Anti-mouse IgG-FITC Research Articles

A novel antigen immunochromatography fluorometric strip for rapid detection and application of pathogenic bacterial high-quality antibody

Unlike traditional immunoassay strips, a novel antigen immunechromatography fluorometric strip (AICFS) using inactivated bacterial antigen instead of an antibody as a test line and goat anti-mouse IgG-FITC as a tracer was developed. The applicability survey of AICFS indicated that E. coli O157:H7 (D3) and Acidovorax citrulli (6F) hybridoma cell cultures could be detected, but Vibrio parahemolyticus (H7, C9) hybridoma cell cultures were missed compared with the indirect enzyme-linked immunosorbent assay (ELISA). The four antibody affinity constants (Ka) were measured and compared, and AICFS could be suitable for high-affinity antibody detection. Compared with the traditional indirect ELISA, the AICFS sensitivity for D3 cell cultures, ascites, and purified antibodies was at least 2-fold more sensitive, the AICFS specific for D3 cell cultures by comparative interpretation was compliant except for the strain ATCC 43895, and the indirect ELISA missed it. More importantly, the AICFS method was confirmed by various real samples that it could be used in different scenarios regarding the antibody, including McAb preparation, the effective antibody use, and high-affinity antibody-secreted hybridoma auxiliary preparation and screening. It could be an excellent alternative method with less than 5% corresponding processing time for indirect ELISA method for pathogenic bacterial high-quality antibody detection. This is the first report of using AICFS for bacterial high-quality antibody detection and application in different samples, which demonstrates a rapid auxiliary tool for high-affinity antibody secreted-hybridoma screening and an excellent alternative method for high-quality antibody application.

Development of label-free plasmonic Au-TiO2 thin film immunosensor devices

This work reports on the development of a label-free immunosensor technology, based on nanoplasmonic Au-TiO2 thin films. The Au-TiO2 thin films were prepared by cost-effective reactive DC magnetron sputtering, followed by a thermal annealing procedure. The latter promoted the growth of the Au nanoparticles throughout the TiO2 matrix and induced some morphological changes, which are the base for the immunosensor device functionality. A posterior plasma etching treatment was required to partially expose the nanoparticles to the biological environment. It gave rise to a 6-fold increase of the total area of gold exposed, allowing further possibilities for the sensor sensitivity enhancement. Experimental results demonstrated the successful functionalization of the films' surface with antibodies, with the immobilization occurring preferentially in the exposed nanoparticles and negligibly on the TiO2 matrix. Antibody adsorption surface coverage studies revealed antibody low affinity to the film's surface. Nevertheless, immunoassay development experiments showed a strong and active immobilized antibody monolayer at an optimized antibody concentration. This allowed a 236 signal-to-noise-ratio in a confocal microscope, using mouse IgG and 100 ng/ml of Fab-specific anti-mouse IgG-FITC conjugated. Label-free detection of the optimized antibody monolayer on Au-TiO2 thin films was also tested, revealing an expected redshift in the LSPR band, which demonstrates the suitability for the development of cost-effective, label-free LSPR based immunosensor devices.

Localization of IgG Antigenic Sites in Goat Testicular Tissue

Testis is the tissue with immune privilege which is maintained by blood-testis barrier to protect autoimmunogenic components of testis. IgG have binding affinity for CD16, CD32 and CD64 receptors present on various immune cells. Present study was conducted to investigate the IgG antigenic sites in the testicular tissue of goat. The paraffin embedded histological sections were processed for immunohistochemistry protocol using Anti Mouse IgG FITC conjugate antibodies and observed under fluorescence microscope. Strong reaction was observed in epithelial lining of seminiferous tubule and moderate reaction was observed in lumen of seminiferous tubules and interstitial space. Present studies indicate that the most of IgG antigenic components i.e. CD16, CD32 and CD64 receptors were located in these sites. Current work will help in understanding of immune privilege and provide a baseline on which further studies can be conducted.

