Separation of the four most important latex allergens from latex gloves: A potential tool for diagnosis and immunotherapy purposes

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"apellidos" => "Peixinho" "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] ] ] 1 => array:3 [ "nombre" => "M.F." "apellidos" => "Gabriel" "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] ] ] 2 => array:3 [ "nombre" => "C." "apellidos" => "Caramelo-Nunes" "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] ] ] 3 => array:3 [ "nombre" => "P." "apellidos" => "Tavares-Ratado" "referencia" => array:3 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "c" "identificador" => "aff0015" ] 2 => array:2 [ "etiqueta" => "d" "identificador" => "aff0020" ] ] ] 4 => array:4 [ "nombre" => "C.T." "apellidos" => "Tomaz" "email" => array:2 [ 0 => "ctomaz@ubi.pt" 1 => "candidatomaz66@gmail.com" ] "referencia" => array:3 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] 2 => array:2 [ "etiqueta" => "¿" "identificador" => "cor0005" ] ] ] ] "afiliaciones" => array:4 [ 0 => array:3 [ "entidad" => "CICS-UBI – Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506, Covilhã, Portugal" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Department of Chemistry, University of Beira Interior, Covilhã, Portugal" "etiqueta" => "b" "identificador" => "aff0010" ] 2 => array:3 [ "entidad" => "Department of Health Sciences, University of Beira Interior, Covilhã, Portugal" "etiqueta" => "c" "identificador" => "aff0015" ] 3 => array:3 [ "entidad" => "Laboratory of Clinical Pathology, Sousa Martins Hospital, ULS Guarda, Portugal" "etiqueta" => "d" "identificador" => "aff0020" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1070 "Ancho" => 3056 "Tamanyo" => 192253 ] ] "descripcion" => array:1 [ "en" => "HIC on Epoxy-Sepharose CL-6B column. Sample: 100μL, 0.4mg/mL of latex glove extract obtained by selective precipitation with ammonium sulphate: A: 25% (P1), B: 50% (P2), C: 75% (P3). Eluent: PBS 0.01M, pH 7.4 containing 0.5M (A); 1.0M (B) and 1.5M ammonium sulphate (C)." ] ] ] "textoCompleto" => "Due to a more wide-spread use of latex products, in particular medical gloves, allergy to natural rubber latex (NRL) had posed serious concerns during the 1980s and 90s. Currently, it is known that this epidemic of allergic reactions is especially prevalent in certain occupational groups repeatedly exposed to latex products, such as health care workers (HCW) and patients with spina bifida (SBP).<a class="elsevierStyleCrossRef" href="#bib0005">1</a>The identification of the patients who become sensitised and are likely to suffer from symptoms upon repeated exposure to latex products is a major goal for prevention of the allergic reactions. It is generally accepted that the diagnosis must be based on the clinical history and on a confirmative assay which may include in vivo tests such as skin prick tests (SPT), provocation tests and/or laboratory-based in vitro analyses.<a class="elsevierStyleCrossRef" href="#bib0010">2</a> Among these, SPT are described as the most reliable in vivo method for diagnosis of sensitisation to latex proteins.<a class="elsevierStyleCrossRef" href="#bib0015">3</a> However, a potential low diagnostic sensitivity of latex SPT is associated to poorly represented and/or denatured allergens, such as Hev b 3 and Hev b 5.<a class="elsevierStyleCrossRefs" href="#bib0020">4,5</a> In fact, to perform an appropriate diagnosis, it is critical that the applied extract contains an adequate amount of all clinically relevant allergen components.<a class="elsevierStyleCrossRef" href="#bib0020">4</a>In the management of latex allergy, specific immunotherapy (SIT) represents the most promising alternative for latex allergy treatment. Nevertheless, SIT is currently associated with a high risk of adverse events and is not available in routine clinical practice. Refined allergen preparations and administration regimens must be developed to allow widespread use. In immunotherapy extracts, unlike diagnostic extracts, the inclusion of allergens that are not clinically relevant can dilute the essential allergen components of extract and decrease its effectiveness.Nowadays, immunotherapy and diagnosis of latex allergy are performed using NRL extracts that are obtained directly from the tree Hevea brasiliensis sap without any purification steps. This crude extract contains several hundred proteins from which 14 (Hev b 1 to Hev b 14) have been recognized by the International Union of Immunological Societies (IUIS) as latex allergens (<a id="intr0005" class="elsevierStyleInterRef" href="http://www.allergen.org/">www.allergen.org</a>). Among the latex allergens, Hev b 1, Hev b 3, Hev b 5 and Hev b 6 are regarded as the most crucial allergens in natural rubber products.<a class="elsevierStyleCrossRef" href="#bib0030">6</a> Here we describe a new approach to obtain and separate the four most relevant latex allergens from the major sensitisation source, the latex gloves.Since the proteins of gloves and other NRL products are responsible for latex sensitisation, we consider that they are the ideal allergen sources of reagents for clinical purposes. Indeed, Yeang et al. reported that the latex allergens for research and clinical applications should theoretically be proteins isolated and purified from latex gloves. These researchers also noted that there are considerable problems in sourcing latex allergens from gloves because they vary widely in their protein content, both qualitatively and quantitatively. However, these problems also exist in crude extracts because there is an inherent variation in allergen content due to genetic environmental and agronomic factors.