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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 ddH2O (100 l TFA in 99.9 ml

ddH2O).

3) Centrifuge samples at 4C/17,000 g for 15-min.

4) Equilibrate Sep-Pak columns with 1 mlacetonitrile (pipet into column).

5) Wash column with 10-25 ml0.1% TFA in ddH2O.

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 ml0.1% TFA in ddH2O

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 NAMESample

(ml)TFA vol. (ml)

AVP

pg/ml

AVP pg/

Plasm

1

2

34

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 Cin the sealed bag provided. The wells should be used in the frameprovided.

3. Care must be taken to minimize contamination by endogenous alkaline phosphatase. Contaminatingalkaline phosphatase activity, especially in the substrate solution, may lead to high blanks. Care should be takennot to touch pipet tips and other items that are used in the assay with bare hands.

4. Prior to the addition of pNpp substra te, ensure that there is no residual wash buffer in the wells. Anyremaining 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 LofAssay Bufferinto tube #1 and 600Linto tubes #2 through #7.3. Add 100 Lof the 10,000 pg/mL standard to tube #1. Vortex thoroughly.4. Add 400 Lof 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 aliquotedand 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 2O.This can be stored at room temperature until the kitexpiration date, or for 3 months, whichever is earlier.

Assay ProcedureBring 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 withthe 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), Bo(Blank standard for Total

bound

max bound)2. Pipet 150 LAssay Buffer into the NSB(no-sample) and 100 Linto the Bo(0 pg/mL Standard) wells.3. Pipet 100 Lof Standards#1 through #7 into the appropriate wells.4. Pipet 100 Lof the Samplesinto the appropriate wells

5. Pipet 50 Lof th e blue Conjugateinto each well, except the Total Activity (TA)and Blank wells.6. Pipet 50 Lof the yel low A nt ibodyinto 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 wellsare 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 Lof wash solut ionto every well. Repeat the wash 2more 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 buffercauses variations in assay results).

10. Add 5 Lof th e blue Conjugateto the TA wells.11. Add 200Lof the pNpp Sub stra te solu t ionto every well. Incubate at 37C in spectrophotometer for 1 hour

without shaking.----------------------------------------------------------------------------------------------------------------------------- -------------------------------------

12. Add 50 Lof Stop Solut ionto 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 correctionbetween 570 and 590 nm. If the plate reader is not able to be blanked against the Blank wells, manually subtractthe 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.

AddedVasopressin

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 50Incub 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