Monoclonal antibody-based enzyme immunoassay for detection of porcine plasma in fish surimi

Detection of porcine plasma using indirect ELISA was developed using mAb B4E1 for the prevention of their usage in human food that creates religious and health conflicts. The immunoassay has a CV<20% and did not cross-react to other meat and non-meat proteins. The sensitivity of the assay is 0.25% (w/w) of porcine plasma in spiked raw and cooked fish surimi. The assay did not produce a false positive result for any of the commercial fish surimi tested that were not contain porcine plasma. Determination of a 60-kDa antigenic protein of porcine blood using Western Blot confirmed its presence in the plasma fraction of the porcine blood. Further proteomic analysis involving liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed the 60-kDa protein to be porcine serum albumin

Authentication approach using enzyme-linked immunosorbent assay for detection of porcine substances

Food manufacturers across the world commonly add animal substances in their food products. Some food products may contain porcine substances including pork, gelatine, blood and pepsin. These substances significantly affect the texture, colour or taste of the end products. Aside from enhancing sensory qualities, additional ingredients also contribute to preservation, bulk and nutrition. However, the inclusion of porcine substances might not be suitable among certain communities. One primary concern is fraud labelling which includes hiding the addition of porcine substances in food. Therefore, analytical techniques such as enzyme-linked immunosorbent assay (ELISA) have been developed to detect the porcine proteins in food. The ELISA delivers specificity and sensitivity in detecting the targeted animal species in food. This review provides an overview of the ELISA technique which has been developed for potential detection of porcine substances in laboratory-prepared food samples and commercial food products

Mechanisms of increased susceptibility to influenza infection in mouse models of chronic lung diseases

Research Doctorate - Doctor of Philosophy (PhD)Influenza infections are of major importance as they have a significant impact on the health of individuals and impart substantial socio-economic ramifications on society. Prevention and treatment of influenza infections are complicated by frequent genetic mutations of the influenza virus. Patients with underlying chronic lung diseases, such as chronic obstructive pulmonary disease (COPD) and asthma are more susceptible to influenza infection, and infection with influenza exacerbates these diseases. Therefore, elucidation of the mechanisms underpinning increased susceptibility to influenza in these patients is vital. Here, we established an experimental mouse model of COPD and utilised an existing ovalbumin-induced allergic airways disease (AAD) model to investigate the effects of influenza infection in COPD and asthma, respectively. Influenza infection in experimental COPD resulted in increased viral titre, exaggerated airway inflammation and further impaired lung function. These effects were accompanied by decreased neutrophil influx into the airways, reduced antiviral interferon responses, and the suppression of a range of cytokines and chemokines, including interferon (IFN)-γ, tumour necrosis factor (TNF)-α, IFN-γ-induced protein (IP)-10, macrophage inflammatory protein (MIP)-1α, keratinocyte-derived chemokine (KC, or IL-8 in humans) and interleukin (IL)-10, as well as increased IL-6. This increased susceptibility was mediated by an increase in phosphoinositide 3-kinase (PI3K) protein expression. The inhibition of PI3K effectively reduced viral titre, enhanced antiviral IFNs and improved lung function. Influenza infection in recombinant IL-13-treated or ovalbumin (Ova)-induced AAD models led to increased viral titre, impaired antiviral responses and increased airway hyper-responsiveness (AHR). It also resulted in exaggerated airway inflammation, more severe histopathology, increased mucus secreting cell numbers and increased IL-13. Importantly, we also showed that inhibition of IL-13 by administration of anti-IL-13 (αIL-13) monoclonal antibody during influenza infection reduced viral titre, AHR, eosinophil infiltration and MSCs, which were associated with improved antiviral IFN responses. In summary, these data highlight the important roles of PI3K and IL-13 in the increased susceptibility to influenza infection in experimental models of COPD and asthma, respectively. Such findings offer evidence for new and promising avenues for influenza disease management in these chronic lung diseases. In fact, both PI3K inhibitors and anti-IL-13 antibodies have already entered clinical trials and may be utilised as novel therapeutic approaches for influenza infections in the future

Current analytical methods for porcine identification in meat and meat products

Authentication of meat products is critical in the food industry. Meat adulteration may lead to religious apprehensions, financial gain and food-toxicities such as meat allergies. Thus, empirical validation of the quality and constituents of meat is paramount. Various analytical methods often based on protein or DNA measurements are utilized to identify meat species. Protein-based methods, including electrophoretic and immunological techniques, are at times unsuitable for discriminating closely related species. Most of these methods have been replaced by more accurate and sensitive detection methods, such as DNA-based techniques. Emerging technologies like DNA barcoding and mass spectrometry are still in their infancy when it comes to their utilization in meat detection. Gold nanobiosensors have shown some promise in this regard. However, its applicability in small scale industries is distant. This article comprehensively reviews the recent developments in the field of analytical methods used for porcine identification

Klebsiella virus UPM2146 lyses multiple drug-resistant Klebsiella pneumoniae in vitro and in vivo.

