Production of a specific monoclonal antibody for 1-naphthol based on novel hapten strategy and development of an easy-to-use ELISA in urine samples.

1-naphthol (1-NAP) is the main metabolite of pesticide carbaryl and naphthalene, and is also a genotoxic and carcinogenic intermediate in the synthesis of organic compound, dyes, pigment and pharmaceutical industry. In this work, two novel haptens were designed and synthesized for developing a competitive indirect enzyme-linked immunosorbent assay (ciELISA) method for 1-NAP in urine samples. The assay showed a limit of detection of 2.21 ng/mL and working range from 4.02 ng/mL to 31.25 ng/mL for 1-NAP in optimized working buffer. The matrix effect of samples was eliminated via 15-fold dilution of optimized working buffer. Good average recoveries (102.4%-123.4%) with a coefficient of variation from 11.7% to 14.7% was obtained for spiked urine samples. Subsequent instrument verification test showed good correlation between the results of ciELISA and high-performance liquid chromatography. The developed ciELISA is a high-throughput tool to monitor 1-NAP in urine, which can provide technical support for the establishment of biological exposure level for the exposure to carbaryl, naphthalene and other related pollutants.

Aspergillus fumigatus Cell Wall Promotes Apical Airway Epithelial Recruitment of Human Neutrophils.

Aspergillus fumigatus is a ubiquitous fungal pathogen capable of causing multiple pulmonary diseases, including invasive aspergillosis, chronic necrotizing aspergillosis, fungal colonization, and allergic bronchopulmonary aspergillosis. Intact mucociliary barrier function and early airway neutrophil responses are critical for clearing fungal conidia from the host airways prior to establishing disease. Following inhalation, Aspergillus conidia deposit in the small airways, where they are likely to make their initial host encounter with epithelial cells. Challenges in airway infection models have limited the ability to explore early steps in the interactions between A. fumigatus and the human airway epithelium. Here, we use inverted air-liquid interface cultures to demonstrate that the human airway epithelium responds to apical stimulation by A. fumigatus to promote the transepithelial migration of neutrophils from the basolateral membrane surface to the apical airway surface. Promoting epithelial transmigration with Aspergillus required prolonged exposure with live resting conidia. Swollen conidia did not expedite epithelial transmigration. Using A. fumigatus strains containing deletions of genes for cell wall components, we identified that deletion of the hydrophobic rodlet layer or dihydroxynaphthalene-melanin in the conidial cell wall amplified the epithelial transmigration of neutrophils, using primary human airway epithelium. Ultimately, we show that an as-yet-unidentified nonsecreted cell wall protein is required to promote the early epithelial transmigration of human neutrophils into the airspace in response to A. fumigatus Together, these data provide critical insight into the initial epithelial host response to Aspergillus.

Ultrasensitive detection of Sudan I in food samples by a quantitative immunochromatographic assay.

An immunochromatographic assay (ICA) based on surface-enhanced Raman scattering (SERS) for ultrasensitive determination of Sudan I in food samples was reported. Gold-silver core-shell bimetallic nanorods (referred to as Au@Ag NRs) were synthesized, characterized and used as the substrate for preparation of the ICA. Polyclonal antibody against Sudan I was immobilized on the surface of the Au@Ag NRs carrying the Raman reporter 5,5'-dithiobis (2-nitrobenzoic acid). The Raman scattering intensity on the test line was used for quantitation of Sudan I. The assay was completed in 15 min. IC50 and limit of detection (LOD) were 30 pg mL-1 and 0.2 pg mL-1, respectively. There was no cross-reactivity (CR) of the assay with Sunset Yellow, Lemon Yellow and Brilliant blue FCF, but only 3.53%-9.74% CR with Sudan II, III and IV. The recoveries of Sudan I from spiked food samples were in the range of 88.9-107.6% with relative standard deviation of 3.7-8.7% (n = 3).

Recognition of DHN-melanin by a C-type lectin receptor is required for immunity to Aspergillus.

Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31+ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.

Immunoassay of red dyes based on the monoclonal antibody of ß-naphthol.

The objective of the present study was to develop a multi-analyte immunoassay for the determination of eight red dyes in food samples. Two dye intermediates (2-hydroxy-1-naphthoic acid and 1-amino-2-naphthol) were used as the haptens to produce the monoclonal antibodies. The obtained monoclonal antibodies recognized Sudan 1-4, Para red, Sudan red G, Sudan red B and Acid orange II simultaneously. After evaluation of different antibody/coating antigen combinations, a heterologous indirect competitive enzyme linked immunosorbent assay was developed to determine the eight red dyes in food samples (chili oil, chili powder, tomato sauce, hotpot seasoning). The crossreactivities to the eight analytes were in the range of 61%-79% (with ß-naphthol as 100%), and the limits of detection were in the range of 1.3-1.9 ng/mL. The recoveries of the eight analytes from the fortified blank samples were in the range of 84.2%-115% with coefficients of variation lower than 18.3%. Therefore, this method could be used as a rapid and simple tool to detect the residues of the eight red dyes in foods.