Characterization of a primary cell culture from lymphoid organ of Litopenaeus vannamei and use for studies on WSSV replication

Abstract Shrimp aquaculture is a booming agro-industry worldwide. Due to intensification of shrimp farming, pathogens emerge. Control of these pathogens especially viral pathogens is essential for a further expansion of this industry. Until now, the lack of shrimp cell lines has limited research on shrimp viral pathogens. In this context, a primary culture from the lymphoid organ of Litopenaeus vannamei was developed and standardized as a platform for further research on white spot syndrome virus (WSSV). Explants from the lymphoid organ of L. vannamei were cultured in 2 × L-15 (Leibovitz-15) medium supplemented with 20% fetal bovine serum (FBS), 10% Chen's salt, penicillin (100 IU/ml) & streptomycin (100 μg/ml), gentamicin (50 μg/ml) and fungizone (0.25 μg/ml) with a pH of 7.5. Gelatin (0.1%)-coated culture plates promoted the migration of cells from the explants and cell survival. 600 μg/ml cholesterol and 1000 μg/ml l -glutathione (GSH) both enhanced cell survival and performance in vitro. Susceptibility of lymphoid organ cells for infection with white spot syndrome virus (WSSV) was determined by indirect immunofluorescence (IIF) staining by monoclonal antibodies against VP28 (W29) and goat anti-mouse IgG-FITC. FITC positive signals in the nuclei starting from 9 h post inoculation (hpi) demonstrated the susceptibility of the cells for WSSV infection in these cultures. This cell culture system will be used in the future as a tool for studying host–virus interactions in WSSV pathogenesis.

BZLF1 Expression of EBV is correlated with PARP1 Regulation on Nasopharyngeal Carcinoma Tissues

Nasopharyngeal carcinomas (NPC) is a cancer that arises in the epithelial tissue that covers the inside of the nasopharyngeal mucosa and nasopharynx. Infected Epstein Barr Virus (EBV) cell in a latent infection associated with the expression of nine latent proteins. Latent Membrane Protein 1 (LMP1) is one of latent proteins, and mayor EBV oncoprotein, with functions including virus growth, and to activate BamHI-Z Leftward Reading Frame 1 (BZLF1)-EBV, which can inhibit p53 to induce apoptotic resistance, metastasis, and immune modulation. The body will respond to the expansion of EBV infection with activation of Poly(ADP-ribose)Polymerase-1 (PARP1). The objective of study is to observe the expression of BZLF1 and determine PARP1 regulation in nasopharyngeal tissues. NPC-T2, NPC-T3 and polyp tissues slides are from Ulin Hospital, Banjarmasin. To characterize the necrotic cells such as pyknosis, karyorrhexsis, and karyolysis, histological slides were stained by HE that the necrotic cells measured by using a BX-53 microscope (Olympus) with CellSens Standard software. Tissues slides were stained by using immunofluorohistochemistry with EBV-BZLF1 antibody-Mouse anti-EBV monoclonal antibody against Goat anti-mouse IgG-FITC and anti-PARP1 antibody (MC-10) against Goat anti-mouse IgG labeled Rhodamin. The expression intensities were measured by Confocal Laser Scanning Microscope (Olympus). The percentage number of necrotic cells and BZLF1 and PARP1 expression intensity were analyzed using SPSS 16.0 by one-way ANOVA test with α = 0.05, beside that we use correlate and regression analyze. The research showed that the amount of karryorhexis higher than pyknosis and karyolysis in both tissues. BZLF1 expression 1.79 INT/sel (in polyp), 2.76 INT/sel (NPC Type 2) and 4.36 INT/sel (NPC Type 3), PARP1 expression 2.25 INT/sel (in polyp), 3.31 INT/sel (NPC Type 2), dan 5.93 INT/sel (NPC Type 3).The high of intensity of expression BZLF1 induced the increasing of PARP1 expression, but not correlated with percentage of necrotic cell. Interestingly, in all tissues (NPC-T1, NPC-T2 and polyp) is increased number of karyolysis cells properly, but not in pyknosis and karryorrhexis cells. The conclusion of this study indicated that the BZLF1 induce PARP1 to repair DNA damage against EBV infection.Keywords: BZLF1, CLSM, EBV, nasopharyngeal carcinoma, PARP1.

