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Extraction from plasma

1) Decapitate and collect trunk blood in chilled vacutainer tubes (K2 EDTA BD #366643)
2) Centrifuge at 4C for 10 min at 3000x rpm for plasma extraction and store at -80C in Cryogenic tubes
Vasopressin extraction from plasma
1) Use 1 extraction column (Sep-Pak 3cc [200mg] C18 cartridgesWaters #WAT054945) per sample, do
no re-use.
2) Defrost sample, record volume, and add equal volume of 0.1% TFA in ddH
2
O (100 l TFA in 99.9 ml
ddH
2
O).
3) Centrifuge samples at 4C/17,000 g for 15-min.
4) Equilibrate Sep-Pak columns with 1 ml acetonitrile (pipet into column).
5) Wash column with 10-25 ml 0.1% TFA in ddH
2
O.
6) Add plasma supernatant to column, do not touch pellet on bottom of centrifuge tube
7) Allow plasma supernatant to flow completely through the column.
8) Wash column with 10-20 ml 0.1% TFA in ddH
2
O
9) Elute samples in 3 ml 60:40 acetonitrile:0.1% TFA mixture.
a. (60% acetonitrile: 40% TFA [0.1%]; or 10 ml Acetonitrile + 8 ml 0.1% TFA)
10) Evaporate samples overnight in lyophilizer vacuum centrifuge.
11) Store @ -20C

SAMPLE NAME
Sample
(ml)
TFA vol. (ml)
AVP
pg/ml
AVP pg/ml
Plasma
1



2


3


4


5


6


7


8


9


10


11


12


13


14


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Procedural Notes
1. Allow all reagents to warm to room temperature for at least 30 minutes before opening.
2. This kit uses break-apart microtiter strips, which allow the user to measure as many samples as desired. Unused
wells must be kept desiccated at 4 C in the sealed bag provided. The wells should be used in the frame
provided.
3. Care must be taken to minimize contamination by endogenous alkaline phosphatase. Contaminating
alkaline phosphatase activity, especially in the substrate solution, may lead to high blanks. Care should be taken
not to touch pipet tips and other items that are used in the assay with bare hands.
4. Prior to the addition of pNpp substrate, ensure that there is no residual wash buffer in the wells. Any
remaining wash buffer may cause variation in the assay results.
Reagent Preparation: Vasopressin Standard
1. Allow the 10,000 pg/mL Vasopressin standard solution to warm to room temperature.
a. Label seven 12 x 75 mm glass tubes #1 through #7.
2. Pipet 900 L of Assay Buffer into tube #1 and 600 L into tubes #2 through #7.
3. Add 100 L of the 10,000 pg/mL standard to tube #1. Vortex thoroughly.
4. Add 400 L of tube #1 to tube #2 and vortex thoroughly.
a. Continue this for tubes #3 --- #7.
5. The concentration of Vasopressin in tubes #1 through #7 will be 1,000, 400, 160, 64, 25.6, 10.24, and 4.10
pg/mL respectively.
6. See the Vasopressin Assay Layout Sheet for dilution details. Diluted standards should be used within 60
minutes of preparation.
Vasopressin Conjugate and Wash buffer
1. AVP Conjugate: Allow the conjugate to warm to room temperature. Any unused conjugate should be aliquoted
and re-frozen at or below -20 C. Avoid repeated freeze/thaws of the aliquots.
2. Wash Buffer: 5 mL of 20x Wash Buffer in 95 mL of ddH
2
O. This can be stored at room temperature until the kit
expiration date, or for 3 months, whichever is earlier.
Assay Procedure
Bring all reagents to room temperature for at least 30 minutes prior to opening.
1. Refer to the Assay Layout Sheet to determine the number of wells to be used and put any remaining wells with
the desiccant back into the pouch and seal the ziploc. Store unused wells at 4 C.
Wells: Blank, TA (total or max bound well), NSB (null susbstrate boundmin bound), B
o
(Blank standard for Total
boundmax bound)
2. Pipet 150 L Assay Buffer into the NSB (no-sample) and 100 L into the B
o
(0 pg/mL Standard) wells.
3. Pipet 100 L of Standards #1 through #7 into the appropriate wells.
4. Pipet 100 L of the Samples into the appropriate wells

5. Pipet 50 L of the blue Conjugate into each well, except the Total Activity (TA) and Blank wells.
6. Pipet 50 L of the yellow Antibody into each well, except the Blank, TA and NSB wells.
NOTE: Every well used should be Green in color except the NSB wells which should be Blue. The Blank and TA wells
are empty at this point and have no color.
7. Tap the plate gently to mix. Seal the plate and incubate at 4 C for 18-24 hours.
----------------------------------------------------------------------------------------------------------------------------------------------------- -------------
8. Empty the contents of the plate and wash by adding 400 L of wash solution to every well. Repeat the wash 2
more times for a total of 3 washes.
9. After the final wash empty the wells and tap the plate dry on a lint free paper towel (RESIDUAL wash buffer
causes variations in assay results).

10. Add 5 L of the blue Conjugate to the TA wells.
11. Add 200 L of the pNpp Substrate solution to every well. Incubate at 37C in spectrophotometer for 1 hour
without shaking.
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12. Add 50 L of Stop Solution to every well. This stops the reaction and the plate should be read immediately.

13. Blank the plate reader against the Blank wells, read the optical density at 405 nm, preferably with correction
between 570 and 590 nm. If the plate reader is not able to be blanked against the Blank wells, manually subtract
the mean optical density of the Blank wells from all readings.

14. To calculate, multiply by ratio of the total reconstituted volume/(assay volume x plasma sample volume)
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Dilution Table For Making Standards 1-9:
Standards 1-7
Assay
Buffer
Vol.
Added
Vasopressin
Vol. (ul) Vol. (ul) Conc. (pg/mL)
1 900
100, Stock
1,000
2 600
400, Std. 1
400
3 600
400, Std. 2
160
4 600
400, Std. 3
64
5 600
400, Std. 4
25.6
6 600
400, Std. 5
10.24
7 600
400, Std. 6
4.1
8 600
400, Std. 7
1.6
9 600
400, Std. 8
0.64

Assay Protocol Flow Chart:


A1, B1 C1, D1 E1, F1 G1, H1 A2-B4 x*2
Blank (ul) TA (ul) NSB (ul) Bo (ul) Standards (ul) Samples (ul)
Assay Buffer 150 100
Std. and/or Sample 100 100
Conjugate 50 50 50 50
Antibody 50 50 50
Incub 24 hrs.
Wash 3x with 200 uL 600 600 600 600 600 600
(conjugate for TA) 5
Substrate 200 200 200 200 200 200
Stop Solution 50 50 50 50 50 50
Well I.D.:
A1 Blank A2 Std 1 A3 Std 5 A4 Std 9 A5 A6 A7 A8 A9 A10 A11 A12 A13
B1 Blank B2 Std 1 B3 Std 5 B4 Std 9 B5 B6 B7 B8 B9 B10 B11 B12 B13
C1 TA
C2 Std 2 C3 Std 6 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13
D1 TA
D2 Std 2 D3 Std 6 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13
E1 NSB
E2 Std 3 E3 Std 7 E4 E5 E6 E7 E8 E9 E10 E11 E12 E13
F1 NSB
F2 Std 3 F3 Std 7 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13
G1 Bo
G2 Std 4 G3 Std 8 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13
H1 Bo
H2 Std 4 H3 Std 8 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13