**3. Antigen-antibody reaction for IgE detection: general principle**

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

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

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 specific-

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

ity and the information obtained in functional allergen-based tests.

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

78 Allergen

sensitization is the SPT.

**2.2. IgE and allergy**

Quantification of IgE is based on the antigen-antibody reaction, in which antigen is recognized specifically by an antibody forming an immune complex (**Figure 1**). In these tests, the antigen corresponds to serum IgE, and the antibody reacts against IgE in the serum sample. To carry out these assays, a specific antibody for the Fc fraction of the IgE is adsorbed into a solid phase usually in polystyrene or cellulose wells. This so-called "primary antibody" or "capture antibody" has the function of binding to the IgE in the sample of serum; this interaction generates a stable bound. Then, this immune complex is measured through a second antibody called "secondary antibody" or "detection antibody" which is coupled to a radioisotope (RAST) or an enzyme which allows the development of a colored (ELISA) or fluorescent (FEIA) substrate in an antigen-dependent manner. Simultaneously, a calibration curve containing known concentrations of the analyte to determine is processed to extrapolate the data of absorbance (in colorimetric methods—ELISA) or the fluorescence (in fluorometric methods—FEIA) to a protein concentration, finally reported in µg, ng, or IU.

**Figure 1.** Antigen-antibody reaction. Each antibody is able to bind its specific antigen, forming antigen-antibody complexes. Different laboratory testing techniques are based on this principle.

## **3.1. Radio allergo sorbent test (RAST)**

RAST was the first laboratory method developed for *in vitro* detection of specific IgE [21]; despite that it is no longer used and its historical importance is evident since it was the second most used test after SPT, and gave rise to development of new methodology to facilitate sIgE detection. The main advantage of this method over SPT lied on the safety of patient. In SPT, the allergen is administered in the cutaneous layer of the skin, which may lead to sensitization to new allergens or in the worst of cases it may trigger anaphylaxis. All of the previous disadvantages were avoided with the introduction of *in vitro* tests like RAST.

As mentioned above, this test is based on the principle of antigen-antibody reaction. In this method, the allergen is adsorbed covalently to a solid particle, then, the serum of a patient is added. IgE antibodies present in the sample binds to the adsorbed allergen. After this, a washing step is needed to remove non-specific weak bindings. Next, a radio-iodinated anti-IgE antibody is added to this reaction, and finally, the radiation detected is directly proportional to the number of antigen-antibody complexes formed (**Figure 2**).

This method was validated in comparison with sIgE in pollen-sensitized individuals, finding 96% of concordance with both tests [22].

#### **3.2. Enzyme-linked immunosorbent assay (ELISA)**

ELISA is currently one of the most common immune-assays used in clinical and experimental procedures. This technique allows detection of allergy-related analytes, e.g., IgE or Th2 cytokines, and screening of different molecules. Advantages of ELISA are fast performance, improved biosafety when compared with radioimmunoassay, low reagent cost, affordability for the patient, and simple methodology [23].

The first step to perform an ELISA is sensitizing the plate. A solid polystyrene plate is coated with an anti-IgE antibody directed against Fc region of the immunoglobulin (capture antibody). This process is achieved pre-treating the plate wells with carbonate buffers or cyanogen bromide allowing a better chance for adsorbing the capture antibody or antigen. Another

**Figure 2.** RAST methodology. An allergen is adsorbed covalently to a solid particle, then serum of patient is added to react with the allergen. Next, a radiolabeled anti-IgE antibody identifies the previous formed immune complexes. The radiation generated is measured by a radiation detector.

strategy is to radiate the polystyrene plate; this permits the breaking of a certain number of benzene rings yielding carboxyl (COOH) and hydroxyl (OH) groups. Radiation of polystyrene increases the chances for hydrophilic interactions with Fc fractions of capture antibody. Protein A from *Staphylococcus aureus* is also a suitable linker for orienting and spacing the capture antibody appropriately, optimizing the space, and homogenizing the coating. In this step, several factors like pH and temperature could affect the proper adsorption of antibody or allergen. Fortunately, commercial kits contain the pre-sensitized plates. Next step is incubation of samples (serum or plasma), although serum samples are preferred over plasma samples since some commercial houses have documented diminishing IgE detection sensitivity in plasma samples.

