PerkinElmer Life Sciences, Inc. Prostaglandin E 2 [ 125 I] RIA Kit Catalog Number NEK020 NEK020A (125 tubes) (250 TUBES) Instruction Manual For Laboratory Use Caution: A research chemical for research purposes only.
I. INTRODUCTION The schematic for the "in vivo" metabolic pathways of the major prostaglandins, shown in Figure 1, represents only the most general outline of a very complex series of biochemical interconversions. Linoleic acid is an essential fatty acid which is metabolized to arachidonic acid, the starting compound of the cascade. Arachidonic acid is stored in cell walls esterified in phospholipids (1). Upon demand, arachidonic acid may be released from the cell wall by phospholipase A 2 (2). Prostaglandin synthetase contains both cyclo-oxygenase and peroxidase activities to convert arachidonic acid to prostaglandin endoperoxide. Cyclo-oxygenase enzymatically metabolizes arachidonic acid to prostaglandin G 2 (PGG 2 ), a cyclic endoperoxide, while peroxidase reduces PGG 2 to another cyclic endoperoxide, prostaglandin H 2 (PGH 2 ) (3, 4). Both PGG 2 and PGH 2 have a half-life of about five minutes at 37 C in aqueous buffer at ph 7.4 (5). Prostaglandin endoperoxide PGH 2 is considered a pivotal compound, since it is metabolized by three different reaction pathways: A. In the presence of thromboxane synthetase, known to be present in large amounts in platelets (4, 6), PGH 2 is converted to thromboxane A 2. Thromboxane A 2 is rapidly hydrolyzed to thromboxane B 2 (TXB 2 ). B. Prostacyclin synthetase, which has been demonstrated in the microsomal fraction of endothelial cells (7, 8), hydrolyzes PGH 2 to prostacyclin. Prostacyclin is unstable and converts to 6-ketoprostaglandin F 1a (6-keto-PGF 1a ). C. Classical prostaglandins, PGE 2, PGF 2a, can be formed from the prostaglandin endoperoxide PGH 2 either spontaneously or enzymatically in the absence of either thromboxane or prostacyclin synthetases (5). In addition, the classical prostaglandins may be formed in association with the other two pathways. 1
Figure 1 Schematic of Arachidonic Acid Metabolic Pathway Linoleic Acid fl Arachidonic Acid fi Phospholipid fl fl PGG2 fl PGH2 Prostaglandin Synthetase Thromboxane Synthetase Prostacyclin Synthetase Isomerases Thromboxane A2 Reductase, or Prostacyclin Spontaneous (PGI2) fl fl fl Thromboxane B2 PGE2, PGF2, PGD2 6-Keto-PGF1 a II. EXPLANATION OF THE TEST Prostaglandin E 2 (PGE 2 ) has been detected in plasma and urine as well as in a variety of human tissues (9). The level of PGE 2, however, is often very low and sensitive procedures are required for accurate measurement. The original methods, bioassay and GC/mass spectrometry (10-12), were either too insensitive, non-specific, or cumbersome for routine use. The development of specific antisera resulted in radioimmunoassays (13, 14), which started to approach the sensitivity required. However, the relatively low specific activity of the tritiated PGE 2 used in these assays limited the sensitivity that could be obtained. It is only recently that high specific activity, iodinated, prostaglandin tracers have been developed and used to increase the sensitivity of these RIA systems (15, 16). The PerkinElmer Life Sciences Prostaglandin E 2 [ 125 I] Radioimmunoassay Kit is based on the use of an iodinated analog of prostaglandin E 2 as tracer and rabbit anti-prostaglandin E 2 as the antiserum (specific antibody). III. PRINCIPLE OF THE METHOD 2
