PerkinElmer Life and Analytical Sciences, Inc. cAMP [125I] RADIOIMMUNOASSAY KIT (ADENOSINE 3', 5' CYCLIC MONOPHOSPHATE) CATALOG NUMBER NEK033 For Laboratory Use CAUTION: A research chemical for research purposes only. 1 TABLE OF CONTENTS I. Proprietary Name 3 II. Intended Use 3 III. Explanation of Test 3 IV. Principle of the Method 3 V. Reagents 4 VI. Sample Collection, Processing and Storage 8 VII. Non-Acetylated Procedure (Urine) 10 VIII. Acetylated Procedure (Plasma) 14 IX. Procedure for Calculating Unknowns in Urine and Plasma Samples 18 X. Precautions 22 XI. Performance Characteristics for the Non-Acetylated (Urine) Procedure 22 Performance Characteristics for Acetylated (Plasma) Procedure 25 XIII. Correlation 27 XIV. References 27 XII. 2 I. PROPRIETARY NAME cAMP [125I] RIA Kit PerkinElmer Life Sciences, Inc. Catalog Number: NEK033, 200 Tubes II. INTENDED USE This kit is designed to measure cAMP levels in plasma, urine and tissues. III. EXPLANATION OF TEST Adenosine 3', 5' cyclic monophosphate (cAMP) plays a critical role in the transmission of hormonal signals by functioning as a “second messenger”1,2. The binding of a hormone to its receptor can either enhance or inhibit the rate at which cAMP is produced. This is accomplished by altering the enzymatic activity of adenyl cyclase, the membrane associated enzyme which catalyzes the production of cAMP from ATP3. By this mechanism, intracellular levels of cAMP are altered in response to hormonal stimulation. In turn, the intracellular level of cAMP regulates the enzymatic activity of a protein kinase which phosphorylates other substances setting off a cascade of cellular events which leads to the expression of the hormone4. The cAMP Assay is a double-antibody RIA which utilizes a prereacted antibody complex. The assay is performed using acetylated or nonacetylated standards and samples. Acetylation of the sample allows for a more sensitive assay of cAMP5,6. The assay is accurate over a wide range of values and has a high degree of specificity. IV. PRINCIPLE OF THE METHOD The basic principle of radioimmunoassay is the competition between a radioactive and a non-radioactive antigen for a fixed number of antibody binding sites. This interaction is illustrated in Figure 17. 3 Figure 1 Labeled Antigen Ag* + Specific Antibody Ab → ← Labeled Antigen Antibody Complex Ag*Ab + Unlabeled Antigen Ag (In standard solutions ↑↓ or unknown samples) Unlabeled Antigen-Antibody Complex AgAb When unlabeled antigen from standards or samples and a fixed amount of the labeled antigen are allowed to react with a constant and limiting amount of antibody, decreasing amounts of the labeled antigen are bound to the antibody as the amount of unlabeled antigen is increased. In the cAMP [125I] Kit, separation of the bound from free antigen is achieved by using a prereacted primary antibody/secondary antibody complex. The second antibody is derived from an animal species different from that used to generate the primary antibody. After incubation and centrifugation, the supernatant is discarded and the antigen-antibody complex is counted to quantitate the bound tracer. The data are used to construct a standard curve from which the values of the unknowns may be obtained by interpolation. V. REAGENTS All necessary reagents are supplied and are intended FOR LABORATORY USE. Kits are shipped at ambient temperatures and must be stored upon receipt at refrigerator temperature (2-8°C). The reagents are stable for the times indicated if the specific precautions given below are followed. Sodium azide has been added as an antibacterial agent where appropriate. 