A sensitive and selective enzyme-linked immunosorbent assay for the analysis of Para red in foods.

Para red is a synthetic dye and a potential genotoxic carcinogen. A hapten mimicking Para red structure was synthesized by introducing a carboxyl to the naphthol part of Para red and coupled to carrier protein to form an immunogen for the production of specific antibodies. A sensitive and selective enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Para red in food samples. The limit of detection and inhibition half-maximum concentrations of Para red in phosphate buffered saline with 10% methanol were 0.06 and 2.2 ng mL(-1), respectively. Cross-reactivity values of the ELISA with the tested compounds including Sudan red I, II, III, IV, and G, sunset yellow, 2-naphthol, and 4-nitroaniline were ≤0.2%. This assay was used to determine Para red in tomato sauce, chilli sauce, chilli powder and sausage samples after ultrasonic extraction, cleanup and concentration steps. The average recoveries, repeatability (intraday extractions and analysis), and intra-laboratory reproducibility (interday extractions and analysis) were in the range 90-108%, 4-12% and 8-17%, respectively. This assay was compared to a high-performance liquid chromatographic method for 28 samples, displaying a good correlation (R(2) = 0.95). Para red residues in 53 real world samples determined by ELISA were below the limit of detection.

A monoclonal antibody-based immunosensor for detection of Sudan I using electrochemical impedance spectroscopy.

Sudan I monoclonal antibodies (Mabs) were prepared by hybridoma technique and firstly used to develop a Sudan I immunosensor by immobilizing the Mabs on a gold electrode. o-Mercaptobenzoic acid (MBA) was covalently conjugated on the gold electrode to form a self-assembled monolayer (SAM). The immobilization of Sudan I Mabs to the SAM was carried out through a stable acyl amino ester intermediate generated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide (NHS), which could condense antibodies reproducibly and densely on the SAM. The changes of the electrode behavior after each assembly step were investigated by cyclic voltammetric (CV) technique. The Sudan I concentration was measured through the increase of impedance values in the corresponding specific binding of Sudan I and Sudan I antibody. A linear relationship between the increased electron-transfer resistance (Ret) and the logarithmic value of Sudan I concentrations was found in the range of 0.05-50 ng mL(-1) with the detection limit of 0.03 ng mL(-1). Using hot chili as a model sample, acceptable recovery of 96.5-107.3% was obtained. The results were validated by conventional HPLC method with good correlation. The proposed method was proven to be a feasible quantitative method for Sudan I analysis with the properties of stability, highly sensitivity and selectivity.

[Synthesis of Sudan I artificial antigen].

OBJECTIVE: To prepare Sudan I-HS conjugates with the carrier protein for developing a new immune assay for Sudan I. METHODS: Sudan I was actived by succinic anhydride and converted into Sudan I hemisuccinate (Sudan I-HS) as the hapten, then Sudan I-HS was linked to carrier protein bovine serum albumin (BSA) as immunogen, and ovalbumin (OVA) as coating antigen all by the active ester method. RESULTS: The results of IR and HPLC-MS indicated that the hapten was synthesized successfully. The conjugation ratios of Sudan I-HS-BSA and Sudan I-HS-OVA confirmed by ultraviolet spectrum were 11 : 1 and 5 : 1, respectively. CONCLUSION: Antigens Sudan I-HS-BSA and Sudan I-HS-OVA were prepared successfully.

Immunoglobulin purification by affinity chromatography using protein A mimetic ligands prepared by combinatorial chemical synthesis.

