Discovery of M Protease Inhibitors Encoded by SARS-CoV-2.

The coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a health threat worldwide. Viral main protease (Mpro, also called 3C-like protease [3CLpro]) is a therapeutic target for drug discovery. Herein, we report that GC376, a broad-spectrum inhibitor targeting Mpro in the picornavirus-like supercluster, is a potent inhibitor for the Mpro encoded by SARS-CoV-2, with a half-maximum inhibitory concentration (IC50) of 26.4 ± 1.1 nM. In this study, we also show that GC376 inhibits SARS-CoV-2 replication with a half-maximum effective concentration (EC50) of 0.91 ± 0.03 µM. Only a small portion of SARS-CoV-2 Mpro was covalently modified in the excess of GC376 as evaluated by mass spectrometry analysis, indicating that improved inhibitors are needed. Subsequently, molecular docking analysis revealed that the recognition and binding groups of GC376 within the active site of SARS-CoV-2 Mpro provide important new information for the optimization of GC376. Given that sufficient safety and efficacy data are available for GC376 as an investigational veterinary drug, expedited development of GC376, or its optimized analogues, for treatment of SARS-CoV-2 infection in human is recommended.

Development of high-growth influenza H7N9 prepandemic candidate vaccine viruses in suspension MDCK cells.

BACKGROUND: Influenza vaccine manufacturers traditionally use egg-derived candidate vaccine viruses (CVVs) to produce high-yield influenza viruses for seasonal or pandemic vaccines; however, these egg-derived CVVs need an adaptation process for the virus to grow in mammalian cells. The low yields of cell-based manufacturing systems using egg-derived CVVs remain an unsolved issue. This study aimed to develop high-growth cell-derived CVVs for MDCK cell-based vaccine manufacturing platforms. METHODS: Four H7N9 CVVs were generated in characterized Vero and adherent MDCK (aMDCK) cells. Furthermore, reassortant viruses were amplified in adherent MDCK (aMDCK) cells with certification, and their growth characteristics were detected in aMDCK cells and new suspension MDCK (sMDCK) cells. Finally, the plaque-forming ability, biosafety, and immunogenicity of H7N9 reassortant viruses were evaluated. RESULTS: The HA titers of these CVVs produced in proprietary suspension MDCK (sMDCK) cells and chicken embryos were 2- to 8-fold higher than those in aMDCK cells. All H7N9 CVVs showed attenuated characteristics by trypsin-dependent plaque assay and chicken embryo lethality test. The alum-adjuvanted NHRI-RG5 (derived from the fifth wave H7N9 virus A/Guangdong/SP440/2017) vaccine had the highest immunogenicity and cross-reactivity among the four H7N9 CVVs. Finally, we found that AddaVax adjuvant improved the cross-reactivity of low pathogenic H7N9 virus against highly pathogenic H7N9 viruses. CONCLUSIONS: Our study indicates that cell-derived H7N9 CVVs possessed high growth rate in new sMDCK cells and low pathogenicity in chicken embryo, and that CVVs generated by this platform are also suitable for both cell- and egg-based prepandemic vaccine production.

Evaluation of disulfide scrambling during the enzymatic digestion of bevacizumab at various pH values using mass spectrometry.

Disulfide linkages play an important role in protein stability and activity. Thus, it is critical to characterize disulfide bonds to ensure the quality and function of protein pharmaceuticals. There are, however, problems associated with maintaining disulfide linkages in the conventional procedures that are used to digest a protein. In order to preserve enzyme activity during the digestion of a protein, it is commonly carried out at neutral to basic environment which increases the possibilities of disulfide bond scrambling. However, it is not easy to differentiate whether the scrambled disulfide linkages are initiated by the sample itself or whether they are induced during the protease digestion process. In this study, the optimum pH for minimizing disulfide bond rearrangements during the digestion process was determined. Three sets of proteases, trypsin plus Glu-C, Lys-C and thermolysin were used, followed by dimethyl labeling and mass spectrometry for a bevacizumab (Avastin) disulfide linkage analysis. No disulfide linkage scrambling was detected at pH6 when Lys-C or trypsin plus Glu-C were used as enzymes. When thermolysin was applied, some scrambled disulfide bonds were identified at pH5, 6 and 7. Nevertheless, there was less disulfide bond scrambling at a lower pH. All correct disulfide bonds on bevacizumab could be identified using this approach. The results demonstrated that by choosing the proper enzymes, using a lower pH environment for the digestion could reduce the degree of artifact disulfide scrambling.

