AbFRONTIER


1.	Put 100 ㎕ of antigen diluted with coating solution¹ into each well in the plate.
2.	Following incubation for 2 hours at 37℃ or overnight at 4℃, pour out the coating solution.
3.	Put 300 ㎕ blocking solution² into each well.
4.	Following incubation for 0.5 ~ 1 hour at 37℃ (or overnight at 4 ℃ ) , , pour out the blocking solution.
5.	After washing out with 300 ㎕ of TBS-T buffer³, completely dry out the remaining solution by patting softly on paper towel.
6.	Put 100 ㎕ diluted primary antibody into each well and incubate for 1 hour at 37℃.
7.	After pouring out the plate, completely dry out the remaining solution by patting softly on paper towel.
8.	After washing out each well with 300 ㎕ of TBS-T buffer, completely dry out the remaining solution. Repeat these procedures three times.
9.	Put 100 ㎕ of HRP-labeled secondary antibody combining primary antibody and incubate for 1 hour at 37℃.
10.	After pouring out the plate, completely dry out the remaining solution by patting softly on paper towel.
11.	1 After washing out each well with 300 ㎕ of TBS-T buffer, completely dry out the remaining solution. Repeat these procedures five times.
12.	Put 100 ㎕ of substrate into each well.
13.	After 10 minutes (adjust according to color development) put 100 ㎕ of stop solution.
14.	Read absorbance at 492nm with ELISA reader.

¹ Coating solution: 32mM Na2CO3, 68mM NaHCO3
² Blocking solution: 1% non fat dry skim milk or 1% Bovine serum albumin in PBS
³ TBS-T buffer: 0.1M Tris-buffered saline (pH7.4) with 0.1% Tween 20

1.	Put 100 ㎕ of capture antibody diluted with coating solution⁴ into each well (250ng/well).
2.	Following incubation for 2 hours at 37℃ (or overnight at 4 ℃ ) , pour out the coating solution.
3.	Put 300 ㎕ blocking solution⁵ into each well.
4.	Following incubation for 0.5 ~ 1 hour at 37℃ (or overnight at 4 ℃ ) , pour out the blocking solution.
5.	After washing out with 300 ㎕ of TBS-T buffer⁶, completely dry out the remaining solution by patting softly on paper towel.
6.	Put 100 ㎕ of titrated antigen solution (diluted with blocking buffer of 3% BSA/PBS) into each well and incubate in high humidity for 2 hours at room temperature.
7.	Pour out the antigen solution and wash out with 300 ㎕ of PBS (repeat 4 times).
8.	Put 100 ㎕ diluted primary antibody into each well and incubate for 1 hour at 37℃.
9.	After pouring out the plate, completely dry out the remaining solution by patting softly on paper towel.
10.	After washing out each well with 300 ㎕ of TBS-T buffer, completely dry out the remaining solution. Repeat these procedures three times.
11.	Put 100 ㎕ of enzyme-labeled secondary antibody and incubate for 1 hour at 37℃.
12.	After pouring out the plate, completely dry out the remaining solution by patting softly on paper towel.
13.	After washing out each well with 300 ㎕ of TBS-T buffer, completely dry out the remaining solution. Repeat these procedures five times.
14.	Put 100 ㎕ of substrate into each well.
15.	After 10 minutes (adjust according to color development) put 100 ㎕ of stop solution.
16	Read absorbance at 492nm with ELISA reader.

⁴ Coating solution: 32mM Na2CO3, 68mM NaHCO
⁵ Blocking solution: 1% non fat dry skim milk or 1% Bovine serum albumin in PBS
⁶ TBS-T buffer: 0.1M Tris-buffered saline (pH7.4) with 0.1% Tween 20