FITC-labeled Protein Research Articles

Features of phagocytic and chemiluminescent activity of neutrophils when exposed to polyprenols <i>in vitro</i>

Aim: Studying of the features of phagocytic activity and the state of the respiratory burst of neutrophils under the influence of polyprenols in vitro.
 Materials and methods. The study involved 30 relatively healthy donors aged 23-59 years from whose peripheral blood neutrophilic granulocytes were isolated to determine phagocytic and chemiluminescent activity. The isolated cells were incubated for 30 minutes with polyprenols in vitro. The level of phagocytosis of immature and mature neutrophils was determined by flow cytometry using FITC-labeled staphylococcal protein A. The state of the respiratory burst was assessed using chemiluminescent analysis.
 Results. It was found that the levels of phagocytic index (PI) and number (PN) of mature neutrophils were reduced after incubation with polyprenols relative to initial and control values. The values of PI and PN of immature neutrophils under the influence of polyprenols and during control incubation were decreased relative to the initial values. The intensity maximum and area under the curve of spontaneous and zymosan-induced lucigenin- and luminol-dependent chemiluminescence of the neutrophils were reduced after incubation with polyprenols.
 Conclusion. The effect of polyprenols on neutrophilic granulocytes in vitro leads to a decrease in activity in respiratory burst cells, which is realized in blocking by polyprenols the interaction of primary and secondary ROS with chemiluminescent indicators by the mechanism of primary antioxidants. The binding of ROS by polyprenols also manifests itself in a decrease in the phagocytic activity of mature neutrophils while maintaining the phagocytic activity of immature cells at the level of control incubation. In general, polyprenols exhibit anti-inflammatory activity when exposed in vitro to neutrophilic granulocytes.

Site-specific internal protein labeling through trans-splicing

Atypical S1 and S11 split inteins have been used for N-terminal or C-terminal protein labeling. Here we reported a novel site-specific internal protein labeling method based on two atypical split inteins, Ter DnaE3 S11 and Rma DnaB S1. Protein-peptide trans-splicing activity was first demonstrated in vitro between a short peptide (Flag tag, FLAG) and two recombinant proteins (Maltose binding protein, MBP, and Thioredoxin, Trx) by trans-splicing between MBP-TE3S11N (MBP-N fragment of Ter DnaE3 S11), TE3S11C-FLAG-RBS1N (C fragment of Ter DnaE3 S11-FLAG-N fragment of Rma DnaB S1), and RBS1C-Trx (C fragment of Rma DnaB S1-Trx). To minimize the middle synthetic peptide (TE3S11C-linker-RBS1N), we reduced the number of native extein amino acids, which may play a role in protein trans-splicing. The results showed at least 3 (CKG) native extein amino acids were required for detectable trans-splicing activity. This method was further demonstrated to be effective in facilitating the incorporation of fluorescent probe (FITC) to the internal site of recombinant protein, generating the FITC-labeled protein. Besides the fluorescent group, these two split inteins can also be useful for adding any desirable chemical groups into a protein of interest, which may include biotin, modified and unnatural amino acids, or drug molecules.

Quality Control Of FITC-labeled Proteins for Interactomics Investigations Using SDS Capillary Gel Electrophoresis and SPR Biosensor

The technology of dye-labeled proteins has many fields of application, especially in interactomics. The aim of this work was to adapt protocol of conjugation of low molecular weight (12 – 15 kDа) heme-containing proteins with fluorescein isothiocyanate, isomer I, (FITC) for subsequent protein-protein interaction studies. We have monitored the quality of FITC-labeling of the target protein and comparative assessment of its binding capacity. Using the cytochrome C (Mw 12 kDа) as an example, it has been shown that using the three step method approach including conventional spectrophotometry, capillary gel electrophoresis and SPR analysis it is possible to assess: (i) the capability of the FITC-labeled target protein to interact with its protein partner and protein material from tissue lysates, (ii) the fact of dye conjugation with the protein, and (iii) the quality of purification for final protein preparation from unreacted free dye molecules