A biphasic response pattern of lipid metabolomics in the stage progression of hepatitis B virus X tumorigenesis.

Metabolic syndrome has closely linked to the development of human hepatocellular carcinoma (HCC). By using the hepatitis B virus (HBV) X (HBx) transgenic mouse model, we studied the dynamic evolution of serum and liver profiles of lipids and global cDNA expression at different stages of HBx tumorigenesis. We observed that the lipid (triglycerides, cholesterol, and fatty acids) profiles revealed a biphasic response pattern during the progression of HBx tumorigenesis: a small peak at early phase and a large peak or terminal switch at the tumor phase. By analyzing cDNA microarray data, the early peak correlated to the oxidative stress and pro-inflammatory response, which then resolved at the middle phase and were followed by the terminal metabolic switch in the tumor tissues. Five lipid metabolism-related genes, the arachidonate 5-lipoxygenase, lipoprotein lipase, fatty acid binding protein 4, 1-acylglycerol-3-phosphate O-acyltransferase 9, and apolipoprotein A-IV were identified to be significantly activated in HBx transgenic HCCs and further validated in human HBV-related HCCs. Inhibition of these lipid genes could reverse the effect of HBx on lipid biosynthesis and suppress HBx-induced cell proliferation in vitro. Our results support the concept that metabolic syndrome plays an important role in HBV tumorigenesis. The dysregulation of lipid metabolic genes may predict the disease progression to HCC in chronic hepatitis B patients.

Acinetobacter baumannii Extracellular OXA-58 Is Primarily and Selectively Released via Outer Membrane Vesicles after Sec-Dependent Periplasmic Translocation.

Carbapenem-resistant Acinetobacter baumannii (CRAb) shelter cohabiting carbapenem-susceptible bacteria from carbapenem killing via extracellular release of carbapenem-hydrolyzing class D ß-lactamases, including OXA-58. However, the mechanism of the extracellular release of OXA-58 has not been elucidated. In silico analysis predicted OXA-58 to be translocated to the periplasm via the Sec system. Using cell fractionation and Western blotting, OXA-58 with the signal peptide and C terminus deleted was not detected in the periplasmic and extracellular fractions. Overexpression of enhanced green fluorescent protein fused to the OXA-58 signal peptide led to its periplasmic translocation but not extracellular release, suggesting that OXA-58 is selectively released. The majority of the extracellular OXA-58 was associated with outer membrane vesicles (OMVs). The OMV-associated OXA-58 was detected only in a strain overexpressing OXA-58. The presence of OXA-58 in OMVs was confirmed by a carbapenem inactivation bioassay, proteomic analysis, and transmission electron microscopy. Imipenem treatment increased OMV formation and caused cell lysis, resulting in an increase in the OMV-associated and OMV-independent release of extracellular OXA-58. OMV-independent OXA-58 hydrolyzed nitrocefin more rapidly than OMV-associated OXA-58 but was more susceptible to proteinase K degradation. Rose bengal, an SecA inhibitor, inhibited the periplasmic translocation and OMV-associated release of OXA-58 and abolished the sheltering effect of CRAb. This study demonstrated that the majority of the extracellular OXA-58 is selectively released via OMVs after Sec-dependent periplasmic translocation. Addition of imipenem increased both OMV-associated and OMV-independent OXA-58, which may have different biological roles. SecA inhibitor could abolish the carbapenem-sheltering effect of CRAb.

Global disulfide bond profiling for crude snake venom using dimethyl labeling coupled with mass spectrometry and RADAR algorithm.

Snake venom consists of toxin proteins with multiple disulfide linkages to generate unique structures and biological functions. Determination of these cysteine connections usually requires the purification of each protein followed by structural analysis. In this study, dimethyl labeling coupled with LC-MS/MS and RADAR algorithm was developed to identify the disulfide bonds in crude snake venom. Without any protein separation, the disulfide linkages of several cytotoxins and PLA2 could be solved, including more than 20 disulfide bonds. The results show that this method is capable of analyzing protein mixture. In addition, the approach was also used to compare native cytotoxin 3 (CTX III) and its scrambled isomer, another category of protein mixture, for unknown disulfide bonds. Two disulfide-linked peptides were observed in the native CTX III, and 10 in its scrambled form, X-CTX III. This is the first study that reports a platform for the global cysteine connection analysis on a protein mixture. The proposed method is simple and automatic, offering an efficient tool for structural and functional studies of venom proteins.

