Development of an ultrasensitive sandwich immunoassay for detecting small molecule semicarbazide.

Ultrasensitive sandwich immunoassays for detecting the small molecule semicarbazide (SEM) were developed based on derivatization. Several SEM derivatizing agents were synthesized by linking o-nitrobenzaldehyde (NBA) and biotin with dihydroxyalkanes (different lengths), which were then used to evaluate the distance effect of two epitopes. Sandwich ELISA for SEM derivatives was developed using an anti-SEM-NBA antibody and horseradish peroxidase-labeled avidin or anti-biotin antibody as a secondary conjugate. The advantageous distances of the two epitopes under the double-antibody sandwich and antibody-avidin sandwich modes were ≥12 and ≥13 Å, respectively. Under the distances, the sensitivities of the sandwich ELISA were no lower than those of competitive ELISA. The obtained optimal EC50 values were 11.2 pg/mL (double-antibody sandwich with the epitope distance ≥16 Å) and 7.3 pg/mL (antibody-avidin sandwich with the epitope distance ≥17 Å). Compared with competitive ELISA, the developed method achieved a 30-fold improvement in sensitivity, with simpler aquatic product pretreatment.

A validated ultra-HPLC-MS/MS method for determination of honokiol in human plasma and its application to a clinical pharmacokinetic study.

Aim: To investigate pharmacokinetics of honokiol after administration of honokiol liposome injection (HKLI) and support the clinical studies of HKLI; it is crucial to determine the concentration of honokiol in human biological samples. Experimental method & results: Human plasma samples were extracted by protein precipitation and analyzed by a new ultra-HPLC-MS/MS (UPLC-MS/MS) method with LLOQ of 0.5 ng/ml. The method was validated according to bioanalytical guidelines from the US FDA and EMA. Successful method validation proved that the method was sensitive and selective, and was suitable for determination of honokiol in clinical plasma samples. Conclusion: The method was successfully applied to evaluate the pharmacokinetics of honokiol after administration of HKLI to Chinese subjects with advanced non-small-cell lung cancer in a first in-human study.

[Effect of exogenous ATP on NLRP3 inflammasome activation in gingival fibroblasts cells].

PURPOSE: To investigate exogenous ATP-dependent activation of NLRP3 inflammasome and interleukin-1ß ( IL-1ß) secretion in P.gingivalis infected and heat-killed P.gingivalis induced gingival fibroblasts cells ( hGFs) in vitro. METHODS: Gingival tissues were obtained from healthy patients and hGFs were cultured in vitro with tissue block method to harvest primary cells. HGFs was simulated by being treated with 100 MOI live P.gingivalis or 100 MOI heat-killed P.gingivalis (HP.gingivalis) after 5 mmol/L ATP pre-treatment. Real-time PCR was carried out to assess mRNA expression of NLRP3, ASC, caspase-1 and IL-1ß. The protein level of NLRP3 , caspase-1 and IL-1ß was evaluated by Western blot. IL-1ß secretion was measured using ELISA. Statistical analysis was performed using Graphpad prism 6 statistical package and the measurement data were analyzed by t test or one-way ANOVA. RESULTS: Compared with the control group, P.gingivalis downregulated NLRP3 mRNA and ASC mRNA while upregulated IL-1ß mRNA. Moreover, the protein expression of NLRP3 and IL-1ß was decreased. The gene and protein expression of NLRP3, ASC and IL-1ß was induced by HP.gingivalis, while caspase-1mRNA and IL-1ßsecretion was free from P.gingivalis or HP.gingivalis stimulus. All those genes as well as intracellular protein expression and IL-1ßsecretion were significantly potentiated with ATP/P.gingivalis or ATP/HP.gingivalis stimuli in hGFs. CONCLUSIONS: Exogenous ATP may be a potential stimulus signal in favour of NLRP3 inflammasome activation of hGFs and mediated inflammatory factor IL-1ß secretion.

High-specificity quantification method for almond-by-products, based on differential proteomic analysis.

A highly specific competitive enzyme-linked immunosorbent assay (ELISA) protocol has been developed to identify and classify almond products based on differential proteomic analysis. We applied two-dimensional electrophoresis to compare the differences between almond and apricot kernels to search for almond-specific proteins. The amino acid of apricot Pru-1 was sequenced and aligned to almond Pru-1. One peptide, RQGRQQGRQQQEEGR, which exists in almond but not in apricot, was used as hapten to prepare monoclonal antibody against almond Pru-1. An optimized ELISA method was established using this antibody. The assay did not exhibit cross-reactivity with the tested apricot kernels and other edible plant seeds. The limit of detection (LOD) was 2.5-100µg/g based on different food samples. The recoveries of fortified samples at levels of twofold and eightfold LOD ranged from 82% to 96%. The coefficients of variation were less than 13.0%. Using 7M urea as extracting solution, the heat-treated protein loss ratios were 2%, 5% and 15% under pasteurization (65°C for 30min), baking (150°C for 30min) and autoclaved sterilization (120°C for 15min), respectively.