Pore-forming mechanism of TUBP1 protein act on Verticillium dahliae

Abstract Background In our previous study, the novel anti-Verticillium dahliae protein TUBP1 was isolated from Bacillus axarquiensis, and the anti-V. dahliae effect was investigated. In the present study, the membrane-active action of the TUBP1 protein on V. dahliae was evaluated. Result Experiments were conducted to examine plasma membrane lesions and membrane depolarization, which were dyed using propidium iodide (PI) and 3,3`-dipropylthiacarbocyanine [DiSC3(5)], respectively. The results showed perturbation of the plasma membrane of the spores and the hyphae of V. dahliae when they were treated with the TUBP1 protein. Atomic absorption spectrometry (AAS) was used to analyze the K+ concentration. The results showed that through releasing K+, the TUBP1 protein induced membrane damage and depolarization. FITC-labeled TUBP1 protein influx, calcein leakage from liposomes, and pore size of the spores and hyphae of V. dahliae experiments were performed with FITC-labeled TUBP1 protein, calcein-encapsulating large unilamellar vesicles (LUV), and edextran (FD) loaded LUVs, respectively. The results demonstrated that the negative effect of the TUBP1 protein on spores and hyphae plasma membranes of V. dahliae was apparent in the formation of pores with radii from 1.4 to 2.3 nm. Finally, further morphological changes in V. dahliae spores and hyphae induced by the TUBP1 protein were observed in the results of flow cytometry and scanning electron microscopy (SEM). Conclusion These results showed that in three-dimensional flow cytometric contour plots, cell sizes were reduced by the pore-forming action of TUBP1 protein.

PHAGOCYTIC ACTIVITY AND BLOOD NEUTROPHILS RESPIRATORY BURST STATE FEATURES AMONGST WIDESPREAD PURULENT PERITONITIS PATIENTS IN THE POSTOPERATIVE PERIOD DYNAMICS

The research aim was to study the phagocytic activity and the blood neutrophils respiratory burst state amongst the patients with widespread purulent peritonitis (WPP) in the dynamics of the postoperative period. The study was involved patients with acute surgical diseases and injuries of the abdominal organs complicated by WPP. Blood sampling was performed prior to surgery (pre-operative period) and at 7, 14 and 24 day post-operative period. The neutrophil phagocytosis level was determined by flow cytometry using FITC-labeled staphylococcal protein A. There was counted the percentage of fluorescent neutrophils (defined as the phagocytic index — PI) and average of the cell fluorescence (phagocytic number — PN). According to the fluorescence intensity divided phagocytic neutrophils in an active (high PN) and weakly phagocytic (low PN). Condition of the respiratory burst in the neutrophils was examined using a chemiluminescent assay. It was found that patients with WPP in the preoperative period had increased phagocytic activity of the blood neutrophils. The maximum phagocytic activity observed on day 7 of the postoperative period and by 24 days the number of phagocytic cells is reduced to control level whereas phagocytic activity of the cells remains at the preoperative level. In the preoperative period in patients with WPP the amount of active phagocytic blood neutrophils 3.3 times higher than the number of weakly phagocytic cells. By the end of the observation period the number of active and weakly phagocytic neutrophils returning to normal. In the study of the state of respiratory burst in the neutrophils by the WPP patients discovered that the primary synthesis levels of reactive oxygen species (ROS) increases only with 7 days of postoperative period and the induced activity of NADPH-oxidase remains elevated to the end of the observation period. At the same time, the synthesis of secondary ROS in the blood neutrophil of the WPP patients increased already in the preoperative period and remains high until the end of the survey. Using correlation analysis it was found that if healthy people had a competitive relationship between the level of synthesis of primary ROS and the activity of phagocytosis then all detected correlations in the WPP patients characterized by high level of coordination between the respiratory burst and the phagocytic activity of the neutrophils. These relationships are identified in the preoperative period, postoperatively do not exist. It can be assumed that the surgery and the subsequent treatment leads to complete incoordination in the neutrophils the mechanisms of phagocytosis and respiratory burst.

