Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 1 Chimeric antigen receptor (CAR) detection methods. Binding mechanisms of the different CAR detection reagents. (1) The APC- conjugated AffiniPure F(ab')2 fragment goat antihuman IgG, Fcγ fragment specific, also called anti-Fc, binds the human IgG1 derived hinge domain. (2–4) These three methods use a recombinant human CD19 protein fragment able to bind the anti-CD19 single-chain fragment variable (scFv). The CD19his (2) is fused to a histidine tag that is recognized by an APC-conjugated antihistidine antibody. The CD19bio (3) is fused to a biotin tag that is recognized by an APC-conjugated antibiotin antibody. The CD19-FITC (4) is directly conjugated to the FITC fluorochrome. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Binding Assay, Derivative Assay, Recombinant

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 2 Comparison between different chimeric antigen receptor (CAR) staining methods. (A) Histograms of CARCIK-CD19 cells staining with the anti-Fc antibody in presence of complete medium (RPMI + 10% fetal bovine serum) or after 1 h incubation in human serum at 100%. Negative controls are shown in the first row and represent the unstained samples. (B) Dot plot of CARCIK-CD19 cells detection on the infusion bag product and in the peripheral blood (PB) patient sample with the three CAR detection methods. Negative controls are shown in the first row and represent the samples stained with only the secondary antibody for the indirect methods (CD19his and CD19bio), while the unstained sample for the direct method (CD19-FITC). (C) Stain index for six independent PB samples stained with the three CAR detection methods. Stain index = ((MFI CAR+ population) – (MFI CAR population))/(2 standard deviation of the CAR population). Bars indicate the mean of each group of samples. (D) Comparison of CD19his, CD19bio, CD19-FITC CAR staining methods in six different PB samples from patients treated with CARCIK-CD19 cells. Statistically significant differences are noted in each figure (*p < 0.05; two-tailed paired t-test). [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Comparison, Staining, Incubation, Standard Deviation, Two Tailed Test

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 3 Specificity and sensitivity of the three chimeric antigen receptor (CAR) staining methods using a recombinant human CD19 protein fragment. (A) The specificity was evaluated by staining healthy donor peripheral blood mononuclear cells with the three CAR detection staining reagents to assess background staining. Data are representative of three different donors acquired in one experiment and bars indicate the mean. (B) The sensitivity was evaluated serially diluting CARCIK-CD19+ cells into untransduced CIK cells at six different dilutions (from 100% to 0%). The graph shows mean values and the standard deviation of three independent experiments for each staining method (CD19his, CD19bio, CD19-FITC). The dotted line represents, in both graphs, the lower limit of quantification (LLOQ) for CD19his, identified at 0.1% CAR+

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Staining, Recombinant, Standard Deviation

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 4 Comparison between chimeric antigen receptor (CAR) expression measured with CD19his flow cytometry and real-time polymerase chain reaction (PCR) data. (A) CAR detection was concurrently assessed in 56 postinfusion peripheral blood samples from 4 patients treated with CARCIK-CD19 cells by flow cytometry using the CD19his method (CD3+CAR+ cell/μL) and qPCR (VCN/μg of DNA). The results show clear inter-method concordance by Pearson correlation test (R = 0.51, p = 0.0008). Data are displayed in a logarithmic scale to better represent values <1 cell/μL. (B,C) Monitoring of CARCIK-CD19 expansion, using CD19his flow cytometry staining and qPCR, in two representative patients. (D–F) CARCIK-CD19 cells were expanded in vitro and purified on a magnetic column for CAR expression. Data are representative of five independent experiments. Untransduced CIK cells, CAR+ and CAR purified cells were then analyzed by qPCR and for their cytotoxic activity. In each histograms columns represent the mean and bars the standard deviation. (D) CAR gene integration measured with quantitative PCR and expressed as vector copy number/μg of DNA. (E) CAR mRNA expression measured with qPCR and expressed as CAR mRNA copies normalized on the reference gene GUS. (F) Cytotoxic activity against a CD19+ REH cell line at a 1:1 effector to target ratio, after 72 h coculture. Statistically significant differences are noted in each figure (*p < 0.05; **p < 0.01; two-tailed paired t-test). [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Comparison, Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction, Staining, In Vitro, Purification, Activity Assay, Standard Deviation, Plasmid Preparation, Two Tailed Test