4 NOTE: The National Institute for Occupational Safety and Health has issued a bulletin citing the potentially explosive hazard due to the reaction of sodium azide with copper, lead, brass, or solder in plumbing systems. Although sodium azide is added at a minimal concentration, it is still recommended that copious amounts of water be used to flush the drain pipeline after disposal of these reagents in the plumbing system. Copper-free and lead-free discharge lines should be used whenever possible. Decontamination procedures should be followed prior to maintenance on drain lines which have been used for azide-containing reagents. Recommended decontamination procedures are available from RIA Technical Services. NOTE: An excess of each reagent beyond the labeled content is added to each container in order to allow the withdrawal of the number of aliquots required to fill the stated quantity of tubes. Any reagent remaining after the stated quantity of tubes have been prepared should be discarded. A. cAMP Sodium Acetate Buffer One vial of concentrated buffer is supplied. Dilute to 500 mL with distilled water. The final solution will contain sodium acetate buffer, pH 6.2, and a stabilizer. The diluted buffer is stable for at least two months when stored at 2-8°C. Refer to vial label for exact expiration date of the concentrated reagent. B. cAMP Standard One vial of liquid standard is supplied. The solution contains: cAMP at a concentration of 50,000 pmol/mL, sodium acetate buffer, and 0.09% sodium azide. The cAMP Standard has been calibrated spectrophotometrically using the molar absorption coefficient, Œ = 14.6 x 103l mol-1 cm-1 at 259 nm, pH 6.9. The standard is stable for at least one year when stored at 2-8°C. Refer to vial label for expiration date of reagent. 5 C. cAMP Antiserum Complex One vial of lyophilized, prereacted, first and second antibody is supplied. Reconstitute with 21 mL of distilled water. The resulting solution will contain the prereacted antibody complex, and an inert ingredient, in sodium phosphate buffer, pH 6.0. This solution may appear cloudy and will settle upon standing. Before use and during prolonged use, mix thoroughly. The reconstituted antiserum complex is stable for at least two months when stored at 2-8°C. Refer to vial label for expiration date of lyophilized reagent. D. cAMP [125I] Tracer (Succinyl cAMP Tyrosine Methyl Ester [125I]) Two vials of concentrated tracer are supplied. Each vial contains less than 74KBq (2 µCi) on calibration date in 1 mL of a 1:1 n-propanol water solution. Use one vial at a time as directed. Add 5.0 mL of distilled water to each vial as required. The concentrate and diluted tracer are stable for at least two months when stored at 2-8°C. This material is radioactive and the user should follow the precautions listed on the following page. 6 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. 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. 7 E. cAMP Carrier Serum Two vials of lyophilized carrier serum are supplied. Use one vial at a time. Reconstitute the contents of each vial with exactly 6.0 mL of distilled water. (One vial of reconstituted carrier serum is sufficient for 100 tubes.) The resulting solution will contain carrier serum, 0.1% sodium azide, a stabilizer, and an inert ingredient in sodium acetate buffer, pH 6.2. Refer to vial label for expiration date of lyophilized reagent. Store at 2-8°C. F. cAMP Acetic Anhydride One vial containing 1 mL is supplied. CAUTION: FLAMMABLE, CORROSIVE, LACHRYMATOR. Store tightly closed in the refrigerator. Allow vial to equilibrate to room temperature before use. Protect from moisture. This material is stable for at least two months under these conditions. Refer to vial label for expiration date. G. cAMP Triethylamine One vial containing 1 mL is supplied. CAUTION: FLAMMABLE, VAPOR HARMFUL. Store tightly closed in the refrigerator. Allow vial to equilibrate to room temperature before use. Refer to vial label for expiration date. H. cAMP Precipitator One bottle containing 100 mL is supplied ready to use and contains a precipitation enhancer and sodium azide in sodium acetate buffer. Refer to bottle label for expiration date. Store at 2-8°C. Shake well before using. VI. SAMPLE COLLECTION, PROCESSING AND STORAGE A. Plasma: EDTA-treated plasma should be used. Collect blood by venipuncture in a 5 or 10 mL glass blood collection tube containing EDTA (lavender top). The usual precautions for venipuncture apply. Samples should