Alexa fluor 647 conjugated goat anti human igg, by Jackson ImmunoResearch - Product details

1

Quantifying Plasmodium Parasitemia with MGD21

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3D7-MGD21⁺ (5% parasitemia, ring stage) was cultured with various concentrations of MGD21 or BKC3 for 2 d. After 2 d, 10× SYBR Green I was added to aliquots of each culture and parasitemia was quantified by flow cytometry. The remaining parasites in each culture were washed to remove the antibodies and incubated for 1 d to allow the parasites to reach the late trophozoite/schizont stage. MGD21 recognition of these cultures was detected using 2.5 μg/mL of Alexa Fluor 647-conjugated goat anti-human IgG (Jackson ImmunoResearch, cat. no 109-606-170).

Tan J., Pieper K., Piccoli L., Abdi A., Perez M.F., Geiger R., Tully C.M., Jarrossay D., Maina Ndungu F., Wambua J., Bejon P., Fregni C.S., Fernandez-Rodriguez B., Barbieri S., Bianchi S., Marsh K., Thathy V., Corti D., Sallusto F., Bull P, & Lanzavecchia A. (2015). A LAIR-1 insertion generates broadly reactive antibodies against malaria variant antigens. Nature, 529(7584), 105-109.

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2

Isolation and Immortalization of Antigen-Specific Memory B Cells

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IgM or IgG memory B cells were isolated from frozen peripheral blood mononuclear cells (PBMCs) by magnetic cell sorting with 0.5 μg ml⁻¹ anti-CD19-PECy7 antibodies (BD, 341113) and mouse anti-PE microbeads (Miltenyi Biotec, 130-048-081), followed by FACS sorting using 3.75 μg ml⁻¹ Alexa Fluor 647-conjugated goat anti-human IgG (Jackson ImmunoResearch, 109-606-170), 5 μg ml⁻¹ Alexa Fluor 647-conjugated goat anti-human IgM (Invitrogen, A21215) and 1/40 PE-labeled anti-human IgD (BD, 555779). As previously described^{47 (link)}, sorted B cells were immortalized with Epstein-Barr virus (EBV) and plated in single cell cultures in the presence of CpG-DNA (2.5 μg ml⁻¹) and irradiated PBMC-feeder cells. Two weeks post-immortalization, the culture supernatants were tested (at a 2/5 dilution) for binding to PfSPZ by flow cytometry using a no-wash protocol. Briefly, cryopreserved PfSPZ were thawed, stained with the supernatants in 6.25× SYBR Green I for 30 min at room temperature, and incubated with 2.5 μg ml⁻¹ Alexa Fluor 647-conjugated goat anti-human IgG or anti-human IgM for 1 hour at 4°C. Only supernatants that did not bind to control beads were selected to exclude polyreactive antibodies.

Tan J., Sack B.K., Oyen D., Zenklusen I., Piccoli L., Barbieri S., Foglierini M., Fregni C.S., Marcandalli J., Jongo S., Abdulla S., Perez L., Corradin G., Varani L., Sallusto F., Sim B.K., Hoffman S.L., Kappe S.H., Daubenberger C., Wilson I.A, & Lanzavecchia A. (2018). A public antibody lineage that potently inhibits malaria infection by dual binding to the circumsporozoite protein. Nature medicine, 24(4), 401-407.

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3

Quantifying NZM antibody binding and neutralization

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Serial dilutions of NZM and NZM variants IgG4 were prepared in MACS
buffer (PBS 1% FBS, 2mM EDTA). T cells isolated from healthy donors were used as
source of the cell adhesion molecule α4-integrin and added (50,000
cell/well) to the plates for 30 min, 4°C. T cells were washed and stained
with 3.75 μg/ml Alexa Fluor 647–conjugated goat anti-human IgG
(Jackson ImmunoResearch, cat. no. 109-606-170) for 30 min, 4°C. Cells
were washed and analyzed by FACS. NZM binding was calculated as percentage of
IgG⁺ stained cells. To study NZM neutralization, NZM-mFc was
diluted to 5 ng/ml (final concentration) in MACS buffer (PBS 1% FBS, 2mM EDTA)
and incubated with titrated monoclonal antibodies for 1 h, 37°C. T cells
were added to the plates for 30 min, 4°C, then washed and stained with
secondary goat anti-mouse-IgG-PE (SouthernBiotech, cat. no. 1030-09) at 1
μg/ml for 30 min, 4°C. Cells were washed and analyzed by FACS. NZM
neutralization was calculated for each well as percentage of inhibition of
binding of NZM-mFc to T cells with the following formula: 1 – % of cells
stained by NZM-mFc. Gates were defined based on negative and positive
controls.