Rad18-mediated Translesion Synthesis of Bulky DNA Adducts Is Coupled to Activation of the Fanconi Anemia DNA Repair Pathway

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.

Synergy between Ficolin-2 and Pentraxin 3 Boosts Innate Immune Recognition and Complement Deposition

The long pentraxin 3 (PTX3) is a multifunctional soluble pattern recognition molecule that is crucial in innate immune protection against opportunistic fungal pathogens such as Aspergillus fumigatus. The mechanisms that mediate downstream effects of PTX3 are largely unknown. However, PTX3 interacts with C1q from the classical pathway of the complement. The ficolins are recognition molecules of the lectin complement pathway sharing structural and functional characteristics with C1q. Thus, we investigated whether the ficolins (Ficolin-1, -2, and -3) interact with PTX3 and whether the complexes are able to modulate complement activation on A. fumigatus. Ficolin-2 could be affinity-isolated from human plasma on immobilized PTX3. In binding studies, Ficolin-1 and particularly Ficolin-2 interacted with PTX3 in a calcium-independent manner. Ficolin-2, but not Ficolin-1 and Ficolin-3, bound A. fumigatus directly, but this binding was enhanced by PTX3 and vice versa. Ficolin-2-dependent complement deposition on the surface of A. fumigatus was enhanced by PTX3. A polymorphism in the FCN2 gene causing a T236M amino acid change in the fibrinogen-like binding domain of Ficolin-2, which affects the binding to GlcNAc, reduced Ficolin-2 binding to PTX3 and A. fumigatus significantly. These results demonstrate that PTX3 and Ficolin-2 may recruit each other on pathogens. The effect was alleviated by a common amino acid change in the fibrinogen-like domain of Ficolin-2. Thus, components of the humoral innate immune system, which activate different complement pathways, cooperate and amplify microbial recognition and effector functions.

RECK Forms Cowbell-shaped Dimers and Inhibits Matrix Metalloproteinase-catalyzed Cleavage of Fibronectin

The membrane-anchored protease regulator RECK plays important roles in mammalian development and tumor suppression. The biochemical bases of these bioactivities, however, remain poorly understood. Here we report on the properties of a recombinant RECK protein expressed in mouse fibroblasts and purified to near homogeneity. Multiple lines of evidence indicate that RECK forms dimers. Single particle reconstruction using transmission electron microscopy revealed a unique cowbell-like shaped RECK dimer. RECK is cleaved by MMP-2 and MMP-7 and competitively inhibits MMP-7-catalyzed cleavage of fibronectin. Forced RECK expression in HT1080 cells, whose endogenous RECK expression is minimal, leads to an increase in the amount of fibronectin associated with the cell. Our data demonstrate the ability of RECK to protect fibronectin from MMP-mediated degradation.

Fabrication of protein chips based on 3-aminopropyltriethoxysilane as a monolayer

Although 3-aminopropyltriethoxysilane (APTES) is widely adopted as a monolayer in biosensors, experimental silanization takes at least 1 h at high temperature. Therefore, the feasibility of the silanization with APTES in a short reaction time and at room temperature was investigated. The surface modification of glass slides using a self-assembled monolayer of APTES with a concentration of 10% was studied by immobilizing FITC. APTES was successfully immobilized on the glass slide. The effect of reaction temperature and time of silanization were investigated. Various silanization conditions of APTES were examined by contact angle measurement and fluorescence microscopy. The surface of glass patterns with a gold thin film as background was characterized by determining the fluorescent intensities following the immobilization of fluorescein isothiocyanate (FITC), protein A-FITC, antimouse IgG-FITC and sheep anti-bovine albumin-FITC. The normalized fluorescent intensity indicated that a short period (4 min) of silanization at 25 degrees C suffices to form an APTES thin film by the immobilization of protein A on a glass surface. Such a condition does not require microheaters and temperature sensors in a microfluidic system, which will significantly reduce the manufacturing process, cost, and reaction time in the future.