be kept on ice after drawing. Separation of cells from plasma should be carried out as soon as possible after collection in a refrigerated centrifuge (15 minutes, approximately 760 x g). Plasma may be stored at -20°C for at least four weeks. Repeated freezing and thawing should be avoided. B. Urine: Voided urine specimens are required for the assay. Urine samples should be centrifuged to remove any particulate 8 matter present. Random, timed, or 24 hour urine collections can be used. For twenty-four hour specimens, it may be necessary to prevent bacterial growth by collecting urine into acid (2 mL of 6 N HCl per 100 mL urine). Urine collected utilizing antibacterial agents should be validated as a suitable specimen by the operator. Urine samples can be stored undiluted at 2-8°C for 24 hours, but should be frozen at -20°C for longer storage. C. Tissue Samples: Precipitation of proteins from plasma or tissues has been accomplished with trichloroacetic acid (TCA), perchloric acid, or ethanol followed, in some cases, by ion exchange or alumina column chromatography. The decision as to which procedure to use depends on the nature of the sample and is left up to the individual investigator. This protocol is applicable to tissue culture cells as well as solid tissue; however, it is the responsibility of the investigator to validate this procedure for each specific application. As part of this validation, we recommend adding phosphodiesterase to some samples and noting the loss of immunoreactivity. 1. Homogenize the frozen tissue sample at 4°C with 6% TCA to make a 1 mL 10% (w/v) homogenate. Add an equal volume of cold 10% TCA to cell culture preparations or supernatants. 2. To determine the recovery of cyclic AMP during extraction, add to each sample extract approximately 4,000 cpm of 3 H-cyclic AMP marker (NET275 [2,8 3H] Cyclic AMP, diluted 1:200 in 50% Ethanol / 50% H2O solution, available from PerkinElmer Life Sciences) to the TCA extract. At a specific activity of approximately 37 Ci/mmol and at a 50% counting efficiency, this represents approximately 0.1 pmol (100 fmol) of cyclic AMP. This amount must be taken into account when calculating the cyclic AMP content of the tissue. 3. Centrifuge TCA extracts at 2,500 x g at 4°C for 15 minutes. 4. Collect the supernatant and extract 4 times with 5x volume of water-saturated ether. Discard the ether phase. 5. Place sample in a water bath at 70-80°C and evaporate to dryness under a stream of air. 9 6. Dissolve the residue in Assay Buffer (volume depends on the amount of c-AMP in the sample) and use 100 µL directly in the Assay or the sample may be diluted. The decision as to which procedure to use for the sample depends on expected levels of cyclic AMP in the sample. If necessary, the TCA extract may be purified further by ion exchange column chromatography. In this example, it is not necessary to remove the TCA with ether as the sample may be applied directly to the column. a. Prepare a 0.6 x 5.0 cm column of Dowex 50w - X8 (H+), 200-400 mesh in water. This is conveniently done in a disposable Pasteur capillary pipet. b. Prior to the sample additions to the column, it should be characterized to locate which fraction contains 3H-cyclic AMP marker. Use water as the eluate. c. After the elution volume containing 3 H-cyclic AMP has been determined, allow the water to drain into the resin bed, pipet 1 mL of the TCA extract onto the column and start collecting 1 mL samples. d. After the TCA extract has drained into the column, add water and continue to collect the effluent. e. Combine the fractions previously determined to contain 3H-cyclic AMP marker and continue at Step 5. We have found recoveries > 90% with this procedure. If an excess of cyclic AMP is suspected in the sample, dilute with Assay Buffer. VII. NON-ACETYLATED PROCEDURE (URINE) A. Reagents Supplied - sufficient for 200 tubes 1. 1 vial cAMP Antiserum Complex, lyophilized 2. 2 vials cAMP [125I]-Tracer, 1 mL (concentrate) 10 B. 3. 2 vials cAMP Carrier Serum, lyophilized 4. 1 vial cAMP Standard, liquid 5. 