Sustained release of rhBMP-2 from microporous tricalciumphosphate using hydrogels as a carrier.

BackgroundTissue engineering and bone substitutes are subjects of intensive ongoing research. If the healing of bone fractures is delayed, osteoinductive materials that induce mesenchymal stem cells (MSCs) to form bone are necessary. The use of Bone Morphogenetic Protein - 2 is a common means to enhance effectiveness and accelerate the healing process. A delivery system that maintains and releases BMP biological activity in controlled fashion at the surgical site while preventing systemic diffusion (and thereby the risk of undesirable effects by controlling the amount of protein implanted) is essential.In this study, we aimed to test a cylindrical TCP-scaffold (porosity ~ 40 %, mean pore size 5 μm, high interconnectivity) in comparison to BMP-2. Recombinant human BMP-2 was dissolved in different hydrogels as a carrier, namely gelatin and alginate cross-linked with CaCl2-solution, or a solution of GDL and CaCO3. FITC-labeled Protein A was used as a model substance for rhBMP-2 in the pre-trials. For loading, the samples were put in a flow chamber and sealed with silicone rings. Using a directional vacuum, the samples were loaded with the alginate-BMP-2-mixture and the loading success monitored by observing changes in a fluorescent dye (FITC labeled Protein A) under a fluorescence microscope. A fluorescence reader and ELISA were employed to measure the release. Efficacy was determined in cell culture experiments (MG63 cells) via Live-Dead-Assay, FACS, WST-1-Assay, pNPP alkaline phosphatase assay and confocal microscopy. For statistical analysis, we calculated the mean and standard deviation and carried out an analysis of variance.ResultsDirectional vacuum makes it possible to load nearly 100 % of the interconnected micropores with alginate mixed with rhBMP-2. Using alginate hardened with CaCl2 as a carrier, BMP-2's release can be decelerated significantly longer than with other hydrogels - eg, for over 28 days. The effects on osteoblast-like cells were an increase of the growth rate and expression of alkaline phosphatase while triggering no toxic effect.ConclusionThe rhBMP-2-loaded microporous TCP scaffolds possess proliferative and osteoinductive potential. Alginate helps to lower the local growth factor dose below the cytotoxic limit, and allows the release period to be lengthened by at least 28 days.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0275-8) contains supplementary material, which is available to authorized users.

Intracellular Trafficking Network of Protein Nanocapsules: Endocytosis, Exocytosis and Autophagy.

The inner membrane vesicle system is a complex transport system that includes endocytosis, exocytosis and autophagy. However, the details of the intracellular trafficking pathway of nanoparticles in cells have been poorly investigated. Here, we investigate in detail the intracellular trafficking pathway of protein nanocapsules using more than 30 Rab proteins as markers of multiple trafficking vesicles in endocytosis, exocytosis and autophagy. We observed that FITC-labeled protein nanoparticles were internalized by the cells mainly through Arf6-dependent endocytosis and Rab34-mediated micropinocytosis. In addition to this classic pathway: early endosome (EEs)/late endosome (LEs) to lysosome, we identified two novel transport pathways: micropinocytosis (Rab34 positive)-LEs (Rab7 positive)-lysosome pathway and EEs-liposome (Rab18 positive)-lysosome pathway. Moreover, the cells use slow endocytosis recycling pathway (Rab11 and Rab35 positive vesicles) and GLUT4 exocytosis vesicles (Rab8 and Rab10 positive) transport the protein nanocapsules out of the cells. In addition, protein nanoparticles are observed in autophagosomes, which receive protein nanocapsules through multiple endocytosis vesicles. Using autophagy inhibitor to block these transport pathways could prevent the degradation of nanoparticles through lysosomes. Using Rab proteins as vesicle markers to investigation the detail intracellular trafficking of the protein nanocapsules, will provide new targets to interfere the cellular behaver of the nanoparticles, and improve the therapeutic effect of nanomedicine.

