Hu-TTR- ELISA PROTOCOL-Sandwich

 

1.-Coat plate with primary antibody (immunolon 4HBX, fisher part # 14-245-153)

100 μl/well

Rabbit anti-human TTR (Dako A0002) at 1:1000 dilution

in 50 mM carbonate buffer, pH 9.6,

4C overnight, or 4 hr at r.t.

2.-Wash plate; Plate washer 5x200 μl in TBST buffer; ELISA program

3.-Block plate with 200 μl/well 5% non-fat dry milk in TBST, pH 7.6 (pH have to be readjusted after the addition of milk), and incubate 1hr at 37C

4.- Wash plate,

5.- Antigen incubation 100 μl/well antigen, appropriately diluted with blocking buffer. Add also the calibration curve (70 ng/ml, 50, 40, 30, 25, 20, 10 to 0 ng/ml). Incubate 1h15min, 37C.

6.- Wash plate.

7.- Secondary antibody incubation:

100 μl/well secondary antibody (anti-hu-TTR-AP; EY laboratories) diluted 1:2000 (4 ul in 12 ml blocking buffer), incubate 1h15min at 37C.

8.- Wash

9.- Develop plate

100 μl/well NPP substrate. Incubate in the dark for ~1hr; read plate at 405 nm. (max OD must be ~1)

 


Buffers and solutions

50 mM carbonate buffer, pH 9.6 (500 ml)

[15 mM Na2CO3, 35 mM NaHCO3]

 

0.8 g Na2CO3 (or 2.15 g Na2CO3.10H20)

1.46 g NaHCO3

475 ml dd water, pH 9.6

adjust to 500 ml

 

10X TBST (1L):

39.3 g Tris HCl

43.8 g NaCl

Adjust to pH 7.5

5 ml Tween 20

 

5% non-fat dry milk in TBST, (100 ml)

5 g non-fat dry milk

100 ml 1X TBST

readjust the pH at 7.6 (up to 7.8 is OK)

 

NPP substrate

1 mg/ml NPP (p-nitrophenylphosphate, Sigma cat#104-0) in NPP buffer. For 1 plate ~20 mg in 20 ml NPP buffer.

 

NPP Buffer [10mM Diethanolamine, 0.5mM MgCl2] (300 ml):

29.1 ml diethanolamine

3 ml MgCl2 (50 mM)

200 ml water

adjust pH at 9.8 with HCl

adjust to 300 ml, keep at 4C

 

 


TTR STANDARDS

Usually we have a solution 0.5 mg/ml WT-TTR at 20C.

 

Table of TTR standard curve to prepare. (two plates), use cluster tubes.

well

TTR (ng/ml)

TTR Stock 2 (μl)

Blocking buffer (μl)

1

0

0

1000

2

10

100

900

3

20

200

800

4

25

250

750

5

30

300

700

6

40

400

600

7

50

500

500

8

70

700

300