The development and application of a surface plasmon ... - DCU

Journal of Immunological Methods 291 (2004) 11 – 25 

Research paper 

The development and application of a surface plasmon 

resonance-based inhibition immunoassay for the determination 

of warfarin in plasma ultrafiltrate 

Brian Fitzpatrick, Richard O’Kennedy* 

School of Biotechnology and National Centre for Sensor Research, Dublin City University, Glasnevin, Dublin 9, Ireland 

Received 5 January 2004; received in revised form 26 February 2004; accepted 8 March 2004 

Available online 5 June 2004 

www.elsevier.com/locate/jim 

Abstract 

Warfarin is the most widely prescribed oral anticoagulant for the management of a wide variety of thromboembolic disorders 

such as atrial fibrillation and deep vein thrombosis. A panel of warfarin–protein conjugates were produced and characterised 

and subsequently used for the production of monoclonal antibodies to warfarin. Following characterisation, the monoclonal 

antibodies were used in the development of a surface plasmon resonance-based inhibition immunoassay for the determination of 

the physiologically active ‘nonprotein’-bound fraction of the drug in plasma ultrafiltrate. The inhibition immunoassay was 

compared with an existing high-performance liquid chromatography (HPLC) chromatographic technique for the determination 

of warfarin in plasma ultrafiltrate, and an excellent correlation was achieved between the two independent analytical techniques. 

The ligand-binding capacity and stability of various immobilised ligands were also compared. The BIACore-based inhibition 

immunoassay demonstrated an assay precision range of approximately 4–250 ng/ml, which is within the clinical range and 

demonstrated good reproducibility and robustness. 

D 2004 Elsevier B.V. All rights reserved. 

Keywords: Warfarin; Inhibition immunoassay; Surface plasmon resonance; Nonprotein bound; Ultrafiltration 

1. Introduction 

Abbreviations: RU, Response units; HPLC, high-performance 

liquid chromatography; BSA, bovine serum albumin; KLH, keyhole 

limpet haemocyanin; CM, carboxymethylated; IR, infrared; NMR, 

nuclear magnetic resonance; ELISA, enzyme-linked immunosorbent 

assay; ANOVA, analysis of variance; DMSO, dimethyl 

sulphoxide; HBS, HEPES-buffered saline; EDC, N-ethyl-N-(dimethylaminopropyl)-carbodiimide 

hydrochloride; NHS, N-hydroxysuccinimide; 

PDA, photo-diode array. 

* Corresponding author. Tel.: +353-17005319; fax: +353- 

17005412. 

E-mail address: r.okennedy@dcu.ie (R. O’Kennedy). 

The oral anticoagulants emerged from veterinary 

research carried out in the 1920s, showing that a 

haemorrhagic disorder affecting cattle was caused by 

the consumption of spoiled sweet clover hay. The 

offending agent was subsequently identified by Link 

(1943–1944) as dicoumarol (3,3V-methylenebis-4- 

hydroxycoumarin), which occurs during the spoilage 

of cured hay, whereby coumarin is oxidised to 4- 

hydroxycoumarin, which, when coupled with formaldehyde 

leads to the production of dicoumarol. Link 

0022-1759/$ - see front matter D 2004 Elsevier B.V. All rights reserved. 

doi:10.1016/j.jim.2004.03.015

12 

B. Fitzpatrick, R. O’Kennedy / Journal of Immunological Methods 291 (2004) 11–25 

and his team synthesised over 100 4-hydroxycoumarin-type 

compounds and found that the minimal 

structural requirements for anticoagulant activity 

were an intact 4-hydroxycoumarin residue with the 

3-position substituted by a carbon residue. Warfarin 

was subsequently synthesised (Ikawa et al., 1944) 

and is currently the most widely prescribed oral 

anticoagulant for the management of a variety of 

thromboembolic disorders including atrial fibrillation, 

deep vein thrombosis and threatened stroke 

(Hirsh et al., 1995). 

Warfarin exerts its anticoagulant effect by inhibiting 

the posttranslational carboxylation of the vitamin 

K-dependent precursor proteins factors II, VII, IX 

and X, resulting in the production of partially carboxylated 

forms of these vitamin K-dependent proteins. 

The presence of these partially carboxylated 

proteins, named PIVKAs (proteins induced by vitamin 

K antagonism), results in a slower activation of 

the coagulation cascade, as the g-carboxyglutamyl 

residues confer unique metal-binding properties to 

the proteins. Upon occupancy of these metal-binding 

sites, these proteins undergo a conformational 

change, which permit interactions with the phospholipid 

bilayers and cell membrane providing a template 

for the clotting mechanism (Cooke et al., 

1997). 

