THROMBOSIS RESEARCH 66; 33-42,1992 0049-3848/92 $5.00 + .OO Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.

DETERMINATION OF R DNA HIRUDIN AND A-HUMAN THROMBIN- HIRUDIN COMPLEX IN PLASMA SAMPLES: ENZYME LINXED INNUNOSORBENT ASSAYS ASSAY FOR HIRUDIN AND COMPLEX VS. CHROMOGENIC TRROMBIN SUBSTRATE J. Piinter, J.Reindl, P. Seipp Hoechst AG, Frankfurt am Main, FRG

G. Berscheid

(Received

20.8.1991;

, H. Grijtsch, H. Neubauer,

accepted

in revised form 20.1 .1992 by Editor F. Markwardt)

ABSTRACT

in man and rhesus monkeys were rDNA hirudin plasma concentrations determined over a period of 15 and 24 h. The plasma concentration of c-human thrombin-hirudin complex was measured after administration of the complex to rhesus monkeys. The complex was also determined after administration of hirudin to man and rhesus monkeys to study a possible formation of a complex with c-human thrombin in blood. The determination of hirudin was performed by a sandwich ELISA, using and the chromogenic thrombin antibodies polyclonal and monoclonal substrate assay. The or-human thrombin-hirudin complex concentration in the plasma of rhesus monkeys was measured over a period of 48 hours. The results of a sandwich ELISA were compared with those of the chromogenic thrombin substrate assay. A good agreement between the total hirudin concentrations analyzed by the hirudin ELISA and the LYhuman thrombin-hirudin complex ELISA and those of the chromogenic thrombin substrate assay, measuring total hirudin, too, was observed.

INTRODUCTION Routine analyses of hirudin in plasma are being increasingly performed by the chromogenic thrombin substrate assay (1, 2), but this assay does not distinguish between free hirudin and the (Yhuman thrombin-hirudin complex because according to Grijtsch et al. (2) the complex is destroyed during the heat denaturation at the released hirudin makes a corresponding 65OC (1) and contribution to free hirudin. Immuno assays are applicable and useful as reference methods as well. Recently, some immuno assays were developed for the determination of r hirudin (3, 4, 5, 6). The aim of this investigation was to establish a sandwich ELISA for routine analyses of r hirudin with high sensitivity, sufficient accuracy, good reproducibility and capacity for rapid analyses of large amounts of samples. For determination of -----_Key words: hirudin; thrombin-hirudin complex; chromogenic thrombin substrate assay.

33

ELISA

for hirudin

and complex;

34

HIRUDIN-COMPLEX

COMPAR. METHODS

Vol. 66, No. 1

c-human thrombin-hirudin complex (THC) . Bichler et al. (7) reported on the development and his experience with an ELISA for research purposes. The analyses are feasible when the method described is used. ELISAs are also useful if interfering substances such as anticoagulants other than hirudin are involved The present study was nerformed to examine the (8). _ correspondence of results of the determination of total hirudin concentrations in plasma, analyzed by both the hirudin and the THC ELISA, with those of the chromogenic thrombin substrate assay.

MATERIALS Materials

AND METHODS

for hirudin ELISA

Microtitre plates (Maxisorb F96, with certificate) were purchased from Nunc, Wiesbaden, FRG, Microplate washer (Titerteke Microplate Washer M96) and microplate photometer (Titertek Multiscan@ Plus. ME II) from ICN FLOW, Meckenheim, FRG. Bovine serum albumin (BSA) was obtained from Serva, Heidelberg, FRG, phosphate buffered saline (PBS) tablets, from Sigma, Miinchen, FRG, 1,2-phenylene diamine from Dakopatts, Hamburg, FRG, and Tween 20 from Riedel de Haen, Seelze, FRG. All other chemicals of analytical grade were purchased from Merck, Darmstadt, FRG. The e-human thrombin was supplied from Behringwerke, Marburg, FRG. The rDNA hirudin (HBW 023) and the c-human thrombin-hirudin complex were obtained from Hoechst AG, Frankfurt, FRG. Materials

