Effects of material interfaces on orientation and function of fibrinogen / 生物医学工程学杂志

Fibrinogen (Fg) in human plasma plays an important role in hemostasis, vascular repair and tissue integrity. The surface chemistry of extracellular matrix or biological materials affects the orientation and distribution of Fg, and changes the exposure of integrin binding sites, thereby affecting its adhesion function to platelets. Here, the quantity, morphology and side chain exposure of Fg adsorbed on hydrophilic, hydrophobic and avidin surfaces were measured by atomic force microscopy (AFM) and flow cytometry (FCM), then the rolling behavior of platelets on Fg was observed through a parallel plate flow chamber system. Our results show that the hydrophobic surface leads to a large amount of cross-linking and aggregation of Fg, while the hydrophilic surface reduces the adsorption and accumulation of Fg while causing the exposure and spreading of the α chain on Fg and further mediating the adhesion of platelets. Fg immobilized by avidin / biotin on hydrophilic surface can maintain the monomer state, avoid over exposure and stretching of α chain, and bind to the platelets activated by the A1 domain of von Willebrand factor instead of inactivated platelets. This study would be helpful for improving the blood compatibility of implant biomaterials and reasonable experimental design of coagulation

MCC1019, a selective inhibitor of the Polo-box domain of Polo-like kinase 1 as novel, potent anticancer candidate

Polo-like kinase (PLK1) has been identified as a potential target for cancer treatment. Although a number of small molecules have been investigated as PLK1 inhibitors, many of which showed limited selectivity. PLK1 harbors a regulatory domain, the Polo box domain (PBD), which has a key regulatory function for kinase activity and substrate recognition. We report on 3-bromomethyl-benzofuran-2-carboxylic acid ethyl ester (designated: MCC1019) as selective PLK1 inhibitor targeting PLK1 PBD. Cytotoxicity and fluorescence polarization-based screening were applied to a library of 1162 drug-like compounds to identify potential inhibitors of PLK1 PBD. The activity of compound MC1019 against the PLK1 PBD was confirmed using fluorescence polarization and microscale thermophoresis. This compound exerted specificity towards PLK1 over PLK2 and PLK3. MCC1019 showed cytotoxic activity in a panel of different cancer cell lines. Mechanistic investigations in A549 lung adenocarcinoma cells revealed that MCC1019 induced cell growth inhibition through inactivation of AKT signaling pathway, it also induced prolonged mitotic arrest-a phenomenon known as mitotic catastrophe, which is followed by immediate cell death apoptosis and necroptosis. MCC1019 significantly inhibited tumor growth in a murine lung cancer model without affecting body weight or vital organ size, and reduced the growth of metastatic lesions in the lung. We propose MCC1019 as promising anti-cancer drug candidate.

Effect of the two-step pretargeting radioimmunotherapy of CD45 monoclonal antibody and 188Re-Avidin on lymphoma Raji cell line / 中华放射医学与防护杂志

Objective To study the effect of two-step pretargeting radioimmunotherapy of CD45 McAb and 188Re-Avidin on lymphoma Raji cell line.Methods The CD45 McAb and Avidin were directly labeled with 188Re,and the labeling efficiency and radiochemical purity were measured by the paper chromatography.The specific binding test and competition binding test between 188 Re-CD45 McAb and Raji cells in vitro were also performed.CCK-8 assay was used to determine the inhibition effect on Raji cell proliferation in the pretargeted group,188Re-CD45 McAb,188Re-Avidin and 188ReO4 groups,then the cell survival and proliferation inhibition rate were calculated.Results The specific cell binding rate of 188Re-CD45 McAb with Raji cells was (70.92 ± 1.91) ％,in the competition group,the binging rate of 188Re-CD45 McAb with Raji cells was only (7.96 ± 0.87)％.The Raji cells proliferation was inhibited in all groups with 188Re radiolabel,and the inhibition rate was positively correlated with the radioactivity dose (r=0.907-0.992,P ＜0.05).However,at the same dose,the inhibition effect in the group of two-step pretargeting at each time point were all stronger than those of 188Re-CD45 McAb,188Re-Avidin and 188 ReO4-alone (t =124.76-607.98,P ＜ 0.05).But there was no significantly statistical difference in the inhibitory effect between the groups of 188Re-Avidin and 188ReO4-(P ＞ 0.05).Conclusions It is confirmed that 188Re-CD45 McAb could be specifically bound to Raji cells,and the two-step pretargeting of CD45 McAb and 188Re-Avidin has obvious inhibitory effect on the Raji cell proliferation.

