Instructions for use

Aldosterone ELISAMS E-5200R

Aldosterone ELISA
1. Introduction
1.1 Intended use
The Aldosterone ELISA is an enzyme immunoassay for the measurement of aldosterone in serum, plasma(EDTA-, heparin- or citrate plasma) and urine.
1.2 Summary and Explanation
The steroid hormone aldosterone is a potent mineral corticoid that is produced by the zona glomerulosa of
the adrenal cortex in the adrenal gland. The synthesis and release are controlled by the renin-angiotensinaldosterone system (RAAS)1, as well as by plasma potassium concentration 2, the pituitary peptide ACTH,
and by the blood pressure via pressure sensitive baroreceptors in the vessel walls of nearly all large arteriesof the body3. Aldosterone binds to mineralocorticoid receptors (MR) and triggers the transcription of
hormone-responsive genes.
2. PRINCIPLE OF THE TESTThe Aldosterone ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA), based on theprinciple of competitive binding.
The microtiter wells are coated with a polyclonal rabbit antibody directed towards an antigenic site of thealdosterone molecule. Endogenous aldosterone of a sample competes with an aldosterone-horseradish
peroxidase conjugate for binding to the coated antibody. After incubation the unbound conjugate is washedoff.
After addition of the substrate solution, the intensity of colour developed is inversely proportional to theconcentration of aldosterone in the sample.
3. WARNINGS AND PRECAUTIONS
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.

This kit is for research use only. For professional use only.
All reagents of this test kit which contain human serum or plasma have been tested and confirmed
negative for HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should betreated as potential biohazards in use and for disposal.
Before starting the assay, read the instructions completely and carefully. Use the valid version of
instructions for use provided with the kit. Be sure that everything is understood.
The microplate contains snap-off strips. Unused wells must be stored at 2 °C - 8 °C in the sealed foil
pouch and used in the frame provided.
Pipetting of samples and reagents must be done as quickly as possible and in the same sequence foreach step.
Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using areservoir for dispensing a substrate solution that had previously been used for the conjugate solution
may turn solution colored. Do not pour reagents back into vials as reagent contamination may occur.
Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reusemicrowells.
Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
Allow the reagents to reach room temperature (21 °C - 26 °C) before starting the test. Temperature
will affect the absorbance readings of the assay. However, values for the samples will not be affected.
Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
Wear disposable latex gloves when handling specimens and reagents. Microbial contamination ofreagents or specimens may give false results.
Handling should be done in accordance with the procedures defined by an appropriate national
biohazard safety guideline or regulation.
Do not use reagents beyond expiry date as shown on the kit labels.
All indicated volumes have to be performed according to the protocol. Optimal test results are only
obtained when using calibrated pipettes and microtiterplate readers.
Do not mix or use components from kits with different lot numbers. It is advised not to exchange wellsof different plates even of the same lot. The kits may have been shipped or stored under different
conditions and the binding characteristics of the plates may result slightly different.
Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns.
Some reagents contain Proclin 300, BND and/or MIT as preservatives. In case of contact with eyes orskin, flush immediately with water.
TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with anabundant volume of water and skin with soap and abundant water. Wash contaminated objects before
reusing them. If inhaled, take the person to open air.

Version: 11.0-r

Effective: 2019-03-05

2/10

20. Chemicals and prepared or used reagents have to be treated as hazardous waste according to thenational biohazard safety guideline or regulation.
21. For information on hazardous substances included in the kit please refer to Safety Data Sheets.
Safety Data Sheets for this product are available upon request directly from manufacturer.
4. REAGENTS
4.1 Reagents provided
Microtiterwells

MS E-5231
Contents:

12 x 8 (break apart) strips; 96 wells; Wells coated with anti-aldosterone antibody(polyclonal rabbit)

Standards and Controls - lyophilized
Concentration

Volume/Vial

MS E-5201

0 pg/ml

1.0 mlMS E-5202

20 pg/ml

1.0 mlMS E-5203

80 pg/ml

1.0 mlMS E-5204

Cat. no.

