i. Prepare the wells in duplicate for Positive and Negative
Controls and in single for Blank and Samples.
ii. Dilute samples in Diluent buffer (A1) to a final
dilution of 1:100. Pipette 100 µl of Controls
and Samples into the corresponding wells.
Pipette 100 µl
of Diluent buffer into the Blank well.
iii. Cover the tray with aluminium foil and incubate
for 1hour at 37?C.
iv. Wash the wells 4 times with 400 µl of diluted
Wash buffer. After the final washing cycle,
turn the strips/plate down onto blotting
paper or clean towel, and tap to remove
any remaining solution.
v. Add 100 µl of Prediluted Anti-Human IgM*HRP
(B1) into each well.
vi. Cover the tray with aluminium foil and incubate
for 1 hour at 37C.
vii. Wash the wells as described in step (iv).
viii. Pipette 100 µl of previously prepared ABTS Substrate
Reagent into all wells. Cover the plate
with aluminium foil and incubate at room
temperature for 10 minutes, avoiding direct
light exposure.
ix. After 10 minutes, add 100 µl of Stop
Solution (D1) into all well.
x. Read the absorbance of the wells at 405 nm
with a bichromatic spectrophotometer, preferably
with reference wavelength at 492 nm
(setting the instrument at zero with
Blank well). Read the absorbance within
5 minutes after stopping the reaction.
The cut-off value for the test is O.D. 0.300. An O.D. value above 0.300 is
taken as positive ELISA result while an O.D. value below 0.300 is a negative
result.