On the possible involvement of bovine serum albumin precursor in lipofection pathway.

Protein factors involved in lipofection pathways remain elusive. Using avidin-biotin affinity chromatography and mass finger printing analysis technique, herein we report the identification of a 70 kDa size protein (bovine serum albumin precursor, BSAP) which binds strongly with lipoplexes and may play role in lipofection pathway. Using multiple cultured animal cells and three structurally different cationic transfection lipids, we show that the efficiencies of liposomal transfection vectors get significantly enhanced (by ~2.5- to 5.0-fold) in cells pre-transfected with lipoplexes of reporter plasmid construct encoding BSAP. Findings in the cellular uptake experiments in A549 cells cultured in DMEM supplemented with 10% (w/w) BODIPY-labelled BSAP are consistent with the supposition that BSAP enters cell cytoplasm from the cell culture medium (DMEM supplemented with 10% FBS) used in lipofection. Cellular uptake studies by confocal microscopy using BODIPY-labelled BSAP and FITC-labelled plasmid DNA revealed co-localization of plasmid DNA and BSAP within the cell cytoplasm and nucleus. In summary, the present findings hint at the possible involvement of BSAP in lipofection pathway.

Biotin-avidin Mediated Competitive Enzyme-linked Immunosorbent Assay for Determination of Ketamine / 分析化学

A rapid and sensitive method based on biotin-avidin mediated competitive enzyme-linked immu nosorbent assay(BA-ELISA) was established for the determination of ketamine.The optimal concentration of coated antigen and anti-ketamine monoclonal antibody were found to be 2.0 and 10.2 mg/L.The concentra tions of biotinylated goat anti-mouse IgG(Biotin-IgG) and streptavidin-horseradish peroxidase(SA-HRP) were optimized and the optimum results were found to be 0.29 and 1.0 mg/L, respectively.The linear range of the presented method was from 0.1 to 1000 μg/L, and the limit of detection was found to be 0.03 μg/L.The recoveries of ketamine spiked in human serum and urine were between 94% and 102%.Comparing the result of traditional ELISA, the present BA-ELISA method had a lower detection limit for ketamine.The experimental results indicated that the present BA-ELISA method was specific and sensitive.

Establishment of ELISA for detection of hepatoma specific γ-glutamyltransferase and its clinical application / 中华检验医学杂志

Objective To prepare monoclonal antibody (McAb) against γ-glutamyhransferase(GGT) firmly bound to datura stramonim (DSA) leetin from primary hepatic carcinoma (PHC) tissue and establish an avidin-biotin enzyme-linked immunosorbent assay (ELISA) for evaluating the diagnostic value of serum DSA-GGT for PHC. Methods Anti-DSA-GGT monoclonal antibodies were obtained by McAb technology and purified by protein G-sepharose affinity chromatography. The McAb was labeled with biotin and avidin-biotin ELISA for measurement of serum DSA-GGT was established. Using the avidin-biotin ELISA, serum DSA-GGT levels was detected in 39 patients with PHC, and 122 patients with non-PHC diseases. The distribution of serum DSA-GGT values of 119 healthy subjects were determined by P-P plots. Optimal cut-off value for the diagnosis of PHC was determined by receiver operating characterstic (ROC) curve. Results The protein levels of McAb in the ascites derived from 5 McAb hybridoma cell strains ranged from 2. 12 to 6. 70 mg, The biotin-labeled rate varied from 48. 6% to 72. 2% respectively. The minimum detection limit of serum DSA-GGT in avidin-biotin ELISA was 2 μg/L. Intra-assay and inter-assay coefficients of variation were 8.9% and 11.5% respectively. The distribution of DSA-GGT values of 119 healthy subjects showed Gaussian distribution and its Mean ± SD was ( 1.50±0. 51 ) μg/L. Optimal cut-off value (3.25 μg/L) in the diagnosis of PHC was determined by ROC curve. DSA-GGT was positive in 26 out of 39 patients with PHC and 10 out of 122 patients with non-PHC diseases were positive. The sensitivity and specificity of this assay for the diagnosis of PHC were 66. 7% and 91.8% respectively. Conclusions The convenient avidin-biotin ELISA method was successfully established in our laboratory and it showed a good reproducibility and reliability. It may be a potential tool in the diagnosis of PHC to achieve higher sensitivity and specificity.