Component

200 pg/ml

1.0 mlMS E-5205

500 pg/ml

1.0 mlMS E-5206

1000 pg/ml

1.0 mlMS E-5251

For control values andranges please refer to viallabel or QC-Report.

MS E-5252
Conversion:

1.0 ml
1.0 ml1 pg/ml corresponds to 2.77 pmol/lContents:
Contain non-mercury preservative.
see “Reagents Preparation”
MS E-5240
Contents:
Volume:Enzyme Conjugate - Ready to use
Aldosterone conjugated to horseradish peroxidase,
Contain non-mercury preservative.
1 x 20 ml
Substrate Solution - Ready to use

SA E-0055
Contents:

Tetramethylbenzidine (TMB).

Volume:1 x 25 ml

FR E-0080

Stop Solution - Ready to use
contains 0.5 M H2SO4
Avoid contact with the stop solution. It may cause skin irritations and burns.
1 x 14 ml

Contents:
Volume:Hazards
identification:

H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
Wash Solution - 40x concentrated

FR E-0030
Volume:1 x 30 ml

See “Preparation of Reagents”.
Note: Additional Standard A for sample dilution is available upon request.
4.2 Materials required but not provided







A microtiter plate calibrated reader (450 ± 10 nm)
Calibrated variable precision micropipettes.
Absorbent paper.
Distilled or deionized water
Timer
Scale paper or semi-logarithmic graph paper or software for data reduction

Version: 11.0-r

Effective: 2019-03-05

3/10





Optional: Reagents for determination of Aldosterone in urine (REF MS U-5200) – Contents:
1) Release Reagent, 1 vial, 3 ml, ready to use. Containing 1M HCl.
Avoid contact with Release Reagent. It may cause skin irritation.
2) Neutralization Buffer, 1 vial, 3 ml, ready to use. Containing Tris buffer, pH 8.5.
3) Dilution Buffer, 2 vials, 25 ml each, ready to use. Containing PBS.
Optional: Plastic tubes (e.g. 0.5 - 1.5 ml) for pre-treatment of urine samples

4.3 Storage Conditions
When stored at 2 °C to 8 °C unopened reagents will retain reactivity until expiration date. Do not use
reagents beyond this date.
Opened reagents must be stored at 2 °C to 8 °C. Microtiter wells must be stored at 2 °C to 8 °C. Once thefoil bag has been opened, care should be taken to close it tightly again.
Opened kits retain activity for two months if stored as described above.
4.4 Reagent Preparation
Bring all reagents and required number of strips to room temperature prior to use.
Standards
Reconstitute the lyophilized contents of the standard vials with 1.0 ml deionized water and let stand for atleast 10 minutes. Mix several times before use.
Note: The reconstituted standards are stable for 8 weeks at 2 °C to 8 °C.
For longer storage freeze - only once - at -20 °C.
Controls
Reconstitute the lyophilized content of the controls with 1.0 ml deionized water and let stand for at least
10 minutes. Mix several times before use.
Note: The reconstituted controls are stable for 8 weeks at 2 °C to 8 °C.
For longer storage freeze - only once - at -20 °C.
Wash Solution
Add deionized water to the 40X concentrated Wash Solution.
Dilute 30 ml of concentrated Wash Solution with 1170 ml deionized water to a final volume of 1200 ml.
The diluted Wash Solution is stable for 2 weeks at room temperature.
4.5 Disposal of the Kit
The disposal of the kit must be made according to the national regulations. Special information for thisproduct is given in the Safety Data Sheet.
4.6 Damaged Test Kits
In case of any severe damage to the test kit or components, the manufacturer has to be informed in writing,at the latest, one week after receiving the kit. Severely damaged single components should not be used fora test run. They have to be stored until a final solution has been found. After this, they should be disposed
according to the official regulations.
5. SPECIMEN COLLECTION AND PREPARATION
Serum or plasma (EDTA-, heparin- or citrate plasma) and urine can be used in this assay.
Do not use haemolytic, icteric or lipaemic specimens.
Please note: Samples containing sodium azide should not be used in the assay.
5.1 Serum / Plasma Samples
5.1.1 Specimen Collection
Serum:
Collect blood by venipuncture (e.g. Sarstedt Monovette for serum), allow to clot, and separate serum bycentrifugation at room temperature. Do not centrifuge before complete clotting has occurred.
Plasma:Whole blood should be collected into centrifuge tubes containing anti-coagulant (e.g. Sarstedt Monovettewith the appropriate plasma preparation) and centrifuged immediately after collection.

