- PA5-65610 - Invitrogen Antibodies ADAM22 antibody

Submitted references Distinctive binding properties of human monoclonal LGI1 autoantibodies determine pathogenic mechanisms.

Ramberger M, Berretta A, Tan JMM, Sun B, Michael S, Yeo T, Theorell J, Bashford-Rogers R, Paneva S, O'Dowd V, Dedi N, Topia S, Griffin R, Ramirez-Franco J, El Far O, Baulac S, Leite MI, Sen A, Jeans A, McMillan D, Marshall D, Anthony D, Lightwood D, Waters P, Irani SR
Brain : a journal of neurology 2020 Jun 1;143(6):1731-1745

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent staining of ADAM22 in human cell line HEK 293 shows localization to cell junctions. Samples were probed using an ADAM22 Polyclonal Antibody (Product # PA5-65610).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 LRR-specific mAbs internalize LGI1 and its receptors. ( A ) LRR-specific mAbs caused internalization of sLGI1-ADAM22/23 complexes on HEK293T cells at 37degC after 4 h ( top : example with ADAM23 staining). Bottom : An internalized pHrodo-labelled LRR-specific mAb. Images were similar to ADAM22 (not shown). ( B ) Gating strategy and quantification of pHrodo-labelled mAb uptake (5 mug/mL) by flow cytometry after 4 h incubations at 37degC. ( C ) The percentage of HEK293T cells with pHrodo fluorescence and the fold increase in pHrodo median fluorescent intensity are shown, after incubation with whole mAbs and Fab' fragments (both at 5 mug/mL). Medians of two to four experiments are shown (Mann-Whitney test ** P < 0.01). ( D ) Corresponding decrease of surface-bound IgG over time. Graphs summarize the per cent of baseline surface-bound human IgG on sLGI1-ADAM22/23 expressing HEK293T cells after 0.5 and 4 h at 37degC, and after 4 h at 4degC. Data from each time point were compared to their own control values at baseline, and are shown across two experiments using seven LRR-directed mAbs [box plots with median, 25th and 75th percentiles, whiskers indicate 10th and 90th percentiles; repeated measures one-way ANOVA (plus Bonferroni correction); * P < 0.05, ** P < 0.01, *** P < 0.001]. ( E ) Quantification of pHrodo fluorescence from conjugated mAbs and Fab' (5 mug/mL) by flow cytometry with and without dynasore. Medians of two experiments are shown (Mann-Whitney test * P < 0.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 EPTP-specific mAbs block the interaction of LGI1 with its receptors. ( A ) Human sLGI1 was preincubated with mAbs, and sLGI1 binding was detected on the surface of ADAM22/23-expressing HEK293T cells with a fluorescently-labelled LRR-specific mAb (mAb02 shown; to assess LRR-specific mAbs within cross-blocking Group 1, a fluorescently-labelled LRR-specific mAb from Group 2 was used). Preincubation with increasing concentrations of all EPTP-specific mAbs (gradient depicted in left four panels), but none of the LRR-specific mAbs ( right -most panel), resulted in a complete loss of fluorescence. ( B ) Individual minimum titres of EPTP-directed mAbs required to achieve complete loss of fluorescence are shown for both ADAM22 and ADAM23-transfected HEK293T cells. No blocking was achieved with LRR-directed mAbs (data from one of three representative experiments are shown; medians were compared using Mann-Whitney test *** P < 0.001). ( C ) Representative images showing EPTP-specific, but not LRR-specific, Fab' fragments (200 ng/mL) blocked the interaction of sLGI1 with ADAM23 in transfected HEK293T cells. ( D ) Representative images showing that preincubation of hippocampal neurons with an excess of each EPTP-specific mAb individually reduced LGI1 internalization, as visualized with a pHrodo-conjugated LRR-specific mAb (mAb01). Preincubation with an excess of the unconjugated LRR-specific mAb (mAb01) was used as a negative control and displaced all of the observed pHrodo-conju

PA5-65610

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