Biotin/avidin sandwich enzyme-linked immunosorbent assay for Culicidae mosquito blood meal identification

The knowledge of mosquitoes Culicidae host feeding patterns is basic to understand the roles of different species and to indicate their importance in the epidemiology of arthropod-borne diseases. A laboratory assay was developed aiming at standardizing the biotin-avidin sandwich enzyme-linked immunosorbent assay, which was unprecedented for mosquito blood meal identification. The enzyme-linked immunosorbent assay (ELISA) activity was evaluated by the detection of titers on each sample of the 28 blood-fed Culex quinquefasciatus. In light of the high sensitivity that the technique permits, by means of small quantities of specific antibodies commercially provided and phosphatase substrate which reinforces additional dilutions, human and rat blood meals were readily identified in all laboratory-raised Culex quinquefasciatus tested. The assay was effective to detect human blood meal dilutions up to 1:4,096, which enables the technique to be applied in field studies. Additionally, the present results indicate a significant difference between the detection patterns recorded from human blood meal which corroborate the results of host feeding patterns.

Effect of Avidin and Biotin in Attachment of Human Adipose Stem Cells to Micronized Acellular Dermal Matrix

PURPOSE: In tissue engineering, it is important that the scaffolds have high affinity with cells for making efficient use of cells. The authors studied the binding affinity of human adipose stem cells(ASCs) to micronized acellular dermal matrix(alloderm) using biotin and avidin linkages. METHODS: Human ASCs were harvested from adipose tissue obtained by abdominoplasty. ASCs(1x10(4), 5x10(4), 1x10(5), 5x10(5), 1x10(6), 5x10(6) cells) were attached to micronized alloderm(1mg) in three groups; 1) control group in which no ASCs and alloderm was treated; 2) serum group in which alloderm was exposed to fetal bovine serum; and 3) biotin group in which biotinylated cells were attached to biotinylated alloderm. The binding affinities were determined 1 day after making ASC-alloderm complexes. The proliferation rates were determined by XTT assays in 4, 7, 14, and 21 days and scanning electron microscopic examination was performed in 7 and 21 days after culture of ASC-alloderm complexes. RESULTS: The binding affinities of the biotin group were significantly increased in all cell concentrations. Maximum binding affinity was observed at 5x10(4)/mg of micronized dermal matrix in biotin group. The viabilities were lowest in biotin group in contrast to binding affinity, but the difference was not significant. SEM showed well attachment of cells to micronized dermal matrix in all groups. CONCLUSION: The use of avidin/biotin facilitated human ASCs attaching to micronized acellular dermal matrix. This attachment would not disturb adipose stem cells viabilities. The present study suggests that avidin/ biotin can be used as making efficient use of cells in adipose tissue engineering.

Electrochemical properties of enzyme electrode in biofuel cell / 国际生物医学工程杂志

Objective To study the electrochemical features of an improved enzyme bioelectrode for biofuel cell. Methods The enzyme electrode was prepared via biotin-avidin technology and its electrochemical features were explored. The conditions such as layers of enzyme films, strength of glucose, scan speed and temperature, which affected the electrochemical behavior of enzyme electrode, were studied. Results The results showed that the electrode had good electric current response to glucose. The enzyme electrode prepared can fulfill the need of biofuel cell and is a promising electrode to be used in human body.

