anti mouse fluorescein isothiocyanate fitc  (Vector Laboratories)

Journal: Cellular & Molecular Biology Letters

Article Title: GPA33 forms a distinct diagnostic target class to Claudin 18.2 in oesophageal adenocarcinoma enabling the development of a novel GPA33 antibody-based detection platform

doi: 10.1186/s11658-025-00852-1

Figure Lengend Snippet: Development of antibodies to detect captured GPA33 in an ELISA format. Evaluation of RSE-05 in capture-sensor ELISA format using three distinct polyclonal antibodies to GPA33. A The indicated sera (from peptide 1 and peptide 2 derived from potential GPA33 surface loops; Supplementary Fig. 6) were validated for presence of peptide specific IgG; the sera was affinity purified on a peptide 1 or peptide 2 column, respectively, and after elution with 0.1 M glycine buffer (pH 2.5), fractions were reduced and separated on an SDS gel and stained with Coomassie Blue to validate the presence and amounts of peptide-specific IgG. B Dot-blotting of transfected HEK293T cell lysates without or with GPA33. 7.5 μg of protein lysate was used in each dot. Left, empty-vector transfections. Right, GPA33-gene transfections. Polyclonal fractions 1.5 and 2.6 were used for peptide 1 and peptide 2 respectively. C Anti-GPA33 activity of polyclonal sera by ELISA format, fractions 1.5 and 2.6. 200 ng of tagged GPA33 was coated, or with pre-treatment using 50 mM DTT to reduce di-sulphide bonds, with uncoated wells as negative control. D Sandwich ELISAs using three different polyclonal antibodies in conjunction with RSE-05 to determine whether RSE-05 can function in a capture-sensor format. The indicated antibodies were coated to the immunoplate wells as capture agents (RSE-05, Atlas polyclonal antibody, CPF1.5 P1 polyclonal antibody or CEG 2.6 P2 polyclonal antibody). Following this, 200 ng of recombinant GPA33 protein (FC tagged) was added then complementary antibodies were added in sensor format, labelled as ‘detection’. Anti-mouse or anti-rabbit HRP secondary antibodies were then added, and signal measured using HRP and luminescence

Article Snippet: Then, cells were incubated with anti-mouse fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Vector Laboratories Inc., Burlingame, CA, USA, cat. no. FI-2000-1.5) in the blocking solution for 30 min in the dark and washed twice in PBS.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Affinity Purification, SDS-Gel, Staining, Transfection, Plasmid Preparation, Activity Assay, Negative Control, Recombinant

Journal: Cellular & Molecular Biology Letters

Article Title: GPA33 forms a distinct diagnostic target class to Claudin 18.2 in oesophageal adenocarcinoma enabling the development of a novel GPA33 antibody-based detection platform

doi: 10.1186/s11658-025-00852-1

Figure Lengend Snippet: The RSE-05 MAB binds to full-length GPA33 protein after transfection into human cells. A Validation of GPA33 expression plasmids in human cells. MCF7 cells were transfected with either HA-tagged (T) (lanes 1 and 3) or non-tagged (nT) full length GPA33 containing the transmembrane domain (lanes 2 and 4) expression plasmids, and after acquiring the cell lysates using urea lysis buffer (8 M urea in PBS), a denaturing immunoblot was processed in lanes that either contain DTT (D) (lanes 1 and 2) or without DTT in the sample loading buffer (lanes 3–5). Lysates from non-transfected MCF7 cells are in lane 5. Samples were immunoblotted with the Atlas pAb (commercial antibody), which can detect all isoforms of GPA33. The presumed monomeric form of GPA33 has a faster mobility in the absence of DTT, which presumably reflects SDS resistance of the two IgG-like lobes to denaturation due to the three di-sulphide bonds. B Analysis of RSE-05 MAB binding to GPA33 in fixed cells using immunofluorescence. C , D Analysis of RSE-05 MAB binding to cell surface localized GPA33 in living cells. MCF7 cells were mock transfected ( C ) or transfected with the GPA33 expression plasmid ( D ) and processed by flow cytometry. The data are plotted as: left panels, FITC-A is plotted as a function of FSC-A; right panels, cell count is plotted as a function of FITC-A (left panels and right panels). E MCF7 cells were stably transfected with the GPA33 expression plasmid and flow cytometry was used to isolate the positive cells (in grey) separated from the GPA33 negative cells (in red). The data are plotted as: FITC-A as a function of FSC-A stable GPA33-positive cells and GPA33-negative negative cells. F Analysis of individual GPA33 + cell clones, 3, 7 and 11. Three GPA33 stably expressing cell clones were processed by flow cytometry to measure GPA33-positive staining with the RSE-05 MAB. The data are plotted as: GPA33-positive cells as a function of FITC-A

Article Snippet: Then, cells were incubated with anti-mouse fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Vector Laboratories Inc., Burlingame, CA, USA, cat. no. FI-2000-1.5) in the blocking solution for 30 min in the dark and washed twice in PBS.

Techniques: Transfection, Biomarker Discovery, Expressing, Lysis, Western Blot, Binding Assay, Immunofluorescence, Plasmid Preparation, Flow Cytometry, Cell Characterization, Stable Transfection, Clone Assay, Staining

Journal: Cellular & Molecular Biology Letters

Article Title: GPA33 forms a distinct diagnostic target class to Claudin 18.2 in oesophageal adenocarcinoma enabling the development of a novel GPA33 antibody-based detection platform

doi: 10.1186/s11658-025-00852-1

Figure Lengend Snippet: The epitope of the RSE-05 IgG is sensitive to reduction. A The indicated antibodies were used in immunoblots using purified His-tagged GPA33 protein without or with DTT in the SDS loading buffer. After incubating with the primary antibodies and adding anti-mouse or anti-rabbit HRP-conjugated secondary antibodies, membranes were stained using TMB. B ELISA was used to measure the binding activity of the RSE-05 MAB compared with the commercially available rabbit polyclonal antibody (Atlas pAb). Using GPA33 treated with the indicated chemicals (0.5% v/v SDS and/or 50 mM DTT), at different temperatures, RSE-05 and Atlas pAb (commercial antibody) binding was evaluated using anti-mouse or anti-rabbit HRP-conjugated secondary antibody, respectively. The binding activity is measured in luminescence, relative light units. C ELISA performed as in B , in this case with DTT titrated

Article Snippet: Then, cells were incubated with anti-mouse fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Vector Laboratories Inc., Burlingame, CA, USA, cat. no. FI-2000-1.5) in the blocking solution for 30 min in the dark and washed twice in PBS.

Techniques: Western Blot, Purification, Staining, Enzyme-linked Immunosorbent Assay, Binding Assay, Activity Assay