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Aldosterone ELISA For the quantitative determination of Aldosterone in serum, plasma, and urine.

For Research Use Only. Not For Use In Diagnostic Procedures.

Catalog Number: 11-AD2HU-E01 Size: 96 wells Version: 5.0 - ALPCO October 23, 2018

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Intended Use The Aldosterone ELISA is for the direct quantitative determination of Aldosterone in human serum, plasma and urine by an enzyme immunoassay. For research use only. Not for use in diagnostic procedures. Principle of the Assay The principle of the following enzyme immunoassay test follows the typical competitive binding scenario. Competition occurs between an unlabeled antigen (present in standards, controls and samples) and an enzyme-labeled antigen (conjugate) for a limited number of antibody binding sites on the microplate wells. The washing and decanting procedures remove unbound materials. After the washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stop solution. The absorbance is measured with a microplate reader. The intensity of the color formed is inversely proportional to the concentration of aldosterone in the sample. A set of standards is used to plot a standard curve from which the amount of aldosterone in samples and controls can be directly read. Research Applications Research demonstrates that aldosterone is a potent mineralocorticoid whose synthesis and release are controlled by the renin-angiotensin system of the body. Aldosterone promotes the reabsorption of sodium in the distal tubules of the kidney resulting in potassium secretion along with sodium retention, which controls the circulating blood volume. Studies show that chronic overproduction and secretion of aldosterone can lead to hypertension. Measurement of aldosterone levels in serum in conjunction with plasma renin activity levels can be used to research the differentiation between primary and secondary aldosteronism as well as the possible causes behind aldosteronism, Procedural Cautions and Warnings 1. This kit is intended for research use only. 2. Practice good laboratory practices when handling kit reagents. This includes: • Do not pipette by mouth. • Do not smoke, drink, or eat in areas where samples or kit reagents are being handled. • Wear protective clothing and disposable gloves when handling the samples and kit reagents. • Wash hands thoroughly after performing the test. • Avoid contact with eyes; use safety glasses; in case of contact with eyes, flush eyes with water immediately and contact a doctor. 3. Users should have a thorough understanding of this protocol for the successful use of this kit. Reliable performance will only be attained by strict and careful adherence to the instructions provided. 4. Avoid microbial contamination of reagents. 5. A calibrator curve must be established for every run. 6. It is recommended to prepare additional control materials or serum pools which should be included in every run at a high and low level for assessing the reliability of results. 7. The controls (included in kit) must be included in every run and the obtained values must fall within the acceptable ranges, as stated in the quality control certificate. 26-G Keewaydin Drive, Salem, NH 03079│P: (800) 592-5726│F: (603) 898-6854│[email protected]│www.alpco.com Page 2 of 11

8. When the use of water is specified for dilution or reconstitution, use deionized or distilled water. 9. All kit reagents and samples should be brought to room temperature and mixed gently but thoroughly before use. Avoid repeated freezing and thawing of samples. 10. Improper procedural techniques, imprecise pipetting, incomplete washing as well as improper reagent storage may be indicated when assay values for the control do not reflect established ranges. 11. When reading the microplate, the presence of bubbles in the microplate wells will affect the optical densities (ODs). Carefully remove any bubbles before performing the reading step. 12. The substrate solution (TMB) is sensitive to light and should remain colorless if properly stored. Instability or contamination may be indicated by the development of a blue color, in which case it must not be used. 13. When dispensing the substrate and stopping solution, do not use pipettes in which these liquids will come into contact with any metal parts. 14. To prevent contamination of reagents, use a new disposable pipette tip for dispensing each reagent, sample, standard and control. 15. Do not use kit components from different kit lots within a test and do not use any component beyond the expiration date printed on the label. 16. Kit reagents must be regarded as hazardous waste and disposed of according to local and/or national regulations. Limitations 1. All the reagents within the kit are calibrated for the direct determination of aldosterone in human serum, plasma and urine. The kit is not calibrated for the determination of aldosterone in other sample types of human or animal origin. 2. Do not use grossly hemolyzed, grossly lipemic, icteric or improperly stored serum, plasma or urine. 3. Any samples or control sera containing azide or thimerosal are not compatible with this kit, as they may lead to false results. 4. Only the serum/plasma diluent provided upon request may be used to dilute any high serum or plasma samples. Safety Cautions and Warnings Potential Biohazardous Material Human serum that may be used in the preparation of the standards and controls has been tested and found to be nonreactive for Hepatitis B surface antigen and has also been tested for the presence of antibodies to HCV and Human Immunodeficiency Virus (HIV) and found to be negative. No test method however, can offer complete assurance that HIV, HCV and Hepatitis B virus or any infectious agents are absent. The reagents should be considered a potential biohazard and handled with the same precautions as applied to any blood sample. All human samples should also be considered a potential biohazard and handled as if capable of transmitting infections and in accordance with good laboratory practices. Chemical Hazards

