G. Gunzer

H Dittmer

H. Fritz

H. Lang

J Schultz

Marianne Jochum

RA Clark

S Neumann

S. Neumann

SJ Klebanoff

T Olofsson

English

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Quantifizierung von Myeloperoxidase aus humanen Granulocyten als Entzündungsparameter mittels Enzymimmunoassay vom Sandwichtyp

Neumann, S.

Gunzer, G.

Lang, H.

Jochum, Marianne

Fritz, Hans

Open Access LMU ( Ludwig-Maximilians-Univ. München)

Quantitation of myeloperoxidase from human granulocytes as an inßammation marker by cnzyme-Iinkcd immunosorbent assay S. Neumann J. G. Gunzer I, H. Lang I, Mariannc J ochum 1, and H . Fritz ~ Biochemische Forschung E. Merek, Fra nHurter Straße 250, 0 -6100 Oarmstadt 1, Federal Republic ofGennany 2 Abteilung für Klinische Chemie und Klinische Biochemie, Chirurgische Klinik der Universität München, Nußbaumstraße 20, 0 -8000 Miinchen 2, Federal Republic of Germany Quantifizierung von Myeloperoxidase aus humanen Granulocyten als [ntlündungsparameter mittels J::nzymimmunoassay \'om SandwiehlYP Myeloperoxidase (donor: H20 2 oxidorcd uctase, EC 1.11.1.7) is a cationic heme enzyme of mol wt of approximately 140,000 whieh is found as a main constituent of the azurophilic granules of polymorphonuclcar lcukoeytes (PMN) and in monocytcs (1]. Myclopcroxidase (MPO) plays an important role in killing bacteria using halide ions as a cofactor [2]. As a consequencc of phagocytosis MPO is discharged into phagolysosomes. Leakagc during phagocytosis or secretion on stimulation by various in-flammatory mediators causes th.e appearance of extraccllular MPO activity. MPO activity outside of the cells may either stimulate inflammatory processes by oxidation ofphysiological substrates Jike IgG, C3b, Cl.1-proteinase inhibitor as weil as latent collagenase and by killing cells or may aLternatively modulate mflammatory activity by inactivation of cbemotactic peptides, lysosomal enzymes as weil as Fc- and C3b-rcceptors on phagocytes \3]. Quantita tive detennination of MPO in serum or plasma has been accomplished by radioimmunoassay [4] and in cell extracts or tissue extracts by enzyme assays. Here we describe a sandwicb-type enzyme-linked immunosorbent assay for quanti tation of MPO concentration iD plasma, broncho-alveolar lavage fluid or extracts from granulocytes. Materials and mcthods MPQ was purified from nOnTIal human granulocytes by a proce-durc involving acid elltraction of granules, copper chelate chromat0f::aphy, cation exchange chromatography on SP-Sephadex M C-50 and size exclusion chromatography on Fractogelll.TId TSK HW·55 . Tbc final enzyme prepamtion was sho:wn to bc ffee of oth~r granular proteins by SOS-polyacryl-amIde gel electrophores!s and immunoelectrophoresis against various polyvalent or monospecifi c autisera against granular components. - Antigen.spccific anlisera to MPO were raised in sheep and rabbilS. The IgG fractions were isolated by ammo-nium sulfate precipitation and ion-exchange chromatography on O EAE cellulose. The specific IgG fractioD from rabbilS was isolatcd by adding immunoadsorption on MPO-agarose, and thcn coupled 10 calf intestinal alkaline pbosphatase with glutar-dialdehyde as describcd previously {5]. For the enzyme immunoassay a procedure as reported pre-~o~s!y fo r tbe compJell of PMN elastase Wilh Cl.1 -proteinase mh!bltor was followed analogously [6]. Brieny, polystyrcne tubcs (10.5 mm x 40 mm) werc coated by incubation wilh O.5 ml containing 50 ).lg sheep TgG 10 MPOJml in phosphate buffered saline pH 7.5 (PBS) at room temperature (20 to 2rq ovem ight and washed with 0.05% (wJv) polyoxyethylene (20)-sorbitan monolaurate scveral times before use. FOT tbe assay 0.5 ml sampie or calibrator was incubated in the coated tubes for I h at 20 10 22°C. Tbe tubes were washed three times, and 0.5 ml enzyme labellcd anlibodies to MPO in saJine Tris/HCI buffer pH 8.0 was added . After incubation for 2 h at 20 to 22~ C and wa~h ing of the tubes 0.5 ml substrate soLution, i.c. 10 mmol/l 4-nitrophenyl phosphate in I