Application of Differential Proteomic Analysis to Authenticate Ophiocordyceps sinensis.

Ophiocordyceps sinensis (Berk.) Sacc. is one of the most well-known fungi in traditional Chinese medicine and is attracting attention because of its nutritious and medicinal properties. The present study aimed to produce a proteomic map to identify common O. sinensis proteins. The caterpillar body and stroma of O. sinensis collected from five locations and four fungal specimens of similar appearance were examined by two-dimensional electrophoresis (2-DE). Five proteins were identified using MALDI-TOF--TOF/MS, and the 2-DE identification pattern was provided. OCS_04585 and ß-lactamase domain-containing protein, the two abundant and characteristic proteins, were separated and purified using liquid-phase isoelectric focusing. The products were high-quality materials that can be used for future protein-function studies and immunoassay development.

Enzyme-linked Fab' fragment based competitive immunoassay for ovalbumin in hot-processed food.

A hen's egg is one of the most common causes of food allergy. The allergen quantitation in hot-processed food is always a difficult task, because the protein in these samples will be denatured, insoluble, and degraded. This article presents a competitive enzyme linked immunosorbent assay (ELISA) for the quantitation of ovalbumin in hot-processed food. Its recovery was improved nearly two times by the assay method as compared with previous sandwich ELISA. The heat and DL-Dithiothreitol treated ovalbumin was used as antigen for monoclonal antibody preparation. A smaller labeled antibody molecule, horseradish peroxidase (HRP) labeled Fab' fragment, was used to replace IgG in ELISA for improving sensitivity and analytical speed of the method. A binding site protection procedure was developed for Fab' fragment labeling with HRP, which prevented damage to the Fab' binding site. The combination and separation steps were efficiently completed in an affinity spin column. Based on the optimized ELISA condition, the IC50 was 1.2 µg/mL and the coefficients of variation were less than 10%.

Development of monoclonal antibodies and quantitative sandwich enzyme linked immunosorbent assay for the characteristic sialoglycoprotein of edible bird's nest.

The article presents a sandwich enzyme linked immunosorbent assay (ELISA) for identification of edible bird's nest. The characteristic sialoglycoproteins were found by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and purified by liquid-phase isoelectric focusing (LIEF). According to the analysis, the molecular weight was 106-128 kDa and the isoelectric point was ≤pH 3.0. Two anti-characteristic sialoglycoprotein monoclonal antibodies were produced. The monoclonal antibodies were examined by western-blot assay. One of the monoclonal antibody was used as coating and the other as the enzyme-labeled antibody after being coupled to horseradish peroxidase (HRP). Based on the optimized ELISA condition, the method was established with IC(50) of 1.5 ng/mL, and low cross-reactivity with various fake materials (<0.01%). ELISA provided a suitable means for screening of a large number of samples. The coefficients of variation were between 2.9% and 5.8%.

Proteomic profile of edible bird's nest proteins.

Edible bird's nest (EBN) is made of the swiftlets' saliva, which has attracted rather more attention owing to its nutritious and medical properties. Although protein constitutes the main composition and plays an important role in EBN, few studies have focused on the proteomic profile of EBN. The purpose of this study was to produce a proteomic map and clarify common EBN proteins. Liquid-phase isoelectric focusing (LIEF) was combined with two-dimensional electrophoresis (2-DE) for comprehensive analysis of EBN proteins. From 20 to 100 protein spots were detected on 2-DE maps of EBN samples from 15 different sources. The proteins were mainly distributed in four taxa (A, B, C, and D) according to their molecular mass. Taxa A and D both contained common proteins and proteins that may be considered another characteristic of EBN. Taxon A was identified using MALDI-TOF-TOF/MS and found to be homologous to acidic mammalian chitinase-like ( Meleagris gallopavo ), which is in glycosyl hydrolase family 18.

Competitive enzyme-linked immunoassay for sialoglycoprotein of edible bird's nest in food and cosmetics.