The basic principle of this radioimmunoassay is competitive binding, where a radioactive antigen competes with a non-radioactive antigen for a fixed number of antibody binding sites. When unlabeled antigen from standards or samples and a fixed amount of tracer (labeled antigen) are allowed to react with a constant and limiting amount of antibody, decreasing amounts of tracer are bound to the antibody as the amount of unlabeled antigen is increased. In the PerkinElmer Prostaglandin E 2 [ 125 I] RIA Kit, separation of the antibody-antigen complexes from free antigen is achieved by precipitation of the antibody-bound tracer with polyethylene glycol in the presence of carrier immunoglobulin. After centrifugation, the supernatant containing the unbound antigen is decanted, and the pellet containing the antibodyantigen complex is counted in a gamma counter. Results obtained for the standards are used to construct a standard (dose-response) curve from which the unknowns are read by interpolation. Figure 2 Labeled Antigen + Specific Antibody fi Labeled Antigen Ag* Ab Antibody Complex Ag*Ab + Unlabeled Antigen Ag (In standard solutions or unknown samples) fl Unlabeled Antigen-Antibody Complex AgAb IV. REAGENT DESCRIPTION AND PREPARATION This kit is intended for research use and not for diagnostic purposes. All necessary reagents are supplied for 125 (NEK020) or 250 (NEK020A) assay tubes if the suggested assay protocol is followed. NEK020 125 tubes NEK020A 250 tubes Kit Components 1 vial 2 vials PGE2 Antibody, lyophilized 1 vial 2 vials PGE2 [ 125 I] Tracer Concentrate, 0.75 ml 1 vial 2 vials PGE 2 Standard Concentrate, 1.0 ml 3
1 bottle 2 bottles Assay Buffer, 125 ml 1 bottle 2 bottles Precipitating Solution, 125 ml 4
The Prostaglandin E 2 Kit is shipped ambient, but upon receipt the individual components should be stored as directed. Component stability and handling precautions are described below. A. PGE 2 [ 125 I] Tracer The tracer concentrate contains < 2 µci of PGE 2, [ 125 I]-, in 0.75 ml of acetonitrile. Stored at -20 C, the tracer concentrate is stable for at least one month from date of receipt. INSTRUCTIONS RELATING TO THE HANDLING, USE, STORAGE, AND DISPOSAL OF THIS RADIOACTIVE MATERIAL This radioactive material may be received, acquired, possessed, and used only by research laboratories for in vitro laboratory tests not involving internal or external administration of the material, or the radiation therefrom, to human beings or animals. Its receipt, acquisition, possession, use, and transfer are subject to the regulations and a general license of the U.S. Nuclear Regulatory Commission or of a State with which the Commission has entered into an agreement for the exercise of regulatory authority. 1. All radioactive materials must be labeled and secured in specifically designated posted areas. Records of receipt and survey must be maintained. 2. All work with these materials must be carried out only in authorized areas. 3. Prohibit mouth pipetting of radioactive materials. 4. There must be no smoking or eating within the work area. 5. Hands must be washed after handling radioactive materials. 6. Any spilled material must be wiped up quickly and thoroughly and the contaminated substances transferred to a suitable receptacle. The surfaces involved must be washed thoroughly with an appropriate decontaminant. Monitor to ensure the area has been effectively decontaminated. 7. When use of the Tracer reagent has been completed, empty and decontaminate the vial. This radioactive material can be discarded into the sanitary sewerage system using copious amounts of water to ensure a minimal discharge concentration. 5
8. Prior to disposal of the empty, uncontaminated Kit and Tracer containers to unrestricted areas, remove or deface the radioactive material labels or otherwise clearly indicate that the containers no longer contain radioactive material. For use in the assay, an appropriate aliquot of the tracer concentrate is diluted 1:20 (v:v) in assay buffer in a siliconized glass or polypropylene tube or vial. (For example, for a 20 tube assay, dilute 0.1 ml of tracer concentrate with 2.0 ml assay buffer.) Dilute only enough tracer for use in each assay. Do not store and reuse. Return the unused portion of tracer concentrate