Warfarin is administered orally as a racemic mixture 

of (R-) and (S-) warfarin and is rapidly absorbed from 

the gastrointestinal tract reaching peak plasma concentrations 

within 60–90 min following administration 

(King et al., 1995). Following absorption, the drug is 

highly bound to plasma proteins (>99%), leaving only 

a small fraction of the free drug available for metabolism. 

It is the ‘nonprotein’-bound or ‘nonprotein’-free 

fraction of the administered drug that dictates its 

pharmacokinetic profile. The primary warfarin-binding 

site on human serum albumin (HSA) has been identified 

on domain II with a secondary binding site on 

domain I (Dockal et al., 1999). The (S-) enantiomer has 

been shown to have a higher affinity for HSA than the 

corresponding (R-) enantiomer (Bertucci et al., 1999). 

This high degree of protein binding predisposes warfarin 

to a range of possible drug interactions, which in 

turn can greatly affect the degree of anticoagulation 

achieved (Wells et al., 1994). Moreover, the (S-) 

enantiomer has also approximately five times the 

anticoagulant potency of the (R-) enantiomer. 

Warfarin has also shown promising results as a 

potent HIV-1 inhibitor in vitro, and this has led to the 

development of a potent orally bioavailable class of 

5,6-dihydro-4-hydroxy-2-pyrones compounds, which 

are undergoing clinical trials (Thaisrivongs and Strohbach, 

1999). Warfarin has also demonstrated potential 

as an antimetastatic agent and has shown particularly 

promising results with small cell carcinoma of the 

lung, a tumour cell type that is characterised by a 

coagulation-associated pathway (Zacharski et al., 

1984; Amirkhosravi and Francis, 1995). 

There is currently a wide range of analytical 

techniques available for the determination of warfarin 

in biological fluids ranging from chromatography to 

phosphorescence-based measurements (Capitán-Vallvey 

et al., 1999; Ring and Bostick, 2000). The 

majority of these analytical techniques involve 

lengthy sample pretreatment, derivatisation schemes 

and postcolumn reactions for the determination of the 

free ‘unbound’ fraction of warfarin in plasma samples 

and, consequently, are not routinely applicable for the 

detection of warfarin in clinical samples. The use of 

chromatographic techniques relies on the inherent 

physicochemical properties of the analyte in question, 

and the measured response is usually directly proportional 

to the concentration of the analyte and is best 

represented by a linear calibration model. The physicochemical 

properties of a given analyte and the lack 

of suitable chromophoric or electrochemically active 

groups can therefore be limiting factors for the detailed 

analysis of the pharmacokinetic profile of 

particular drug molecules. Immunoassays rely on the 

specific interaction between antibody and antigen for 

analyte determination, and the measured response 

usually gives rise to a calibration plot that is inherently 

sigmoidal in nature and best represented by a 

four-parameter logistic fit (Findlay et al., 2000). 

Recent advances in antibody technology have permitted 

the standardisation of antibody preparations and 

revolutionised their use as clinical and diagnostic 

tools. The capability of producing antibodies and 

fragments thereof with enhanced affinities (i.e., 

K D =10 

15 M; Boder et al., 2000) allied with developments 

in transduction technologies have greatly 

advanced the potential of antibody-based techniques 

for the detection of low molecular weight analytes in 

solution. Similarly, given the unique specificity of 

antibodies and the use of antibody libraries, common

B. Fitzpatrick, R. O’Kennedy / Journal of Immunological Methods 291 (2004) 11–25 13 

biosensor formats can now be easily and quickly 

adapted for the detection of specific analytes in 

solution without the need for developing individual 

chromatographic assay separation techniques. The 

current trend in warfarin therapy is towards lower 

intensity treatment. Consequently, there is the need to 

develop more sensitive analytical techniques capable 

of detecting lower concentrations of warfarin in 

biological fluids for the accurate determination of 

the physiological-free fraction of warfarin in plasma 

ultrafiltrate. 

Surface plasmon resonance-based techniques using 

BIACore have been successfully described for the 

detection of low molecular weight analytes (Crooks 

et al., 1998; Daly et al., 2000; Brennan et al., 2003). 

