Manual

MPO ELISA Kit
For the in vitro determination of myeloperoxidase (MPO)
in serum and plasma

Valid from 2014-10-23+8 °C

K 6631B96+2 °C

Immundiagnostik AG, Stubenwald-Allee 8a, D-64625 Bensheim
Tel.: +49 6251 70190-0

Fax: + 49 6251 849430

e.mail: info@immundiagnostik.com

www.immundiagnostik.com

ManualTable of Contents
1.

INTENDED USE ___________________________________________________ 17

2.

INTRODUCTION___________________________________________________ 17

3.

MATERIAL SUPPLIED ______________________________________________ 18

4.

MATERIAL REQUIRED BUT NOT SUPPLIED ____________________________ 18

5.

STORAGE AND PREPARATION OF REAGENTS _________________________ 19

6.

STORAGE AND PREPARATION OF SAMPLES __________________________ 20

7.

ASSAY PROCEDURE _______________________________________________ 21
Principle of the test __________________________________________________ 21
Test procedure______________________________________________________ 21

8.

RESULTS _________________________________________________________ 22

9.

LIMITATIONS _____________________________________________________ 23

10. QUALITY CONTROL________________________________________________ 23
Reference range ____________________________________________________ 24
11. PERFORMANCE CHARACTERISTICS _________________________________ 24
Precision and reproducibility __________________________________________ 24
Spiking Recovery ____________________________________________________ 24
Dilution recovery____________________________________________________ 25
Analytical Sensitivity _________________________________________________ 25
Specificity _________________________________________________________ 25
12. PRECAUTIONS ____________________________________________________ 26
13. TECHNICAL HINTS ________________________________________________ 26
14. GENERAL NOTES ON THE TEST AND TEST PROCEDURE ________________ 27
15. REFERENCES _____________________________________________________ 27
General literature ___________________________________________________ 27
Literature using Immundiagnostik MPO ELISA _____________________________ 28

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ManualMPO

1. INTENDED USE
The described Enzyme-Linked-Immuno-Sorbent-Assay (ELISA) is intended for thequantitative determination of MPO (myeloperoxidase) in serum and plasma. For invitro diagnostic use only.

2. INTRODUCTION
MPO is part of the defence mechanism of the polymorphonuclear leukocytes against
exogenic substances. During bacterial infection, these leukocytes, are stimulated bychemotactically effective substances (leukotrienes, complement factors, bacterial toxins etc.). They move to the site of the infection and encapsulate the foreign
substances. If the foreign agent is located in an intracellular vacuole, different substances are used for the intracellular digestion. Amongst these are MPO, cationic proteins, lysozyme, lactoferrin and some acidic hydrolases. A strong surge of oxidative
metabolism takes place, producing a high number of oxygen radicals which leads
to the destruction of foreign proteins. Some of these molecules can leak into theextracellular space during this process. This happens to a greater extent, when theleukocytes cannot encapsulate the foreign body because of its size or in cases where
the neutrophils are destroyed (by bacterial toxins, crystalline substances etc.).
MPO, together with hydrogen peroxide and a halogen, forms a very strong anti microbial system, which can effectively combat a number of microorganisms. MPO ispresent at high concentration in neutrophil granulocytes, whereas hydrogen peroxide is produced during infection/ inflammation. The MPO system is inhibited bycatalase, excess of hydrogen peroxide and other reducing substances (e.g. ascorbic acid, glutathione). In the absence of these agents other cells in the extracellularspace can be affected (e.g. spermatocyte, erythrocytes, leukocytes, and tumor cells)
Apart from its implications in host defence, involvement of MPO has been described in numerous non-infectious diseases such as atherosclerosis, lung cancer,
Alzheimer´s disease, and multiple sclerosis. MPO is present and active within atherosclerotic lesions. Numerous lines of evidence suggest mechanistic links between
myeloperoxidase, inflammation and both acute and chronic manifestations of cardiovascular disease.
Brennan et al. (2003) showed that in 604 sequentially ascertained patients presenting
with chest pain, a single initial measurement of plasma myeloperoxidase was an independent early predictor of myocardial infarction, as well as the risk of major adverse cardiac events in ensuing 30-day and 6-month periods. In contrast to troponinT, creatine kinase MB isoform, and C-reactive protein levels, MPO levels may identify
patients at risk for cardiac events in the absence of myocardial necrosis.
Summary: The inflammatory protein myeloperoxidase is present, active and me17