to -20 C storage. Pipets and/or pipet tips used to transfer the tracer solution must be of polypropylene or siliconized glass. Do not use those made of unsiliconized glass. B. PGE 2 Antibody The lyophilized rabbit anti-pge 2 antibody is stable for at least two months at 2-8 C. It should be reconstituted with 13 ml of assay buffer for use. The reconstituted solution is stable for at least two months when stored at 2-8 C. C. PGE 2 Standard Concentrate This solution contains 100 ng/ml of prostaglandin E 2 in acetonitrile. Immediately before use in the assay, dilute an aliquot of the stock solution to prepare standards. A suggested procedure for preparing diluted standards appears in Section VI. Do not store diluted standards. Pipets and/or pipet tips used to transfer diluted standard must be made of polypropylene or siliconized glass. Do not use those made of unsiliconized glass. Store the remaining standard concentrate at -20 C. Under these conditions, the solution is stable for at least two months. Extreme care must be taken to avoid evaporation of the solvent and resulting concentration of the standard. Avoid unnecessary exposure to air and ambient temperature. Use promptly, cap, and return to -20 C storage. D. Assay Buffer This solution consists of 0.9% NaCl, 0.01M EDTA, 0.3% bovine g-globulin, 0.005% Triton-X-100, and 0.05% sodium azide, 0.0255M NaH 2 PO 4 H 2 O, 0.0245M Na 2 HPO 4 X7H 2 O, ph 6.8. Stored at 6
2-8 C, the assay buffer is stable for at least two months. E. Precipitating Reagent The solution contains 16% polyethylene glycol (PEG 6000) and 0.05% sodium azide in 50 mm phosphate buffer, ph 6.8. Stored at 2-8 C, it is stable for at least two months. This reagent is quite viscous and the use of a positive displacement device facilitates dispensing. V. SAMPLE HANDLING A. Collection and Storage It is recommended that all samples be processed immediately after collection and assayed as soon as possible. PGE 2 is reported to be stable in urine (17) if kept frozen, but significant decreases in PGE 2 have been observed after plasma was stored at -20 C for a week. Blood samples, at least 2 ml, should be collected in pre-chilled siliconized glass or polypropylene test tubes coated with a solution of 4.5mM EDTA containing a prostaglandin synthetase inhibitor such as indomethacin or aspirin (18). Indomethacin has been reported to be very effective at concentrations of up to 10 µg/ml. The plasma fraction should be isolated from the whole blood as soon as possible after collection and frozen at -70 C if it is not assayed on same day. Urine should be stored at -20 C immediately after collection. Urinary PGE 2 levels are reported to show a circadian rhythm, and a single sample may not be representative of the true value (19). If tissues are not analyzed immediately after collection, they should be stored at -70 C or lower. Tissue samples should be processed in the presence of prostaglandin synthetase inhibitors (20), such as indomethacin, at concentrations up to 10 µg/ml. It is good practice to assay, in each run, a method blank consisting of distilled water which has been extracted along with samples. This practice will assure the user that non-specific interfering substances have not been introduced from solvents, etc. It is also good practice to use [3H]-PGE 2 (NET-428) as recovery marker in any extraction procedure. 7