Version: 11.0-r

Effective: 2019-03-05

4/10

5.1.2 Specimen Storage and Preparation
Specimens should be capped and may be stored for up to 5 days at 2 °C to 8 °C prior to assaying.
Specimens held for a longer time (up to two months) should be frozen only once at -20 °C prior to assay.
Thawed samples should be inverted several times prior to testing.
5.1.3 Specimen Dilution
If in an initial assay, a specimen is found to contain more than the highest standard, the specimens can be
diluted with Standard A and reassayed as described in Assay Procedure.
For the calculation of the concentrations this dilution factor has to be taken into account.
Example:a) dilution 1:10: 10 µl sample + 90 µl Standard A (mix thoroughly)b) dilution 1:100: 10 µl dilution a) 1:10 + 90 µl Standard A (mix thoroughly).
5.2 Urine Samples
Aldosterone concentration can also be determined from urine samples. However, urine samples must be pretreated before analysis. This will need additional reagents that are not included in this kit, but can beordered separately (MS U-5200).
5.2.1 Sample Collection
First clean genital area with mild disinfectant to prevent contamination. Then collect clean-catch midstream
urine in an appropriate sterile container. Directly after collection, the urine should be centrifuged for
5 - 10 minutes (e.g. at 2,000 g) to remove cellular debris. Use supernatant for analyte quantification. The
supernatant may be stored for up to 8 hours at 2 °C to 8 °C prior to assaying. Specimens held for a longer
time should be frozen at -20 °C. Thawed supernatant should be inverted several times prior to testing.
5.2.2 Protocol for Urine Sample Pre-treatment
1. Secure the desired number of vials (e.g. 0.5 - 1.5 ml plastic tubes; not included in this kit).
2. Dispense 25 µl of urine with new disposable tips into appropriate tubes.
3. Dispense 25 µl Release Reagent into each tube
Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.
4. Incubate over night at 2 °C to 8 °C.
5. Add 25 µl Neutralization Reagent to each tube and mix thoroughly.
6. Add 400 µl Dilution Buffer to each tube and mix thoroughly(This pre-treatment leads to a 1:19 dilution. Therefore the dilution factor 19 has to be taken into accountfor calculation of the final concentration of the urine sample.)
7. Transfer 50 µl of pre-treated and diluted urine samples directly to the microtiter well and continue
with step 3 of Test Procedure (Chapter 6.2).
5.2.3 Storage of pre-treated urine samples
Pre-treated and diluted urine samples should be capped and may be stored for up to 7 days at 2 °C to 8 °C
prior to assaying.
Samples held for a longer time (up to two months) should be frozen only once at -20 °C prior assay.
Thawed samples should be inverted several times prior to testing.
5.2.4 Specimen Dilution
If in an initial assay, an urine sample is found to contain more than the highest standard, the pre-treated
and diluted urine sample can be further diluted with Dilution Buffer and reassayed as described in Assay
Procedure.
For the calculation of the concentrations this dilution factor has to be taken into account too.
Example:a) dilution 1:10:

10 µl pre-treated and diluted urine sample + 90 µl Dilution Buffer (mix thoroughly)(final dilution factor = 19 x 10 = 190)

6. ASSAY PROCEDURE
6.1 General remarksAll reagents and specimens must be allowed to come to room temperature before use. All reagents must
be mixed without foaming.
Once the test has been started, all steps should be completed without interruption.

