We developed a method for accurate quantification of the intact virus particles in inactivated avian influenza virus feedstocks. To address the problem of impurities interference in the detection of inactivated avian influenza virus feedstocks by direct high performance size exclusion chromatography (HPSEC), we firstly investigated polyethylene glycol (PEG) precipitation and ion exchange chromatography (IEC) for H5N8 antigen purification. Under the optimized conditions, the removal rate of impurity was 86.87% in IEC using DEAE FF, and the viral hemagglutination recovery was 100%. HPSEC was used to analyze the pretreated samples. The peak of 8.5-10.0 min, which was the characteristic adsorption of intact virus, was analyzed by SDS-PAGE and dynamic light scattering. It was almost free of impurities and the particle size was uniform with an average particle size of 127.7 nm. After adding antibody to the IEC pretreated samples for HPSEC detection, the characteristic peak disappeared, indicating that IEC pretreatment effectively removed the impurities. By coupling HPSEC with multi-angle laser scattering technique (MALLS), the amount of intact virus particles in the sample could be accurately quantified with a good linear relationship between the number of virus particles and the chromatographic peak area (R²=0.997). The established IEC pretreatment-HPSEC-MALLS assay was applied to accurate detection of the number of intact virus particles in viral feedstocks of different subtypes (H7N9), different batches and different concentrations, all with good applicability and reproducibility, Relative standard deviation < 5%, n=3.
Animals ; Reproducibility of Results ; Influenza A Virus, H7N9 Subtype ; Influenza in Birds ; Chromatography, Gel ; Virion ; Lasers

This paper aims to detect the antigens in porcine circovirus type 2 (PCV2) vaccines by high-performance size-exclusion chromatography (HPSEC) coupled with multi-angle laser light scattering (MALLS). With purified inactivated PCV2 and PCV2 virus-like particles (VLP) as references, two inactivated vaccines (a and b) and two VLP vaccines (c and d) for PCV2 from four manufacturers were analyzed by HPSEC-MALLS after demulsification. The antigen peaks in HPSEC-MALLS were identified by PCV2 antigen test strips, Western blotting and transmission electron microscope (TEM). The repeatability and linearity of the method were investigated. The results showed the virus antigens in the two inactivated vaccines were eluted at about 13.3 min in HPSEC. The molecular weight of these antigens was 2.61×10⁶ (±4.34%) Da and 2.40×10⁶ (±2.51%) Da, respectively, as calculated by MALLS. The antigen peaks of the two VLP vaccines also appeared at 13.3 min and the molecular weight was 2.09×10⁶ (±2.94%) Da and 2.88×10⁶ (±11.85%) Da, respectively, which was close to the theoretical molecular weight of PCV2. Moreover, an antigen peak of VLP vaccine c was observed at 11.4 min and the molecular weight was 4.37×10⁶ (±0.42%) Da. The antigen was verified to be the dimer of VLP by TEM. Vaccine d and purified Cap VLP antigens were tested repeatedly, and the RSD of the peak area (n=3) was all < 1.5%, indicating that the method was repeatable. The purified VLP were diluted in serial and tested for linearity. The result suggested good linear relationship between the peak area of VLP or VLP aggregates and the protein concentration of the sample with R² of 0.999 and 0.997, respectively. Thus, the method met the requirement for quantification and aggregate analysis. This method is accurate and efficient in in vitro quality evaluation and improvement of PCV2 vaccine.
Animals ; Antibodies, Viral ; Capsid Proteins ; Chromatography, Gel ; Circoviridae Infections/prevention & control* ; Circovirus ; Lasers ; Swine ; Vaccines, Inactivated ; Vaccines, Virus-Like Particle ; Viral Vaccines

