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Immunological Characteristics Associated with the Protective Efficacy of Antibodies to Ricin

Massimo Maddaloni, Corrie Cooke, Royce Wilkinson, Audrey V. Stout, Leta Eng and Seth H. Pincus
J Immunol May 15, 2004, 172 (10) 6221-6228; DOI: https://doi.org/10.4049/jimmunol.172.10.6221
Massimo Maddaloni
*Department of Microbiology and
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Corrie Cooke
*Department of Microbiology and
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Royce Wilkinson
*Department of Microbiology and
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Audrey V. Stout
*Department of Microbiology and
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Leta Eng
† Animal Resources Center, Montana State University, Bozeman, MT 59717; and
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Seth H. Pincus
*Department of Microbiology and
‡ Research Institute for Children, Children’s Hospital, Louisiana State University Health Sciences Center, New Orleans, LA 70118
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  •            FIGURE 1.
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    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.
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    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.
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    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.
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    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.
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    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.
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    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.
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    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.
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    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.
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    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.
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    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.

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    Table I.

    Subclass and specificity of anti-ricin Absa

    IsotypeA ChainHolotoxinB ChainBSAWestern Blot Reactivity
    Fusion 1: immunize with A chain, select for A chain binding
     RAC mAb 1IgG2b0.331.000.110.02+
     RAC mAb 3IgG12.262.290.070.03+
     RAC mAb 4IgG10.360.240.030.01+
     RAC mAb 5IgG10.220.950.020.01+
     RAC mAb 6IgG2a0.150.180.020.03+
     RAC mAb 8IgG12.532.430.090.02+
     RAC mAb 9IgG2b2.302.400.580.08+
     RAC mAb 10IgG10.831.510.020.03+
     RAC mAb 11IgG10.790.540.030.03+
     RAC mAb 12IgG2b1.100.990.450.04+/−
     RAC mAb 14IgG10.421.040.020.02+
     RAC mAb 16IgG12.302.270.060.03+
     RAC mAb 17IgG12.382.330.130.05+
     RAC mAb 18IgG2a2.292.560.060.03+
     RAC mAb 19IgG10.611.410.030.02+/−
     RAC mAb 20IgG2b0.591.350.050.01+
     RAC mAb 21IgG10.180.430.040.03−
     RAC mAb 22IgG12.542.450.110.05+
     RAC mAb 23IgG12.192.380.040.03+
    Fusion 2: immunize with A chain, then holotoxin, select for survival in ricin
     RAC mAb 24IgG2a0.741.510.030.01+/−
     Holo mAb 1IgG10.180.570.020.01−
     HAB mAb 1IgG10.851.130.930.01−
    Fusion 3: immunize with B chain, select for B chain binding
     RBC mAb 1IgG10.140.991.170.06−
     RBC mAb 2IgG10.010.440.350.03−
     RBC mAb 3IgG10.010.070.250.03−
     RBC mAb 4IgG2a0.481.952.140.03+
     RBC mAb 5IgG10.191.501.640.03+
     RBC mAb 6IgG10.020.480.930.04−
     RBC mAb 7IgG2b0.100.991.370.05+
     RBC mAb 8IgG2b0.011.731.820.02−
     RBC mAb 9IgG2a0.000.070.140.02−
     RBC mAb 10IgG2b0.581.962.020.03+
     RBC mAb 11IgG2b0.502.092.030.04+
     RBC mAb 12IgG2a0.000.220.370.08−
     RBC mAb 13IgG10.010.160.160.04−
     RBC mAb 14IgG2b0.491.962.150.03+
     RBC mAb 15IgG2a0.181.761.600.08+
     RBC mAb 16IgG2a0.020.881.350.05+
     RBC mAb 17IgG10.401.901.790.01+
     RBC mAb 18IgG10.171.211.350.01−
    Medium0.020.040.010.01
    Irrel mAb0.040.070.020.02
    • 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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The Journal of Immunology: 172 (10)
The Journal of Immunology
Vol. 172, Issue 10
15 May 2004
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Immunological Characteristics Associated with the Protective Efficacy of Antibodies to Ricin
Massimo Maddaloni, Corrie Cooke, Royce Wilkinson, Audrey V. Stout, Leta Eng, Seth H. Pincus
The Journal of Immunology May 15, 2004, 172 (10) 6221-6228; DOI: 10.4049/jimmunol.172.10.6221

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Immunological Characteristics Associated with the Protective Efficacy of Antibodies to Ricin
Massimo Maddaloni, Corrie Cooke, Royce Wilkinson, Audrey V. Stout, Leta Eng, Seth H. Pincus
The Journal of Immunology May 15, 2004, 172 (10) 6221-6228; DOI: 10.4049/jimmunol.172.10.6221
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