Version: 11.0-r

Effective: 2019-03-05

5/10





Use new disposal plastic pipette tips for each standard, control or sample in order to avoid crosscontamination.
Absorbance is a function of the incubation time and temperature. Before starting the assay, it isrecommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will
ensure equal elapsed time for each pipetting step without interruption.
As a general rule the enzymatic reaction is linearly proportional to time and temperature.

6.2 Test Procedure
Each run must include a standard curve.
1.

Secure the desired number of Microtiter wells in the frame holder.

2.

Dispense 50 µl of each Standard, Control and samples with new disposable tips into appropriate
wells.
For urine samples dispense 50 µl of the pre-treated and diluted urine samples (see chapter 5.2.2
Protocol for Urine Sample Pre-treatment, step 7).

3.

Incubate for 30 minutes at room temperature.

4.

Dispense 150 µl Enzyme Conjugate into each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.

5.

Incubate for 60 minutes at room temperature.

6.

Briskly shake out the contents of the wells. Rinse the wells
5 x with 400 µl diluted Wash Solution per well (if a plate washer is used) - or.
5 x with 300 µl/well for manual washing.
Strike the wells sharply on absorbent paper to remove residual droplets.
Important note:
The sensitivity and precision of this assay is markedly influenced by the correct performance of thewashing procedure!

7.

Add 200 µl of Substrate Solution to each well.

8.

Incubate for 30 minutes at room temperature.

9.

Stop the enzymatic reaction by adding 100 µl of Stop Solution to each well.

10.

Determine the absorbance (OD) of each well at 450 ± 10 nm with a microtiter plate reader.
It is recommended that the wells be read within 10 minutes after adding the Stop Solution.

6.3 Calculation of Results
1. Calculate the average absorbance values for each set of standards, controls and samples.
2. Using scale paper or semi-logarithmic graph paper, construct a standard curve by plotting the mean
absorbance obtained from each standard against its concentration with absorbance value on the vertical(Y) axis and concentration on the horizontal (X) axis.
3. Using the mean absorbance value for each sample determine the corresponding concentration from thestandard curve.
4. Automated method: The results in the Instructions for Use have been calculated automatically using a
4 Parameter curve fit. (4 Parameter Rodbard or 4 Parameter Marquardt are the preferred methods.)
Other data reduction functions may give slightly different results.
5. The concentration of the serum / plasma samples can be read directly from this standard curve.
For urine samples the concentration read from the standard curve, has to be multiplied with thedilution factor 19 (see chapter 5.2.2).
6. Samples with concentrations higher than that of the highest standard have to be further diluted orreported as > 1000 pg/ml. For the calculation of the concentrations this dilution factor has to be taken
into account too.
6.3.1 Example of Typical Standard Curve
The following data is for demonstration only and cannot be used in place of data generations at the time of
assay.
Standard
Standard A (0 pg/ml)
Standard B (20 pg/ml)
Standard C (80 pg/ml)
Standard D (200 pg/ml)
Standard E (500 pg/ml)
Standard F (1000 pg/ml)
Version: 11.0-r

Optical Units (450 nm)
2.11
1.90
1.55
1.15
0.76
0.54
Effective: 2019-03-05

6/10

6.4 Final Calculation for Urine Samples
Calculate the 24 hours excretion for each urine sample: µg/24 h = µg/l x l/24 h
Example:Concentration for urine sample read from the standard curve
Result after correction with the dilution factor 19
9500 pg/l / 1000 = 9.5 µg/l==

500 pg/ml
9500 pg/ml

Total volume of 24 h-urine = 1.3 l (example)
9.5 µg/l × 1.3 l/24 h=

12.35 µg/24 h

7. EXPECTED NORMAL VALUES
It is strongly recommended that each laboratory should determine its own normal and abnormal values.
7.1 Serum/Plasma
In a study conducted with EDTA plasma samples of apparently normal healthy adults, using the
Aldosterone ELISA the following values are observed:
Healthy AdultsnMean(pg/ml)Median(pg/ml)2.5th - 97.5th
percentile (pg/ml)

