Urea avidity ELISA

Urea serum IgG avidity assay
 

Protocol
Prepare enough plates/wells to assay all serum samples in presence and absence of
Urea, and include wells for assessing background signal.

Coat ELISA plates (Nunc Maxisorp, Fisher Scientific, UK) overnight at 4°C with 1
μg/ml antigen protein in 0.1M bicarbonate buffer pH 9.3. Wash plates three times in
phosphate-buffered saline pH 7.4(PBS) with 0.05% Tween-20 (Sigma, UK) (PBST)
and block for 60 minutes at room temperature with PBST/2% bovine serum albumin
(BSA, Sigma). Wash plates three times with PBST and incubate with serum samples
diluted 1/200 in PBST/1.0% BSA for two hours at room temperature.

After incubation with diluted serum, wash plates once with PBST and then incubate
for 10 minutes at room temperature with PBST or PBST/7M Urea (see notes). Wash
plates a further two times with PBST, incubate with alkaline-phosphatase conjugated
goat anti-mouse IgG (Sigma) for one hour at room-temperature, wash three times
with PBST and develop with pNPP substrate (Sigma) for one hour at room
temperature. Measure absorbance at 405 nm.

Calculate the avidity index for each serum sample by dividing readings from 7M Urea
treatment by readings from PBST-only treatment, after subtraction of background
absorbance.
 

Notes
This is a polyclonal serum IgG avidity assay using a chaotropic substance to
destabilize the antibody/antigen interaction. Whilst Urea is commonly used in these
assays, other chaotropes such as Sodium thiocyanate can be used if more suitable.
Likewise, it is worth determining the most appropriate concentration of Urea to use
for the particular antigen/antibody system. Perform a series of test avidity assays
with varying concentrations of Urea treatment e.g. 5M, 7M and 9M, to determine the
lowest Urea concentration that gives good discrimination with serum samples known
to have differences in avidity.

Adapted from: Puschnik et al., 2013, PLoS Neglected Tropical Diseases, 7(6) e2274
doi:10.1371/journal.pntd.0002274

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