Klebsiella pneumoniae are opportunistic bacteria found in the gut. In recent years they have been associated with nosocomial infections. The increased incidence of multiple drug-resistant K. pneumoniae makes it necessary to find new alternatives to treat the disease. In this study, phage UPM2146 was isolated from a polluted lake which can lyse its host K. pneumoniae ATCC BAA-2146. Observation from TEM shows that UPM2146 belongs to Caudoviriales (Order) based on morphological appearance. Whole genome analysis of UPM2146 showed that its genome comprises 160,795 bp encoding for 214 putative open reading frames (ORFs). Phylogenetic analysis revealed that the phage belongs to Ackermannviridae (Family) under the Caudoviriales. UPM2146 produces clear plaques with high titers of 1010 PFU/ml. The phage has an adsorption period of 4 min, latent period of 20 min, rise period of 5 min, and releases approximately 20 PFU/ bacteria at Multiplicity of Infection (MOI) of 0.001. UPM2146 has a narrow host-range and can lyse 5 out of 22 K. pneumoniae isolates (22.72%) based on spot test and efficiency of plating (EOP). The zebrafish larvae model was used to test the efficacy of UPM2146 in lysing its host. Based on colony forming unit counts, UPM2146 was able to completely lyse its host at 10 hours onwards. Moreover, we show that the phage is safe to be used in the treatment against K. pneumoniae infections in the zebrafish model

Detection of porcine pepsin in model cheese using polyclonal antibody-based ELISA

The usage of porcine pepsin or other porcine derivatives in food products is a common practice in European, American and certain Asian countries although it creates issues in religious and personnel health concerns. In this study, porcine pepsin was detected using indirect ELISA that involved the anti-pep80510 polyclonal antibody raised against a specific peptide of porcine pepsin, pep80510. The sensitivity of the assay for standard porcine pepsin was 0.008 µg/g. The immunoassay did not cross-react to other animal rennet and milk proteins except for microbial coagulant from Mucor miehie. The recovery of porcine pepsin in spiked cheese curd within the range of CV < 20% while for porcine pepsin in spiked cheese whey the recovery is also within the range of CV% < 20%

Nanoplate-based digital PCR for highly sensitive pork DNA detection targeting multi-copy nuclear and mitochondrial genes

The inclusion of ingredients derived from pigs in highly processed consumer products poses a significant challenge for DNA-targeted analytical enforcement, which could be overcome by using digital PCR. However, most species detection methods use digital PCR to target single-copy nuclear genes, which limits their sensitivity. In this work, we examined the performance of a nanoplate-based digital PCR method that targets multi-copy nuclear (MPRE42) and mitochondrial (Cytb) genes. Poor separation of positive and negative partitions, as well as a ‘rain effect’ were obtained in the porcine-specific MPRE42 assay. Among the optimization strategies examined, the inclusion of restriction enzymes slightly improved the separation of positive and negative partitions, but a more extensive ‘rain effect’ was observed. The high copy number of the MPRE42 amplicon is hypothesized to contribute to the saturation of the positive signal. In contrast, the porcine-specific Cytb assay achieved perfect separation of positive and negative partitions with no ‘rain effect’. This assay can detect as little as 0.4 pg of pork DNA, with a sensitivity of 0.05 (w/w) in a pork-chicken mixture, proving its applicability for detecting pork in meat and meat-based products. For the MPRE42 assay, potential applications in highly degraded products such as gelatin and lard are anticipated

Targeting PI3K-p110α suppresses influenza virus infection in chronic obstructive pulmonary disease

Rationale: Chronic obstructive pulmonary disease (COPD) and influenza virus infections are major global health issues. Patients with COPD are more susceptible to infection, which exacerbates their condition and increases morbidity and mortality. The mechanisms of increased susceptibility remain poorly understood, and current preventions and treatments have substantial limitations. Objectives: To characterize the mechanisms of increased susceptibility to influenza virus infection in COPD and the potential for therapeutic targeting. Methods: We used a combination of primary bronchial epithelial cells (pBECs) from COPD and healthy control subjects, a mouse model of cigarette smoke-induced experimental COPD, and influenza infection. The role of the phosphoinositide-3-kinase (PI3K) pathway was characterized using molecular methods, and its potential for targeting assessed using inhibitors. Measurements and Main Results: COPDpBECs were susceptible to increased viral entry and replication. Infected mice with experimental COPD also had more severe infection (increased viral titer and pulmonary inflammation, and compromised lung function). These processes were associated with impaired antiviral immunity, reduced retinoic acid-inducible gene-I, and IFN/cytokine and chemokine responses. Increased PI3K-p110α levels and activity inCOPDpBECs and/or mice were responsible for increased infection and reduced antiviral responses. Global PI3K, specific therapeutic p110α inhibitors, or exogenous IFN-β restored protective antiviral responses, suppressed infection, and improved lung function. Conclusions: The increased susceptibility of individuals with COPD to influenza likely results from impaired antiviral responses, which are mediated by increased PI3K-p110α activity. This pathway may be targeted therapeutically in COPD, or in healthy individuals, during seasonal or pandemic outbreaks to prevent and/or treat influenza

TLR7 promotes smoke-induced lung damage through the activity of mast cell tryptase

Toll-like receptor (TLR)7 is known for eliciting immunity against single-stranded RNA viruses. TLR7 was increased in both human and cigarette smoke (CS)-induced experimental chronic obstructive pulmonary disease (COPD). Severity of CS-induced emphysema and COPD was reduced in TLR7-deficient mice whilst inhalation of imiquimod (TLR7-agonist) induced emphysema in naïve mice. Imiquimod-induced emphysema was reduced in mice treated with mast cell stabilizer cromolyn or deficient in mast cell protease-6. Therapeutic treatment with anti-TLR7 monoclonal antibody suppressed CS-induced emphysema, experimental COPD and accumulation of pulmonary mast cells. We demonstrate an unexpected role for TLR7 in mediating emphysema and COPD through mast cell activity