for THC ELISA

Microtitre plates (Linbro II Plus) were purchased from ICN FLOW, Meckenheim, FRG. All other materials used are described above. Preparation

of the a-human thrombin-hirudin

The c-human thrombin-hirudin complex was 5 fold surplus of r hirudin (HBW 023, thrombin (Behringwerke). The complex was hirudin by chromatography according to activity of 0.2 c(g of free hirudin per mg

complex

synthesized by adding a Hoechst AG) to o-human separated from the free Grijtsch et al. (9). An complex was measured.

Immunogen for hirudin ELISA P3CSG*-hirudin was synthesized by coupling HBW 023, to P CSG-NDep. of 8 rganic hydroxysuccinimide (Wiesmtiller and Jung, Chemistry, University of Tiibingen, FRG) according to Bessler et al.(lO). Antibodies

for hirudin ELISA

Polyclonal antibodies were developed by immunizing sheep with P3CSG-hirudin. Monoclonal antibodies were obtained by fusion of ------* P3CSG = Tripalmitoyl-S-glyceryl-cysteinyl

Seryl-glyCin

HIRUDIN-COMPLEX

Vol. 66, No. 1

COMPAR. METHODS

35

mouse myeloma cells with spleen cells isolated from balb/c mice that had been immunized with P3CSG-hirudin. IgG fractions were fluid, respectively by serum and ascites isolated from precipitation with saturated ammonium sulfate solution. After centrifugation the precipitate was redissolved in PBS to the starting volume and used without further purification. Antibodies

for

THC ELISA

Affinity purified rabbit anti o-human thrombin and horseradish labelled sheep anti-hirudin antibodies were peroxidase-(HRP) kindly provided by Behringwerke, Marburg, FRG. ELISA

procedure

for

the determination

of rDNA

hirudin

The Elisa was performed according to the principle of Voller (11) with modifications. All a1.(4, and Spinner et 5) incubation steps were performed for 90 min at 25°C. The washing steps after each incubation were carried out three times with PBS/Tween, pH 7.4. Microtitre plates were coated with 100 ~1 of monoclonal mouse anti-hirudin antibody (1.5 pg/ml) in O.lM sodium carbonate buffer pH 9.6. Citrated plasma samples were diluted with PBS according to the expected concentration of r hirudin. Standard and control stock solutions were diluted with PBS to yield final concentrations of 0.01, 0.163, 0.625, 1.25, 2.5, and 5.0 ng/ml for standards and 0.17, 1,7 and 3.3 ng/ml for controls in plasma, respectively. Standards, controls and samples (50 ~1 each) were added to the wells in triplicates. Next, polyclonal sheep anti-hirudin (100 ~1) was added, followed by 100 1.11HRPlabelled anti-sheep IgG. The substrate reaction was performed and diamine/H202 optical absorbance was with 1,2-phenylene measured at 492 nm. Concentrations were calculated from a hirudin standard curve. ELISA

procedure

for

the determination

of THC

The ELISA for the determination of THC in the plasma of rhesus monkeys was performed as decribed by Bichler et al. (7). The anti-o-human was thrombin absorbed to the surface of the microtitre plate and the HRP-labelled anti-hirudin antibodies were used to sandwich the complex at its hirudin site. Substrate reaction, measurement of optical absorbance and calculation of plasma concentrations were performed as described above. Chromogenic

thrombin

substrate

assay

The chromogenic thrombin substrate assay was performed according to Griesbach et al. (l), modified by Grotsch et al. (2) who used hirudin standards (50, 75, 125, 175 ng/ml) in the blank plasma of each individuum instead of aqueous standards. The evaluation of hirudin concentrations was performed by an regression line calculated from the measured optical densities vs. the concentrations of the individual plasma standards.