The majority of these techniques generally employ 

inhibition-based assay formats, whereby an equilibrium 

antibody–antigen mixture is passed over the 

surface of a functionalised chip surface (see Jönsson 

et al., 1991 for a more detailed description on the 

mode and operation of BIACore). Unbound antibody 

in the equilibrated mixtures is available for binding to 

the derivatised chip surface as the equilibrium mixtures 

pass over the chip surface. The measured binding 

response is therefore inversely proportional to the 

concentration of free analyte in solution. Here, we 

describe the development and application of a surface 

plasmon resonance-based inhibition immunoassay for 

the detection of the free fraction of warfarin in plasma 

ultrafiltrate and comparison of the inhibition immunoassay 

with a chromatographic technique. The suitability, 

ligand-binding capacity and assay performance 

characteristics of various immobilised ligands are also 

addressed. 

2. Materials and methods 

2.1. Equipment 

A Brucker 400 MHz AC400 nuclear magnetic 

resonance (NMR) spectrometer (Coventry, England) 

was used for NMR analysis of 4V-aminowarfarin. A 

Nicloet infrared (IR) spectrometer (Madison, WI, 

USA) was used to generate the IR spectra of 4V-aminowarfarin. 

A Beckman System Gold high-performance 

liquid chromatography (HPLC) apparatus with photodiode 

array (PDA) detection (Fullerton, CA, USA) was 

used for the analysis of the drug–protein conjugates. 

Protein fractions were analysed using a Shimadzu UV- 

160A spectrophotometer (Kyoto, Japan). Concentration 

of plasma ultrafiltrate and tissue culture supernatant 

was carried using an Amicon Ultrafiltration Stirred 

Cell 8400 (Beverley, MA, USA). A BIACorek 3000 

biosensor instrument and CM5 Chips (Pharmacia Biosensor, 

St. Albans, Hertfordshire, England) controlled 

with BIAEvaluation software version 3.1 were used to 

perform the biosensor study. Warfarin detection by 

HPLC was carried out using a Supelco Discoveryk 

column (Bellefonte, PA, USA) and a Varian Star 9030 

pump, 9012 UV detector, fluorescence detector and AI- 

200 autosampler system modules obtained from JVA 

Analytical (Dublin, Ireland). 

2.2. Reagents 

4V-Nitrowarfarin, acenocoumarin (SintromR) was 

kindly donated by Ciba-Geigy (Dublin, Ireland). 

Zinc granules, acetic acid, sodium chloride, Tris, 

Freund’s Complete adjuvant, N-hydroxy-succinimide 

(NHS), N-ethyl-N-(dimethylaminopropyl)-carbodiimide 

hydrochloride (EDC), Ethanolamine, EDTA, 

Tween-20, sodium nitrite, bovine serum albumin 

(BSA), thyroglobulin, keyhole limpet haemocyanin 

(KLH), 10 kDa dialysis tubing, protein-G Sepharose 

4B and warfarin were all purchased from Sigma 

(Poole, Dorset, England). BALB/c mice were purchased 

from Harlan UK (Bicester, Oxon, England). 

TLC plates and silica gel grade 40 were purchased 

from Riedel de Haën (Hanover, Germany). Deuterated 

dimethyl sulphoxide (DMSO) was purchased 

from Aldrich Chemical (Gillingham, Dorset, England). 

Filters (0.2 Am) were purchased from AGB 

Scientific (Dublin, Ireland). Acetonitrile and HPLC 

vials were purchased from JVA Analytical (Dublin, 

Ireland). 

2.3. Preparation and characterisation of 

4V-aminowarfarin 

4V-Nitrowarfarin (2.00 g) was reduced in the 

presence of 3.00 g zinc and 50 ml of 30% (v/v) 

dilute acetic acid with constant stirring to yield the 

‘putative’ 4V-aminowarfarin product. 4V-Aminowarfarin 

was partially purified from the initial reaction 

mixture by filtration through Whatman filter paper

14 


(grade number 1). The partially purified reaction 

mixture following filtration was then purified to 

homogeneity by silica gel column chromatography 

through a 20-cm glass column containing silica gel 

grade 40 using a mobile phase of methanol/chloroform 

(2:98, v/v), and 10 ml fractions was collected. 

Fractions were analysed by thin layer chromatography 

(TLC), and those fractions containing 4V-aminowarfarin 

were evaporated to dryness using a rotary 

evaporator. The reaction product 4V-aminowarfarin 

was characterised by a combination of IR and NMR 

spectral studies. 

