Journal: Microbiology Spectrum

Article Title: Rapid Point-of-Care Tests Using Staphylococcal Protein A Can Detect Early IgM Responses in HIV-1 and Treponema pallidum Infections

doi: 10.1128/spectrum.03309-22

Figure Lengend Snippet: Antibody detection in HIV-1 seroconversion panel PRB-927 and effect of sample pretreatment with 0.1 M 2-ME. (A) Micro Reader reflectance values generated with the DPP HIV 1/2 (red lines) and DPP HIV 1/2 IgM assays (blue lines) are shown for samples tested before (solid lines) and after 2-ME treatment (dashed lines). Framed are test results for samples 03 and 04 collected during initial IgM seroconversion (days 33 and 35 since 1st bleed), in which antibody detection by each of the two immunoassays was diminished after 2-ME treatment by at least 50% signal reduction. (B) Results show differentially evolving susceptibility to the 2-ME treatment of postseroconversion samples tested by the DPP HIV 1/2 (red line) and DPP HIV 1/2 IgM assays (blue line). Framed is the same data subset as that included in panel A but results are shown in percent signal reduction over time.

Article Snippet: DPP HIV 1/2 and DPP HIV-Syphilis tests were converted into investigational IgM-detecting assays, named DPP HIV 1/2 IgM and DPP HIV-Syphilis IgM, respectively, by replacing SpA coupled to gold nanoparticles with mouse monoclonal antibody to human IgM (HyTest Ltd., Turku, Finland), a specific reagent for IgM detection validated in commercial DPP products ( , ).

Techniques: Generated

Journal: Microbiology Spectrum

Article Title: Rapid Point-of-Care Tests Using Staphylococcal Protein A Can Detect Early IgM Responses in HIV-1 and Treponema pallidum Infections

doi: 10.1128/spectrum.03309-22

Figure Lengend Snippet: IgM antibody detection by SpA-based rapid point-of-care tests in HIV-1 seroconversion panels ^a

Article Snippet: DPP HIV 1/2 and DPP HIV-Syphilis tests were converted into investigational IgM-detecting assays, named DPP HIV 1/2 IgM and DPP HIV-Syphilis IgM, respectively, by replacing SpA coupled to gold nanoparticles with mouse monoclonal antibody to human IgM (HyTest Ltd., Turku, Finland), a specific reagent for IgM detection validated in commercial DPP products ( , ).

Techniques:

Journal: Microbiology Spectrum

Article Title: Rapid Point-of-Care Tests Using Staphylococcal Protein A Can Detect Early IgM Responses in HIV-1 and Treponema pallidum Infections

doi: 10.1128/spectrum.03309-22

Figure Lengend Snippet: Antibody detection in AccuVert Syphilis Seroconversion Panel PSS901 and effect of sample pre-treatment with 0.1 M 2-ME. (A) Micro Reader reflectance values generated with the DPP HIV-Syphilis (red lines) and DPP HIV-Syphilis IgM assays (blue lines) are shown for samples tested before (solid lines) and after 2-ME treatment (dashed lines). Framed are test results for samples 06 to 09 collected during the initial IgM response (days 45 to 59 since 1st bleed), in which antibody detection with each of the two immunoassays was diminished after 2-ME treatment by at least 50% signal reduction. (B) Results show differentially evolving susceptibility to the 2-ME treatment of postseroconversion samples (same as framed in panel A) tested by the DPP HIV-Syphilis (red line) and DPP HIV-Syphilis IgM assays (blue line).

Article Snippet: DPP HIV 1/2 and DPP HIV-Syphilis tests were converted into investigational IgM-detecting assays, named DPP HIV 1/2 IgM and DPP HIV-Syphilis IgM, respectively, by replacing SpA coupled to gold nanoparticles with mouse monoclonal antibody to human IgM (HyTest Ltd., Turku, Finland), a specific reagent for IgM detection validated in commercial DPP products ( , ).

Techniques: Generated

Journal: Microbiology Spectrum

Article Title: Rapid Point-of-Care Tests Using Staphylococcal Protein A Can Detect Early IgM Responses in HIV-1 and Treponema pallidum Infections

doi: 10.1128/spectrum.03309-22

Figure Lengend Snippet: IgM antibody detection by DPP HIV-Syphilis assay in active syphilis ^a

Article Snippet: DPP HIV 1/2 and DPP HIV-Syphilis tests were converted into investigational IgM-detecting assays, named DPP HIV 1/2 IgM and DPP HIV-Syphilis IgM, respectively, by replacing SpA coupled to gold nanoparticles with mouse monoclonal antibody to human IgM (HyTest Ltd., Turku, Finland), a specific reagent for IgM detection validated in commercial DPP products ( , ).

