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Rosette inhibition of antibodies depleted by absorption against NTS-DBL1α.

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posted on 2013-02-20, 20:53 authored by Ashfaq Ghumra, Pongsak Khunrae, Ricardo Ataide, Ahmed Raza, Stephen J. Rogerson, Matthew K. Higgins, J. Alexandra Rowe

Immunoblotting (A) and rosette inhibition (B) by pairs of antibodies that were either non-absorbed, or absorbed on NTS-DBL1α recombinant protein coupled to sepharose. A) Recombinant NTS-DBL1α protein was spotted onto nitrocellulose membrane at doubling dilutions, starting from 2 µg/ml, and incubated with 1/1000 dilution of absorbed or non-absorbed antibody. 1) non-absorbed anti-NTS-DBL1α, 2) absorbed anti-NTS-DBL1α, 3) non-absorbed anti-NTS-DBL1α-CIDR1γ, 4) absorbed anti-NTS-DBL1α-CIDR1γ, 5) non-absorbed anti-DBL3ε, 6) absorbed anti-DBL3ε, 7) non-absorbed anti-DBL4δ and 8) absorbed anti-DBL4δ. Non-absorbed antibodies to DBLα (lanes 1 and 3) and DBL4δ (lane7) recognized NTS-DBL1α recombinant protein. After absorption, however, this activity was lost (lanes 2, 4 and 8). Antibodies to DBL3ε did not recognize NTS-DBL1α recombinant protein (lanes 4 and 5). B) Rosette inhibition assays showed that the anti-rosetting activity of NTS-DBL1α antibodies was lost after absorption. Antibodies to DBL3ε and DBL4δ retained rosette-inhibitory activity after absorption, showing that their anti-rosetting effects are likely to be independent of DBL1α. Antibodies to NTS-DBL1α-CIDR1γ also retained inhibitory effects after absorption on NTS-DBL1α protein, suggesting that antibodies to the CIDR1γ domain of ITvar9 also have anti-rosetting effects. Data shown are the mean and standard deviation of triplicate determinations of rosette frequency after overnight incubation with absorbed or non-absorbed antibody diluted 1/10 from the 1 mg/ml stock used for absorption. The control (with binding medium only added) had more than 50% of infected erythrocytes in rosettes.

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