共查询到20条相似文献,搜索用时 31 毫秒
1.
Yeon-Gu Kim Byoungwoo Park Jung Oh Ahn Joon-Ki Jung Hong Weon Lee Eun Gyo Lee 《BMC biotechnology》2012,12(1):1-6
Background
The establishment of high producer is an important issue in Chinese hamster ovary (CHO) cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS)-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production.Results
An internal ribosome entry site (IRES) was introduced for using two green fluorescence protein (GFP) fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (q Ab) than that of the unsorted pool. The q Ab was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and q Ab in individual selected clones.Conclusions
This study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of q Ab with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system. 相似文献2.
Birgit C. Schultes Richard P. Baum Andreas Niesen Antoine A. Noujaim R. Madiyalakan 《Cancer immunology, immunotherapy : CII》1998,46(4):201-212
Intravenous injection of the murine monoclonal anti-CA125 antibody B43.13 (Ovarex: Ab1) into ovarian cancer patients led to the induction of an idiotypic network. Of the 75 patients who received one to ten injections
of a 2-mg dose of the antibody, 48 developed anti-(mAb B43.13) antibodies (Ab2); 18 of these patients also had elevated levels of anti-[anti-(mAb B43.13)] antibodies (Ab3; = anti-CA125 antibodies) compared to pre-injection values. Characterization of these antibodies revealed that the binding
to CA125 could be inhibited by mAb B43.13 in most samples. Human anti-CA125 antibodies or Ab3 purified from patient serum samples specifically recognized human ovarian tumor cells and tissues expressing CA125. In addition,
these anti-CA125 antibodies were able to conduct Fc-mediated tumor cell killing (antibody-dependent cell-mediated cytotoxicity). This raises the possibility of using an Ab1 for anti-idiotype induction immunotherapy of cancer.
Received: 14 October 1997 / Accepted: 9 January 1998 相似文献
3.
Edwards BM Barash SC Main SH Choi GH Minter R Ullrich S Williams E Du Fou L Wilton J Albert VR Ruben SM Vaughan TJ 《Journal of molecular biology》2003,334(1):103-118
It is well established that the humoral immune response can generate antibodies to many different antigens. The antibody diversity required to achieve this is believed to be substantial. However, the extent to which the immune repertoire can generate structural diversity against a single target antigen has never been addressed. Here, we have used phage display to demonstrate the extraordinary capacity of the human antibody repertoire. Over 1000 antibodies, all different in amino acid sequence, were generated to a single protein, B-lymphocyte stimulator (BLyS™ protein). This is a highly diverse panel of antibodies as exemplified by the extensive heavy and light chain germline usage: 42/49 functional heavy chain germlines and 19/33 Vλ and 13/35 Vκ light chain germlines were all represented in the panel of antibodies. Moreover, a high level of sequence diversity was observed in the VH CDR3 domains of these antibodies, with 568 different amino acid sequences identified. Thus we have demonstrated that specific recognition of a single antigen can be achieved from many different VDJ combinations, illustrating the remarkable problem-solving ability of the human immune repertoire. When studied in a biochemical assay, around 500 (40%) of these antibodies inhibited the binding of BLyS to its receptors on B-cell lines. The most potent antibodies inhibited BLyS binding with sub-nanomolar IC50 values and with sub-nanomolar affinities. Such antibodies provide excellent choices as candidates for the treatment of BLyS-associated autoimmune diseases. 相似文献
4.
Marie Pancera Yongping Yang Mark K. Louder Jason Gorman Gabriel Lu Jason S. McLellan Jonathan Stuckey Jiang Zhu Dennis R. Burton Wayne C. Koff John R. Mascola Peter D. Kwong 《PloS one》2013,8(2)
Dozens of broadly neutralizing HIV-1 antibodies have been isolated in the last few years from the sera of HIV-1-infected individuals. Only a limited number of regions on the HIV-1 spike, however, are recognized by these antibodies. One of these regions (N332) is characterized by an N-linked glycan at residue 332 on HIV-1 gp120 and is recognized by antibody 2G12 and by the recently reported antibodies PGT121-137, the latter isolated from three donors. To investigate the diversity in mode of antibody recognition at the N332 site, we used functional complementation between antibody heavy and light chains as a means of assessing similarity in mode of recognition. We examined a matrix of 12 PGT-heavy chains with each of 12 PGT-light chains. Expression in 96-well format for the 144 antibodies (132 chimeric and 12 wild-type) was generally consistent (58±10 µg/ml). In contrast, recognition of HIV-1 gp120 was bimodal: when the source of heavy and light chains was from the same donor, recognition was good; when sources of heavy and light chains were from different donors, recognition was poor. Moreover, neutralization of HIV-1 strains SF162.LS and TRO.11 generally followed patterns of gp120 recognition. These results are consistent with published sequence, mutational, and structural findings, all of which indicate that N332-directed neutralizing antibodies from different donors utilize different modes of recognition, and provide support for a correlation between functional complementation of antibody heavy and light chains and similarity in antibody mode of recognition. Overall, our results add to the growing body of evidence that the human immune system is capable of recognizing the N332-region of HIV-1 gp120 in diverse ways. 相似文献
5.
