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Preprint

Antibodies

JO Ji Eun Oh
AI Akiko Iwasaki

Last updated date: Feb 2, 2022 Views: 695 Forks: 0

original An abbreviated version of this protocol was published in Science Immunology in Dec, 2021
Intranasal priming induces local lung-resident B cell populations that secrete protective mucosal antiviral IgA
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*original volume: serum (100-200ul), BAL fluid (1ml)
<Virus-specific antibody ELISA>.

1. Coating with PFA-inactivated (PR8 virus incubated in 0.1% PFA in PBS for 1 wk at 4'c) PR8 virus (106 pfu equivalent per 100ul

2. Sample dilution: indicated in the figure (need to be serially diluted)

3. Capture with HRP-conjugated anti-mouse lgG2b, lgG2c, IgA....
Anti-mouse lgM-HRP (1020-05)(SouthernBiotec) 1/1000
Anti-mouse lgG1-HRP(1070-05): 1/1000
Anti-mouse lgG2c-HRP(1079-05): 1/1000
Anti-mouse lgG3-HRP(1100-05):1/1000
Anti-mouse lA-HRP(1040-05): 1/1000
Anti-mouse lgG2b-HRP(1090-05):1/1000

 

<Secretory IgA ELISA>
1. Coating with 2ug/ml goat anti-mouse plgR purified Ab (R&D systems, #AF2800)
2. Sample (BAL fluid) dilution: 1:50~1:200
3. Capture with HRP-conjugated anti-mouse lgA (SothernBiotech #1040-05, 1:1000)
 

<pIgR(SC) ELISA>
1. Coating with BAL fluid (1:50/ for standard curve. coatina with recombinant plR protein (R&D systems, #2800-PG-050)

2. Sample: 2ug/ml goat anti-mouse plgR purified Ab (R&D systems, #AF2800)

3. Capture with 1:100 HRP Donkey anti-goat lgG (H+L) (Jackson lmmunoresearch, #705-035-147)

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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Oh, J and Iwasaki, A(2022). Antibodies. Bio-protocol Preprint. bio-protocol.org/prep1524.
  2. Oh, J. E., Song, E., Moriyama, M., Wong, P., Zhang, S., Jiang, R., Strohmeier, S., Kleinstein, S. H., Krammer, F. and Iwasaki, A.(2021). Intranasal priming induces local lung-resident B cell populations that secrete protective mucosal antiviral IgA. Science Immunology 6(66). DOI: 10.1126/sciimmunol.abj5129

Category

Immunology

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