Journal of Immunological Methods (J IMMUNOL METHODS )

Publisher: Association of Medical Laboratory Immunologists, Elsevier


The Journal of Immunological Methods is devoted to covering techniques for: (1) Quantitating and detecting antibodies and/or antigens and haptens based on antigen-antibody interactions. (2) Fractionating and purifying immunoglobulins, lymphokines and other molecules of the immune system. (3) Isolating antigens and other substances important in immunological processes. (4) Labelling antigens and antibodies with radioactive and other markers. (5) Localizing antigens and/or antibodies in tissues and cells, in vivo or in vitro. (6) Detecting, enumerating and fractionating immunocompetent cells. (7) Assaying for cellular immunity. (8) Detecting cell-surface antigens by cell-cell interactions. (9) Initiating immunity and unresponsiveness. (10) Transplanting tissues. (11) Studying items closely related to immunity such as complement, reticuloendothelial system and others. In addition the journal will publish articles on novel methods for analysing the organization, structure and expression of genes for immunologically important molecules such as immunoglobulins, T cell receptors and accessory molecules involved in antigen recognition, processing and presentation. Submitted full length manuscripts should describe new methods of broad applicability to immunology and not simply the application of an established method to a particular substance - although papers describing such applications may be considered for publication as a short Technical Note The Recombinant Technology section will contain articles relating to modification by recombinant techniques of molecules of immunological interest; isolation of novel binding proteins by phage display; gene therapy; transfection; and expression. Immunology Protocols is a section providing detailed, step-by-step descriptions of new and established techniques in immunology. Articles on the molecular biological analysis of immunologically relevant receptor binding sites are also invited.

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  • Website
    Journal of Immunological Methods website
  • Other titles
    Journal of immunological methods, Immunological methods, JIM
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  • Material type
    Periodical, Internet resource
  • Document type
    Journal / Magazine / Newspaper, Internet Resource

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    • Author can archive a post-print version
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    • Voluntary deposit by author of pre-print allowed on Institutions open scholarly website and pre-print servers
    • Voluntary deposit by author of authors post-print allowed on institutions open scholarly website including Institutional Repository
    • Deposit due to Funding Body, Institutional and Governmental mandate only allowed where separate agreement between repository and publisher exists
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    • Must link to journal home page or articles' DOI
    • Publisher's version/PDF cannot be used
    • Articles in some journals can be made Open Access on payment of additional charge
    • NIH Authors articles will be submitted to PMC after 12 months
    • Authors who are required to deposit in subject repositories may also use Sponsorship Option
    • Pre-print can not be deposited for The Lancet
  • Classification
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Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: Introduction: Despite extensive vaccinations, there have been pertussis epidemics in many countries including the Netherlands, the UK, Australia and the USA. During these epidemics Bordetella pertussis strains not producing the vaccine antigen pertactin (Prn) are emerging and increasing in numbers. However, methods for confirming PRN production of B. pertussis isolates are combined PCR or PCR-based sequencing tests and western blotting. Furthermore, data about production of pertussis toxin (PT) and filamentous hemagglutinin (FHA) of these isolates are scarce. Fimbriae (Fim) production is usually determined by agglutination and reported as serotype. In this study we developed an easy, accurate and rapid method for screening PT and FHA production. Methods for Prn and Fim production have been published earlier. Methods: We analyzed altogether 109 B. pertussis strains, including 103 Finnish B. pertussis strains collected during 2006-2013, international strain Tohama I, French strains FR3496 (PT-negative), FR3693 (Prn-negative) and FR4624 (FHA-negative) and Fim-serotype reference strains S1 (producing only Fim2) and S3 (producing only Fim3). An indirect ELISA with whole bacterial cells as coating antigen was developed and used for rapid screening of the B. pertussis strains. Production of different antigens (PT, FHA, Prn, Fim2 and Fim3) was detected with specific monoclonal antibodies (mAbs). Results: From the 103 Finnish B. pertussis strains tested, all were positive for PT, FHA and Fim. Four were found negative for Prn, and they were isolated during 2011-2013. Conclusions: The newly developed method proved to be useful and simple for rapid screening of different antigen production of B. pertussis isolates.