The Perforin Pore Facilitates the Delivery of Cationic Cargos

Cytotoxic lymphocytes eliminate virally infected or neoplastic cells through the action of cytotoxic proteases (granzymes). The pore-forming protein perforin is essential for delivery of granzymes into the cytoplasm of target cells; however the mechanism of this delivery is incompletely understood. Perforin contains a membrane attack complex/perforin (MACPF) domain and oligomerizes to form an aqueous pore in the plasma membrane; therefore the simplest (and best supported) model suggests that granzymes passively diffuse through the perforin pore into the cytoplasm of the target cell. Here we demonstrate that perforin preferentially delivers cationic molecules while anionic and neutral cargoes are delivered inefficiently. Furthermore, another distantly related pore-forming MACPF protein, pleurotolysin (from the oyster mushroom), also favors the delivery of cationic molecules, and efficiently delivers human granzyme B. We propose that this facilitated diffusion is due to conserved features of oligomerized MACPF proteins, which may include an anionic lumen.

A Neutralization scFv Antibody against IL-1β Isolated from a NIPA-based Bacterial Display Library

RA is one of autoimmune diseases, has drawn great attention of the world. Currently, the anti- IL-1β monoclonal antibody Canakinumab (ACZ885) for treatment of RA has entered into clinical trials. However, Full length antibody has large molecular weight, and is difficult to penetrate the tissue or the nidus. In contrast, scFv has low molecular weight and strong penetration ability, and is favorable to increase the drug concentration in the indus, hence improving the efficacy of the drug. The aim of this study is to obtain a neutralizing scFv antibody from a combinatorial scFv library against hIL-1β by the modified NLPA-based bacterial display system, for further development of the small molecule antibody drug for treatment of RA. The modified NIPA-based bacterial display system was used to construct the combinatorial scFv library derived from the spleen cDNA of immunized mice with hIL-1β. FACS was used to screen hIL- 1β-binding clones with FITC-labeled hIL-1β protein. Three clones were randomly selected from the third round of screening, and their nucleotide sequences were aligned with mouse immunoglobulin genes. The single chain antibody genes of the hIL-1β-binding clones were subcloned into the prokaryotic expression vector pET-27b for expression. The molecular mass of the purified anti-hIL-1β single chain antibody was about 28ku. The hIL-1β-binding ability of antibody were examined by ELISA and Western blot assays. Ability of the scFv antibody to neutralize hIL-1β was evaluated by the MTT test. In this study, it is the first time to use the NIPA-based bacteria display system to construct and screen the combinatorial scFv library. Three scFvs against hIL-1β were obtained from the scFv library of the immunized mice. Prokaryotically expressed and purified scFvs demonstrate binding ability with hIL-1β. Among the three clones. The MTT test suggests that scFv-20 is a neutralization antibody against hIL-1β. The study provides a lead candidate for further development of small molecule therapeutic antibodies for treatment of RA.

Hepatopoietin Cn suppresses apoptosis of human hepatocellular carcinoma cells by up-regulating myeloid cell leukemia-1

To investigate the role of hepatopoietin Cn (HPPCn) in apoptosis of hepatocellular carcinoma (HCC) cells and its mechanism. Two human HCC cell lines, SMMC7721 and HepG2, were used in this study. Immunostaining, Western blotting and enzyme linked immunosorbent assay were conducted to identify the expression of HPPCn and the existence of an autocrine loop of HPPCn/HPPCn receptor in SMMC7721 and HepG2. Apoptotic cells were detected using fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide. The HPPCn was highly expressed in human HCC cells and secreted into culture medium (CM). FITC-labeled recombinant human protein (rhHPPCn) could specifically bind to its receptor on HepaG2 cells. Treatment with 400 ng/mL rhHPPCn dramatically increased the viability of HCC-derived cells from 48.1% and 36.9% to 85.6% and 88.4%, respectively (P < 0.05). HPPCn silenced by small-interfering RNA reduced the expression and secretion of HPPCn and increased the apoptosis induced by trichostatin A. Additionally, HPPCn could up-regulate the expression of myeloid cell leukemia-1 (Mcl-1) in HCC cells via mitogen-activated protein kinase (MAPK) and sphingosine kinase-1. HPPCn is a novel hepatic growth factor that can be secreted to CM and suppresses apoptosis of HCC cells by up-regulating Mcl-1 expression.