1 vial cAMP Buffer, 25 mL (concentrate) 6. 1 bottle cAMP Precipitator, 100 mL Equipment and Reagents Required In addition to the reagents supplied with the kit, the following materials are required: C. 1. Pipettors and/or pipets that can accurately and precisely deliver the required volumes. 2. Gamma scintillation counter. 3. Laboratory vortex mixer. 4. Test tube rack. 5. Distilled water. 6. Centrifuge - refrigerated (swing bucket head). 7. Test tubes - 12 x 75 mm - glass. 8. Test tube - 13 x 100 mm - glass. 9. Absorbent paper for blotting. 10. Radioactive waste container. 11. 2-8°C refrigerator or equivalent. Urinary Radioimmunoassay Protocol NOTE: A full understanding of the Protocol and Precautions is necessary for successful completion of the RIA. Read these two sections carefully before proceeding with the assay. Seven levels of cAMP standards are recommended, each point to be run in duplicate. 1. Prepare standard solutions as follows, mixing each solution thoroughly before adding it to the next tube. Prepare the standards fresh each day. 11 Pipet mL cAMP Std. From Tube Add mL Assay Buffer Into Labeled Tube Concentration (pmol/mL) 0.1 0.2 1.0 1.0 1.0 1.0 1.0 1.0 * A B C D E F G 9.9 1.8 1.0 1.5 1.0 1.0 1.5 1.0 A B C D E F G H 500 50 25 10 5 2.5 1.0 0.5 *50,000 pmol/mL (stock standard reagent) 2. Prepare the urine sample as follows: First, dilute the urine specimen tenfold by adding 100 µL of urine to 900 µL of Assay Buffer. Then, take 100 µL of the tenfold dilution and add it to 4.9 mL of Assay Buffer. This produces a 1/500 dilution of the sample. 3. Prepare Working Tracer Solution by adding one volume of diluted cAMP [125I]-Tracer to one volume of the reconstituted cAMP Carrier Serum. Make enough of the Working Tracer Solution to run the desired number of tubes (e.g., 1.0 mL of cAMP Carrier Serum and 1.0 mL of cAMP [125I]-Tracer will provide 2.0 mL of solution which is theoretically sufficient for 20 tubes). Any remaining volume is to be discarded appropriately. Do not store and reuse this solution. 4. Number a series of 20 tubes to be used for the standard curve, plus two additional tubes for each sample. The assay may be set up at room temperature. 5. Tubes 1 and 2 measure the total counts added and receive only the Working Tracer Solution. 6. Add 200 µL of Assay Buffer to tubes 3 and 4 (blank tubes). 7. Add 100 µL of Assay Buffer to tubes 5 and 6 (zero standard tubes). 12 8. Add 100 µL of each standard solution or sample to the appropriate tubes (see Table I). 9. Add 100 µL of the Working Tracer Solution to all tubes (see Step 3). 10. Thoroughly mix Antiserum Complex and add 100 µL to all tubes, except Total Count tubes and Blank tubes. 11. Mix all tubes, except 1 and 2, by using a vortex mixer. 12. Cover and incubate overnight (16-18 hours) at 2-8°C. 13. Set tubes 1 and 2 aside, and add 500 µL of 2-8°C cAMP Precipitator to all the other tubes. Mix well with a vortex mixer and centrifuge 2-8°C for 15 minutes at approximately 1,200 x g. 14. Decant by gently inverting all tubes once, preferably at the same time, discarding the supernatant into a radioactive waste container. Keeping the tubes inverted, place them on absorbent paper for blotting. To facilitate removal of remaining droplets, gently tap the rims of the tubes on the paper. Allow tubes to drain for 20-30 seconds. 15. Count all tubes, including Tubes 1 and 2, in a gamma counter. At the usual counting efficiency of 50-70%, a counting time of one minute should be sufficient. 16. Calculations are described in Section IX. 13 TABLE I - PROTOCOL FOR NON-ACETYLATED STANDARD CURVE (All volumes are in microliters) Tube Total Cts. Blank “0” Std. 0.5 Std. 1 Std. 2.5 Std. 5 Std. 10 Std. 25 Std. 50 Std. Sample 1, 2 3, 4 5, 6 7, 8 9, 10 11, 12 13, 14 15, 16 17, 18 19, 20 21, 22 Assay Stds. SamBuffer ple 200 100 - 100 100 100 100 100 100 100 - 100 Working Tracer Antiserum Complex 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Precipitator Mix. Cover and incubate 16-18 hours at 2 8°C. 500 500 500 500 500 500 500 500 500 500 Centrifuge at 1,200 x g for 15 minutes at 2-8°C. Decant and blot tubes. Count pellets. VIII. ACETYLATED PROCEDURE (PLASMA) A. B. Reagents Supplied - sufficient for 200 tubes 1. 