Norbert Dr Hennrich

Bestimmung der Aktivität von Creatinkinase MB im Serum unter Verwendung inhibierender Antikörper

SummaryA new method for the determination of creatine kinase-MB activity in the serum is presented. The principle of this method is the direct measurement of the activity of creatine kinase M subunits by inhibiting antibodies. The total test procedure takes 15 min. In the sera of all the 83 patients tested, who have clinically proven myocard infarction, creatine kinase-MB activity can be measured between the 6th and 28th hour after infarction. At the time of maximum total creatine kinase activity the percentage of creatine kinase-MB activity is between 6 and 17%, the mean value being 8%. In cases of emergency this method can be used for the differential diagnosis of elevated total creatine kinase activities of unknown origin.ZusammenfassungEs wird über eine neue Methode zur quantitativen Bestimmung der Creatinkinase MB-Aktivität im Serum berichtet. Die Methode beruht auf einer direkten Messung der Aktivität der Creatin-kinase-Untereinheit B nach Hemmung der Aktivität der Creatinkinase-Untereinheit M durch inhibierende Antikörper und benötigt zur Durchführung 15 min. Bei allen 83 untersuchten Patienten mit klinisch gesichertem Myokardinfarkt konnten zwischen der 6. und 28. Stunde nach Infarkteintritt Creatinkinase MB-Aktivität gemessen werden. Der Creatinkinase MB-Anteil zum Zeitpunkt der höchsten Creatinkinase-Gesamtaktivität betrug 6–17%, im Mittel 8%. Diese Methode ermöglicht daher in der Notfalldiagnostik eine Differentialdiagnose unklarer Creatinkinase-Gesamtaktivitäts-Erhöhungen.A new method for the determination of creatine kinase-MB activity in the serum is presented. The principle of this method is the direct measurement of the activity of creatine kinase M subunits by inhibiting antibodies. The total test procedure takes 15 min. In the sera of all the 83 patients tested, who have clinically proven myocard infarction, creatine kinase-MB activity can be measured between the 6th and 28th hour after infarction. At the time of maximum total creatine kinase activity the percentage of creatine kinase-MB activity is between 6 and 17%, the mean value being 8%. In cases of emergency this method can be used for the differential diagnosis of elevated total creatine kinase activities of unknown origin.

Enzyme-Linked Immunoassay for Human Granulocyte Elastase in Complex with α1 -Proteinase Inhibitor

Granulocyte elastase in complex with its main inhibitor in plasma, i.e. alpha 1-proteinase inhibitor, was quantitatively determined by incubating the sample with solid-phase fixed antibodies against elastase first and reacting then with alkaline phosphatase-labelled antibodies against alpha 1-proteinase inhibitor. In normal plasma a level of 97.5 +/- 25.8 micrograms elastase/1 (mean +/- s.d., n = 43) was found, whereas moderately to markedly increased plasma concentrations were demonstrated in a variety of patients with inflammatory diseases like septicemia or rheumatoid arthritis.

[Determination of creatine phosphokinase-MB in the serum of patients with myocardial infarction by an immunological method (author's transl)].

SummaryThe immunological method of determining creatine phosphokinase-MB in the serum of patients with myocardial infarction described here is based on the differential measurements of CK-activities before and after a specific immuno-precipitation of the CK-MB-type. The minimum activity of the CK-MB-type which it is possible to determine with this method is 4% of the total activity. In patients with clinically confirmed myocardial infarctions 1–15% (mean 7.8%) of the total activity can be calculated as CK-activity of the MB-type on the first/second day(s) after the infarction. In patients with increased total CK-activity and without verified infarction the CK-MB content does not differ significantly from zero. The differences between the two groups are statistically significant. In patients with myocardial reinfarction the CK-MB-activity is higher than that after the first infarction. The immunological method to determine creatine-phosphokinase isoenzyme MB is of differential-diagnostic value in myocardial infarction.ZusammenfassungEs wird über den Nachweis von Creatinkinase-MB im Serum beim Myokardinfarkt mit einer immunologischen Methode berichtet. Die Methode beruht auf der differentiellen Bestimmung der Creatinkinase-Aktivitäten vor und nach spezifischer Immunpräcipitation von Creatinkinase-MB. Die Nachweisgrenze der Creatinkinase-MB-Aktivität mit dieser Methode liegt bei 4% der Creatinkinase-Gesamtaktivität. Bei Patienten mit klinisch gesichertem Myokardinfarkt kann am 1./2. Tag nach dem Infarkt 1–15% (Mittel 7,8%) der Creatinkinase-Gesamtaktivität als CK-MB-Aktivität nachgewiesen werden. Bei Patienten mit erhöhter CK-Gesamtaktivität ohne gesicherten Infarkt ist der CK-MB-Anteil nicht signifikant von Null unterschieden. Die Unterschiede der beiden Gruppen sind statistisch signifikant. Bei Patienten mit Reinfarkt findet sich ein höherer Anteil von CK-MB-Aktivität als nach Erstinfarkt. Die immunologische Nachweismethode für Creatinkinase-MB hat differentialdiagnostische Bedeutung beim Myokardinfarkt.

