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Search results for: Mouse IgG2a Negative Control, Biotinylated

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#27185103   2016/05/16 To Up

Rapid characterization of hybridomas producing monoclonal antibodies against platelet β3 integrin using ELIspot.

Generally, B-cell responses against human platelet antigens are assessed by the serological detection of specific platelet antibodies, mostly against β3 integrin. However, this approach seems to be of low sensitivity, since platelet autoantibodies against αIIbβ3 are detected in only 50% of all patients with immune thrombocytopenia (ITP). In this study, a novel B-cell ELIspot method was established to characterize the specificity of mouse monoclonal antibodies (moabs) against human β3 integrin. Moabs produced by hybridomas were immobilized on membrane and bound antibodies were visualized as spots using biotinylated recombinant proteins αIIbβ3 or αvβ3 and the enzyme labeled streptavidin-substrate system. Three hybridomas, Gi5, Gi16 and AP3, designated previously as anti-αIIbβ3, anti-αIIb and anti-β3, respectively, were investigated. Hybridoma producing moab against CD177 was used as the negative control. Whereas AP3 reacted with αIIbβ3 and αvβ3, Gi5 only formed spots with αIIbβ3. Titration analysis showed that the number of spots correlated significantly with the number of seeded cells. Approximately 15 antibody producing hybridoma cells could be identified among 10 nonproducing B-cells. Furthermore, superior correlation with the total number of IgG producing cells was obtained. Analysis of the third hybridoma, Gi16 (anti-αIIb), showed only few spots with αIIbβ3, indicating that this hybridoma contained different clones (producer and non-producer). Significant increased number of spots could be identified after re-cloning of these clones by limiting dilution method. Our results demonstrate that this B-cell ELIspot assay can be used for the identification of a small number of hybridoma cells producing moabs against β3 integrin, verification of their monoclonality, productivity and for determining their specificity in the early state of workup steps. In the future, this approach may be useful to define B-cell clones in patients who developed platelet antibodies against different β3-integrins and to differentiate their diversities.
Denis Maenner, Silke Werth, Gregor Bein, Sentot Santoso

1045 related Products with: Rapid characterization of hybridomas producing monoclonal antibodies against platelet β3 integrin using ELIspot.

100.00 ug100.00 ug1 mg100 ug200.00 ug100.00 ug100 ug1 mg100 ug100.00 ug100.00 ug1 mg

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#22787191   2012/07/11 To Up

No evidence for xenotropic murine leukemia-related virus infection in Sweden using internally controlled multiepitope suspension array serology.

Many syndromes have a large number of differential diagnoses, a situation which calls for multiplex diagnostic systems. Myalgic encephalomyelitis (ME), also named chronic fatigue syndrome (CFS), is a common disease of unknown etiology. A mouse retrovirus, xenotropic murine leukemia-related virus (XMRV), was found in ME/CFS patients and blood donors, but this was not corroborated. However, the paucity of serological investigations on XMRV in humans prompted us to develop a serological assay which cover many aspects of XMRV antigenicity. It is a novel suspension array method, using a multiplex IgG assay with nine recombinant proteins from the env and gag genes of XMRV and 38 peptides based on known epitopes of vertebrate gammaretroviruses. IgG antibodies were sought in 520 blood donors and 85 ME/CFS patients and in positive- and negative-control sera from animals. We found no differences in seroreactivity between blood donors and ME/CFS patients for any of the antigens. This did not support an association between ME/CFS and XMRV infection. The multiplex serological system had several advantages: (i) biotinylated protein G allowed us to run both human and animal sera, which is essential because of a lack of XMRV-positive humans; (ii) a novel quality control was a pan-peptide positive-control rabbit serum; and (iii) synthetic XMRV Gag peptides with degenerate positions covering most of the variation of murine leukemia-like viruses did not give higher background than nondegenerate analogs. The principle may be used for creation of variant tolerant peptide serologies. Thus, our system allows rational large-scale serological assays with built-in quality control.
Jonas Blomberg, Fredrik Blomberg, Anna Sjösten, Ali Sheikholvaezin, Agnes Bölin-Wiener, Amal Elfaitouri, Sanna Hessel, Carl-Gerhard Gottfries, Olof Zachrisson, Christina Ohrmalm, Magnus Jobs, Rüdiger Pipkorn

2608 related Products with: No evidence for xenotropic murine leukemia-related virus infection in Sweden using internally controlled multiepitope suspension array serology.



