Mouse Anti-CD4 Recombinant Antibody (clone Leu3a) (CAT#: FAMAB-0048CQ)

Figure 1 Immunofluorescence flow cytometry analysis of FcYRI, FcyRIl, FcYRIII, and CD4 expression in U937 cells with MAbs 32.2, IV.3, anti-Leulla, and anti-Leu3a, respectively.

(A) U937 cells were stained for FcYRI, FcYRII, and FcYRIII. Dotted lines depict negative controls treated with the second antibody alone (for MAbs 32.2 and IV.3) or control mouse IgGl (for MAb anti-Leulla). (B) U937 cells were stained for dual parameter analysis of FcyRl and CD4.

Takeda, A., Sweet, R. W., & Ennis, F. A. (1990). Two receptors are required for antibody-dependent enhancement of human immunodeficiency virus type 1 infection: CD4 and Fc gamma R. Journal of virology, 64(11), 5605-5610.

Figure 2 Infection enhancement in cultured macrophages and the effect of anti-CD4 antibody.

Adherent macrophages (5 x 10⁵) were treated with anti-Leu3a antibody at 5 μg/ml (○) or left untreated (●) for 30 min at room temperature and then incubated for 2 h at 37 centigrade with 5× 10⁴ TCID50 of HIV-1 (MOI, 0.1) or HIV-1 complexed with various concentrations of IgG from an HIV-1 antibody-positive serum in the absence or presence of ariti-Leu3a antibody. The infected macrophages were gently washed three times and cultured with the same medium. Supernatants were harvested on day 6 for determination of p24. Each plot represents the means ± standard deviations of duplicate cultures.

Takeda, A., Sweet, R. W., & Ennis, F. A. (1990). Two receptors are required for antibody-dependent enhancement of human immunodeficiency virus type 1 infection: CD4 and Fc gamma R. Journal of virology, 64(11), 5605-5610.

Figure 3 Staining of mouse T-cell lines with and without human CD4 on surface with a monoclonal antibody (Leu3a-FITC) and the affinity-enriched RNA library directly labeled with fluorescein.

Mouse T-cell line (BW5147) (a-1–a-3) and the same cell line transfected with human CD4 (B6) (b-1–b-3) were incubated at room temperature for 20 min (50000 cells in 50 µl final volume) in PBS, 2 mM MgCl₂ and 0.1% BSA. Cells were washed with 2 ml of buffer and suspended in 0.5 ml of buffer for analysis. In each plot the ordinate represents the frequency of events (or number of cells), whereas the abscissa indicates the fluorescence intensity. (a-1 and b-1) Autofluorescence; (a-2 and b-2) staining with 60 nM Leu 3a-FITC that stains human CD4; (a-3 and b-3) staining with 200 nM fluoresceinated affinity-enriched RNA aptamer library (round 15).

Davis, K. A., Abrams, B., Lin, Y., & Jayasena, S. D. (1998). Staining of cell surface human CD4 with 2′-F-pyrimidine-containing RNA aptamers for flow cytometry. Nucleic acids research, 26(17), 3915-3924.

Figure 4 Interference (or blocking) of the binding of RNA aptamer library (round 15) to CD4 on beads by different CD4-specific antibodies.

Beads were preincubated with 1 µg of an unlabeled antibody for 10 min at room temperature prior to the addition of fluoresceinated aptamer library to a final concentration of 100 nM. CD4 on L200 beads was used for experiments with L113, L117 and L121, whereas CD4 on SA beads was used for the rest.

Davis, K. A., Abrams, B., Lin, Y., & Jayasena, S. D. (1998). Staining of cell surface human CD4 with 2′-F-pyrimidine-containing RNA aptamers for flow cytometry. Nucleic acids research, 26(17), 3915-3924.

Figure 5 Staining of T lymphocytes in a human PBMC preparation with either a monoclonal antibody to CD4 (Leu 3a-PE) or an RNA aptamer selected for CD4 binding (Aptamer-9:SA-PE).

PBMCs were incubated in a buffer consisting of PBS, 2 mM MgCl₂ and 0.1% BSA either with two antibodies: CD3-Cy5-PE and CD4(Leu 3a)-PE, or with an antibody and RNA complex: CD3-Cy5-PE and Aptamer-9: SA-PE for 15 min at room temperature. Cells were washed with 2 ml and suspended in 0.5 ml of the same buffer for analysis. In all three panels abscissas indicate the staining intensity of CD3-CY5-PE that binds to T cells. (a) Staining with 14 nM CD3-Cy5-PE alone; (b) double staining with 14 nM CD3-Cy5-PE (abscissa) and 5 nM CD4(Leu 3a)-PE (ordinate); and (c) double staining with 14 nM CD3-Cy5-PE (abscissa) and 150 nM Aptamer-9:SA-PE (ordinate). Gate R1 (in green) includes CD3⁺ T cells, whereas gate R2 (in red) includes T cells that are stained with either CD4(Leu 3a)-PE or Aptamer-9:SA-PE and represent CD4⁺ T cells. The observed percentage of CD4⁺ T cells in each panel is also shown. This figure only shows cells selected by gating on lymphocytes by scatter.

Davis, K. A., Abrams, B., Lin, Y., & Jayasena, S. D. (1998). Staining of cell surface human CD4 with 2′-F-pyrimidine-containing RNA aptamers for flow cytometry. Nucleic acids research, 26(17), 3915-3924.

Figure 6 Simultaneous staining of human PBMCs with an affinity-selected RNA aptamer and an antibody to CD4.

PBMCs (∼1.2 × 10⁵) in 50 µl of PBS containing 2 mM MgCl₂ and 0.1% BSA were incubated with 25 nM Aptamer-12:SA-PE, 2.5 nM CD4 (Leu3a)-FITC and 15 nM CD14 (LeuM3)-PerCP at room temperature for 30 min. Cells were washed with 2 ml of buffer, suspended in 0.5 ml of buffer, analyzed and cell staining with these three reagents compared. (a) Staining pattern observed with CD4 (Leu3a)-FITC [staining CD4⁺ T cells (red) and monocytes (green)] and CD14 (LeuM3)-PerCP [staining monocytes only (green)]; (b) staining pattern observed with (Aptamer-12:SA-PE) [staining CD4⁺ T cells (red) and monocytes (green)] and CD14 (LeuM3)-PerCP [staining monocytes only (green)]; (c) staining pattern observed with (Aptamer-12:SA-PE) and CD4 (Leu3a)-FITC [staining CD4⁺ T cells (red) and monocytes (green)].

Davis, K. A., Abrams, B., Lin, Y., & Jayasena, S. D. (1998). Staining of cell surface human CD4 with 2′-F-pyrimidine-containing RNA aptamers for flow cytometry. Nucleic acids research, 26(17), 3915-3924.