Increased thermoregulation in cold-exposed transgenic mice overexpressing lipoprotein lipase in skeletal muscle: an avian phenotype?

LPL is an enzyme involved in the breakdown and uptake of lipoprotein triglycerides. In the present study, we examined how the transgenic (Tg) overexpression of human LPL in mouse skeletal muscle affected tolerance to cold temperatures, cold-induced thermogenesis, and fuel utilization during this response. Tg mice and their nontransgenic controls were placed in an environmental chamber and housed in metabolic chambers that monitored oxygen consumption and carbon dioxide production with calorimetry. When exposed to 4 degrees C, an attenuation in the decline in body temperature in Tg mice was accompanied by an increased metabolic rate (15%; P < 0.001) and a reduction in respiratory quotient (P < 0.05). Activity levels, the expression of uncoupling proteins in brown fat and muscle, and lean mass failed to explain the enhanced cold tolerance and thermogenesis in Tg mice. The more oxidative type IIa fibers were favored over the more glycolytic type IIb fibers (P < 0.001) in the gastrocnemius and quadriceps muscles of Tg mice. These data suggest that Tg overexpression of LPL in skeletal muscle increases cold tolerance by enhancing the capacity for fat oxidation, producing an avian-like phenotype in which skeletal muscle contributes significantly to the thermogenic response to cold temperatures.

N-Glycosylation as Novel Strategy to Improve Pharmacokinetic Properties of Bispecific Single-chain Diabodies

The therapeutic efficacy of recombinant antibodies such as single-chain Fv fragments and small bispecific or bifunctional molecules is often limited by rapid elimination from the circulation because of their small size. Here, we have investigated the effects of N-glycosylation on the activity and pharmacokinetics of a small bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. We could show that the introduction of N-glycosylation sequons into the flanking linker and a C-terminal extension results in the production of N-glycosylated molecules after expression in transfected HEK293 cells. N-Glycosylated scDb variants possessing 3, 6, or 9 N-glycosylation sites, respectively, retained antigen binding activity and bispecificity for target and effector cells as shown in a target cell-dependent IL-2 release assay, although activity was reduced approximately 3-5-fold compared with the unmodified scDb. All N-glycosylated scDb variants exhibited a prolonged circulation time compared with scDb, leading to a 2-3-fold increase of the area under curve (AUC). In comparison, conjugation of a branched 40-kDa PEG chain increased AUC by a factor of 10.6, while a chimeric anti-CEA IgG1 molecule had the longest circulation time with a 17-fold increase in AUC. Thus, N-glycosylation complements the repertoire of strategies to modulate pharmacokinetics of small recombinant antibody molecules by an approach that moderately prolongs circulation time.

Development of a flow cytometry based method for rapid and sensitive detection of a novel marine fish iridovirus in cell culture

A sensitive and accurate flow cytometry (FCM) based method has been developed to detect and quantitate a novel marine fish iridovirus (Singapore grouper iridovirus, SGIV) after amplification in cell cultures. Confluent grouper cell (GP) monolayers were infected with SGIV. When advanced cytopathic effect (CPE) appeared, the cell cultures were fixed and permeabilized, and then reacted with monoclonal antibodies specific against SGIV, followed by a second antibody conjugated with FITC (anti-mouse IgG-FITC). A Coulter ® EPICS Elite ESP flow cytometer was used to directly detect and analyze the percentage of virus-infected cells. Three fixation and permeabilization methods were evaluated. The kinetics of the virus infection process was determined. The FCM procedure enables large amounts of cells to be screened rapidly for infectivity, and it can also detect low levels of virus infection. As early as 8 h after inoculation with the virus, 0.34% of infected cells were detected in cell culture. The maximum level of infection was obtained at 72 h. The efficiency and reliability of the FCM procedure were compared with those of the standard methods of immunofluorescence microscopy and PCR.

Predictive Diagnostic and Prognostic Value of Platelets Microparticles (PMP), but Not of Reticulated Platelets (RP) or CD36 Expression in Essential Thrombocythaemia.