    Journal of Immunological Methods 06/2014;
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    ABSTRACT: The enzyme-linked immunospot (ELISpot) assay is a widely used method for immune monitoring in cancer immunotherapy trials. In the ELISpot assay, peripheral blood mononuclear cells (PBMC) are stimulated with specific antigens, and cytokines of interest produced by individual cells are detected. In the standard procedure, T cells rely for antigen presentation on other cells like the monocyte/macrophage population present among the PBMC. Whereas oligopeptides can be added directly to the ELISpot assay without the necessity of a pre-incubation step, protein antigens must be internalized and processed by antigen-presenting cells to accomplish efficient presentation via HLA class I or II. We have studied the impact of sources for different antigen-presenting cell (i.e. PBMC-resident monocytes and monocyte-derived dendritic cells maturated with Poly I:C and PGE-2 based cocktails) on ELISpot assay performance and defined an optimized dendritic cell-based ELISpot protocol. This protocol is suitable for monitoring immune responses directed to protein antigens with higher sensitivity than the standard procedure.
    Journal of Immunological Methods 03/2013;
  • Journal of Immunological Methods 01/2011; 367:63-69.
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    ABSTRACT: The most common method for the generation of monoclonal antibodies involves the identification and isolation of hybridomas from polyclonal populations. The discovery of new antibodies for biochemical and immunohistochemical assays in a rapid and efficient manner, however, remains a challenge. Here, a series of experiments are described that realize significant improvements to an approach for screening large numbers of single cells to identify antigen-specific monoclonal antibodies in a high-throughput manner (105–106 cells in less than 12 h). The soft lithographic process called microengraving yields microarrays of monoclonal antibodies that can be correlated to individual hybridomas; the cells can then be retrieved and expanded to establish new cell lines. The factors examined here included the glass slide used for the microarray, the buffer used to deposit capture antibodies onto the glass, the type of polyclonal antibodies used to capture the secreted antibodies, and the time required for microengraving. Compared to earlier reports of this method, these studies resulted in increased signal-to-noise ratios for individual elements in the microarrays produced, and a considerable decrease in the time required to produce one microarray from a set of cells (from 2–4 h to 3–10 min). These technical advances will improve the throughput and reduce the costs for this alternative to traditional screening by limiting serial dilution.
    Journal of Immunological Methods 01/2009;
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    ABSTRACT: The masking effects of antigens by chemical fixation, processing, embedding media interactions, represent a serious problem for immunohistochemical purposes. Fortunately, different approaches in antigen retrieval exist. These techniques are relatively recent and continuously expanding. This review focuses on the present state of the art in antigen retrieval methods for immunohistochemistry in light and electron microscopy. Moreover, a brief discussion on the chemical aspects of fixation, mechanism of retrieval, as well as its efficacy, is given.
    Journal of Immunological Methods 01/2009; 341(1-2):1-18.
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    ABSTRACT: Orthopoxviruses code for numerous immunomodulatory proteins, the structure and function of which are clarified inadequately. Antibodies constitute a potent tool to study such proteins, enabling conclusions on protein location and time course of expression. However, common antibody production in mice or rabbits requires tedious protein expression and injection, as well as blood collection at regular intervals. To simplify this procedure, IgY antibodies specific for poxviral proteins (F1L and p28) were generated by immunisation of chickens, because antibody retrieval from eggs allows the non-invasive generation of huge amounts of antibodies. The main intentions were (i) to decrease invasiveness, (ii) to immunise with native forms of proteins and (iii) to circumvent previous protein expression and purification. Therefore, chicken were immunised with DNA expression vectors coding for conserved domains of the selected proteins delivered for the first time by a gene gun. Four weeks after initial immunisation specific antibodies were found in the egg yolk as proven by immunofluorescence staining of poxvirus-infected cells. The specific IgY titre rose to 1:80,000 and was stable for more than 120 days. With this investigation we present an universal procedure for IgY design and production that can be applied for various issues in the future.
    Journal of Immunological Methods 01/2009; 341(1-2):146-53.