<a class="elsevierStyleCrossRef" href="#bib0035">7</a>As a first step of the present method, six different glove brands were collected from health institutions in Portugal, aiming to reduce the effect of inherent protein variation that results from glove manufacture and processing. Latex proteins were extracted from the gloves and lyophilized as described previously.<a class="elsevierStyleCrossRef" href="#bib0040">8</a> Then, 300mg of lyophilized latex glove extract were dissolved in 20mM tris–HCl, pH 7.5 and a salt precipitation was applied for both enrichment and concentration of latex allergens. This step was first performed by using 25% ammonium sulphate. After being stirred for 30min, at 4°C, the precipitated extract (denoted P1) was centrifuged at 12,000×g for 15min, at 4°C. The same process was repeated adding 50% and 75% of ammonium sulphate (extracts denoted P2 and P3, respectively). The three precipitated latex extracts (P1, P2 and P3) were then fractionated by Hydrophobic Interaction Chromatography (HIC). This technique was employed to exploit the differences in protein hydrophobicity between the four major latex allergens and to accomplish their separation. In fact, it is known that Hev b 5 and Hev b 6 have mainly hydrophilic characteristics while Hev b 1 and Hev b 3 are hydrophobic allergens.<a class="elsevierStyleCrossRefs" href="#bib0045">9,10</a> HIC was applied using a FPLC system (GE Healthcare Biosciences, Uppsala, Sweden) on a Sepharose CL-6B column modified with butyl-1,4-bis-(2,3-epoxy-propoxyd). The gel was packed in a XK column 16/20 (GE Healthcare Biosciences, Uppsala, Sweden) and equilibrated in a series of experiments using as eluent PBS 0.01M, pH 7.4 with different ammonium sulphate concentrations. The best separation conditions were achieved using 0.5M ammonium sulphate for precipitate P1, 1.0M for P2 and 1.5M for P3 (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>). At these conditions, two resolved peaks corresponding to unbound proteins were eluted (peaks 1 and 2). Bound proteins were then eluted (peak 3) by subsequently decreasing of ammonium sulphate concentration in eluent buffer to 0M, in a stepwise mode. Results showed different chromatographic profiles which suggest qualitative and quantitative differences in protein content of precipitated extracts (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>A–C).<elsevierMultimedia ident="fig0005"></elsevierMultimedia>Quantification of Hev b 1, Hev b 3, Hev b 5 and Hev b 6.02 in each HIC fraction was performed by capture enzyme immunoassay (EIA) using a commercial kit (FITkit™,Icosagen, Estonia) according to the manufacturer's instructions. FITkit™ tests are based on the use of specific monoclonal antibodies developed against the four major NRL allergens, providing individual results for each NRL allergen. All assays were performed in triplicate and when allergen levels were below the detection limit they were denoted as zero (<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>). In this quantitative analysis was observed that HIC fractionation of P1 (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>A) promoted a selective separation of hydrophobic allergens, Hev b 3 (2.53μg/L) and Hev b 1 (1.40μg/L), in peaks 1 and 3 respectively (<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>). Moreover, HIC of P2 (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>B) showed a separation of Hev b 6.02 (3.43μg/L) in peak 1 (<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>), whereas in peak 2 a mixture of Hev b 5 and Hev b 6.02 was obtained. It was also interesting to observe a high concentration of Hev b 1 in peak 3, despite the slight contamination of Hev b 6.02. On the other hand, separation of Hev b 5 (0.53 and 0.50μg/L) was achieved in peaks 2 and 3 (<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>) of the HIC assay applied to the P3 precipitate (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>C). It is known that although latex sensitisation is mainly acquired at health institutions for both SBP and HCW, in general, these risk groups present different sensitisation profiles, being that Hev b 1 and Hev b 3 are considered major allergens for SBP, while Hev b 5 and Hev b 6.02 are the major allergens for HCW.