1. Urine PGE 2 of renal origin is present in urine (25), in addition to at least eight metabolites derived from PGE 2 in the peripheral circulation (26). Successful direct assay of urine for immunoreactive PGE 2 levels has been reported (27). The urine may be extracted by conventional methods. We have employed a modification of the method of Frölich (25) with good success. The urine sample is acidified to ph 4.0, and extracted two times with an equal volume of chloroform. The extracts are pooled, dried under nitrogen and reconstituted with assay buffer. It is recommended that serial dilutions be assayed because of the wide range of expected values. We have observed a reduction in method blanks with filtration of the reconstituted sample through 0.22 µm Millex filter. 2. Tissue and Plasma Plasma and tissue extraction procedures have been reported in references 20, 23, and 24. Due to the wide variety of sample types and extraction procedures used by our customers, validation of these procedures and any matrix effects remain the responsibility of the individual researcher. VI. PROCEDURE A. Materials Required In addition to the reagents supplied with the kit, the following materials are required: 1. Pipettors and/or pipets that accurately and precisely deliver the required volumes. Pipets and/or pipet tips used to transfer diluted standards, tracer, or samples should be made of polypropylene or siliconized glass. Do not use those made of unsiliconized glass. Positive-displacement* pipettors or repipet devices may be most convenient for dispensing the precipitating reagent. 2. Siliconized glass test tubes (these can be prepared by treating borosilicate glass tubes with Sigmacote**), or polypropylene 8
test tubes. 3. Test tube rack. 4. Ice bath. 5. Beakers or flasks. 6. Vortex mixer. 7. Centrifuge, refrigerated, with swinging bucket rotor. 8. Gamma counter. 9. Extraction devices and reagents. 10. Dry-down apparatus and nitrogen. 11. Repipeting device. *Positive-displacement pipettors such as Repipet, Dispensette, and Oxford dispensers are readily available through Fisher and other chemical supply companies. **Sigmacote is the trade name of a siliconizing agent which is commercially available from Sigma Chemical Co., St. Louis, MO 63178. B. Preparation of Prostaglandin E 2 Working Standards An aliquot of the PGE 2 standard concentrate (tube "a" in the dilution scheme below) is diluted with assay buffer in order to prepare a series of working prostaglandin E 2 standards (tubes b - h in the dilution scheme below). A suggested dilution scheme to cover a standard curve range of 1.0 pg to 100 pg added (per 0.1 ml) is shown below. Other dilution schemes which cover this range can be used, but the assay buffer provided with the kit must be used for dilution of the standards. The assay buffer contains additives which minimize the non-specific adsorption of prostaglandin E 2 to the wall of the test tubes. NOTE: Working standards must be freshly prepared on the day of use. Working standards should not be stored overnight before use. Use only siliconized glass test tubes, or polypropylene tubes. Use polypropylene or siliconized glass 9
pipets or pipet tips for transferring standard solutions. Do not use pipets of unsiliconized glass. 10
Table I - Suggested Dilution Scheme Tube a b c d e f g h 0.1 ml standard concentrate+ 1.9 ml Assay Buffer 0.5 ml of dilution a + 2.0 ml Assay Buffer 1.0 ml of dilution b + 1.0 ml Assay Buffer 1.0 ml of dilution c + 1.0 ml Assay Buffer 1.0 ml of dilution d + 1.5 ml Assay Buffer 1.0 ml of dilution e + 1.0 ml Assay Buffer 1.0 ml of dilution f + 1.0 ml Assay Buffer 1.0 ml of dilution g + 1.5 ml Assay Buffer *This concentration represents actual mass added to assay tube. Dilutions b through h should be used for the standard curve. Concentration (pg/0.1 ml)* 500 100 50 25 10 5 2.5 1 C. Radioimmunoassay Protocol 1. Prepare all reagents according to directions in Section IV. 2. Equilibrate all reagents to room temperature and mix before use. 3. Label duplicate tubes for total counts, blank, each standard, and each sample. 4. Place tubes in a suitable test tube rack. (Refer to Table II for a synopsis of steps 5-18.) 5. Pipet 200 µl of assay buffer into tubes 3-4 (blank tubes). 6. Pipet 100 µl of assay buffer into tubes 5-6 (zero standard tubes). 7. Pipet 100 µl of each diluted standard (b - h) into tubes 7-20. 8. Pipet 100 µl of each reconstituted sample extract in duplicate into the appropriate tubes starting with tube 21. 9. Pipet 100 µl of tracer solution into each tube and mix. 10. Pipet 100 µl of antiserum into all tubes beginning with tube 5 and vortex thoroughly for 2-5 seconds. 11. Incubate overnight (20-24 hours) at 2-8 C. 11