Binding of heat shock protein 70 to extracellular phosphatidylserine promotes killing of normoxic and hypoxic tumor cells

Hypoxia is well known to limit curability of tumors by ionizing radiation. Here, we show that hypoxia treatment of tumor cells causes coexpression of heat shock protein 70 (Hsp70) and phosphatidylserine (PS) on the cell surface. Colocalization of Hsp70 and PS, as determined by confocal microscopy, also occurs when exogenous FITC-labeled Hsp70 protein is added to normoxic and hypoxic tumor cells. Moreover, the interaction of Hsp70 with PS was demonstrated in artificial unilamellar phosphatidylcholine/ phosphatidylserine (PC/PS) liposomes at the physiological ratio of 8/2. Indeed, the Hsp70-liposome interaction gradually increased with elevating PS molar ratios (8/2 > or = 7/3 < 5/5 < 4/6 < 3/7 < 2/8). In contrast, only a weak Hsp70 interaction was detected in phosphatidylcholine/phosphatidylglycerol (PC/PG) liposomes, thus demonstrating that the interaction was not a charge-related effect. The interaction of Hsp70 with surface PS significantly reduces clonogenic cell survival in normoxic (EC(50) of Hsp70=85 microg/ml) and hypoxic (EC(50) of Hsp70=55 microg/ml) tumor cells. The radiation-induced tumor cell killing was significantly enhanced by the addition of Hsp70 protein (50 microg/ml). Since apoptosis was not significantly enhanced in normoxic and hypoxic tumor cells by the addition of Hsp70, we hypothesize that the Hsp70 protein-induced reduction in clonogenic cell survival might be through necrosis rather than apoptosis.

Hyaluronan Binding to Link Module of TSG-6 and to G1 Domain of Aggrecan Is Differently Regulated by pH

The physiological functions of hyaluronan (HA) in the extracellular matrix of vertebrate tissues involve a range of specific protein interactions. In this study, the interaction of HA with the Link module from TSG-6 (Link_TSG6) and G1 domain of aggrecan (G1), were investigated by a biophysical analysis of translational diffusion in dilute solution using confocal fluorescence recovery after photobleaching (confocal FRAP). Both Link_TSG6 and G1 were shown to bind to polymeric HA and these interactions could be competed with HA(8) and HA(10) oligosaccharides, respectively. Equilibrium experiments showed that the binding affinity of Link_TSG6 to HA was maximal at pH 6.0, and reduced dramatically above and below this pH. In contrast, G1 had maximum binding at pH 7.0-8.0 and moderate to strong binding affinity over a much broader pH range (5.5-8.0). The K(D) determined for Link_TSG6 binding to HA showed a 100-fold increase in binding affinity between pH 7.4 and 6.0, whereas G1 showed a 75-fold decrease in binding affinity over the same pH range. The sharp difference observed in their pH binding suggests that pH controls the physiological function of TSG-6, with a low affinity for HA at neutral pH, but with increased affinity as the pH falls below pH 7. TSG-6 and aggrecan interact with HA through structurally homologous domains and the difference in pH-dependent binding can be understood in terms of differences in the presence and topographical distribution of key regulatory amino acids in Link_TSG6 and in the related tandem Link domains in aggrecan G1.