1 vial cAMP Antiserum Complex, lyophilized 2. 2 vials cAMP [125I]-Tracer, 1 mL (concentrate) 3. 2 vials cAMP Carrier Serum, lyophilized 4. 1 vial cAMP Standard, liquid 5. 1 vial cAMP Buffer, 25 mL (concentrate) 6. 1 vial cAMP Triethylamine 7. 1 vial cAMP Acetic Anhydride 8. 1 bottle cAMP Precipitator, 100 mL Equipment and Reagents Required In addition to the reagents supplied with the kit, the following materials are required: 14 C. 1. Pipettors and/or pipets that can accurately and precisely deliver the required volumes. 2. Gamma scintillation counter. 3. Laboratory vortex mixer. 4. Test tube rack. 5. Distilled water. 6. Centrifuge - refrigerated (swing bucket head). 7. Test tubes - 12 x 75 mm - glass. 8. Test tube - 13 x 100 mm - glass. 9. Absorbent paper for blotting. 10. Radioactive waste container. 11. 2-8°C refrigerator or equivalent. Acetylated (Plasma) Radioimmunoassay Protocol NOTE: A full understanding of the Protocol and Precautions is necessary for successful completion of the RIA. Read these two sections carefully before proceeding with the assay. 1. Dilute the cAMP stock standard reagent (50,000 pmol/mL) fifty-fold in a 13 x 100 mm test tube by adding 100 µL of the 50,000 pmol/mL solution to 4.9 mL of Assay Buffer. The resulting solution will be 1,000 pmol/mL. Prepare a 40 pmol/mL solution by adding 0.1 mL of the 1,000 pmol/mL solution to 2.4 mL of Assay Buffer. Place 200 µL of the 40 pmol/mL solution in a marked glass tube. 2. Prepare the plasma samples for acetylation as follows: Pipet 100 µL of the plasma sample into 400 µL of Assay Buffer (1:5 dilution). Place 100 µL of the 1:5 dilution in a marked glass test tube. 3. Prepare 150 µL of acetylation reagent by mixing together in a glass test tube 100 µL of Triethylamine and 50 µL of Acetic Anhydride. Vortex before using. Make up a fresh solution each time. 15 4. Prepare 10 mL of Modified Assay Buffer by adding 50 µL of the acetylation reagent (Step 3) to 10 mL of Assay Buffer. Mix well and let incubate at room temperature for at least three minutes. Modified Assay Buffer is used for the preparation of the cAMP standards and in the “blank” and “zero standard” tubes (see Table II). 5. Acetylate the 40 pmol/mL standard prepared in Step 1 (i.e., the 200 µL aliquot) by adding to it 10 µL of the freshly prepared acetylation reagent. Allow the reaction to proceed for at least three minutes at room temperature and then add 1.8 mL of Assay Buffer. Label this 4 pmol/mL standard as tube A. The amount of acetylation reagent used is sufficient to acetylate 25,000 pmols of cAMP. 6. Acetylate the previously prepared 100 µL aliquot of diluted sample (Step 2) by adding 5 µL of acetylation reagent to each tube. Immediately vortex and let the sample incubate for at least three minutes at room temperature. Then add 900 µL of Assay Buffer to each tube. This makes a 1:50 dilution of the sample. 7. Dilute the 4 pmol/mL standard with Modified Assay Buffer (see Step 4) as shown below. The standards should be prepared fresh each day. Do not store for reuse. Pipet mL cAMP Std. From Tube Add mL Modified Assay Buffer Into Labeled Tube Concentration (pmol/mL) 1.0 1.0 1.0 1.0 1.0 1.0 A B C D E F 1.0 1.0 1.0 1.0 1.5 1.0 A B C D E F G 4.0 2.0 1.0 0.5 0.25 0.10 0.05 8. Prepare Working Tracer Solution by adding one volume of diluted cAMP [125I]-Tracer to one volume of the reconstituted cAMP Carrier Serum. Make enough of the Working Tracer Solution to run the desired number of tubes (e.g., 1.0 mL of cAMP Carrier Serum and 1.0 mL of cAMP [125I]-Tracer will provide 2.0 mL of solution which is theoretically sufficient for a maximum of 20 tubes). Any 16 remaining volume is to be discarded appropriately. Do not store and reuse this solution. 9. Number a series of 20 tubes to be used for the standard curve, plus two additional tubes for each sample. The assay may be set up at room temperature. 