Abstract：
:We describe a method for the generation of high-affinity monoclonal antibodies, which combines the power of natural immune responses with in vitro panning, B cell culture, RT-PCR and expression of the recombinant product. B cells from immunised rabbits were incubated at approximately 1000-10,000 cells per well with solid phase antigen coated on the surface of 96-well ELISA plates. Extensive washing removed non-binding cells as well as those B cells, which bound with low affinity. Retained B cells were cultured for 7 days in the presence of activated rabbit splenocyte supernatant and irradiated EL-4-B5 mouse thymoma cells, to induce proliferation and secretion of immunoglobulin. Supernatants were screened to confirm the presence of specific antibody, before the cells were harvested en masse from individual positive wells. Single heavy- and light-chain variable region genes were recovered from individual wells by RT-PCR, critically without the need for isolation of single B cells. Paired VH and VL genes were subsequently expressed as recombinant antibodies and shown to retain the original activity and specificity of the B cell culture supernatants. The method has also been successfully applied to the generation of high-affinity antibodies to antigen expressed on the surface of target cells.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Lightwood DJ,Carrington B,Henry AJ,McKnight AJ,Crook K,Cromie K,Lawson ADdoi
10.1016/j.jim.2006.08.010subject
Has Abstractpub_date
2006-10-20 00:00:00pages
133-43issue
1-2eissn
0022-1759issn
1872-7905pii
S0022-1759(06)00236-5journal_volume
316pub_type
杂志文章abstract：:A monoclonal antibody prepared against the murine interleukin-2 receptor (IL-2R) was employed to develop an ELISA method for measuring the immunological activation of T-cells. The assay detects an increase in IL-2R expression on activated lymphocytes. Stimulated splenic lymphocytes displayed markedly higher IL-2R expr...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(87)90114-1
更新日期：1987-02-26 00:00:00
abstract：:This paper describes a new method, with high specificity and sensitivity, for evaluating cell surface markes such as differentiation antigens and cytokine receptors on immunoglobulin-secreting cells. Mononuclear cells, freshly derived from peripheral blood or following stimulation in vitro with pokeweed mitogen or Sta...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(90)90405-k
更新日期：1990-08-28 00:00:00
abstract：:Household contacts of tuberculosis patients are at high risk of infection and development of active disease. In this study we evaluated the cytokine production and mRNA expression of IFN-γ, TNF-α, IL-10&IL-6 stimulated with r32kDa M. bovis BCGAg in active pulmonary tuberculosis patients (APTB), household contacts (HHC...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2016.02.012
更新日期：2016-05-01 00:00:00
abstract：:A new method of mouse B cell isolation has been developed. It is based on the differences in buoyant density of free lymphocytes and cells bound within liposome-cellular complexes (LCC). LCC arise during the interaction of B cells bearing surface immunoglobulins (Ig) with liposomes loaded with affinity purified rabbit...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(89)90074-4
更新日期：1989-12-20 00:00:00
abstract：:By pre-stimulating the lymphocytes of cancer patients with BCGF plus anti-mu chain specific antibody, we have generated human X human hybridomas. The resulting hybridomas preferentially produced IgG class antibody compared with those obtained from non-stimulated lymphocytes. With non-stimulated lymph-node lymphocytes,...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(90)90280-9
更新日期：1990-12-31 00:00:00
abstract：:Functional analysis of antigen-specific CD8(+) T cells is important for understanding the immune response in various immunological disorders. To analyze CD8(+) T cell responses to a variety of antigens with no readily defined peptides available, we developed a system using CD4(+) phytohemagglutinin (PHA) blasts transd...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2006.05.009
更新日期：2006-07-31 00:00:00
abstract：:Rises in antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) have predictive potential for a relapse of Wegener's granulomatosis (WG). To assess the value of image analysis for monitoring ANCA levels, we measured PR3-ANCA in a cohort of PR3-ANCA positive patients with WG that were prospect...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(02)00273-9
更新日期：2003-03-01 00:00:00
abstract：:We have developed an enzyme-linked immunosorbent assay (ELISA) for the quantitation of C4 nephritic factor (C4NeF). Incubation of the C4NeF-positive serum from patient M.I. with normal human serum (NHS) in the presence of human aggregated IgG (AHG) resulted in the formation of stable C4-C2 complex. No complex was form...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(90)90468-b
