Suggested General Method for Blood Collection

Blood Withdrawal

Collect blood samples into the Lavender Vacutainer tubes (#VT-6450) which contain EDTA and can collect up to 7 ml blood/tube. Gently rock the Lavender Vacutainer tubes several times immediately after collection of blood for anti-coagulation. Transfer the blood from the lavender vacutainer tubes to centrifuge tubes containing aprotinin (0.6TIU/ml of blood) and gently rock several times to inhibit the activity of proteinases. Centrifuge the blood at 1,600 x g for 15 minutes at 4°C and collect the plasma. Plasma kept at -70°C may be stable for one month.

Units of conversion factor for Aprotinin:

1 TIU (Trypsin Inhibitor Unit) = 1025~1300 KIU (Kallikrein Inhibitor Unit)

We recommend using 0.6 TIU of Aprotinin (100µl) [600~750 KIU of Aprotinin] (Catalog No.: RK-APRO, $85/5ml) per 1 ml of blood sample.

One Trypsin Inhibitor Unit (TIU) will decrease the activity of two trypsin units by 50%, where one trypsin unit will hydrolyze 1.0 µmole of N-alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA) per minute at pH 7.8 and 25°C.

Suggested General Method for the Extraction of Peptides from Plasma or Serum

Extraction procedure of Peptide from Plasma or Serum:

1. Acidify the plasma/serum with an equal amount of buffer A. For example, if you are using 1ml of plasma/serum, add 1 ml of buffer A. Mix and centrifuge at 6,000 to 17,000 x g for 20 minutes at 4°C.

2. Equilibrate a SEP-COLUMN containing 200 mg of C18 (Catalog No.: RK-SEPCOL-1) by washing with buffer B (1 ml, once) followed by buffer A (3 ml, 3 times).

NOTE: From steps 3-5, no pressure should be applied to the column.

3. Load the acidified plasma/serum solution onto the pre-treated (Step 2) C-18 SEP-COLUMN.

4. Slowly wash the column with buffer A (3 ml, twice) and discard the wash.

5. Elute the peptide slowly with buffer B (3 ml, once) and collect eluant into a polystyrene tube.

6. Evaporate eluant to dryness in a centrifugal concentrator or by a suitable substitute method.

7(i). For EIA kits, Fluorescent EIA kits and Chemiluminescent EIA kits, keep the dried extract at -20°C and perform the assay as soon as possible. For normal subject extracted from 1 ml original plasma/serum, use 125 ul 1x assay buffer to reconstitute the dried extract. Aliquot 50 ul into two designated assay wells (25 ul is left over). the concentration factor in this case is 8. (1 ml / 125 ul = 8). The original plasma peptide level is 1/8 of the level of final extracted plasma.
(If the level of the final extracted plasma is 100 pg/ml, then the total level of peptide in the original plasma = (100 pg/ml) / 8 = 12.5 pg/ml.

After performing assay, if the peptide value exceeds or does not fall in the range of detection, dilute or concentrate the samples accordingly.)

 

7(ii). For RIA kits, Ultrasensitive RIA kits, and Magnetic Bead RIA kits, dissolve the residue in RIA buffer for radioimmunoassay as follows: For normal subject extracted from 1 ml original plasma, dissolve in 250 ul RIA buffer for a two-tube assay. Aliquot 100 ul into each tube (50 ul is left over). The concentration factor in this case is 4. (1 ml / 250 ul = 4)
(If the level of the final extracted plasma is 100 pg/ml, then the total level of peptide in the original plasma = (100 pg/ml) / 4 = 25 pg/ml. If upon assay the peptide value exceeds or does not fall in the range of detection, dilute or concentrate the samples accordingly.)

It is ideal to extract at least 1 ml of plasma. It may be possible to perform extraction on smaller volumes, yet a smaller volume of buffer should be used for reconstitution. Always remember to add the equal amount of buffer A into the sample. 125 ul is sufficient rehydration volume for an original plasma volume of 500 ul (concentration factor of 4). This would provide sufficient extracted plasma volume for a duplicate EIA assay, and sufficient volume for a single tube RIA.

8. After the extraction, follow the procedure listed in the protocol.

Elution Solvents:

1. Buffer A (Code RK-BA-1)

2. Buffer B (Code RK-BB-1)

References:

1. Berson, S.A. and Yalow, R.S. Kinetics of reaction between insulin and insulin binding antibody. J. Clin Invest 36: 873, 1957.

2. Patrono, C. and Peskar, B.A. (eds) Radioimmunoassay in basic and clinical pharmacology. Heidelberg, Springer-Verlag, 1987

3. Reuter, A., Vrindts-Gevaerts, Y., Meuleman-Gathy, R., Joris, J., Chretien, M. and Franchimont, P. A radioimmunoassay for beta-endorphins. (BETA-END) and (BETA-LPH) in plasma. Horm Res 25: 236, 1987.