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    ABSTRACT: Natural killer (NK) cells represent the first line of defense against transformed or virally infected cells. Upon triggering of activating receptors NK cells can respond by secreting cytokines such as interferon-gamma or tumor necrosis factor-alpha and by the release of cytotoxic granules, resulting in the lysis of susceptible target cells. The importance of NK cells becomes clear in patients with impaired NK cell function or development. These patients suffer from recurrent illness and have particular problems in controlling viral infections despite their functional adaptive immune response. A detailed analysis of NK cell function is therefore of great importance. Here we describe a fast and comprehensive NK cell assay. The assay is performed in whole blood samples, eliminating the need for the isolation of PBMC or pure NK cells, while still allowing for the stimulation of the samples with cytokines. In each sample the absolute NK cell number is determined. The cytolytic activity is assayed by the lysis of (51)Cr labeled target cells and by determining the externalization of CD107a in the NK cells. Furthermore, cytokine production is detected by intracellular FACS analysis. Due to the strong reduction of required material this approach utilizes less than 3.5 ml of heparinized whole blood and is particularly applicable for frequent monitoring the immune function of adult and especially of pediatric patients.
    Journal of Immunological Methods 01/2009; 341(1-2):154-64.
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    ABSTRACT: This paper presents automated methods to quantify dynamic phenomena such as cell–cell interactions and cell migration patterns from time-lapse series of multi-channel three-dimensional image stacks of living specimens. Various 5-dimensional (x, y, z, t, λ) images containing dendritic cells (DC), and T-cells or thymocytes in the developing mouse thymic cortex and lymph node were acquired by two-photon laser scanning microscopy (TPLSM). The cells were delineated automatically using a mean-shift clustering algorithm. This enables morphological measurements to be computed. A robust multiple-hypothesis tracking algorithm was used to track thymocytes (the DC were stationary). The tracking data enable dynamic measurements to be computed, including migratory patterns of thymocytes, and duration of thymocyte-DC contacts. Software was developed for efficient inspection, corrective editing, and validation of the automated analysis results. Our software-generated results agreed with manually generated measurements to within 8%.
    Journal of Immunological Methods 01/2009;
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    ABSTRACT: Exploring the intricacies of CD8(+) T-cell epitope recognition using emerging technologies to combine assessment of affinity, phenotype and resulting polyfunctional efficacy advances our understanding of HIV-1 immunopathogenesis and disease progression. Complexities within T-cell antigen recognition, such as epitope:MHC binding, stability and affinity, appear to influence the distinction between protective and ineffective anti-HIV-1 immune responses, which are thought to govern rate of disease progression. This study utilises the novel ProImmune REVEAL and ProVE(R) technology of rapid peptide synthesis, binding and affinity assays, and pentamer synthesis in conjunction with flow cytometry and simultaneous assessment of multiple CD8(+) T-cell effector functions in response to HLA-B3501-restricted HIV-1 Gag peptides, to discover new T-cell epitopes. The predicted HLA-B3501-restricted peptides, HPVHAGPIA and YPLTSLRSL, and relevant pentamers were used in parallel to validate T-cell epitopes on clinical HIV-1(+) samples, confirming correlation between the expected superior immunogenicity of newly discovered epitopes and the ex vivo T-cell response. Such a platform should be employed in prophylactic and therapeutic vaccine settings.
    Journal of Immunological Methods 01/2009; 341(1-2):76-85.
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    ABSTRACT: Micron scale latex beads are well established as highly biocompatible reagents. Imbibing two fluorescent dyes into the interior of the beads enables the creation of a family of combinatorially colored labels. Previous use of such beads, in flow cytometry for example, has focused on beads of approximately 5 microm diameter. We show here that 280 nm combinatorially labeled particles can be used to create ELISA-style assays in 200 microm scale virtual wells, using digital microscopy as the readout. The utility of this technique is illustrated by profiling the secreted cytokine footprints of peripheral blood mononuclear cells in a multiparametric version of the popular Elispot assay. Doing so reveals noncanonical classes of T lymphocytes. We further show that the secreting cell type can be concurrently identified by surface staining with a cell type specific antibody conjugated to the same multiplexed beads.
    Journal of Immunological Methods 01/2009; 341(1-2):127-34.