**3.1. Radio allergo sorbent test (RAST)**

80 Allergen

96% of concordance with both tests [22].

**3.2. Enzyme-linked immunosorbent assay (ELISA)**

for the patient, and simple methodology [23].

radiation generated is measured by a radiation detector.

RAST was the first laboratory method developed for *in vitro* detection of specific IgE [21]; despite that it is no longer used and its historical importance is evident since it was the second most used test after SPT, and gave rise to development of new methodology to facilitate sIgE detection. The main advantage of this method over SPT lied on the safety of patient. In SPT, the allergen is administered in the cutaneous layer of the skin, which may lead to sensitization to new allergens or in the worst of cases it may trigger anaphylaxis. All of the previous

As mentioned above, this test is based on the principle of antigen-antibody reaction. In this method, the allergen is adsorbed covalently to a solid particle, then, the serum of a patient is added. IgE antibodies present in the sample binds to the adsorbed allergen. After this, a washing step is needed to remove non-specific weak bindings. Next, a radio-iodinated anti-IgE antibody is added to this reaction, and finally, the radiation detected is directly proportional

This method was validated in comparison with sIgE in pollen-sensitized individuals, finding

ELISA is currently one of the most common immune-assays used in clinical and experimental procedures. This technique allows detection of allergy-related analytes, e.g., IgE or Th2 cytokines, and screening of different molecules. Advantages of ELISA are fast performance, improved biosafety when compared with radioimmunoassay, low reagent cost, affordability

The first step to perform an ELISA is sensitizing the plate. A solid polystyrene plate is coated with an anti-IgE antibody directed against Fc region of the immunoglobulin (capture antibody). This process is achieved pre-treating the plate wells with carbonate buffers or cyanogen bromide allowing a better chance for adsorbing the capture antibody or antigen. Another

**Figure 2.** RAST methodology. An allergen is adsorbed covalently to a solid particle, then serum of patient is added to react with the allergen. Next, a radiolabeled anti-IgE antibody identifies the previous formed immune complexes. The

disadvantages were avoided with the introduction of *in vitro* tests like RAST.

to the number of antigen-antibody complexes formed (**Figure 2**).

Incubation let the captured antibody bind to IgE through Fc fraction (in total IgE determination) or allows the specific IgE contained in the sample bind to the allergen adsorbed in the solid phase (in specific IgE determination). After incubation time, a washing step is performed to remove weak and unspecific binding. Then, a second anti-Fc antibody is added to the well to detect the immune complexes formed in the previous step. This "secondary or detection antibody" is linked to an oxidative enzyme that acts on its substrate which once oxidized develops a color that can be measured trough a spectrophotometer (**Figure 3**).

There are various enzymes and substrates commonly used in ELISA (**Table 1**); the biotinstreptavidin system is the most often employed in detection methods. A washing step is followed next to eliminate the excess of not bound antibodies. In addition to samples, control or standard curve is processed with increasing concentrations of protein. The goal of this standard curve is to extrapolate the absorbance obtained from samples into a curve of known concentration through a linear regression, and obtaining an estimated concentration of the analyte (**Figure 4**).

The last step consists in adding and incubating the substrate for 10–20 min and reading the absorbance obtained after stopping the reaction. Some substrates may be read without stopping

**Figure 3.** ELISA assays. Direct ELISA mostly used for antigen detection. Indirect ELISA mainly used for antibodies detection. Sandwich ELISA used to detect total IgE.


