Instructions for use

Plasma Renin Activity (PRA) ELISAMS E-5600R

Plasma Renin Activity (PRA) ELISAINTENDED USE
For the quantitative determination of Plasma Renin Activity (PRA) in human plasma by an enzyme
immunoassay.
PRINCIPLE OF THE TESTThis kit measures PRA and the results are expressed in term of mass of angiotensin-I (Ang-I) generated pervolume of human plasma in unit time (ng/ml.h).
The blood sample is collected in a tube that contains EDTA. The plasma is separated and either stored frozen or
kept at room temperature for immediate use, samples should not be chilled on ice or stored at temperatures
between 0 and 10°C during collection or processing before adjustment of pH, this could lead to overestimationof renin activity. Before the start of immunoassay a protease inhibitor and the Generation buffer is added to theplasma sample, which will prevent Angiotensin-I (Ang-I) in plasma from degradation. The pH of the plasma
sample should be around 6.0 after the addition of the supplied Generation buffer. The plasma sample is split in
two and the fractions are incubated at 0–4°C (in ice bath) and 37°C respectively for 90 minutes or longer, toallow the generation of Ang-I by plasma renin at 37°C. Optionally, the pH can be adjusted to 6.5 or 7.4.
Adjustment of pH is a critical step during the assay, acidification of plasma to pH 3.3 or lower for prolonged
time with subsequent return to neutral pH causes irreversible activation of the renin (Derkx et al., 1987), on
the other side incubation at pH higher than 8.0 can destroy renin. During the immunoassay incubation, anotherset of protease inhibitors are involved, which function to stop the new generation as well as degradation of AngI to smaller peptides.
The immunoassay of Ang-I is a competitive assay that uses two incubations, with a total assay incubation timeof less than two hours. During the first incubation unlabelled Ang-I (present in the standards, controls andplasma samples) competes with biotinylated Ang-I to bind to the anti-Ang-I antibody. In the second incubation
the labelled Streptavidin- HRP conjugate, binds to the immobilized Ang-I-Biotin. The washing and decanting
procedures remove unbound materials. The colorimetric HRP substrate is added and, after stopping
the color development reaction, the light absorbance (OD) is measured in a microplate reader. The absorbancevalues are inversely proportional to the concentration of Ang-I in the sample. A set of standards is used to plota standard curve from which the concentrations of Ang-I in the samples and controls can be directly read.
PROCEDURAL CAUTIONS AND WARNINGS
1. 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.
2. Ang-I is presently not included in any external QC schemes. Therefore, each laboratory is suggested toestablish its own internal QC materials and procedure for assessing the reliability of results.
3. When the use of water is specified for dilution or reconstitution, use deionized or distilled water.
4. All kit reagents and specimens should be at room temperature and mixed gently but thoroughly before use.
Avoid repeated freezing and thawing of reagents and plasma specimens.
5. A standard curve must be established for every run. The kit controls should be included in every run and fallwithin established confidence limits.
6. Do not mix various lot numbers of kit components within a test and do not use any component beyond theexpiration date printed on the label.
7. The substrate (TMB) solution is sensitive to light and should always be stored in dark bottles away fromdirect sunlight.
8. To prevent contamination of reagents, use a new disposable pipette tip for dispensing each reagent, sample,
standard and controls.
9. Improper procedural techniques, imprecise pipetting, incomplete washing as well as improper reagent
storage may be indicated when assay values for the controls do not reflect established ranges. The
performance of this assay is markedly influenced by the correct execution of the washing procedure!
LIMITATIONS
1. This kit is specifically designed and validated for the determination of renin activity/Ang-I generation in
EDTA plasma. Other sources of material should be validated before being applied.
2. The Ang-I level depends on multiple factors, including renin activity, renin substrate concentration, theplasma pH, temperature and selection of inhibitors. Therefore, only carefully prepared plasma samples are
suitable for this test.
Bacterial contaminations, repeated freeze and thaw cycles and dilution of plasma samples may affect theassay result.
3. The interpretation of the results should recognise the conditions that can affect renin secretion, such as
sodium and potassium intake, posture, medications like diuretics, chlonidine, beta-blockers,
estroprogestogens and peripheral vasodilators.
4. Do not use grossly haemolysed, lipaemic, icteric plasma, and any sample that was not handled properly
according to the instruction.

