Yao and coworkers[28]described the application of nonfluorescent nanocrystals for for IgE determination. the surface of mast cells and basophils; upon interactions with allergens they cause cell degranulation, leading to the release of mediators Erythrosin B that induce allergic reactions[1],[2]. Normally, the content of IgE in human blood is usually less than 0.001% of the total amount of all immunoglobulins[3]. IgE concentration is typically expressed in kU/L, where 1 kU/L corresponds to 2.4 g/L[4],[5]. The concentration of total IgE in a healthy adult is about 80 kU/L[6],[7]. In the case of allergic disease or myeloma, the IgE content in blood is usually increased 430 fold[8],[9]. Therefore, methods for determining the total IgE concentration are necessary for primary care providers to assess the state of the immune system and quickly refer patients for further examination. Clinical diagnostic laboratories mainly use the enzyme-linked immunosorbent assay (ELISA) to determine the total IgE[10]. Available ELISA packages determine total IgE in the range of 51000 kU/L; however, Col4a4 the assay requires at least 2 hours for obtaining the results[11][13]. Immunochromatographic assays are attractive alternatives to ELISA because they are faster and less labor-intensive[14]. Existing commercially available assessments can determine the total IgE in human serum in 725 moments[15],[16]. These assessments use colloidal gold as a label; its binding is usually detected visually or by an optical detector as the appearance of coloration in target zones of the strip. However, when working with complex matrices such as bodily fluids, the labels and sample components can cause significant unspecific staining of the test strip, hampering reliable measurements of low analyte concentrations. Currently available immunochromatographic assessments for human serum Erythrosin B can detect total IgE with qualitative precision. For example, the ALFA Total IgE test from Dr. Fooke Laboratorien (Neuss, Germany)[16]indicates the levels of total IgE in human serum or plasma by the appearance from one to three colored lines. The quantitative determination of total IgE is usually, however, the most relevant for decisions to pursue further therapy[17]; this information, at present, can only be obtained via the Milenia Biotec (Giessen, Germany) MQTE 1 immunochromatographic test, based on a colloidal platinum label and makes use of a portable reader with software to rapidly obtain quantitative results. However, this system has a relatively high detection limit of 30 kU/L[15]. The use of other labels, such as fluorescent markers, in immunochromatographic assay may help decrease the detection limit and decrease the influence of the matrix. Several studies have reported low detection limits for assays based on fluorescent nanoparticles[18],[19]. Quantum dots (QDs) have also been investigated as fluorescent labels for immunoassays[20][25]. QDs are semiconductor nanocrystals whose diameter varies from 2 to 10 nm; their fluorescence emission peak strongly depends on their size as well as their composition. Water-soluble QDs have a surface covering enriched with carboxyl or amine groups, thus facilitating their conjugation with antibodies[22],[26]. QDs, in comparison with organic fluorescent labels, are more stable, have a thin and symmetrical emission maximum, and are resistant to photobleaching[27]. QDs thus show promise as bioanalytical labels. There are several publications describing QD-based immunodetection of human IgE by using such techniques as ELISA[28]and phosphorescent immunoassay[29]. However, the use of QDs in immunochromatographic assays has not been previously reported. The purpose of our study was to develop an immunochromatographic test, using QDs as a label, for the determination of total IgE in human serum. Levels of IgE were assessed with a portable fluorescence detector REFLEKOM-UV, made up of a UV light source[30], which is amenable to clinical work or field studies as well as laboratory conditions. The paper explains results of a technical development, including the optimization of the assay conditions and screening of the immunochromatographic assay using serum samples. == Materials and Methods == == 1. Reagents == Water-soluble quantum dots were obtained from Invitrogen (Eugene, OR, USA). The QDs were composed of CdSe/ZnS and coated with carboxylated polymer, and their emission maximum was 625 nm. The non-ionic detergent Tween 20 and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO, USA). Clone 4F4 of anti-human IgE monoclonal antibodies was obtained from Polygnost (St. Petersburg, Russia) and clone ME-113 was obtained from GeneTex (Irvine, CA, USA). Goat anti-mouse Erythrosin B IgG polyclonal antibodies were obtained from IMTEK (Moscow, Russia). N-(4-dimethylaminopropyl)-N-ethyl-carbodiimide hydrochloride (EDC) and sodium N-hydroxysulfosuccinimide (sulfo-NHS) were obtained from Fluka (St. Gallen, Switzerland). Sephadex G-100 was obtained from MP Biomedicals (Solon, OH, USA). Standard solutions of IgE with concentrations of 5, 20, 50, 100, 200 and 1000 kU/L were obtained from Dr. Fooke Laboratorien (Neuss, Germany). All other reagents were obtained from Chimmed (Moscow,.
H1 Receptors