更新日期：1990-03-27 00:00:00
abstract：:alpha-2-macroglobulin (alpha-2-M), a plasma proteinase inhibitor, plays an important role in the pathogenesis of lung inflammation. The purpose of this study was to develop a sensitive ELISA assay for rabbit alpha-2-M to allow us to define the role of this protein in a rabbit model of lung inflammation/injury. Therefo...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(02)00278-8
更新日期：2002-12-15 00:00:00
abstract：:An immunoaffinity method for the purification of hepatitis B surface antigen (HBsAg) using monoclonal anti-idiotype (anti-Id) as a specific eluent is described. Ammonium sulfate fractions of HBsAg-containing plasma were absorbed on monoclonal idiotype (Id) immunoadsorbents. The bound material was eluted from the colum...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(90)90019-r
更新日期：1990-01-24 00:00:00
abstract：BACKGROUND:Antibodies that target a single tumor antigen fail to cure stage IV cancer patients due to tumor heterogeneity and variable expression of antigen. Tumor cells with insufficient binding of antibody will not undergo antibody induced cytotoxicity. We describe targeting multiple tumor-specific antigens that resu...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2019.112720
更新日期：2020-03-01 00:00:00
abstract：:Hepatocellular carcinoma (HCC) accounts for >700,000 deaths worldwide, largely related to poor rates of diagnosis. Our previous work identified glycoproteins with increased levels of fucosylation in HCC. Plate-based assays to measure this change were compromised by increased levels of heterophilic antibodies with glyc...
journal_title：Journal of immunological methods
pub_type： 临床试验,杂志文章
doi：10.1016/j.jim.2018.08.010
更新日期：2018-11-01 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：:A simple apparatus for measuring chemotaxis is described which contains thirty chambers per unit. Only very few cells and small volumes of chemotactic factor solution are required per chamber. For satisfactory results the apparatus must be centrifuged briefly prior to incubation. Under these conditions results can be ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(97)90008-9
更新日期：1977-01-01 00:00:00
abstract：:Establishment of humoral immunity against pathogens is dependent on events that occur in the germinal center and the subsequent induction of high-affinity neutralizing antibodies. Quantitative assays that allow monitoring of affinity maturation and duration of antibody responses can provide useful information regardin...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2013.11.026
更新日期：2014-02-01 00:00:00
abstract：:A method is described for identification of the animal origin of mosquito bloodmeals (MBM). The immunoassay is named DOT-PAP, it makes use of nitrocellulose as solid phase and of peroxidase-anti-peroxidase soluble complexes as detectors. DOT-PAP includes a built-in absorbing system to remove possible cross-reactivitie...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(86)90257-7
更新日期：1986-01-22 00:00:00
abstract：:Cytokines are critical regulators of the development and maturation of hematopoietic cells. Signal transduction via cytokine receptors proceeds through activation of the JAK-STAT pathway to stimulate cell proliferation, differentiation and effector functions. Phosphorylation of intracellular STAT molecules by the rece...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(03)00177-7
更新日期：2003-07-01 00:00:00
abstract：:Nowadays many reagents are available for the biotinylation of proteins. As most of them bind to amino groups of the protein the degree of labelling differs from batch to batch and the possibility exists that the biological activity of the target protein may be affected by the labelling procedure. In the present study ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(00)00172-1
更新日期：2000-06-23 00:00:00
abstract：:Picloram-specific variable fragments (V(HH)s) of heavy chain antibodies (HCAbs) were selected from a nai;ve-llama library using ribosome display technology. A cDNA library of V(HH)s was constructed from lymphocytes of a non-immunized llama and engineered to allow in vitro transcription and translation. With no stop co...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2003.07.011
更新日期：2003-10-01 00:00:00
abstract：:The 7th International Workshop on Human Leucocyte Differentiation Antigens (HLDA7) studied a number of newly characterised molecules relevant to human leucocyte differentiation and function. The HLDA organisation, which devised and continues to maintain the CD nomenclature, is responsible, under the auspices of IUIS a...