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SAFETY CAUTIONS AND WARNINGS
POTENTIAL BIOHAZARDOUS MATERIAL
All reagents in this kit should be considered a potential biohazard and handled with the same precautions asapplied to any blood specimen. Human plasma samples should be handled as if capable of transmitting
infections and in accordance with good laboratory practices.
CHEMICAL HAZARDS
Avoid contact with reagents containing PMSF and hydrogen peroxide. If contacted with any of these or otherreagents in this kit, wash with plenty of water.
REAGENTS AND EQUIPMENT NEEDED BUT NOT PROVIDED
1. Disodium EDTA (2 mg/ml blood) collection tubes
2. Single and multi-channel pipettes and disposal tips
3. Distilled or deionized water
4. Disposable test tubes (glass or polypropylene)
5. Plate shaker
6. Microplate absorbance reader equipped with a 450 nm filter
7. 37°C incubator
8. Ice bath
9. 95% Ethanol
REAGENTS PROVIDED
1. AA E-0030

Wash Buffer Concentrate – Requires Preaparation X10

Contents:

Two bottles containing buffer with a non-ionic detergent and a non-mercury preservative.

Volume:2x50 ml/bottle

Storage:

Refrigerate at 2-8oC

Stability:

12 months or as indicated on label.

Preparation:

Dilute 1:10 in distilled or deionized water before use. If one whole plate is to be used dilute
50 ml of the wash buffer concentrate in 450 ml of water.
TMB Substrate - Ready To Use.

2. MS E-5655
Contents:

One bottle containing tetramethylbenzidine and hydrogen peroxide in a non-DMF or DMSO
containing buffer.

Volume:32 ml/vial

Storage:

Refrigerate at 2-8oC

Stability:

12 months or as indicated on label.
Stopping Solution - Ready To Use.

3. MS E-5680
Contents:

One bottle containing 1M sulfuric acid.

Volume:12 ml/bottle

Storage:

Refrigerate at 2-8oC

Stability:

12 months or as indicated on label.

Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.

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4. Standards and Controls- Ready To Use.
Listed below are approximate concentrations, please refer to vial labels for exact concentrations:Cat. no.

SymbolStandard

MS E-5601

Standard A

Concentration*
0 ng/ml

MS E-5602

Standard B

0.2 ng/ml

0.7 mlMS E-5603

Standard C

0.5 ng/ml

0.7 mlMS E-5604

Standard D

1.5 ng/ml

0.7 mlMS E-5605

Standard E

4 ng/ml

0.7 mlMS E-5606

Standard F

10 ng/ml

0.7 mlStandard G

25 ng/ml

0.7 mlStandard H
Control 1

60 ng/ml

0.7 ml
0.7 mlMS E-5607
MS E-5608
MS E-5651

Control 2

Volume/Vial

Refer to vial labels for
acceptable range!

MS E-5652* Approximate value - please refer to vial labels for exact concentrations.

2.0 ml

0.7 mlContents:

Synthetic angiotensin-I peptide in a protein-based buffer with a non-mercury preservative.
The standards are calibrated against the World Health Organization reference reagent
NIBSC code 86/536.

Storage:

Refrigerate at 2-8oC

Stability:

12 months in unopened vials or as indicated on label.

5. MS E-5613
Contents:

Assay Buffer - Ready To Use.
One bottle containing protein-based buffer with a non-mercury preservative.

Volume:40 ml/bottle

Storage:

Refrigerate at 2-8oC

Stability:

12 months or as indicated on label.

6. MS E-5631

Rabbit Anti-Ang-I Antibody Coated Microplate - Ready To Use.

Contents:

Two 96 well pre-coated microplates in a resealable pouch with desiccant.

Storage:

Refrigerate at 2-8oC

Stability:

12 months or as indicated on label.

7. MS E-5640

Streptavidin-Horseradish Peroxidase Conjugate Concentrate –Requires Preparation X100

Contents:

Streptavidin-HRP conjugate in a protein-based buffer with a non-mercury preservative.

Volume:

0.5 ml/vial

Storage:

Refrigerate at 2-8oC

Stability:
Preparation:

12 months or as indicated on label.
Dilute the conjugate concentrate 1:100 in assay buffer before use. The working conjugate
solution is stable for 8 hours; discard the unused solution after this period.

