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Preprint

Enzyme-linked immunosorbent assay

JO Ji Eun Oh
AI Akiko Iwasaki

Last updated date: Feb 2, 2022 Views: 785 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 (10^6 pfu equivalent per 100ul)

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

3.Capture with HRP-conjugated anti-mouse IgG2b, IgG2c, IgA....

-Anti-mouse IgM-HRP (1020-05) (SouthernBiotec) 1/1000

Anti-mouse IgG1-HRP (1070-05): 1/1000

Anti-mouse IgG2c-HRP (1079-05): 1/1000

Anti-mouse IgG3-HRP (1100-05): 1/1000

Anti-mouse IgA-HRP (1040-05): 1/1000

Anti-mouse IgG2b-HRP (1090-05): 1/1000

 

<Secretory IgA ELISA>

1.Coating with 2ug/ml goat anti-mouse pIgR purified Ab (R&D systems, #AF2800)

2.Sample (BAL fluid) dilution: 1:50~1:200

3.Capture with HRP-conjugated anti-mouse IgA (SothernBiotech #1040-05, 1:1000)

 

<pIgR (SC) ELISA>

1.Coating with BAL fluid (1:50)/ for standard curve, coating with recombinant pIgR protein (R&D systems, #2800-PG-050)

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

3.Capture with 1:100 HRP Donkey anti-goat IgG (H+L) (Jackson Immunoresearch, #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). Enzyme-linked immunosorbent assay. Bio-protocol Preprint. bio-protocol.org/prep1523.
  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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