Abstract:
:Melanins are notoriously difficult to work with because of their unique physical and chemical properties. The study of melanins is hampered by the scarcity of melanin-specific reagents and serological techniques. In this study we describe modifications to the standard method for the isolation of melanins from in vitro-melanized fungal cells and detail the optimization of serological techniques for the study of melanin compounds. The isolation procedure involves the digestion of melanized cells with a combination of proteolytic and glycolytic enzymes, denaturant, organic extractions, and boiling in 6.0 M HCl. Elemental quantitative analyses suggest that this procedure does not significantly affect the relative elemental composition of melanins. For the serological assays, our goal was to achieve a homogenous distribution of melanin particles on a solid support to maximize their recognition by melanin-binding antibodies. The results from enzyme-linked immunosorbent assays (ELISAs) demonstrate that melanins, in general, disperse more efficiently on, and adhere better to, medium-binding polystyrene surfaces, especially in the presence of trace amounts of salt. Blocking the melanin-coated ELISA plates with the commercially available SuperBlock((R)) Blocking Buffer for 4 h was more efficient at reducing non-specific binding of a negative control monoclonal antibody (mAb) compared to blocking with 2% bovine serum albumin (BSA) and 5% milk. Increasing the ionic strength of the antibody solutions reduced binding to the melanins, indicating that binding is in part mediated by electrostatic interactions. These conditions were also applied to immunofluorescence (IF) analyses of melanins, and the results were consistent with those obtained by ELISA.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Rosas AL,Nosanchuk JD,Gómez BL,Edens WA,Henson JM,Casadevall Adoi
10.1016/s0022-1759(00)00255-6keywords:
subject
Has Abstractpub_date
2000-10-20 00:00:00pages
69-80issue
1-2eissn
0022-1759issn
1872-7905pii
S0022-1759(00)00255-6journal_volume
244pub_type
杂志文章abstract::A simple, rapid and reproducible method is presented for direct determination of the substitution ratio of a carrier protein with a synthetic nonradioactively labeled peptide. The peptide was covalently linked by a thiol group of a cysteine residue to the immunogenic carrier protein using the heterobifunctional reagen...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(88)90421-8
更新日期:1988-04-06 00:00:00
abstract::The basic method for evaluating the efficacy of antibody-mediated immunity (AMI) dates from the 1890s and involves the administration of specific Ab to an immunologically naïve host followed by microbial or toxin challenge. Other methods used to evaluate AMI involve correlating the presence of specific Ab with resista...
journal_title:Journal of immunological methods
pub_type: 杂志文章,评审
doi:10.1016/j.jim.2004.04.027
更新日期:2004-08-01 00:00:00
abstract::Ia molecule (Ek,b beta:Ek alpha or Ak beta:Ak alpha)-containing planar membranes were constructed with cholesterol and a 9:1 molar ratio of the phospholipids dipalmitoyl phosphatidylcholine and dilinoleoyl phosphatidylcholine. This lipid composition was found to be optimal for the stimulation of T cell hybridomas of d...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(87)90432-7
更新日期:1987-04-02 00:00:00
abstract::We have studied the primate cellular requirements in the antibody forming cell (AFC) assay, using a 185,000 molecular mass streptococcal antigen. Cultures of B cells alone stimulated with the antigen were unable to develop into AFC. Reconstitution of B cells with T cells resulted in a significant increase in the numbe...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(90)90026-r
更新日期:1990-09-14 00:00:00
abstract::Different numbers of Chinese hamster lymphocytes were cultured in microtiter plates with flat-, round- and V-bottomed wells for different culture times. The smallest number of cells could be stimulated in plates with V-bottomed wells. At least 25,000-50,000 cells and a longer culture time than for round- or V-bottomed...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(80)90102-7
更新日期:1980-01-01 00:00:00
abstract::To date, studies on T cells in acute myeloid leukemia (AML) have been limited to flow cytometric analysis of whole peripheral blood mononuclear cell (PBMC) specimens or functional work looking at the impact of AML myeloblasts on normal or remission T cells. This lack of information on T cells at the time of presentati...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2009.06.010
更新日期:2009-08-31 00:00:00
abstract::The Raji cell assay for circulating immune complexes (CIC) is frequently the method of choice when the detection of large, complement fixing complexes is desired. We have developed an enzyme-linked immunosorbent assay modification (Raji-ELISA) of the Raji cell technique which is easy to perform, uses commercially avai...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(83)90046-7
