Journal: bioRxiv

Article Title: Development of a LAG-3 Immunohistochemistry Assay for Melanoma

doi: 10.1101/2022.02.25.481964

Figure Lengend Snippet: Identification of LAG-3 in human tissues using the LAG-3 IHC assay. A, Detection of LAG-3 in human tonsil tissue. Left-hand image depicts LAG-3 staining pattern in tonsil tissue showing moderate to strong plasma membrane/cytoplasmic staining in lymphocytes in germinal centers and interfollicular region. The crypt epithelium is negative. No staining is seen with negative reagent control (right-hand image). B, Staining of FFPE melanoma samples with negative reagent control (upper) or LAG-3 antibody (lower) before (left) and after (right) melanin removal procedure at 10× magnification. C, Examples of LAG-3 staining in FFPE melanoma samples before (upper) and after (lower) the melanin removal procedure at 20× magnification. FFPE, formalin-fixed paraffin-embedded; IHC, immunohistochemistry; LAG-3, lymphocyteactivation gene 3.

Article Snippet: FFPE melanoma specimens and control tonsil tissues were obtained from commercial vendors (Boca Biolistics, Pompano Beach, FL; BioIVT, Westbury, NY; and Avaden Biosciences, Seattle, WA).

Techniques: Staining, Clinical Proteomics, Membrane, Control, Formalin-fixed Paraffin-Embedded, Immunohistochemistry

Journal: bioRxiv

Article Title: Development of a LAG-3 Immunohistochemistry Assay for Melanoma

doi: 10.1101/2022.02.25.481964

Figure Lengend Snippet: Detection of a range of LAG-3 expression levels using the LAG-3 IHC assay. Bar chart showing scoring distribution across LAG-3–positive samples (defined as those with LAG-3–positive IC content ≥1%) from a set of 100 commercially procured human FFPE melanoma specimens. Of the 100 samples, 38 were LAG-3–positive and 62 were LAG-3–negative. FFPE, formalin-fixed paraffin-embedded; IC, immune cell; IHC, immunohistochemistry; LAG-3, lymphocyte-activation gene 3.

Article Snippet: FFPE melanoma specimens and control tonsil tissues were obtained from commercial vendors (Boca Biolistics, Pompano Beach, FL; BioIVT, Westbury, NY; and Avaden Biosciences, Seattle, WA).

Techniques: Expressing, Formalin-fixed Paraffin-Embedded, Immunohistochemistry, Activation Assay

Journal: PLoS Neglected Tropical Diseases

Article Title: Antigenicity, stability, and reproducibility of Zika reporter virus particles for long-term applications

doi: 10.1371/journal.pntd.0008730

Figure Lengend Snippet: (A) Schematic of the ZIKV RVP, composed of capsid, prM/M, and E proteins from a defined ZIKV strain (SPH2015), and an RNA reporter genome replicon. (B) Luciferase ZIKV RVPs were tested for infectivity with cell lines that are commonly used for flavivirus infectivity studies, including BHK-DC-SIGN, Vero, and Raji-DC-SIGN-R cells. At 72 h after infection, cells were lysed and analyzed for luciferase activity (RLU). Values for BHK-DC-SIGN cells (filled symbols) are plotted on the left y-axis. Values for Raji-DC-SIGN-R and Vero cells (open symbols) are plotted on the right y-axis. All values represent n = 3 replicate wells, and error bars represent SD. Regression analysis indicates a linear relationship for the three datasets with RVP volume (R 2 > 0.99). (C) Serial dilutions of the indicated MAbs were incubated with ZIKV RVPs for 1 h, followed by infection of BHK-DC-SIGN cells. After 72 h, cells were lysed and analyzed for RLU. All neutralization results are shown as normalized % infection (% of luciferase signal in the absence of MAb). For 1N5, 4E8, and 1C19, n = 2 and error bars indicate range. For all other data sets n = 7 or 8 and error bars represent SD. Gray symbols represent non-neutralizing MAbs, black symbols represent neutralizing MAbs. (D) Epitopes of 11 anti-ZIKV MAbs used in this paper, mapped onto the ZIKV structure. MAbs that neutralize ZIKV are indicated in bold and underlined.

Article Snippet: ZIKV positive IgG serum was obtained from Boca Biolistics (donor D000013779).

Techniques: Luciferase, Infection, Activity Assay, Incubation, Neutralization

Journal: PLoS Neglected Tropical Diseases

Article Title: Antigenicity, stability, and reproducibility of Zika reporter virus particles for long-term applications

doi: 10.1371/journal.pntd.0008730

Figure Lengend Snippet: (A) ELISA was performed using ZIKV RVPs with various conformational MAbs. Detection MAbs were two anti-DENV MAbs with ZIKV cross-neutralizing activity (C8, C10), five neutralizing anti-ZIKV MAbs (ZIKV-117, ZIKV195, ZIKV116, A9E, LM-081), and two negative control MAbs (CHIKV-specific CKV063 and DENV1-specific 1F4). The capture MAb was a mouse anti-fusion loop MAb (4G2). Mean signal to background ratio (S:B) of three concentrations of detection MAb (1, 2, and 4 μg/mL) is shown with the SD (error bars). Dashed horizontal line represents S:B = 5. ( B) Flow cytometry analysis of LM-081 binding to viral envelope proteins expressed in human cells shows that LM-081 binds specifically to ZIKV and not to other flaviviruses, including DENV serotypes 1–4. (C) Shotgun mutagenesis epitope mapping of LM-081 revealed two critical residues in the fusion loop on one E monomer (N103, G106), and one critical residue on Domain III of an adjacent monomer (I317). (D) Shotgun mutagenesis epitope mapping of anti-DENV EDE MAbs C8 and C10 reveal critical residues (green spheres), shown on the ZIKV E ectodomain (PDB # 5IRE; ).

Article Snippet: ZIKV positive IgG serum was obtained from Boca Biolistics (donor D000013779).

Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay, Negative Control, Flow Cytometry, Binding Assay, Mutagenesis, Residue