In the present study,fresh Guangdilong( GD),originating from Pheretima aspergillum,was taken as the object. The total proteins from GD were firstly separated by SDS-PAGE according to their molecular weights and in-gel digestion was then performed.After that,the peptides were analyzed by nano LC/orbitrap fusion lumos high resolution mass spectrometry( nano LC/orbitrap fusion lumos HR-MS). Protein identification was implemented by comparison with Annelida. fasta database using Proteome Discoverer software.As a result,386 proteins were tentatively identified,including chain F,globin B chain,glyceraldehyde-3-phosphate dehydrogenase,fibrinolytic protein,and so on. Most of the proteins took part in cell structure and energy metabolism,and fibrinolytic protein and lombricine kinase might be related to fibrinolytic activity. Protein classification based on gene ontology was carried out using PANTHER and KEGG for metabolic pathway enrichment. The results indicated that these proteins were related to diverse signal transduction pathways,including metabolic pathways,central carbon metabolism,biosynthesis of amino acids,ribosome,glycolysis,citrate cycle( TCA cycle),and so on. This study would lay the foundation for the further research on the proteins in GD and also their functions.
Animals ; Chromatography, Liquid ; Electrophoresis, Polyacrylamide Gel ; Gene Ontology ; Mass Spectrometry ; Oligochaeta ; chemistry ; Proteome ; Proteomics

We developed a pre-treatment method to remove interfering substances during quantification of 146S antigens in foot-and-mouth disease (FMD) vaccines by high performance size exclusion chromatography (HPSEC). Three methods, including ultracentrifugation, PEG precipitation and nuclease digestion, were optimized and compared for removal efficiency of the interfering impurities in FMD vaccines. Under optimized conditions, the 146S contents in two batches of FMD vaccines were determined to be 7.1 and 7.6 μg/mL by ultracentrifugation, 9.7 and 10.4 μg/mL by PEG precipitation, and 10.5 and 10.4 μg/mL by nuclease digestion. The optimal condition for nuclease digestion using Benzonase determined by response surface method was as follows: appending Benzonase into 200 μL of antigen phase to a final concentration of 421 U/mL and incubating at 25.1 °C for 1.29 h. This method has advantages including efficient removal of the interfering impurities, fast processing speed, and mild operating conditions. Then 12 bathes of FMD vaccines with different serotypes produced by 4 manufacturers were tested to verify the established treatment method. Results showed the method was applicable to various FMD vaccines with good reproducibility (RSD<5.3%, n=3). The developed method removed interference from impurities during quantification of 146S, and therefore would broaden the application of HPSEC in vaccine quality control and ensure the accuracy and reliability.
Animals ; Chromatography, Gel ; Foot-and-Mouth Disease ; Foot-and-Mouth Disease Virus ; Reproducibility of Results ; Viral Vaccines

Neuraminidase (NA) cleaves the glycosidic bond linkages of sialic acids to release the mature virions from infected cells and has been an attractive therapeutic target for anti-influenza agents. In our ongoing investigation of NA inhibitors in mushroom extracts, we found that the extract the fruiting body of Glaziella splendens potently inhibited neuraminidase. The fruiting bodies of G. splendens were extracted and partitioned successively with hexane, ethyl acetate, and butanol. The ethyl acetate soluble-layer was subjected to silica gel and Sephadex LH-20 column chromatographies, and MPLC to obtain five compounds (1–5). Their structures were determined by spectroscopic methods. NA inhibitory activity of these compounds was evaluated using NAs from recombinant rvH1N1, H3N2, and H5N1 influenza A viruses. One compound (1) was elucidated as a new azaphilone derivative, and four compounds (2–5) were identified as entonaemin A, comazaphilone D, rubiginosin A, and entonaemin B, respectively. Compounds 3 and 4 showed considerable inhibitory activity against three types of neuraminidases with the IC₅₀ values of 30.9, 41.8, and 35.7 µM for 3 and 46.5, 50.4, and 29.9 µM for 4, respectively. This study reveals that the fruiting bodies of G. splendens possess azaphilone derivatives with the NA inhibitory activity. This is the first report on the isolation of neuraminidase inhibitors from the fruiting bodies of G. splendens.
Agaricales ; Chromatography ; Fruit ; Influenza A virus ; N-Acetylneuraminic Acid ; Neuraminidase ; Sialic Acids ; Silica Gel ; Virion