G I Bell 《Journal of theoretical biology》1971,33(2):339-378
Several modifications are proposed to a recent mathematical model (Bell, 1970) of the clonal selection and antibody production which take place when an adult animal is injected with an antigen. In the original model, antigen molecules were assumed univalent, i.e. to have only one combining site per molecule, and the first modification is an allowance for multivalent antigens by permitting an antigen molecule to interact with only one cell at a time. It is found that, for antigens with few (? 10) sites per molecule this modification is not important while for many sites per molecule, the modification will reduce the response as compared to that from the same number of independent antigenic sites. In section 3, it is seen that by restricting the number of cells which can arise in an immune response, much more realistic responses to high antigen doses are obtained. Moreover, the response is made more predictable by assuming that both target and proliferating cells are stimulated by antigen when a fraction of their receptors, between fmin and fmax, is bound to antigen. The parameter fmin is shown to determine the extent to which a molecule which can be recognized by antibodies will also serve as an immunogen giving rise to cellular proliferation and antibody production. In section 4, it is found that if more plasma cells than memory cells result from antigen stimulation, then weak stimulation will lead to a depletion of target plus memory cells and to at least partial immunological paralysis. Optimum antigen doses and times for the induction of such paralysis are examined. In section 5, precipitation of antigen-antibody mixtures is considered and it is shown that the number of doubly bound antibody molecules per antigen site determines whether precipitation will occur. This number is easily computed for heterogeneous bivalent antibodies. 相似文献
6.
Yves Moné Benjamin Gourbal David Duval Louis Du Pasquier Sylvie Kieffer-Jaquinod Guillaume Mitta 《PLoS neglected tropical diseases》2010,4(9)
For many decades, invertebrate immunity was believed to be non-adaptive, poorly specific, relying exclusively on sometimes multiple but germ-line encoded innate receptors and effectors. But recent studies performed in different invertebrate species have shaken this paradigm by providing evidence for various types of somatic adaptations at the level of putative immune receptors leading to an enlarged repertoire of recognition molecules. Fibrinogen Related Proteins (FREPs) from the mollusc Biomphalaria glabrata are an example of these putative immune receptors. They are known to be involved in reactions against trematode parasites. Following not yet well understood somatic mechanisms, the FREP repertoire varies considerably from one snail to another, showing a trend towards an individualization of the putative immune repertoire almost comparable to that described from vertebrate adaptive immune system. Nevertheless, their antigenic targets remain unknown. In this study, we show that a specific set of these highly variable FREPs from B. glabrata forms complexes with similarly highly polymorphic and individually variable mucin molecules from its specific trematode parasite S. mansoni (Schistosoma mansoni Polymorphic Mucins: SmPoMucs). This is the first evidence of the interaction between diversified immune receptors and antigenic variant in an invertebrate host/pathogen model. The same order of magnitude in the diversity of the parasite epitopes and the one of the FREP suggests co-evolutionary dynamics between host and parasite regarding this set of determinants that could explain population features like the compatibility polymorphism observed in B. glabrata/S. mansoni interaction. In addition, we identified a third partner associated with the FREPs/SmPoMucs in the immune complex: a Thioester containing Protein (TEP) belonging to a molecular category that plays a role in phagocytosis or encapsulation following recognition. The presence of this last partner in this immune complex argues in favor of the involvement of the formed complex in parasite recognition and elimination from the host. 相似文献
7.