Techniques:

Journal: Microbiology Spectrum

Article Title: Rapid Point-of-Care Tests Using Staphylococcal Protein A Can Detect Early IgM Responses in HIV-1 and Treponema pallidum Infections

doi: 10.1128/spectrum.03309-22

Figure Lengend Snippet: Correlation between antibody levels detected by SpA or anti-IgM reagent in early HIV-1 seroconversion and syphilis samples. (A) Micro Reader reflectance values generated with the DPP HIV-1/2 and DPP HIV-1/2 IgM assays are shown for the HIV-1 seroconversion samples included in Table 1 , Groups 2 and 3 (circles), and matching preseroconversion members (triangles). (B) Micro Reader reflectance values generated for treponemal antibody levels with the DPP HIV-Syphilis and DPP HIV-Syphilis IgM assays are shown for the syphilis-positive samples listed in Table 2 (circles) and preseroconversion panel members (triangles). Horizontal and vertical dashed lines indicate cutoff values used for test interpretation: DPP HIV-1/2, 10 RLU; DPP HIV-1/2 IgM, 40 RLU; DPP HIV-Syphilis, 7 RLU; DPP HIV-Syphilis IgM, 60 RLU.

Article Snippet: DPP HIV 1/2 and DPP HIV-Syphilis tests were converted into investigational IgM-detecting assays, named DPP HIV 1/2 IgM and DPP HIV-Syphilis IgM, respectively, by replacing SpA coupled to gold nanoparticles with mouse monoclonal antibody to human IgM (HyTest Ltd., Turku, Finland), a specific reagent for IgM detection validated in commercial DPP products ( , ).

Techniques: Generated

Journal: Frontiers in Immunology

Article Title: Natural Human Immunity Against Staphylococcal Protein A Relies on Effector Functions Triggered by IgG3

doi: 10.3389/fimmu.2022.834711

Figure Lengend Snippet: Opsonizing anti-SpA antibodies in human sera are predominantly IgG3. (A) Detection of SpA opsonizing antibodies in sera via soluble biotinylated FcγR1A coupled with streptavidin-PE. Each line represents the titration-dependent trend of median fluorescence for each tested serum. ISA subjects are identified by a progressive number, with v2 indicating a second visit. Healthy subjects are identified by the nomenclature HC = healthy control or PHC = pediatric healthy control. Two exemplificative negative controls are shown, PHC13, and 44. The data represent the mean ± SD of three independent experiments. (B) Detection of subclass 3 opsonizing antibodies deposited on SpAwt coated beads from positive FcγR1A hits. The data represent the mean ± SD of three independent experiments. (C) Detection of subclass 1 anti-SpA opsonizing antibodies deposited on SpAmut coated beads. The data represent the mean ± SD of three independent experiments. (D) Phagocytic score (calculates as the mean fluorescence intensity of cells of PE+ cells, multiplied by the number of PE+ cells, divided by 100) indicating the efficiency of internalization of SpAwt beads by THP-1 monocytes for a subgroup of opsonizing sera from ISA patients used, compared to three representative anti-SpA IgG negative sera 35, 50v1, 34 in gray. The data represent one experiment.

Article Snippet: IgG3 antibodies were detected with a Monoclonal Mouse Anti-Human IgG3 Secondary Antibody (Fab’2) (LSBio) at a final concentration of 0.8 μg/mL, and the presence of IgG1 antibodies was detected with a Rat anti-Human IgG1 Heavy Constant antibody (Biolegend), at a final concentration of 0.15 μg/mL.

Techniques: Titration, Fluorescence, Control

Journal: Frontiers in Immunology

Article Title: Natural Human Immunity Against Staphylococcal Protein A Relies on Effector Functions Triggered by IgG3

doi: 10.3389/fimmu.2022.834711

Figure Lengend Snippet: THP-1 phagocytosis of SpAwt beads and USA300 lac opsonized with anti-SpA IgG3 and IgG1 mAbs. Mean fluorescence of total THP-1 population engulfing fluorescent FITC-labelled SpAwt beads (A) or GFP-expressing S. aureus USA300 lac (B) opsonized with increasing concentrations of four model anti-SpA antibodies. In the upper panels, targets are opsonized with mAbs expressed in the IgG3 scaffold ( full dots ), while the lower panels targets are opsonized with mAbs expressed in the IgG1 scaffold ( empty dots ). The data represent the mean ± SEM of three independent experiments.

