Article Figures & Data
Figures
- FIGURE 1.
Protection against ricin challenge by mice immunized with either RAC or RBC. Mice were immunized with RAC, RBC, or saline (10 μg of A or B chain sc in CFA, followed 1 mo later by 10 μg i.p. in IFA; challenge was performed 1 mo later). ELISA results are on the left; the response to challenge is on the right. ELISA was performed with a 1/5000 dilution of serum. Results are the mean and SEM of triplicate samples. The challenge dose in micrograms per kilogram is indicated above the arrow. Crosses indicate euthanized mice. No control mice or B chain-immune mice survived the initial challenge, whereas all A chain-immune animals survived three successive challenges with 200, 600, and 2000 μg/kg. This is one of two different experiments yielding essentially the same observations.
- FIGURE 2.
Relative avidity of anti-ricin Abs. Anti-ricin mAbs were purified on protein G. Purified Abs were titrated in ELISA for binding to intact ricin holotoxin (50 ng/well). Abs with the highest binding at the lowest concentrations have the greatest relative avidity for ricin. Similar titration curves were performed two to five times for each Ab; the data are representative of those experiments.
- FIGURE 3.
Cross-inhibition among RAC mAbs. Microtiter plates were coated with 50 ng/well of ricin holotoxin. Unconjugated RAC mAbs (5 μg/ml), indicated on the horizontal axis, were added to the coated microtiter wells and incubated for 60 min, the alkaline phosphatase-conjugated Ab indicated at the top of the graph was added, and the mixture was incubated for 4 h. The plates were washed, and colorimetric substrate was added. Abs were tested in up to four different experiments, of which this is representative.
- FIGURE 4.
Epitope mapping of RAC mAbs. Epitopes defined by mapping using peptide display phage libraries were plotted onto the three-dimensional structure of ricin. A, Yellow indicates RAC mAb 17 (AA66–69 HAEL), green RAC mAb 18 (Q173, A178), red enzyme active site (E177, R180, N209), and white residue present in the active site and bound by RAC mAb 18 (W211). B, The two potential sites for RAC mAb 23 binding: green, G213, T217, and S222; and yellow, S156, G159, and T160. The molecule has been rotated on all three axes from A to B to allow best visualization of the epitopes.
- FIGURE 5.
Inhibition of ricin enzymatic and binding activities by Abs. A fixed concentration of ricin holotoxin was mixed with varying concentrations of Ab. A, The ability of Abs to block ricin’s inhibition of cell-free protein synthesis was measured. B, Flow cytometry was used to measure binding of ricin to cells. The results are reported as the ratio of binding (mean fluorescence) in the presence of Ab/binding in the absence of Ab. Each Ab was tested one to three times in the different assays; these are the data from the most complete experiment of each.
- FIGURE 6.
Ab-mediated inhibition of ricin cytotoxicity. H9 cells were incubated with ricin (2 ng/ml) and the indicated Ab. Three days later cell viability was assayed by MTT dye reduction. Viability was also measured in the absence of ricin and in the presence of ricin without Ab. Some Abs were tested in protection assays up to 15 times, but at least twice. The data are representative of these experiments.
- FIGURE 7.
Protection of hybridoma cells from ricin-mediated toxicity by secreted Ab. Hybridoma cell lines secreting the indicated Ab (or the nonsecretor parental fusion partner SP2/0) were incubated in the presence of various concentrations of ricin for 3 days, and viability was tested by MTT dye reduction. Cell lines were tested two or three times, and representative data are shown.
- FIGURE 8.
In vivo effect of RAC mAbs on ricin toxicity. Mice were injected with 30 μg/kg of ricin holotoxin and 0.8 mg/kg of the indicated Ab. Blood sugar was determined 18 h later. Mice were sacrificed if they became moribund or if their blood sugar level fell below 25 mg/dl. Mean blood sugar levels (±SEM) and survival are shown.
- FIGURE 9.
In vivo effects of Abs to ricin A chain. Mice were injected with 15 μg/kg of ricin and 0.8 mg/kg of Ab. Blood sugar was determined 18 and 34 h later. Mean (±SEM) blood sugar levels and survival are shown.