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Avoid contact with reagents containing TMB, hydrogen peroxide and sulfuric acid. If contacted with any of these reagents, wash with plenty of water. TMB is a suspected carcinogen. Sample Collection and Storage Serum: Approximately 0.2 mL of serum is required per duplicate determination. Collect 4 – 5 mL of blood into an appropriately labelled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at 4°C for up to 24 hours or at -10°C or lower if the analyses are to be done at a later date. Plasma: Approximately 0.2 mL of plasma is required per duplicate determination. Collect 4 – 5 mL of blood into EDTA plasma tubes. Store at 4°C for up to 24 hours or at -10°C or lower if the analyses are to be done at a later date. Urine: 24-hour urine into a sample collection container. Store at 4°C for up to 24 hours or at -10°C or lower if the analyses are to be done at a later date. Consider all human samples as possible biohazardous materials and take appropriate precautions when handling. Sample Pretreatment Serum and plasma: Serum and plasma are loaded directly to the microplate wells; no sample pretreatment is necessary. Urine: Dilute urine samples 1:50 in urine diluent right before the test. Do not store diluted urine samples. Example: To 0.98 mL of urine diluent, add 20 μL of the urine sample. Reagents and Equipment Needed But not Provided Precision pipettes to dispense 20, 50, 100, 150 and 350 μL Disposable pipette tips Distilled or deionized water Plate shaker Microplate reader with a filter set at 450 nm and an upper OD limit of 3.0 or greater* (see assay procedure step 10) 6. Urine Diluent – Required for the dilution of urine samples before assaying. Available in any quantity. 7. Serum and Plasma Diluent – Required if high samples (> 1000 pg/mL) are to be tested again. Available in any quantity. 8. Timer 1. 2. 3. 4. 5.

Reagents Provided 1. Anti-Aldosterone Polyclonal Antibody-Coated Break Apart Well Microplate- Ready to use Contents: One 96-well (12x8) polyclonal antibody-coated microplate in a resealable pouch with desiccant. Storage: Refrigerate at 2–8°C 26-G Keewaydin Drive, Salem, NH 03079│P: (800) 592-5726│F: (603) 898-6854│[email protected]│www.alpco.com Page 4 of 11

Stability:

12 months or as indicated on label

2. Aldosterone-Horse Radish Peroxidase (HRP) Conjugate- Ready to use Contents: Aldosterone-HRP conjugate in a protein-based buffer with a non-mercury preservative. Volume: 15 mL/bottle Storage: Refrigerate at 2–8°C Stability: 12 months or as indicated on label 3. Aldosterone Calibrators- Ready to Use Contents: Six vials containing aldosterone in a protein based buffer with a non-mercury preservative. Prepared by spiking buffer with a defined quantity of aldosterone. Calibrator concentrations*: 0, 15, 50, 200, 500 and 1000 pg/mL. * Approximate values - please refer to vial labels for exact concentrations. Volume: Calibrators A–F: 1 mL/vial Storage: Refrigerate at 2–8°C Stability: 12 months in unopened vials or as indicated on label 4. Aldosterone Controls- Ready to Use Contents: Two vials containing aldosterone in a protein based buffer with a non-mercury preservative. Prepared by spiking buffer with defined quantities of aldosterone. Refer to vial labels for acceptable ranges. Volume: 1 mL/vial Storage: Refrigerate at 2–8°C Stability: 12 months in unopened vials or as indicated on label 5. Wash Buffer Concentrate- Requires Preparation (X10) Contents: One bottle containing buffer with a non-ionic detergent and a non-mercury preservative. Volume: 50 mL/bottle Storage: Refrigerate at 2–8°C Stability: 12 months or as indicated on label Preparation: Dilute the wash buffer concentrate 1:10 in distilled or deionized water to prepare the working wash buffer. If one whole plate is to be used, dilute 50 mL of the wash buffer concentrate in 450 mL of water. 6. TMB Substrate- Ready to Use Contents: One bottle containing tetramethylbenzidine and hydrogen peroxide in buffer. Volume: 16 mL/bottle Storage: Refrigerate at 2–8°C Stability: 12 months or as indicated on label

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7. Stop Solution- Ready to Use Contents: One bottle containing 1M sulfuric acid. Volume: 6 mL/bottle Storage: Refrigerate at 2–8°C Stability: 12 months or as indicated on label Assay Procedure All reagents must reach room temperature before use. Calibrators, controls and samples should be assayed in duplicate. Once the procedure has been started, all steps should be completed without interruption. 1. After all kit components have reached room temperature, mix gently by inversion. If serum or plasma samples are being used there is no sample preparation required. If urine samples are being used, they must be diluted prior to use (see Sample Pretreatment section). Prepare the working wash buffer (see wash buffer concentrate under the Reagents Provided section). 2. Remove the required number of strips from the microplate and assemble into a plate frame. Reseal the bag and return any unused strips to the refrigerator. 3. Pipette 50 µL of each calibrator, control and sample (serum, plasma or diluted urine) into correspondingly labelled wells in duplicate. 4. Pipette 100 μL of the aldosterone-HRP conjugate into each well (the use of a multichannel pipette is recommended). 5. Incubate on a plate shaker (~200 rpm on a linear shaker or ~600 rpm on an orbital shaker) for 60 minutes at room temperature. 6. Wash the wells 3 times each time with 350 µL/well of working wash buffer solution. After washing tap the plate firmly against absorbent paper to remove any residual liquid (the use of an automatic plate washer is strongly recommended). The performance of this assay is markedly influenced by the correct execution of the washing procedure. 7. Pipette 150 µL of the TMB substrate into each well at timed intervals (the use of a multichannel pipette is recommended). 8. Incubate on a plate shaker (~200 rpm on a linear shaker or ~600 rpm on an orbital shaker) for 20 minutes at room temperature or until calibrator A attains dark blue color for desired OD. 9. Pipette 50 µL of stopping solution into each well at the same timed intervals as in step 7 (the use of a multichannel pipette is recommended). Mix thoroughly by gently shaking the plate by hand. 10. Measure the absorbance at 450 nm in all wells with a microplate reader, within 20 minutes after addition of the stopping solution. Calculations 1. Calculate the mean optical density of each calibrator duplicate. 2. Draw a calibrator curve on semi-log paper with the mean optical densities on the Y-axis and the calibrator concentrations on the X-axis. If immunoassay software is being used, a 4-parameter or 5-parameter curve is recommended. 3. Calculate the mean optical density of each unknown duplicate. 4. Read the values of the serum and plasma samples directly off the calibrator curve.