《Journal of molecular biology》1996,260(3):359-368
Human antibodies can now be isolated from antibody repertoires displayed on the surface of filamentous bacteriophage in a process that mimics the primary immune response. Here we have attempted to mimic the secondary response, the natural process of affinity maturation of antibodies occurring in germinal centres, by multiple cycles of random mutation and selection. Phage displaying a human antibody fragment recognising the hapten 2-phenyl-5-oxazolone were grown in a mutator strain of bacteria (Escherichia coli: mutD5) to generate a large repertoire of antibodies that should include the majority of possible single nucleotide point mutations. The repertoire of phage antibody mutants was then selected by binding to hapten. By multiple rounds of growth in the mutator strain, and increasingly stringent selection, we succeeded in isolating mutants with improved binding affinities; furthermore, the distribution of mutations and nucleotide substitution preferences strongly resembled those of somatic hypermutation. We then constructed a genealogical tree from the sequences of mutants taken at different rounds, and identified four sequentially acquired mutations that together improve the binding affinity of the antibody by a factor of 100-fold (fromKd320 nM to 3.2 nM). 相似文献
8.
The relative affinity of specific antibody secreted by mouse spleen cells following primary immunization with SRBC was estimated by competitive inhibition assay of antibody secreted by PFC as well as by inhibition of observed PFC number. Inhibition of direct and of indirect anti-SRBC plaque assays by the addition of specific antigen (SRBC stromata) gave sigmoid inhibition profiles from which the concentration of antigen required to inhibit 50% of the plaques (PI50) was determined, Alternatively, the sum of the cube of individual plaque diameters (Σd3) provided a measure of total anti-SRBC antibody secreted by PFCs from which the concentration of antigen required to inhibit 50% of the antibody (Ab50) was determined. Ab50, rather than PI50: (a) was a more sensitive measure of inhibition by antigen; (b) decreased following immunization indicating a progressive increase in mean antibody affinity; and (c) correlated with the results of hemolysin transfer experiments, an independent measure of mean affinity of circulating anti-SRBC antibody. From theoretical considerations, estimation of mean antibody affinity requires quantitative analysis of fractional antibody inhibition by antigen. Determination of Ab50, rather than PI50, provides an estimate of bound and of free antibody and therefore should provide a more valid estimate of the relative antibody affinity at the cellular level. Experimentally, utilizing Ab50 analysis, the IgM and IgG responses of C3H mice to immunization with SRBC demonstrated a progressive increase in affinity during maturation of the immune response. 相似文献
9.
To investigate the repertoire of molecules which are associated with cytolytic T-lymphocyte (CTL)-mediated killing, function-blocking monoclonal antibodies (MAb) have been selected and characterized. Spleen cells from rats immunized with secondary mouse CTL were fused with mouse myeloma cells. Antibodies secreted by 2400 hybrid cultures were selected solely by their ability to block CTL-mediated killing in a mouse anti-rat xenogeneic system. Fifteen cultures with antibodies which blocked CTL-mediated killing were chosen for cloning and further characterized by immunoprecipitation and immunofluorescence flow cytometry. One group of five monoclonal antibodies recognized the Lyt-2,3 molecule of 35,000 Mr. The second group of six MAb recognized the LFA-1 antigen containing two subunits of 180,000 and 95,000 Mr. One MAb giving only partial inhibition of killing was an IgM anti-Thy-1. It strongly agglutinated CTL. The target antigens defined by three other MAb were not definitively identified. Competition in cell binding between anti-Lyt-2,3 and anti-LFA-1 MAb suggested that their blocking effect in cytolysis is due to binding to distinct and spatially separate molecules on effector cells. The results of direct screening for functional blockade support the important role of Lyt-2,3 and LFA-1 molecules in T-cell-mediated cytolysis. 相似文献
10.
11.