Insect Cell-Based Quadrivalent Seasonal Influenza Virus-like Particles Vaccine Elicits Potent Immune Responses in Mice.

Influenza viruses can cause highly infectious respiratory diseases, posing noteworthy epidemic and pandemic threats. Vaccination is the most cost-effective intervention to prevent influenza and its complications. However, reliance on embryonic chicken eggs for commercial influenza vaccine production presents potential risks, including reductions in efficacy due to HA gene mutations and supply delays due to scalability challenges. Thus, alternative platforms are needed urgently to replace egg-based methods and efficiently meet the increasing demand for vaccines. In this study, we employed a baculovirus expression vector system to engineer HA, NA, and M1 genes from seasonal influenza strains A/H1N1, A/H3N2, B/Yamagata, and B/Victoria, generating virus-like particle (VLP) vaccine antigens, H1N1-VLP, H3N2-VLP, Yamagata-VLP, and Victoria-VLP. We then assessed their functional and antigenic characteristics, including hemagglutination assay, protein composition, morphology, stability, and immunogenicity. We found that recombinant VLPs displayed functional activity, resembling influenza virions in morphology and size while maintaining structural integrity. Comparative immunogenicity assessments in mice showed that our quadrivalent VLPs were consistent in inducing hemagglutination inhibition and neutralizing antibody titers against homologous viruses compared to both commercial recombinant HA and egg-based vaccines (Vaxigrip). The findings highlight insect cell-based VLP vaccines as promising candidates for quadrivalent seasonal influenza vaccines. Further studies are worth conducting.

MDCK-Adaptive Mutation of A169S Changes Glycosylation Pattern of Hemagglutinin and Enhances MDCK-Based H7N9 Vaccine Virus Production without Loss of Antigenicity and Immunogenicity.

The adaptation of egg-derived H7N9 candidate vaccine virus (CVV) in the mammalian cell line is an approach to developing a high-growth virus strain for the mass production of vaccine manufacturing. The adaptive mutations that occur in hemagglutinin (HA) are critical to the activity and potency of the vaccine virus. Previously, we identified a new mutation of A169S in the HA protein of an MDCK-adapted H7N9 vaccine virus (A/Anhui/2013, RG268); however, whether and how this mutation affects vaccine potency remain to be investigated. In this study, we serially passaged RG268 in MDCK cells and found that the HA titer and the TCID50 of the passaged virus RG268-M5 were 4-fold (HA units/50 µL) and 3.5-fold (log10 TCID50/mL) higher than those of the original CVV. By inspecting tandem MS spectra, we identified a new glycosylation site at N167 near the receptor binding site of the HA protein of RG268-M5. Flow cytometry results revealed that RG268-M5 could efficiently infect MDCK cells and initiate viral protein replication as well as that of RG268. Though the new glycosylation site is in the antigenic epitope of viral HA protein, the HI assay result indicated that the antigenicity of RG268-M5 was similar to RG268. Additionally, immunizing mice with RG268-M5 mixed aluminum hydroxide could induce potent antibody responses against the homologous and heterologous H7N9 viruses in vitro whereas the titers were comparable with those from the RG268 group. These results provide in-depth structural information regarding the effects of site-specific glycosylation on virus properties, which have implications for novel avian influenza vaccine development.

Subunit vaccines with a saponin-based adjuvant boost humoral and cellular immunity to MERS coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) outbreaks have constituted a public health issue with drastic mortality higher than 34%, necessitating the development of an effective vaccine. During MERS-CoV infection, the trimeric spike protein on the viral envelope is primarily responsible for attachment to host cellular receptor, dipeptidyl peptidase 4 (DPP4). With the goal of generating a protein-based prophylactic, we designed a subunit vaccine comprising the recombinant S1 protein with a trimerization motif (S1-Fd) and examined its immunogenicity and protective immune responses in combination with various adjuvants. We found that sera from immunized wild-type and human DPP4 transgenic mice contained S1-specific antibodies that can neutralize MERS-CoV infection in susceptible cells. Vaccination with S1-Fd protein in combination with a saponin-based QS-21 adjuvant provided long-term humoral as well as cellular immunity in mice. Our findings highlight the significance of the trimeric S1 protein in the development of MERS-CoV vaccines and offer a suitable adjuvant, QS-21, to induce robust and prolonged memory T cell response.