Range(min - max)
(pg/ml)Supine position

60

56.14

39.71

14.21 - 156.47

8.58 - 272.30

Upright position

60

77.48

58.00

13.37 - 233.55

12.87 - 358.50

These values are also valid for serum, heparin plasma and citrate plasma.
These results correspond well to published reference ranges 8,9.
In a study conducted with apparently normal healthy adults, using the Aldosterone ELISA and the Renin
ELISA the following Aldosterone-Renin Ratios were determined in plasma:
Ratio Aldosterone Renin (pg/ml / pg/ml)
Healthy Adults

n
89

Mean
8.68

Median5.30

2.5th - 97.5th Percentile
0.52 - 37.83

7.2 Urine Samples
In a study conducted with urine samples of apparently normal healthy adults, using the Aldosterone ELISA
the following values are observed:

Healthy AdultsnMean(µg/24h)Median(µg/24h)

2.5th - 97.5th
percentile (µg/24h)

Range (min - max)(µg/24h)

8

11.34

9.40

3.31 - 25.09

3.06 - 27.17

These results correspond well to published reference ranges 8.
8. QUALITY CONTROLGood laboratory practice requires that controls be run with each standard curve. A statistically significant
number of controls should be assayed to establish mean values and acceptable ranges to assure proper
performance.
It is recommended to use control samples according to state and federal regulations. The use of control
samples is advised to assure the day to day validity of results. Use controls at both normal and pathologicallevels.
The controls and the corresponding results of the QC-Laboratory are stated in the QC Report added to thekit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for
direct comparison of the results.
It is also recommended to make use of national or international Quality Assessment programs in order toensure the accuracy of the results.
Employ appropriate statistical methods for analysing control values and trends. If the results of the assay do
not fit to the established acceptable ranges of control materials results should be considered invalid.
In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration
dates of reagents, storage and incubation conditions, aspiration and washing methods.
After checking the above mentioned items without finding any error contact your distributor or the
manufacturer directly.

Version: 11.0-r

Effective: 2019-03-05

7/10

9. PERFORMANCE CHARACTERISTICS
9.1 Assay Dynamic Range
For serum and plasma the range of the assay is between 7.75 pg/ml - 1000 pg/ml.
For urine the range of the assay is between 9.81 pg/ml - 1000 pg/ml.
9.2 Specificity of Antibodies (Cross Reactivity)
The following substances were tested for cross reactivity of the assay:
Substance

Cross Reactivity (%)
17.2 %
0.12 %

3 β, 5 α Tetrahydroaldosterone
3 β, 5 β Tetrahydroaldosterone

The following steroids were tested but cross-reacted at less than 0.1%:
androstenedione, androsterone, corticosterone, cortisol, cortisone, 11-deoxycortisol, DHEA, estradiol, estriol,
estrone, 17-hydroxyprogesterone, prednisolone, prednisone, progesterone and testosterone.
9.3 Sensitivity
The analytical sensitivity of the ELISA was calculated by subtracting 2 standard deviations from the mean of
20 replicate analyses of the Standard A (Std. A) and was found to be < 5.7 pg/ml.
Serum / Plasma
7.75 pg/ml

Urine
9.81 pg/ml

The Limit of Detection (LoD)

12.07 pg/ml

16.94 pg/ml

The Limit of Quantification (LoQ)

14.78 pg/ml

23.66 pg/ml

The Limit of Blank (LoB)

9.4 Reproducibility
9.4.1 Intra Assay
The within assay variability is shown below:
SamplenMean (pg/ml)

CV (%)

Serum 1

40

61.93

4.63

Serum 240

247.50

2.02

Serum 340

560.33

1.80

Urine 1

40

79.12

5.91

Urine 240

229.994.85

Urine 340495.82

5.09

9.4.2 Inter Assay
The within assay variability is shown below:
SamplenMean (pg/ml)

CV (%)