10. Tubes 1 and 2 measure the total counts added and receive only the Working Tracer Solution. 11. Add 200 µL of Modified Assay Buffer to Tubes 3 and 4 (blank tubes). 12. Add 100 µL of Modified Assay Buffer to Tubes 5 and 6 (zero standard tubes). 13. Add 100 µL of each standard solution or sample to the appropriate tubes (see Table II). 14. Add 100 µL of Working Tracer Solution to all tubes. 15. Thoroughly mix Antiserum Complex and add 100 µL to all tubes, except Total Count tubes and Blank tubes. 16. Mix all tubes, except 1 and 2, by using a vortex mixer. 17. Cover and incubate overnight (16-18 hours) at 2-8°C. 18. Set Tubes 1 and 2 aside, and add 500 µL of 2-8°C cAMP Precipitator to all the other tubes. Mix well with a vortex mixer and centrifuge at 2-8°C for 15 minutes at approximately 1,200 x g. 19. Decant by gently inverting all tubes once, preferably at the same time, discarding the supernatant into a radioactive waste container. Keeping the tubes inverted, place them on absorbent paper for blotting. To facilitate removal of remaining droplets, gently tap the rims of the tubes on the paper. Allow tubes to drain for 20-30 seconds. 20. Count all tubes, including Tubes 1 and 2, in a gamma counter. At the usual counting efficiency of 50-70%, a counting time of one minute should be sufficient. Include an instrument blank. 21. Calculations are described in Section IX. 17 TABLE II - PROTOCOL FOR ACETYLATED STANDARD CURVE (All volumes are in microliters) Tube Assay Buffer Stds. Sample Working Tracer Antiserum Complex Precipitator Total Cts. 1, 2 - - - 100 - Blank 3, 4 200 - - 100 - “0” Std. 5, 6 100 - - 100 100 0.05 Std. 7, 8 - 100 - 100 100 0.10 Std. 9, 10 - 100 - 100 100 500 0.25 Std. 11, 12 - 100 - 100 100 500 0.5 Std. 13, 14 - 100 - 100 100 500 1.0 Std. 15, 16 - 100 - 100 100 500 2.0 Std. 17, 18 - 100 - 100 100 500 4.0 Std. 19, 20 - 100 - 100 100 500 Sample 21, 22 - - 100 100 100 500 Mix. Cover and incubate 16-18 hours at 2-8°C. - 500 500 500 Centrifuge at 1,200 x q for 15 minutes at 2 - 8°C. Decant and blot tubes. Count pellets. IX. PROCEDURE FOR CALCULATING UNKNOWNS IN URINE AND PLASMA SAMPLES NOTE: Urine and plasma sample values are determined in an identical manner, i.e., by interpolation from their respective standard curves. The range of standard concentrations on a non-acetylated (urine) standard curve is from 0-50 pmol/mL. The range of standard concentrations on an acetylated (plasma) standard curve is from 0-4 pmol/mL. 18 A. If all tubes have been counted for the same period of time, use the total accumulated counts, otherwise, correct all counts to a common count rate. B. Average the counts for each set of duplicates. C. Calculate the average Net Counts for all standards and samples by subtracting from each the average blank counts. D. Express the average Net Counts for each standard and sample as a percentage of the average Net Counts for the zero standard. (This is termed “normalized” percent bound or % B/Bo.) % B/Bo = Average Net Counts of Standard or Sample x 100 Average Net Counts of Zero Standard E. Using semi-logarithmic or log-logit graph paper, plot % B/Bo for each standard against the corresponding concentration of cAMP in pmol/mL. F. Determine the concentration of cAMP in the samples by interpolation from the standard curve. Since identical volumes are used for standards and samples, and the standard curve is expressed as pmol/mL of cAMP, samples can be read as pmol/mL and then multiplied by the appropriate dilution factor (i.e., 50 for plasma samples, 500 for urine samples) to calculate the sample concentration. Any samples with concentrations above the range of the standard curve must be diluted and re-assayed. The values are then multiplied by the appropriate dilution factor. See Tables III and IV for sample calculations, and Figure 2 for typical standard curve. 