The proliferation of fake and inferior edible bird's nest (EBN) products has recently become an increasingly serious concern. To identify and classify EBN products, a competitive enzyme-linked immunoassay (ELISA) was developed to quantitate sialoglycoprotein in EBN used in food and cosmetic applications. The characteristic sialoglycoprotein in EBN was found, extracted, purified, and analyzed. Sialoglycoprotein, considered the main carrier of sialic acid in EBN, consisted of 106 and 128 kDa proteins. A monoclonal antibody that could recognize both proteins was prepared. The heat-treated process did not change the affinity of sialoglycoprotein with the antibody. An optimized ELISA method was established with a cross-reactivity of less than 0.1% and an IC(50) of 3.3 µg/mL. On the basis of different food and cosmetic samples, the limits of detection (LOD) were 10-18 µg/g. Recoveries of fortified samples at levels of 20 and 80 µg/g ranged from 81.5 to 96.5%, respectively. The coefficients of variation were less than 8.0%.

Characterization of a deep-sea sediment metagenomic clone that produces water-soluble melanin in Escherichia coli.

To access to the microbial genetic resources of deep-sea sediment by a culture-independent approach, the sediment DNA was extracted and cloned into fosmid vector (pCC1FOS) generating a library of 39,600 clones with inserts of 24-45 kb. The clone fss6 producing red-brown pigment was isolated and characterized. The pigment was identified as melanin according to its physico-chemical characteristics. Subcloning and sequences analyses of fss6 demonstrated that one open reading frame (ORF2) was responsible for the pigment production. The deduced protein from ORF2 revealed significant amino acid similarity to the 4-hydroxyphenylpyruvate dioxygenase (HPPD) from deep-sea bacteria Idiomarina loihiensis. Further study demonstrated that the production of melanin was correlated with homogentistic acid (HGA). The p-hydroxyphenylpyruvate produced by the Escherichia coli host was converted to HGA through the oxidation reaction of introduced HPPD. The results demonstrate that expression of DNA extracted directly from the environment might generate applicable microbial gene products. The construction and analysis of the metagenomic library from deep-sea sediment contributed to our understanding for the reservoir of unexploited deep-sea microorganisms.

Fungal communities from methane hydrate-bearing deep-sea marine sediments in South China Sea.

To elucidate fungal diversity in methane hydrate-bearing deep-sea marine sediments in the South China Sea, internal transcribed spacer (ITS) regions of rRNA genes from five different sediment DNA samples were amplified and phylogenetically analyzed. Total five ITS libraries were constructed and 413 clones selected randomly were grouped into 24 restriction patterns by Amplified Ribosomal DNA Restriction Analysis (ARDRA). ITS sequences of 44 representative clones were determined and compared with the GenBank database using gapped-BLAST. The phylogenetic analysis showed that the ITS sequences (71-97% similarity) were similar to those of Phoma, Lodderomyces, Malassezia, Cryptococcus, Cylindrocarpon, Hortaea, Pichia, Aspergillus and Candida. The remaining sequences were not associated to any known fungi or fungal sequences in the public database. The results suggested that methane hydrate-bearing deep-sea marine sediments harbor diverse fungi. This is the first report on fungal communities from methane hydrate-bearing deep-sea marine sediments in South China Sea.

[Expression of a DNA fragment encoding the active domain of human TNF related apoptosis inducing ligand in pichia pastoris].

Human tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) is a member of the tumor necrosis factor (TNF) family of ligands which has been reported in 1995. The TRAIL protein induces apoptosis of certain types of target cells, such as transformed cells that include but are not limited to cancer cells and virus-infected cells but the normal cells. It is a type II transmembrane protein and the extracellular domain of TRAIL is the functional domain in induction of cell apoptosis. A gene fragment encoding for the active domain of TRAIL was modified with oligo-nucleotide directed mutagenesis according to the characters of Pichia pastoris expressing vector. Arginine at the position of 149 corresponding to the amino acid residue 531 which might be a potential Kex2 protease processing sites was substituted with Lysine to prevent the expressed protein from the digestion by the protease. After proved with DNA sequencing. the modified gene fragment coding soluble TRAIL domain was inserted into the Pichia pastoris expression vector pPIC9K in the same reading frame with alpha-factor secreting signal peptide. The recombinant plasmid pPIC9K - TRAIL was transferred into P. pastoris cell by spheroplast transformation. The recombinant yeasts were identified by antibiotic G418 and Southern dot blot. The transformants (His+ Mut(s)) containing multi-copy gene fragment of TRAIL were selected with increasing concentration of G418 and induced with 0.5% methanol in shaking flask to expression the active domain of TRAIL. After inducing for 3 - 4 days, the proteins in the culture supernatant was assayed with SDS-PAGE and Western blot. Two expressed protein bands whose appearant molecular weight were 19kD and 38kD, respectively, could be specifically recognized by polyclonal antibodies against human TRAIL. The 38kD protein might be a dimers of TRAIL in the culture supernatant. The amount of expressed foreign protein made up to 36% of the total proteins in the culture suprenatant. Biological activity assay, in vitro, indicated that the expressed protein could induce tumor cells apoptosis.