At the present time, Essential Thrombocythaemia (ET) remains an exclusion diagnostic since no diagnostic finding can definitely distinguish between clonal and reactive thrombocytosis (RT). In addition, no biological marker predicts readily the thrombotic risk of ET. We investigated by flowcytometry the diagnosis and prognosis value of PMP, RP and CD36 expression, as platelets parameters easily available in routine laboratory in 34 ET patients and 49 controls. Membrane platelet CD36 expression were stained by indirect immunofluorescence with specific anti-CD36 antibody and anti mouse IgG FITC (Biocytex, France), analyzed on FACSCalibur cytometer (BD), quantified using the calibration beads (Platelet Calibrator, Biocytex) and expressed as antigen molecules/platelet. RP were determined after RNA thiazole orange labeling (10 ng/ml, 45 min, at room temperature, in the dark). Assays performed after TRAP (Thrombin receptor activating peptide) stimulation demonstrated the specificity of the labeling (data not shown). PMP were identified on forward and side scatter after CD41a-PE or isotype control (Immunotech) labeling. ET was diagnosed according to revised PVSG criteria in 11 men and 23 women. The median age was 63 y (24–85) and the median platelet count was 674 x109/l (249–1324). At the time of evaluation, 19 patients received antiplatelets therapy, 9 oral chemotherapy, 10 exhibit arterial thrombosis (AT), 1 venous thrombosis and one, included in the AT group, experimented venous thrombosis as well. RP, CD36 and PMP were significantly increased in ET as compared to controls (P =.01, 10−4, <10−5, respectively). RP and CD36 expression were not significantly different between patients according to AT, antiplatelets agents and oral chemotherapy patterns. However PMP were significantly higher in patients with AT than in patients without thrombosis history (P =.02). We could not observe any decrease of PMP in patients with antiplatelets drugs since all patients with thrombosis had also antiplatelets therapy. In conclusion, RP, CD36 and PMP are increased in ET. These platelets parameters are currently evaluated in RT. The effects of antiplatelets agents on PMP and the thrombotic risk will have to be evaluated prospectively.NRP (platelet %)CD36 (Nb/platelet)PMP (Nb/platelet)control493.86 (1.16–21.2)9164 (4141–16254)4632 (2073–13225)ET345.78 (2.24–15.52)10734 (5155–19920)27928 (10905–117669)Arterial Thrombosis106.73 (2.93–12.52)9613 (5441–16096)29948 (10905–117669)No thrombosis235.78 (2.24–15.5)11024 (5155–19920)26173 (12377–72895)aspirin/clopidogrel196.14 (2.93–13.75)10543 (5155–16275)32280 (10905–117669)no aspirin155.78 (2.24–15.52)10925 (8992–19929)23586 (12377–72895)hydroxyurea95.02 (2.24–13.84)14478 (8560–19924)37451 (17315–117669)no hydroxyurea256.84 (2.91–15.52)10012 (5155–16275)26171 (12377–72895)

Cholesterol-independent Interactions with CD47 Enhance αvβ3 Avidity

Expression in OV10 cells of either wild-type CD47 or its extracellular IgV domain linked to a glycosylphosphatidylinositol anchor-(IgV-GPI) enhanced ligand-induced alpha(v)beta(3) activation as detected by the binding of LIBS1 and LIBS6 mAbs. The amplitude of LIBS binding was greater with both CD47 and IgV-GPI expression, indicating an increase in the population of "activable" integrin molecules. Expression of either CD47 species also increased alpha(v)beta(3)-mediated adhesion to vitronectin, and to surfaces coated with the anti-beta(3) antibody AP3, because of enhanced clustering of alpha(v)beta(3) as confirmed by chemical cross-linking. Cholesterol depletion with methyl-beta-cyclodextrin did not prevent the increase in anti-LIBS binding, but reduced cell adhesion to vitronectin and AP3. However, cells expressing CD47 were partially insulated against this disruption, and IgV-GPI was even more effective. Both CD47 and IgV-GPI were found in cholesterol-rich rafts prepared in the absence of detergent, but only CD47 could recruit alpha(v)beta(3) and its associated signaling molecules to these domains. Thus CD47-alpha(v)beta(3) complexes in cholesterol-rich raft domains appear to engage in G(i)-dependent signaling whereas CD47-alpha(v)beta(3) interactions that lead to integrin clustering are also detergent resistant, but are insensitive to cholesterol depletion and do not require the transmembrane region of CD47.

