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Microbial Immunity and Vaccines

Anti-Lipid A Monoclonal Antibody Centoxin (HA-1A) Binds to a Wide Variety of Hydrophobic Ligands

E. J. Helmerhorst, J. J. Maaskant, B. J. Appelmelk
E. J. Helmerhorst
Department of Oral Biochemistry and
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J. J. Maaskant
Department of Medical Microbiology, Vrije Universiteit, 1081 BT Amsterdam, The Netherlands
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B. J. Appelmelk
Department of Medical Microbiology, Vrije Universiteit, 1081 BT Amsterdam, The Netherlands
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DOI: 10.1128/IAI.66.2.870-873.1998
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    Fig. 1.

    (a) Western blot analysis of S. minnesota R5 bacteria, R5 OMPs, and proteolytic digests with MAb 40. Blots were incubated for 2 h at 37°C with MAb 40 (1.8 μg/ml) in phosphate-buffered saline supplemented with 0.1% Tween 80. Lane 1,S. minnesota R5 OMPs (7.5 μl) incubated with proteinase K (500 μg/ml); lane 2, S. minnesota R5 OMPs (7.5 μl); lane 3, S. minnesota R5 bacteria (OD = 3; 7.5 μl); lane 4,S. minnesota R5 bacteria (OD = 3; 7.5 μl) treated with proteinase K (500 μg/ml); lane 5, molecular weight markers; lane 6, proteinase K (12.5 μg). kD, kilodaltons. (b) Western blot analysis of S. minnesota R5 bacteria, R5 LPS, and proteolytic digests with anti-lipid A and anti-Rc MAb. Left panel: blot incubated with MAb HA-1A (20 μg/ml); right panel: blot incubated with α-Rc MAb 3 (3 μg/ml). Lanes 1, R5 LPS (7.5 μg); lanes 2, S. minnesotaR5 bacteria (OD = 3; 7.5 μl) treated with proteinase K (500 μg/ml); lanes 3, S. minnesota R5 bacteria (OD = 3; 7.5 μl); lanes 4, proteinase K (12.5 μg).

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    Fig. 2.

    Dot spot analysis of BSA (1 mg/ml) preincubated at various denaturing conditions (DTT, urea, boiling) with anti-lipid A MAb 43 (1.8 μg/ml) (A), without a first antibody (B), and with MAb 40 (1.8 μg/ml) (C). Spot 1, 8 M urea plus 200 mM DTT; spots 2, 3, and 4, BSA incubated for 24 h in 2, 5, and 8 M urea, respectively; spots 5, 6, and 7, BSA incubated in 2, 5, and 8 M urea, respectively, plus 200 mM DTT; spot 8, BSA incubated in 200 mM DTT; spot 9, 200 mM DTT; spot 10, native BSA; spot 11, boiled BSA.

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    Fig. 3.

    Binding of anti-lipid A MAbs to immobilized dodecyl-BSA by ELISA. Ninety-six-well microtiter plates were coated with dodecyl-BSA (1 μg/ml). Data for twofold dilution series for MAbs 28 (+), 37 (▴), 40 (⧫), and HA-1A (×) and control MAb 38 (▾) are shown. The OD was measured at 492 nm and expressed as a function of antibody concentration. Data are representative for two independent experiments.

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Anti-Lipid A Monoclonal Antibody Centoxin (HA-1A) Binds to a Wide Variety of Hydrophobic Ligands
E. J. Helmerhorst, J. J. Maaskant, B. J. Appelmelk
Infection and Immunity Feb 1998, 66 (2) 870-873; DOI: 10.1128/IAI.66.2.870-873.1998

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Anti-Lipid A Monoclonal Antibody Centoxin (HA-1A) Binds to a Wide Variety of Hydrophobic Ligands
E. J. Helmerhorst, J. J. Maaskant, B. J. Appelmelk
Infection and Immunity Feb 1998, 66 (2) 870-873; DOI: 10.1128/IAI.66.2.870-873.1998
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KEYWORDS

Antibodies, Monoclonal
Immunoglobulin M
lipid A

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