Avidity ELISA Protocol for Antibody Characterization

For avidity ELISA, serum samples were applied to wells in duplicate. After one-hour incubation, one set of samples was incubated with wash buffer and another with 4M urea (Sigma-Aldrich, St. Louis, MO, USA) for 5 min and washed twice with wash buffer, and the ELISA protocol was completed as described. The avidity index (AI) was calculated as previously described32 (link) for the lowest dilution (1:2500) as AI = (U+/U-) ×100, where ‘U+’ is the OD₄₉₀ for wells washed with urea and ‘U-‘ is the OD₄₉₀ for wells washed with PBST.

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Blanchfield K., Kamal R.P., Tzeng W.P., Music N., Wilson J.R., Stevens J., Lipatov A.S., Katz J.M, & York I.A. (2014). Recombinant influenza H7 hemagglutinins induce lower neutralizing antibody titers in mice than do seasonal hemagglutinins. Influenza and Other Respiratory Viruses, 8(6), 628-635.

Publication 2014

4M urea

Buffer Dilution Elisa Serum Urea

Corresponding Organization : National Center for Immunization and Respiratory Diseases

Other organizations : Carter Center, Battelle

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Variable analysis

independent variables

Wash buffer
4M urea (Sigma-Aldrich, St. Louis, MO, USA)

dependent variables

Avidity index (AI)
OD490 for wells washed with urea (U+)
OD490 for wells washed with PBST (U-)

control variables

Serum samples applied in duplicate
One-hour incubation time
Wash buffer and urea incubation time (5 min)
Two washes with wash buffer after incubation with wash buffer or urea
ELISA protocol completed as described

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