Biochemistry

Oxidative stress is associated with numerous diseases, and markers of oxidative stress in biological material are becoming a mainstay of both experimental and clinical/epidemiological research. Lipid peroxidation is a major form of oxidative stress, but due to their rapid degradation and instability, lipid peroxides are notoriously difficult to measure, particularly in biological specimens where their production and removal are continuously occuring. Thus, a commonly used surrogate marker of lipid peroxidation is protein adducts of 4-Hydroxynonenal (HNE), an α, β-unsaturated hydroxyalkenal (i.e., a reactive aldehyde) formed via degradation of oxidized polyunsaturated fatty acids (PUFAs). HNE adducts can be measured via commercially-available immunosorbent assays, but these have their limitations due to excessive costs, and reproducibility among laboratories is challenging due to variability in assay sensitivity, procedure, and reagents. Here we present a reproducible, facile, and economically conservative protocol for quantifying HNE protein adducts. The key to this protocol is to generate HNE-adduct standards by incubating bovine serum albumin (BSA) with HNE. These standards are then adsorbed to immunsorbent plastic in a multi-well plate format alongside biological samples. An enzyme-linked immunosorbent assay (ELISA) is then performed on the multi-well plate using commercially-available primary and secondary antibodies, and a peroxide-based fluorescent developing reagent. This protocol is highly sensitive and offers advantages to commercial sources in that it allows for reproducible, high-throughput quantitation of HNE adducts in a large number of samples.

Rice yellow mottle virus (RYMV), a mechanically transmitted virus that causes serious damage to cultivated rice plants, is endemic to Africa. Varietal selection for resistance is considered to be the most effective and sustainable management strategy. Standardized resistance evaluation procedures are required for the identification and characterization of resistance sources. This paper describes a protocol for mechanical inoculation of rice seedlings with RYMV and two methods of resistance evaluation – one based on a symptom severity index and the other on virus detection through double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA).

The Ribo-ELISA was originally developed to elucidate the basis for the ribopuromycylation method (RPM)-based detection of ribosome bound nascent chains. The Ribo-ELISA enables characterization of the translational status of ribosomes, and can be applied to the discovery of super-ribosomal complexes with novel ribosome associated macromolecules that are isolated by physical fractionation in sucrose gradients or other methods.

Drug-induced mitochondrial injury can be caused by many different mechanisms including inhibition of mitochondrial DNA replication, transcription, translation, and altered protein function. Determination of the level of mitochondrial protein synthesis, or mitochondrial biogenesis, relative to the cellular protein synthesis, provides important information on potential mitochondrial toxicity.

Lipopolysaccharide is the major constituent of the outer membrane of gram-negative bacteria and, once released from the bacterial surface into the bloodstream, is a potent activator of the host immune system, which can lead to septic shock. LPS has a hydrophilic region consisting of a repeating oligosaccharide that is strain-specific (O-antigen) and a core polysaccharide, which is covalently linked to a hydrophobic lipid moiety (lipid A). Lipid A is the most conserved part and is responsible for the toxicity of LPS. Therefore, finding molecules able to bind to this region and neutralize LPS toxicity is of relevant interest as it may provide new therapies to prevent septic shock (Chen et al., 2006). Several proteins and peptides were reported to bind LPS and alter its toxicity towards reduction and even enhancement (Brandenburg et al., 1998), such as serum albumin (Ohno and Morrison, 1989), lipopolysaccharide binding protein (LBP) (de Haas et al., 1999), casein (López-Expósito et al., 2008), lysozyme, the antibiotic polymyxin B and antimicrobial peptides (Chen et al., 2006). Although some of these proteins are neutral and even anionic/acidic (pI<7) (Jang et al., 2009), due to the amphipathic structure of LPS and the presence of negatively charged phosphate groups on the lipid A, the most important factors that are considered for optimal binding to LPS are a cationic/basic (pI>7) and amphipathic nature (Chen et al., 2006).

Here we describe a competitive ELISA that can be used to identify proteins or peptides that bind LPS, as a first approach before analyzing the possible activity in vitro and in vivo. In this ELISA, serial dilutions of the protein or peptide to be tested are preincubated with a fixed concentration of fluorescein isothiocyanate (FITC)-labeled LPS from Escherichia coli serotype O111:B4 and then added to wells of a microtitre plate which are blocked with a casein hydrolysate that binds LPS (Martínez-Sernández et al., 2014). Binding of the protein to LPS displaces LPS from binding to the casein, which is revealed using a horseradish peroxidase (HRP)-labeled anti-FITC polyclonal conjugate. This method allows simultaneous analysis of several proteins or peptides in a short period of time and no recognizing molecules (e.g., antibodies) to a specific protein or peptide are needed.