CE‐based noncompetitive immunoassay for immunoglobulin G in bovine colostrum products

A CE-based noncompetitive immunoassay for IgG in bovine colostrum products was established. FITC-labeled protein G (FITC-PrG) was tagged through noncovalent bindings to the Fc region of the mouse monoclonal antibovine IgG (Ab). The FITC-PrG, Ab, and IgG formed a sandwiched immunocomplex FITC-PrG-Ab-IgG under optimal incubation conditions. The immunocomplex was separated and analyzed by CZE with LIF detection in less than 2 min in an uncoated fused-silica capillary. Addition of PEG 20,000 (PEG 20M) in the running buffer significantly suppressed analyte adsorption and thus improved the reproducibility and the resolution. The precision of the method was 5.1% (n = 7). A linear relationship was established for the IgG concentration in the range of 1-5 mg/L with a linear correlation coefficient (r = 0.9917). The LOD was 0.1 mg/L (S/N = 3). The method was successfully applied for the determination of IgG in bovine colostrum products and satisfactory results were achieved.

Abstract 1007: Platelet Glycoprotein VI (GPVI) Deletion Reduces Myocardial Infarction in Mice

Fibrillar collagen, exposed by damage of vascular endothelium, is the most thrombogenic component of the subendothelium, which, in addition to supporting platelet adhesion, is also a potent platelet activator. Platelet glycoprotein VI (GPVI) plays a major role in collagen-induced platelet activation, leading to thrombus formation. Inhibition of GPVI function may be a novel anti-platelet therapy with minimal bleeding risk. Recent evidence in FcRγ-chain-knockout (KO) mice, which also lack GPVI, shows attenuated myocardial infarction after ischemia and reperfusion. Unlike GPVI, which is exclusively expressed in platelets and megakaryocytes, FcRγ-chain also exists in white blood cells and immune cells. Thus, it is unclear whether GPVI deletion is cardioprotective. In this study, we compared myocardial infarction in GPVI-KO and age-matched wild-type (WT) mice. In parallel FcRγ-chain-KO mice were compared with age-matched WT mice. A major branch of left coronary artery was ligated for 30 min followed by reperfusion for 24 hrs. Myocardial infarction was measured by tetrozolium staining. Hemodynamics were not statistically different among the groups. Risk zone sizes were also similar between WT and GPVI-KO mice (0.020 ± 0.004 cm 3 and 0.022 ± 0.005 cm 3 , respectively) (Mean ± SD). However, percentage infarction of the risk zone was significantly smaller in GPVI-KO mice (averaged 22 ± 8 % vs. 45 ± 18 % in WT). Comparable protection was observed in FcRγ-chain-KO mice (12 ± 6 % vs. 46 ± 13 % in WT). Significant exposure of vascular collagen in the ischemia/reperfusion region of myocardium was revealed by FITC-labeled recombinant GPVI protein, injected prior to ischemia, indicating endothelium injury. Furthermore, there was significantly less expression of P-selectin, a marker of activated platelets, in the myocardium from GPVI-KO and FcRγ-KO mice when compared with WT mice. It is concluded that deletion of GPVI reduces myocardial infarction induced by ischemia and reperfusion, likely due to the attenuation of the platelet-collagen interaction. With its known anti-thrombotic effect and minimal risk of bleeding in animals, anti-GPVI therapy may offer a promising novel treatment for cardiovascular diseases.

Collagen–chondroitin sulfate-based PLLA–SAIB-coated rhBMP-2 delivery system for bone repair

