Avidin-biotin complex-based capture coating platform for universal Influenza virus immobilization and characterization

Influenza virus mutates quickly and unpredictably creating emerging pathogenic strains that are difficult to detect, diagnose, and characterize. Conventional tools to study and characterize virus, such as next generation sequencing, genome amplification (RT-PCR), and serological antibody testing, are not adequately suited to rapidly mutating pathogens like Influenza virus where the success of infection heavily depends on the phenotypic expression of surface glycoproteins. Bridging the gap between genome and pathogenic expression remains a challenge. Using sialic acid as a universal Influenza virus binding receptor, a novel virus avidin-biotin complex-based capture coating was developed and characterized that may be used to create future diagnostic and interrogation platforms for viable whole Influenza virus. First, fluorescent FITC probe studies were used to optimize coating component concentrations. Then atomic force microscopy (AFM) was used to profile the surface characteristics of the novel capture coating, acquire topographical imaging of Influenza particles immobilized by the coating, and calculate the capture efficiency of the coating (over 90%) for all four representative human Influenza virus strains tested.

Highly sensitive and rapid detection of porcine circovirus 2 by avidin-biotin complex based lateral flow assay coupled to isothermal amplification

In this study, a new platform for the detection of porcine circovirus 2 was developed by avidin-biotin complex based lateral flow assay (LAMP-LFA). Improved detection sensitivity was attained by using...

Evaluation of Immunohistochemical Reactivity with Avidin-Biotin Complex (ABC) Method

Immunohistochemistry is any technique that can detect cellular and extracellular components in situ by means of specific antibodies, using enzymatic detection systems. Among immunohistochemical methods, the technique of avidin - biotin complex (ABC) is widely used because of its high sensitivity. The aim of this study was to evaluate the immunohistochemical reactivity of the 4C4.9 antibody for detection of S-100 protein using the ABC method. For the evaluation of immunohistochemical reactivity 2 biopsies of human skin were used with histopathological diagnosis of ulcerated malignant melanoma and melanocytic intradermal nevi from the Research Laboratory on Animal Biotechnology of the Universidad de La Frontera, Chile. The Kit VECTASTAIN® was used as detection method, the dilution the 4C4.9 antibody was 1/250 and incubation temperature was at 4 ° C or 37 °C for 18 hours. To validate the technique, a positive control and a negative for 4C4.9 was performed. The results of immunohistochemical staining by the method of ABC complex showed positive staining for protein S-100 both in ulcerated malignant melanoma and melanocytic intradermal nevi, incubated for 18 hours at 4 ° C or 37 ° C. However, immunostaining was more intense when the primary antibody was incubated at 37° C. For a correct interpretation of the results, it is necessary to take into consideration that the antigen-antibody reaction is influenced by various factors such as the concentration of antibody, time and temperature of incubation. In conclusion, our results suggest incubating the samples with the first antibody (4C4.9) at 1/250 dilution in distilled water, incubating for 18 h at 37 oC. However, immunostaining was more intense when the primary antibody was incubated at 37° C. For a correct interpretation of the results, it is necessary to take into consideration that antigen- antibody reaction is influenced by various factors such as the concentration of antibody, time and temperature of incubation. In conclusion, our results suggest incubating the samples with the first antibody (4C4.9) at 1/250 dilution in distilled water, incubating for 18 h at 37 oC. The use of the antibody 4C4.9 is recommended to support the diagnosis and differential diagnosis.