更新日期:1983-01-01 00:00:00
abstract::The common marmoset is a small New World Primate that has been used as an immunological model for a number of human diseases. Dendritic cells (DC) have not been extensively characterised in this species and in particular protocols to derive DC from living donors without the need for animal sacrifice are presently lack...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2009.11.008
更新日期:2010-01-31 00:00:00
abstract::Anti-melatonin antibodies of high titer and good sensitivity were obtained by coupling melatonin to bovine serum albumin with formaldehyde and by immunizing rabbits with multifocal intra-dermal injections. A systematic study of cross-reactions with 14 compounds (indole, aromatic and imidazole derivatives) showed that ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(80)90026-5
更新日期:1980-01-01 00:00:00
abstract::The immune system is characterized by high combinatorial complexity that necessitates the use of specialized computational tools for analysis of immunological data. Machine learning (ML) algorithms are used in combination with classical experimentation for the selection of vaccine targets and in computational simulati...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2011.07.007
更新日期:2011-11-30 00:00:00
abstract::The chemiluminescence (CL) assay has been used to measure the reactive oxygen species (ROS)-generating capacity of phagocytes. To achieve more optimal measurement conditions for a multi-channel microplate photon-counting CL analyzer with the cooled charge-coupled device (CCD) camera which offers enhanced sensitivity, ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(97)00159-2
更新日期:1997-12-15 00:00:00
abstract::MicroRNAs are small regulatory molecules that demonstrate useful biomarker potential. They have been recognised in biofluids, where they are protected from degradation by encapsulation into extracellular vesicles (EVs). A number of commercial products are available for the isolation of EVs and their RNA content; howev...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2015.12.011
更新日期:2016-02-01 00:00:00
abstract::We studied the binding of dog immunoglobulins G, A, M and E to protein A and protein G. Passive cutaneous anaphylaxis (PCA) testing was used for the measurement of dog IgE and enzyme-linked immunosorbent assays (ELISA) were used for the measurements of dog IgG, IgA and IgM. Protein A from lyophilized cells of Staphylo...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(91)90335-d
更新日期:1991-12-15 00:00:00
abstract::A novel bioanalysis system based on immunochromatography was developed in connection with a nitrocellulose resin modified micropipette tip, such as ZipTip. The sandwich-type immunoassay was applied to our bioanalysis system. The simple handling of the micropipette enabled us to increase the sample volume and detect lo...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2006.02.011
更新日期:2006-05-30 00:00:00
abstract::Anti-drug neutralizing antibodies (NAbs) formed due to unwanted immunogenicity of a therapeutic protein point towards a mature immune response. NAb detection is important in interpreting the therapeutic's efficacy and safety in vivo. In vitro cell-based NAb assays provide a physiological system for NAb detection, howe...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2015.02.006
更新日期:2015-04-01 00:00:00
abstract::Hapten-carrier protein conjugates were made using five different small haptens (MW < 1000), two carrier proteins and two methods of conjugation. Nondenaturing agarose gel electrophoresis was used to demonstrate that when as few as two molecules of a small hapten are attached to the carrier, the conjugate band migrates...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(93)90317-z
更新日期:1993-09-15 00:00:00
abstract::Radioactive double labeling was adapted for the analysis of mediators of cellular immunity. Two identical lymphocyte cultures were simultaneously labeled with [3H]- or [14C]leucine. Each of the cultures was stimulated with antigen or mitogen. The combined supernatants were then subjected to various fractionation proce...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(78)90177-1
更新日期:1978-01-01 00:00:00
abstract::Giant liposomes (mean diameter 5.5 microns) composed of egg phosphatidylcholine or distearoyl phosphatidylcholine, phosphatidyl glycerol, cholesterol and triolein were prepared by a double emulsion technique. They were then mixed with model particulate (killed Bacillus subtilis, and killed Bacille Calmette-Guérin) and...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(93)90368-h
更新日期:1993-12-03 00:00:00