Bone morphogenetic proteins (BMPs) are osteoinductive proteins used intensively in clinical investigations involving various bone-related treatments. Owing to their high potential in new bone formation they require local application at the treatment site. For this purpose various controlled delivery systems with BMPs as the excipients have been prepared in recent years. Focusing on this clinical need a disc-shaped BMP carrier was designed as a local delivery system using soluble collagen and chondroitin sulfate. In situ release studies carried out with a model protein (FITC-labeled Protein A) presented a very high rate of release; with most of the protein content being released within 24 h. This rate could be decreased by providing a poly( l-lactide) (PLLA) and sucrose acetate isobutyrate-based (SAIB-based) coat around the release system, applied after BMP loading. In this way, it was possible to extend the release period from 24 h to about 12 days. In situ release of BMP from the same carriers, as quantitated using an ELISA kit, was even slower, with 50% of the protein being released in 15 days. In order to be able to secure the BMP delivery system at the bone defect site and to provide support a mesh knitted using Vicryl sutures and bonded with poly( l-lactide- co-glycolide) (PLGA) was tested in in vivo. Two time periods, 1 and 3 weeks, were used to evaluate the healing process. Osteoinduction by the BMP carrier system was assessed by histology-based bone scoring and X-ray examinations. PLLA–SAIB-coated collagen discs containing BMP presented good biocompatibility and optimum osteogenic stimulation. Structural changes in histological micrographs at week 1 indicated dose-dependent periosteal ossification. At the end of week 3 histological findings with both BMP (1 and 2 μg) doses were almost the same.

Whole‐column fluorescence‐imaged capillary electrophoresis

An axially illuminating whole-column fluorescence imaging capillary electrophoresis (CE) experimental setup was developed. A 6 cm long Teflon tube with an inside diameter (ID) of 42 microm was used as separation column. Excitation light of 488 nm from Ar+ laser was introduced to one end of the separation column by an optical fiber. The excitation light propagated inside the separation column by total internal reflection, since the refractive index of the buffer solution was larger than that of the Teflon tube. The fluorescence from the whole separation column was imaged with a charge-coupled device (CCD) camera. Fluorescent compounds such as fluorescein isothiocyanate (FITC), 5-carboxyfluorescein, and FITC-labeled protein were used to test the basic performance of the experimental setup. Experimental results illustrate that the whole-column-fluorescence imaging CE is a fast and sensitive separation method for fluorescent compounds and fluorescent-labeled proteins. Furthermore, it could be used for simple, fast, and easy comparisons of the resistance to photodegradation for various fluorescent compounds.

Reagentless and regenerable immunosensor for monitoring of immunoglobulin G based on non-separation immunoassay

Based on the enhancement of fluorescein isothiocyanate (FITC) fluorescence caused by reactions between proteins, we developed a reagentless, regenerable and rapid immunosensing system to determine immunoglobulin G (IgG). Fluorescence intensity of the immobilized FITC depends on IgG concentration, ranging from 10 to 50 μg/ml, specifically, even with co-existing proteins. The response time is 30 min during steady-state measurement and is less than a minute during transient measurement. When the FITC-labeled protein A binds to IgG, the surrounding atmosphere of FITC becomes hydrophobic. Since the fluorescence intensity of fluorescent substances generally increases at a hydrophobic environment, FITC fluorescence intensity increases with the concentration of protein A bonding to IgG. This system is regenerable because the fluorescence enhancement repeatedly occurs every time the immobilized fluorescent reagent is immersed in sample solutions.

A new reagentless immunosensor for measuring IgG concentration in human plasma based on fluorescence-enhancement immunoassay

Based on the enhancement of fluorescein isothiocyanate (FITC) fluorescence caused by binding to proteins, we developed a reagentless immunosensor to directly determine immunoglobulin G (IgG) in human plasma. The fluorescence-enhancement immunoassay (FEI) is used in only limited conditions in spite of its simplicity, because the homogeneous reagent cannot be reused. In the present study, a new FEI was evaluated with an immobilized reagent. FITC was used to label protein A, which has specific reactivity with IgG. The FITC-labeled protein A was immobilized on nylon membrane and placed in a fluorometer cell. It produced fluorescence enhancement corresponding to IgG concentration in phosphate-buffered saline (PBS) and bovine serum solution, and it responded to IgG also in human plasma. Thus the IgG concentration can be determined directly using the reagentless immunosensor based on FEI.

S100A8, S100A9 and the S100A8/A9 heterodimer complex specifically bind to human endothelial cells: identification and characterization of ligands for the myeloid-related proteins S100A9 and S100A8/A9 on human dermal microvascular endothelial cell line-1 cells.