2.4. Preparation and characterisation of 4V-azowarfarin–protein 

conjugates 

4V-Aminowarfarin (350 mg) was dissolved in 50 ml 

of a solution containing 0.1 M HCl and 7 mM 

potassium bromide. Ten milliliters of a 0.014 M 

solution of sodium nitrite was added dropwise to the 

solution containing 4V-aminowarfarin, and continuous 

stirring was maintained on an ice bath for 1 h. The 

activated 4V-aminowarfarin solution was then added 

dropwise to 10 ml of a 5% (w/v) solution of bovine 

serum albumin (BSA), the pH maintained between 9.0 

and 9.5 with the simultaneous addition of 0.1 M NaOH 

and was stirred overnight at 4 jC (Satoh et al., 1982). 

The resulting dark brown solution was then dialysed 

against several changes of PBS, pH 7.4 and lyophilised 

prior to further studies. While BSA binds warfarin 

under physiological conditions, our studies indicated 

that it gave excellent levels of chemical conjugation to 

4V-azowarfarin. For the production of keyhole limpet 

haemocyanin (KLH) conjugates, the protein solution 

was made up in PBS, pH 7.4, containing 0.9 M NaCl, 

to allow for solubilisation of KLH. 4V-Aminowarfarin 

(100 mg) was activated and added to 40 mg of KLH 

and the reaction followed as described above. The 

resulting drug–protein solution was dialysed against 

several changes of PBS, pH 7.4 with 1.0 M NaCl 

added. The resulting dark brown solution was lyophilised 

for further studies. The drug–protein conjugates 

following lyophilisation were characterised by UV 

spectroscopy by direct comparison of the reference 

spectra obtained for the drug–protein conjugate, 4Vaminowarfarin 

and a ‘control’ protein (i.e., a protein 

solution which had undergo the same chemical procedure 

as the conjugate solution except that the drug 

solution had been omitted from the coupling step) 

from 200 to 400 nm. HPLC-PDA studies using a 

Biosep SEC 4000 column were also recorded from 

200 to 400 nm using PBS, pH 7.4, as mobile phase at a 

flow rate of 1.0 ml/min. 

2.5. Monoclonal antibody production and purification 

of murine IgG by protein-G affinity chromatography 

Male BALB/c mice, 5–10 weeks of age, were used 

for the production of monoclonal antibodies to warfarin, 

as previously described (Fitzpatrick, 2001). All 

procedures involving the use of animals were approved 

and licensed by the Department of Health. 

Every precaution was taken to ensure the minimum 

distress to the animals. Hybridoma clones were 

screened for specific antibody production by direct 

enzyme-linked immunosorbent assay (ELISA), and 

‘positive’ wells were propagated and cloned by limiting 

dilution. ‘Spent’ hybridoma supernatants from 

hapten-specific clones were collected and concentrated 

10-fold using an Amicon concentration device. 

Concentrated supernatant (10 ml) was passed through 

a column containing protein-G immobilised on 

Sepharose 4B, the eluate collected and passed through 

the column a second time. The column was then 

washed with 25 ml of PBS containing Tween 

(0.05%, v/v) and the retained protein eluted with 0.1 

M glycine–HCl (pH 2.5). Fractions (850 Al) were 

collected in eppendorf tubes containing 150 Al of 

Tris–HCl (pH 8.5). The absorbance of each fraction 

was then measured at 280 nm for protein concentration 

determination. 

2.6. Sample preparation 

Blank plasma was filtered through an Amicon 

concentration device containing a 1-kDa molecular 

weight cut-off membrane and collected under positive 

pressure and stored at 20 jC until required for 

further use. Antibody preparations were diluted in 

HEPES-buffered saline solution (HBS running buffer, 

pH 7.4) prior to sample analysis (i.e., nominally a 

1:100 dilution) and filtered through a 0.2-Am filter 

prior to use. A 10 mg/ml solution of warfarin was 

initially prepared in DMSO. Serial dilutions of warfarin 

were then prepared in plasma ultrafiltrate ranging 

from 0.48 to 500 ng/ml. An additional set of indepen-


dent calibration standards of various concentrations 

spanning the range of the calibration plot was also 

prepared in plasma ultrafiltrate for each assay procedure 

carried out. 

2.7. Preparation of running buffer used in BIACore 

analyses 

HEPES-buffered saline (HBS), containing 50 mM 

NaCl, 10 mM HEPES, 3.4 mM EDTA and 0.05% (v/v) 

Tween-20, was prepared by dissolving 8.76 g of NaCl, 

2.56 g of HEPES, 1.27 g of EDTA and 500 Al of 

Tween-20 in 600 ml of ultrapure water. The pH of this 

solution was adjusted to 7.4 with the addition of 2 M 

NaOH and the final volume adjusted to 1000 ml in a 

volumetric flask. The solution was then filtered 

through a 0.2-Am filter and degassed prior to use. 