8. MS E-5610
Contents:
Volume:Storage:
Stability:

Version: 6.0-r

Angiotensin-I-Biotin Conjugate - Ready To Use.
One bottle containing buffer, protease inhibitors, Angiotensin-I-Biotin conjugate and a nonmercury preservative.
30 ml/bottle
Refrigerate at 2-8°C
12 months in unopened vial or as indicated on label.

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PMSF – Requires Preparation - Ready To Use.

9. MS E-5614
Contents:
Storage:
Stability:
Preparation:

One bottle containing phenylmethylsulfonyl fluoride (PMSF).
Refrigerate at 2-8°C
12 months or as indicated on label.
Reconstitute by adding 0.5 ml of 95% ethanol to the bottle and vortex for two minutes tocompletely dissolve the PMSF. Refrigerate after first use, vortex again to re-dissolve contents.
Do not keep the bottle open unnecessarily.

10. MS E-5515

Generation Buffer - Ready To Use.

Contents:

Buffer and non-toxic antibiotic.

Volume:5 ml/bottle

Storage:

Refrigerate at 2-8°C

Stability:

12 months or as indicated on label

SPECIMEN COLLECTION AND STORAGE
A minimum of 0.5 ml of plasma is required per duplicate determination. Appropriate sample collection isessential to the accurate determination of angiotensin-I. The generation and degradation of angiotensin-I canbe minimized by the following recommended collection procedure:
1. Collect 2 ml of blood into an EDTA venipunture tube or syringe.
2. Centrifuge blood for 15 minutes at 5000 rpm at room temperature.
3. Transfer plasma sample to a test tube at room temperature.
4. If samples are to be assayed now proceed to the Angiotensin-I generation procedure, otherwise freeze
samples immediately at -20°C or less. Avoid freezing and thawing samples more than once.
ANGIOTENSIN-I GENERATION PROCEDURE
1. If a freshly drawn plasma sample is being used proceed to step 2.
If frozen plasma samples are being used thaw them as follows. Quickly bring frozen plasma samples to room
temperature by placing the tubes in a container with room temperature water.
2. Transfer 0.5 ml of the plasma sample into a test tube.
3. Add 5 μl of the PMSF solution to the 0.5 ml of plasma sample (1:100 ratio). Vortex the tube to mix
thoroughly.
4. Add 50 μl of the generation buffer to the treated sample from step 3 (1:10 ratio). Vortex the tube again to
mix thoroughly.
5. Divide the treated sample from step 4 equally into two aliquots by transferring 0.25 ml into two test tubes.
Incubate one aliquot for 90 minutes or longer (do not exceed 180 minutes) at 37°C, place the second aliquot
on an ice bath (0°C). Be sure to record the incubation time used for the aliquots as this is used for
calculations.
6. At the end of the incubation period place the 37°C aliquot on the ice-bath for 5 minutes to cool it down
quickly.
7. Bring both aliquots to room temperature by placing in a bath with room temperature water for 5–10 minutes(do not exceed 10 minutes).

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ASSAY PROCEDURE
1.
2.
3.

Allow all kit components to reach room temperature. Remove the required number of well strips andassemble into the plate frame.
Pipette 50 μl of each standard, control and treated plasma sample (both 37°C and 0°C aliquots)
into correspondingly labelled wells in duplicate.
Pipette 100 μl of the angiotensin-I-biotin conjugate into each well(the use of a multichannel pipette is recommended).

4.

Incubate on a plate shaker (~200 rpm) for 60 minutes at room temperature.

5

Wash the wells 5 times each time with 300 μl/well of diluted wash buffer. After washing tap theplate firmly against absorbent paper to remove any residual liquid(the use of an automatic strip washer is strongly recommended).
The performance of this assay is markedly influenced by the correct execution of the washingprocedure!
Pipette 150 μl of the streptavidin-HRP conjugate working solution into each well(the use of a multichannel pipette is recommended).

6.
7.

Incubate on a plate shaker (~200 rpm) for 30 minutes at room temperature.

8.

Wash the wells 5 times with the same procedure as in step 5.

9.