Affinity Improvement of Antibody-Avidin Fusion Proteins for Biotin / 대한면역학회지

To generate drug delivery vector to locales in the body, genetic engineering and expression techniques have been used to produce antibody avidin fusion proteins. Chicken avidin has been fused to mouse-human chimeric IgG3 immediately after the hinge with a flexible linker (H-Flex-Av) and at the end of CH2 (CH2-Av). Fusion heavy chains were expressed with the expected molecular weight, assembled as H2L2 forms with a co-expressed light chain, and were secreted. The expression level of H- Flex-Av was 1~10 ug/ml/10(8)/24 hrs, but that of C2-Av was a very little (0.08~0.9 ug/ ml/10(8)/24 hrs). The resulting H-Flex-Av and CH2-Av fusion proteins continued to bind antigen dansyl and also bound biotinylated bovine serum albumin; both H-Flex-Av and CH2-Av had shown to retain 3-4 times higher relative affinity than that of CH3-Av in ELISA. Importantly the fact that both avidin fusion proteins had a higher relative affinity suggests that these avidin fusion proteins can be effectively used to deliver biotinylated ligands such as drugs and peptides to a certain locale, such as the brain.

Distribution of Neuropeptides in Nasal Polyp and Polypoid Mucosa / 대한이비인후과학회지

BACKGROUND: The pathogenesis of nasal polyp is not well understood, however the common pathway of nasal polyp formation is tissue edema. Factors that are known to predispose tissue edema include histamine, arachidonic acid metabolites, serotonin and peptidergic neuro-transmitters. OBJECTIVES: To elucidate the pathophysiological roles of peptidergic neurotransmitters on the polyp formation, we investigated the distribution of several immunoreactive nerve fibers. MATERIALS AND METHODS: We obtained normal middle turbinate mucosa, edematous middle turbinate mucosa, polypoid middle turbinate mucosa and nasal polyp tissue from 5 patients at the time of surgery and we determined the distribution of substance P(SP) immunoreactive, vasoactive intestinal peptide(VIP) immunoreactive and neuropeptide-Y(NPY) immunoreactive nerve fibers in the above tissues using avidin-biotin complex(ABC) immunohistochemical method. RESULTS: Fine varicose peptidergic nerve fibers were found numerously in normal mucosa and they were decreased in number in the edematous mucosa. However the peptidergic nerve fibers were hardly found in the polypoid mucosa and the pedicle of polyp, and no nerve fibers were found in the apex of polyp except VIP-immunoreactive nerve fibers. CONCLUSION: Decreasing tendency of distribution of these peptidergic nerve fibers in the order of edematous mucosa, polypoid mucosa, and polyp may indicate the denervation of autonomic nervous system. These phenomenon suggests the important role of peptidergic neurotransmitters in the early stage of polyp formation. However, once the polyp has been formed, contribution of the peptidergic neurotransmitters is considered to be negligible.

Detection of Chromosome Aberration in Interphase Nuclei of Tumor Cells by Nonradioactive In Situ Hybridization Using Chromosome-specific Probes

An improved protocol for in situ hybridization(ISH) to routinely processed, paraffin-embedded tissue sections from lung carcinoma is presented. For this study, DNA probes for alpha-satellite chromosome 7 and 17 were used. The protocol to detect numerical chromosome aberrations involved treatment of sections with 1 M sodium thiocyanate prior to pepsin digestion, resulting in reproducible ISH reactions. The effect of avidin-biotin detection system. Four layer avidin methods and triple biotin methods, using avidin-PO, goat antiavidin, biotinylated antigoat IgG, avidin-PO or anti-biotin, biotinylated antirabbit IgG, avidin-PO, markedly enhanced the intensity of positive signals. More than 80% of the tumor and stromal cells showed distinct chromosome hybridization signals in 6 micrometer-thick sections. Lung carcinoma cells showed multiple chromosome signals(2~5 spots), contrasted by one or two signals in the stromal cells in the same section. These results suggest that chromosome polysomy can be reliably detected in tissue sections using in situ hybridization. This capability will prove to be an important tool for determining the underlying genetic basis for tumor development, tissue phenotype heterogeneity and progression by allowing genetic determination to be made on paraffin-embedded tissue sections where tumor histologic architecture is preserved.