<a class="elsevierStyleCrossRef" href="#bib0005">1</a> Although the sensitisation profile of these risk groups may be more complex than this general rule, since it is common that SBP are also sensitised to Hev b 5 and Hev b 6, it is interesting to note that two different fractions containing specific allergens for each risk group were also obtained with this methodology: Peak 2 of P1 with Hev b 1 (1.60μg/L) and Hev b 3 (4.40μg/L), and Peak 2 of P2 containing Hev b 5 (0.68μg/L), Hev b 6.02 (2.80μg/L) (<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>). These extracts and the separated allergens can be used to adjust the content of each major allergen that is present in the diagnosis reagents and also can provide a specific extracts for a more effective immunotherapy. In fact, the success of allergen-specific diagnosis and treatment is dependent on the allergen composition of the used material. It is recognized that the commercially available latex extracts vary significantly in their allergen composition and concentration and this heterogeneity could strongly affect not only the diagnosis but also the immunotherapy, due to the lack of correct identification of patient sensitisation profile and to the poor quality of the therapeutic preparations. In order to achieve balanced allergen content, commercially available diagnosis reagents should be enriched in trace latex allergens. Thus, the improvement of NRL reagents using allergen preparation such our extracts could favour the correct diagnosis of clinical allergy that is crucial to guide subsequent investigations and treatment. On the other hand, in terms of immunotherapy purposes, the complex NRL extracts should be replaced by refined protein preparations, containing the clinically relevant allergens for the sensitised patient to treat, with minimal protein contamination. In this regard, we showed that the different hydrophobicity presented by the most clinically relevant latex allergens can be exploited to obtain allergen preparations which can be useful to formulate specific immunotherapy reagents, based on the individual reactivity profile of each latex-allergic patient to treat. It is also important to stress that the minor NRL allergen content of our fractions is unknown and a high concentration of some minor allergens in each fraction cannot be excluded. For this reason, the use of such extracts in immunotherapy trials should raise safety concerns, especially in patients presenting co-sensitisation to minor NRL allergens.<elsevierMultimedia ident="tbl0005"></elsevierMultimedia>To summarize, in this work it was described a reproducible procedure that allowed the separation of the four most important latex allergens directly from rubber gloves and its enrichment in specific fractions which can potentially be applied for diagnostic or clinical purposes.Ethical disclosuresProtection of human subjects and animals in researchProtection of human and animal subjects. The authors declare that no experiments were performed on humans or animals for this investigation.Patients’ data protectionConfidentiality of data. The authors declare that no patient data appears in this article.Right to privacy and informed consentRight to privacy and informed consent. The authors declare that no patient data appears in this article." 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Sample: 100μL, 0.4mg/mL of latex glove extract obtained by selective precipitation with ammonium sulphate: A: 25% (P1), B: 50% (P2), C: 75% (P3). Eluent: PBS 0.01M, pH 7.4 containing 0.5M (A); 1.0M (B) and 1.5M ammonium sulphate (C)." ] ] 1 => array:7 [ "identificador" => "tbl0005" "etiqueta" => "Table 1" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:2 [ "leyenda" => "The detection limit levels of allergens on FITkit™ were: Hev b 1, 1.2μg/L; Hev b 3, 2.3μg/L; Hev b 5, 0.5μg/L; Hev b 6.02, 0.1μg/L. Values below these detection limits were denoted as zero." "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " colspan="3" align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">P1</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " colspan="3" align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">P2</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " colspan="3" align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">P3</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " colspan="3" align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">Peaks (μg/L)</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " colspan="3" align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">Peaks (μg/L)</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " colspan="3" align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">Peaks (μg/L)</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">2 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">3 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">2 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">3 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">2 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">3 \t\t\t\t\t\t\n \t\t\t\t</td></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Hev b 1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">1.60 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">1.40 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">5.01 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">1.30 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Hev b 3 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">2.53 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">4.40 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Hev b 5 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.68 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.57 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.53 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.50 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Hev b 6.02 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n 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Research letter