molfl diethanolamine/HCI buffer pH 9.8 witb 0.5 mmol MgCI2/1, was dclivered to each tube and incubated for 1 hat 20 to 2rc. 0.5 ml 2 mol/l NaOH wasaddcd witb vortexing. Absorbance was read at 405 nm . A40s was plotted against MPO concentration on a linear seale. As a routine citrated pLasma was used in a final dilution of one part plasma plus 20 or 50 parts of PBS containing 1 mg bovine serum albumin/mi and 20 mmol EOTA/I. Calibrators wcre used in tbe same diluent pLus 2"/" (viv) citrated normal sheep plasma_ Results and discussion A linear calibration curve was obtained for thc range of 0 to 20llg MPO/I. Unknowns werc detcrmined by reference 10 the calibration curve as calculated by linear regression analysis. Detection limit, i.e. mean absorbance of tbe blank plus 3 x standard deviation, was abOllI 0.25 ).Ig/l. Sensitivi ty cor-rcsponded to LlA = 0.004 in the calibration curve and approached O. I ).1g MPOjl. Within-run imprecision (coefficient of variation fo r 15 parallel detenninations) was in the range of 8.4 to 6.8% with plasma sampies in the range of 32 to 143 Ilg MPO/I, respectively. Between-run imprecision (CV for aseries of 9 independent tests) was from 5.2 10 12.2% for 3 different commercially obtainable control plasma specimen. The assay was shown to be specific for myeloperoxidase as lactoferrin as weil as otbcr granular proteins when added to the sampie did not interfere. However, plasma conccntrations higher than 5% (vIv) impaired recovery, possibly by matrix effect(s). As the assay showed high sensitivity and reasonable precision with control plasma specimen we went on to measure myclo-peroxidase levels in normal and pathological sampies. In a group of 156 apparently healthy persons the MPO concentration in citrated plasma was 36 ± 1 t.5)lg MPO/l (mean ± SO). In another group of 25 nonnal persons simila rly a range of 20 to 60 IlgJI was fouo d. Patients sufTering from multiple trauma were studied longitudinally wilh 6 h intervals, measuring in citrated plasma MPO as weil as the complex of PMN clastase with Cl.1 -proteinase inhibitor whicb was previously shown to reOect tbe severity of the disease as weIL as complication by infections P}. Plasma levels of MPO sbowed a perfcct correlation with tbc PMN elastase complex levels with regard to rise aod fall, they were, however, lowcr in terms of concentration. Quite differently, in palients with adult respiratory dis tress syndrome (ARDS) in the analyses of bronehoalveolar lavage flu id high levels of MPO were observed which werc equal to or in severe eases even higher than theconcentration of tbe PMN elastasecomplex. 11 is argucd that the availabili ty of activc Cll -proleinase inhibitor for inactivation of PMN elaslase may bc Iimited here locally, and high. levels of MPO may botb signal the acuilY of the disease process and indicate the oxidative potential at the site of in-nammation. Rererenccs 1. Schultz J (1980) In : Sbarra AJ, Straus RR (eds) The rc-ticuloendothelial system, vol2 . Plenum Press, New York, pp 23 J -254 2. Klebanoff SJ (1975) Sem Hematol 12: 117 - 142 3. Clark RA (1983) In: Weismann G (ed) Advances in in-flammation research, vol 5. Raven Press, New York, pp 107- 146 4. Olofsson T, Olsson I, Venge P, Elgefors 8 (1977) Scand J HaematoL 18:73 - 80 5. Neumann S, Jochum M (1984) In: ßergmeyer HU (cd) Methods of enzymatic analysis, 3rd cdn, vol V. Verlag Chemie, Weinheim, pp 184 -195 6. Neumann S, Gunzer G, Hennrich N, Lang H (1 984) J Clin Chem Clin Biochem 22 : 693 - 697 7_ Dittmer H, Jochum M, Schmit-Neuerburg KP (1985) Chirurg 56: 723 - 727 

Quantification of myeloperoxidase from human granulocytes as an inflammation marker by enzyme.linked immunosorbent assay

Universität München: Elektronischen Publikationen

https://epub.ub.uni-muenchen.de/9491/1/quantitation_of_myeloperoxidase_from_human_9491.pdf

Quantification of myeloperoxidase from human granulocytes as an inflammation marker by enzyme.linked immunosorbent assay

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