Immunogistochemical Studies with Carcinoembryonic Antigen in Various Tumors of Skin / 대한피부과학회지

We performed an immunohistochemical study, using bioti-navidin system to clarify the pattern of carcinoembryonic antigen(CEA)immunoreactivity in various tumors of the skin, according to its histogenetic origin. Positive reaction was observed in syringorna, eccrine spiradenoma, eccrine angiomatous hamartoma, eccrine carcinoma, syringocystadenoma papilliferum, metastatic breast carcinoma and mammary Pagets disease. But in epithelial tumors, metastatic skin cancer from maxillary sinus, tumors derived from pilar and sebaceous gland structure, mesenchymal tumors and neuroectodermal tumors, CEA was not observed. These results suggest that immunohistochemical study for CEA could be used as an adjunct for the differential diagnosis of sweat gland tumors from others, especially pilar structure and sebaceous-gland-derived tumors, for the detection of the histogenesis of metastatic tumors, and for the diagnosis of Pagets clisease.

In vitro immunohistochemical localization of S-phase cells by a monoclonal antibody to bromodeoxyuridine

Bromodeoxyuridine, an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase of the cell cycle. In vitro immunohistochemical application of the BrdU/anti-BrdU-MAb method permits a quantitative assessment of the proliferative activity of a tissue as well as the direct location of the actively replicating cells in histological sections. In this paper, a method for the detection of the labeling index of S-phase cells in normal and neoplastic tissues with in vitro BrdU labeling and standard immunohistochemical techniques using anti-BrdU-MAb and avidin-biotin peroxidase complex is described. We have employed this method in 47 human solid tumor samples, including squamous cell carcinomas of head and neck and cervix uteri, adenocarcinomas and malignant lymphomas, and also evaluated the possible application of the BrdU labeling index to estimate the cycling S-phase cells in neoplastic cell populations. In our data, the in vitro labeling index varied greatly in an individual case (3.56-29.2%) and from an area to an area within the same case. Squamous cell carcinomas of the head and neck showed higher LI than those of the cervix uteri. A case of metastatic carcinoma to the lung from ductal carcinoma of the breast had the highest LI (29.2%), in contrast to the low LI (3.6%) in the primary ductal carcinoma of breast.

Detection of Treponema pallidum in Tissues by the Avidin - Biotin - Peroxidase Complex Techniques / 대한피부과학회지

Using the avidin-biotin-peroxidase complex(ABC) technique, 36 skin specimens from 30 patients with primary and secondary syphilis and a gastric mucosal specimen from a patient with secondary syphilis which were confirmed by clinical history, physical examination, the VDRL, FTA-ABS, TPHA, and 19s(IgM)-FTA test, were examined. The results were compared with that of the indirect mmunoperoxidase technique which was done by authors previously with same specimens. The following results were obtained. 1. Of the 37 specimens, 35(95%) were positive in ABC technique and 33 of the 37 specirnens(89%) were positive in the indirect immunoperoxidase technique. 2. The ratio of agreement of the results between the ABC and the indirect immunoperoxidase technique was 89%. 3. Most of the treponemes were located in the upper dermis, epidermis, and blood vessel walls in the arder named, and rarely in the lower dermis of the syphilitic skin lesions. There was no remarkable difference in histologic distribution of treponemes between the clinical stages and types of syphilitic skin lesions. :From the results, the ABC technique is considered to be an excellent method for detecting the treponemes in the suspected syphilitic lesions.