- FIGURE 10.
Potentiation of ricin toxicity by RAC mAb 23. Mice were injected with 30 μg/kg of ricin and the indicated dose of RAC mAb 23 or 1.6 mg/kg of an irrelevant isotype-matched Ab. Blood sugar was determined 13 h later, and survival was determined at 18 h.
Tables
Isotype A Chain Holotoxin B Chain BSA Western Blot Reactivity Fusion 1: immunize with A chain, select for A chain binding RAC mAb 1 IgG2b 0.33 1.00 0.11 0.02 + RAC mAb 3 IgG1 2.26 2.29 0.07 0.03 + RAC mAb 4 IgG1 0.36 0.24 0.03 0.01 + RAC mAb 5 IgG1 0.22 0.95 0.02 0.01 + RAC mAb 6 IgG2a 0.15 0.18 0.02 0.03 + RAC mAb 8 IgG1 2.53 2.43 0.09 0.02 + RAC mAb 9 IgG2b 2.30 2.40 0.58 0.08 + RAC mAb 10 IgG1 0.83 1.51 0.02 0.03 + RAC mAb 11 IgG1 0.79 0.54 0.03 0.03 + RAC mAb 12 IgG2b 1.10 0.99 0.45 0.04 +/− RAC mAb 14 IgG1 0.42 1.04 0.02 0.02 + RAC mAb 16 IgG1 2.30 2.27 0.06 0.03 + RAC mAb 17 IgG1 2.38 2.33 0.13 0.05 + RAC mAb 18 IgG2a 2.29 2.56 0.06 0.03 + RAC mAb 19 IgG1 0.61 1.41 0.03 0.02 +/− RAC mAb 20 IgG2b 0.59 1.35 0.05 0.01 + RAC mAb 21 IgG1 0.18 0.43 0.04 0.03 − RAC mAb 22 IgG1 2.54 2.45 0.11 0.05 + RAC mAb 23 IgG1 2.19 2.38 0.04 0.03 + Fusion 2: immunize with A chain, then holotoxin, select for survival in ricin RAC mAb 24 IgG2a 0.74 1.51 0.03 0.01 +/− Holo mAb 1 IgG1 0.18 0.57 0.02 0.01 − HAB mAb 1 IgG1 0.85 1.13 0.93 0.01 − Fusion 3: immunize with B chain, select for B chain binding RBC mAb 1 IgG1 0.14 0.99 1.17 0.06 − RBC mAb 2 IgG1 0.01 0.44 0.35 0.03 − RBC mAb 3 IgG1 0.01 0.07 0.25 0.03 − RBC mAb 4 IgG2a 0.48 1.95 2.14 0.03 + RBC mAb 5 IgG1 0.19 1.50 1.64 0.03 + RBC mAb 6 IgG1 0.02 0.48 0.93 0.04 − RBC mAb 7 IgG2b 0.10 0.99 1.37 0.05 + RBC mAb 8 IgG2b 0.01 1.73 1.82 0.02 − RBC mAb 9 IgG2a 0.00 0.07 0.14 0.02 − RBC mAb 10 IgG2b 0.58 1.96 2.02 0.03 + RBC mAb 11 IgG2b 0.50 2.09 2.03 0.04 + RBC mAb 12 IgG2a 0.00 0.22 0.37 0.08 − RBC mAb 13 IgG1 0.01 0.16 0.16 0.04 − RBC mAb 14 IgG2b 0.49 1.96 2.15 0.03 + RBC mAb 15 IgG2a 0.18 1.76 1.60 0.08 + RBC mAb 16 IgG2a 0.02 0.88 1.35 0.05 + RBC mAb 17 IgG1 0.40 1.90 1.79 0.01 + RBC mAb 18 IgG1 0.17 1.21 1.35 0.01 − Medium 0.02 0.04 0.01 0.01 Irrel mAb 0.04 0.07 0.02 0.02 -
a The specifity of each Ab was tested in two to nine different experiments, subclass in one to three. Data shown are representative of those experiments.
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