Cassotta A., Mikol V., Bertrand T., Pouzieux S., Le Parc J., Ferrari P., Dumas J., Auer M., Deisenhammer F., Gastaldi M., Franciotta D., Silacci-Fregni C., Fernandez Rodriguez B., Giacchetto-Sasselli I., Foglierini M., Jarrossay D., Geiger R., Sallusto F., Lanzavecchia A, & Piccoli L. (2019). A single T cell epitope drives the neutralizing anti-drug antibody response to natalizumab in multiple sclerosis patients. Nature medicine, 25(9), 1402-1407.

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4

Quantifying Antibody Binding to PfSPZ

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Cryopreserved PfSPZ (Sanaria^®) were thawed and stained with different concentrations of Tanzanian sera or monoclonal antibodies in 3.3× SYBR Green I (ThermoFisher Scientific) for 30 min at 4°C. The PfSPZ were washed twice by centrifugation at 3220 × g for 5 min. Human serum antibody binding was detected using 2.5 μg ml⁻¹ Alexa Fluor 647-conjugated goat anti-human IgG (Jackson ImmunoResearch, 109-606-170) or Alexa Fluor 647-conjugated goat anti-human IgM (Jackson ImmunoResearch, 109-606-129). Mouse serum antibody binding was detected using 1 μg ml⁻¹ PE-Cy7-conjugated goat anti-mouse IgG (Biolegend, 405315) or PE-Cy7-conjugated rat anti-mouse IgM (BD Biosciences, 552867). FACS Diva (version 6.2) was used for acquisition of samples and Flow-Jo (version 10.1) was used for FACS analysis. The PfSPZ were gated based on high fluorescence in the FITC channel. Median fluorescence intensity (MFI) of the PfSPZ in the Alexa Fluor 647 or PE-Cy7 channel was calculated to quantify IgG or IgM binding. The concentration of antibody needed to achieve MFI 10,000 (Conc₁₀₀₀₀) was calculated by interpolation of binding curves fitted to a sigmoidal curve model (Graphpad Prism 7) as a measure of affinity. The gating strategy can be found in Supplementary Figure 1.

Tan J., Sack B.K., Oyen D., Zenklusen I., Piccoli L., Barbieri S., Foglierini M., Fregni C.S., Marcandalli J., Jongo S., Abdulla S., Perez L., Corradin G., Varani L., Sallusto F., Sim B.K., Hoffman S.L., Kappe S.H., Daubenberger C., Wilson I.A, & Lanzavecchia A. (2018). A public antibody lineage that potently inhibits malaria infection by dual binding to the circumsporozoite protein. Nature medicine, 24(4), 401-407.

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5

ALPPL2 Expression and Antibody Binding

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CHO-K1 or HEK293 cells were transfected with plasmids expressing human ALPPL2, ALPP, ALPI, and ALPL using Lipofectamine® 2000 (Life Technologies/Thermo Fisher Scientific), and incubated with anti-ALPPL2 human antibodies followed by Alexa Fluor® 647-conjugated goat anti-human IgG (Jackson ImmunoResearch Laboratories). Binding was analyzed by flow cytometry (BD Accuri™ C6, BD Biosciences) that generated median fluorescence intensity (MFI) values.

Su Y., Zhang X., Bidlingmaier S., Behrens C.R., Lee N.K, & Liu B. (2020). ALPPL2 is a highly specific and targetable tumor cell surface antigen. Cancer research, 80(20), 4552-4564.