C.M. Peixinhoa,b, M.F. Gabriela,b, C. Caramelo-Nunesa,b, P. Tavares-Ratadoa,c,d, C.T. Tomaza,b,

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ctomaz@ubi.pt
candidatomaz66@gmail.com

Corresponding author.

a CICS-UBI – Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506, Covilhã, Portugal

b Department of Chemistry, University of Beira Interior, Covilhã, Portugal

c Department of Health Sciences, University of Beira Interior, Covilhã, Portugal

d Laboratory of Clinical Pathology, Sousa Martins Hospital, ULS Guarda, Portugal

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          "en" => "<p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">HIC on Epoxy-Sepharose CL-6B column&#46; Sample&#58; 100<span class="elsevierStyleHsp" style=""></span>&#956;L&#44; 0&#46;4<span class="elsevierStyleHsp" style=""></span>mg&#47;mL of latex glove extract obtained by selective precipitation with ammonium sulphate&#58; A&#58; 25&#37; &#40;P1&#41;&#44; B&#58; 50&#37; &#40;P2&#41;&#44; C&#58; 75&#37; &#40;P3&#41;&#46; Eluent&#58; PBS 0&#46;01<span class="elsevierStyleHsp" style=""></span>M&#44; pH 7&#46;4 containing 0&#46;5<span class="elsevierStyleHsp" style=""></span>M &#40;A&#41;&#59; 1&#46;0<span class="elsevierStyleHsp" style=""></span>M &#40;B&#41; and 1&#46;5<span class="elsevierStyleHsp" style=""></span>M ammonium sulphate &#40;C&#41;&#46;</p>"
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    "textoCompleto" => "<span class="elsevierStyleSections"><p id="par0005" class="elsevierStylePara elsevierViewall">Due to a more wide-spread use of latex products&#44; in particular medical gloves&#44; allergy to natural rubber latex &#40;NRL&#41; had posed serious concerns during the 1980s and 90s&#46; Currently&#44; it is known that this epidemic of allergic reactions is especially prevalent in certain occupational groups repeatedly exposed to latex products&#44; such as health care workers &#40;HCW&#41; and patients with spina bifida &#40;SBP&#41;&#46;<a class="elsevierStyleCrossRef" href="#bib0005"><span class="elsevierStyleSup">1</span></a></p><p id="par0010" class="elsevierStylePara elsevierViewall">The identification of the patients who become sensitised and are likely to suffer from symptoms upon repeated exposure to latex products is a major goal for prevention of the allergic reactions&#46; It is generally accepted that the diagnosis must be based on the clinical history and on a confirmative assay which may include <span class="elsevierStyleItalic">in vivo</span> tests such as skin prick tests &#40;SPT&#41;&#44; provocation tests and&#47;or laboratory-based <span class="elsevierStyleItalic">in vitro</span> analyses&#46;<a class="elsevierStyleCrossRef" href="#bib0010"><span class="elsevierStyleSup">2</span></a> Among these&#44; SPT are described as the most reliable <span class="elsevierStyleItalic">in vivo</span> method for diagnosis of sensitisation to latex proteins&#46;<a class="elsevierStyleCrossRef" href="#bib0015"><span class="elsevierStyleSup">3</span></a> However&#44; a potential low diagnostic sensitivity of latex SPT is associated to poorly represented and&#47;or denatured allergens&#44; such as Hev b 3 and Hev b 5&#46;<a class="elsevierStyleCrossRefs" href="#bib0020"><span class="elsevierStyleSup">4&#44;5</span></a> In fact&#44; to perform an appropriate diagnosis&#44; it is critical that the applied extract contains an adequate amount of all clinically relevant allergen components&#46;<a class="elsevierStyleCrossRef" href="#bib0020"><span class="elsevierStyleSup">4</span></a></p><p id="par0015" class="elsevierStylePara elsevierViewall">In the management of latex allergy&#44; specific immunotherapy &#40;SIT&#41; represents the most promising alternative for latex allergy treatment&#46; Nevertheless&#44; SIT is currently associated with a high risk of adverse events and is not available in routine clinical practice&#46; Refined allergen preparations and administration regimens must be developed to allow widespread use&#46; In immunotherapy extracts&#44; unlike diagnostic extracts&#44; the inclusion of allergens that are not clinically relevant can dilute the essential allergen components of extract and decrease its effectiveness&#46;</p><p id="par0020" class="elsevierStylePara elsevierViewall">Nowadays&#44; immunotherapy and diagnosis of latex allergy are performed using NRL extracts that are obtained directly from the tree <span class="elsevierStyleItalic">Hevea brasiliensis</span> sap without any purification steps&#46; This crude extract contains several hundred proteins from which 14 &#40;Hev b 1 to Hev b 14&#41; have been recognized by the International Union of Immunological Societies &#40;IUIS&#41; as latex allergens &#40;<a id="intr0005" class="elsevierStyleInterRef" href="http://www.allergen.org/">www&#46;allergen&#46;org</a>&#41;&#46; Among the latex allergens&#44; Hev b 1&#44; Hev b 3&#44; Hev b 5 and Hev b 6 are regarded as the most crucial allergens in natural