Escherichia coli glutamine synthetase (GS) preparations composed of 12 adenylylated subunits (GS12?) are almost completely precipitated by sheep Anti-AMP immunoglobulin G (IgG), whereas glutamine synthetase preparations containing 6 adenylylated subunits (GS6?) are only partially precipitated by the antibodies (R.J. Hohman, S.G. Rhee, and E.R. Stadtman, 1980, Proc. Nat. Acad. Sci. USA77, 7410–7414). By means of 125I-labeled anti-AMP antibodies and double immunoprecipitation techniques, in which rabbit antiserum to sheep IgG or anti-GS antibodies were used to precipitate soluble immune complexes, it was demonstrated that under optimal conditions, both the soluble and insoluble immune complexes obtained with either GS6? or GS12? contain 0.5 mol antibody/mol adenylylated subunit. In agreement with the lattice theory of immuno-precipitation, soluble immune complexes are formed in antibody excess. Scatchard plots of binding data indicate that under conditions of antibody excess, one antibody molecule is bound to each AMP moiety of GS12?, whereas GS6? binds a maximum of only 0.68 antibody molecule/adenylylated subunit. We propose that with some species of GS6?, the distribution of adenylylated subunits favors monogamous interactions of the bivalent antibody with two subunits within the same GS molecule and thereby leads to the formation of small, soluble, immune complexes. Other explanations are considered. Only 30% of the antibody population that recognizes unconjugated 5′-AMP binds to the AMP moiety of adenylylated GS. Anti-AMP antiserum can be fractionated on a GS12?-Sepharose matrix into two subpopulations of antibody with strikingly different immunoprecipitation characteristics. Conversely, species of GS with various states of adenylylation ranging from 0 to 8 were separated from a GS6? preparation by means of affinity chromatography on an anti-AMP antibody-Sepharose matrix. Under optimal conditions, antibodies purified by affinity chromatography precipitated a smaller fraction of a GS6? preparation than did unfractionated antiserum. Competence of the purified antibody was nearly restored to that of the unfractionated serum by the addition of an enhancement factor present in the IgG fraction of nonimmune serum. The enhancement factor was not required for complete precipitation of GS?12 by purified antibodies. Contrary to most antibody-antigen reactions, immunoprecipitation of GS6? with anti-AMP antibodies is greater at 30 °C than at 4 °C. 相似文献
12.
Russell W. Anderson Avidan U. Neumann Alan S. Perelson 《Bulletin of mathematical biology》1993,55(6):1091-1131
A Cayley tree model of idiotypic networks that includes both B cell and antibody dynamics is formulated and analysed. As in
models with B cells only, localized states exist in the network with limited numbers of activated clones surrounded by virgin
or near-virgin clones. The existence and stability of these localized network states are explored as a function of model parameters.
As in previous models that have included antibody, the stability of immune and tolerant localized states are shown to depend
on the ratio of antibody to B cell lifetimes as well as the rate of antibody complex removal. As model parameters are varied,
localized steady-states can break down via two routes: dynamically, into chaotic attractors, or structurally into percolation
attractors. For a given set of parameters percolation and chaotic attractors can coexist with localized attractors, and thus
there do not exist clear cut boundaries in parameter space that separate regions of localized attractors from regions of percolation
and chaotic attractors. Stable limit cycles, which are frequent in the two-clone antibody B cell (AB) model, are only observed
in highly connected networks. Also found in highly connected networks are localized chaotic attractors. As in experiments
by Lundkvistet al. (1989.Proc. natn. Acad. Sci. U.S.A.
86, 5074–5078), injection ofAb
1 antibodies into a system operating in the chaotic regime can cause a cessation of fluctuations ofAb
1 andAb
2 antibodies, a phenomenon already observed in the two-clone AB model. Interestingly, chaotic fluctuations continue at higher
levels of the tree, a phenomenon observed by Lundkvistet al. but not accounted for previously. 相似文献
13.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1991,18(2):187-189
We report on the superiority of a radioimmunoassay (RIA) system wherein the second antibody (Ab2) is incorporated as a pre-incubated complex with the primary antibody (Ab1) for the assay of haptens like triiodothyronine (T3) and thyroxine (T4). Separation of the antibody bound and free antigen (Ag) was accomplished by 8% polyethylene glycol (PEG) (final concentration) following a single incubation of less than 1 h. The other advantages of this system are a 15-fold reduction in the quantity of Ab2 (without any need for increasing the concentration of PEG) and the consequent savings in cost. 相似文献
14.
Margaret Doherty Jonathan Bones Niaobh McLoughlin Jayne E. Telford Bryan Harmon Michael R. DeFelippis Pauline M. Rudd 《Analytical biochemistry》2013
Oligosaccharides attached to Asn297 in each of the CH2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5-h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies. 相似文献
15.
