Validate EIA Kit
To validate the EIA kits we obtained water samples from 10 non-experimental animal using
water borne hormone collection method. Evaporated samples were re-suspended in ?200 µl EIA
buffer and combined in a concentrated pool of all samples. The pools will be diluted to 1:2 for
the serial dilutions and the quantitative recovery .
For Parallelism of the serial dilution curve, use 230 µl of the pooled (1:2) control. Place 115
µl of this sample in a 1.5 ml microcentrifuge tube and mix (vortex) with 115 µl of EIA buffer to
create a 1:4 dilution; 115 µl of the 1:4 dilution mix with an equal volume of EIA buffer to create
a 1:8 dilution and so on until 1:128 (or one less).
Quantitative recovery place 70 µl of the pooled 1:2 sample into each of eight 1.5 ml
microcentrifuge tubes and mix with an equal volume of each of the eight standards from the EIA
kit. Place 115 µl of the unmanipulated pooled 1:2 sample into a ninth 1.5 ml microcentrifuge
tube for the control sample. Expect recovery concentrations based on the known hormone in
control sample =((known sample concentration + standard 1 pg/ml) / 2).
smpl 1
smpl 16
smpl r
con
Smpl r 4
smpl r8
1:1
1:1
1:1
1:1
1:1
smpl2
smpl 32
smpl r 1
smpl r 5
1:1
1:1
1:1
1:1
1:1
smpl 4
smpl 64
smpl r 2
smpl r 6
extra
1:1
1:1
1:1
1:1
1:?
smpl 8
smpl 128
smpl r 3
smpl r 7
Smpl pool
1:1
1:1
1:1
1:1
1:1
Serial (260ul)
Recovery (690 ul)
smpl1 =1:1
70 ul Standard 1 +70ul smpl hormone
130 ul smpl hormone
1:2
130 ul +130 buffer
70 ul Standard 2 +70
1:4
130 of 1:2 +130 buff
70 ul Standard 3 +70
1:8
130 of 1:4 +130 buff
70 ul Standard 4 +70
1:16
130 of 1:8 +130 buff
70 ul Standard 5 +70
1:32
130 of 1:16 +130 buff
70 ul Standard 6 +70
1:64
130 of 1:32 +130 buff
70 ul Standard 7 +70
1:128 130 of 1:64 +130 buff
70 ul Standard 8 +70
or not 128
Control 130 ul hormone = pl r con
1:1
1:?