journal_title：Journal of immunological methods
pub_type：
doi：10.1016/s0022-1759(03)00057-7
更新日期：2003-04-01 00:00:00
abstract：:Utilization of compounds that enhance the innate immune response against Mycobacterium tuberculosis is an attractive strategy for combating tuberculosis in the post-antibiotic era. Thus, it is crucial to develop methods that can be used to screen for such compounds and to investigate their mechanisms of action. Here, ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2015.10.003
更新日期：2015-12-01 00:00:00
abstract：:A method is presented for the preparation of electron dense markers of defined size for use in immunoelectron microscopy, The electron dense markers were formed by the covalent binding of rabbit IgG and horse spleen ferritin mediated by glutaraldehyde. We demonstrated that the conjugate size, i.e. the number of ferrit...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(82)90124-7
更新日期：1982-03-26 00:00:00
abstract：:A rapid whole blood test has been developed for circulating antibodies to human immunodeficiency virus type 1 (HIV-1), based on agglutination of autologous red blood cells. Evaluation of the test revealed that 100% of seropositive HIV-1 patients (both asymptomatic and AIDS cases) were detected (n = 94) with a specific...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(91)90070-v
更新日期：1991-04-08 00:00:00
abstract：:Neisseria meningitidis is a leading cause of morbidity and mortality worldwide. The presently available capsular polysaccharide vaccine is poorly immunogenic in children under the age of 2 due to its T-independent (TI) nature. Efforts to overcome the TI response elicited by the polysaccharide vaccine have led to the d...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2003.08.005
更新日期：2004-02-01 00:00:00
abstract：:Phage-displayed random peptide libraries are important tools in identifying novel epitopes/mimotopes that may lead to the determination of antigen specificity. In this approach, high-affinity phage peptides are enriched by affinity selection (panning) on a monoclonal antibody. To facilitate identification of all poten...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2006.08.006
更新日期：2006-10-20 00:00:00
abstract：:The group I allergen of cockroach is found in both American and German cockroaches, designated as Per a 1 and Bla g 1, respectively. Members of these allergens so far identified are composed of tandem repeats that may cause the high allergenicity of Per a 1 allergen. In this study, we used monoclonal antibodies HW-8 a...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(03)00105-4
更新日期：2003-05-01 00:00:00
abstract：:Phagocyte functions such as chemotaxis and phagocytosis involve the rapid and transient development of cellular polarity. Study of this highly complex spatial and temporal cellular remodelling has been limited by the static nature of immunofluorescence and immunogold microscopy and because biochemical techniques are n...
journal_title：Journal of immunological methods
pub_type： 杂志文章,评审
doi：10.1016/s0022-1759(99)00155-6
更新日期：1999-12-17 00:00:00
abstract：:The production and characterisation of 17 monoclonal antibodies to human parathyroid hormone-related protein (PTH-rP) 1-34 is described. Five of the antibodies were shown to be of high avidity (Ka 4 X 10(10)-1.9 X 10(11) L/M) and able to detect 15-100 pg PTH-rP 1-34 per tube by RIA. None cross-reacted with PTH 1-34, a...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(90)90346-w
更新日期：1990-02-20 00:00:00
abstract：:A previously described assay for neutrophil phagocytosis of oil droplets labeled with the lipophilic red dye, oil red O, has been adapted to a microtiter plate format. Oil laden neutrophils are fixed to the plate with glutaraldehyde, washed free of uningested oil, and evaluated for degree of oil red O uptake by direct...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(91)90237-a
更新日期：1991-11-05 00:00:00
abstract：:Western blotting is one of the most commonly and widely used methods for identifying the presence of an antigen in biochemical studies. The present study describes a novel 'coupled blotting' approach that allows antigen on dot and Western blots to be simultaneously probed with primary and secondary probing reagents. T...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(96)00102-0
更新日期：1996-09-09 00:00:00