Temporary disturbance of actin stress fibers in swine kidney cells during pseudorabies virus infection

Rounding and loosening of cells is a consequence of infection with pseudorabies virus (PrV), both in vitro and in vivo. These changes in the normal structure of the cell may be the result of cytoskeletal changes. Immunofluorescence staining of actin filaments and microtubule bundles was performed to examine whether PrV induces a reorganization of these cytoskeletal components in infected swine kidney (SK) cells. Every 2 h until 12 h post-inoculation (p.i.), cells were washed in cytoskeleton stabilizing buffer (CSB), fixed with paraformaldehyde and washed again with CSB. Cells were permeabilized with a 1/1000 dilution of Triton X-100 and actin filaments were stained by incubating cells with phalloidin-Texas Red. Staining of microtubules was done by incubating the cells subsequently with mouse monoclonal anti-α-tubulin and goat anti-mouse IgG-FITC. During the course of infection, actin fibers of SK cells were rearranged in the following sequence: (1) disappearance of thick actin stress fibers between 4 and 6 h p.i., (2) complete loss of stress fibers between 6 and 8 h p.i., and (3) reappearance of thin stress fibers starting from 10 h p.i. In contrast to herpes simplex virus 1 (HSV1) or equine herpesvirus 1 (EHV1), PrV infection did not induce changes in the cellular microtubule network. PrV infection induces a temporary disassembly of actin stress fibers.

Flow cytometry and RT-PCR for rotavirus detection in artificially seeded oyster meat

A flow cytometry (FC)-based method was developed for the detection of rotavirus in oyster meat using simian rotavirus SA11 as a model. To study virus recovery, oyster meat was injected with rotavirus and the oyster extract used to infect MA104 cell monolayers. Following varying periods of infection, the cells were recovered and reacted with the monoclonal antibody M60 which is specific for the rotavirus group A serotypes 1–4 outer capsid protein, VP7, followed by a second antibody (anti mouse IgG-FITC). A FACScan ® FC was used to estimate the number of infected cells as well as the level of infection. To evaluate the sensitivity of the method, non-inoculated oysters were processed following the same extraction protocol and, at the end, they were seeded with the same amount of virus used for oyster inoculation. This seeded oyster extract was then used to infect MA104 cells and the number of infected cells determined using the same FC procedure. A semi-nested two-step PCR for detection of rotavirus nucleic acid was undertaken to compare the sensitivity of FC with RT-PCR. Using FC, as little as 0.02 flow cytometry units (fcu) (number of infected cells counted by FC) could be detected after 72 h of cell infection. This is a very similar limit of sensitivity to that obtained with RT-PCR. Both methods are approximately 100 times more sensitive than the plaque-forming units (pfu) assay.

Fibrin monomer increases platelet adherence to tumor cells in a flowing system: A possible role in metastasis?