Automatic disulfide bond assignment using a1 ion screening by mass spectrometry for structural characterization of protein pharmaceuticals.

An automatic method for disulfide bond assignment using dimethyl labeling and computational screening of a(1) ions with customized software, RADAR, is developed. By utilization of the enhanced a(1) ions generated from labeled peptides, the N-terminal amino acids from disulfide-linked peptides can be determined. In this study, we applied this method for structural characterization of recombinant monoclonal antibodies, an important group of therapeutic proteins. In addition to a(1) ion screening and molecular weight match, new RADAR is capable of confirming the matched peptide pairs by further comparing the collision-induced dissociation (CID) fragment ions. With the N-terminal amino acid identities as a threshold, the identification of disulfide-linked peptide pairs can be achieved rapidly at a higher confidence level. Unlike most current approaches, prior knowledge of disulfide linkages or a high-end mass spectrometer is not required, and tedious work or deliberate interpretation can be avoided in this study. Our approach makes it possible to analyze unknown disulfide bonds of protein pharmaceuticals as well as their degraded forms without further protein separation. It can be used as a convenient quality examination tool during biopharmaceutical development and manufacturing processes.

Cytotoxicity of Venoms and Cytotoxins from Asiatic Cobras (Naja kaouthia, Naja sumatrana, Naja atra) and Neutralization by Antivenoms from Thailand, Vietnam, and Taiwan.

Envenoming by cobras (Naja spp.) often results in extensive local tissue necrosis when optimal treatment with antivenom is not available. This study investigated the cytotoxicity of venoms and purified cytotoxins from the Monocled Cobra (Naja kaouthia), Taiwan Cobra (Naja atra), and Equatorial Spitting Cobra (Naja sumatrana) in a mouse fibroblast cell line, followed by neutralization of the cytotoxicity by three regional antivenoms: the Thai Naja kaouthia monovalent antivenom (NkMAV), Vietnamese snake antivenom (SAV) and Taiwanese Neuro bivalent antivenom (NBAV). The cytotoxins of N. atra (NA-CTX) and N. sumatrana (NS-CTX) were identified as P-type cytotoxins, whereas that of N. kaouthia (NK-CTX) is S-type. All venoms and purified cytotoxins demonstrated varying concentration-dependent cytotoxicity in the following trend: highest for N. atra, followed by N. sumatrana and N. kaouthia. The antivenoms moderately neutralized the cytotoxicity of N. kaouthia venom but were weak against N. atra and N. sumatrana venom cytotoxicity. The neutralization potencies of the antivenoms against the cytotoxins were varied and generally low across NA-CTX, NS-CTX, and NK-CTX, possibly attributed to limited antigenicity of CTXs and/or different formulation of antivenom products. The study underscores the need for antivenom improvement and/or new therapies in treating local tissue toxicity caused by cobra envenomings.

Local Cytotoxic Effects in Cobra Envenoming: A Pilot Study.

The cobra (genus Naja (N.)) is one of the most common venomous snakes. Due to its frequency and deadly complications of muscle paralysis, local necrosis, and chronic musculoskeletal disability, it should not be ignored. The pathology of devastating tissue destruction, even though specific antivenoms exist, is not fully clear. Here, we attempted to dig in envenomed tissues to study the clinical toxicology of cobra venom. Four cases of N. atra snake envenomation, in which the subjects developed advanced tissue injury, were involved in this study. We used enzyme-ligand sandwich immunoassay (ELISA) to assay the whole venom, cytotoxin A3 and short-chain neurotoxin (sNTX) in blood, bullae, wound discharge, and debrided tissue. We found that persistently high concentrations of venom and toxins, especially cytotoxin A3, were detected in bullae, wound discharge fluid and necrotic tissue of these patients even after large doses of specific antivenom treatment, and wide excision and advanced debridement could largely remove these toxins, lessen the size of necrosis, and promote wound healing. We also found that the point-of-care apparatus, ICT-Cobra kit, might be used to promptly monitor the wound condition and as one of the indicators of surgical intervention in cases of cobra envenomation in Taiwan.