Pipette 150 μl of the TMB substrate into each well(the use of a multichannel pipette is recommended).
Incubate on a plate shaker (~200 rpm) for 10 to 15 minutes at room temperature.

10.

Add 50 μl of stopping solution to each well and mix thoroughly by gently tapping the plate.

11.

Measure the absorbance at 450 nm in all wells with a microplate reader between 0–20 minutes after
addition of the stopping solution.

CALCULATIONSPERFORMANCE CHARACTERISTICS
1. Using immunoassay software, choose either a 4-parameter or 5-parameter curve fitting method for
calculating results.
2. If a sample reads more than 60 ng/ml then dilute the sample (that has undergone the angiotensin-I
generation procedure) with Standard A at a dilution of no more than 1:10 and rerun the sample. The result
obtained should be multiplied by the dilution factor.
Note: Samples must be diluted only after they have undergone the angiotensin-I generation procedure; do
not dilute any samples before performing the angiotensin-I generation procedure.
3. Calculate the plasma renin activity (PRA) in each sample using the following equation:

Where time (hrs) is the incubation time used during the generation step.
TYPICAL TABULATED DATA
Sample data only. Do not use to calculate results
Standard

Mean OD (450 nm)

Ang-I (ng/ml)

A

2.303

0

B

2.156

0.2

C

1.937

0.5

D

1.552

1.5

E

1.066

4

F

0.591

10

G

0.311

25

H

0.122

60

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TYPICAL STANDARD CURVE
Sample curve only, do not use to calculate results.

PERFORMANCE CHARACTERISTICSSENSITIVITY
The limit of detection (LoD) was determined from the analysis of 40 samples of the blank and a low value
sample and it wascalculated 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 theblank.
LoD = 0.14 ng/mL of Angiotensin I
SPECIFICITY (CROSS-REACTIVITY)
The following compounds were tested for cross-reactivity using the Abraham method with angiotensin-I crossreacting at 100%:
AntigenSequence% Cross-Reactivity

Angiotensin-I

DRVYIHPFHL

100

Angiotensin 1-9

DRVYIHPFH

0.015

Angiotensin-II

DRVYIHPF<0.001

Angiotensin-III

RVYIHPF<0.001

Angiotensin 1-5

DRVYI<0.001

Renin Substrate
human

DRVYIHPFHLVIHN

0.001

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RECOVERY
Spiked samples were prepared by adding defined amounts of angiotensin-I to three plasma samples. The
results (in ng/ml) are tabulated below:
Sample1.Unspiked
+0.48
+1.92
+5.77
+11.53
1.Unspiked
+0.48
+1.92
+5.77
+11.53
1.Unspiked
+0.48
+1.92
+5.77
+11.53Observed Result
0.86
1.43
2.82
6.47
10.58
2.84
3.30
5.34
8.84
13.08
9.45
9.92
11.35
13.82
17.62

Expected Result
1.34
2.78
6.63
12.40
3.32
4.77
8.61
14.38
9.93
11.37
15.22
20.99

Recovery %
107
101
98
85
99
112
103
91
100
100
91
84

LINEARITY
Three plasma samples were diluted with Standard A. The results (in ng/ml) are tabulated below:
Obs. Result

Exp. Result

Recovery %

1
1:2
1:4
1:8
1:16

Sample

10.96
5.739
2.718
1.423
0.776

5.48
2.74
1.37
0.685

105
99
104
113

2
1:2
1:4
1:8
1:16

15.798
8.273
3.934
1.948
1.146

7.899
3.950
1.975
0.987

105
100
99
116

3
1:2
1:4
1:8
1:16

30.7
16.142
7.477
3.542
1.574

15.350
7.675
3.838
1.919

105
97
92
82

INTERFERENCEInterference testing was per formed according to CLSI guideline EP7-A2. Plasma samples with varying levels ofangiotensin-I were spiked with potential interfering substances at recommended levels and analyzed. Results
were compared to the same plasma samples with no extra substances added to calculate the % interference.