The natural ligands of the S100 EF hand proteins S100A8 and A9 [myeloid-related proteins 8 and 14] have long been searched for in order to further the understanding of the role of the S100A8/A9-expressing monocyte subpopulation in progressing inflammatory processes. We demonstrate that S100A8, S100A9 and the S100A8/A9 heterodimeric complex bind to human dermal microvascular endothelial cell line (HMEC)-1 with an increasing binding capacity progressing from S100A8 < or = S100A9 < or = S100A8/A9. Similar results were obtained in the apolipoprotein E knockout mouse model, where preferably recombinant S100A9 but no S100A8 bound to the endothelium of the aorta ascendens. The binding of the S100A8/A9 heterodimer complex to activated HMEC-1 is specific as demonstrated by a dose-responding and satiable binding curve and the competition of FITC-labeled versus unlabeled protein. The protein character of the binding site was proven by treatment with trypsin. S100A8/A9 binding to HMEC-1 is inducible by lipopolysaccharide and tumor necrosis factor-alpha, and in the presence of calcium. A 163-kDa protein was isolated from a cell lysate of activated HMEC-1 cells using an affinity-chromatography protocol. The endothelial cell-associated ligand proteins isolated by the use of the S100A9 monomer and the S100A8/A9 dimer were subjected to mass spectrometry for protein identification. Clearly, alpha(2)-macroglobulin was identified as a binding partner for the S100A9 monomer, whereas no protein could be identified from the database for the ligand of the S100A8/A9 dimer.

α2-Macroglobulin-mediated Clearance of Proteases from the Plasma of the American Horseshoe Crab, Limulus polyphemus

Because proteases free in the body are damaging to the tissues, animals have evolved various agents for their inactivation and clearance. Mammals, for instance, have a diverse array of active site protease inhibitors in the plasma. In addition, mammals have alpha 2-macroglobulin (alpha 2M), which binds active proteases, and the alpha 2M-protease complex is then cleared from the plasma by a receptor-mediated endocytotic process. alpha 2M is also present in the plasma of many invertebrates, and in the American horseshoe crab, Limulus polyphemus, it is the only protease inhibitor in the plasma. To search for a clearance process for proteases in Limulus, fluorescein isothiocyanate (FITC)-labeled proteins were injected into the blood, and the fluorescence in the plasma and associated with the blood cells was determined. FITC-labeled trypsin was cleared with an initial mixing period (0-10 min) and a rapid clearance period (10-30 min), followed by the reappearance of FITC in the plasma (45-90 min). Before and during the clearance process, the labeled trypsin was associated with a complex having a molecular mass identical to that of Limulus alpha 2M, and that was precipitated by antibodies directed against Limulus alpha 2M. The fluoresceinated material that reappeared in the plasma after 45 min was of low molecular mass (< 10 kDa) and thus appears to have experienced degradation. The clearance of trypsin requires its protease activity, since phenylmethylsulfonyl fluoride-inactivated, FITC-labeled trypsin was cleared only very slowly if at all (t1/2 > 180 min). FITC-labeled, trypsin-reacted Limulus alpha 2M was cleared rapidly from the plasma of Limulus, whereas FITC-labeled, native Limulus alpha 2M persisted undiminished in excess of 400 min. The blood cells of Limulus bound FITC-labeled trypsin-reacted Limulus alpha 2M, and the peak of recovery from the blood cells coincided with the minimum concentration of FITC-labeled protein in the plasma, suggesting that the blood cells participate in the clearance of alpha 2M-protease complex from the plasma. Thus, we have demonstrated the existence of a clearance pathway in Limulus that operates selectively on enzymatically active proteases and have shown that Limulus alpha 2M is the probable agent for protease clearance. This is the first documentation of a protease clearance pathway in invertebrates and represents the first identified physicological function for alpha 2M in invertebrates.