Two proteins of similar molecular weight (named as ASPR-C-1 and ASPR-C-2) from the crude drug of Angelica sinensis were purified and characterized by 80% ammonium sulfate precipitation, Sephadex G-50 gel filtration chromatography, and DEAE-Sepharose anion exchange chromatography. The molecular weight of ASPR-C-1 and ASPR-C-2 on SDS-PAGE was 17.33 kDa and 17.18 kDa, respectively. They were mainly monomeric in solution, but partially formed dimers and they were glycoproteins with glycosyl content of 2.6% and 8.2%, respectively. Both ASPR-C-1 and ASPR-C-2 were identified to be members of pathogenesis-related 10 family of proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and have ribonuclease activities with the specific activity of 73.60 U/mg and 146.76 U/mg, respectively. The optimum pH of the two isoforms was similar, at about 5.6, while their optimum temperatures were different. The optimum temperature of ASPR-C-1 was 50 ℃, and that of ASPR-C-2 was 60 ℃. Both isoforms presented highest thermal stability at 60 ℃. However, ASPR-C-2 was more thermotolerant than ASPR-C-1. The latter was rapidly inactivated and retained only about 20% residual activity while the former still maintained about 80% of its original activity at a higher treatment temperature (80 to 100 ℃). In addition, Fe²⁺ had an activating effect on the ribonuclease activities of two isoforms while Ca²⁺, Mg²⁺, Zn²⁺, Mn²⁺, Ag⁺, Cu²⁺, EDTA (Elhylene diamine tetraacetic acid), dithiothreitol and sodium dodecylsulphate showed different degrees of inhibition of the enzyme activities. Our findings provide a foundation for further research on the biological function of PR-10 protein from Angelica sinensis.
Angelica sinensis ; Chromatography, Gel ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Enzyme Stability ; Hydrogen-Ion Concentration ; Kinetics ; Molecular Weight ; Protein Isoforms ; Temperature

Globally, peptide-based anticancer therapies have drawn much attention. Marine organisms are a reservoir of anticancer peptides that await discovery. In this study, we aimed to identify cytotoxic oligopeptides from Sarcophyton glaucum. Peptides were purified from among the S. glaucum hydrolysates produced by alcalase, chymotrypsin, papain, and trypsin, guided by a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay on the human cervical cancer (HeLa) cell line for cytotoxicity evaluation. Purification techniques adopted were membrane ultrafiltration, gel filtration chromatography, solid phase extraction (SPE), and reversed-phase high-performance liquid chromatography (RP-HPLC). Purified peptides were identified by de novo peptide sequencing. From papain hydrolysate, three peptide sequences were identified: AGAPGG, AERQ, and RDTQ (428.45, 502.53, and 518.53 Da, respectively). Peptides synthesized from these sequences exhibited cytotoxicity on HeLa cells with median effect concentration (EC₅₀) values of 8.6, 4.9, and 5.6 mmol/L, respectively, up to 5.8-fold stronger than the anticancer drug 5-fluorouracil. When tested at their respective EC₅₀, AGAPGG, AERQ, and RDTQ showed only 16%, 25%, and 11% cytotoxicity to non-cancerous Hek293 cells, respectively. In conclusion, AERQ, AGAPGG, and RDTQ are promising candidates for future development as peptide-based anticancer drugs.
Amino Acid Sequence ; Animals ; Anthozoa/chemistry* ; Antineoplastic Agents/pharmacology* ; Chromatography, Gel ; Chromatography, High Pressure Liquid ; Chromatography, Reverse-Phase ; Cytotoxins/pharmacology* ; Drug Discovery ; HEK293 Cells ; HeLa Cells ; Humans ; Hydrolysis ; Marine Toxins/pharmacology* ; Oligopeptides/pharmacology* ; Solid Phase Extraction ; Tandem Mass Spectrometry