12. At the end of the overnight incubation, place all tubes in an ice bath. 13. Pipet 1 ml of cold precipitating reagent into all tubes beginning with tube 3. Vortex each tube thoroughly for 2-5 seconds. (Use of a repipettor makes this step simpler to perform.) 14. Allow tubes to incubate in an ice bath at 2-8 C for 20-30 minutes. 15. Centrifuge the tubes in a refrigerated centrifuge at 1000-2000 x g for 30 minutes. 16. Decant the supernatants of all tubes beginning with tube 3 and allow to drain for ~ one minute on absorbent paper. Blot the tubes to remove residual liquid. It may be more convenient, but is not necessary, to employ a rack so that all tubes may be decanted simultaneously. 17. Count all tubes in a gamma counter. At normal efficiencies, a one minute count time is sufficient. 18. Calculate results as described in Section VIII. Table II - Prostaglandin E 2 Assay Protocol Schematic Total Counts Blank "0" Standard Standards (All volumes are in microliters) Tube No. 1-2 3-4 5-6 7-20 Buffer Standard Samples Tracer Antibody --- --- --- 100 --- 200 --- --- 100 --- 100 --- --- 100 100 --- 100 --- 100 100 Incubate overnight (20-24 hours) at 2-8 C. Add 1 ml of cold precipitating Reagent to all tubes except total counts. Mix, incubate for 20-30 minutes at 2-8 C, and centrifuge at refrigerated temperatures for 30 minutes at 1000-2000 x g. Decant all tubes except total count tubes into hot waste, blot and count. D. Precautions 1. WARNING: THIS PRODUCT CONTAINS A CHEMICAL KNOWN TO THE STATE OF CALIFORNIA TO CAUSE CANCER. (NOTE: [ 125 I] TRACER) 12
2. Pipetting must be performed reproducibly and accurately. Prostaglandin E 2 has the tendency to adhere to many surfaces. To minimize assay interference from this "sticking" problem, use only polypropylene or siliconized glass pipets, or pipet tips when transferring diluted materials or incubating. 3. Inadequate incubation time with the precipitating reagent or centrifugation time or speed may result in incomplete precipitation of bound counts. 4. Inadequate centrifugation speed or prolonged inversion after decanting may cause the pellets to become dislodged from the bottom of the tubes. 5. Since PGE 2 can be converted to PGA 2 as an artifact of sample handling, it is suggested that the samples be extracted as soon as possible after collection and that exposure to ambient temperatures be minimized. VII. PROCEDURE FOR CALCULATING UNKNOWNS After counting has been completed, the concentration of prostaglandin E 2 in the samples is determined from a standard curve. The following method is suggested. (See Table III for sample calculations.) 1. If all tubes have been counted for the same period of time, use the total accumulated counts; otherwise, correct all raw counts to counts per minute (CPM). 2. Average the counts for each set of duplicates. 3. Calculate the average NET counts for all standards and samples by subtracting from each the average blank counts (tubes 3-4). 4. Determine the normalized percent bound (% B/B o ) for each standard and sample as follows: % B/Bo = Net cpm of Standard or Sample x 100 Net cpm of "0" Standard 5. Using semi-logarithmic graph paper, plot % B/B o for each standard versus the corresponding amounts of prostaglandin E 2 added in 13
