERG-O-LIQ and ImmunoCAP system. Noteworthy ALFA detected no sIgE to BV in WV allergic patients and vice versa. In case of specificity, a high degree of correlation was found for ALFA and ALLERG-O-LIQ, whereas differing results were obtained using the ImmunoCAP system.
We conclude that the Allergy Lateral Flow assay together with the novel scanner-based system represents a versatile and reliable tool for the measurement of sIgE to insect venom allergens meeting the growing demand for digital documentation of laboratory results. Further studies with more allergens (e.g., recombinant insect venom allergens) are mandatory to further proof the applicability of the ALFA test system.
This study was supported in part by a research grant to T. Jakob by Dr. Fooke Laboratorien GmbH. N. Pfender declares that he has no conflict of interests. R. Lucassen, N. Offermann, J. Schulte-Pelkum, and M. Fooke are employees of Dr. Fooke Laboratorien GmbH.
N. Pfender performed the experiments. N. Offermann, R. Lucassen, J. Schulte-Pelkum, and M. Fooke developed the test system. T. Jakob supervised the experiments. All authors have contributed equally to writing and corrections of the manuscript.
The authors thank Andrea Komann for excellent technical assistance.