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Concentration% Interference

1 g/ l

-3.0

2 g/l

-3.8

20 µM (12 mg/l)

0500 µM (300 mg/l)

020 µM (16 mg/l)+3.0

500 µM (400 mg/l)+13.5

1 g/l + 20 µM-0.4

1 g/l + 500 µM-0.1

2 g/l + 20 µM-3.9

2 g/l + 500 µM-12.4

Triglycerides(2C-10 C)

3.7 mM

+4.8

37 mM

+16.9

Triglycerides(8C-16 C)

3.7 mM

-0.6

37 mM

+2.2

40 g/l-2.2

60 g/l-9.6

Interferent
Haemoglobin

Bilirubin UnconjugatedBilirubin Conjugated*

Haemoglobin +Bilirubin

HSA*Taurobilirubin

INTRA-ASSAY PRECISION
Four samples were assayed 14 times each on the same standard curve.
below:
Sample
1
2
3
4

Mean
2.3
3.6
7.0
13.3

SD
0.2
0.2
0.4
0.9

The results (in ng/ml) are tabulated

CV%
8.7
6.8
6.3
7.0

INTER-ASSAY PRECISION
Four samples were assayed in ten different tests. The results (in ng/ml) are tabulated below:
Sample
1
2
3
4

Mean
0.48
0.82
9.46
11.70

SD
0.03
0.04
0.45
0.64

CV%
7.12
5.32
4.81
5.44

COMPARATIVE STUDIESThe PRA ELISA kit (y) was compared with a competitor’s PRA RIA kit (x). The comparison of 73 plasma samples
yielded the following linear regression results:
y = 0.93x - 0.08,
r = 0.97
EXPECTED NORMAL VALUESEach laboratory should collect data and establish their own range of expected normal values. Data presented
here were from samples incubated at pH 6.0 during the generation step (Brossaud and Corcuff, 2009).
N

PRA
Mean (ng/ml.h)

PRA
Range (10th-90th percentile) (ng/ml.h)

533

0.75

0.06-4.69

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REFERENCES
1. Brossaud J, Corcuff JB. Pre-Analytical and Analytical Considerations for the Determination of Plasma Renin
Activity. Clin Chim Acta. 2009; 410(1–2):90–2.
2. Bystrom CE, et al. Plasma Renin Activity by LC-MS/MS: Development of a Prototypical Clinical Assay
Reveals a Subpopulation of Human Plasma Samples With Substantial Peptidase Activity. Clin Chem. 2010;
56(10):1561–9.
3. Campbell DJ, et al. Activity Assays and Immunoassays for Plasma Renin and Prorenin: Information
Provided and Precautions Necessary for Accurate Measurement. Clin Chem. 2009; 55(5):867–77.
4. Cartledge S, Lawson N. Aldosterone and Renin Measurements. Ann Clin Biochem. 2000; 37(Pt 3):262–78.
5. Derkx FH, et al. Two-step Prorenin-Renin Conversion. Isolation of an Intermediary Form of ActivatedProrenin. J Biol Chem. 1987; 262(6):2472–7.
6. Hartman D, et al. Direct Renin Assay and Plasma Renin Activity Assay Compared. Clin Chem. 2004;
50(11):2159–61.
7. Pimenta E, Calhoum D. Response to “Effective” Plasma Renin Activity: A Derived Measure for Assessing
Residual Plasma Renin Activity in Patients Taking Angiotensin- Converting Enzyme Inhibitors or Angiotensin
Receptor Blockers. Hypertension. 2010; 55:e17.
8. Reudelhuber TL. Prorenin, Renin, and Their Receptor: Moving Targets. Hypertension. 2010; 55(5):1071–4.
9. Sealey JE, et al. Plasma Renin Methodology: Inadequate Sensitivity and Accuracy of Direct Renin Assay for
Clinical Applications Compared With the Traditional Enzymatic Plasma Renin Activity Assay. J Hypertens.
1995; 13(1):27–30.
10. Sealey JE. Plasma Renin Activity and Plasma Prorenin Assays. Clin Chem. 1991; 37(10 Pt 2):1811–9.
11. Sealey JE, et al. Plasma Renin and Aldosterone Measurements in Low Renin Hypertensive States. Trends
Endocrinol Metab. 2005; 16(3):86–91.
12. Ulmer PS, Meikle AW. Sample Requirements for Plasma Renin Activity and Immunoreactive Renin. Clin
Chem. 2000; 46(9):1442–4.

Symbols:

Version: 6.0-r

Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContent

CautionCatalogue
numberEffective: 2018-10-10

Contains sufficient for<n> tests

For research use
only!
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