**5. Functional tests**

**3.5. Western blot**

86 Allergen

two different ways:

estimated by densitometer analysis.

Detection of reaginic antibodies is identified by chemiluminescence.

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

(a) Developing color. The enzyme oxidizes the substrate and precipitates (e.g., when using a secondary antibody conjugated to horse radish peroxidase (HRP) and 4-cloronaphthol).

(b) Releasing light. The substrate is dephosphorylated by an enzyme, releasing light (chemiluminescence), that is later detected by a photographic film or autoradiography (i.e., when using a secondary antibody conjugated to alkaline phosphatase (AP) and adamantyl-1,2-dioxetane phosphate or HRP and luminol) (**Figure 9**). Finally, concentration can be

**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. The techniques above described answer two simple requests: is the sIgE present in the sample? So, if there, how much sIgE is present? The answer to these questions and the analysis of the clinical history allows the allergist/immunologist to initiate treatments centered on allergenspecific desensitization in every single patient in a personalized way. However, sometimes answer to these questions is not enough, and functional tests are needed to understand some clinical manifestations, e.g. allergy to a particular drug.

#### **5.1. Flow cytometry and fluorescence-activated cell sorting (FACS)**

Early in the 1950s, Coulter developed a technology able to read size and complexity of blood cells based on diffraction of light laying the fundamentals for automatized blood counting used in our days. Exploiting this innovation, Bonner, Sweet, Hulett, Herzenberg invented the Fluorescence Activated Cell Sorter (FACS) in the late 1960s to achieve flow cytometry and cell sorting of viable cells. Becton Dickinson with Bernie Shoor introduced the commercial cytometers in the early 1970s, utilizing a Stanford patent and the expertise supplied by the Herzenberg Laboratory [41]. Today, isolation of cells by FACS is performed in complete sterility, and sorted cells could be used as an adoptive transfer for therapeutical interventions [42].

Flow cytometry detects and analyzes optical signals (angular light scatter or emitted fluorescence) to identify individual characteristics of cells or in biological samples. Inside the flow cytometer, the suspended cells are conducted in a fluidic system ensuring cells travel at a uniform velocity in a laminar form. Here, the cells are directed to a specific point in which a laser passes through cells. The light is diffracted in all directions, the emitted light is recovered in filters, and photodetectors collect the detection signals. The optical detection system obtains information about forward light scatter (FSC), side light scatter (SSC), and fluorescence channels (FL1, FL2, FL3). Then, the luminous signal is detected in photomultiplier tubes; information recollected is digitalized that is to be analyzed by a computer system. Information obtained is showed in histograms or dot plots. The quality of both systems (optical and fluidic) is critical for performance and reliability of this technique [43] (**Figure 10**).

Flow cytometry could be used to determine the expression of cell surface markers, to know absolute or relative numbers of cells, to determine intracellular proteins, to quantify soluble proteins, or combine all of these possibilities.


Allergen-Based Diagnostic: Novel and Old Methodologies with New Approaches http://dx.doi.org/10.5772/intechopen.69276 89

answer to these questions is not enough, and functional tests are needed to understand some

Early in the 1950s, Coulter developed a technology able to read size and complexity of blood cells based on diffraction of light laying the fundamentals for automatized blood counting used in our days. Exploiting this innovation, Bonner, Sweet, Hulett, Herzenberg invented the Fluorescence Activated Cell Sorter (FACS) in the late 1960s to achieve flow cytometry and cell sorting of viable cells. Becton Dickinson with Bernie Shoor introduced the commercial cytometers in the early 1970s, utilizing a Stanford patent and the expertise supplied by the Herzenberg Laboratory [41]. Today, isolation of cells by FACS is performed in complete sterility, and sorted cells could be used as an adoptive transfer for therapeutical

Flow cytometry detects and analyzes optical signals (angular light scatter or emitted fluorescence) to identify individual characteristics of cells or in biological samples. Inside the flow cytometer, the suspended cells are conducted in a fluidic system ensuring cells travel at a uniform velocity in a laminar form. Here, the cells are directed to a specific point in which a laser passes through cells. The light is diffracted in all directions, the emitted light is recovered in filters, and photodetectors collect the detection signals. The optical detection system obtains information about forward light scatter (FSC), side light scatter (SSC), and fluorescence channels (FL1, FL2, FL3). Then, the luminous signal is detected in photomultiplier tubes; information recollected is digitalized that is to be analyzed by a computer system. Information obtained is showed in histograms or dot plots. The quality of both systems (optical and flu-