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chanistically poised to participate in the initiation and progression of cardiovasculardisease. The many links between myeloperoxidase, oxidation and cardiovascular disease suggest this leukocyte protein may have clinical utility in risk stratification for
cardiovascular disease status and outcomes.
Indications• Marker for inflammatory activities in the gastrointestinal tract (Stool)• Renal transplant rejection (Urine)• Oxidative stress (Serum)• For the differentiation between allergic and infectious asthma (bronchial lavage, respiratory condensate, sputum)• Prediction of risk in patients with acute coronary syndromes (Serum)

3. MATERIAL SUPPLIED
Cat. No.

Label

Kit components

Quantity

K 6631B MTPPLATEOne holder with precoated strips

12 x 8 wells

K 6631B WPWASHBUFELISA wash buffer concentrate 10x1 x 100 ml

K 6631B K

CONJConjugate, rabbit anti-MPO peroxidaselabelled antibody, concentrate

1 x 50 µl

K 6631B ST

STD

MPO-Standard, lyophilized(see specification for concentration )4 vials

K 6631B KO1

CTRL

Control, lyophilized

4 vials

K 6631B KO2

CTRLControl, lyophilized

4 vials

K 6631B PV

SAMPLEBUF

Sample dilution buffer, ready to use

1 x 50 ml

K6631B TMBSUBTMB substrate (Tetramethylbenzidine),
ready to use

1 x 15 ml

K6631B ACSTOPELISA stop solution, ready to use

1 x 15 ml

4. MATERIAL REQUIRED BUT NOT SUPPLIED• Ultra pure water*• Laboratory balance• Calibrated precision pipettors and 10–1000 µl tips
• Foil to cover the microtiter plate• Horizontal microtiter plate shaker• Multi-channel pipets or repeater pipets
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ManualMPO• Centrifuge, 3000 g• Vortex• Standard laboratory glass or plastic vials, cups, etc.
• Microtiter plate reader (required filters see chapter 7)* Immundiagnostik AG recommends the use of Ultra Pure Water (Water Type 1; ISO 3696), whichis free of undissolved and colloidal ions and organic molecules (free of particles > 0.2 µm) with anelectrical conductivity of 0.055 µS/cm at 25 °C (≥ 18.2 MΩ cm).

5. STORAGE AND PREPARATION OF REAGENTS• To run the assay more than once, ensure that reagents are stored at the conditions stated on the label. Prepare only the appropriate amount necessaryfor each run. The kit can be used up to 4 times within the expiry date statedon the label.
• Reagents with a volume less than 100 µl should be centrifuged before use to
avoid loss of volume.
• The ELISA wash buffer concentrate (WASHBUF) should be diluted 1:10 inultra pure water before use (100 ml concentrate + 900 ml ultra pure water),mix well. Crystals could occur due to high salt concentration in the stock solutions. The crystals must be redissolved at room temperature or 37 °C before
dilution of the buffer solutions. The buffer concentrate is stable at 2–8 °C until the expiry date stated on the label. Diluted buffer solution (wash buffer)
can be stored in a closed flask at 2–8 °C for one month.
• The lyophilized STD (standard) and the lyophilized CTRL (controls) are stable at 2–8°C until the expiry date stated on the label. Standard and control
have to be reconstituted with SAMPLEBUF (sample dilution buffer) (volume,
concentration and dilution schema for the calibration curve see product specification). Allow the vial content to solve for 10 minutes and then mix thoroughly by gentle inversion to insure complete reconstitution. Reconstituted
standard and controls are not stable.
• The conjugate concentrate (CONJ) must be diluted 1:301 in wash buffer(40 µl CONJ + 12 ml wash buffer). The concentrate is stable at 2–8 °C until the
expiry date stated on the label. Diluted conjugate is not stable and cannot
be stored.
• All other test reagents are ready to use. Test reagents are stable until the expiry date (see label of test package) when stored at 2–8 °C.

