- Quantification of sheep polyclonal IgG concentration by Cap-
ture ELISA. A 96-well flat bottomed ELISA plate is incubated
with 100μL per well of 1μg/mL anti-sheep IgG antibody and
incubated at 37C for 1 h. After washing the plates three times
with 200μL of PBS containing 0.1% (vol/vol) Tween 20, and
three times with 200μL of PBS, the wells are blocked with
200 μL of MPBS and incubated at 37C for 1 h. - Purified polyclonal sheep serum fromstep 3is diluted 1:1000
in PBS, and 200μL of this solution are added to desired wells
(in duplicate) and double diluted across the plate. - In order to produce a standard curve, sheep IgG standard with
a starting concentration of 4μg/mL is double diluted in PBS
across the plate. - The plate is incubated at room temperature for 1 h and washed
as described instep 4. - Dilute anti-sheep IgG antibody HRP conjugate 1:1000 in PBS
and add 100μL of this dilution per well. - Incubate for 1 h at room temperature, wash the plate as
described instep 4. - The plate is developed by adding 100μL per well of 1-Step
Ultra TMB-ELISA substrate solution (Thermo Fisher Scien-
tific). The reaction is stopped by adding 50μL per well of 1 M
H 2 SO 4 and the optical density of the wells is measured at
450 nm wavelength with a multiplate spectrophotometer.
The polyclonal IgG concentration is calculated based on the
standard curve plotted from the absorbance values of Sheep
IgG at known concentrations. - Polyclonal sheep sera Binding ELISA. 100μL aliquots of PBS
containing 1μg/mL AHL-TG conjugates are dispensed in a
high-binding affinity polystyrene 96-well flat bottomed ELISA
plate. The plate is incubated at 37C for 1 h (or at 4C
overnight). A control plate incubated with 1μg/mL BSA and
TG is included to check the nonspecific binding of the poly-
clonal sera. The plates are washed as described instep 4,
blocked with 200μL of MPBS per well and incubated at
37 C for 1 h. The washing step is repeated as before and
then 200 μL of crude or purified sheep polyclonal serum
(obtained instep 3) are added to designated wells, double
diluted in PBS across the plate, and incubated at room temper-
ature for 1 h. The plates are washed as described instep 4, and
100 μL of anti-sheep IgG antibody HRP conjugate diluted
1:1000 in PBS is added to the wells as secondary antibody.
The plate is incubated for 1 h at room temperature. Afterwards
the wells are washed as indicated instep 4and developed as
described instep 10.
334 Soumya Palliyil