rubber products&#46;<a class="elsevierStyleCrossRef" href="#bib0030"><span class="elsevierStyleSup">6</span></a> Here we describe a new approach to obtain and separate the four most relevant latex allergens from the major sensitisation source&#44; the latex gloves&#46;</p><p id="par0025" class="elsevierStylePara elsevierViewall">Since the proteins of gloves and other NRL products are responsible for latex sensitisation&#44; we consider that they are the ideal allergen sources of reagents for clinical purposes&#46; Indeed&#44; Yeang et al&#46; reported that the latex allergens for research and clinical applications should theoretically be proteins isolated and purified from latex gloves&#46; These researchers also noted that there are considerable problems in sourcing latex allergens from gloves because they vary widely in their protein content&#44; both qualitatively and quantitatively&#46; However&#44; these problems also exist in crude extracts because there is an inherent variation in allergen content due to genetic environmental and agronomic factors&#46;<a class="elsevierStyleCrossRef" href="#bib0035"><span class="elsevierStyleSup">7</span></a></p><p id="par0030" class="elsevierStylePara elsevierViewall">As a first step of the present method&#44; six different glove brands were collected from health institutions in Portugal&#44; aiming to reduce the effect of inherent protein variation that results from glove manufacture and processing&#46; Latex proteins were extracted from the gloves and lyophilized as described previously&#46;<a class="elsevierStyleCrossRef" href="#bib0040"><span class="elsevierStyleSup">8</span></a> Then&#44; 300<span class="elsevierStyleHsp" style=""></span>mg of lyophilized latex glove extract were dissolved in 20<span class="elsevierStyleHsp" style=""></span>mM tris&#8211;HCl&#44; pH 7&#46;5 and a salt precipitation was applied for both enrichment and concentration of latex allergens&#46; This step was first performed by using 25&#37; ammonium sulphate<span class="elsevierStyleInf">&#46;</span> After being stirred for 30<span class="elsevierStyleHsp" style=""></span>min&#44; at 4<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; the precipitated extract &#40;denoted P1&#41; was centrifuged at 12&#44;000<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 15<span class="elsevierStyleHsp" style=""></span>min&#44; at 4<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The same process was repeated adding 50&#37; and 75&#37; of ammonium sulphate &#40;extracts denoted P2 and P3&#44; respectively&#41;&#46; The three precipitated latex extracts &#40;P1&#44; P2 and P3&#41; were then fractionated by Hydrophobic Interaction Chromatography &#40;HIC&#41;&#46; This technique was employed to exploit the differences in protein hydrophobicity between the four major latex allergens and to accomplish their separation&#46; In fact&#44; it is known that Hev b 5 and Hev b 6 have mainly hydrophilic characteristics while Hev b 1 and Hev b 3 are hydrophobic allergens&#46;<a class="elsevierStyleCrossRefs" href="#bib0045"><span class="elsevierStyleSup">9&#44;10</span></a> HIC was applied using a FPLC system &#40;GE Healthcare Biosciences&#44; Uppsala&#44; Sweden&#41; on a Sepharose CL-6B column modified with butyl-1&#44;4-bis-&#40;2&#44;3-epoxy-propoxyd&#41;&#46; The gel was packed in a XK column 16&#47;20 &#40;GE Healthcare Biosciences&#44; Uppsala&#44; Sweden&#41; and equilibrated in a series of experiments using as eluent PBS 0&#46;01<span class="elsevierStyleHsp" style=""></span>M&#44; pH 7&#46;4 with different ammonium sulphate concentrations&#46; The best separation conditions were achieved using 0&#46;5<span class="elsevierStyleHsp" style=""></span>M ammonium sulphate for precipitate P1&#44; 1&#46;0<span class="elsevierStyleHsp" style=""></span>M for P2 and 1&#46;5<span class="elsevierStyleHsp" style=""></span>M for P3 &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>&#41;&#46; At these conditions&#44; two resolved peaks corresponding to unbound proteins were eluted &#40;peaks 1 and 2&#41;&#46; Bound proteins were then eluted &#40;peak 3&#41; by subsequently decreasing of ammonium sulphate concentration in eluent buffer to 0<span class="elsevierStyleHsp" style=""></span>M&#44; in a stepwise mode&#46; Results showed different chromatographic profiles which suggest qualitative and quantitative differences in protein content of precipitated extracts &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>A&#8211;C&#41;&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0035" class="elsevierStylePara elsevierViewall">Quantification of Hev b 1&#44; Hev b 3&#44; Hev b 5 and Hev b 6&#46;02 in each HIC fraction was performed by capture enzyme immunoassay &#40;EIA&#41; using a commercial kit &#40;FITkit&#8482;&#44;Icosagen&#44; Estonia&#41; according to the manufacturer&#39;s instructions&#46; FITkit&#8482; tests are based on the use of specific monoclonal antibodies developed