Considerable evidence exists linking hemostasis and malignancy. Platelet adhesion to tumor cells has been implicated in the metastatic process. Plasma fibrinogen (Fg) and fibrin (Fn) monomer, increased in cancer, may play a role in tumor biology. Binding of Fn monomer to tumor cells and its effect on platelet-tumor cell adhesion in a flowing system were studied. Fn monomer was produced by adding thrombin (1 μ/mL) to FXIII- and plasminogen-free Fg in the presence of Gly-Pro-Arg-Pro (GPRP) amide. Fn monomer binding to live A375 cells was visualized by confocal laser scanning microscopy (CLSM). Adherent cells were perfused for 1h with Fn monomer, washed and stained in situ with anti-human Fn (American Biogenetic Sciences, Inc.) followed by goat anti-mouse IgG FITC. Platelet adherence to Fn monomer treated A375 cells was performed under flow conditions by passing platelets (5×10 4/μl; 0.25 mL/min; labeled with the carbocyanine dye DiI) over the tumor cells for 30 min. CLSM images were obtained after washing. There was considerable binding of Fn monomer, but not Fg alone. Platelets adhered relatively weakly to untreated A375 cells and this was not significantly affected by pre-treatment of the tumor cells with fibrinogen or thrombin. However, pre-treatment with Fn monomer resulted in extensive platelet binding to tumor cells, suggesting that coagulation activation and the subsequent increase in circulating Fn monomer may enhance platelet adhesion to circulating tumor cells and thereby facilitate metastatic spread.

Development of a rapid and sensitive quantitative assay for rotavirus based on flow cytometry

A very sensitive and accurate flow cytometry (FC) based method have developed to quantitate rotavirus infection in MA104 cells. Confluent cell monolayers were infected with serial dilutions of rotavirus SA11. After infection, the cells were recovered with the aid of trypsin and then reacted with monoclonal antibody M60 (specific for the rotavirus outer capsid protein, VP7), followed by a second antibody (anti-mouse IgG-FITC). A FACScan FC was used to estimate the number of infected cells, as well as the level of infection. Viral infection was optimised by varying the concentration of trypsin used in the maintenance medium. The FC method enables many cells to be screened quickly for infectivity, and can detect low levels of virus. This method can be adapted to monitor the presence of other viruses in clinical and environmental samples without the need for prolonged periods of adaptation to growth in tissue culture.

Detection of the Variant Frequency in Human Erythrocytes using Glycophorin a Locus Mutation Assay

Glycophorin A (GPA) is the major glycoprotein on human erythrocyte membranes, and bears M and N blood group determinants. Genotoxic exposure causes erythroid precursor cells to mutate at the glycophorin. A locus, producing phenotypes of rare variant erythrocytes that lack the expression of one form of the protein. The BR6 assay is now the most widely used method to determine the variant frequency (Vf) by immunolabelling and flow cytometry. In this study, two kinds of labelling were used. Second antibody labelling, namely BRIC157-sheep anti-mouse IgG-FITC binding, increased the sensitivity, while the specificity and the precision decreased. NO/NN Vf of 10 normal samples conform with previous reports, 7.96 ± 3.05/2.24 ± 1.05 (x10-6) and 10.12 ± 3.2/3.95 ± 1.02 (x10-6) of direct and indirect dyeing respectively. Some factors affect the dyes emitting fluorescence, and the optimal temperature of immunolabelling is under 20(C.

Diffusion and transfer of antibody proteins from a sugar-based hydrogel.

Diffusion of antibody protein from hydrogel films and hydrogel encapsulated in a microcapillary was studied. Thin hydrogel films were formed by crosslinking 6-acryloyl-B-O-methylgalactoside with N,N'-methylene-bis-acrylamide and the diffusive transport of monoclonal antimouse IgG-FITC into and out of the hydrate films was measured. Diffusion coefficients in 2 and 4% crosslinked hydrogel films were measured. The measured diffusion constants determined for IgG in both the 2 and 4% hydrogel films were comparable to the free diffusion of IgG in bulk water (Dmean approximately 10(-7) cm2/s). In addition, 2% crosslinked hydrogels were prepared in a capillary tube and the transport of antimouse IgG-FITC into and out of the hydrated hydrogel was measured. Kinetic analysis indicated that the protein transport through the capillary hydrogel was faster than would be expected for a simple diffusion process. Finally, by utilizing the diffusion of antibody from the capillary hydrogel, transfer of antibody to a silica surface was demonstrated. A capillary hydrogel loaded with antimouse IgG-FITC was used to transfer the protein to a silica surface forming a 30-micron spot of antibody, which was imaged using fluorescence microscopy. These results may lead to the development of a nonlithographic method of patterning antibodies on surfaces for use in integrated microimmunosensors.