Characterization and Immunogenicity of Influenza H7N9 Vaccine Antigens Produced Using a Serum-Free Suspension MDCK Cell-Based Platform.

Human infections with avian-origin H7N9 influenza A viruses were first reported in China, and an approximately 38% human mortality rate was described across six waves from February 2013 to September 2018. Vaccination is one of the most cost-effective ways to reduce morbidity and mortality during influenza epidemics and pandemics. Egg-based platforms for the production of influenza vaccines are labor-intensive and unable to meet the surging demand during pandemics. Therefore, cell culture-based technology is becoming the alternative strategy for producing influenza vaccines. The current influenza H7N9 vaccine virus (NIBRG-268), a reassortant virus from A/Anhui/1/2013 (H7N9) and egg-adapted A/PR/8/34 (H1N1) viruses, could grow efficiently in embryonated eggs but not mammalian cells. Moreover, a freezing-dry formulation of influenza H7N9 vaccines with long-term stability will be desirable for pandemic preparedness, as the occurrence of influenza H7N9 pandemics is not predictable. In this study, we adapted a serum-free anchorage-independent suspension Madin-Darby Canine Kidney (MDCK) cell line for producing influenza H7N9 vaccines and compared the biochemical characteristics and immunogenicity of three influenza H7N9 vaccine antigens produced using the suspension MDCK cell-based platform without freeze-drying (S-WO-H7N9), the suspension MDCK cell-based platform with freeze-drying (S-W-H7N9) or the egg-based platform with freeze-drying (E-W-H7N9). We demonstrated these three vaccine antigens have comparable biochemical characteristics. In addition, these three vaccine antigens induced robust and comparable neutralizing antibody (NT; geometric mean between 1016 and 4064) and hemagglutinin-inhibition antibody (HI; geometric mean between 640 and 1613) titers in mice. In conclusion, the serum-free suspension MDCK cell-derived freeze-dried influenza H7N9 vaccine is highly immunogenic in mice, and clinical development is warranted.

Rapid isolation and characterization of bacterial lipopeptides using liquid chromatography and mass spectrometry analysis.

The structural analysis of post-translational modifications (PTMs) of lipoproteins is difficult due to the hydrophobic properties of their fatty acid moieties. At the present time, the relative positions of fatty acid components on the N-acyl-S-diacylglycerylcysteine core structure has not been specifically identified in any natural or bacterial expressed recombinant lipoproteins. In this study, we describe a rapid solid-phase extraction using acetonitrile and isopropanol method that can be performed manually to isolate large amounts of relatively pure lipopeptides generated by the limited tryptic-digestion of recombinant lipoproteins. Using these lipopeptides and LC/MS mass spectra analysis, two groups of N-terminal lipidated (diacyl or triacyl) molecules that differ by one fatty acid unit were successfully identified. This LC/MS method also provided the separation of lipopeptides differing by 14 Da for the on-line MS identification. Multiple-stage fragmentation analyses of the di- and triacyl lipopeptides using both the positive and negative ion modes enabled to identify the putative structure of the N-acyl-S-diacylglycerylcysteine containing an amide bond to palmitic acid at the N-terminal cysteine, a palmitic acid at sn1 position, and an unsaturated fatty acid of either hexadecenoic acid, cyclopropaneoctanoic acid, oleic acid and nonadecenoic acid at sn2 position of diacylglycerol residue through ester bonding. For diacyl lipoprotein, the saturated palmitoyl fatty acid group is absent at sn1 position of glycerol-derived lipid residue of lipopeptide.

Development of a Broad-Spectrum Antiserum against Cobra Venoms Using Recombinant Three-Finger Toxins.