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 5 Inclusion of CD19his chimeric antigen receptor (CAR) staining method in a 12 colors flow cytometry panel. Representation of CD3 cells staining in a single patient treated with CARCIK-CD19 cells at different time point after infusion, using viSNE map visualization tool. To interpret high-dimensional single-cell data that were produced by multicolor flow cytometry panel, we used a tool based on the viSNE algorithm [32], which allows visualization of high-dimensional cytometry data on a two-dimensional map at single-cell resolution and preserve the nonlinearity [33]. In the viSNE map, cell position reflects their proximity in high-dimensional space based on the similarity of marker expression. The first column shows the distribution of the four major T-cell populations: CD4+ CAR+ cells (blue), CD4+ CAR cells (orange), CD8+ CAR+ cells (green), and CD8+ CAR cells (red). All the other columns show the expression level of each marker included in the panel, excluding CD45 and CD3 used for the identification of the T cells. D, day; M, month after CARCIK infusion. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Staining, Flow Cytometry, Produced, Cytometry, Marker, Expressing

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 6 Application of the CD19his staining method on different commercial chimeric antigen receptor (CAR)-T products and within different sample sources. (A) Representative dot plots of three peripheral blood (PB) samples from patients previously infused with tisagenlecleucel (Kymriah®), axicabtagene ciloleucel (Yescarta®),brexucabtagene autoleucel (Tecartus®) and lisocabtagene maraleucel (Breyanzi®). (B) Representative dot plots of CAR detection on different sample sources: bone marrow (BM), pleural effusion and cerebrospinal fluid (CSF) in patients infused with CARCIK-CD19 cells. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Staining

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Histogram showing in-silico analysis of CAR T cell-treated patients ( n = 4219) revealed a high relapse rate, with 42.11% ( n = 216 of n = 513 overall relapse patients) experiencing CD19-negative recurrence after monospecific CAR Therapy ( n = 2916). b Schematic overview of the CAR design strategy showing mono, bi, and trispecific constructs targeting CD19, CD20, and CD22. c Experimental workflow illustrating CAR screening: 1452 CARs were transduced into primary T cells and analyzed for signal-1 (activation), signal-2 (exhaustion), and signal-3 (cell death). Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . d Categorization of CARs into low (L), medium (M), and high (H) levels based on fluorescence intensity cutoffs determined by CD19 CARs as reference. e bar graph showing the distribution of 1452 screened CARs across L-, M-, and H-CARMSeD categories using the CARMSeD scoring system. f AI model development pipeline for CAR dysfunction risk prediction, based on 1,452 CAR constructs with an 80:20 split for training and testing. g–j Performance metrics of AI model predicting CARMSeD scores using 1452 CAR constructs. g ML learning curve of model accuracy over 50 epochs, achieving a training accuracy of 0.98 and validation accuracy of 0.95. h Scatter plot comparing measured versus predicted CARMSeD scores for training ( R 2 = 0.87) and validation ( R 2 = 0.83) sets. i Predicted versus measured CARMSeD scores on the validation set, categorized into low (blue), medium (orange), and high (green) CARMSeD. j Box plots show the median (center line), the 25th–75th percentiles (box), and whiskers extending to the minimum and maximum non-outlier values; individual points denote outliers. Numbers above each box indicate sequence counts. k Molecular dynamics simulation of CAR constructs with varying linker lengths, assessing scFv-scFv interaction. Structural conformations at 0 ns, 50 ns and 200 ns for different CAR scFv arrangements highlighting CDR regions (surface transparency 30%), Root Mean Square Deviation (RMSD) plots over 200 ns for both constructs, respectively, indicating structural stability and conformational changes. l Bar graph showing in vitro receptor binding affinity validation for top humanized scFvs of CD19, CD20, and CD22 CARs ( n = 6 biologically independent samples). Data represent mean ± SEM. ** p < 0.01; **** p < 0.001; ns: not significant. A non-parametric t-test was used for statistical analysis between groups. Source data are provided as a file.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Miltenyi Biotec, #130-129-550) and CD22 (Miltenyi Biotec, #130-126-727).