Affinity chromatography using protein A from Staphylococcus aureus as the ligand has been widely used for the isolation of immunoglobulin G (IgG) from various species. Since ligand leakage from the affinity support can occur, time consuming analytical controls are required to detect the presence of contaminants associated with the isolated IgG prior to its use for therapeutic purpose in humans. Besides, protein A is an expensive bacterial product, whose isolation involves complex and labor intensive procedures. Combinatorial chemistry enables the synthesis of a wide variety of ligands within a short period of time. Therefore, chemically defined, stable and inexpensive ligands, which can mimic the action of protein A, have been developed to isolate immunoglobulins. Two different types of ligands, synthesized following the techniques of combinatorial chemistry, have been used to isolate immunoglobulins. One of them is a synthetic peptide (TG 19318) comprising four identical tripeptide chains linked to a central polylysine core. Immobilized TG 19318 has been used to isolate polyclonal and monoclonal antibodies of different classes (IgG, IgM, IgA, IgE) from different sources (serum, ascities and cell supernatants) and species. The ligand has a binding capacity that can reach upto 25mg IgG/mL of the support. It is stable when treated with sanitation agents such as ethanol and 0.1 M sodium hydroxide. Computer-aided molecular design and combinatorial chemistry have been used to develop an IgG binding affinity ligand (22/8), which consists of two organic aromatic amines (3-aminophenol and 4-amino-1-napthol) linked to a scaffold of cyanuric chloride (triazine). Ligand 22/8 displayed wider specificity than protein A, as it isolated IgG from a number of species, the order of adsorption being human> chicken > cow > rabbit > pig > horse > rat > goat > sheep > mouse. It showed an apparent binding capacity of 51.9 mg IgG/g moist gel and can isolate human IgG from plasma in 60% yield with a purity of 92%. The ligand is stable, as it withstood incubation in 1M NaOH for a week without loss of binding capacity for IgG. These findings suggest that synthetic affinity ligands, which are inexpensive, stable and specific can facilitate the purification of immunoglobulins in a cost-effective manner.

T-lymphocyte responses to plicatic acid-human serum albumin conjugate in occupational asthma caused by western red cedar.

BACKGROUND: T cells are known to play a major role in the pathogenesis of atopic allergic asthma, but it is less clear whether they are involved in occupational asthma caused by low molecular weight chemicals such as plicatic acid. OBJECTIVES: We sought to determine whether peripheral blood T cells from patients with western red cedar asthma (WRCA) recognize plicatic acid (PA) conjugated to human serum albumin (HSA) as judged by proliferation or cytokine production and to analyze the response to PA inhalation with flow cytometry. RESULTS: Significant proliferative responses to PA-HSA were observed in eight of 33 patients with WRCA, none of 10 exposed nonasthmatic cedar workers, and one of 18 nonasthmatic control subjects. Two of 25 patients with WRCA also showed proliferative responses to unconjugated PA. All the WRCA responders were either currently exposed to cedar or had ceased exposure within the preceding 2 years. None of the four patients receiving oral steroids responded, but inhaled steroids did not seem to influence responsiveness. No correlations were found between the maximum stimulation response and any of the current FEV1 values, the current PC20 methacholine values, or the magnitude of the late asthmatic response to PA. Peripheral blood T-cell subset proportions and their degree of activation were similar in patients with WRCA and exposed control subjects. There was no change in T-cell phenotypes or their activation markers after PA inhalation challenge. In vitro, PA-HSA stimulation did not affect subset ratios but led to release of small amounts of IL-5 and IFN-gamma, with no detectable increase in IL-4. CONCLUSIONS: PA-HSA-specific T lymphocytes seem to be present in small numbers in the peripheral blood of patients with WRCA and may respond to antigenic exposure by producing IFN-gamma and IL-5. However, the proportion of responding cells would appear to be lower than in comparable studies of atopic asthma.

Is tyrosine kinase activation involved in basophil histamine release in asthma due to western red cedar?

Occupational asthma due to western red cedar is associated with histamine release from basophils and mast cells on exposure to plicatic acid (PA), but the mechanisms underlying this response remain unclear. Specific kinase inhibitors were used to study the role of tyrosine and serine/threonine kinases in PA-induced histamine release from human basophils. Pretreatment with the tyrosine kinase inhibitor methyl 2,5-dihydroxy-cinnamate (MDHC) attenuated histamine release from basophils triggered by anti-IgE (29.8% inhibition; n = 15; P < 0.01) or grass pollen (48% inhibition; n = 6; P < 0.01). Inhibition was concentration-dependent and could be reversed by washing the cells in buffer, while the inactive stereoisomer of MDHC did not affect histamine release. In contrast, the protein kinase C inhibitor staurosporine did not affect histamine release by either anti-IgE or grass pollen. Pretreatment with MDHC partially inhibited PA-induced histamine release from basophils of 6/9 patients with red cedar asthma (25.4% vs 33.8%; P = NS). Staurosporine gave a similar level of inhibition of PA-induced histamine release (25.3% vs 33.8%; P = NS). Thus, signal transduction of the human basophil Fc epsilon RI appears to depend upon tyrosine kinase activation, but not on protein kinase C (serine/threonine kinase) activation. The lack of specific effect on plicatic acid-induced histamine release in basophils obtained from patients with occupational asthma due to western red cedar suggests that tyrosine kinases are not as important in this disease as in atopic asthma, and is consistent with the view that histamine release in red cedar asthma is largely IgE-independent.

