During the course of raising monoclonal antibodies, reagents are often produced that are not directed against the immunising antigen. These may pass unnoticed unless a screening step based on immunostaining of human tissue is included. Many of these reagents are auto-antibodies, often directed against intracellular targets (e.g. nuclear components), and the process of hybridoma production serves to "rescue" these self-reactive B cell clones. Such unexpected antibodies of this sort may prove of interest if their distribution is analysed on normal tissues and within the spectrum of leukaemia/lymphoma samples showing specific patterns of staining. However, before they can be used diagnostically or therapeutically, their target molecule needs to be identified, and this can be technically demanding. We describe a novel approach that can be used to define the targets of new monoclonal antibodies to intracellular molecules. The technique involves screening against protein arrays comprising thousands of recomb
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January 2018
Large-scale screening in Drosophila cultured or primary cells facilitates rapid genome-scale interrogation of gene function. Many factors influence screen outcomes, including cell line selection, screen format, type of reagent, coverage of genes by the reagent library, cell assay, and screen data analysis. Approaches to the design and analysis of screens are discussed. Protocols for small- or
... [Show full abstract] large-scale production of a common screening reagent, double-stranded RNA (dsRNA), as well as for immunostaining of cells in microwell format are also presented. Read more January 2005 · Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology
To obtain monoclonal antibodies (McAbs) against severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) nucleocapsid (N) protein to develop diagnostic test for SARS and study the pathogenesis of the disease.
BALB/c mice were immunized with purified N protein of SARS-CoV. Hybridoma cell lines secreting monoclonal antibodies against SARS-associated coronavirus nucleocapsid were
... [Show full abstract] established after cell fusion with mouse splenic cells and SP2/0 cells. The specificity of the McAbs obtained was examined by Western blot and indirect fluorescence assay. Epitopes reacted with the McAbs were preliminarily located through Western blot by expressing truncated N proteins.
After cell fusion and three rounds of cell cloning, six hybridoma cell lines secreting monoclonal antibodies specifically against SARS-CoV nucleocapsid were obtained. Western blot and indirect fluorescence assay showed that the McAbs reacted specifically with nucleocapsid protein and SARS-CoV. Among the six McAbs, three recognize the epitopes located in the N-terminus of the protein, whereas the others reacted with those located in the C-terminus.
The anti-SARS-CoV nucleocapsid McAbs were developed and these McAbs may be useful in the development of diagnosis assays and basic research of SARS. Read more January 2005 · Japanese Journal of Clinical Chemistry
The trace characterization of physiologically active substances with a low molecular weight (e.g., steroids, catecholamines and prostaglandins) is an important subject in clinical analysis, and specific antibodies against such "haptens" work as an essential analytical reagent. However, conventional antisera contain various antibody species derived from different B cell clones, whose nature
... [Show full abstract] prevent the constant supply of an antibody reagent with a defined quality. The hybridoma technology that was invented in 1975 by Köhler and Milstein principally resolved these problems; the cell fusion of immune spleen cells and myeloma generated the hybridoma clones, each secreting monoclonal antibody molecules with a defined and unique amino acid sequence and binding property. However, it is still not very easy to establish practical monoclonal anti-hapten antibodies, because this establishment requires an adequate cell culture, a reasonable design of hapten-carrier conjugate (immunogen), an effective immunization, an efficient antibody screening in hybridoma microcultures, etc. We succeeded in the generation of monoclonal antibodies against various haptens including steroids, endocrine-disrupting chemicals and drugs, which have been useful for developing practical immunoassays or immunoaffinity extraction systems. This article summarizes our biomedical analytical studies highlighting the technical aspect of the monoclonal antibody generation. Cloning of the antibody variable region genes and antibody engineering by gene manipulation are also discussed. Read more Article
Full-text available
February 2005 · Journal of Biomaterials Science Polymer Edition
The aim of this study was to assess the influence of different seeding densities on the function of hybridoma cells (clone 1B5, IgG 2alpha) producing an anti-angiogenic monoclonal antibody (mAb), microencapsulated using a high-voltage electrostatic field. Viable cells were microencapsulated in alginate/poly-L-lysine/alginate (APA) capsules and maintained in tissue culture. Cellular growth rates,
... [Show full abstract] production and release of mAb from the capsules were assessed. This study shows that hybridoma cells survive, proliferate and remain functionally competent for over one month in vitro after microencapsulation in APA capsules generated in an electrostatic field. However, the cell seeding density had to be at least 10(7) cells/ml for the microencapsulated cells to be viable and to produce and release mAb through the capsule membrane. The maximum monoclonal antibody concentration in this culture was 29.1 microg/ml by day 17, with a tendency to increase, but capsule breakage impeded the follow-up of this determination. View full-text December 2007 · Sheng wu gong cheng xue bao = Chinese journal of biotechnology
Unlabelled:
To reduce the huge labor-cost in the screening in traditional monoclonal antibody generation, We established a new system for monoclonal antibody generation integrating with protein array. BALB/c mice were immunized by eight recombinant proteins respectively, and the positive hybridoma cells were obtained by cell fusion and ELISA screening. All the eight kinds of positive hybridoma
... [Show full abstract] cells were mixed, cloned, screened by protein array, and definite dilution cloned.