Three-finger toxins (3FTXs) are the most clinically relevant components in cobra (genus Naja) venoms. Administration of the antivenom is the recommended treatment for the snakebite envenomings, while the efficacy to cross-neutralize the different cobra species is typically limited, which is presumably due to intra-specific variation of the 3FTXs composition in cobra venoms. Targeting the clinically relevant venom components has been considered as an important factor for novel antivenom design. Here, we used the recombinant type of long-chain α-neurotoxins (P01391), short-chain α-neurotoxins (P60770), and cardiotoxin A3 (P60301) to generate a new immunogen formulation and investigated the potency of the resulting antiserum against the venom lethality of three medially important cobras in Asia, including the Thai monocled cobra (Naja kaouthia), the Taiwan cobra (Naja atra), and the Thai spitting cobra (Naja Siamensis) snake species. With the fusion of protein disulfide isomerase and the low-temperature settings, the correct disulfide bonds were built on these recombinant 3FTXs (r3FTXs), which were confirmed by the circular dichroism spectra and tandem mass spectrometry. Immunization with r3FTX was able to induce the specific antibody response to the native 3FTXs in cobra venoms. Furthermore, the horse and rabbit antiserum raised by the r3FTX mixture is able to neutralize the venom lethality of the selected three medically important cobras. Thus, the study demonstrated that the r3FTXs are potential immunogens in the development of novel antivenom with broad neutralization activity for the therapeutic treatment of victims involving cobra snakes in countries.

Functional fluorinated modifications on a polyelectrolyte coated polydimethylsiloxane substrate for fabricating antibody microarrays.

Fluorinated compounds exhibit hydrophobic, nonstick, and self-cleaning properties, making them attractive for use as the coating material for biochips. In this study, we copolymerized the fluorinated compound 1H,1H,2H-perfluoro-1-decene (FD) with acrylic acid (AA) and bonded the resulting copolymer with protein G on the surface of polyelectrolyte coated polydimethylsiloxane (PDMS) to form a functional surface that captures antibodies. We demonstrated that the modified PDMS surface remained hydrophobic while becoming resistant to nonspecific protein binding. Thus, aqueous sample solutions formed the droplets (4 µL) on the surface without spreading and drying during the sample printing. Contact angle measurements showed that this functionalized surface was as hydrophobic as the native PDMS with a virtually constant contact angle over seven days of the study under dried condition at 4 °C. Spectroscopic measurements revealed that FD/AA copolymerization formed a homogeneous and highly dense multilayer (50 mg/mm(2)) with a fluorine coverage of 35.4%. Moreover, protein G was shown to covalently bind to AA molecules on the surface at a binding density of 0.24 µg/mm(2). We demonstrated that the fluorinated coating withstood nonspecific binding with extremely low background emission, leading to bioassays that, without the need of blocking agents, exhibited six times more sensitivity than PEG coatings. The fluorinated PDMS antibody microarrays were further applied to accurately determine the absolute concentration of ERα in MCF-7 cells. In conclusion, the unique properties of fluorinated compounds, such as withstanding wetting, nonspecific binding and contamination, make them an excellent coating material for use in sensitive and simple on-chip assays.

A Rapid and International Applicable Diagnostic Device for Cobra (Genus Naja) Snakebites.

Cobra snakes (genus Naja) are some of the most dangerous snake species in Asia and Africa, as their bites cause severe life-threatening respiratory failure and local tissue destruction, especially in the case of late diagnosis. The differential diagnosis of snakebite envenomation still mainly relies upon symptomatology, the patient's description, and the experience of physicians. We have designed a rapid test, immunochromatographic test of cobra (ICT-Cobra), which obtained fair results in improving the diagnosis and treatment of Naja (N.) atra snakebites in Taiwan. In this study, we further investigated the feasibility of applying the kit for the detection of other cobra venoms based on the potential interspecies similarity. We firstly demonstrated the cross-reactivity between eight venoms of medically important cobra species and the rabbit anti-N. atra IgG that was used in ICT-Cobra by Western blotting and sandwich enzyme-linked immunosorbent assay. Then, ICT-Cobra was used to detect various concentrations of the eight venoms to elucidate its performance. Noticeable correlations between the cross-reactivity of venoms from genus Naja snakes and existing geographical characteristics were found. ICT-Cobra could detect venoms from other Asian cobras with variable detection limits comparable to those observed for N. atra, but the kit was less successful in the detection of venom from African cobras. The similar but slightly different venom components and the interaction between venom and rabbit anti-N. atra IgG led to variations in the detection limits. The transcontinental usage of ICT-Cobra might be possible due to the cross-reactivity of antibodies and similarities among the larger-sized proteins. This study showed that the close immunological relationships in the genus Naja could be used to develop a venom detection kit for the diagnosis of cobra envenomation in both Asian and African regions. Additional clinical studies and technical adjustments are still needed to improve the efficacy and broadening the application of ICT-Cobra in the future.

Development and characterization of standard reagents for cell-based prepandemic influenza vaccine products.