PURPOSE: Japanese hop (Humulus japonicus) is a major cause of weed pollinosis in East Asia. However, supplies of commercial allergen extract from this plant have not met clinical demand. The pollen of common hop (Humulus lupulus), a closely related species, may provide an alternative source if there is strong IgE cross-reactivity between these two species. We aimed to compare the IgE cross-reactivity and allergenicity of common hop and Japanese hop pollen. MATERIALS AND METHODS: Cross-reactivity was measured by inhibition ELISA. One- and two-dimensional (2D) gel analyses combined with IgE immunoblotting and mass spectrometry [liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)] were performed to detect IgE-reactive pollen components. RESULTS: Up to 16.7% of IgE reactivity to Japanese hop was inhibited by common hop. A 12-kDa protein component of Japanese hop pollen that showed the most potent IgE reaction was absent from common hop. Six IgE-reactive components from Japanese hop were detected by 2D gel electrophoresis and LC-ESI-MS/MS, but showed low Mascot scores, preventing positive identification. CONCLUSION: No significant IgE cross-reaction was observed for Japanese and common hop pollen allergens. Development of allergy diagnostic and immunotherapeutic reagents based on Japanese hop pollen are urgently needed.
Allergens ; Asian Continental Ancestry Group ; Chromatography ; Electrophoresis, Gel, Two-Dimensional ; Enzyme-Linked Immunosorbent Assay ; Equipment and Supplies ; Far East ; Humans ; Humulus* ; Hypersensitivity ; Immunoblotting ; Immunoglobulin E* ; Indicators and Reagents ; Mass Spectrometry ; Plants ; Pollen ; Rhinitis, Allergic, Seasonal ; Tandem Mass Spectrometry

The aim of this study is to validate an integrated air monitoring approach for assessing airborne formaldehyde (FA) in the workplace. An active sampling by silica gel impregnated with 2,4-dinitrophenylhydrazine, a passive solid phase microextraction technique using O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine as on-fiber derivatization reagent, an electrochemical direct-reading monitor, and an enzyme-based badge were evaluated and tested over a range of 0.020–5.12 ppm, using dynamically generated FA air concentrations. Simple linear regression analysis showed the four methods were suitable for evaluating airborne FA. Personal and area samplings in 12 anatomy pathology departments showed that the international occupational exposure limits in the GESTIS database were frequently exceeded. This monitoring approach would allow a fast, easy-to-use, and economical evaluation of both current work practices and eventual changes made to reduce FA vapor concentrations.
Chromatography ; Formaldehyde* ; Humans ; Linear Models ; Occupational Exposure ; Pathology ; Silica Gel ; Solid Phase Microextraction

Brucella abortus is a bacterium that causes brucellosis and is the causative agent of worldwide zoonoses. Pathogenesis of the B. abortus infection is complicated, and several researchers have attempted to elucidate the infection mechanism of B. abortus. While several proteins have been revealed as pathogenic factors by previous researchers, the underlying mechanism of B. abortus infection is unresolved. In this study, we identified proteins showing different expression levels in B. abortus mutants with different biological characteristics that were generated by random insertion of a transposon. Five mutants were selected based on biological characteristics, in particular, their growth features. Total proteins of mutant and wild-type B. abortus were purified and subjected to two-dimensional gel electrophoresis. Thirty protein spots of each mutant with expression increases or decreases were selected; those with a change of more than 2-fold were compared with the wild-type. Selected spots underwent liquid chromatography tandem mass spectrometry for peptide analysis. DnaK and ClpB, involved in protein aggregation, increased. SecA and GAPDH, associated with energy metabolism, decreased in some mutants with a growth rate slower than that of the wild-type. Mutants with slower growth showed a decrease in energy metabolism-related proteins, while mutants with faster growth showed an increase in pathogenicity-related proteins.
Brucella abortus ; Brucella ; Brucellosis ; Chromatography, Liquid ; Electrophoresis, Gel, Two-Dimensional ; Energy Metabolism ; Population Characteristics ; Sequence Analysis, Protein ; Tandem Mass Spectrometry ; Zoonoses