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#15820778   // To Up

Human heterophilic antibodies display specificity for murine IgG subclasses.

The study investigated heterophilic antibodies: the human immunoglobulin classes involved and their specificity for different murine IgG subclasses.
Johan Bjerner, Kari Hauge Olsen, Ole P Børmer, Kjell Nustad

1210 related Products with: Human heterophilic antibodies display specificity for murine IgG subclasses.

100 TESTS0.2 mg1 mg0.1 mg0.2 mg0.5 mg1 mg1 mg1 mg

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#9776294   // To Up

Biological properties of biotin-chelate conjugates for pretargeted diagnosis and therapy with the avidin/biotin system.

Three-step pretargeting increases target-to-background ratios in radioimmunodetection and can potentially decrease harmful radiation to normal tissues in radioimmunotherapy. We studied four biotin-chelate conjugates (BCCs) for use in the avidin/biotin pretargeting system.
D A Goodwin, C F Meares, M Osen

1857 related Products with: Biological properties of biotin-chelate conjugates for pretargeted diagnosis and therapy with the avidin/biotin system.

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#7682210   // To Up

Immunocytochemical double staining of cytokeratin and prostate specific antigen in individual prostatic tumour cells.

Early dissemination of malignant cells is the main cause for metastatic relapse in patients with solid tumours. By use of monoclonal antibodies (mAbs) specific for cytokeratins, disseminated individual epithelial tumour cells can now be identified in mesenchymal organs such as bone marrow. Further to characterize such cells in patients with prostate cancer, an immunocytochemical procedure was developed for simultaneous labelling of cytokeratin component no. 18 (CK18) and prostate specific antigen (PSA). In a first step, cells were incubated with mAb ER-PR8 against PSA and secondary gold-conjugated goat anti-mouse antibodies. In a second step, biotinylated mAb CK2 to CK18 was applied as primary antibody and subsequently incubated with complexes of streptavidin-conjugated alkaline phosphatase, which were developed with the Newfuchsin substrate. The binding of gold-labelled antibodies was visualized by silver enhancement. The sensitivity and specificity of the technique was demonstrated on cryostat sections of hyperplastic prostatic tissue, and cytological preparations of LNCaP prostatic tumour cells. Double staining was restricted to cells derived from the secretory epithelium of the prostate. Cross-reactivity between both detection systems was excluded by several controls, including the use of unrelated antibodies of the same isotype and the staining of CK18+/PSA- HT29 colon carcinoma cells. CK18+ cells co-expressing PSA were found in bone marrow aspirates from 5 out of 13 patients with carcinomas of the prostate, a finding that is consistent with the relative fraction of double-positive LNCaP cells. The specificity of CK18 for epithelial tumour cells in bone marrow was supported by negative staining of 12 control aspirates from patients with benign prostatic hypertrophy.(ABSTRACT TRUNCATED AT 250 WORDS)
R Riesenberg, R Oberneder, M Kriegmair, M Epp, U Bitzer, A Hofstetter, S Braun, G Riethmüller, K Pantel

1694 related Products with: Immunocytochemical double staining of cytokeratin and prostate specific antigen in individual prostatic tumour cells.

25 ml Ready-to-use 25 ml Ready-to-use 2 ml Ready-to-use 6 ml Ready-to-use 2 ml Ready-to-use 0.05 mg 6 ml Ready-to-use 100 1 kit(96 Wells)

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