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6. STORAGE AND PREPARATION OF SAMPLES
Preanalytic handlingSignificant differences in the MPO levels can be observed due to different sample
preparation procedures, e.g. analysis of plasma or serum samples. The reasons are
as follows:• The granulocytes are activated during the serum clotting and release granulocyte-activating markers. The time between serum collecting and analysis as
well as repeated freeze-thaw cycles don‘t cause a MPO concentration shift.
• On the contrary, in the case of plasma samples, varying the time between
sampling and analysis or the number of freeze-thaw cycles will cause variation in the observed MPO levels. Therefore, the preanalytical conditions ofplasma samples should be held constant. This is a general requirement independent of the test-system used.
• Fresh collected Serum/Plasma should be centrifuged within one hour. Store
samples at -20 °C if not assayed on the same day. Lipemic or hemolytic samples
may give erroneous results. Samples should be mixed well before assaying.
We recommend duplicate analyses for each sample.
• The preanalytical handling is critical for accurate and consistent/reproducible
MPO measurement results.
• Shih et al. (2008) report that MPO concentrations were consistently higher
in serum and heparin plasma samples than in samples in EDTA or citrate andrecommend the analysis of EDTA plasma samples. Furthermore, the authors
investigated the effects of preanalytical handling, storage temperature andtime for EDTA plasma, lithium-heparin and citrate preparation. Less than 10%
differences were found after storage of samples at room temperature for 2days, after storage at 2–8 °C for 8 days, and after 3 freeze-thaw cycles for all
sample types• Videm (1996) describes at heparin concentrations, as applied in clinical practice, a dose-dependent increase in granulocyte activation as measured by MPO
release, quantitated in enzyme-immunoassay. Thus, direct effects of heparinon granulocytes, e.g. MPO release and concentration, should be taken into
consideration for the evaluation of MPO results of samples from patients receiving systemic heparin therapy.
Serum samples
Prior to analyses the serum samples should be diluted 1:40 with SAMPLEBUF (sample
dilution buffer).
For example: 25 µl sample + 975 µl SAMPLEBUF.
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EDTA-plasma samples
Prior to analyses the EDTA-plasma samples should be diluted 1:10 with SAMPLEBUF(sample dilution buffer).
For example: 100 µl sample + 900 µl SAMPLEBUF.

7. ASSAY PROCEDURE
Principle of the test
The assay utilizes the two-site “sandwich” technique with two selected polyclonal antibodies that bind to human MPO.
Assay standards, controls and prediluted patient samples containing human MPO
are added to wells of microplate that was coated with a high affine polyclonal antihuman MPO antibody. After the first incubation period, antibody immobilized on thewall of microtiter wells captures human MPO in the sample. Then a peroxidase-conjugated polyclonal anti-human MPO antibody is added to each microtiter well and a“sandwich” of capture antibody - human MPO – Peroxidase conjugate is formed. Tetramethylbenzidine (TMB) is used as a substrate for peroxidase. Finally an acidic stop
solution is added to terminate the reaction. The color changes from blue to yellow.
The intensity of the yellow color is directly proportional to the concentration of MPO
in the sample. A dose response curve of the absorbance unit (optical density, OD at
450 nm) vs. concentration is generated, using the values obtained from the standard.
MPO present in the patient samples, is determined directly from this curve.

Test procedure
Bring all reagents and samples to room temperature (15–30 °C) and mix well.
Take as many microtiter strips as needed from kit. Store unused strips in the aluminium foil bag with desiccant at 2–8 ° C. Strips are stable until expiry date stated on
the label.
We recommend to carry out the tests in duplicate.
Wash each well 5 x by dispensing 250 µl of diluted WASHBUF (wash1. buffer) into each well. After the final washing step remove residual
buffer by tapping the plate on absorbent paper.
2.

Add 100 µl of STD (Standard) SAMPLE (Sample) CTRL (Controls) into
respective well.

3.

Cover the plate tightly and incubate for 1 hour at room temperature(15-30°C) on a horizontal shaker.
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Discard the contents of each well. Wash each well 5 x by dispensing
250 µl of diluted WASHBUF (washbuffer) into each well. After the
4.
final washing step remove residual buffer by tapping the plate on
absorbent paper.
5. Add 100 µl diluted CONJ (conjugate) into each well.
6.