Article Snippet: IgG3 antibodies were detected with a Monoclonal Mouse Anti-Human IgG3 Secondary Antibody (Fab’2) (LSBio) at a final concentration of 0.8 μg/mL, and the presence of IgG1 antibodies was detected with a Rat anti-Human IgG1 Heavy Constant antibody (Biolegend), at a final concentration of 0.15 μg/mL.

Techniques: Fluorescence, Expressing

Journal: Frontiers in Immunology

Article Title: Natural Human Immunity Against Staphylococcal Protein A Relies on Effector Functions Triggered by IgG3

doi: 10.3389/fimmu.2022.834711

Figure Lengend Snippet: Human neutrophils phagocytosis of USA300 opsonized with anti-SpA IgG3 and IgG1 mAbs and human complement. Mean fluorescence of total neutrophil population engulfing S. aureus USA300 opsonized with anti-SpA mAbs in the absence (A) or presence (B) of a human complement source depleted of IgG and IgM (ΔIgGΔIgM serum). In the upper panels, bacteria are opsonized with mAbs expressed in the IgG3 scaffold ( full dots ), while in the lower panels, bacteria are opsonized with mAbs expressed in the IgG1 scaffold ( empty dots ). The data represent the mean ± SEM of three independent experiments.

Article Snippet: IgG3 antibodies were detected with a Monoclonal Mouse Anti-Human IgG3 Secondary Antibody (Fab’2) (LSBio) at a final concentration of 0.8 μg/mL, and the presence of IgG1 antibodies was detected with a Rat anti-Human IgG1 Heavy Constant antibody (Biolegend), at a final concentration of 0.15 μg/mL.

Techniques: Fluorescence, Bacteria

Journal: Frontiers in Immunology

Article Title: Natural Human Immunity Against Staphylococcal Protein A Relies on Effector Functions Triggered by IgG3

doi: 10.3389/fimmu.2022.834711

Figure Lengend Snippet: THP-1 phagocytosis of USA300 lac opsonized with anti-SpA IgG3 and IgG1 mAbs in the presence of human IgGs. Mean fluorescence of total THP-1 population engulfing S. aureus USA300 opsonized with anti-SpA mAbs in IgG3 scaffold (A) or IgG1 scaffold (B) in the presence of a saturating concentration of human IgGs. The data represent the mean ± SEM of three independent experiments.

Article Snippet: IgG3 antibodies were detected with a Monoclonal Mouse Anti-Human IgG3 Secondary Antibody (Fab’2) (LSBio) at a final concentration of 0.8 μg/mL, and the presence of IgG1 antibodies was detected with a Rat anti-Human IgG1 Heavy Constant antibody (Biolegend), at a final concentration of 0.15 μg/mL.

Techniques: Fluorescence, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Natural Human Immunity Against Staphylococcal Protein A Relies on Effector Functions Triggered by IgG3

doi: 10.3389/fimmu.2022.834711

Figure Lengend Snippet: Displacement of human IgGs from SpAwt beads by anti-SpA monoclonal antibodies. Mean fluorescence associated to PE-conjugated human IgGs attached to SpA wild type on beads after incubation with IgG3 anti-SpA monoclonal antibodies and an isotype control ( Figure S10 ). The data represent the mean ± SEM of two independent experiments.

Article Snippet: IgG3 antibodies were detected with a Monoclonal Mouse Anti-Human IgG3 Secondary Antibody (Fab’2) (LSBio) at a final concentration of 0.8 μg/mL, and the presence of IgG1 antibodies was detected with a Rat anti-Human IgG1 Heavy Constant antibody (Biolegend), at a final concentration of 0.15 μg/mL.

Techniques: Bioprocessing, Fluorescence, Incubation, Control

Journal: Frontiers in Immunology

Article Title: Natural Human Immunity Against Staphylococcal Protein A Relies on Effector Functions Triggered by IgG3

doi: 10.3389/fimmu.2022.834711

Figure Lengend Snippet: Approximate epitope mapping of IgG3 anti-SpA mAbs using SpA AA and SpA KK mutant proteins.

Article Snippet: IgG3 antibodies were detected with a Monoclonal Mouse Anti-Human IgG3 Secondary Antibody (Fab’2) (LSBio) at a final concentration of 0.8 μg/mL, and the presence of IgG1 antibodies was detected with a Rat anti-Human IgG1 Heavy Constant antibody (Biolegend), at a final concentration of 0.15 μg/mL.