against the four major NRL allergens&#44; providing individual results for each NRL allergen&#46; All assays were performed in triplicate and when allergen levels were below the detection limit they were denoted as zero &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41;&#46; In this quantitative analysis was observed that HIC fractionation of P1 &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>A&#41; promoted a selective separation of hydrophobic allergens&#44; Hev b 3 &#40;2&#46;53<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;L&#41; and Hev b 1 &#40;1&#46;40<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;L&#41;&#44; in peaks 1 and 3 respectively &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41;&#46; Moreover&#44; HIC of P2 &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>B&#41; showed a separation of Hev b 6&#46;02 &#40;3&#46;43<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;L&#41; in peak 1 &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41;&#44; whereas in peak 2 a mixture of Hev b 5 and Hev b 6&#46;02 was obtained&#46; It was also interesting to observe a high concentration of Hev b 1 in peak 3&#44; despite the slight contamination of Hev b 6&#46;02&#46; On the other hand&#44; separation of Hev b 5 &#40;0&#46;53 and 0&#46;50<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;L&#41; was achieved in peaks 2 and 3 &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41; of the HIC assay applied to the P3 precipitate &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>C&#41;&#46; It is known that although latex sensitisation is mainly acquired at health institutions for both SBP and HCW&#44; in general&#44; these risk groups present different sensitisation profiles&#44; being that Hev b 1 and Hev b 3 are considered major allergens for SBP&#44; while Hev b 5 and Hev b 6&#46;02 are the major allergens for HCW&#46;<a class="elsevierStyleCrossRef" href="#bib0005"><span class="elsevierStyleSup">1</span></a> Although the sensitisation profile of these risk groups may be more complex than this general rule&#44; since it is common that SBP are also sensitised to Hev b 5 and Hev b 6&#44; it is interesting to note that two different fractions containing specific allergens for each risk group were also obtained with this methodology&#58; Peak 2 of P1 with Hev b 1 &#40;1&#46;60<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;L&#41; and Hev b 3 &#40;4&#46;40<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;L&#41;&#44; and Peak 2 of P2 containing Hev b 5 &#40;0&#46;68<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;L&#41;&#44; Hev b 6&#46;02 &#40;2&#46;80<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;L&#41; &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41;&#46; These extracts and the separated allergens can be used to adjust the content of each major allergen that is present in the diagnosis reagents and also can provide a specific extracts for a more effective immunotherapy&#46; In fact&#44; the success of allergen-specific diagnosis and treatment is dependent on the allergen composition of the used material&#46; It is recognized that the commercially available latex extracts vary significantly in their allergen composition and concentration and this heterogeneity could strongly affect not only the diagnosis but also the immunotherapy&#44; due to the lack of correct identification of patient sensitisation profile and to the poor quality of the therapeutic preparations&#46; In order to achieve balanced allergen content&#44; commercially available diagnosis reagents should be enriched in trace latex allergens&#46; Thus&#44; the improvement of NRL reagents using allergen preparation such our extracts could favour the correct diagnosis of clinical allergy that is crucial to guide subsequent investigations and treatment&#46; On the other hand&#44; in terms of immunotherapy purposes&#44; the complex NRL extracts should be replaced by refined protein preparations&#44; containing the clinically relevant allergens for the sensitised patient to treat&#44; with minimal protein contamination&#46; In this regard&#44; we showed that the different hydrophobicity presented by the most clinically relevant latex allergens can be exploited to obtain allergen preparations which can be useful to formulate specific immunotherapy reagents&#44; based on the individual reactivity profile of each latex-allergic patient to treat&#46; It is also important to stress that the minor NRL allergen content of our fractions is unknown and a high concentration of some minor allergens in each fraction cannot be excluded&#46; For this reason&#44; the use of such extracts in immunotherapy trials should raise safety concerns&#44; especially in patients presenting co-sensitisation to minor NRL allergens&#46;</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><p id="par0040" class="elsevierStylePara elsevierViewall">To summarize&#44; in this work it was described a reproducible procedure that allowed the separation of the four most important latex allergens directly from rubber gloves and its enrichment in specific fractions which can potentially be applied for diagnostic or clinical purposes&#46;</p><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0005">Ethical disclosures</span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Protection of human subjects and animals in research</span><p id="par0045" class="elsevierStylePara elsevierViewall">Protection of human and animal subjects&#46; 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            "Ancho" => 3056
