How Exactly Do AIEgens Target Bacteria? Leveraging the Targeting Mechanism to Design Sensitive Fluorescent Immunosensors.

Aggregation-induced emission luminogens (AIEgens) are promising candidates for bacterial imaging and detection because they can "Light-Up" pathogenic bacteria without complicated labeling or washing steps. However, there have been few in-depth analyses of the intrinsic mechanism underlying their utility as fluorescence probes for targeting bacteria. Therefore, using large-scale molecular dynamics simulations, we investigated the mechanism of their bacterial "Light-Up" behavior with N,N-diphenyl-4-(7-(pyridin-4-yl)benzo[c][1,2,5]thiadiazol-4-yl) aniline functionalized with 1-bromoethane (TBP-1). We propose that the triphenylamine motif of TBP-1, rather than the positively charged pyridine group, first contacts the cell membrane. After TBP-1 completely inserts into the cell membrane, the hydrophobic triphenylamine motif localizes in the hydrophobic core of the cell membrane, restricting the molecular variation of TBP-1, which induces the fluorescent "turn-on" and bacterial "Light-Up." On this basis, we established a heterogeneous lateral flow immunoassay (LFIA) for the detection of foodborne pathogens. The LFIA system showed improved sensitivity with a limit of detection as low as 103 CFU mL-1 and strong specificity. Our protocol opened an effective shortcut to the design of more efficient AIEgens and novel AIEgens-based immunoassays.

Anti-Metatype Antibody Screening, Sandwich Immunoassay Development, and Structural Insights for ß-Lactams Based on Penicillin Binding Protein.

Theoretically, sandwich immunoassay is more sensitive and has a wider working range than that of competitive format. However, it has been thought that small molecules cannot be detected by the sandwich format due to their limited size. In the present study, we proposed a novel strategy for achieving sandwich immunoassay of ß-lactams with low molecular weights. Firstly, five ß-lactam antibiotics were selected to bind with penicillin binding protein (PBP)2x* to form complexes. Then, monoclonal and polyclonal antibodies against PBP2x*-ß-lactams complexes were produced by animal immunization. Subsequently, the optimal pairing antibodies were utilized to establish sandwich immunoassay for detection of 18 PBP2x*-ß-lactam complexes. Among them, ceftriaxone could be detected at as low as 1.65 ng/mL with working range of 1-1000 ng/mL in milk. To reveal the detection mechanism, computational chemistry and molecular recognition study were carried out. The results showed that ß-lactams with a large size and complex structures maybe conducive to induce conformational changes of PBP2x*, and then exhibit greater possibility of being detected by sandwich immunoassay after combination with PBP2x*. This study provides insights for subsequent investigations of anti-metatype antibody screening and sandwich immunoassay establishment for small-molecule detection.

Advances in hybridoma preparation using electrofusion technology.

As a rapidly developing cell engineering technique, cell electrofusion has been increasingly applied in the field of hybridoma preparation in recent years. However, it is difficult to completely replace the polyethylene glycol-mediated cell fusion using electrofusion due to the high operation requirements, high cost of electrofusion instruments, and lack of prior reference research work. The key elements limiting electrofusion in the field of hybridoma preparation also introduce practical complications, such as the use/choice of electrofusion instruments, setup/optimization of electrical parameters, and precise control of cells. This review summarizes the state of the art of cell electrofusion in hybridoma preparation based on recent published literature, mainly focusing on electrofusion instruments and their components, process control and characterization, and cell treatment. It also provides new information and insightful commentary critically important for further electrofusion development in the field of hybridoma preparation.

Molecular Recognition Mechanism of an Anti-Amatoxins mAb and Its Application in Centrifugal Disk-Based Immunoassay.

Amatoxins are polypeptides that cause 90% of fatalities from accidental ingestion of poisonous mushrooms. Unfortunately, there are no specific antidotes against amatoxins poisoning, hence preparation of high-affinity antibodies, understanding the receptor (amatoxins) and ligand (antibody) mechanism, and establishing a straightforward screening approach are of great significance for confirming poison agents and clinical diagnosis. Here, anti-amatoxins monoclonal antibody (mAb) 9B2 was prepared and the recognition mechanism was investigated. The approach is useful for designing desirable immunogens, developing new antibodies with improved performance, and constructing effective immunoassays. Based on the mAb, we designed a centrifugal disk-like microfluidics chip and developed a fully automated immunoassay capable of detecting amatoxins poisoning in various samples including serum, urine, and mushrooms. The whole detection process could be automatically accomplished within 30 min, with a limit of detection of 0.08 to 0.12 µg/L for real samples, ∼30-fold more sensitive than conventional enzyme-linked immunosorbent assay (ELISA). Our platform not only provided a practical approach for performing poison agent confirmation and clinical diagnosis but also had important implications for improving the survival of patients with mushroom poisoning.

An Automated and Highly Sensitive Chemiluminescence Immunoassay for Diagnosing Mushroom Poisoning.

Mushrooms containing Amanita peptide toxins are the major cause of mushroom poisoning, and lead to approximately 90% of deaths. Phallotoxins are the fastest toxin causing poisoning among Amanita peptide toxins. Thus, it is imperative to construct a highly sensitive quantification method for the rapid diagnosis of mushroom poisoning. In this study, we established a highly sensitive and automated magnetic bead (MB)-based chemiluminescence immunoassay (CLIA) for the early, rapid diagnosis of mushroom poisoning. The limits of detection (LODs) for phallotoxins were 0.010 ng/ml in human serum and 0.009 ng/ml in human urine. Recoveries ranged from 81.6 to 95.6% with a coefficient of variation <12.9%. Analysis of Amanita phalloides samples by the automated MB-based CLIA was in accordance with that of HPLC-MS/MS. The advantages the MB-based CLIA, high sensitivity, repeatability, and stability, were due to the use of MBs as immune carriers, chemiluminescence as a detection signal, and an integrated device to automate the whole process. Therefore, the proposed automated MB-based CLIA is a promising option for the early and rapid clinical diagnosis of mushroom poisoning.