Techniques: Mutagenesis

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: Overview of suppliers. Chemiluminescence immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA), nucleocapsid protein (N-protein), spike protein (S-protein).

Article Snippet: The most sensitive SARS-CoV-2 antibody test was the Euroimmun IgA test with 87%, followed by the Epitope Diagnostics IgG test with 83%, the Shenzhen YHLO Biotech IgG test with 77%, the Roche IgM/IgG test with 77%, the Euroimmun IgG test with 75%, the Diasorin IgG test with 53%, the Epitope Diagnostics IgM test with 52%, the Snibe IgG Test with 47%, the Shenzhen YHLO Biotech IgM test with 35%, and the Snibe IgM test with 26%.

Techniques: Chemiluminescence Immunoassay, Enzyme-linked Immunosorbent Assay, Diagnostic Assay

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: Precision box-whiskers plot of the inter-assay and intra-assay variations in the IgM and IgG SARS-CoV-2 assay from Shenzhen YHLO Biotech and Roche Elecsys Anti-SARS-CoV-2 ECLIA.

Article Snippet: The most sensitive SARS-CoV-2 antibody test was the Euroimmun IgA test with 87%, followed by the Epitope Diagnostics IgG test with 83%, the Shenzhen YHLO Biotech IgG test with 77%, the Roche IgM/IgG test with 77%, the Euroimmun IgG test with 75%, the Diasorin IgG test with 53%, the Epitope Diagnostics IgM test with 52%, the Snibe IgG Test with 47%, the Shenzhen YHLO Biotech IgM test with 35%, and the Snibe IgM test with 26%.

Techniques: Inter Assay, Intra Assay

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: Z-Score comparison. Comparison between the IgM and IgG SARS-CoV-2 assay from Shenzhen YHLO Biotech and the IgM/IgG assay by Roche Diagnostics. Data were transformed to Z-scores, as described in the materials and methods section, and the cut-off values were uniformly set to log (1). Measurement of qRT-PCR-confirmed COVID-19 cases (n = 59) and pre-pandemic negative controls (n = 50).

Article Snippet: The most sensitive SARS-CoV-2 antibody test was the Euroimmun IgA test with 87%, followed by the Epitope Diagnostics IgG test with 83%, the Shenzhen YHLO Biotech IgG test with 77%, the Roche IgM/IgG test with 77%, the Euroimmun IgG test with 75%, the Diasorin IgG test with 53%, the Epitope Diagnostics IgM test with 52%, the Snibe IgG Test with 47%, the Shenzhen YHLO Biotech IgM test with 35%, and the Snibe IgM test with 26%.

Techniques: Transformation Assay, Quantitative RT-PCR

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: AUROC curves. AUROC graphs for the highest ((A) Shenzhen YHLO Biotech IgG, AUC: 0.97) and lowest ((B) Snibe IgM, AUC: 0.66) values; detailed information can be found in .

Article Snippet: The most sensitive SARS-CoV-2 antibody test was the Euroimmun IgA test with 87%, followed by the Epitope Diagnostics IgG test with 83%, the Shenzhen YHLO Biotech IgG test with 77%, the Roche IgM/IgG test with 77%, the Euroimmun IgG test with 75%, the Diasorin IgG test with 53%, the Epitope Diagnostics IgM test with 52%, the Snibe IgG Test with 47%, the Shenzhen YHLO Biotech IgM test with 35%, and the Snibe IgM test with 26%.

Techniques:

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: AUROC calculations. Diagnostic performance of all immunosorbent assays listed in Table 2 , as calculated by AUROC values.

Article Snippet: The most sensitive SARS-CoV-2 antibody test was the Euroimmun IgA test with 87%, followed by the Epitope Diagnostics IgG test with 83%, the Shenzhen YHLO Biotech IgG test with 77%, the Roche IgM/IgG test with 77%, the Euroimmun IgG test with 75%, the Diasorin IgG test with 53%, the Epitope Diagnostics IgM test with 52%, the Snibe IgG Test with 47%, the Shenzhen YHLO Biotech IgM test with 35%, and the Snibe IgM test with 26%.