Flow cytometry could be used to determine the expression of cell surface markers, to know absolute or relative numbers of cells, to determine intracellular proteins, to quantify soluble

(a) Expression of cell surface markers. Information obtained by analysis of expression of cell surface markers could be useful to know the cellular phenotype and some functions of labeled cells. A few examples include the state of activation of a particular cell [e.g., CD63 on basophils after drug exposition (see the next section of this chapter for a deeper explanation of basophils activation test)], to know absolute numbers of circulating cells (e.g., 1700 CD4/µL), or combining information (e.g., patients with ocular allergy have increas-

(b) Determination of intracellular proteins. This procedure is useful to assess specific functions of the cell. First, isolation of cells is needed prior incubation with a stimulus (e.g., allergens as specific stimulus). Culture or incubation conditions must be standardized to ensure reproducibility of results. It is important to note that if studied protein is secreted (e.g., cytokines) protein secretion must be inhibited (e.g., brefeldin-A that blocks internal protein transport) to allow retention of proteins inside the cytoplasm. Labeling of intracellular proteins is performed after cells were fixed and permeabilized with detergents (e.g.,

idic) is critical for performance and reliability of this technique [43] (**Figure 10**).

ing percentage of circulating helper activated CD4+CD25+ T cells) [44].

proteins, or combine all of these possibilities.

clinical manifestations, e.g. allergy to a particular drug.

interventions [42].

88 Allergen

**5.1. Flow cytometry and fluorescence-activated cell sorting (FACS)**

**Figure 10.** Flow cytometer and fluorescent activated cell sorter. The figure resumes the optical and the fluidic systems working together to analyze biophysical characteristics of cells, expression of molecules detected by monoclonal antibodies, and sorting of cells expressing selected characteristics.

saponin). Permeabilization process ensures that monoclonal antibodies (mAbs) labeled with fluorochromes enter into the cell and react with their specific antigens (**Figure 11**) [45]. The determination of intracellular proteins has significantly contributed to the understanding of physiopathology induced by allergens (e.g., Allergen-activation induces cytokines related to the damage of IL-25 in asthma, IL-31 in atopic dermatitis, and IL-5 in vernal conjunctivitis) [46–48].

(c) Quantification of soluble proteins. The determination of soluble proteins could be used to know normal ranges of proteins in human fluids or to assess cellular functions. Multiplex technology has been developed to detect several proteins in the same sample, and it is named cytometric bead arrays (CBA). The advantage of this test is the low volume of sample letting to process a broad range of human fluids/secretions (e.g., tears, synovial fluid, aqueous humor, and serum) and cell supernatants [49–52].

Multiple determinations of soluble proteins by flow cytometry are based in microspheres, all of them conjugated with a specific antibody against protein we wish to determine. After bead interacts with its antigen, a second antibody labeled with a fluorochrome is added; usually, this secondary antibody is conjugated to phycoerythrine (PE). However, the real innovation of this assay is that each bead is also labeled with a different fluorochrome in a range of intensity, from low intensity to high intensity, and detected by near infrared (NIR) lasers [53, 54] (**Figure 12**).

**Figure 11.** Determination of intracellular proteins by flow cytometry. Identification of intracellular proteins allows studying cellular functions.

**Figure 12.** Cytometric bead arrays. Multiplex technology permits determination of various soluble proteins at the same time, and in the same sample.

Changes in intensity of fluorescence are expressed as median fluorescence intensity (MFI) and directly correlate with concentration of protein in the sample expressed in pg/mL or ng/mL (**Figure 13**).

#### *5.1.1. Basophils activation test (BAT)*

Adverse drug reactions (ADR) constitute a major health problem worldwide with high morbidity and mortality rates, the incidence of fatal ADR occurs in 5% in hospitalized patients in Europe [55]. ADR may be classified as Type A (augmentation of normal drug effects), Type B

**Figure 13.** Standard-curve and median fluorescence intensity (MFI). Changes in MFI correlate with concentration of soluble protein.