36

HIRUDIN-COMPLEX

COMPAR. METHODS

Vol. 66, No. 1

RESULTS The aim of this study was to evaluate an ELISA for routine determination of rDNA hirudin (HBW 023) and to compare the results of the Elisa methods for hirudin and THC with those of the established chromogenic thrombin substrate assay. In addition it should be examined by the THC ELISA whether r hirudin passes the body unchanged or is complexed by o-human thrombin giving an explanation for the reduced excretion of free hirudin compared to the applied dose (12). The hirudin ELISA developed has a working range between 0.06 and 5.0 ng/ml. Repeated measurements revealed an average coefficient of variation of 7.0%. For reproducibility the coefficient of variation ranged from 4.0-7.6%. The results are presented in Tables l-3 and Figures l-3. The plasma concentrations of hirudin and of THC in three patients after intravenous administration of r hirudin (O.lmg/kg b.wt.) are listed in Table 1. Only a low THC concentration was detected by the THC ELISA (column 4) and calculation of the hirudin content of THC (column 5) contributes very little to the hirudin concentration analyzed by the hirudin ELISA (column 3). Hirudin concentrations of columns 3 and 5 were added to give the total hirudin concentration determined by both ELISAs (column 6) which was compared with the total hirudin concentration measured by the chromogenic thrombin substrate assay (column 2). Comparing the total hirudin concentrations measured, a good agreement between the hirudin ELISA and the chromogenic thrombin substrate assay was observed as demonstrated in Fig. 1. The results of the hirudin determination in the plasma of rhesus monkeys after i.v. administration of r hirudin (0.1 mg/kg b.wt.) are presented in TABLE 3. As shown in Fig. 3, there is a good correspondence between both methods. The THC concentrations in the plasma of rhesus monkeys after i.v. administration of 0.4 mg/kg b.wt. (rhesus monkey no.2) and 0.6 mg/kg b.wt. (rhesus monkeys no.4 and no.6) are summarized in Table 2. The determination was performed directly by the THC ELISA (column 2) and indirectly in terms of hirudin by the chromogenic thrombin substrate assay (2) measuring the released hirudin together with unbound hirudin (column 3). Additionally the concentrations of free hirudin were determined by the hirudin ELISA (column 5). For comparison of methods the hirudin content of THC was calculated from the known molecular weights of THC (M.W. = 43.000) and HBW 023 (M.W. = 7.000) (column 4).The concentrations of free hirudin measured by the hirudin ELISA were added to those calculated from THC ELISA to (column 6) which was result the total hirudin concentration compared with the total hirudin concentration measured by the chromogenic thrombin substrate assay.(column 3) As pointed out in Fig. 2 the results of the THC analysis in terms of hirudin demonstrate the good correspondence of the two methods. In addition the hirudin ELISA developed makes it possible to determine free r hirudin in the presence of o-human thrombinwith the chromogenic This is not possible hirudin complex. thrombin substrate assay as demonstrated above.

Vol. 66, No. 1

HIRUDIN-COMPLEX

COMPAR. METHODS

TABLE

1

Plasm~fw&xzien~ratio,n pf r h.irudin of . . admlnlstratlon Hirudin Time

Chromog. assay

and 0.1

THC ELISA

37

ELISA

Hirudin calculated from THC ELISA

Total hirudin ELISAs

'atient 1 2'::

342 536

331 592

17.78 10.59

2.89 1.72

334 595

::: z::

208 172 108 126

218 167 105

5.21 3.55 3.18 5.49

0.58 0.85 0.89 0.52

219 168 106

::: 1%

42 63

Determination of r DNA hirudin and A-human thrombin- hirudin complex in plasma samples: enzyme linked immunosorbent assays for hirudin and complex vs. chromogenic thrombin substrate assay.

rDNA hirudin plasma concentrations in man and rhesus monkeys were determined over a period of 15 and 24 h. The plasma concentration of alpha-human thr...
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