Techniques: Diagnostic Assay

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: Longitudinal monitoring. Kinetics of antibody concentrations were analysed for 15 patients with qRT-PCR-confirmed COVID-19 infections. Multiple blood samplings were performed for up to 14 weeks after qRT-PCR. The cut-off values for the Shenzhen YHLO Biotech assays (10 AU/mL) are indicated with horizontal lines. The courses of antibody concentrations were grouped into four categories: (I) decreasing (blue), (II) increasing (orange), (III) steady (green), and (IV) variable (yellow). Most interestingly, antibody formation could not be detected for IgM or IgG in one case, even 9 weeks after a positive qRT-PCR result (triangle symbols, Patient 1).

Article Snippet: The most sensitive SARS-CoV-2 antibody test was the Euroimmun IgA test with 87%, followed by the Epitope Diagnostics IgG test with 83%, the Shenzhen YHLO Biotech IgG test with 77%, the Roche IgM/IgG test with 77%, the Euroimmun IgG test with 75%, the Diasorin IgG test with 53%, the Epitope Diagnostics IgM test with 52%, the Snibe IgG Test with 47%, the Shenzhen YHLO Biotech IgM test with 35%, and the Snibe IgM test with 26%.

Techniques: Quantitative RT-PCR

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: Overview of suppliers. Chemiluminescence immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA), nucleocapsid protein (N-protein), spike protein (S-protein).

Article Snippet: Overall diagnostic sensitivities were: Euroimmun (IgA) 87%; Epitope Diagnostics (IgG) 83%; YHLO (IgG) 77%; Roche (IgM/IgG) 77%; Euroimmun (IgG) 75%; Diasorin (IgG) 53%; Epitope Diagnostics (IgM) 52%; Snibe (IgG) 47%; YHLO (IgM) 35%; and Snibe (IgM) 26%.

Techniques: Chemiluminescence Immunoassay, Enzyme-linked Immunosorbent Assay, Diagnostic Assay

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: Precision box-whiskers plot of the inter-assay and intra-assay variations in the IgM and IgG SARS-CoV-2 assay from Shenzhen YHLO Biotech and Roche Elecsys Anti-SARS-CoV-2 ECLIA.

Article Snippet: Overall diagnostic sensitivities were: Euroimmun (IgA) 87%; Epitope Diagnostics (IgG) 83%; YHLO (IgG) 77%; Roche (IgM/IgG) 77%; Euroimmun (IgG) 75%; Diasorin (IgG) 53%; Epitope Diagnostics (IgM) 52%; Snibe (IgG) 47%; YHLO (IgM) 35%; and Snibe (IgM) 26%.

Techniques: Inter Assay, Intra Assay

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: Z-Score comparison. Comparison between the IgM and IgG SARS-CoV-2 assay from Shenzhen YHLO Biotech and the IgM/IgG assay by Roche Diagnostics. Data were transformed to Z-scores, as described in the materials and methods section, and the cut-off values were uniformly set to log (1). Measurement of qRT-PCR-confirmed COVID-19 cases (n = 59) and pre-pandemic negative controls (n = 50).

Article Snippet: Overall diagnostic sensitivities were: Euroimmun (IgA) 87%; Epitope Diagnostics (IgG) 83%; YHLO (IgG) 77%; Roche (IgM/IgG) 77%; Euroimmun (IgG) 75%; Diasorin (IgG) 53%; Epitope Diagnostics (IgM) 52%; Snibe (IgG) 47%; YHLO (IgM) 35%; and Snibe (IgM) 26%.

Techniques: Transformation Assay, Quantitative RT-PCR

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: AUROC curves. AUROC graphs for the highest ((A) Shenzhen YHLO Biotech IgG, AUC: 0.97) and lowest ((B) Snibe IgM, AUC: 0.66) values; detailed information can be found in .

Article Snippet: Overall diagnostic sensitivities were: Euroimmun (IgA) 87%; Epitope Diagnostics (IgG) 83%; YHLO (IgG) 77%; Roche (IgM/IgG) 77%; Euroimmun (IgG) 75%; Diasorin (IgG) 53%; Epitope Diagnostics (IgM) 52%; Snibe (IgG) 47%; YHLO (IgM) 35%; and Snibe (IgM) 26%.

Techniques:

Journal: International Journal of Infectious Diseases

Article Title: Clinical evaluation of commercial automated SARS-CoV-2 immunoassays

doi: 10.1016/j.ijid.2020.12.003

Figure Lengend Snippet: Longitudinal monitoring. Kinetics of antibody concentrations were analysed for 15 patients with qRT-PCR-confirmed COVID-19 infections. Multiple blood samplings were performed for up to 14 weeks after qRT-PCR. The cut-off values for the Shenzhen YHLO Biotech assays (10 AU/mL) are indicated with horizontal lines. The courses of antibody concentrations were grouped into four categories: (I) decreasing (blue), (II) increasing (orange), (III) steady (green), and (IV) variable (yellow). Most interestingly, antibody formation could not be detected for IgM or IgG in one case, even 9 weeks after a positive qRT-PCR result (triangle symbols, Patient 1).