(bizarre effects), Type C (chronic effects), Type D (delayed effects), and Type E (end of drug use effects). The most frequent ADR are Type A and are related to genetics, age, sex, and disease, and they have low mortality and high morbidity; in contrast, Type B are 25% of ADR and are unpredictable, with high mortality and low morbidity. The pathophysiological mechanisms of Type B reactions are not well understood. Some cases are mediated by type I hypersensitivity (true allergy), but other cases are related with the generation of reactive metabolites that react nonenzymatically on multiple proteins to form immunogenic-drugs complexes that induce a cascade of cell-based reactions and result in a wide range of severe clinical symptoms [56]. Due to the complexity of ADR, only Type B reactions could be explored by basophil activation test (BAT).

Principle of this test is simple, basophils are activated *in vitro* by the suspicious drug; if basophils are sensitized to the drug, they become active, upregulating on their surface two molecules CD63 and CD203c [57]. CD63 is an intracellular lysosomal protein whose surface expression is upregulated after activation. CD63 is also expressed on activated platelets, degranulated neutrophils, monocytes, macrophages, and endothelium [58]. On the other hand, CD203c is an ectoenzyme located both on the plasma membrane and in the cytoplasmic compartment of basophils. Cross-linking of the FcεRI by an allergen or anti-IgE antibody results in a rapid upregulation of intracellular CD203c molecules to the cell surface and is accompanied by mediator release [59] (**Figure 14**).

Changes in intensity of fluorescence are expressed as median fluorescence intensity (MFI) and directly correlate with concentration of protein in the sample expressed in pg/mL or ng/mL

**Figure 12.** Cytometric bead arrays. Multiplex technology permits determination of various soluble proteins at the same

**Figure 11.** Determination of intracellular proteins by flow cytometry. Identification of intracellular proteins allows

Adverse drug reactions (ADR) constitute a major health problem worldwide with high morbidity and mortality rates, the incidence of fatal ADR occurs in 5% in hospitalized patients in Europe [55]. ADR may be classified as Type A (augmentation of normal drug effects), Type B

(**Figure 13**).

time, and in the same sample.

studying cellular functions.

90 Allergen

*5.1.1. Basophils activation test (BAT)*

**Figure 14.** Basophils activation test. (a) After activation basophils upregulate CD63 and CD203c on membrane surface. Both CD63 and CD203c are detected by antibodies conjugated to fluorochromes; (b) the histogram shows increased expression of CD203c on gated basophils.

Reports about sensitivity and specificity for BAT indicate that determination of both, CD63 and CD203c, considerably increases the sensitivity up to 92% and specificity in a range of 86–90% [60, 61]. Today, BAT is also used to determine sensitization to several allergens such as diverse types of pollen and house dust mites. It has been reported that BAT has the same sensitivity but lower specificity when compared with FEIA. BAT could be used as an alternative to SPT in some patients with allergy to aeroallergens [62] and as a useful test preventing preoperative anaphylaxis [63].

BAT assay is performed with 100 µL of peripheral blood; the drug is incubated with the blood at 1 mg/mL, in 36.5°C of temperature and atmosphere of 5% CO<sup>2</sup> during 1 h; as an internal control, the same volume of blood is incubated with negative or positive controls. N-formyl-methionyl-leucyl-phenylalanine (f-MLP) is used as positive control. f-MLP is an N-formylated tripeptide that functions as a chemotactic peptide for polymorphonuclear (PMN) cells but is a potent activator of basophils too. After incubation, cells are labeled with monoclonal antibodies for 30 min, and then erythrocytes are lysed and results are analyzed by flow cytometry. To ensure that CD63 expressing cells are basophils, analyzed cells are also labeled against CD123 and Human leukocyte antigen-DR (HLA-DR). CD123 is the IL-3Rα, granulocytes including basophils, that constitutively express this cluster of differentiation [64]; whereas HLA-DR is expressed on B lymphocytes, monocytes, macrophages, activated T lymphocytes, activated natural killer (NK) lymphocytes but is absent in basophils. First, we analyzed cells by their complexity (SSC) and expression of CD123 and HLA-DR, basophils would be CD123+HLA-DR−, and only if activated by allergen or drug-medication, basophils would be CD63+ CD203c+ (SSC/CD123+HLA-DR−CD63+CD203c+) (**Figure 15**).

Allergen-Based Diagnostic: Novel and Old Methodologies with New Approaches http://dx.doi.org/10.5772/intechopen.69276 93

**Figure 15.** Representative dot plot of flow cytometry analysis of a basophils activated test. Upper panel shows nonstimulated cells. The lower panel shows stimulated cells with f-MLP. Percentage of activated basophils is showed at the squares next to dot plots.