picograms (pg). (See Figure 3 for a typical standard curve using the standard protocol.) 6. Determine the pg prostaglandin E 2 in each sample by interpolation from the standard curve. Since the standard curve is expressed as pg prostaglandin E 2 added, sample values must then be corrected for aliquots, dilution, recovery, etc. to determine the original concentration in the sample. NOTE: Any samples with concentrations which are above the range of the standard curve may be diluted with assay buffer and reassayed. The values obtained are then multiplied by the appropriate dilution factor. Table III - Sample Calculations Tube No. CPM Average CPM Net CPM %B/Bo 14
Total Counts 1 2 15541 15274 15408 14859 - Blank 3 4 528 571 549 0 - "0" Standard 5 6 6575 6802 6688 6139 100 1 pg 7 8 6231 6109 6170 5621 91.6 2.5 pg 9 10 5453 5568 5510 4961 80.8 5 pg 11 12 4736 4770 4753 4204 68.5 10 pg 13 14 3720 3794 3757 3208 52.3 25 pg 15 16 2493 2612 2552 2003 32.6 50 pg 17 18 1829 1765 1797 1248 20.3 100 pg 19 20 1318 1433 1375 826 13.5 15
Figure 3 Typical Standard Curve Do not use to calculate samples 100 90 80 70 60 %B/B0 50 40 30 20 10 0 1 10 100 Prostaglandin E2, pg/0.1 ml 16
VIII. LIMITATIONS A. The following compounds have been checked for cross-reactivity. The percentages are calculated at the 50% B/B o point. Compound PGE2 PGE1 DHKPGE2 PGA2 PGF1a Thromboxane B2 PGF2a Table IV % Cross- Reactivity Compound 100 30 0.02 0.8 0.7 0.01 0.9 Arachidonic Acid DHKF2a PGA1 PGB2 6KPGF1a Linoleic Acid PGD2 % Cross- Reactivity 0.01 0.005 0.08 0.07 1 0.002 B. The prostaglandin E 2 standards are prepared in the assay phosphate buffer. The effect of other sample matrices upon the assay system must be determined by the investigator. C. Sensitivity Defined as the mass corresponding to twice the standard deviation of the zero binding, the sensitivity of the system was found to be approximately 0.44 pg added. 0.3 IX. REFERENCES 1. Ramwell, P. W., Biol. Reprod., 16: 70 (1977). 2. Flower, R. J. and Blackwell, G. J., Biochem. Pharmacol., 25: 285 (1976). 3. Moncada, S. and Vane, J. R., Pharmacol. Rev., 30: 293 (1979). 4. Sameulsson, B., et. al., Ann. Rev. Biochem., 47: 997 (1978). 5. Nugteren, D. H. and Hazelhof, E., Biochim. Biophys. Acta, 326: 448 (1973). 6. Hamberg, M., Biochim. Biophys. Acta, 431: 651 (1976). 7. Moncada, S., et. al., Nature, 263: 663 (1976). 8. Moncada, S. and Korbut, R., Lancet, 1: 1286 (1978). 17
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9. Samuelsson, B., Granstrom, E., Green, K., Hamberg, M., and Hammarstrom, S., Ann. Rev. Biochem., 44: 669 (1975). 10. Ferreira, S. H. and Vane, J. R., Nature 216: 868 (1967). 11. Samuelsson, B., Hamberg, M., and Sweeley, C., Anal. Biochem. 38: 301 (1970). 12. Green, K., Granstrom, E., and Samuelsson, B., Anal. Biochem. 54: 434 (1973). 13. Levine, L., Pharmacol. Rev. 25: 293 (1973). 14. Jaffe, B. M., Behrman, H., and Parker, C. W., J. Clin. Invest. 52: 398 (1974). 15. Ohki, S., et al., Prostaglandins 6: 137 (1974). 16. Maclouf, J., et al., Biochim. Biophys. Acta 431: 139 (1976). 17. Ciabattoni, G., et al., Adv. in Prostaglandin & Thromboxane Res. 6: 207 (1980). 18. Yamamoto, S., et al., Biochemical Aspects of Prostaglandins and Thromboxanes, N. Kharasch and J. Fried, (Eds.), Academic Press, New York, pp 1-13 (1977). 19. Bowden, R., et al., Prostaglandins 14: 151 (1977). 20. Shaw, J. E., and Ramwell, P. W., Methods of Biochem. Analysis 17: 325 (1969). 21. Dray, F., Charbonnel, B., and Maclouf, J., European J. Clin. Invest. 5: 311 (1975). 22. Morris, H. G., Sherman, N. A., and Shepperdson, F. T., Prostaglandins 21: 771 (1981). 23. Green, K., Hamberg, M., Samuelsson, B. and Frolich, J. C., Adv. in Prostaglandin and Thromboxane Res., 5: 15 (1978). 24. Skrinska, V. and Lucas, F., Prostaglandins, 22: 365 (1981). 25. Frolich, J., et al., J. Clin. Invest. 55: 763 (1975). 26. Hamberg, M. and Wilson, M., International Conf. on Prostaglandins, S. Bergstrom, (Ed.), Pergamon Press (1972). 19
27. Korteweg, M., et al., Adv. in Prostaglandin and Thromboxane Res. 6: 201 (1980). 20
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