**2. Historical perspective of allergen-based diagnostic methodologies**

### **2.1. Skin prick test (SPT)**

Skin prick testing is an essential clinical test to confirm sensitization in IgE-mediated allergic diseases. Historically, we can found an early report in 1850, in a textbook of Henry Salter, a physician from London´s Charing Cross Hospital who described the formation of wheals following scratches in patients with asthma and exposed to cats [3]. In 1907, Clemens von Pirquet reported a modification of Koch´s subcutaneous procedure based in abrasion of the skin to evaluate tuberculin response [4]. This procedure was rapidly adopted by others as a prototype for prick-puncture testing, and in 1909, the first case of anaphylactic response after scarification and exposition to an allergen was reported [5]. Practical application of a standardized procedure was suggested by Schloss [6] who described a correlation of time with clinical signs, reporting 5–15 min of erythematous reaction after abrasion of the skin in a child with rhinitis, asthma, and eczema. Since then, several techniques to evaluate allergenic sensitization have been described, e.g., intracutaneous test, [7] conjunctival test, [8] intracutaneous test by serial dilutions [9]. Nowadays the best technique to evaluate with safety allergenic sensitization is the SPT.

The standardized method of prick testing includes the appropriate selection of allergens, i.e., allergens tested are according to the country, the geographic location inside the same country, and even with seasons [10, 11]. SPT is based on the presence of sensitized cells, mainly mast cells in the skin, and the resulted cutaneous reactivity is used by the clinician as a surrogate biomarker for sensitization in eyes, nose, lung, gut, and skin. During the test, positive and negative controls must be included, a positive result is defined with a wheal ≥3 mm diameter after 15–20 min; reproducible results are obtained with standardized mixtures [12, 13]. In the early years of use, skin prick testing did not have with the entire approbation of the medical community, and their clinical relevance was questioned [14]. That vision has changed, and in the last years, it has been recognized a concordance between the clinical manifestations and allergen-specific wheal size [15]. Thus, skin prick test is considered as a fundamental technique to explore allergen sensitization in patients, but if it is true, why we need other methods to study sensitization in allergic/hypersensitivity conditions? In the following paragraphs, we will explain applications of the most common laboratory assays used to evaluate IgE specificity and the information obtained in functional allergen-based tests.

#### **2.2. IgE and allergy**

The discovery of the reaginic activity in the IgE antibody by Ishizaka in 1967 [16] developed a revolution in the knowledge of allergy affecting not only in basic research but also in applied research resulting in innovative diagnostic tools. It is well known that patients with allergy have a tendency to produce high levels of IgE antibodies due to its atopic condition. Usually, the concentration of total IgE in serum from healthy individuals ranges below 1 µg/mL. It is worthy of note that this is a very low concentration of protein so many laboratories rather use IU/mL or kU/L instead of µg/mL to report IgE levels, but understanding that 1 kU/L equals to 2.4 ng/mL [17]. Total IgE does not correlate with clinical manifestations, and is preferably to measure specific IgE (sIgE) [18, 19]. Total IgE concentration is the addition of all the specific IgE (sIgE) to the different allergens the individual has been exposed to; in non-allergic subjects, sIgE levels are below the limits of detection (0.35 kU/L) [20]. Thus, to identify the triggering antigen of allergic manifestations, one of the most common laboratory test requirement is the determination of sIgE concentration in serum. The quantification of sIgE can be performed through several methods based on antigen-antibody reaction, e.g., radio allergo sorbent test (RAST), enzyme-linked immunosorbent assay (ELISA) and fluorescence enzyme immune assay (FEIA).
