Instructions for use

Active Renin ELISAMS E-5300R

Active Renin ELISA
1. INTRODUCTION
1.1 Intended Use
The Renin ELISA is an enzyme immunoassay for the quantitative measurement of active Renin in humanserum and EDTA plasma.
2. PRINCIPLE OF THE TESTThe Renin ELISA is a solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich
principle.
The microtiter wells are coated with a monoclonal [mouse] antibody directed towards a unique antigenic siteof the human active Renin molecule. An aliquot of sample containing endogenous Renin is incubated in thecoated well together with Assay Buffer. After incubation, unbound components are washed off. Finally,
Enzyme Conjugate, which is a monoclonal anti-Renin antibody conjugated with horseradish peroxidase, isadded, and after incubation, unbound enzyme conjugate is washed off.
The amount of bound peroxidase is proportional to the concentration of Renin in the sample.
Having added the substrate solution, the intensity of color developed is proportional to the concentration ofactive Renin in the sample.
3. WARNINGS AND PRECAUTIONS
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This kit is for research 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 to 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 to 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 microtiter plate 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.
Chemicals and prepared or used reagents have to be treated as hazardous waste according to thenational biohazard safety guideline or regulation.

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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 the manufacturer.
4. REAGENTS
4.1 Reagents provided
MS E-5331
Content:

Microtiterwells
12 x 8 (break apart) strips, 96 wells;
Wells coated with anti-human Renin antibody (monoclonal).

Standards and Controls - lyophilized
Cat. no.

Component

Concentration

Volume/Vial

0 pg/ml

MS E-5301

1 ml

MS E-5302

4 pg/ml

1 ml

MS E-5303

16 pg/ml

1 ml

MS E-5304

32 pg/ml

1 ml

MS E-5305

64 pg/ml

1 ml

MS E-5306

128 pg/ml

1 ml

MS E-5351

For control values and ranges
please refer to vial label or QCReport!

MS E-5352
Conversion:

1 pg/ml = 1.44 µlU/ml

Content:

Contain non-mercury preservative.

1 ml
1 ml

The standards are calibrated against WHO 1st International Standard for Renin 68/356
See “Reagent Preparation”
MS E-5313

Assay Buffer - Ready to use

Content:

Contains non-mercury preservative.

Volume:1 x 20 ml

MS E-5340

Enzyme Conjugate - Ready to use

Content:anti-human Renin antibody (monoclonal); HRP conjugated.
Contains non-mercury preservative.

Volume:1 x 14 ml

FR E-0055

Substrate Solution - Ready to use

Content:

Tetramethylbenzidine (TMB).

Volume:1 x 14 ml
Stop Solution - Ready to use

FR E-0080
Content:

contains 0.5 M H2SO4
Avoid contact with the stop solution. It may cause skin

Volume:1 x 14 ml

Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
FR E-0030
Volume:Wash Solution - 40x concentrated
1 x 30 ml

See “Preparation of Reagents”
Note: Additional Assay Buffer for sample dilution is available upon request.

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4.2 Materials required but not provided
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A microtiter plate calibrated reader (450 nm)
Calibrated variable precision micropipettes
Absorbent paper
Distilled or deionized water
Timer
Linear graph paper or software for data reduction
Microplate shaker (300 - 700 rpm)

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 8 weeks 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 vial with 1.0 ml distilled water and let stand for
10 minutes in minimum. Mix the standards several times before use.
Note: The reconstituted standards are stable for 14 days at 2 °C - 8 °C. For longer storage (up to 12
months) aliquot and freeze at -20 °C.
Controls
Reconstitute the lyophilized content with 1.0 ml distilled water and let stand for 10 minutes in minimum. Mix
the controls several times before use.
Note: The reconstituted controls are stable for 14 days at 2 °C - 8 °C. For longer storage (up to 12 months)
aliquot and freeze 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 EDTA plasma can be used in this assay.
Do not use hemolytic, icteric or lipaemic specimens.
Please note: Samples containing sodium azide should not be used in the assay.
Conditions under which samples are collected must be carefully controlled, since a number of physiologicalfactors can influence the renin secretion. These include:- Posture: the donor must have been lying down for more than 1 hour or upright for more than 1 hour
- Daily Renin oscillations: sampling is to be done between 7 AM and 10 AM if possible.
- Diet: sodium content in the diet must be known and eventually verified by the measurement of natriuria
over a period of 24 hours- Medication: the level of active renin can be affected by antihypertensive medication (e.g. diuretics, ACE
inhibitors, beta adrenergic blocking agents, vasodilators, renin inhibitors)- Pregnancy: the level of inactive and active renin increases during pregnancy- Menstrual cycle: the level of active renin increases on the second phase of the cycle (sampling is to bedone if possible during the first phase)- Age: active renin level decreases with age
NOTE:
Samples from tumor donors may contain elevated levels of Renin.
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5.1 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. Donors
receiving anticoagulant therapy may require increased clotting time.
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.
5.2 Specimen Storage and Preparation
Specimens should be capped and stored at room temperature and NOT stored at 2 °C - 8 °C prior toprocessing, since cryoactivation of prorenin may occur in the temperature range of 2 °C - 8 °C, giving false
positive active renin values (12,13).
If samples cannot be tested within 4 hours of primary collection, store frozen (up to 12 months) at -20 °C or
below.
It is recommended to rapidly freeze and thaw processed samples avoiding the temperature range of
2 °C - 8 °C.
A dry ice/ethanol bath can be used for rapid freezing procedures.
5.3 Specimen Dilution
If in an initial assay, a specimen is found to contain more analyte than the highest standard, the specimen
can be diluted with Assay Buffer and reassayed as described in the assay procedure.
For calculation of concentrations this dilution factor has to be taken into account.
If a dilution is required, the specimen must be diluted at least 1:10 with Assay Buffer.
Example:a) dilution 1:10:b) dilution 1:20:

20 µl sample + 180 µl Assay Buffer (mix thoroughly)
10 µl sample + 190 µl Assay Buffer (mix thoroughly).

6. ASSAY PROCEDURE
6.1 General Remarks
1. All reagents and specimens must be allowed to come to room temperature before use. All reagents must
be mixed without foaming.
2. Once the test has been started, all steps should be completed without interruption.
3. Use new disposal plastic pipette tips for each standard, control or sample in order to avoid crosscontamination.
4. 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.
5. As a general rule the enzymatic reaction is linearly proportional to time and temperature.

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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 150 µl of Assay Buffer in all wells.

3.

Dispense 50 µl of each Standard, Control and samples with new disposable tips into appropriate
wells.

4.

Incubate for 90 minutes at room temperature on a plate shaker with 300 - 700 rpm.

5.

Briskly shake out the contents of the wells.
Rinse the wells 4 times with 300 µl diluted Wash Solution. 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!

6.

Dispense 100 µl Enzyme Conjugate in all wells.

7.

Incubate for 90 minutes at room temperature on a plate shaker with 300 - 700 rpm.

8.

Briskly shake out the contents of the wells.
Rinse the wells 4 times with 300 µl diluted Wash Solution. Strike the wells sharply on absorbent
paper to remove residual droplets.

9.

Add 100 µl of Substrate Solution to each well.

10.

Incubate for 15 minutes at room temperature.

11.

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

12.

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 linear graph paper, construct a standard curve by plotting the mean absorbance obtained fromeach standard against its concentration with absorbance value on the vertical (Y) axis and concentrationon 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 samples can be read directly from this standard curve. Samples withconcentrations higher than that of the highest standard have to be further diluted or reported as> 128 pg/ml. For the calculation of the concentrations this dilution factor has to be taken into account.
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

Optical Units (450 nm)
0 pg/ml

0.09

Standard B

4 pg/ml

0.19

Standard C

16 pg/ml

0.44Standard D

32 pg/ml

0.78

Standard E

64 pg/ml

1.14Standard F

128 pg/ml

2.48

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7. EXPECTED NORMAL VALUES
It is strongly recommended that each laboratory should determine its own normal and abnormal values.
In a study conducted with apparently normal healthy adults, using the Renin ELISA the following values areobserved in EDTA plasma:
Healthy AdultsnSupine position
Upright position

59
59

Mean
Median(pg/ml) (pg/ml)
16.23
12.40
19.73
16.18

2.5th - 97.5th Percentile(pg/ml)2.14 - 53.83
2.79 - 61.83

Range (min - max)
(pg/ml)2.13 - 58.78
1.63 - 95.56

These values are also valid for serum.
In a study conducted with apparently normal healthy adults, using Aldosterone ELISA and Renin ELISA thefollowing Aldosterone-Renin Ratio were determined in EDTA 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

These values are also valid for serum.
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 sample 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.
9. PERFORMANCE CHARACTERISTICS
9.1 Assay Dynamic Range
The range of the assay is between 0.80 – 128 pg/ml.
9.2 Specificity of Antibodies (Cross-Reactivity)
The following substances were tested for cross-reactivity of the assay:
Mean cross reactivity with Prorenin was 0.71% (mean value when prorenin was spiked in a concentration
range from 256 – 4096 pg/ml). However, the observed cross reactivity may only represent a contaminationof the recombinant prorenin preparation with active renin due to auto-activation.
Cross-reactivity was not detectable against human serum albumin, human gamma globulin, human hepcidin,
and pepsin.
9.3 Sensitivity
The analytical sensitivity of the ELISA was calculated by adding 2 standard deviations to the mean of 20
replicate analyses of the Standard A and was found to be 0.80 pg/ml.
The Limit of Blank (LoB) is 2.50 pg/ml.
The Limit of Detection (LoD) is 4.31 pg/ml.
The Limit of Quantification (LoQ) is 6.02 pg/ml.