Pengecatan Imunohistokimia HER2 Menggunakan Susu Skim dan Normal Serum

HER2 (Human Epidermal Growth Factor Receptor 2) merupakan suatu reseptor pada permukaan sel yang berpengaruh pada proliferasi jaringan, mutasinya dapat menjadi onkogen. Over ekspresi dari HER2 pada kasus kanker dapat dilihat dengan teknik imunohistokimia (IHC). Protein blocking merupakan salah satu langkah dalam pengecatan IHC yang berfungsi menghalangi ikatan non spesifik pada jaringan dengan menggunakan normal serum dan protein solution (susu skim). Tujuan penelitian mengetahui gambaran hasil pengecatan IHC menggunakan normal serum dan susu skim. Penelitian secara eksperimental dengan pendekatan cross sectional. Sampel penelitian jaringan kanker payudara HER2 positif dengan stadium +2 dari satu organ dan pasien yang sama. Pengecatan IHC menggunakan teknik Strep (Avidin) Biotin Complex. Pengecatan menggunakan normal serum didapatkan hasil +2, menggunakan susu skim 1% didapatkan hasil +3, sedangkan menggunakan susu skim 2% dan 3% didapatkan hasil +2. Terdapat perbedaan yang signifikan antara normal serum dengan susu skim 1%. Tidak terdapat perbedaan yang signifikan antara normal serum dengan susu skim 2% dan susu skim 3%. Simpulan adalah normal serum dapat diganti dengan susu skim 2%.

Microfluidic Molecular Trap: Probing Extracellular Signaling by Selectively Blocking Exchange of Specific Molecules in Cell-Cell Interactions

Communication among cell populations is achieved via a wide variety of soluble, extracellular signaling molecules [1]. In order to investigate the role of specific molecules in a cellular process, researchers often utilize in vitro cell culture techniques in which the molecule under question has been removed from the signaling pathway. Traditionally, this has been accomplished by eliminating the gene in the cell that is responsible for coding the targeted ligand/receptor by using modern DNA technology such as gene knockout; however, this process is expensive, time-consuming, and labor intensive. Previously, we have demonstrated a microfluidic platform that uses a semi-permeable barrier with embedded receptor-coated nanoparticles to selectively remove a specific molecule or ligand from the extracellular signaling pathway in a cell co-culture environment [2]. This initial proof-of-principle was conducted using biotinylated nanoparticles and fluorescently tagged avidin molecules, as the avidin/biotin complex is the strongest known non-covalent interaction between a protein and a ligand (Dissociation constant kd = 10−15 M). Also, the trap was only effective for short time periods (<15 min) because the high concentration of fluorescently tagged avidin molecules required for visualization quickly saturated the barrier. However, nearly all biologically relevant ligand-receptor interactions have lower binding affinities than the avidin-biotin complex, with dissociation constants that are larger by several orders of magnitude. In addition, many in vitro cell culture experiments are conducted over multiple hours or days. Thus, a practically useful molecular trap device must be able to operate in a lower binding affinity regime while also lasting for extended time periods. Here we present results in which a biotinylated-particle barrier was used to successfully block lower concentrations of fluorescently tagged avidin for multiple days, showcasing the applicability of the device for long term experiments. In addition, we introduce a modified molecular trap in which the protein A/goat IgG complex was used to demonstrate the effectiveness of the platform for lower binding affinity protein-ligand interactions. These results indicate the potential usefulness of the microfluidic molecular trap platform for probing extracellular signaling pathways.

The expression of caspase-3, caspase-7, caspase-9 and cytokeratin AE1/AE3 in goats with enzootic nasal adenocarcinoma: an immunohistochemical study

The aim of this study was to examine the expression of caspase-3, caspase-7, caspase-9 and cytokeratin AE-1/AE-3 using the avidin-biotin complex (ABC) immunoperoxidase technique in 20 goats with enzootic nasal adenocarcinoma (ENA). Clinically, dyspnoea and nasal discharge were observed in all cases. Macroscopically, polypoid and sessile masses were seen in the ethmoidal area. At the histopathological examination, tubular, papillary and mixed patterns of ENA were diagnosed. Immunohistochemically, strong positive reactions were generally seen for caspase-3, while strong to moderate and slight reactions were observed for caspase-7 and caspase-9 in the cytoplasm of the tumour cells. Positive reactions for cytokeratin AE-1/AE-3 were only seen in epithelial cells. In addition, the causative agent of ENA, retrovirus, was detected immunohistochemically in tumour cells. &nbsp;