Analytical Principle UniCAP Specific IgE

The UniCAP-specific IgE FEIA is an in vitro quantitative assay that measures the concentration of circulating allergen-specific IgE in human blood samples.

Fig. 1. Schematic of original radioallergosorbent test reported in 1967. In the first assay incubation, human antibodies of different isotypes (e.g., IgG, IgE) in reference and unknown sera bind to antigens coupled to the sorbent (allergosorbent). After isotonic buffer washes to remove unbound serum proteins, bound IgE antibody is detected with 125I-labeled rabbit anti-human IgE detection antibody. The percentage of total radioactivity in counts per minute (CPM) bound to the allergosorbent is then measured in a gamma counter and plotted against the serum dilution analyzed. The amount of specific IgE in reference and original sera is proportional to the amount of radioactivity bound. Interpolated units of antibody are corrected for sample dilution and reported as the number of arbitrary units of allergen-specific IgE in the original serum. (Reproduced with permission from ref. 18.)

Fig. 1. Schematic of original radioallergosorbent test reported in 1967. In the first assay incubation, human antibodies of different isotypes (e.g., IgG, IgE) in reference and unknown sera bind to antigens coupled to the sorbent (allergosorbent). After isotonic buffer washes to remove unbound serum proteins, bound IgE antibody is detected with 125I-labeled rabbit anti-human IgE detection antibody. The percentage of total radioactivity in counts per minute (CPM) bound to the allergosorbent is then measured in a gamma counter and plotted against the serum dilution analyzed. The amount of specific IgE in reference and original sera is proportional to the amount of radioactivity bound. Interpolated units of antibody are corrected for sample dilution and reported as the number of arbitrary units of allergen-specific IgE in the original serum. (Reproduced with permission from ref. 18.)

Table 1

Relative Concentration of Specific IgE

Class

Relative activity

kUA/L

Class 0

Undetectable

<0.35

Class 1

Low

0.35-0.70

Class 2

Moderate

0.71-3.50

Class 3

High

3.5-17.5

Class 4

Very high

17.6-50

Class 5

Very high

51-100

Class 6

Very high

>100

The calibration reflects total IgE bound and not specific IgE. Specific IgE to ryegrass, short ragweed, and cat extract would be ideal for use as a standard for each assay. However, such specific standards for over 400 assays would be technically impractical. For that reason, bound IgE is used as a relative calibrator for both specific and total IgE.

The calibration reflects total IgE bound and not specific IgE. Specific IgE to ryegrass, short ragweed, and cat extract would be ideal for use as a standard for each assay. However, such specific standards for over 400 assays would be technically impractical. For that reason, bound IgE is used as a relative calibrator for both specific and total IgE.

In principle, the allergen of interest, covalently coupled to ImmunoCAP, captures the specific IgE in the patient specimen. Nonspecific IgE is washed away and enzyme-labeled antibodies against IgE are then added to form a complex. After incubation, unbound enzyme-anti-IgE is washed away and the bound complex is incubated with a developing agent. The reaction is then stopped and the fluorescence of the eluate is measured. To evaluate the test results, the response units for patient samples are compared directly to the response of the calibrators.

The IgE calibrators are traceable to the Second International Reference Preparation 75/502 of Human Serum Immunoglobulin E from the World Health Organization (WHO). The UniCAP 1000 Instrument calculates the concentration in kU/L and the instrument software automatically calculates the concentration.

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