Summary:A solid phase, enzyme-linkcd immunoassay is described for the quantitative determination of the complex of human granulocyte elastase (EC 3.4.21.37) with αι-proteinase inhibitor. The assay employs antibody-coated test tubes and it is suitable for routine use in clinical chemistry laboratories. Data for sample stability and test characteristics are given. A reference r nge of 20-180 μg/l elastase in plasma was determined. The diagnostic significance of granulocyte elastase levels in plasma in inflammatory diseases is discussed. "PMN-Elastase Assay": Enzymimmunassay zur Bestimmung des Komplexes von menschlicher GranulocytenElastase mit a/-ProteinaseinhibitorZusammenfassung: Es wird ein Festphasen-Enzymimmunoassay zur quantitativen Bestimmung des Komplexes aus menschlicher Granulocyten-Elastase und arProteinaseinhibitor beschrieben. Es handelt sich um eine routinef hige Testversion mit Antik rper-beschichteten R hrchen. Daten zur Stabilit t von Proben und die Testcharakteristica werden angegeben. Als Referenzbereich f r Elastase im Plasma wurden 20-180 μο/1 ermittelt. Die Bedeutung der Granulocyten-Elastase im Plasma f r die Diagnose und Verlaufskontrolle von entz ndlichen Prozessen wird diskutiert.

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Federal Rep ublic of Germany Q uantifizierung von Myeloperoxidase aus humanen Granulocyten als [ntlündungsparameter mittels J::nzymimmunoassay \'om SandwiehlYP Myeloperoxidase (donor: H 2 0 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 Materials and mcthodsMPQ was purified from nOnTIal human granulocytes by a procedurc involving acid elltraction of gran ules, copper chelate chromat0f::aphy, cation exchange chromatography on SPSephadex 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-polyacrylamIde 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. T he IgG fractions were isolated by ammonium 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 al kaline pbosphatase with glutardialdehyde as describcd previously {5].For the enzyme immunoassay a p rocedure as repo rted p reo~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 T gG 10 MPOJml in p hosphate buffered saline pH 7.5 (PBS) at room te mperature (20 to 2rq ovem ight and washed with 0.05% (wJv) polyoxyethylene (20)-sorbitan monolaurate scveral ti mes before use. FO T 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 i ng 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 MgCI 2 /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 agains t MPO concentration on a linear seale. As a rou tine citrated pL asma 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 pL us 2"/" (viv) citrated normal sheep plasma_ Results and discussion A linear calibration curve was obtained for thc range of 0 to 20llg MPO/ I. Unk nowns 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....