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6

Isolation and Immortalization of Antigen-Specific Memory B Cells

Cited 1 time

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IgM or IgG memory B cells were isolated from frozen peripheral blood mononuclear cells (PBMCs) by magnetic cell sorting with 0.5 μg ml⁻¹ anti-CD19-PECy7 antibodies (BD, 341113) and mouse anti-PE microbeads (Miltenyi Biotec, 130-048-081), followed by FACS sorting using 3.75 μg ml⁻¹ Alexa Fluor 647-conjugated goat anti-human IgG (Jackson ImmunoResearch, 109-606-170), 5 μg ml⁻¹ Alexa Fluor 647-conjugated goat anti-human IgM (Invitrogen, A21215) and 1/40 PE-labeled anti-human IgD (BD, 555779). As previously described^{47 (link)}, sorted B cells were immortalized with Epstein-Barr virus (EBV) and plated in single cell cultures in the presence of CpG-DNA (2.5 μg ml⁻¹) and irradiated PBMC-feeder cells. Two weeks post-immortalization, the culture supernatants were tested (at a 2/5 dilution) for binding to PfSPZ by flow cytometry using a no-wash protocol. Briefly, cryopreserved PfSPZ were thawed, stained with the supernatants in 6.25× SYBR Green I for 30 min at room temperature, and incubated with 2.5 μg ml⁻¹ Alexa Fluor 647-conjugated goat anti-human IgG or anti-human IgM for 1 hour at 4°C. Only supernatants that did not bind to control beads were selected to exclude polyreactive antibodies.

Tan J., Sack B.K., Oyen D., Zenklusen I., Piccoli L., Barbieri S., Foglierini M., Fregni C.S., Marcandalli J., Jongo S., Abdulla S., Perez L., Corradin G., Varani L., Sallusto F., Sim B.K., Hoffman S.L., Kappe S.H., Daubenberger C., Wilson I.A, & Lanzavecchia A. (2018). A public antibody lineage that potently inhibits malaria infection by dual binding to the circumsporozoite protein. Nature medicine, 24(4), 401-407.

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7

Quantifying Plasmodium Parasitemia with MGD21

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3D7-MGD21⁺ (5% parasitemia, ring stage) was cultured with various concentrations of MGD21 or BKC3 for 2 d. After 2 d, 10× SYBR Green I was added to aliquots of each culture and parasitemia was quantified by flow cytometry. The remaining parasites in each culture were washed to remove the antibodies and incubated for 1 d to allow the parasites to reach the late trophozoite/schizont stage. MGD21 recognition of these cultures was detected using 2.5 μg/mL of Alexa Fluor 647-conjugated goat anti-human IgG (Jackson ImmunoResearch, cat. no 109-606-170).

Tan J., Pieper K., Piccoli L., Abdi A., Perez M.F., Geiger R., Tully C.M., Jarrossay D., Maina Ndungu F., Wambua J., Bejon P., Fregni C.S., Fernandez-Rodriguez B., Barbieri S., Bianchi S., Marsh K., Thathy V., Corti D., Sallusto F., Bull P, & Lanzavecchia A. (2015). A LAIR-1 insertion generates broadly reactive antibodies against malaria variant antigens. Nature, 529(7584), 105-109.

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8

Quantifying Antibody Binding to PfSPZ

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Cryopreserved PfSPZ (Sanaria^®) were thawed and stained with different concentrations of Tanzanian sera or monoclonal antibodies in 3.3× SYBR Green I (ThermoFisher Scientific) for 30 min at 4°C. The PfSPZ were washed twice by centrifugation at 3220 × g for 5 min. Human serum antibody binding was detected using 2.5 μg ml⁻¹ Alexa Fluor 647-conjugated goat anti-human IgG (Jackson ImmunoResearch, 109-606-170) or Alexa Fluor 647-conjugated goat anti-human IgM (Jackson ImmunoResearch, 109-606-129). Mouse serum antibody binding was detected using 1 μg ml⁻¹ PE-Cy7-conjugated goat anti-mouse IgG (Biolegend, 405315) or PE-Cy7-conjugated rat anti-mouse IgM (BD Biosciences, 552867). FACS Diva (version 6.2) was used for acquisition of samples and Flow-Jo (version 10.1) was used for FACS analysis. The PfSPZ were gated based on high fluorescence in the FITC channel. Median fluorescence intensity (MFI) of the PfSPZ in the Alexa Fluor 647 or PE-Cy7 channel was calculated to quantify IgG or IgM binding. The concentration of antibody needed to achieve MFI 10,000 (Conc₁₀₀₀₀) was calculated by interpolation of binding curves fitted to a sigmoidal curve model (Graphpad Prism 7) as a measure of affinity. The gating strategy can be found in Supplementary Figure 1.