Involvement of immunologic mechanisms in a guinea pig model of western red cedar asthma.

Western red cedar asthma is the most common form of occupational asthma in the Pacific Northwest. Plicatic acid (PA) is the chemical component of Western red cedar that causes asthma. The role of immunologic processes involved in the PA-induced asthmatic reaction has not been established. To characterize the mechanisms of PA-induced asthmatic reaction, guinea pigs were sensitized to PA through biweekly injection of PA-ovalbumin conjugate with aluminum hydroxide as an adjuvant for a period of 6 months. Specific IgG1 antibodies to PA were detected in the blood 3 months after sensitization of animals. The level of specific IgG1 antibodies to ovalbumin after 6 months was about two times the level of specific IgG1 to PA. At 6 months, tracheal tissue from PA-ovalbumin-sensitized guinea pigs contracted after exposure to either PA or ovalbumin in vitro. The degree of contraction induced by PA was two to three times less than the contraction induced by ovalbumin. PA caused histamine, prostaglandin D2, and leukotriene D4 release from both lung mast cells and blood basophils. The amount of histamine and eicosanoids released by PA was also two to three times less than the amount of mediators released by ovalbumin. When the trachea of normal guinea pigs was passively sensitized with serum from PA-ovalbumin-sensitized guinea pigs, it contracted in response to PA or ovalbumin in an organ bath. When the serum of PA-ovalbumin-sensitized guinea pigs was depleted of immunoglobulins and then used for passive sensitization of normal trachea, no contraction was observed when challenged with PA, suggesting that IgG1 antibodies mediate the tracheal reaction to PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of occupational asthma due to western red cedar (Thuja plicata).

Occupational asthma due to Western red cedar is the most common form of occupational asthma in the Pacific Northwest and affects 4-13.5% of the exposed population. It has been shown to be caused by plicatic acid, a low molecular weight compound present uniquely in the wood. The mechanism of asthma induced by plicatic acid is not known, as specific IgG antibodies were found only in about 20% of patients. Sera from patients with red cedar asthma failed to passively sensitize human lung fragments of human basophils. Basophils from patients with this disease released histamine when challenged directly with plicatic acid in a specific manner. Immunologic mechanisms other than Type I hypersensitivity reaction are likely to be involved.

Lack of role for mononuclear cell-derived histamine releasing factors in occupational asthma due to western red cedar.

Occupational asthma due to Western Red Cedar (WRCA) is attributed to sensitization to plicatic acid (PA), but does not appear to be dependent on PA-specific IgE antibodies. Exposure to PA induces histamine release in vivo and in vitro, so if IgE is not important, other mechanisms of histamine release must presumably operate in WRCA. To explore the possible role of histamine-releasing factors in WRCA, peripheral blood mononuclear cells were obtained and cultured with PA, PA-albumin conjugate plicatic acid-human serum albumin (PA-HSA), grass pollen or Concanavalin A using a standard histamine releasing factor (HRF) generation protocol. Supernatants were dialysed to remove endogenous histamine and then assayed for histamine releasing activity using human basophils as targets and a Con A-induced bulk supernatant as an internal HRF standard. In contrast to some previous reports, spontaneous HRF release from the peripheral blood mononuclear cells (PBMC) of WRCA patients (n=9) and atopic asthmatic subjects (n=5) was not elevated compared with the non-asthmatic controls (n=11; five atopic and six non-atopic). Both PA and PA-HSA induced the production of small amounts of HRF by PBMC of WRCA patients, but a similar degree of HRF generation was also observed in PBMC from the atopic asthmatic, atopic nonasthmatic, and non-atopic subjects. In contrast, grass pollen induced the production of HRF by PBMC from the subjects with positive skin tests to grass pollen but not by PBMC of non-atopic subjects, confirming that our methods and assay were capable of detecting antigen-specific HRF production. Since neither PA nor PA-HSA induced significantly elevated HRF production from PBMC of WRCA patients, it seems unlikely that PA-induced HRFs play a substantial role in the pathogenesis of WRCA.

Immunologic studies of the mechanisms of occupational asthma caused by western red cedar.

