Fragment antigen-binding (Fab) antibody-based lateral flow immunoassay for rapid and sensitive detection of potent phytoestrogen,deoxymiroestrol

Pueraria candollei is an ingredient of Thai herbal medicine, dietary supplements, and cosmetics. The in vitro and in vivo studies of this plant supported anti-osteoporotic activity and used for hormone replacement therapy. Deoxymiroestrol shows the most potent phytoconstituent in tuberous root of P. candollei with estrogenic activity. The quality controls are important for good agricultural practice (GAP) and good manufacturing practice (GMP) of plant-derived raw materials. The rapid detection of lateral flow immunoassay (LFIA) using colloidal gold is simply method, easy visualize detection and produce less waste than conventional chromatographic detection. In this study, LFIA for qualitative detection of deoxymiroestrol using antigen-binding fragment antibody (Fab) was developed. The result showed that the developed LFIA displays specific detection of deoxymiroestrol. Cross reactivity of this method was analyzed with miroestrol, isomiroestrol and methylisomiroestrol which showed 39.97%, 7.71% and 5.72%, respectively. After optimal condition, limit of detection (LOD) for deoxymiroestrol is 250 ng/ml. Plant samples were applied to strip test compare with indirect competitive ELISA using polyclonal antibody to confirm the application of LFIA. The results of LFIA method were comparable with those from ELISA. This developed lateral flow immunoassay can apply to detect deoxymiroestrol for the rapid testing. The developed method can use for quality control in plant samples as deoxymiroestrol is biomarker compound in P. candollei.

Graphic abstract

     

Development of a simple and rapid method for the detection of isomiroestrol in Pueraria candollei by an immunochromatographic strip test

Pueraria candollei (P. candollei) is a traditional Thai herb widely used for estrogen replacement therapy because it contains many unique chromenes that possess potent estrogenic activity, one of which is known as isomiroestrol. Since isomiroestrol is a promising compound that is solely present in P. candollei, it can be used as an identifying marker for standardization of P. candollei. Here, we developed a lateral-flow immunochromatographic strip (ICS) test using a colloidal gold nanoparticle-conjugated anti-isomiroestrol monoclonal antibody (12C1-mAb) for the detection of isomiroestrol in plant samples and products of P. candollei. The advantages of the developed ICS over an enzyme-linked immunosorbent assay are its simplicity and rapidity, as the ICS test can be completed 15 min after dipping the strip into the analyte solution. The detectable concentration of isomiroestrol was 7.0 µg/mL. Considering the demand for the standardization of P. candollei due to concerns regarding its quality, our ICS test using isomiroestrol as an identifying marker would be effective and useful to assess the presence of isomiroestrol.

     

Characterization of the circulating hemocytes in mud crab (Scylla olivacea) revealed phenoloxidase activity

This study focused on an isolation and characterization of the circulating hemocytes in mud crab, Scylla olivacea. Isolation of specific cell types of hemocytes from crab hemolymph was accomplished by using 60% Percoll density gradient centrifugation. Four separated bands of the hemocytes were successfully obtained. Characterization of these isolated hemocytes by light microscope using trypan blue-rose bengal staining, rose bengal–hematoxilin staining, and phase contrast revealed four distinct types of hemocyte cells. Using their specific morphology and granularity, they were identified as hyaline cell (HC), small granular cell (SGC), large granular cell (LGC) and mixed granular cell (MGC). Transmission electron microscopy (TEM) revealed more details on specific cell size, size of cytoplasmic granule, and nuclear to cytoplasmic ratio, and confirmed the classification. Relative abundance of these cells types in the hemolymph of an adult crab were 15.50 ± 8.22% for HC, 55.50 ± 7.15% for SGC, 13.50 ± 5.28% for LGC, and 15.50 ± 3.50% for MGC. Proteomic analysis of protein expression for each specific cell types by two-dimensional electrophoresis identified two highly abundant proteins, prophenoloxidase (ProPO) and peroxinectin in LGC. Determination of phenoloxidase (PO) activity in each isolated cell types using in vitro and in situ chemical assays confirmed the presence of PO activity only in LGC. Based on an increased PO activity of crab hemolymph during the course of White Spot Syndrome Virus (WSSV) infection, these results suggest that prophenoloxidase pathway was employed for host defense mechanism against WSSV and it may link to the role of large granular hemocyte.     

Mesoscale modeling technique for studying the dynamics oscillation of Min protein: Pattern formation analysis with lattice Boltzmann method

We presented an application of the Lattice Boltzmann method (LBM) to study the dynamics of Min proteins oscillations in Escherichia coli. The oscillations involve MinC, MinD and MinE proteins, which are required for proper placement of the division septum in the middle of a bacterial cell. Here, the LBM is applied to a set of the deterministic reaction diffusion equations which describes the dynamics of the Min proteins. This determines the midcell division plane at the cellular level. We specifically use the LBM to study the dynamic pole-to-pole oscillations of the Min proteins in two dimensions. We observed that Min proteins’ pattern formation depends on the cell's shape. The LBM numerical results are in good agreement with previous findings, using other methods and agree qualitatively well with experimental results. Our results indicate that the LBM can be an alternative computational tool for simulating the dynamics of these Min protein systems and possibly for the study of complex biological systems which are described by reaction-diffusion equations. Moreover, these findings suggest that LBM could also be useful for the investigation of possible evolutionary connection between the cell's shape and cell division of E. coli. The results show that the oscillatory pattern of Min protein is the most consistent with experimental results when the dimension of the cell is 1×2. This suggests that as the cell's shape is close to being a square, the oscillatory pattern no longer places the cell division of E. coli at the proper location. These findings may have a significant implication on why, by natural selection, E. coli is maintained in a rod shape or bacillus form.