            "Tamanyo" => 192253
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">HIC on Epoxy-Sepharose CL-6B column&#46; Sample&#58; 100<span class="elsevierStyleHsp" style=""></span>&#956;L&#44; 0&#46;4<span class="elsevierStyleHsp" style=""></span>mg&#47;mL of latex glove extract obtained by selective precipitation with ammonium sulphate&#58; A&#58; 25&#37; &#40;P1&#41;&#44; B&#58; 50&#37; &#40;P2&#41;&#44; C&#58; 75&#37; &#40;P3&#41;&#46; Eluent&#58; PBS 0&#46;01<span class="elsevierStyleHsp" style=""></span>M&#44; pH 7&#46;4 containing 0&#46;5<span class="elsevierStyleHsp" style=""></span>M &#40;A&#41;&#59; 1&#46;0<span class="elsevierStyleHsp" style=""></span>M &#40;B&#41; and 1&#46;5<span class="elsevierStyleHsp" style=""></span>M ammonium sulphate &#40;C&#41;&#46;</p>"
        ]
      ]
      1 => array:7 [
        "identificador" => "tbl0005"
        "etiqueta" => "Table 1"
        "tipo" => "MULTIMEDIATABLA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "tabla" => array:2 [
          "leyenda" => "<p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">The detection limit levels of allergens on FITkit&#8482; were&#58; Hev b 1&#44; 1&#46;2<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;L&#59; Hev b 3&#44; 2&#46;3<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;L&#59; Hev b 5&#44; 0&#46;5<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;L&#59; Hev b 6&#46;02&#44; 0&#46;1<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;L&#46; Values below these detection limits were denoted as zero&#46;</p>"
          "tablatextoimagen" => array:1 [
            0 => array:2 [
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                  \t\t\t\t">0&#46;53&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</td></tr></tbody></table>
                  """
              ]
              "imagenFichero" => array:1 [
                0 => "xTab522880.png"
              ]
            ]
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Quantification of Hev b 1&#44; Hev b 3&#44; Hev b 5&#44; Hev b 6&#46;02 by EIA in latex glove fractions obtained by HIC of P1&#44; P2 and P3 &#40;precipitation with 25&#37;&#44; 50&#37; and 75&#37; ammonium sulphate&#44; respectively&#41;&#46;</p>"
        ]
      ]
    ]
    "bibliografia" => array:2 [
      "titulo" => "References"
      "seccion" => array:1 [
        0 => array:2 [
          "identificador" => "bibs0005"
          "bibliografiaReferencia" => array:10 [
            0 => array:3 [
              "identificador" => "bib0005"
              "etiqueta" => "1"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Latex allergy&#58; position paper"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:3 [
                            0 => "N&#46; Caba&#241;es"
                            1 => "J&#46;M&#46; Igea"
                            2 => "B&#46; de la Hoz"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:1 [
                      "Revista" => array:6 [
                        "tituloSerie" => "J Investig Allergol Clin Immunol"
                        "fecha" => "2012"
                        "volumen" => "22"
                        "paginaInicial" => "313"
                        "paginaFinal" => "330"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/23101306"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            1 => array:3 [
              "identificador" => "bib0010"
              "etiqueta" => "2"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Clinical and laboratory-based methods in the diagnosis of natural rubber latex allergy"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:3 [
                            0 => "R&#46;G&#46; Hamilton"
                            1 => "E&#46;L&#46; Peterson"
                            2 => "D&#46;R&#46; Ownby"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:1 [
                      "Revista" => array:6 [
                        "tituloSerie" => "J Allergy Clin Immunol"
                        "fecha" => "2002"
                        "volumen" => "110"
                        "paginaInicial" => "S47"
                        "paginaFinal" => "S56"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/12170243"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
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Article information