Purification of α1-proteinase inhibitor by triazine dye affinity chromatography, ion-exchange chromatography and gel filtration on fractogel TSK

The purification of plasma proteins by affinity chromatography on triazine dye matrices can be optimised with regard to the triazine dye used as a group-specific ligand. A comparison by electrophoretic and immunological means of the results of affinity chromatography with human plasma on Fractogel TSK-Blue and Fractogel TSK-Red demonstrated that the Procion Red HE-3B-containing gel was able to adsorb more plasma constituents than the Cibacron Blue F3-GA gel. The preparation of alpha 1-proteinase inhibitor obtained after chromatography on Fractogel TSK-Red showed a higher degree of purity and could easily be further purified by ion-exchange chromatography on Fractogel TSK DEAE-650 and gel filtration on Fractogel TSK HW 55, without significant loss of biological activity.

Isolation and properties of bromelin protease

Bromelin (EC. 3.4.4.24) is a mixture of several enzymes obtained from the stems of Ananas CU~OSUS [l]. Several authors, using different methods, succeeded in separating this mixture into various components [2-S]. Besides proteases bromclin contains acid phosphatase and ribonuclease [6]. This report is concerned with the enzymatic properties of bromelin main peak protease (denoted “bromelin protease” in this paper).

Determination of reactive groups of polymer carriers with amino acid 4-nitroanilides

Carrier bound biologically active substances (e.g. enzymes, enzyme effecters) have attracted great interest for preparative and analytical application in biochemistry [ 1,2]. They are prepared by using lnsoluble hydrophilic natural or synthetic polymers with free reactive groups, e.g. anhydride-, azide-, cyanate-, diazonium-, epoxide-, vinyl-groups. The choice of suitable reactive groups and of optimum conditions for the binding reaction is difficult since methods for the quantitative determination of bound enzymes or effecters e.g. measurement of activity or affinity, titration of reactive groups, elementary analysis are time consuming and insufficient. In this respect we found that the use of amino acid 4-nitroanilides is very favourable. It is possible to bind the amino acid 4nitroanilides like proteins or peptides to the active groups of the polymers, e.g. by means of the free amino group. Amino acid 4-nitroanilides, therefore like amino acids or dipeptides [3] are useful model substances for proteins provided that factors such as steric hindrance influence of lipophilic or hydrophilic sites are not to be measured. The particular advantage in using amino acid 4nitroanilides is the easy quantification: after alkaline hydrolysis

Immunologischer Schnelltest zur Bestimmung der Kreatinkinase MB-Aktivität im Serum

A rapid and sensitive method for the quantitative determination of creatine kinase (ATP: creatine phosphotransferase, EC 2.7.3.2, short term CK) isoenzyme MB activity is presented. This method utilises inhibiting anti-CK-MM, which is produced by immunisation of goats with reactivated human CK-MM isoenzyme. The resulting antiserum completely inhibits the activity of CK-M subunits without affecting the activity of CK-B subunits. Complete inhibition of CK-M activity is achieved within a few minutes and persists over a prolonged time. Table I shows, that in accord with the specificity of the antibodies for M subunits, within experimental error the activity of CK-MM is inhibited to 100 ~o, the activity of CK-MB to 50 ~o, and the activity of CK-BB to 0 ~. The effect can be reproduced in a CK activity range from less than 100 to 1000 U/l, Data from experiments with purified human CK isoenzymes are presented, which show the activity inhibiting action of anti-CK-MM in dependence from reaction time, from different CK substrates, and from different CK activators. These inhibiting anti-CK-MM sera can be utilised in a quantitative test for measurement of CK-MB Table 1. Inhibition of creatine kinase isoenzyme activity by inhibiting anti-CK-MM. Inactivated human sera with addition of purified CK isoenzymes. Mean values + 1 s from 5 fold measurements