Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used.
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.
22 Related JoVE Articles!
High-throughput Detection Method for Influenza Virus
Institutions: Blood Research Institute, Mount Sinai School of Medicine , Blood Research Institute, City of Milwaukee Health Department Laboratory, Medical College of Wisconsin , Medical College of Wisconsin .
Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3
to test the efficacy of this technique using MDCK cells 4
. MDCK cells (104
or 5 x 103
per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5
and hemagglutinin 1
are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102
PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102
PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.
Immunology, Issue 60, Influenza virus, Virus titer, Epithelial cells
Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats
Institutions: University of California, Irvine (UCI).
Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin (KLH).
Here we demonstrate how to induce an active DTH reaction in Lewis rats. We will first prepare a water-in-oil emulsion of KLH, our antigen of interest, in complete Freund's adjuvant and inject this emulsion subcutaneously to rats. This will prime the immune system to develop memory T cells directed to KLH. Seven days later we will challenge the rats intradermally on the back with KLH on one side and with ovalbumin, an irrelevant antigen, on the other side. The inflammatory reaction will be visible 16-72 hours later and the red and swollen area will be measured as an indication of DTH severity.
Cell Biology, Issue 6, Immunology, Immune Response, Inflammation, lymphocyte, inflammatory reaction, skin test, video protocol
Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding
Institutions: School of Medicine, New York University.
The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' surface envelope glycoprotein. We previously proposed that the crown of the V3 loop, although dynamic and sequence variable, is constrained throughout the population of HIV-1 viruses to an immunologically relevant β-hairpin tertiary structure. Importantly, there are thousands of different V3 loop crown sequences in circulating HIV-1 viruses, making 3D structural characterization of trends across the diversity of viruses difficult or impossible by crystallography or NMR. Our previous successful studies with folding of the V3 crown1, 2
used the ab initio
accessible in the ICM-Pro molecular modeling software package (Molsoft LLC, La Jolla, CA) and suggested that the crown of the V3 loop, specifically from positions 10 to 22, benefits sufficiently from the flexibility and length of its flanking stems to behave to a large degree as if it were an unconstrained peptide freely folding in solution. As such, rapid ab initio
folding of just this portion of the V3 loop of any individual strain of the 60,000+ circulating HIV-1 strains can be informative. Here, we folded the V3 loop of the R2 strain to gain insight into the structural basis of its unique properties. R2 bears a rare V3 loop sequence thought to be responsible for the exquisite sensitivity of this strain to neutralization by patient sera and monoclonal antibodies4, 5
. The strain mediates CD4-independent infection and appears to elicit broadly neutralizing antibodies. We demonstrate how evaluation of the results of the folding can be informative for associating observed structures in the folding with the immunological activities observed for R2.
Infection, Issue 43, HIV-1, structure-activity relationships, ab initio simulations, antibody-mediated neutralization, vaccine design
High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots
Institutions: Akonni Biosystems, Inc., Akonni Biosystems, Inc., Akonni Biosystems, Inc., Akonni Biosystems, Inc..
TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).
Genetics, Issue 76, Bioengineering, Biomedical Engineering, Molecular Biology, Automation, Laboratory, Clinical Laboratory Techniques, Molecular Diagnostic Techniques, Analytic Sample Preparation Methods, Clinical Laboratory Techniques, Molecular Diagnostic Techniques, Genetic Techniques, Molecular Diagnostic Techniques, Automation, Laboratory, Chemistry, Clinical, DNA/RNA extraction, automation, nucleic acid isolation, sample preparation, nasopharyngeal aspirate, blood, plasma, high-throughput, sequencing
Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System
Institutions: Icahn School of Medicine at Mount Sinai, Icahn School of Medicine at Mount Sinai, Icahn School of Medicine at Mount Sinai.
The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.
Infection, Issue 81, Influenza A virus, Orthomyxoviridae Infections, Influenza, Human, Influenza in Birds, Influenza Vaccines, hemagglutinin, neuraminidase, H7N9, baculovirus, insect cells, recombinant protein expression
Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems
Institutions: George Mason University.
Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional overlays for certain heat labile viruses.
Virology, Issue 93, Plaque Assay, Virology, Viral Quantification, Cellular Overlays, Agarose, Avicel, Crystal Violet Staining, Serial Dilutions, Rift Valley fever virus, Venezuelan Equine Encephalitis, Influenza
Generation of Recombinant Influenza Virus from Plasmid DNA
Institutions: University of Rochester School of Medicine and Dentistry, Mount Sinai School of Medicine .
Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2
plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3
Microbiology, Issue 42, influenza viruses, plasmid transfection, recombinant virus, reverse genetics techniques, HA assay
ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza
DNA microarrays have emerged as a powerful tool for pathogen detection.1-5
For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11
The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1
replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11
, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is ˜1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.
Immunology, Issue 52, microarrays, colorimetric detection, ampliPHOX, diagnostic, low-density, pathogen detection, influenza
Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
Institutions: New York University School of Medicine, Mount Sinai Medical Center, Mount Sinai Medical Center.
Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2
. Interleukin-1β is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1β secretion6
. Pro-IL-1β protein expression is limited in resting cells; therefore a priming signal is required for IL-1β transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1β secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells (moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro
human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.
Immunology, Issue 87, NLRP3, inflammasome, IL-1beta, Interleukin-1 beta, dendritic, cell, Nigericin, Toll-Like Receptor 8, TLR8, R848, Monocyte Derived Dendritic Cells
Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface
Institutions: The University of North Carolina at Chapel Hill, The University of North Carolina at Chapel Hill, The University of North Carolina at Chapel Hill, The University of North Carolina at Chapel Hill.
models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens.
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc.
models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo
Cellular Biology, Issue 80, Epithelium, Cell culture models, ciliated, air pollution, co-culture models, nasal epithelium
A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example
Institutions: Vrije Universiteit Brussel.
In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1
. Since poliovirus is sensitive to conformational conversion2
when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4
is the micro protein A-immunoprecipitation test5
. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7
. However, as another opportunity, these interesting and stable single-domain antibodies8
can be easily engineered with different tags. The widely used (His)6
-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35
S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.
Molecular Biology, Issue 63, Virology, Poliovirus, VHH, nanobody, magnetic beads, affinity capture, liquid phase based assay, protein interaction
Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge
Institutions: St. John-Providence Health System, Wayne State University School of Medicine.
Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b
) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 μg/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 μg/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.
Immunology, Issue 62, Autoimmune diseases, experimental autoimmune encephalomyelitis, immunization, myelin basic protein, adoptive transfer, paralysis
An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium
Institutions: University of Georgia.
Current commercial PCRs tests for identifying Salmonella
target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella
serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies
IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica
subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.
We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica
subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella
serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella
serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica
subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.
Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella
Immunology, Issue 53, PCR, Salmonella, multiplex, Serovar
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana
plants with Agrobacteria
carrying launch vectors. Optimization of Agrobacterium
cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana
, N. excelsiana
× N. excelsior
) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium
harboring pBID4-GFP (Tobacco mosaic virus
-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium
laboratory strain GV3101 showed the highest protein production compared to Agrobacteria
laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria
strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana
resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Plant Biology, Issue 86, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
Perform plating procedures without contaminating media.
Isolate single bacterial colonies by the streak-plating method.
Use pour-plating and spread-plating methods to determine the concentration of bacteria.
Perform soft agar overlays when working with phage.
Transfer bacterial cells from one plate to another using the replica-plating procedure.
Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
Test Samples for Optimizing STORM Super-Resolution Microscopy
Institutions: National Physical Laboratory.
STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.
Molecular Biology, Issue 79, Genetics, Bioengineering, Biomedical Engineering, Biophysics, Basic Protocols, HeLa Cells, Actin Cytoskeleton, Coated Vesicles, Receptor, Epidermal Growth Factor, Actins, Fluorescence, Endocytosis, Microscopy, STORM, super-resolution microscopy, nanoscopy, cell biology, fluorescence microscopy, test samples, resolution, actin filaments, fiducial markers, epidermal growth factor, cell, imaging
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data
Institutions: Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory.
Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g.
, signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation.
The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.
Bioengineering, Issue 90, 3D electron microscopy, feature extraction, segmentation, image analysis, reconstruction, manual tracing, thresholding
Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice
Institutions: University of Melbourne, Radboud University Nijmegen Medical Centre, The Walter and Eliza Hall Institute for Medical Research.
During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae
(the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2
). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae
infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae
. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo
Infection, Issue 50, Bioluminescent imaging, influenza A virus, Streptococcus pneumoniae, mice, intranasal infection, otitis media, co-infections
Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling
Institutions: University of California, Los Angeles.
Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.
Cellular Biology, Issue 5, mosquito, malaria, popuulation, replacement, modeling, infectious disease
Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats
Institutions: University of California, Irvine (UCI).
Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.
Immunology, Issue 8, Rodent, Hypersensitivity, Mouse, Skin, Immune Reaction, Blood Draw, Serum, Video Protocol, Vaccination, Adjuvant