Results:
175 single cell clones were obtained by complex cloning, and 119 of those were positive clones. Then 8 positive cell lines were generated by the following 2 rounds definite dilution cloning. By comparing with the traditional method, we got 8 monoclonal antibodies using the combined protein array screening and multiplex cloning method in 1 cycle, and fewer amounts of antigens were used. As a result, the combined protein array and multiplex cloning method could be used as an economical, rapid and simple tool applying in high throughput monoclonal antibody generation. Read more September 1983 · Journal of Experimental Medicine
Two antisera and a monoclonal antibody raised in BALB.K mice against cloned, major histocompatibility complex (MHC)-restricted, antigen-specific helper T cell lines are described. These antibodies are specific for individual cloned T cell lines and are potent inducers of T cell proliferation. The induction of T cell proliferation by these antibodies requires the presence of an adherent accessory
... [Show full abstract] cell. There is no H-2 restriction between this accessory cell and the cloned T cell, nor is this antibody-induced proliferation blocked by a monoclonal anti-Fc receptor antibody. The requirement for an accessory cell, however, is eliminated in the presence of an IL-1- or IL-2-rich supernatant. Thus this system allows the analysis of helper T cell activation with only a single cell type present. Anti-T cell sera also induce T cell-dependent B cell proliferation and immunoglobulin secretion. The induction of T cell-dependent B cell activation by these sera does not require H-2-matched T cells and B cells. The specificity of these antibodies and their ability to stimulate cloned helper T cells in the absence of antigen and antigen-presenting cells strongly suggest that these antibodies are directed against antigen and/or Ia recognition sites on the T cell. Read more June 2012 · Hybridoma (2005)
The UL16-binding proteins (ULBPs) are a novel family of human MHC class I-related, cell surface proteins that function as ligands for NKG2D. In this study, the gene encoding human ULBP3 was cloned into prokaryotic expression vector pQE30, resulting in a recombinant plasmid pQE30-ULBP3. The pQE30-ULBP3 was transformed into Escherichia coli M15 and induced the expression of recombinant protein
... [Show full abstract] ULBP3 (rec-ULBP3). The purified rec-ULBP3 as an antigen was used to immunize BALB/c mice. Through cell fusion, sub-cloning, and screening approach, three hybridoma cell clones expressing monoclonal antibodies (MAb) were acquired. The results from Western blot analysis, flow cytometry, and enzyme-linked immunosorbent assay showed that the hybridoma clones B2-F1-F1 and B4-C5-D11, and not G2-A4-A12, reacted with rec-ULBP3 and nature ULBP3 expressed on the cell surface of the tumor cells. In conclusion, the new MAb described here provides a valuable tool for further investigating ULBP3 function and clinical application. Read more April 1995 · The Journal of Immunology
We have previously described a set of secondary (2 degrees) hybridomas that are specific for the Sb site of influenza hemagglutinin (HA(Sb)) and share the H37-68 ld (68ld). Cells producing 68ld Abs dominated the 2 degree response of one BALB/c mouse, but were virtually absent from the primary (1 degree) and 2 degree responses of other BALB/c mice. To understand the basis for the idiosyncratic
... [Show full abstract] nature of this response, we have assessed the functional importance of a conserved DJ junctional sequence. We find that substitutions of conserved residues within this sequence drastically reduced HA(Sb) binding, providing an explanation for the absence of this ld from 1 degree responses. We have also examined intraclonal affinity maturation by generating Abs inferred to have been produced by the unmutated precursors and branchpoint intermediates of the largest 68ld clone. Interestingly, unmutated Abs of this clone bound HA(Sb) well, comparable to that of some 2 degree 68ld hybridoma Abs. Abs produced by branchpoint intermediates of this clone have a lower affinity for HA(Sb) than their unmutated precursor, despite containing parallel amino acid replacement mutations. Thus, expansion of this clone did not follow the paradigm of stepwise increases in affinity. Together, these data underscore the interconnective nature of precursor frequency and somatic mutation in the formation of the 2 degree response to a given Ag. Read more December 1988 · International Journal of Immunopharmacology