Techniques: In Silico, Construct, Activation Assay, Fluorescence, Biomarker Discovery, Sequencing, In Vitro, Binding Assay

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Schematic illustration of the K562 cell line model expressing individual or triple combinations of CD19 (purple), CD20 (red), and CD22 (yellow) antigens. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . b Bar chart depicting the percentage expression of each antigen in K562 cell lines, both individually and in combination. c–f Line graph of cytotoxicity assays showing antigen-specific killing of K562 target cells. All tested constructs surpassed the performance of second-generation monospecific CD19 (m19) CAR T cells ( n = 3 biologically independent samples). g Heatmap showing comparison of proliferation rates for bispecific; b20/19 or b22/19, and trispecific; t20/19/22 CAR T cells, represented as fold expansion up to Day 17 with respect to the baseline at the time of cell seeding. h Schematic of the Raji WT cell line platform expressing CD19 (purple), CD20 (red), and CD22 (yellow) antigens, edited using CRISPR-Cas9 to generate knockout variants. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . i , j Line graph of cytotoxicity assays demonstrating the superior efficacy of b20/19 CAR T cells in eliminating antigen-negative Raji variants, compared to m19 CARs ( n = 5 biologically independent samples). k Schematic representation of the tumor rechallenge (TR) model using the Raji WT cell line (Raji WT ). Gray circles represent initial engraftment and monitoring phases, pink circle shows the first incubation with Raji WT , while purple circles indicate the timing of the RajiCD19 −/− rechallenge. l Heatmap representation of TR model showing IFN-γ secretion (pg/mL), percentage of tumor lysis (1:10; T: E), and the number of CAR T cells detected on days 7, 9, 11, 15, and 17 post-rechallenge ( n = 5 biologically independent samples). Data represent mean ± SEM. Source data are provided as a file.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Miltenyi Biotec, #130-129-550) and CD22 (Miltenyi Biotec, #130-126-727).

Techniques: Expressing, Construct, Comparison, CRISPR, Knock-Out, Incubation, Lysis

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Schematic timeline of in vivo lymphoma model for evaluation of monospecific and bispecific CAR T cells. Mice were xenografted with RajiWT cells (expressing CD19, CD20, and CD22) (day 0), followed by administration of m19 or b20/19 CAR T cells on day 5 and subsequent RajiCD19 −/− TR on day 12, 19 and 26. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . b Bioluminescent imaging and ( c ) stacked area plot showing tumor burden quantification show effective tumor control by b20/19 CAR T cells versus m19 CARs ( n = 5). d CAR T cell survival over time ( n = 5 mice). e Kaplan-Meier survival curves showing survival outcomes over 70 days ( n = 5 mice). f Analysis of residual tumor CD19 or CD20 tumor cells over time ( n = 5 mice). g , h Bar plot showing Granzyme B and IFN-γ secretion from human CD8 + CAR T cells isolated b20/19 post-treatment to confirm functional cytotoxicity of b20/19 against CD19⁻ targets ( n = 5). The CAR T cells isolated from mice that received conventional monospecific (m)CD19 CAR T cells served as the control for comparison. i , j TR induced upregulation of exhaustion markers PD-1 and LAG-3 ( n = 5 mice). k Immunophenotyping of CAR T cells post-TR shows loss of central memory (T cm ) populations and increased PD-1 expression, consistent with functional exhaustion and limited engraftment ( n = 5 mice). Data represents mean ± SEM. ** p < 0.01; *** p < 0.005; **** p < 0.001. A non-parametric t-test was used for statistical analysis between groups, and for ( k ), a Two-way ANOVA followed by post-hoc testing was applied. Source data are provided as a file.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Miltenyi Biotec, #130-129-550) and CD22 (Miltenyi Biotec, #130-126-727).

Techniques: In Vivo, Expressing, Imaging, Control, Isolation, Functional Assay, Comparison