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5. Read the values of the urine samples directly off the curve and multiply by a factor of 50. Next, multiply by the volume of collected 24-hour urine (in mL) to obtain values in pg/24 hour. Finally, divide the pg/24 hour values by 1,000,000 to obtain values in μg/24 hour. 6. If a serum or plasma sample reads greater than 1000 pg/mL then dilute it with the Serum and Plasma Diluent (available separately) at a dilution of no more than 1:8. The result obtained must be multiplied by the dilution factor. If a urine sample reads more than 1000 pg/mL then dilute it with the urine diluent at a dilution of no more than 1:8 (from the original 1:50 dilution). The result obtained must be multiplied by the dilution factor. Typical Tabulated Data Sample data only. Do not use to calculate results. Calibrator

Mean OD (450 nm)

Aldosterone (pg/mL)

A

1.804

0

B

1.63

15

C

1.43

50

D

1.05

200

E

0.71

500

F

0.51

1000

Unknown

1.11

162

Typical Calibrator Curve Sample curve only. Do not use to calculate results.

Aldosterone (pg/mL)

Performance Characteristics Sensitivity The limit of detection (LoD) was determined from the analysis of 60 samples of the blank and a low value sample and it was calculated as follows: LoD = µB + 1.645σB + 1.645σS, where σB and σS are the standard deviation of the blank and low value sample and µB is the mean value of the blank.

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The Limit of Detection (LoD) was determined to be 9.1 pg/mL.

Specificity (Cross Reactivity) The following compounds were tested for cross-reactivity with aldosterone cross-reacting at 100%: Steroid

% Cross-Reactivity

Aldosterone

100

Androsterone

0.01

Cortisol

0.01

Dihydrotestosterone

0.01

11-Deoxycorticosterone

0.075

Testosterone

0.009

The following compounds were tested and cross-reacted at less than 0.001%: Caffeine, Cholesterol, Cortisone and DHEAS. Interference The following substances were tested and did not show significant interference in the assay: hemoglobin up to 4 g/L, bilirubin conjugated and free up to 125 mg/L and triglycerides up to 30 mg/mL. Recovery Spiked samples were prepared by adding defined amounts of aldosterone to three serum and urine samples. The results are tabulated below: Serum Sample

Observed Result (pg/mL)

Expected Result (pg/mL)

Recovery %

1 + 100 pg/mL + 200 pg/mL + 400 pg/mL

45.8 146.5 232.5 376.1

145.8 245.8 445.8

100.5 94.6 84.4

2 + 100 pg/mL + 200 pg/mL + 400 pg/mL

77.8 203.5 302.4 444.2

177.8 277.8 477.8

114.5 108.9 93.0

3 + 100 pg/mL + 200 pg/mL + 400 pg/mL

82.7 183.1 260.9 401.3

182.7 282.7 482.7

100.2 92.3 83.1

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Urine Sample

Observed Result (pg/mL)

Expected Result (pg/mL)

Recovery %

1 + 100 pg/mL + 200 pg/mL + 400 pg/mL

32.6 151.9 261.0 374.6

132.6 232.6 432.6

114.6 112.2 86.6

2 + 100 pg/mL + 200 pg/mL + 400 pg/mL

59.7 185.0 297.3 436.0

159.7 259.7 459.7

115.8 114.5 94.8

3 + 100 pg/mL + 200 pg/mL + 400 pg/mL

73.6 190.5 316.6 449.8

173.6 273.6 473.6

109.7 115.7 95.0

Linearity Serum samples were diluted with Serum and Plasma Diluent. Urine samples were diluted with Urine Diluent after an initial dilution of 1:10 in Urine Diluent. The results are tabulated below: Serum Sample

Observed Result (pg/mL)

Expected Result (pg/mL)

Recovery %

1 1:2 1:4 1:8

241.8 127.8 65.0 31.6

120.9 60.5 30.2

105.7 107.6 104.5

2 1:2 1:4 1:8

840.8 456.7 217.1 125.0

420.4 210.2 105.1

108.6 103.3 118.9

3 1:2 1:4 1:8

1152 624.3 282.3 123.2

575.9 287.9 144.0

108.4 98.1 85.6

Observed Result (pg/mL)

