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Suggested General Method for Blood Collection
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.
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
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.
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 for Immunoassay Studies
Note: Because tissue pretreatment can significantly affect immunoassay results, each laboratory should determine their own appropriate procedure based on (1) the tissue type and (2) the target peptide to be analyzed. In most cases, peptide extraction via C-18 columns (Protocol A) is acceptable.1,2 However, for some specific peptides (e.g. somatostatin3, calcitonin4, IGF-15, kisspeptin6), it may be preferable to follow Protocol B to optimize peptide recovery from samples.
1. Weigh out an appropriate amount of tissue. Dissect tissue with clean tools into small pieces, preferably on ice. Combine this tissue with 3 parts lysis buffer (e.g. 50% HoAc) and boil for 10 minutes at 100°C.
2. Homogenize tissue in lysis buffer, usually with a low pH buffer (e.g. 50% HoAC, or 1% trifluoroacetic acid).
3. Centrifuge the tissue homogenate at 12,000rpm (~2,000x g) for 30 minutes at 4°C.
4. Take 1mL of supernatant and combine with 1mL of Buffer A (Catalog# RK-BA-1) to acidify sample.
Note: If a separate protein assay is required, designate and remove an aliquot before addition of Buffer A. This buffer contains materials which may interfere with protein analysis.
5. Centrifuge at 6,000 – 17,000 x g for 20 minutes, and collect the supernatant. This will be loaded onto the C-18 SEP-COLUMN.
Note: Performing the centrifugation on ice helps to inhibit peptidases.
6. Equilibrate a SEP-COLUMN containing 200mg of C18 (Catalog# RK-SEPCOL-1). Wash with 1mL of Buffer B (Catalog# RK-BB-1) once, followed by 3mL of Buffer A three (3) times.
7. Load the acidified tissue/sample solution onto the pre-equilibrated C-18 SEP-COLUMN.
8. Slowly wash the column with 3mL of Buffer A twice and discard the wash.
9. Elute the peptide slowly with 3mL of Buffer B once and collect the eluant into a polystyrene tube.
Note: Ensure there is a constant flow (by gravity) for all solutions during the extraction procedure. For optimal sample processing and recovery, do not allow air bubbles to enter the C-18 matrix.
10. Evaporate eluant to dryness in a centrifugal concentrator or by a suitable substitute method.
Note: A combination of a centrifugal concentrator (i.e. Speedvac) and a lyophilizer (freeze-dryer) produces the best results when drying samples after extraction. If a centrifugal concentrator is not available, the eluent should be frozen at least three hours before using being placed in the lyophilizer.
11. Keep the dried extract at -20°C and perform the assay as soon as possible. Use the 1x assay buffer to reconstitute the dried extract to the desired concentration. If measured peptide concentrations do not fall within the linear range of the assay kit, dilute or concentrate samples accordingly.
Note: For example, if 1mL of tissue homogenate was extracted, dried, and then reconstituted in 250µl of 1x assay buffer, then the original sample would have now have undergone a 4x concentration.
Protocol B: Pretreatment using acid-ethanol
1. Prepare an acid-ethanol solution by combining 1 part concentrated HCl with 7 parts pure ethanol. Your final solution should contain 87.5% ethanol and 12.5% HCl (2mol/L).
2. Weigh out your tissue. Dissect the tissue with clean tools into small pieces, preferably on ice. Resuspend the tissue with an equal volume of the acid-ethanol solution and homogenize.
3. Combine 5 parts acidic ethanol with 1 part tissue homogenate and incubate for 30 minutes at 20°C. After incubation, centrifuge this mixture at 3,000 x g for 30 minutes at 4°C.
4. Transfer the supernatant into new tubes and evaporate to dryness, either using a centrifugal concentrator or by a suitable substitute method.
Note: A combination of a centrifugal concentrator (i.e. Speedvac) and a lyophilizer (freeze-dryer) produces the best results when drying samples after extraction. A lyophilizer alone may not be sufficient as the ethanol solution could stay in liquid form and spill. If no centrifugal concentrator is available, please only fill tubes to ¼ of its maximum volume.
5. Store the dried extract at -20°C and perform the assay as soon as possible. Use the 1x assay buffer to reconstitute the dried extract to the desired concentration. If measured peptide concentrations do not fall within the linear range of the assay kit, dilute or concentrate samples accordingly.
Note: For example, if 1mL of tissue homogenate was extracted, dried, and then reconstituted in 250µl of 1x assay buffer, then the original sample would have now have undergone a 4x concentration.
References:
- Scott AP, Lowry PJ. Adrenocorticotrophic and melanocyte-stimulating peptides in the human pituitary. Biochemical Journal. 1974;139(3):593-602.
- Hofbauer KH, Jensen BL, Kurtz A, Sandner P. Tissue hypoxygenation activates the adrenomedullin system in vivo. American Journal of Physiology. 2000;278(2):R513-9.
- Peeters TL, Depraetere Y, Vantrappen GR. Simple extraction method and radioimmunoassay for somatostatin in human plasma. Clinical Chemsitry. 1981;27(6):888-91.
- Bucht E, Granberg B, Sjöstedt U, Tørring O. A rapid extraction method for serum calcitonin. Clinica Chimica Acta. 1991;195(3):115-24.
- Mohan S, Baylink DJ. Development of a simple valid method for the complete removal of insulin-like growth factor (IGF)-binding proteins from IGFs in human serum and other biological fluids: comparison with acid-ethanol treatment and C18 Sep-Pak separation. Journal of Clinical Endocrinology and Metabolism. 1995;80(2):637-47.
- Kinsey-Jones JS, Beale KE, Cuenco J, et al. Quantification of rat kisspeptin using a novel radioimmunoassay. PLoS ONE. 2014;9(5):e97611.
Suggested Method of Peptide Extraction from CSF (Cerebrospinal Fluid) in Immunoassay studies
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.
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