John de Kruif Arjen Kramer Therèse Visser Carina Clements Roy Nijhuis Freek Cox Vanessa van der Zande Renate Smit Daniel Pinto Mark Throsby Ton Logtenberg 《Journal of molecular biology》2009,387(3):548-4235
To study the contribution of antibody light (L) chains to the diversity and binding properties of immune repertoires, a phage display repertoire was constructed from a single human antibody L chain and a large collection of antibody heavy (H) chains harvested from the blood of two human donors immunized with tetanus toxoid (TT) vaccine. After selection for binding to TT, 129 unique antibodies representing 53 variable immunoglobulin H chain (VH) gene rearrangements were isolated. This panel of anti-TT antibodies restricted to a single variable immunoglobulin L chain (VL) could be organized into 17 groups binding non-competing epitopes on the TT molecule. Comparison of the VH regions in this VL-restricted panel with a previously published repertoire of anti-TT VH regions with cognate VH-VL pairing showed a very similar distribution of VH, DH and JH gene segment utilization and length of the complementarity-determining region 3 of the H chain. Surface plasmon resonance analysis of the single-VL anti-TT repertoire unveiled a range of affinities, with a median monovalent affinity of 2 nM. When the single-VL anti-TT VH repertoire was combined with a collection of naïve VL regions and again selected for binding to TT, many of the VH genes were recovered in combination with a diversity of VL regions. The affinities of a panel of antibodies consisting of a single promiscuous anti-TT VH combined with 15 diverse VL chains were determined and found to be identical to each other and to the original isolate restricted to a single-VL chain. Based on previous estimates of the clonal size of the human anti-TT repertoire, we conclude that up to 25% of human anti-TT-encoding VH regions from an immunized repertoire have promiscuous features. These VH regions readily combine with a single antibody L chain to result in a large panel of anti-TT antibodies that conserve the expected epitope diversity, VH region diversity and affinity of a natural repertoire. 相似文献
16.
Background
The development of a gonorrhea vaccine is challenged by the lack of correlates of protection. The antigenically variable neisserial opacity (Opa) proteins are expressed during infection and have a semivariable (SV) and highly conserved (4L) loop that could be targeted in a vaccine. Here we compared antibodies to linear (Ablinear) and cyclic (Abcyclic) peptides that correspond to the SV and 4L loops and selected hypervariable (HV2) loops for surface-binding and protective activity in vitro and in vivo.Methods/Findings
AbSV cyclic bound a greater number of different Opa variants than AbSV linear, including variants that differed by seven amino acids. Antibodies to the 4L peptide did not bind Opa-expressing bacteria. AbSV cyclic and AbHV2 cyclic, but not AbSV linear or AbHV2 linear agglutinated homologous Opa variants, and AbHV2BD cyclic but not AbHV2BD linear blocked the association of OpaB variants with human endocervical cells. Only AbHV2BD linear were bactericidal against the serum resistant parent strain. Consistent with host restrictions in the complement cascade, the bactericidal activity of AbHV2BD linear was increased 8-fold when rabbit complement was used. None of the antibodies was protective when administered vaginally to mice. Antibody duration in the vagina was short-lived, however, with <50% of the antibodies recovered 3 hrs post-administration.Conclusions
We conclude that an SV loop-specific cyclic peptide can be used to induce antibodies that recognize a broad spectrum of antigenically distinct Opa variants and have agglutination abilities. HV2 loop-specific cyclic peptides elicited antibodies with agglutination and adherence blocking abilities. The use of human complement when testing the bactericidal activity of vaccine-induced antibodies against serum resistant gonococci is also important. 相似文献17.
Sophie M. Thompson David G. Fernig Edwin C. Jesudason Paul D. Losty Els M. A. van de Westerlo Toin H. van Kuppevelt Jeremy E. Turnbull 《The Journal of biological chemistry》2009,284(51):35621-35631
Heparan sulfate (HS) binds and modulates the transport and activity of a large repertoire of regulatory proteins. The HS phage display antibodies are powerful tools for the analysis of native HS structure in situ; however, their epitopes are not well defined. Analysis of the binding specificities of a set of HS antibodies by competitive binding assays with well defined chemically modified heparins demonstrates that O-sulfates are essential for binding; however, increasing sulfation does not necessarily correlate with increased antibody reactivity. IC50 values for competition with double modified heparins were not predictable from IC50 values with corresponding singly modified heparins. Binding assays and immunohistochemistry revealed that individual antibodies recognize distinct epitopes and that these are not single linear sequences but families of structurally similar motifs in which subtle variations in sulfation and conformation modify the affinity of interaction. Modeling of the antibodies demonstrates that they possess highly basic CDR3 and surrounding surfaces, presenting a number of possible orientations for HS binding. Unexpectedly, there are significant differences between the existence of epitopes in tissue sections and observed in vitro in dot blotted tissue extracts, demonstrating that in vitro specificity does not necessarily correlate with specificity in situ/vivo. The epitopes are therefore more complex than previously considered. Overall, these data have significance for structure-activity relationships of HS, because the model of one antibody recognizing multiple HS structures and the influence of other in situ HS-binding proteins on epitope availability are likely to reflect the selectivity of many HS-protein interactions in vivo. 相似文献
18.