ISSN: 03010546
Original language: English

DOI:10.1016/j.aller.2013.03.003

The statistics are updated each day

Year/Month	Html	Pdf	Total

2024 November	3	0	3
2024 October	21	14	35
2024 September	18	6	24
2024 August	23	6	29
2024 July	29	8	37
2024 June	15	1	16
2024 May	18	14	32
2024 April	26	0	26
2024 March	39	6	45
2024 February	44	9	53
2024 January	65	17	82
2023 December	48	12	60
2023 November	51	11	62
2023 October	72	16	88
2023 September	36	6	42
2023 August	33	8	41
2023 July	42	15	57
2023 June	14	0	14
2023 May	49	8	57
2023 April	35	7	42
2023 March	24	6	30
2023 February	13	10	23
2023 January	25	17	42
2022 December	17	17	34
2022 November	27	15	42
2022 October	24	11	35
2022 September	37	20	57
2022 August	10	14	24
2022 July	9	7	16
2022 June	13	13	26
2022 May	35	12	47
2022 April	12	11	23
2022 March	9	11	20
2022 February	5	6	11
2022 January	17	5	22
2021 December	9	9	18
2021 November	8	4	12
2021 October	5	14	19
2021 September	5	16	21
2021 August	8	8	16
2021 July	7	11	18
2021 June	9	12	21
2021 May	8	7	15
2021 April	37	40	77
2021 March	7	11	18
2021 February	8	17	25
2021 January	3	9	12
2020 December	1	1	2
2020 November	0	3	3
2020 September	0	1	1
2020 August	0	1	1
2020 July	0	1	1
2020 May	0	1	1
2020 April	0	1	1
2019 December	0	4	4
2019 October	0	1	1
2019 September	0	1	1
2019 July	0	2	2
2019 June	0	4	4
2019 May	0	19	19
2019 April	0	5	5
2018 March	1	0	1
2018 February	5	3	8
2018 January	8	2	10
2017 December	6	35	41
2017 November	6	2	8
2017 October	10	0	10
2017 September	8	1	9
2017 August	11	5	16
2017 July	12	1	13
2017 June	16	14	30
2017 May	28	5	33
2017 April	16	2	18
2017 March	26	30	56
2017 February	19	2	21
2017 January	13	1	14
2016 December	20	12	32
2016 November	20	7	27
2016 October	37	7	44
2016 September	28	6	34
2016 August	17	6	23
2016 July	9	1	10
2016 June	12	7	19
2016 May	13	6	19
2016 April	18	17	35
2016 March	18	15	33
2016 February	13	15	28
2016 January	17	11	28
2015 December	15	7	22
2015 November	12	6	18
2015 October	20	6	26
2015 September	14	4	18
2015 August	11	6	17
2015 July	6	4	10
2015 May	0	2	2
2015 April	1	1	2
2015 March	0	5	5
2015 February	1	1	2
2015 January	1	0	1
2014 November	1	0	1
2014 October	1	0	1
2014 September	2	0	2
2014 August	2	0	2
2014 July	2	2	4

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Allergologia et Immunopathologia is no longer published on Elsevier since the 2021 year.Transferred to Codon Publications

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Allergologia et Immunopathologia is no longer published on Elsevier since the 2021 year.Transferred to Codon Publications

Indexed in:

Follow us:

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