Expected Result (pg/mL)

Recovery %

1 1:2 1:4 1:8

320.1 178.7 92.8 34.1

160.1 80.0 40.0

111.7 116.0 85.1

2 1:2 1:4 1:8

442.3 231.9 126.5 48.5

221.1 110.6 55.3

104.9 114.9 87.8

3 1:2 1:4 1:8

572.2 290.2 149.4 65.2

286.1 143.0 71.5

101.4 104.4 91.1

Urine Sample

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Intra-Assay Precision Four serum and four urine samples were assayed 24 times each on the same calibrator curve. The results are tabulated below: Serum Sample

Mean (pg/mL)

SD (pg/mL)

CV %

1

81.2

7.6

9.4

2

284.5

25.9

9.1

3

403.0

22.2

5.5

4

529.5

36.5

6.9

Urine Sample

Mean (pg/mL)

SD (pg/mL)

CV %

1

41.2

5.1

12.5

2

335.8

24.9

7.4

3

604.7

36.4

6.0

4

865.8

61.4

7.1

Inter-Assay Precision Five serum samples were assayed in 20 different tests in the span of at least ten days. The results are tabulated below: Sample

Mean (pg/mL)

SD(pg/mL)

CV %

1

80.8

10.4

12.8

2

209.0

22.5

10.7

3

454.5

51.9

11.4

4

677.3

79.1

11.7

5

902.3

68.4

7.6

Comparative Studies The compared with a leading competitor ELISA kit (x). The comparison of 42 serum samples yielded the following linear regression results: y = 0.84x + 3.50, r = 0.96 References 1. Varsano-Aharon N, Ulick S. Further Simplifications in the Immunoassay of Plasma Aldosterone. J Clin Endocrinol Metab. 1974; 39(2):375–9.Tel: (519) 681-8731. 2. Himathongkam T, et al. Potassium-Aldosterone-Renin Interrelationships. J Clin Endocrinol Metab. 1975; 41(1): 153–9. 3. Lun S, et al. A Direct Radioimmunoassay for Aldosterone in Plasma. Clin Chem. 1983; 29(2):268–71. 4. Cartledge S, Lawson N. Aldosterone and Renin Measurements. Ann Clin Biochem. 2000; 37:262–78.

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5. Sequeira SJ, et al. Evaluation of an Aldosterone Radio-immunoassay: The ReninAngiotensin-Aldosterone Axis as a function of Sex and Age. Ann Clin Biochem. 1986; 23:65–75. 6. Stabler TV, Siegel AL. Chemiluminescence Immunoassay of Aldosterone in Serum. Clin Chem. 1991; 37(11):1987–9. 7. Miller MA, et al. Extraction Method and Non-extracted Kit Comparison for Measuring Plasma Aldosterone. Clin Chem. 1997; 43(10):1995–7. 8. Vallotton MB. Primary Aldosteronism. Part 1. Diagnosis of Hyperaldosteronism. Clin Endocrinol. 1996; 45:47–52. 9. Oelkers W, et al. Diagnosis, Therapy Surveillance in Addison’s Disease: Rapid Adrenocorticotrophin (ACTH) Test and Measurement of Plasma ACTH, Renin Activity and Aldosterone. J Clin Endocrinol Metab. 1992; 75:259–64. 10. Ad-Dujaili EA, Edwards CR. Optimization of a Direct Radioimmunoassay for Plasma Aldosterone. J Steroid Biochem. 1981; 14(5):481–7. 11. Corry DB, Tuck ML. Secondary Aldosteronism. Endocrinol Metab Clin North Am. 1995; 24:511–28. 12. Check JH, et al. Falsely Elevated Steroidal Assay Levels Related to Heterophile Antibodies Against Various Animal Species. Gynecol Obstet Invest. 1995; 40(2):139–40.

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