abstract::A novel technique for establishing short term clones of antigen- or mitogen-activated splenic B lymphocytes is described. Spleen cells are plated onto the surface of filter paper discs and subsequently stimulated by antigen or mitogen in situ; activated B cells proliferate and differentiate into pure colonies of cells...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(85)90312-6
更新日期:1985-02-11 00:00:00
abstract::The influence of human hepatocyte growth factor (HGF) on the transforming growth factor beta (TGF-beta) bioassay CCL-64 was examined. HGF induced proliferation of the CCL-64 cells and potently counteracted TGF-beta-induced growth inhibition. HGF was not inactivated by transient acidification to pH 2, a commonly used p...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(95)00228-6
更新日期:1996-01-16 00:00:00
abstract::An antibody binding-inhibition test is described, which allows the detection of P. falciparum in red blood cells (RBC) infected in vitro, using a crossreacting, monoclonal anti-P. berghei antibody and P. berghei coated microtiter plates. Experiments carried out to determine the coating efficiency of various P. berghei...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(85)90231-5
更新日期:1985-09-03 00:00:00
abstract::We describe here a spot test to detect antibodies to non-erythroid cell surface antigens, using ethidium bromide staining to visualize areas of target cell lysis. This assay allows rapid screening of large numbers of samples of culture supernatants, antisera, or ascitic fluids. Antibody titers determined by this spot ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(82)90367-2
更新日期:1982-01-01 00:00:00
abstract::Granulocytes and monocytes/macrophages represent key effector cells of the innate immune system. While human monocytes have been recognized as capable of secreting a broad spectrum of cytokines, the situation has been less clear in granulocytes with studies often showing conflicting results. In this study, lipopolysac...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2009.04.001
更新日期:2009-07-31 00:00:00
abstract::A colored or fluorescent signal is generally evaluated with the naked eye, or by means of different more or less sophisticated and costly instruments. Photodensitometry is an additional technique which is both inexpensive and simple to perform. This technique can satisfactorily quantify a signal without the use of eit...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(87)90235-3
更新日期:1987-10-23 00:00:00
abstract::Conventional culture conditions for GM-CSF expanded murine bone marrow derived dendritic cells (BMDCs) uses ambient (hyperoxic) oxygen pressure (20% v/v, 152 Torr) and medium supplemented with the thiol 2-mercaptoethanol (2-Me). Given the redox activities of O2 and 2-Me, the effects of 2%, 5%, 10%, and 20% v/v O2 atmo...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2005.10.012
更新日期:2006-01-20 00:00:00
abstract::A new simple multi-well plate-based assay to determine monocyte differentiation-inducing activity was developed. It is based on the increase of macrophage adherence after the induction of monocyte differentiation. The assay was conducted using a human monocytic leukemia cell line, THP-1, treated with either 12-O-tetra...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2007.09.004
更新日期:2007-12-01 00:00:00
abstract::Visceral leishmaniasis, which is also known as Kala-Azar, is one of the most severely neglected tropical diseases recognized by the World Health Organization (WHO). The threat of this debilitating disease continues due to unavailability of promising drug therapy or human vaccine. An extensive research is undergoing to...
journal_title:Journal of immunological methods
pub_type: 杂志文章,评审
doi:10.1016/j.jim.2015.03.017
更新日期:2015-07-01 00:00:00
abstract::Accurately predicting peptides binding to major histocompatibility complex (MHC) I molecules is of great importance to immunologists for elucidating the underlying mechanism of immune recognition and facilitating the design of peptide-based vaccine. Various computational methods have been developed for MHC I-peptide b...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2010.09.007
更新日期:2011-11-30 00:00:00
abstract::Biological and biochemical characteristics of monoclonal antibodies (MABs) raised against human interleukin-10 (IL-10) are described as well as their use in the design of a specific ELISA for the measurement of the cytokine. 21 murine anti-human interleukin-10 (IL-10) MABs were obtained by fusion of splenocytes from m...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(94)90160-0
更新日期:1994-12-28 00:00:00
abstract::A semiautomated modification of the membrane immunofluorescence (MF) technique was developed employing units of the Microtiter system. This new rapid technique, which allows the performance of several hundred samples within a few hours is exemplified on a MF test proving the specific reaction to anti-chicken bursa cel...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(76)90165-4
更新日期:1976-03-01 00:00:00