T. Roy Ittyerah Razia Rawala Robert W. Colman 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,714(3):524-529
Bovine coagulation factor V has been examined immunochemically to ascertain whether the coagulant polypeptide (h) with Mr = 290 000–330 000 is complexed in plasma with a second immunochemically distinct polypeptide (I2) of Mr = 400 000. Antiserum containing antibodies to h and l2 detects the l2 polypeptide eluting earlier than the h chain on gel filtration of plasma with either added calcium or EDTA, consistent with the behavior of a higher molecular weight noninteracting species. An immobilized monospecific antibody to l2 removes only the l2 polypeptide from a purified factor V preparation containing both h and l2. Moreover, while a monospecific antibody to the h chain was able to precipitate purified radioactively labelled h chain alone or mixed with plasma, the l2 antibody was unable to precipitate radioactively labelled h chain even after attempted recombination of the h chain with l2 present in plasma. These studies indicate that the l2 polypeptide is not complexed to the h chain in a purified system or in plasma and reinforce the conclusion that factor V is a single polypeptide chain uncomplexed in plasma. 相似文献
19.
Jinjin Guo Xiaowei HanJunchun Wang Junqing ZhaoZilin Guo Yuzhong Zhang 《Analytical biochemistry》2015
In this study, a novel tracer, horseradish peroxidase (HRP) functionalized gold nanorods (Au NRs) nanocomposites (HRP–Au NRs), was designed to label the signal antibodies for sensitive electrochemical measurement of alpha-fetoprotein (AFP). The preparation of HRP–Au NRs nanocomposites and the labeling of secondary antibody (Ab2) were performed by one-pot assembly of HRP and Ab2 on the surface of Au NRs. The immunosensor was fabricated by assembling carbon nanotubes (CNTs), Au NRs, and capture antibodies (Ab1) on the glassy carbon electrode. In the presence of AFP antigen, the labels were captured on the surface of the Au NRs/CNTs via specific recognition of antigen–antibody, resulting in the signal intensity being clearly increased. Differential pulse voltammetry (DPV) was employed to record the response signal of the immunosensor in phosphate-buffered saline (PBS) containing hydrogen peroxide (H2O2) and 3,3′,5,5′-tetramethylbenzidine (TMB). Under optimal conditions, the signal intensity was linearly related to the concentration of AFP in the range of 0.1–100 ng ml−1, and the limit of detection was 30 pg ml−1 (at signal/noise [S/N] = 3). Furthermore, the immunoassay method was evaluated using human serum samples, and the recovery obtained was within 99.0 and 102.7%, indicating that the immunosensor has potential clinical applications. 相似文献
20.
Milan Raska Kazuo Takahashi Lydie Czernekova Katerina Zachova Stacy Hall Zina Moldoveanu Matt C. Elliott Landon Wilson Rhubell Brown Dagmar Jancova Stephen Barnes Jana Vrbkova Milan Tomana Phillip D. Smith Jiri Mestecky Matthew B. Renfrow Jan Novak 《The Journal of biological chemistry》2010,285(27):20860-20869
Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the interaction between a variably glycosylated envelope glycoprotein (gp120) and host-cell receptors. Approximately half of the molecular mass of gp120 is contributed by N-glycans, which serve as potential epitopes and may shield gp120 from immune recognition. The role of gp120 glycans in the host immune response to HIV-1 has not been comprehensively studied at the molecular level. We developed a new approach to characterize cell-specific gp120 glycosylation, the regulation of glycosylation, and the effect of variable glycosylation on antibody reactivity. A model oligomeric gp120 was expressed in different cell types, including cell lines that represent host-infected cells or cells used to produce gp120 for vaccination purposes. N-Glycosylation of gp120 varied, depending on the cell type used for its expression and the metabolic manipulation during expression. The resultant glycosylation included changes in the ratio of high-mannose to complex N-glycans, terminal decoration, and branching. Differential glycosylation of gp120 affected envelope recognition by polyclonal antibodies from the sera of HIV-1-infected subjects. These results indicate that gp120 glycans contribute to antibody reactivity and should be considered in HIV-1 vaccine design. 相似文献