Article Snippet: Overall diagnostic sensitivities were: Euroimmun (IgA) 87%; Epitope Diagnostics (IgG) 83%; YHLO (IgG) 77%; Roche (IgM/IgG) 77%; Euroimmun (IgG) 75%; Diasorin (IgG) 53%; Epitope Diagnostics (IgM) 52%; Snibe (IgG) 47%; YHLO (IgM) 35%; and Snibe (IgM) 26%.

Techniques: Quantitative RT-PCR

Journal: Nature Communications

Article Title: Differential organization of tonic and chronic B cell antigen receptors in the plasma membrane

doi: 10.1038/s41467-019-08677-1

Figure Lengend Snippet: Arrangement of mIgM-containing BCRs in the plasma membrane of human B cells. a Schematic depiction of the staining procedure and the generation of plasma membrane sheets. b , e , h Images of membrane sheets of unstimulated Ramos B cells ( b ), unstimulated primary B cells ( e ), and Ramos cells stimulated with anti-IgM F(ab’) fragments for 30 min on ice ( h ). Membrane sheets were stained with the membrane marker R18 (confocal images pseudo-colored in green) and anti-human IgM affibody-Star635P (STED images displayed in red). White squares on central images indicate the zoomed regions that are displayed in the images to their right. Scale bars represent 5 µm and 1 µm for the low and high zoom images respectively. c , f , i Example histograms showing the fluorescence intensity distributions of single Star635P-conjugated affibodies on coverslips (black curves, control) and mIgM-spots present in membrane sheets (red curves, mIgM-BCRs). d , g , j Percentages of different mIgM-BCR arrangements in plasma membrane sheets were calculated after pooling the spot intensities of dozens of membrane sheets together and then fitting the intensity distribution to a sum of Gaussian curves (Supplementary Fig. ). Data were obtained from four and three independent experiments including a total of 30 membranes from unstimulated and 15 membranes from F(ab’) -stimulated Ramos B cells, respectively. The experiments with primary human B cells were performed three independent times from two different donors and 30 membrane sheets were analyzed in total. Error bars represent the 68% confidence interval, corresponding to 1 standard deviation. Histogram’s source data are provided as a Source Data file

Article Snippet: The anti-IgM affibody K d was measured with a NanoTemper Microscale Thermophoresis Monolith NT.115Pico (NanoTemper Technologies GmbH, Munich, Germany).

Techniques: Staining, Marker, Fluorescence, Standard Deviation

Journal: Nature Communications

Article Title: Differential organization of tonic and chronic B cell antigen receptors in the plasma membrane

doi: 10.1038/s41467-019-08677-1

Figure Lengend Snippet: mIgM-BCRs do not arrange in large protein islands at the plasma membrane. a Schematic depiction of the TIRF/dSTORM setup imaging depth. b Representative TIRF/dSTORM image of an intact Ramos B cell stained with CF TM 647-conjugated anti-IgM affibodies. The dotted white trace delineates the area of the plasma membrane close to the glass coverslip imaged under TIRF/dSTORM mode. The white square indicates the zoomed area shown in the image to the right. Scale bars represent 1 µm and 0.5 µm for the low and high zoom, respectively. c Scatter plot displaying the spot size (FWHM) distribution unstimulated Ramos cells and as resolution reference the scatter plot of single CF TM 647- affibodies spread on a glass coverslip

Article Snippet: The anti-IgM affibody K d was measured with a NanoTemper Microscale Thermophoresis Monolith NT.115Pico (NanoTemper Technologies GmbH, Munich, Germany).

Techniques: Imaging, Staining

Journal:

Article Title: Identification of the Tumor Cells in Peripheral T-Cell Lymphomas by Combined Polymerase Chain Reaction-Based T-Cell Receptor ? Spectrotyping and Immunohistological Detection with T-Cell Receptor ? Chain Variable Region Segment-Specific Antibodies

doi:

Figure Lengend Snippet: Antibodies Used for Single and Double Stains

Article Snippet: CD 8 , 5C2–2H2 , Dianova , 1:20 , , Donkey anti-mouse IgM Cy5.

Techniques: Conjugation Assay, Avidin-Biotin Assay