Risk Of Cross-reactivity Research Articles

Assessment of the potential allergenicity and toxicity of Pichia proteins in a novel leghemoglobin preparation

The production of soy leghemoglobin C2 (LegH) by Pichia pastoris (syn. K. phaffii) was developed by Impossible Foods to serve as a sustainable source of flavor and aroma in plant-based meats. The potential allergenicity and toxicity of a LegH from a new production process was analyzed using bioinformatics, proteomics and a pepsin digestion assay on leghemoglobin, and residual host proteins. LegH in the new preparation had the same proteoform as in the previous preparations as well as in soy root nodule extracts. Results of seven Pichia proteins, each representing ≥1% of the total protein content, showed no significant sequence matches to any known allergens with the exception of one, which matched the highly conserved wheat GAPDH, whose protein homolog is found in fungi and humans. Based on the data, it is unlikely that there is any risk of cross reactivity between LegH Prep and GAPDH. Pichia protein sequences showed very good alignment to homologous proteins from many common yeasts including Saccharomyces sp. In addition, LegH and Pichia proteins were all rapidly digested in a pepsin digest assay. In conclusion, LegH Prep from this P. pastoris production process is unlikely to pose a risk of food allergenicity.

Rapid Development and Validation of a Novel Laboratory-Derived Test for the Detection of SARS-CoV-2.

ObjectivesTo increase testing capability for SARS-CoV-2 during a rapidly evolving public health emergency, we aimed to deploy a validated laboratory-developed real-time reverse transcription polymerase chain reaction (RT-PCR) diagnostic test for SARS-CoV-2 on an accelerated timeline and using reagent supply chains that were not constrained.MethodsA real-time RT-PCR assay that detects the structural envelope (E) gene of SARS-CoV-2 was developed and validated on the Roche cobas 6800 instrument platform with the omni Utility channel reagents, which performs automated nucleic acid extraction and purification, PCR amplification, and detection. In silico analysis was performed for both inclusivity of all SARS-CoV-2 variants and cross reactivity with other pathogenic organisms. Positive control material was used to determine the Limit of Detection (LOD) and patient samples (positive and negative) confirmed by another authorized assay were used for clinical validation. Experiments were carried out at the Christiana Care Health System’s Molecular Diagnostics Laboratory (Newark, DE) between April 1 and April 4, 2020.ResultsA real-time RT-PCR assay for SARS-Cov-2 was developed and validated in just two weeks. For all oligonucleotides, 100% homology to the available SARS-CoV-2 sequences was observed. Greater than 80% homology between one or more oligonucleotides was observed for SARS-Cov (Urbani strain) and Influenza A, however risk of cross reactivity was deemed to be low. The limit of detection (LOD) of the assay was 250 copies/mL. The assay identified 100% of positive patient samples (30/30) and 100% of negative patient samples (29/29 patient negatives and 1/1 saline). Up to 92 samples can be run on a single plate and analysis takes approximately 3.5 hours.ConclusionsIn this work, we demonstrate the development and validation of a single target laboratory-developed test for SARS-CoV-2 in two weeks. Key considerations for complementary supply chains enabled development on an accelerated timeline and an increase in testing capability.

Tools for Community-Based Pharmacist Patient Care Services to Support Optimal Opioid Use

Tools for Community-Based Pharmacist Patient Care Services to Support Optimal Opioid Use

Effect of manufacturing temperature and storage duration on stability of chemically defined media measured with LC‐MS/MS

AbstractBACKGROUNDChemically defined media (CDM) are used routinely in industrial settings for the production of biopharmaceuticals from mammalian cells, so attention has shifted to understanding their chemical behavior and impact on process robustness. In this context, one particular problem is the risk of cross reactivity and instability in complex mixtures of chemicals at high concentrations.RESULTSIn order to characterize potentially unstable medium compounds in feed media, we developed and validated a novel liquid chromatography tandem mass spectrometry (LC‐MS/MS) method to simultaneously quantify the majority of medium compounds. We used this application advantage to provide the following insights into molecular processes occurring in CDM prepared at different temperatures and during storage: Concentration profiles of critical compounds in CDM are independent of preparation temperature and storage material but show significant alterations over time. In particular l‐cysteine, l‐proline, vitamin B6, thiamine and cyanocobalamin were found to be rather unstable. Using mixed mode chromatography allows expansion of the method to cover additional analytes of interest, as demonstrated by inclusion of seven additional CDM reaction products.CONCLUSIONThe successful development of a new LC‐MS/MS method allowed us to show the impact of storage duration on the stability of CDM compounds. Furthermore, the results suggest that essential parameters for medium preparation (e.g. temperature) and storage upscaling (e.g. vessel material) are not impacting upon the chemical composition of CDM. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Sandwich Enzyme-linked Immunosorbent Assay (ELISA) Analysis of Plant Cell Wall Glycan Connections

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.

Lack of cross-reaction between DNCB and chloramphenicol.

Although one study has reported a cross-reactivity between DNCB and chloramphenicol in 40% of patients, other investigators have been unable to confirm the existence of any cross reaction. The purpose of this study is to determine whether sensitization of patients to DNCB poses a risk of cross reaction to chloramphenicol. In the present study, 100 consecutive patients who were sensitized to DNCB failed to show a single reaction to patch testing with chloramphenicol in a 1% ointment. The discrepancy between these results may be explained by a possible misinterpretation of a primary irritant reaction as an allergic contact reaction in the original report.