BACKGROUND: Occupational asthma caused by western red cedar (Thuja plicata) is a common problem in sawmill industries. The objective of this study was to examine the cellular and immunologic mechanisms of western red cedar asthma (WRCA) more closely. METHODS: Bronchial biopsy specimens, bronchoalveolar lavage (BAL) mast cells and peripheral blood basophils from patients with WRCA, patients with atopic asthma, and nonatopic control subjects were challenged in vitro with plicatic acid (PA), PA-human serum albumin conjugate (PA-HSA), grass pollen, or calcium ionophore. RESULTS: PA (100 micrograms/ml) released histamine from the basophils of 9 of 11 patients with WRCA, 1 of 7 patients with atopic asthma, and 2 of 7 normal subjects. PA triggered histamine release from 10 of 11 bronchial biopsy specimens and 8 of 8 BAL samples from patients with WRCA. Interestingly, PA released histamine from BAL cells and bronchial biopsy specimens from 3 of 7 normal subjects but in none of the patients with atopic asthma. PA-HSA-induced histamine release from basophils and biopsy specimens was confined to patients with WRCA. PA-specific IgE was not detectable in serum from most patients with WRCA, and their serum did not transfer PA sensitivity to human lung fragments or lactate-stripped basophils. After pretreatment with anti-IgE in the absence of calcium, basophils from 14 subjects with WRCA still responded to PA (mean 64% to 67% of pretreatment response), whereas responses to grass pollen or anti-IgE were abolished. CONCLUSIONS: This study confirms that PA releases histamine from bronchial mast cells of most patients with WRCA but not from those of patients with atopic asthma. The PA response of some normal subjects suggests that PA may have both specific and nonspecific actions on mast cells and basophils, whereas the serologic studies indicate histamine release in WRCA cannot simply be attributed to PA-specific IgE.

An enzyme immunoassay of phaseolinone and its application in estimation of the amount of toxin in Macrophomina phaseolina-infected seeds.

A microtiter plate-based enzyme immunoassay has been developed for phaseolinone, a phytotoxin isolated from the culture filtrate of the plant-pathogenic fungus Macrophomina phaseolina (Tassi) Goid. The smallest amount of phaseolinone detectable by the method is 5 pg per well. The method is validated by comparison with high-performance liquid chromatography and used to confirm and estimate phaseolinone production in seeds infected with the fungus. The degree of seed inhibition correlated well with the amount of toxin produced in infected seeds, 50% inhibition being observed at a toxin concentration of 0.60 micrograms/g of wet tissue.

Highly specific polyclonal antisera against estriol: cross-reactivity restriction following affinity chromatography.

An immunosorbent technique was developed to attenuate cross-reactivity of a polyclonal antiserum against a 4(2) (rho-carboxyphenylazo)-1,3,5[10]-estratrien-3,16 alpha,17 beta-triol-bovine serum albumin conjugate. The chromatographic separation of antiserum through stationary phases having either rho(carboxymethyl)phenylazo-phenol or rho(carboxymethyl)-phenylazo-2-naphthol side residues reduced the antiserum avidity, while increasing the apparent antiserum affinity and decreasing the residual cross-reactivities against heterologous ligands. The highly specific antiserum obtained allowed the development of a competitive binding assay over an extended analytical range, which opens up the possibility of direct measurement of estriol from the early pregnancy to delivery. The significance of the attenuation of antiserum cross-reactions after affinity chromatography is discussed with reference to epitope-paratope interaction in the case of small endogenous molecules like estrogens.

Pattern of specific airway response in asthma due to western red cedar (Thuja plicata): relationship with length of exposure and lung function measurements.

In order to investigate the relationship between the pattern of response (immediate, late and dual) to specific bronchial challenge test with plicatic acid or red cedar extract and the clinical features of asthma, 332 patients with asthma induced by western red cedar dust were examined at the time of diagnosis. Specific challenge test induced in thirty-one patients (9.3%) an isolated immediate reaction, in 144 patients (43.4%) an isolated late reaction and in 157 patients (47.3%) a dual reaction. Patients with a dual reaction had a longer period of exposure to red cedar dust between the onset of the respiratory symptoms and the time of the definitive diagnosis, a lower FEF 25-75% and a greater degree of non-specific bronchial hyperresponsiveness compared to patients with isolated immediate or isolated late reactions; the difference in bronchial hyperresponsiveness to methacholine among the three groups persisted when the values were adjusted for the different baseline value of FEV1. There was no difference in the prevalence of specific serum IgE antibodies to plicatic acid-human serum albumin conjugate among the three groups of patients with different type of response to red cedar. Except for the greater degree of non-specific bronchial hyperresponsiveness, patients with isolated late reactions were not different from those with isolated immediate reactions in other clinical findings. These findings indicate that a dual reaction in patients with occupational asthma due to simple chemical agents is indicative of a greater severity of disease at diagnosis. The pathogenetic mechanism of various types of asthmatic reaction is unknown and it is likely to be different between isolated immediate and isolated late reactions.