**Table 1.** Enzymes and substrates used in ELISA assays.

**Figure 4.** Absorbance-concentration standard curve. In order to determine sIgE concentration in a sample, a standard curve is run using known concentrations of total or allergen-specific IgE. By plotting the absorbance from patient samples into the standard curve, we can determine sIgE concentration.

the reaction. Results are read in a spectrophotometer; this equipment works emitting a light beam that is filtered through a wavelength selector or filter; then, the filtered light will strike the sample, which will absorb a certain amount of light and let some light pass and reach the detector. Absorbance is the negative logarithm of transmittance, so the absorbance obtained will be proportional to the concentration of the measured antigen-antibody complexes in the samples that may reflect and refract the light at a certain wavelength (**Figure 5**). Optical density (OD) is a common term used to refer to absorbance (see **Figures 4** and **5**).

ELISA rapidly substituted radiolabeled methods, due to its safer and faster performance, with a similar sensibility and specificity when compared to RAST and paper radio immuno-sorbent test (PRIST) for total or specific IgE quantification [24, 25].

**Figure 5.** Spectrophotometer basis. A light beam is generated by a halogen lamp, and this light is filtered through a wavelength selector. Light hits the sample and the transmittance measured is reported as absorbance units. Absorbance is directly proportional to the amount of antibody-antigen complexes formed previously.

#### **3.3. Fluorescent enzyme immune assay (FEIA)**

**Figure 4.** Absorbance-concentration standard curve. In order to determine sIgE concentration in a sample, a standard curve is run using known concentrations of total or allergen-specific IgE. By plotting the absorbance from patient

**Enzyme Substrate Wavelength after stop solution (nm)**

(3-Ethylenbenzothiazoline-6-sulfonic acid) diamonium salt, ABTS

o-phenylenediamine dyhidocloride,

450

490

450

Horse radish peroxidase (HRP) 3,3′,5,5′-tetramethylbenzidine, TMB 450

OPD

Alkaline phosphatase (AP) p-Nitrophenyl phospate disodium salt

**Table 1.** Enzymes and substrates used in ELISA assays.

82 Allergen

2,2′-Azinobis

the reaction. Results are read in a spectrophotometer; this equipment works emitting a light beam that is filtered through a wavelength selector or filter; then, the filtered light will strike the sample, which will absorb a certain amount of light and let some light pass and reach the detector. Absorbance is the negative logarithm of transmittance, so the absorbance obtained will be proportional to the concentration of the measured antigen-antibody complexes in the samples that may reflect and refract the light at a certain wavelength (**Figure 5**). Optical density (OD) is

ELISA rapidly substituted radiolabeled methods, due to its safer and faster performance, with a similar sensibility and specificity when compared to RAST and paper radio immuno-sor-

samples into the standard curve, we can determine sIgE concentration.

a common term used to refer to absorbance (see **Figures 4** and **5**).

bent test (PRIST) for total or specific IgE quantification [24, 25].

Fluorescent enzyme immune assays are based on the same principle used for ELISA and RAST, the antigen-antibody reaction, but differs in the way the read out is made. In FEIA, the secondary antibody is linked to an enzyme that permits the activation of a fluorochrome. The most common enzyme used in fluorometric assays is β-galactosidase, which acts on its substrate 4-methylumbelliferyl-β-D-galactoside transforming it into a 4-methylumbelliferone. When 4-methylumbelliferone is excited at 365 nm, it emits fluorescence at 445 nm. This fluorescence is later measured by a fluorometer [26]. Simultaneously, a standard curve is processed to extrapolate the relative fluorescence units obtained from samples into the known concentration curve (**Figure 6**).

Fluorometric assays have permitted the development of automatized systems, resulting in improved reproducibility, diminished operator involvement, with reduction of mistakes, and increased sensitivity and specificity when compared with other innovative methods based on chemiluminescence [27, 28]. FEIA technology opens the possibility to screen sIgE to several allergens at the same time and with few volume of sample [29]. **Table 2** shows a comparison between antigen-antibody reaction-based methods for quantification of sIgE.

#### **3.4. ImmunoCAP-ISAC (immuno solid-phase allergen chip)**

Innovative and non-invasive techniques led to the identification of many sIgE to different allergens at the same time, with a minimum sample volume (~50 µL), allowing test allergens not limited to a geographical region, and without risk of sensitization, or anaphylaxis for the patient, as has been reported for SPT [30].

