Workshop proceeding - final.pdf - Faculty of Information and ...

Fractions (1 ml) were dried and rehydrated with 50μl of EIA buffer (1.42g/l Na 2 HPO 4 , 0.2g/l KH 2 PO 4 ,
5g/l BSA, 1ml/l Tween 20 in water, pH 7.4) and assayed to identify the different forms of α-MSH
using Enzyme immunoassay (EIA).
Enzyme immunoassay
The EIA plates were first coated with Sheep anti-αMSH (1:10000; Bachem, Bubendorf, Switzerland)
in EIA coating buffer (4.3g NaHCO 3 , 5.3g Na 2 CO 3 , in 1L water, pH 9.4) over night at 4°C. The plate
was then washed in EIA wash buffer (1.42g Na 2 HPO 4 ,0.2g KH 2 PO 4 , 1ml Tween 20 in 1L water,
pH7.4) and blocked (8g NaCl, 1.42g KH 2 PO 4 , 0.2g/KCl, 5.0g BSA in 1L water, pH 7.4) for 1h at
room temperature. After 3 washes, Standards (acetylated αMSH , 0.5 – 1024pg/50μl; Bachem,
Torrance, CA)and samples were added at 50μl/well for 90mins at room temperature. After removing
the supernatant, 50μl of biotinylated αMSH (1:100000, Bachem, Torrance, CA) was added to all wells
except blank and non specific binding wells for 90mins. The plate was washed 3 times followed by
50μl of streptavidin-HRP (BD Pharmingen, Franklin Lakes, NJ) for 1h. Finally, after 3 washes, 50μl
tetramethylbenzidine (TMB) (Pierce, Rockford, IL) was added to each well until a strong blue reaction
developed in the total binding wells (30 mins). The reaction was terminated by the addition of 50μl 2N
H 2 SO 4 and the plates were read at 450nm. The sensitivity of this assay was 4pg/100μl.
Statistical analysis
Results are expressed as mean ± standard deviation. Comparison of means was performed by means of
the analysis of variance procedure (Student–Newman–keuls test, SPSS 13.0 for Windows). P