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Pathway analysis of proteins involved in AKT3 interaction, modifications or regulation of its expression with emphasis on FOXO4. b Relative mRNA expression levels (normalized to beta actin; ACTB) of key genes show upregulation of FOXO4 mRNA in b20/19-AKT3 PROTAC CAR T ( n = 6 biologically independent samples). c Flow cytometry histograms of total FOXO4 and phosphorylated FOXO4 (p-FOXO4) in CAR T cells after TR with RajiCD19 −/− cells. d Histogram analysis of the flow cytometry plots ( n = 10 biologically independent samples). e Bar graph shows the percentage of CD8 + CAR T cells expressing different phenotypes. Pie charts illustrate the proportional distribution of these subsets across conditions ( n = 5 biologically independent samples). f Survival of CAR T cells over 15 days under various conditions ( n = 4 biologically independent samples). g Violin plots showing the percentage of mTOR activity (% mTOR activity) in various conditions, with shRNA based FOXO4 knockdown elevated mTOR activity ( n = 6 biologically independent samples). h Bar plots show the percentage of MFI of autophagy from autophagic flux assay ( n = 8 data points from three independent experiments). i Dot plot showing ECAR in NTP PROTAC+Scram , NTP PROTAC+shFOXO4 , AKT3 PROTAC+Scram , and AKT3 PROTAC+shFOXO4 conditions, with FOXO4 knockdown increasing shift from OXPHOS to glycolysis ( n = 12 data points from three independent experiments). j Similarly, OCR with FOXO4 knockdown decreases mitochondrial respiration. Individual data points are shown for each condition ( n = 12 data points from three independent experiments). k Box-and-whisker plot showing percentage of expression of CD19 (yellow), CD20 (blue), and CD22 (purple) across 129 ALL patient samples, with varying expression levels for each marker ( n = 63 patient samples). l Bar graph showing the number of patient samples categorized as Negative/Dim, Moderate, or Bright for CD19, CD20, and CD22 expression. m Schematic illustration of K562 WT and CD20 expressing K562 stable cells transduced with different MOIs to obtain three populations: CD20 L (low), CD20 M (medium), and CD20 H (high), which were further FACS sorted. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . n Violin plots showing the percentage of CD20 expression (% CD20 expression) in the sorted CD20-expressing K562 cell populations, confirming distinct expression levels ( n = 10 data flow cytometry points from three independent experiments). o Representative super-resolution microscopy images of differential CD20 surface expression in K562 cells. Images show CD20 (red) in K562-CD20 L (low), K562-CD20 M (medium), and K562-CD20 H (high) cell. p–r Survival curves of K562 cells expressing varying CD20 expression levels under CAR T cell treatments. The line graph shows the percentage of CD20 + cell survival when treated with Rituximab-based monospecific CAR (Rtx-m20, dark green), in-house humanized anti-CD20 CAR (AB21-m20, green) ( n = 4 biologically independent samples). s Survival of CAR T cells with varying CD20-targeting CAR constructs over 15 days ( n = 5). Data represents mean ± SEM. **** p < 0.001. A nonparametric t-test was used for statistical analysis between groups. For e , f and s , a Two-way ANOVA followed by post-hoc testing was applied. Scale bar: 5 μm. Source data are provided as a file.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Miltenyi Biotec, #130-129-550) and CD22 (Miltenyi Biotec, #130-126-727).

Techniques: Expressing, Flow Cytometry, Activity Assay, shRNA, Knockdown, Flux Assay, Whisker Assay, Marker, Transduction, Super-Resolution Microscopy, Construct

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Schematic of the strategy for trispecific CAR T cells, integrating b20/19-AKT3 PROTAC with a secretory BiTE module consisting of nanobodies targeting CD3 and CD22 (nbCD3/22). b Correlation of expression of nbCD3, nbCD22, CD19 CAR, and CD20 CAR at various MOIs. The cells were treated with Brefeldin, and data were obtained using intracellular flow cytometry ( n = 7 data points from three independent experiments). c Experimental setup for T cell activation, using Jurkat-GFP cells and Dynabeads (db) coated with CD3 to assess secreted nbCD3/22 functionality via flow cytometry. d Dose-dependent T cell activation (CD69 expression) in response to culture supernatants (used at various ratios with culture media) with nbCD3/22, using db coated with CD3 for validation ( n = 6 data points from three independent experiments). e Line graph of HEK293T synNotch reporter assay showing dose-dependent inhibition of CD22-CAR signaling by nbCD22 in CAR T cell supernatants, confirming BiTE functionality under two condition 1 and condition 2. f Experimental timelines for in vitro T cell engineering, transduction, and co-culture with Raji cells (WT or knockout for CD19, CD20, or CD22). Anti-tumor assays were performed on days 9, 11, and 13. g , h Functional assay of CAR T cells against Raji cells (WT or knockout for CD19, CD20, or CD22) demonstrates that b20/19AKT3 PROTAC CAR T cells co-expressing nbCD3/22 exhibit stronger antitumor activity compared to b20/19-AKT3 PROTAC or mCD19 CAR T cells at Day 7 and Day 14. Data represent mean ± SEM. **** p < 0.001; ns: not significant. A nonparametric t-test was used for statistical analysis between groups. Source data are provided as a file.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Miltenyi Biotec, #130-129-550) and CD22 (Miltenyi Biotec, #130-126-727).

Techniques: Expressing, Flow Cytometry, Activation Assay, Biomarker Discovery, Reporter Assay, Inhibition, In Vitro, Transduction, Co-Culture Assay, Knock-Out, Functional Assay, Activity Assay