Tan J., Sack B.K., Oyen D., Zenklusen I., Piccoli L., Barbieri S., Foglierini M., Fregni C.S., Marcandalli J., Jongo S., Abdulla S., Perez L., Corradin G., Varani L., Sallusto F., Sim B.K., Hoffman S.L., Kappe S.H., Daubenberger C., Wilson I.A, & Lanzavecchia A. (2018). A public antibody lineage that potently inhibits malaria infection by dual binding to the circumsporozoite protein. Nature medicine, 24(4), 401-407.

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9

Immunofluorescent Visualization of ADC Internalization

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M28 and VAMT-1 cells (5,000 per well) were seeded in glass chamber slides (Thermo Fisher Scientific) and grown at 37°C overnight, incubated with 15 μg/ml M25 IgG or M25ADCs for 4h or 24h, washed with PBS, fixed with 4% formaldehyde, and permeabilized with PBS containing 0.1% Triton X-100 and 1% BSA. M25 IgG was detected by Alexa Fluor® 647-conjugated goat anti-Human IgG (Jackson ImmunoResearch Laboratories). Cell nuclei were counterstained with Hoechst (Life Technologies/Thermo Fisher Scientific). Lysosomes were marked by anti-LAMP1 antibodies (Clone D2D11, Cell Signaling Technology) followed by detection with FITC–labeled donkey anti-Rabbit IgG (Jackson ImmunoResearch Laboratories). Images were taken and analyzed by an automated FluoView confocal microscope (Olympus).

Su Y., Zhang X., Bidlingmaier S., Behrens C.R., Lee N.K, & Liu B. (2020). ALPPL2 is a highly specific and targetable tumor cell surface antigen. Cancer research, 80(20), 4552-4564.

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10

CD38+ Cell Line Binding Assay

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CD38⁺ cells including multiple myeloma cell lines (RPMI 8226, NCI-H929, MM.1S), Burkitt’s lymphoma cell lines (Ramos, Daudi, Raji), diffuse large B lymphoma cell line (Toledo), acute B lymphoid leukemia cell lines (RS4;11, NALM-6), and acute T lymphoid leukemia cell lines (CCRF-CEM, TALL-1, HuT 78) were obtained from American Type Culture Collection. Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 2 mM L-Glutamine (HyClone) at 37°C in a humidified 5% CO₂ incubator. 5×10⁴ tumor cells were incubated with serial dilutions of CM313 or isotype control for 45 min. After washing and incubation with Alexa Fluor 647-conjugated goat anti-human IgG (Jackson ImmunoResearch) for a further 45 min, the cells were resuspended in propidium iodide (PI). Cell-associated fluorescence was analyzed using a FACSCelesta with FACSDiva™ software (BD Biosciences).

Liu W., Yu J., Sun K., Song Q., Li Y., He Y., Wang Y., Xu G., Wang C, & Chen B. (2024). Preclinical characterization of a novel investigational monoclonal antibody CM313 with potent CD38-positive cell killing activity. Frontiers in Immunology, 15, 1410457.

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Alexa fluor 647 conjugated goat anti human igg

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11 protocols using alexa fluor 647 conjugated goat anti human igg

Quantifying Plasmodium Parasitemia with MGD21

Isolation and Immortalization of Antigen-Specific Memory B Cells

Quantifying NZM antibody binding and neutralization

Quantifying Antibody Binding to PfSPZ

ALPPL2 Expression and Antibody Binding

Isolation and Immortalization of Antigen-Specific Memory B Cells

Quantifying Plasmodium Parasitemia with MGD21

Quantifying Antibody Binding to PfSPZ

Immunofluorescent Visualization of ADC Internalization

CD38+ Cell Line Binding Assay

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