Demonstration of Monoclonal Antibodies

Peters, J. H.; Schulze, Matthias; Grol, Michael; Schiefer, Samira; Baumgarten, H.; Endl, Josef; Xu, Hao; Cells, Living; Lenzner, S.; Beuche, Wolfgang; Thomas, R.; Denden, Mounir; Friedrichs, Ulrike; Werfel, T.; Hebell, T.; Essig, Ulrich; Hempelmann, Ernst; Oppermann, Marc; Würzner, Reinhard

doi:10.1007/978-3-642-74532-4_10

Cited by 5 publications

(4 citation statements)

References 1 publication

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“…A standard sandwich ELISA 62 was used for measurements of antibody concentration. Mouse IgM B Cell ELISpot Development Module (R&D Systems) and a standard of mouse IgM (clone MM30, Abcam) were used for the quantification of IgMs.…”

Section: Methodsmentioning

confidence: 99%

Agglutinating mouse IgG3 compares favourably with IgMs in typing of the blood group B antigen: Functionality and stability studies

Klaus

Bzowska

Kulesza

et al. 2016

Sci Rep

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Mouse immunoglobulins M (IgMs) that recognize human blood group antigens induce haemagglutination and are used worldwide for diagnostic blood typing. Contrary to the current belief that IgGs are too small to simultaneously bind antigens on two different erythrocytes, we obtained agglutinating mouse IgG3 that recognized antigen B of the human ABO blood group system. Mouse IgG3 is an intriguing isotype that has the ability to form Fc-dependent oligomers. However, F(ab′)2 fragments of the IgG3 were sufficient to agglutinate type B red blood cells; therefore, IgG3-triggered agglutination did not require oligomerization. Molecular modelling indicated that mouse IgG3 has a larger range of Fab arms than other mouse IgG subclasses and that the unique properties of mouse IgG3 are likely due to the structure of its hinge region. With a focus on applications in diagnostics, we compared the stability of IgG3 and two IgMs in formulated blood typing reagents using an accelerated storage approach and differential scanning calorimetry. IgG3 was much more stable than IgMs. Interestingly, the rapid decrease in IgM activity was caused by aggregation of the molecules and a previously unknown posttranslational proteolytic processing of the μ heavy chain. Our data point to mouse IgG3 as a potent diagnostic tool.

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Section: Methodsmentioning

confidence: 99%

Agglutinating mouse IgG3 compares favourably with IgMs in typing of the blood group B antigen: Functionality and stability studies

Klaus

Bzowska

Kulesza

et al. 2016

Sci Rep

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“…Three days prior to euthanasia and dissection of the spleens, the animals displaying the highest serum titer were CLDN1 RE-EXPRESSION IN BREAST CANCER CELLS boosted intravenously with 30 mg CLDN1 protein puri®ed from Sf9 cells (see above). The mice were sacri®ced at day 129, and the spleens were used to generate hybridomas following standard procedures (Peters and Baumgarten, 1992). High-producer hybridoma clones were generated by¯uorescence-activated cell sorting cloning of CLDN1 positive hybridoma mass cultures, via anti-l-NGFR immuno¯uorescence (Machl et al, 1997).…”

Section: Monoclonal Antibody (Mab) Productionmentioning

confidence: 99%

Expression and targeting of the tight junction protein CLDN1 in CLDN1‐negative human breast tumor cells

Hoevel

Macek

Mundigl

et al. 2002

Journal Cellular Physiology

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Claudins and occludin constitute the major transmembrane proteins of tight junctions (TJs). We have previously identified the human homologue of the murine Cldn1, CLDN1 (SEMP1) that is expressed in normal, mammary gland-derived epithelial cells but is absent in most human breast cancer cell lines. To investigate the potential functions of CLDN1 protein in tumor and normal epithelial cells, we developed an I-NGFR retroviral vector and monoclonal anti-CLDN1 antibody. In subconfluent and confluent breast cancer cells, MDA-MB-435 and MDA-MB-361, endogenous CLDN1 expression was not detected by an anti-CLDN1 monoclonal antibody by Western blot analysis or quantitative RT-PCR. When CLDN1-negative breast cancer cell lines were transduced with a CLDN1 retrovirus the cells express CLDN1 mRNA constitutively as shown by quantitative RT-PCR. Immunofluorescence analyses of the CLDN1 retroviral transduced breast tumor cells using monoclonal antibodies against CLDN1 reveals a subcellular distribution at cell-cell contact sites similar to the CLDN1 homing pattern in T47-D cells, which express endogenous CLDN1. This cell-cell contact co-localization of CLDN1 was evident in CLDN1-transduced breast tumor cells which fail to express occludin protein (MDA-MB-361 and MDA-MB-435) and express relatively little ZO-1 protein (MDA-MB-435), suggesting that other proteins may be responsible for targeting of CLDN1 to cell-cell contact sites. The re-expression of CLDN1 decreases the paracellular flux of 3 and 40 kDa dextran despite the absence of occludin in the MDA-MB-361 tumor cells. Our findings indicate that in CLDN1-negative breast tumor cells, the basal protein partner requirements for physiological homing of the CLDN1 protein are intact, and that CLDN1 gene transfer and protein expression itself might be sufficient to exert a TJ-mediate gate function in metastatic tumor cells even in the absence of other TJ-associated proteins, such as occludin.

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“…Cell culture supernatants usually contained between 10-20 µg/ml antibody as determined by sandwich ELISA according to standard protocols (Peters and Baumgarten, 1992). The IgG subclass was determined using a commercially available kit (Boehringer Mannheim, Germany).…”

Section: Production Of Monoclonal Hybridomamentioning

confidence: 99%

Identification and characterization of differentiation‐dependent Schwann cell surface antigens by novel monoclonal antibodies: Introduction of a marker common to the non‐myelin‐forming phenotype

Wewetzer

Grothe

Christ

et al. 1997

Glia

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Demonstration of Monoclonal Antibodies

Cited by 5 publications

References 1 publication

Agglutinating mouse IgG3 compares favourably with IgMs in typing of the blood group B antigen: Functionality and stability studies

Agglutinating mouse IgG3 compares favourably with IgMs in typing of the blood group B antigen: Functionality and stability studies

Expression and targeting of the tight junction protein CLDN1 in CLDN1‐negative human breast tumor cells

Identification and characterization of differentiation‐dependent Schwann cell surface antigens by novel monoclonal antibodies: Introduction of a marker common to the non‐myelin‐forming phenotype

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