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2.18. Detection of Human Antibodies Against AdcA

NM Nishanth Makthal
KN Kimberly Nguyen
HD Hackwon Do
MG Maire Gavagan
PC Pete Chandrangsu
JH John D. Helmann
RO Randall J. Olsen
MK Muthiah Kumaraswami
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Blood was collected from patients with invasive GAS infections or no known recent GAS exposure (Streptolysin O negative). Samples were collected from patients with necrotizing fasciitis at the time of presentation to our hospital. Sera were separated by centrifugation and transferred to sterile cryovials for storage at − 80 °C. This study was approved by the Institutional Review Board at Houston Methodist Hospital and Houston Methodist Research Institute.

Purified AdcA-NTD was resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membrane was incubated with individual human serum sample at 1:50,000 dilution in TBS-T for 1 h at room temperature followed by additional 1 h incubation with goat anti-human HRP–conjugated secondary antibodies (Thermo scientific) diluted at 1:10,000. Antibody-antigen interaction was visualized by chemiluminescence.

Copyright and License information:  ©2017 The Authors
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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