Cover the plate tightly and incubate for 1 hour at room temperature(15-30°C) on a horizontal shaker.

Discard the contents of each well. Wash each well 5 x by dispensing
250 µl of diluted WASHBUF (washbuffer) into each well. After the7.
final washing step remove residual buffer by tapping the plate on
absorbent paper.
8. Add 100 µl of SUB (substrate) into each well.
9. Incubate for 10 - 20 min.* at room temperature (15-30°C) in the dark.
10. Add 100 µl of STOP (stop solution) into each well, mix.
Determine absorption immediately with an ELISA reader at 450 nm
against 620 nm (or 690 nm) as a reference. If no reference wavelength is
11. available, read only at 450 nm. If the extinction of the highest standard
exceeds the range of the photometer, absorption must be measured
immediately at 405 nm against 620 nm as a reference.
* The intensity of the color change is temperature sensitive. We recommend observing the color
change and stopping the reaction upon good differentiation.

For automated ELISA processors the given protocol may need to be adjusted according to the specific features of the respective automated platform.
For further details please contact your supplier or Immundiagnostik AG.

8. RESULTS
The following algorithms can be used alternatively to calculate the results. We recommend using the “4 parameter algorithm“.
1.

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4 parameter algorithm
It is recommended to use a linear ordinate for the optical density and a logarithmic abscissa for the concentration. When using a logarithmic abscissa, the zerostandard must be specified with a value less than 1 (e. g. 0.001).

Manual2.

MPO

Point-to-point calculation
We recommend a linear ordinate for the optical density and a linear abscissa forthe concentration.

3.

Spline algorithm
We recommend a linear ordinate for the optical density and a linear abscissa forthe concentration.
The plausibility of the duplicate values should be examined before the automatic
evaluation of the results. If this option is not available with the programme used, theduplicate values should be evaluated manually.
Serum
The obtained MPO levels of serum samples have to be multplied with the dilution
factor of 40.
PlasmaThe obtained MPO levels of EDTA plasma samples have to be multplied with thedilution factor of 10.
In case another dilution factor has been used, multiply the obtained result with thedilution factor used.

9. LIMITATIONS
Samples with concentrations above the measurement range must be further diluted
and re-assayed. Please consider this greater dilution when calculating the results.
Samples with concentrations lower than the measurement range cannot be clearly
quantified.
The upper limit of the measurement range can be calculated as:
highest concentration of the standard curve × sample dilution factor to be used
The lower limit of the measurement range can be calculated as:
LoB × sample dilution factor to be used

10. QUALITY CONTROL
Immundiagnostik recommends the use of external controls for internal quality control, if possible.
Control samples should be analysed with each run. Results, generated from the analysis of control samples, should be evaluated for acceptability using appropriate sta23

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tistical methods. The results for the patient samples may not be valid if within thesame assay one or more values of the quality control sample are outside the acceptable limits.

Reference range
Based on Immundiagnostik studies of serum and EDTA-plasma samples of apparently healthy persons the following reference range was estimated.
MPO from serum (n = 20)
median = 444 ng/ml
MPO from EDTA-plasma (n = 20)
median = 108 ng/ml
We recommend each laboratory to establish its own reference range.

11. PERFORMANCE CHARACTERISTICS
Precision and reproducibility
Intra-Assay (n = 40)
SampleMPO [ng/ml]

VK [%]

1

465,9

2,6

2

198,9

2,3

SampleMPO [ng/ml]

VK [%]

1

459,0

4,2

2

198,3

4,1

Inter-Assay (n = 13)

Spiking Recovery
Two samples were spiked with different MPO concentrations and measured using
this assay (n = 2).
SampleA

24

Unspikedsample[ng/ml]Spike[ng/ml]MPO expected[ng/ml]MPO measured[ng/ml]

5,1

2,6

7,7

6,6

5,0

4,3

9,3

8,1

4,8

7,9

12,7

10,6

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SampleUnspikedsample[ng/ml]Spike[ng/ml]MPO expected[ng/ml]MPO measured[ng/ml]

5,0

2,6

7,6

6,8

4,9

4,3

9,2

8,8

4,7

7,9

12,6

11,7

B

Dilution recovery
Two samples (1 serum, 1 EDTA-plasma) were diluted and analyzed. The results areshown below (n = 2):
SampleDilution

MPO expected[ng/ml]MPO measured[ng/ml]

1:80

-

8,9

1:160

4,5

5,0

1:320

2,5

2,7

1:640

1,4

1,4

1:1280

0,7

0,8

1:20

-

6,5

1:40

3,2

3,6

1:80

1,8

2,0

1:160

1,0

1,1

1:320

0,5

0,6

A

B

Analytical Sensitivity
Limit of blank, LoB
Limit of detection, LoD

0.161 ng/ml
0.294 ng/ml

The evaluation was performed according to the CLSI-Guideline:EP-17-A.