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    ABSTRACT: The secreted immunoglobulin footprint of single hybridoma cells, containing ~10 fg of antibody purified in situ, has been probed for 9 properties concurrently by use of detection labels comprising 280 nm combinatorially colored fluorescent latex beads functionalized with proteins. Specificity of each individual hybridoma cell's product has thereby been assessed in a primary screen. Varying the density of antigen on beads to modulate the avidity of the interaction between bead and secreted antibody footprint allowed rank ordering by affinity in the same primary screen. As more criteria were added to the selection process, the frequency of positive cells went down; in some cases, the favorable cell was present at <1/50,000. Recovery of the cell of interest was accomplished by plating the cells in a viscous medium on top of a membrane. After collecting the antibody footprint on a capture surface beneath the membrane, the immobilized cells were transferred to an incubator while the footprints were analyzed to locate the hybridoma cells of interest. The desired cells were then cloned by picking them from the corresponding locations on the membrane.
    Journal of Immunological Methods 12/2008; 341(1-2):135-45.
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    ABSTRACT: The spontaneous formation of the neurotoxic carcinogen acrylamide in a wide range of cooked foods has recently been discovered, leading to dietary exposure estimates of 30.8 microg of acrylamide day(-1) for an average 77 kg human male. This is considerably higher than the European legal limit of acrylamide in drinking water, which is approximately 0.2 microg of acrylamide person(-1) day(-1). A recent study of 62,573 women over 11.3 years has observed an increased risk of postmenopausal endometrial and ovarian cancer (but not breast cancer) with increasing dietary acrylamide intake, demonstrating significant risk to human health. As individual acrylamide exposure is affected by dietary habits, cooking methods, and cigarette consumption; accurate extrapolation from estimated dietary exposure is extremely difficult. Quantifying biomarkers of acrylamide exposure therefore remains the most effective means of rapidly determining individual exposure to acrylamide, and correlating exposure with lifestyle choices. Current methodologies for the analysis of blood biomarkers of acrylamide are focused on expensive, slower chromatographic techniques such as GC and LC coupled to mass spectrometry. This paper describes the first successful development of two monoclonal antibodies specific to acrylamide-adducted haemoglobin (IC(50) of 94 ng ml(-1) and 198 ng ml(-1)), that are suitable for use in a high-throughput biomarker immunoassay to determine individual acrylamide exposure. Further development of acrylamide-haemoglobin standards with defined levels of acrylamide adduction will enable a fully quantitative assay, and allow sensitivity comparisons with alternative chromatographic methods of analysis.
    Journal of Immunological Methods 12/2008; 341(1-2):19-29.
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    ABSTRACT: Generation of therapeutic antibodies against human proteins is hampered by the difficulty of obtaining large quantities of correctly folded immunogens when following classic immunization procedures. Here we compared several genetic immunization protocols for their potential ability to generate high levels of antibodies against proteins expressed in their native form. We chose as a model the prion protein (PrP) because it has been demonstrated that the recognition of the native conformation of PrP is an absolute prerequisite for anti-PrP antibodies to be used as therapeutic tools for prion diseases, a group of lethal neurodegenerative disorders. We designed two human PrP-DNA vectors, containing or not a stimulatory T cell epitope, which were injected into mice following four different protocols: in the naked form with or without electroporation, or protected by cationic polymers or block copolymers. For comparison, other animals received conventional injections of recombinant human PrP with Freund's adjuvant or alum. We found that genetic immunization, carried out especially through DNA electroporation and, to a lesser extent, through injection of block copolymer-protected DNA, was able to generate high amounts of antibodies recognizing native PrP as expressed on the cell surface. Conversely, protein immunizations led to very high levels of antibodies against PrP immobilized on microtiter plates, but unable to recognize the native cell membrane-bound PrP. This clearly demonstrates the usefulness of genetic immunization, when performed under well defined conditions, in raising antibodies to native proteins. These results are of interest not only in view of passive immunotherapy of prion diseases, but also, more generally, in view of generating antibodies to human membrane proteins for immunotherapeutic or immunodiagnostic purposes.
    Journal of Immunological Methods 12/2008; 341(1-2):41-9.

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