Outbreaks of infection by novel avian influenza virus strains in humans cause public health issues worldwide, and the development of vaccines against such novel strains is the most effective method for the prevention of these virus outbreaks. All types of vaccines must be tested for potency before use; thus, quantitative potency assays are needed for influenza vaccines. The single radial immunodiffusion (SRID) assay is considered the gold standard for quantification of influenza virus antigens, and the SRID reference reagents are essential for the determination of vaccine potency. However, it remains debatable whether reference reagents derived from egg-based vaccine platforms can be used to precisely quantify non-egg-derived vaccines; thus, influenza vaccine production using cell-based platforms has attracted increasing attention. To evaluate the utility of reference reagents derived from a cell-based influenza vaccine platform, we prepared cell-based reference reagents from MDCK cell-grown viruses and compared them with egg-derived reference reagents. A primary liquid standard (PLS) was purified from cell-derived candidate influenza vaccine viruses, and hemagglutinin (HA) antigen content was determined by a densitometric method. The produced PLS could be stored at 4°C for more than 10 months. We also established a simple HA protein purification method for goat antiserum preparation, and the performance of the resulting antiserum was compared to that of standard reagents obtained using different production platforms. The results of this study indicate that these reference reagents can be used for both cell-based and egg-based production platforms and that the differences between these two types of platforms are negligible.

Heme protein assisted dispersion of gold nanoparticle multilayers on chips: from stabilization to high-density double-stranded DNAs fabricated in situ for protein/DNA binding.

Heme proteins in general are shown to be an effective linking agent in stabilizing gold nanoparticles (AuNPs) and thus facilitate the fabrication of three-dimensional (3D) AuNP multilayers on a chip, resulting in a higher coating density than that on polymer linker anchored surfaces for analytical applications. With the use of electron spectroscopy for chemical analysis (ESCA) measurements, a lower oxidation state of Au(0) and dramatic changes among multiple chemical states of N1s are detected upon coating AuNPs with heme proteins but not detected upon coating AuNPs with non-heme proteins. Thus, we propose that the stabilization power arises from pi-conjugation between AuNPs and the heme group. We also propose that such conjugation must be facilitated by the exposure of the heme group through a conformational change of the protein as well as interactions of other functional groups with AuNPs to bring the heme moiety to a close face-to-face distance with the AuNPs. A high-density double-stranded DNA (dsDNA) composed of a sequence of estrogen response element (ERE) is then fabricated on heme protein anchored chips. An in situ hybridization and tracking method is developed based on hybridization-induced fluorescence restoration associated with AuNPs and assists in the subsequent detection of DNA/protein binding on the same chip. The AuNP ERE chips are shown to have high sensitivity and specificity for quantitative detection of ERE binding with its two transcription factor isoforms, estrogen receptors alpha and beta (ERalpha and ERbeta), in cell lysates with reduced reagents and reaction time.

Functionalized 3D-hydrogel plugs covalently patterned inside hydrophilic poly(dimethylsiloxane) microchannels for flow-through immunoassays.

Integration of a hydrogel and polydimethylsiloxane (PDMS)-based microfluidic device can greatly reduce the cost of developing channel-based devices. However, there are technical difficulties including the hydrophobic and inert surface properties associated with PDMS as well as back pressure and fragile material associated with the use of hydrogel in microchannels. In this study, a strategy to covalently photopattern 3-D hydrogel plugs with functionalized protein G inside microfluidic channels on a hydrophilic PDMS substrate coated with polyelectrolyte multilayers (PEMS) is presented. In this process, a UV-light microscope is applied to initiate the protein G-poly(acryl amide) copolymerization from the bulk substrate to solution areas via the deeply implanted photoinitiator (PI), resulting in sturdy 3D plugs covalently bonded to the upper and lower channel wall, while leaving open spaces in the channel width for the fluid to flow through. In addition, the long-term hydrophilicity and low nonspecific binding property associated with PEMS surface can be conserved for the nonpatterned area, leading to hydrogel plugs in extremely hydrophilic and permeable environment in a restricted channel space for bubble-free fluid transport and affinity interaction. By immobilization of well-oriented antibodies via protein G on the hydrogel plugs in the channel, estrogen receptor alpha (ERalpha) is demonstrated to be captured quantitatively with high loading capacity and high specificity.