fibrin. comparison with coatest BIA t-PA

122

POSTER PRESENTATIONS P-11: Assay Methodology

341 MEASUREMENT OF tPA ANTIGEN:

COMPARISON OF TINTELIZE@ AND IMUBIND@ METHODS

‘Strooo DM. 2Glueck CJ. ’Glueck HI, 2Hamer T, 2Tracy T, and ‘Schumacher HR.

‘Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine and the 2Cholesterol Center, Jewish Hospital, Cincinnati, OH, USA Our specific aim was to compare two commercially available tissue-Plasminogen Activator (tPA) antigen ELISA kit assays (the Biopool AB, Ltd. TintElizeB tPA and the American Diagnostica, Inc. Imubind8 total tPA ELISA) in 70 patients sequentially studied for thrombotic disorders. Each method measures bound or unbound tPA antigen immobilized to a solid matrix (the walls of a plastic microtiter plate) previously coated with polyclonal antihuman tPA IgG antibodies. A second horseradish oeroxidase-labled monoclonal liaand is added which binds to other free antigenic deierminants on the tPA antigen immobilized to the wells by the coat antibody,

342 BIO-IMMUNO ASSAY FOR TISSUE-TYPE PLASMINOGEN ACTIVATOR (t-PA) IN (STABILYTE@) PLASMA. Bos R. Revet M and Nieuwenhuizen W. Gauhius Laboratory, TNO-PG, Leiden, The Netherlands. A low level of active tissue-type plasminogen activator (t-PA) in plasma is associated with (the risk for) thrombo-embolic complications. Determination of t-PA activity may be hindered by protease inhibitors, i.e. plasminogen activator inhibitor type-l (PAI-I), and various other plasma components. We developed a two-step Bio-lmmuno Assay (BIA) for the specific assessment of t-PA activity, especially suited for acidified (Stabilytb) plasma’s, The assay employs a t-PA specific monoclonal antibody immobilised to a microtiter plate. Plasma samples, diluted in TrislTween 80 pH 6, are incubated at 4°C overnight (step 1). After a thorough wash with Tris/Tween 80 pH 6, the wells are rinsed once with Tris/Tween 80 pH 8. To quantitate bound t-PA activity, Glu-plasminogen (optimum 80 nM); CNBr digested fibrinogen (optimum 80 pglml); and the chromogenic plasmin substrate D-Val-Leu-Lys-pNA (optimum 0.8 mM) in TrislTween 80 pH 8 are added and the plate is incubated at 37°C (step 2).

343

INFLUENCE USING

OF ENDOGENOUS

COASET

WITH COATEST mWeikum. E.

t-PA AND BIA

SC&PA

IN t-PA DETERMINATIONS

SPECTROLYSE@

/FIBRIN.

forming a “sandwich” (coat antibody:tPA antigen:conjugate antibody). The within run coefficients of variation for the TintElize (n=15) and lmubind (n=ll) methods were 9.0% and 7.2%, respectively. Between run coefficients of variation (n=5) were 14.5% for TintElize and 9.2% for Imubind. In duplicate aliquots, the intraclass correlation for TintElize vs lmubind methods was 0.85 (p
The release of pNA is followed in time by repeated measurements of the absorbance (A) at 405 nm. The AA/t2 is an accurate measure for the active t-PA concentration in the sample. The assay has a lowest detection level of 3 pg active t-PA/ml and does not discriminate between recombinant, one- or twochain t-PA. The intra-assay coefficients at relatively high and low t-PA concentrations were 5.5 % and 4.3 %, respectively. Active t-PA values determined in samples (N=66) with this assay and with the t-PA BIA from Chromogenix correlated well (r=0.78). With a subset of samples (N= 18), a negative correlation with both t-PA/PAI-I (r=0.79) and PAI-I antigen (r=0.78) values was observed. Surprisingly, no correlation was found with values for t-PA antigen (r=0.21). Samples of citrate4l plasma gave a response of 10% or less, as compared with their corresponding Stabilyte@ plasma sample. Immu~w t-PA depleted plasma gave no response in the BIA. Urokinase. added to t-PA depleted plasma up to 100 nglml, gave no response. Omittance of any component in step 2 of the assay, resulted in an elimination of the response.

To test this hypothesis, acidified

COMPARIX?N

with HCI)

absence of a monoclonal

t-PA and S. Rosen. Chromogenix

t-PA).

AB, Miilndnl,

In parallel,

values without Homogeneous

t-PA activity

and SpectroIyse%ibrin

t-PA in plasma is allowed sensitive chromogenic concentration

methods such as COASET

(BioPool)

presence of a stimulator.

to convett added plasminogen

microtiterplate

where

to plasmin in the

The generated plasmin is detected by a plasmin substrate resulting in release of pNA. The

of pNA

COATEST

t-PA (Chromogenix)

are based on the same principle

should reflect the amount of active t-PA contained in

the plasma and is measured spectrophotometrically. BIA

In the principally

t-PA, t-PA in plasma is immobilized

wells prccoated with a monwlonal

antibody

by binding to

against t-PA.

After binding is complete, unbound substances are washed off followed addition of plasminogen and chromogenic substrate allowing plasmin formation Comparing

by

and pNA release. t-PA with COATEST

BIA

t-PA, we

explanation

for this discrepancy

to u-PA by the plasmin

could be conversion of endogenous scu-PA

formed during assay in the COASET

t-PA system.

This does not occur in the BIA t-PA system since the speciticity of the immobilized monoclonal antibody eliminates influence from endogenous

plasmas further

t-PA.

in presence or

against scu-PA (not cross reacting with

the same plasmas

anti-scu-PA

almost identical

were analysed using the BIA t-PA

antibody

were compared. The COASET

t-PA

were 2-3 times the BIA t-PA values but

to the latter in presence of the scu-PA antibody. t-PA activities, IU/mL

COASET

t-PA+MAb

0.33

0.28 0.29

0.48

0.51 0.59

COASET

t-PA+MAb

0.76

0.87

I .47

1.46

BIA

0.28

t-PA

0.45

The effect of endogenous scu-PA can be general for this type of assays since a similar

effect was found analysing

using Spectrolyse/tibrin scu-PA antibody without

t-PA activities

Thusthe resulting

were approximately

in three patient plasmas

t-PA activities

in presence of

half of the corresponding

activities

the antibody.

In conclusion.

t-PA values from COASET

found that the COASET t-PA values were approximately twice the BIA t-PA values (BIA t-PA = 0.14 + 0.45 COASET t-PA, r = 0.90, n = 37 ). A possible

scu-PA.

antibody

system and the obtained t-PA activities

SU%deo.

different

four patient plasmas (Stabilyte@

were analysed using COASET

the results strongly suggest that t-PA activity

determinations

using COASET t-PA or Spcctrolysc/ tibrin will bc influenced by endogenous scu-PA and consequently ovcrestimace the t-PA activity plasma.

in the