Innate immune cells sense pathogen and danger-associated molecular patterns (PAMPs and DAMPs) through a range of innate immune pattern recognition receptors (PRRs). One type of PRRs are the Nod-like receptors (NLRs), which form inflammasomes; a molecular platform required for the recruitment and activation of Caspase-1, which in turn cleaves and activates IL-1β, IL-18. Examples of inflammasome forming NLRs are NLRP3, NLRP1, NAIP and NLRC4. We can easily identify new inflammasome activators by performing the following protocol.

The gammaherpesvirus murid herpesvirus 4 (MuHV-4) enters cells by endocytosis from the cell surface and fusion of the viral envelope with the membrane of late endosomes. The viral envelope glycoproteins undergo antigenic changes both upon virion endocytosis and upon fusion of the viral envelope with the endosomal membrane. These changes in virion antigenicity during virus entry were first described by immunofluorescence of infected cells. Although immunofluorescence provides valuable information on the subcellular distribution of the viral glycoproteins, the quantification of immunofluorescence signals in a large number of cells is not only dependent on relatively expensive microscopy equipment, but is also relatively time-consuming. In order to quantify the antigenicity of MuHV-4 virions entering NMuMG epithelial cells in a reliable, as well as time- and cost-effective way, we have developed an ELISA with infected cells as the solid phase. In this assay, cells are grown on 96-well tissue culture plates, exposed to virions at 4 °C, followed by incubation at 37 °C allowing virion endocytosis. Cells are fixed either directly after virion binding at 4 °C or after incubation at 37 °C. After subsequent permeabilization, the cells are incubated with monoclonal antibodies specific for the viral envelope glycoproteins, followed by detection with an alkaline phosphatase-coupled secondary antibody. Upon incubation of cells with p-nitrophenyl phosphate substrate, the absorbance is measured on a conventional ELISA microplate reader. The different ways of data interpretation are discussed.

Anti-virulence agents against MRSA inhibit the production of disease-causing virulence factors, such as alpha-hemolysin, but are neither bacteriostatic nor bactericidal. Here we discuss a rapid method to screen for MRSA anti-virulence agents by measuring alpha-hemolysin production through ELISA. This protocol can be used with other alpha-hemolysin producing bacteria or for other excreted toxins to which antibodies exist.

Sandwich ELISA is a highly sensitive method that can be used to determine if two epitopes are part of the same macromolecule or supramolecular complex. In the case of plant cell wall glycans, it can reveal the existence of inter-polymers linkages, leading to better understanding of overall cell wall architectures. This development of a conventional sandwich ELISA protocol uses a carbohydrate-binding module (CBM), a small protein domain found in some carbohydrate catalysing or activating enzymes, and rat monoclonal antibodies (mAbs) which can be combined in the same ELISA plate without risk of cross reaction; the secondary anti-rat HRP antibody being only able to bind to the rat mAb and not the CBM. This protocol was developed and modified in the Prof. J. Paul Knox lab at the University of Leeds.

Upon rupture of Plasmodium falciparum (P. falciparum) schizonts in vitro (an event known as egress), merozoites are released into the culture medium. The merozoites invade fresh red blood cells, a process that involves shedding of a microneme protein called apical membrane antigen-1 (AMA1) from the merozoite surface. This shedding, which takes place even in the absence of invasion, is therefore a surrogate marker for the degree of egress taking place in a culture, and can be measured using a specific capture ELISA to quantify AMA1 levels in culture supernatants (Collins et al., 2013). The assay uses a monoclonal antibody specific for AMA1 (called 4G2dc1) (Kocken et al., 1998; Collins et al., 2009) to capture and immobilize the protein from culture supernatants, then uses a specific rabbit polyclonal antiserum to detect the immobilized antigen. A phosphatase-conjugated goat anti-rabbit antibody is finally used to quantify the binding of the second antibody. Egress is absolutely dependent upon the activity of a parasite cGMP-dependent protein kinase, PKG, and so is influenced by levels of intracellular cGMP (Collins et al., 2013). This is regulated by the interplay between guanylate cyclases and phosphodiesterases. The latter enzymes may also degrade cAMP, so it may also be informative to measure intracellular cAMP levels.