Stick enzyme-linked immunosorbent assay using the avidin–biotin system for detection of circulating antigen in bancroftian filariasis.

Detection of filarial antigen in different groups of sera was carried out by sandwich as well as inhibition enzyme-linked immunosorbent assays using antibody-coated sticks. Both systems were found to be equally sensitive in detecting antigen in 90% of microfilariae carriers. Incorporation of avidin-biotin in the sandwich assay system increased the sensitivity of antigen detection from 10–6 to 10–16 pg. A 67% decrease in the number of false negative results was observed when the sensitive avidin-biotin inhibition enzymelinked immunosorbent assay system was used for analysis of filaria blood samples.

Research of The Titer Critical Value of Anti-Corneum Antibodies in Active Stage of Psoriatic Patients / 昆明医科大学学报

The serous anti-corneum antibodies(ACAs)of 50 psoriatic patients and 32 normal con- trols were measured by Biotin Avidim Enzyme Linked Immunosorbent Assay (BA-ELISA).The results showed that ACAs of patients were significantly lower than that of the controls(P

Immunohistochemical Identification of Keratin in Cutaneous Tumors / 대한피부과학회지

Sixty cutaneous tumors of different types were investigated by means of immunoperoxidase technique(avidin-biotm-peroxidase complex method) with the use of antikeratin antibody. The results were as follows: 1. Squamous cell carcinomas, keratoacanthomas, and Bowen's disease exhibited strong reactivity with antikeratin antibody. 2. In squamous cell carcinomas and keratoacanthomas, the most heaviest staining patterns were detected in areas of keratinization and horn pearl formation. 3. Basal cell carcinomas and seborrheic keratosis were moderately stained, whereas Paget'a disease and metastatic carcinoma(oat cell carcinoma) did not contain keratin. 4. In some cases of basal cell carcinomas, the tumor cells were partially positive. 5. Malignant melanomas, nevi, malignant.lymphomas, neural tumors, hemangiomas, and dermatofibromas were uniformly negative.

Factor VIII Related Antigen in Granuloma Pyogenicum / 대한피부과학회지

Using Avidin-Biotin immunoperaxidase method, we investigate the distribution patterns of factor VIII related antigen in various areas of skin lesions of 14 granuloma pyogenicum. and in the skin lesions of 5 capillary hemangioma and 5 normal skin. The results are summarized as follows: 1. Factor VIII related antigen was detected in 9 of 14 cases of granuloma pyogenicum, in 4 of 5 cases of capillary hemangioma, and in 5 of 5 cases of normal skin. 2. In the lesions of granuloma pyogenicum the areas with prominent capillary endothelial cell proliferation had less positivity than the vascular areas. And the areas with relatively large vesels in the lesions of granuloma pyogenicum had similar positivity as capillary hemangioma. 3. The lesions of granuloma pyogenicum and capillary hemangioma had less positivity than the vessels of normal derrnis.

Solid-phase enzyme. linked immunosorbent assay and its clinical significance for human neutrophil elastase / 中国病理生理杂志

The role of neutrophil in the pathogenesis of many diseases attracts more attentions than before. Neutrophil elastase (NE), a powerful neutrol proteinase capable of causing major tissue destruction in many diseases, is mainly present in the azurophil granules of neutrophil. A solid-phase enzyme-linked immunosorbent-biotin assay for human NE has been developed. Only 20?l of plasma sample is needed. Non-smoking healthy adults have 33.7?6.7ng NE/ml plasma; patients with chronic obstructive pulmonary diseases, 48.0+ 10.9 ng NE/ml; patients with pyogenic dermatisis, 69.1?15.7 ng NE/ml; patients with systemic lupus erythematosus, 61.0?13.9 ng NE/ml; patients with aplastic anemia, only 12.3?5.3 ng NE/ml. All of these results have significant differences compared with the result of healthy adults. The results of assay showed that this quantitative assay with good specificity and accuracy may be a better criterion for investigating the effect of NE on pathogenesis of some diseases.