Read more January 2007
The theory of nonlinear dynamical systems is reviewed, with the aim of showing that it has a range of useful tools to offer
to both immunologists and computer scientists. The theory is illustrated with a simple model for the interaction of B cell
clones, namely the B-model of Perelson and Weisbuch. A simple model is provided for the dynamics of an artificial immune response,
and this is analysed
... [Show full abstract] using optimal control theory. A new model for the interaction of signalling molecules (cytokines) with
immune cells is also outlined. Read more July 1989 · Virology
MT-4 cells persistently infected with human immunodeficiency virus type 1 (HIV-1) (MT-4/HIV-1) were recently isolated (K. Ikuta, C. Morita, M. Nakai, N. Yamamoto, and S. Kato, Japan. J. Cancer Res. (Gann), 79, 418-423, 1988). Mouse hybridoma cell clones producing monoclonal antibodies (MoAbs) to HIV-1 gag p24 and p18, and pol reverse transcriptase (RT) were isolated by using this MT-4/HIV-1 cell
... [Show full abstract] line for the screening of MoAb production by the immunofluorescence (IF) test. By indirect IF tests of acetone-fixed cells with these MoAbs, the IF intensities in MT-4/HIV-1 cells were found to be higher than those in the other HIV-1 infected cells, such as MOLT-4/HIV-1, HL-60/HIV-1, and U937/HIV-1 cells. Cell surface expression of the HIV-1 gag p24 and p18 antigens examined by IF and radioimmune techniques with these MoAbs revealed the p24 and p18 antigens to be expressed strongly on the cell surface of MT-4/HIV-1 cells and faintly on the cell surface of MOLT-4/HIV-1 cells, respectively. However, monoclonal antibody isolated in the present study failed to detect pol RT antigen on the surface of MT-4/HIV-1 cells. These results indicate that the gag p24 and p18 antigens are expressed, at least in part, on the surface of HIV-1-infected cells. Read more July 1977 · The Journal of Immunology
B cell subpopulations have been defined by various morphologic criteria and differences in functional capacity. In a recent report from this laboratory, the in vitro splenic focus system was utilized to define a subpopulation of neonatal splenic B cells on the basis of their susceptibility to tolerance induction. Since this susceptibility to tolerance induction progressively decreases with age,
... [Show full abstract] tolerance susceptibility in this system has been attributed to the immaturity of the cells which constitute developing B cell clones. In the present study, the splenic focus technique was utilized to characterize the maturational state(s) of neonatal and adult bone marrow B cells as assessed by the susceptibility of these cells to tolerance induction, and to compare these responses to the responses of splenic B cells. The results demonstrate that the majority of neonatal bone marrow B cells reach maturity, by the criterion of tolerance susceptibility, later than splenic B cells. Furthermore, in contrast to the spleen, a portion (25%) of bone marrow B cells remain susceptible to tolerance induction even in the adult. Thus, the generative pool of B cells appears to shift from the spleen to the bone marrow after the first few days of neonatal life and resides primarily in the bone marrow thereafter. Finally, the susceptibility of B cells to tolerance induction may serve as a marker for the functional maturation of any given B cell population, and consequently may help to define distinct B cell subpopulations within the B cell pool. Read more Last Updated: 06 Nov 2020
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