19 TABLE III - TYPICAL DATA FOR NON-ACETYLATED (URINE) ASSAY Tube Number Counts Bound Average Counts Bound Net Counts Bound Normalized % Bound Blank 1 2 1016 950 983 - - “0” Standard 3 4 16892 17078 16985 16002 100 0.5 pmol/mL 5 6 15340 15662 15501 14518 91 1.0 pmol/mL 7 8 14305 14369 14337 13354 83 2.5 pmol/mL 9 10 12436 12461 12448 11466 72 5.0 pmol/mL 11 12 10385 10508 10446 9463 59 10 pmol/mL 13 14 7837 8034 7936 6952 43 25 pmol/mL 15 16 5287 5502 5394 4412 28 50 pmol/mL 17 18 3764 3844 3804 2812 18 20 TABLE IV - TYPICAL DATA FOR ACETYLATED (PLASMA) ASSAY Tube Number Counts Bound Average Counts Bound Net Counts Bound Normalized % Bound Blank 1 2 923 1003 963 - - “0” Standard 3 4 15856 16227 16042 15079 100 0.05 pmol/mL 5 6 14942 15008 14975 14012 93 0.10 pmol/mL 7 8 13671 13452 13562 12598 84 0.25 pmol/mL 9 10 10839 11179 11009 10046 67 0.50 pmol/mL 11 12 8003 8050 8026 7064 47 1.0 pmol/mL 13 14 5412 5571 5492 4528 30 2.0 pmol/mL 15 16 3589 3539 3564 2601 17 4.0 pmol/mL 17 18 2493 2578 2536 1572 10 Figure 2 cAMP Typical Standard Curve 100 90 80 nonAcet 70 Acet %B/B0 60 50 40 30 20 10 0 0.01 0.1 1 10 cAMP, pmol/mL 21 100 1000 X. PRECAUTIONS A. Incubation conditions should be standardized for proper day to day internal quality control. B. As with all radioimmunoassay procedures, pipetting is crucial. It is essential that pipetting be accurate and reproducible. C. Samples with concentrations above the range of the standard curve may be reassayed after dilution with Assay Buffer (urine samples) or Modified Assay Buffer (plasma samples). The values obtained are then multiplied by the appropriate dilution factor. During the preparation of plasma samples, it is important to make an initial 1:50 dilution of the acetylated sample as previously specified, and then to dilute further with Modified Assay Buffer. D. The use of grossly hemolyzed or lipemic samples should be avoided. E. The presence of exogenous radioactivity in clinical samples may lead to erroneous results. F. Avoid using a centrifuge which may overheat due to prolonged use. G. The reagents in this kit should be used as a unit. Do not mix different lots of any component within a given assay. H. This product has not been tested for use with any methods other than those stated in this Instruction Manual. I. XI. The average Net Counts for the “0” Standard represents the counts bound to the antibody in the absence of added cAMP. This figure should fall between 40-60% of the average net total counts. The acetylated procedure will generally have a slightly lower Bo than the non-acetylated procedure. PERFORMANCE CHARACTERISTICS ACETYLATED (URINE) PROCEDURE A. FOR THE NON- Recovery To demonstrate the accuracy of the method, recovery studies were performed by adding known quantities of cAMP to 22 aliquots of Assay Buffer and three urine samples. The resulting solutions were re-assayed and the recovery of added cAMP was determined. Typical results are given below. + 5 nmol/mL cAMP Sample Assay Buffer A B C B. nmol/mL Base Value nmol/mL Measured % Rec 0 2.4 1.4 3.0 5.4 7.6 6.2 8.5 108 104 96 110 + 15 nmol/mL cAMP +45 nmol/mL cAMP nmol/mL % Rec nmol/mL % Rec Measured Measured 15.6 17.5 18.2 19.0 104 101 112 107 44.1 49.5 51.2 48.8 98 105 111 102 Reproducibility Precision was determined by multiple duplicate analyses of urine pools. Typical results are given in nmol/mL. Within Assay Variation Sample n Mean + 1 S.D. A B C D 10 10 9 10 5.4 + 0.19 2.4 + 0.07 1.3 + 0.03 4.0 + 0.07 Between Assay Variation Coeff. Sample n of Var. (%) 3.5 2.9 2.3 1.8 23 A B C D E F G H 6 6 6 6 6 6 6 6 Mean + 1 S.D. Coeff. of Var. (%) 9.5 + 0.89 3.4 + 0.16 7.1 + 0.69 3.2 + 0.22 3.8 + 0.25 3.5 + 0.2 5.0 + 0.56 1.9 + 0.18 9.0 4.7 9.7 6.8 6.5 5.7 11.0 9.4 C. Linearity To demonstrate the absence of non-specific urine interferences, five samples were assayed by the standard procedure and diluted to varying degrees with Assay Buffer. The recommended 100 µL aliquot was used for all samples. Typical results are given in nmol/mL, corrected for dilution Sample 1:100 1:250 1:500 1:1000 A B C D 2.5 1.4 6.0 2.5 5.8 1.4 5.8 2.4 5.8 1.4 5.8 2.5 5.2 1.3 D. Sensitivity The mean and standard deviation were determined for 8 duplicate measurements of the zero standard binding. The sensitivity of the method, defined as the cAMP