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

1

2

3

4

5

6

106.03

71.01

48.52

30.13

20.27

10.62

CV (%)

1.5

1.5

2.0

2.4

1.9

4.8

n=

40

40

40

40

40

40

9.4.2 Inter Assay
The between assay variability is shown below:
Sample

1

2

3

4

5

6

106.03

71.01

48.52

30.13

20.27

10.62

CV (%)

2.5

2.1

2.8

3.6

4.1

6.9

n=

80

80

80

80

80

80

Mean (pg/ml)

9.4.3 Inter Lot
The inter-assay (between-lots) variation was determined by measuring each sample 6 times with 3 different
kit lots (n = 18):
SampleMean (pg/ml)

1

2

3

4

5

6

101.72

71.46

49.32

27.65

18.98

10.09

CV (%)

1.2

1.7

2.0

1.2

1.5

5.5

n=

18

18

18

18

18

18

9.5 Recovery
Samples have been spiked by adding Renin solutions with known concentrations.
The recovery (%) was calculated by multiplying the ratio of measured and expected values with 100.
1

2

3

4

Concentration (pg/ml)

8.93

15.97

55.27

100.61

Average Recovery

100.2

96.0

108.4

99.0

Range of Recovery (%)

from

95.5

86.8

106.2

97.3

to

103.3

105.3

111.9

102.8

9.6 Linearity
Samples were measured undiluted and in serial dilutions. The recovery (%) was calculated by multiplying
the ratio of expected and measured values with 100.

Concentration (pg/ml)
Average Recovery
Range of Recovery (%)

1

2

3

4

45.16

53.20

67.35

126.0

101.7

102.8

110.0

98.5

from

96.7

95.6

105.0

94.9

to

108.6

114.6

114.6

100.8

10 LIMITATIONS 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
Haemoglobin (up to 4 mg/ml), Bilirubin (up to 0.5 mg/ml) and Triglyceride (up to 30 mg/ml) have noinfluence on the assay results.
10.2 Drug Interferences
The renin inhibitor aliskiren will increase active renin immunoreactivity in a dose-dependant manner,from 0.54 µM (+ 121%) up to 540 µM (+151%).
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In addition, the level of active renin in plasma may be affected by antihypertensive medication (e.g.
diuretics, ACE inhibitors, beta adrenergic blocking agents, or vasodilators).
10.3 High-Dose-Hook EffectNo hook effect was observed in this test up to 8,200 pg/mL of Renin.
11 REFERENCES / LITERATURE

1.

Imai T, Miyazaki H, Hirose S, et al. Cloning and sequence analysis of cDNA for human renin precursor.
Proc. Natl. Acad. Sci. (1983) 80, 7405–7409.
2. Reudelhuber TL, Ramla D, Chiu L, et al. Proteolytic processing of human prorenin in renal and non-renal
tissues. Kidney Int. (1994) 46, 1522–1524.
3. Neves FA, Duncan KG, Baxter JD. Cathepsin B is a prorenin processingenzyme. Hypertension (1996) 27,
514 –517.
4. Müller DN, Luft FC. Direct renin inhibition with aliskiren in hypertension and target organ damage.
Clin J Am Soc Nephrol. (2006) 1, 221-8.
5. Toffelmire EB, Slater K, Corvol P, et al. Response of plasma prorenin and active renin to chronic and acutealterations of renin secretion in normal humans. Studies using a direct immunoradiometric assay. J Clin
Invest. (1989) 83, 679–687.
6. Carey RM, Padia SH. Angiotensin AT2 receptors: control of renal sodium excretion and blood pressure.
Trends Endocrinol Metab. (2008) 19, 84-7.
7. Koeppen BM, Stanton BA. Renal Physiology (4th ed.). Philadelphia, PA. Mosby Physiology Monograph
Series, 2007.
8. Navar LG, Inscho EW, Majid DSA, et al. Paracrine regulation of the renal microcirculation.
Physiol. Rev. (1996) 76, 425–536.
9. Müller MW, Todorov V, Krämer BK, Kurtz A. Angiotensin II inhibits renin gene transcription via the protein
kinase C pathway. Pflugers Arch. (2002) 444, 499-505.
10. Spät A, Hunyady L. Control of aldosterone secretion: a model for convergence in cellular signaling
pathways. Physiol Rev. (2004) 84, 489-539.
11. Nguyen G., Delarue F., Burcklé C., et al. Pivotal role of the renin/prorenin receptor in angiotensin II
production and cellular responses to renin. J Clin Invest. (2002) 109, 1417–1427.
12. Pitarresi TM., Rubattu S, Heinrikson R, Sealey JE. Reversible cryoactivation of recombinant humanprorenin. J.Biol.Chem. (1992) 267, 11753-9.
13. Nicar MJ. Specimen processing and renin activity in plasma. Clin. Chem. (1992) 38, 598.

Symbols:

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For research use
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