ISAC is the first multiplex diagnostic tool commercially available to evaluate sIgE directed against 112 well-characterized antigens. In fact, the allergens presented in the solid phase are recombinant proteins ensuring specific interaction of serum IgE with higher accuracy when

**Figure 6.** Fluorescence-concentration standard curve. Quantification of sIgE is measured plotting fluorescence units


**Table 2.** Methods for sIgE quantification based on the antigen-antibody reaction.

obtained from samples into a standard curve.

compared with FEIA and SPT [31]. The assay consists of various steps. First, the sIgE from serum samples interacts with the recombinant allergen previously adsorbed to the solidphase; then, a secondary anti-human IgE antibody labeled with fluorochrome recognizes sIgE-recombinant allergen complex (**Figure 7**). Fluorescence is measured using a biochip; and results are scanned and analyzed in specialized software, reporting results in arbitrary units named ISAC Standardized Units (ISU) (**Figure 8**) [32].

Allergen-Based Diagnostic: Novel and Old Methodologies with New Approaches http://dx.doi.org/10.5772/intechopen.69276 85

**Figure 7.** Immuno CAP-ISAC. The recombinant allergens are recognized by sIgE from serum samples; a secondary antibody fluorescent-labeled interacts with IgE. Fluorescence is measured by a biochip and results are analyzed in specialized software. Recombinant allergen diminishes the risk of cross-reactivity.

**Figure 8.** ISAC biochip layout. Results are reported in arbitrary units named ISAC-Standardized Units (ISU).

compared with FEIA and SPT [31]. The assay consists of various steps. First, the sIgE from serum samples interacts with the recombinant allergen previously adsorbed to the solidphase; then, a secondary anti-human IgE antibody labeled with fluorochrome recognizes sIgE-recombinant allergen complex (**Figure 7**). Fluorescence is measured using a biochip; and results are scanned and analyzed in specialized software, reporting results in arbitrary units

High Non or minor Non or minor

**Figure 6.** Fluorescence-concentration standard curve. Quantification of sIgE is measured plotting fluorescence units

High High

**Test RAST ELISA FEIA**

Sensitivity High High High Reproducibility Acceptable Acceptable High Automatization Partially Partially Totally Relative cost per test Low Affordable High Shelf life of reagents Long Low Long

named ISAC Standardized Units (ISU) (**Figure 8**) [32].

**Table 2.** Methods for sIgE quantification based on the antigen-antibody reaction.

obtained from samples into a standard curve.

Specificity Depends on antigen

Health hazards for laboratory personnel

84 Allergen

preparation

ISAC multiplex assay has been proposed to guide therapeutic decisions, e.g., the discontinuation of restrictive diets, the content of allergen-specific desensitization immunotherapy that may be useful to discriminate structurally similar allergens and cross-reactivity, and even to analyze the real sensitization profile in multi-sensitized patients to define whether they can receive a specific immunotherapy [33].

#### **3.5. Western blot**

Western blot combines different techniques to identify new antigens related to allergy. In this method, the antigens are separated according to their molecular weight in a sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), and then transferred to a polyvinylidene difluoride or nitrocellulose membrane, which will function as the solid phase for the antigen-antibody reaction. Then, the membrane is incubated with the patient serum, if sIgE is present in the sample it will react against the allergens found. A secondary anti-IgE antibody coupled to an enzyme is added (**Table 1**). Detection of sIgE becomes evident by the formation of bands in two different ways:


**Figure 9.** Western-blot methodology. Allergen mixtures are separated in a SDS-PAGE according to the molecular size. The separated allergens are transferred to a nitrocellulose or PVDF membrane. Then, by adding the antibodies from the serum samples sIgE will bind to their specific antigen. An enzyme conjugate secondary antibody identifies Fcɛ IgE. Detection of reaginic antibodies is identified by chemiluminescence.

This method has been useful in the identification of clinically relevant immunogenic epitopes after enzymatic digestion of allergens and is also used to identify cross-reacting peptides [34–36].