concentration corresponding to the mean cpm minus twice the standard deviation, is < 0.025 pmol/mL. E. Specificity The following compounds have been checked for crossreactivity. The percentages are calculated at the 50% B/Bo point. Compound % Cross Reactivity CGMP GMP ATP ADP AMP ~ 0.02 < 0.01 < 0.01 < 0.01 < 0.01 24 XII. PERFORMANCE CHARACTERISTICS FOR ACETYLATED (PLASMA) PROCEDURE A. Recovery To demonstrate the accuracy of the method, recovery studies were performed by adding known quantities of cAMP to aliquots of Modified Assay Buffer and three plasma samples. The resulting solutions were re-assayed and the recovery of added cAMP was determined. Typical results are given on below. +25 pmol/mL cAMP Sample pmol/mL pmol/mL Base Measured Value Modified Assay Buffer A B 0 10 7.5 B. 24 34 50 +50 pmol/mL cAMP +100 pmol/mL cAMP % Rec pmol/mL Measured % Rec pmol/mL Measured % Rec 96 94 90 44 57 54 88 94 93 94 96 100 94 86 92 Reproducibility Precision was determined by multiple duplicate analyses of plasma pools. Typical results are given in pmol/mL. Within Assay Variation Sample n A B C D E 10 10 10 10 10 Between Assay Variation Mean + 1 S.D. Coeff. Of Var. (%) 16 + 0.49 6.3 + 0.26 12.8 + 0.24 35.9 + 0.88 24.7 + 0.48 3 4.1 1.9 2.4 1.9 25 Sample n A B C D E F 6 6 6 6 6 6 Mean + 1 S.D. Coeff. of Var. (%) 16 + 0.98 5.6 + 0.51 27 + 3.2 16 + 2.4 11.6 + 1.6 11 + 1.5 6.1 9.1 11.8 15 14 14 C. Linearity To demonstrate the absence of non-specific serum interferences, five plasma samples were assayed by the standard procedure and diluted to varying degrees with Modified Assay Buffer. The recommended 100 µL aliquot was used for all samples. Typical results are given in pmol/mL, corrected for dilution. Sample 1:50 1:100 1:200 A B C D E 13 16 18 9 20 11 14 18 8 20 12 13 16 17 D. Sensitivity The mean and standard deviation were determined for 8 duplicate measurements of the zero standard binding. The sensitivity of the method, defined as the cAMP concentration corresponding to the mean cpm minus twice the standard deviation, is < 0.025 pmole/mL. E. Specificity The following compounds have been checked for crossreactivity. The percentages are calculated at the 50% B/Bo point. Compound % Cross Reactivity CGMP GMP ATP ADP AMP ~ 0.02 < 0.01 < 0.01 < 0.01 < 0.01 26 XIII. CORRELATION The cAMP concentraion of plasma, urine, and cell culture samples were measured. Summarized below is the correlation obtained comparing the present with the previous antibody. Sample Type N Range (pmole/mL) Slope Y-intercept Correlation Coefficient Plasma 40 8 to 75 0.99 -0.01 0.98 Urine 16 60 to 9070 0.96 -141 0.99 Cell Culture 12 876 to 1942 1.04 -192 0.98 XIV. REFERENCES 1. Sutherland, E. W., Robison, G. A., and Butcher, R. W. Some aspects of the biological role of adenosine 3', 5'monophosphate (cAMP). Circulation, 37 (1968), 279-306. 2. Jost, J. P. and Rickenburg, H. A. cAMP. Annual Review of Biochemistry, 40 (1971), 741-774. 3. Perkins, J. P. (1973): Adenyl cyclase. In: Greengaard, P. and Robison, G. A. (Eds.) Advances in Cyclic Nucleotide Research, Vol. 3:1-64. Raven Press, NY. 4. Langan, T. A. (1973): Protein kinase and protein kinase substrates. In: Greengaard, P. and Robison, G. A. (Eds.). Advances in Cyclic Nucleotide Research, Vol. 3:99-153, Raven Press, NY. 5. Harper, J. F. and Brooker, G. J. (1975): Femtomole Sensitive Radioimmunoassay for cAMP and cGMP after 2'0 acetylation by Acetic Anhydride in Aqueous Solution. Cyclic Nucleotide Research 1 (4) 207-218. 6. Frandsen, E. K. and Krishna, G. (1976): A simple ultrasensitive method for the assay of cyclic AMP and cyclic GMP in tissues. Life Sciences, 18:529-541. 7. Yallow, R. S. and Berson, S. A. in Principles of Competitive Protein Binding Assays. Eds. Odell, W. D. and Daughaday, W. H. J. B. Lippincott Co., Philadelphia. 1971, Ch. 1. 27 PerkinElmer Life and Analytical Sciences, Inc. 710 Bridgeport Avenue Shelton, CT 06484-4794 USA (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com For a complete listing of our global offices, visit www.perkinelmer.com/lasoffices 99972-0604 28