4. Dwenger, A. Radioimmunoassay: an overview. J Clin Biochem 22:883, 1984.

5. Wang, Y.N., Chou J., Chang, D., Chang, J.K., Avila, C. and Romero, R. Endothelin-1 in human plasma and amniotic fluid. In Endothelin-derived contracting factors, edited by G. Rubanyi and P. Vanchoutte, Karger, Basel, pp. 143, 1990.

Suggested Methods of Peptide Extraction from Tissues in Immunoassay Studies

Extraction of Adrenomedullin from various tissues

ADM protein levels from plasma and organs: Blood samples were collected in a centrifuge tube (stabilized with EDTA) and, after centrifugation, plasma was stored at 20°C. Livers, lungs, kidneys, hearts, and brains were removed and immediately frozen in liquid nitrogen and stored by 80°C. ADM levels from plasma and organs were measured from extracted samples. For extraction, 1 ml of plasma was used and 1 ml of buffer A was added. After mixing and centrifugation at 10,000 g for 20 min at 4°C, samples were loaded on a equilibrated Sep-Pak C18 cartridge (Millipore).

For extraction from the different organs, tissues were homogenized in 5 ml/g lysis buffer (10 mM Tris, pH 7.4). After centrifugation (1,600 g for 15 min at 4°C) the supernatant was also loaded on a equilibrated SEP-Pak C18 cartridge (Millipore). Equilibration was performed by washing once with 1 ml of buffer B and three times with 3 ml of buffer A. Then the peptides were eluted with 3 ml of buffer B and collected in a 15-ml centrifuge tube, evaporated to dryness, and the residue was dissolved in 250 µl of RIA buffer. ADM levels were measured on the pre-extracted (Sep-Pak C18) plasma, and tissues were sampled by a sensitive RIA employing an antibody against rat ADM (Phoenix Pharmaceuticals). For the RIA, 100 µl of each sample were used.

Hofbauer KH, Jensen BL, Kurtz A, Sandner P. Tissue hypoxygenation activates the adrenomedullin system in vivo. Am J Physiol Regul Integr Comp Physiol. 2000 Feb;278(2):R513-9.

The concentrations of immunoreactive alpha-MSH was quantitated in the PVN, using commercially available 125 I RIA kit [alpha-MSH (acetylated alpha-MSH); Phoenix Pharmaceuticals].

For extraction of PVN peptides, 1 ml of 0.1 M acetic acid was added to each tissue sample, which was then transferred to a boiling water bath for 10 min. After cooling on ice, samples were homogenized in polypropylene tubes. The homogenates were centrifuged at 13,000 g for 15 min. The pellet was re-suspended in 3 N NaOH and analyzed for total protein content. To assay for protein, 150 µl of supernatant were taken from each sample, and the remainder of supernatant was lyophilized and later used for alpha-MSH RIA. The RIA kit was validated before use with tissue extracts. Dose-response curves for PVN tissue extract, and increasing concentrations of the alpha-MSH added to a rat PVN tissue extract were parallel (P < 0.05) to the standard curve.

Alpha-MSH (ranging from 1 to 32 pg) added to rat PVN tissue extract was consistently recovered from 100 µl of extract (90-100%). The assay sensitivity was 4 pg/tube. The cross-reactivity test, pro-vided by Phoenix Pharmaceuticals, indicated 0% cross-reactivity with opioid peptides, 0% cross-reactivity with Beta- and Gamma-MSH, and 0.02% cross-reactivity with ACTH for alpha-MSH assay.

Kim, Eun-Mee, Martha K. Grace, Catherine C. Welch,Charles J. Billington, and Allen S. Levine. STZ-induced diabetes decreases and insulin normalizes POMC mRNA in arcuate nucleus and pituitary in rats. Am. J. Physiol. (Regulatory Integrative Comp. Physiol. 45) 1999; 276: R1320-R1326.

Suggested Method of Peptide Extraction from CSF (Cerebrospinal Fluid) in Immunoassay studies

Extraction of Urotensin II:

During spinal anesthesia, drops of CSF were collected (total of >/=1 ml), 1 ml was dispensed into a 5-mL polypropylene tube, and 1 ml of Buffer A was immediately added. All samples were then frozen and stored at -20°C until extraction and assayed as a single batch. After sample collection, anesthesia and surgery continued.

Two-milliliter samples (1:1 sample/Buffer A) were thawed and centrifuged at 12,000g for 15 min at 4°C and the supernatant was loaded (0.3 ml/min) onto equilibrated (2 ml of Buffer B, followed by three 3-mL washes with Buffer A at a flow rate of 1 ml/min) Strata C18 solid-phase extraction cartridges. The column was then washed twice with 3 ml of Buffer A at a flow rate 1mL/min. Finally, urotensin II was eluted with 2.5 ml of Buffer B and evaporated to dryness in a centrifugal evaporator. Before assay, the sample was reconstituted in 0.25 ml of immunoassay buffer.

Thompson JP et al. A comparison of cerebrospinal fluid and plasma urotensin II concentrations in normotensive and hypertensive patients undergoing urological surgery during spinal anesthesia: A pilot study. Anesth Analg 2003; 97:1501-3.