1

80

61.93

10.06

2

80

547.50

5.07

3

80

560.33

4.65

Urine 1

80

79.12

13.92

Urine 280229.99

9.44

Urine 380495.82

9.65

Version: 11.0-r

Effective: 2019-03-05

8/10

9.5 Recovery
Samples have been spiked by adding aldosterone solutions with known concentrations in a 1:1 ratio.
The % recovery has been calculated by multiplication of the ratio of the measurements and the expectedvalues with 100 (expected value = (endogenous aldosterone + added aldosterone) / 2; because of a 1:2
dilution of serum with spike material).
Urine 1Urine 2Urine 3

585.3

70.59

232.55516.52

98.2

102.1

99.9

95.1

97.4

92.7

91.0

108.0

108.2

99.1

Urine 2Urine 3

Serum 1

Serum 2

Serum 3

Concentration [pg/ml]

94.6

211.6

Average Recovery

91.3

107.4

from

85.1

104.5

91.3

to

96.2

110.9

102.8

Serum 1

Serum 2

Serum 3Urine 1

Concentration [pg/ml]

296.3

406.0

631.2

569.8

655.5

683.7

Average Recovery

101.2

101.6

95.5

98.2

102.7

105.3

Range of Recovery [%]9.6 Linearity

Range of Recovery [%]

from

96.7

93.6

90.6

95.4

91.0

101.8

to

105.6

109.4

98.6

105.5

111.3

110.6

10. LIMITATION OF USE
Reliable and reproducible results will be obtained when the assay procedure is performed with a complete
understanding of the package insert instruction and with adherence to good laboratory practice.
Any improper handling of samples or modification of this test might influence the results.
10.1 Interfering Substances
The following substances have no influence on the assay results up to the below stated concentrations.
Serum

Urine

Concentration up to
Haemoglobin

6 mg/ml

Bilirubin

0.4 mg/ml

Triglyceride

20 mg/ml

Cholesterol

2.84 mg/ml

Total protein

120 mg/ml

Glucose10 mg/ml

Creatinine

0.05 mg/ml

5 mg/ml

10.2 Drug Interferences
Until today no substances (drugs) are known to us, which have an influence to the measurement ofaldosterone in a sample.
10.3 High-Dose-Hook Effect
A High-Dose-Hook Effect is not known for competitive assays.

Version: 11.0-r

Effective: 2019-03-05

9/10

11. REFERENCES / LITERATURE
1. Brown RD, Strott CA, and Liddle GW. Site of stimulation of Aldosterone biosynthesis by angiotensin andpotassium.
J Clin Invest. (1972), 51 (6), 1413–8.
2. Bauer JH, Gauntner WC. Effect of potassium chloride on plasma renin activity and plasma aldosterone
during sodium restriction in normal man.
Kidney Int. (1979), 15 (3): 286–93.
3. Williams GH, Dluhy RG. Aldosterone biosynthesis. Interrelationship of regulatory factors.
Am J Med. (1972), 53 (5), 595–605.
4. Tiu SC et al. The use of aldosterone-renin ratio as a diagnostic test for primary hyperaldosteronism andits test characteristics under different conditions of blood sampling.
J Clin Endocrinol Metab. (2005), 90 (1), 72-8.
5. Mulatero P et al. Confirmatory tests in the diagnosis of primary aldosteronism.
Horm Metab Res. (2010), 42 (6), 406-10.
6. Quillo AR. Primary aldosteronism: results of adrenalectomy for nonsingle adenoma.
J Am Coll Surg. (2011), 213 (1), 106-12.
7. Grossmann C and Gekle M. New aspects of rapid aldosterone signaling.
Mol Cell Endocrinology (2009), 308 (1-2), 53-62.
8. Thomas L (editor). Renin-Angiotensin-Aldosteron-System (RAAS).
Labor und Diagnose (2005); 1406-24.
9. Perschel FH et al. Rapid Screening test for primary hyperaldosteronism: ratio of plasma aldosterone torenin concentration determined by fully automated chemiluminescence immunoassays.
Clin. Chemistry (2004); 50 (9), 1650-55.

For updated literature or any other information please contact your local supplier.
Symbols:

Version: 11.0-r

Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContent

CautionCatalogue
numberEffective: 2019-03-05

Contains sufficient for<n> tests

For research use
only!

10/10