Specificity
No cross reactivity with other plasma proteins in serum/plasma was observed.
• Alpha-1-Antitrypsin
0%• Albumin
0%
• CRP
0%
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ManualMPO• Lysozym0%• sIgA0%• PMN-Elastase0%• Calprotectin0%
No cross reactivity with MPO in mouse serum was observed.

12. PRECAUTIONS• All reagents in the kit package are for in vitro diagnostic use only.
• Human materials used in kit components were tested and found to be negative for HIV, Hepatitis B and Hepatitis C. However, for safety reasons, all kit
components should be treated as potentially infectious.
• Kit reagents contain sodium azide or ProClin as bactericides. Sodium azide
and ProClin are toxic. Substrates for the enzymatic color reactions are toxic
and carcinogenic. Avoid contact with skin or mucous membranes.
• The stop solution consists of diluted sulphuric acid, a strong acid. Although
diluted, it still must be handled with care. It can cause burns and should be
handled with gloves, eye protection, and appropriate protective clothing. Any
spill should be wiped up immediately with copious quantities of water. Do notbreath vapour and avoid inhalation.

13. TECHNICAL HINTS• Do not interchange different lot numbers of any kit component within thesame assay. Furthermore we recommend not assembling wells of different
microtiter plates for analysis, even if they are of the same batch.
• Control samples should be analyzed with each run.
• Reagents should not be used beyond the expiration date stated on kit label.
• Substrate solution should remain colourless until use.
• To ensure accurate results, proper adhesion of plate sealers during incubation
steps is necessary.
• Avoid foaming when mixing reagents.
• Do not mix plugs and caps from different reagents.
• The assay should always be performed according the enclosed manual.
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14. GENERAL NOTES ON THE TEST AND TEST PROCEDURE• This assay was produced and distributed according to the IVD guidelines of
98/79/EC.
• The guidelines for medical laboratories should be followed.
• Incubation time, incubation temperature and pipetting volumes of the components are defined by the producer. Any variation of the test procedure,
which is not coordinated with the producer, may influence the results of thetest. Immundiagnostik AG can therefore not be held responsible for any damage resulting from incorrect use.
• Warranty claims and complaints regarding deficiencies must be logged within
14 days after receipt of the product. The product should be send to Immundiagnostik AG along with a written complaint.

15. REFERENCES
General literature
1. Klebanoff SJ (1999) Proc Assoc Am Physicians 111(5):383-9
2. Oremek et al. (1995) MTA 4: 273-278
3. Markant et al. Pharmazeutische Zeitung 26/1995, 140. Jahrgang: 9-25
4. Saiki (1998) Kurume Med J 45: 69-73
5. Zhang R et al. (2001) JAMA 286 : 2136-2142
6. Brennan M et al. (2003) N Engl J Med 349 : 1595-1604
7. Baldus S et al. (2003) Circulation 108 : 1440-1445
8. Shih et al. (2008) Affect of Collection Tube Type and Preanalytical Handling on
Myeloperoxidase Concentrations Clinical Chemistry 54:6 1076–1079
9. Videm V. (1996) Heparin in clinical doses ‚primes‘ granulocytes to subsequent activation as measured by myeloperoxidase release. Scand J Immunol. Apr;43(4):38590

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Literature using Immundiagnostik MPO ELISA
10. Exner M et al. (2006) JACC 47 (11) 2212-2218
11. Holz O et al. (2005) J Clin Pharmacol 45(5):498-503
12. Stepan H et al. (2003) Hypertens Pregnancy 22(3):239-45
13. Stepan H et al. (2002) Poster zum 10. Kongress der DGPG.

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