2.8. Coupling of 4V-aminowarfarin to CM-dextran 

sensor chip surface 

Initially direct immobilisation of 4V-aminowarfarin 

to the carboxymethylated (CM)-dextran sensor chip 

surface was performed external to the BIACore instrument, 

but it was also carried out in situ. HBS (40 Al) 

buffer was initially added to the well of the sensor chip 

for 5 min; this served to prime the chip surface and was 

removed using ‘lint-free’ absorbent paper. The chip 

surface was then activated by preparing the NHS/EDC 

reaction mixture, as described above, and adding 20 

Al of the reaction mixture to the well of the sensor chip 

for a period of 10 min. The EDC/NHS mixture was 

then removed from the sensor chip well using ‘lintfree’ 

tissue paper. A solution of 50 Ag/mlof4V-aminowarfarin 

in 10 mM sodium acetate buffer (pH 4.5) was 

placed in the sensor chip well for 10 min. The drug 

solution was removed from the sensor chip well using 

‘lint-free’ tissue paper. The remaining unreacted sites 

on the chip surface were deactivated as described 

above. The chip was then washed extensively with 

water and dried under nitrogen. 

2.9. Coupling of 4V-azowarfarin-BSA to CM-dextran 

sensor chip surface 

The carboxymethylated dextran (CM-dextran) surface 

was activated by mixing equal volumes of 100 

mM N-hydroxy-succinimide (NHS) and 400 mM N- 

ethyl-N-(dimethylaminopropyl)-carbodiimide hydrochloride 

(EDC) and injecting the mixture over the 

sensor chip surface for 7 min at a flow rate of 5 Al/ 

min. A solution containing 50 Ag/ml of 4V-azowarfarin–BSA 

in 10 mM acetate buffer (pH 4.5) was then 

injected over the surface for 10 min at a flow rate of 5 

Al/min. Unreacted sites on the sensor chip surface were 

then deactivated by injecting 1 M ethanolamine (pH 

8.5) for 7 min at a flow rate of 5 Al/min. 

2.10. Stability of the immobilised ligand/regeneration 

procedures 

The stability of the immobilised ligands, namely, 

4V-azowarfarin-BSA and 4V-aminowarfarin, was 

assessed by performing consecutive binding–regeneration 

cycles. Antibody (40 Al) at the requisite 

dilution was injected over the derivatised chip 

surface at a flow rate of 10 Al/min and the binding 

response was recorded. Two successive injections of 

5 Al of 30 mM HCl, at a flow rate of 10 Al/min, 

were used to dissociate the bound antibody/antigen 

complex from the chip surface. Greater than 100 

binding–regeneration cycles were conducted over 

each immobilised chip surface, and the measured 

binding response for each antibody injection was 

used to assess the stability (i.e., antibody-binding 

capacity) of the immobilised surface. 

2.11. Salt concentration assay 

Salt solutions ranging from 0.120 to 0.155 M 

NaCl were prepared in ultrapure water, sterile-filtered 

and were degassed prior to use. Antibody (80 Al) 

solution was mixed with 20 Al of the respective salt 

concentration using the BIACore autosampler and 

was allowed to equilibrate for 5 min. The equilibrated 

mixtures were then randomly injected in duplicate 

over the derivatised chip surface at a flow rate of 10 

Al/min for a period of 4 min, and the binding 

response was recorded. The bound antibody complex 

was then dissociated using 30 mM HCl. 

2.12. Nonspecific binding studies 

Nonspecific binding studies were carried out by 

passing antibody at the requisite dilution over both 

the blank CM-dextran chip surface and an immobi-

16 


lised BSA chip surface. The binding response following 

each injection was used to determine the degree of 

nonspecific binding of antibody to the blank dextran 

and/or immobilised BSA surface. 

2.13. BIACore inhibition immunoassay 

Antibody solution (80 Al) was mixed with 20 

Al of warfarin standard using the BIACore autosampler 

and was allowed to equilibrate for a period 

of 5 min. Samples were then injected in duplicate in 

random order at a flow rate of 10 Al/min for a 

period of 4 min over the derivatised chip surface, 

and the binding response was recorded. The bound 

antibody was then dissociated from the derivatised 

chip surface using two 30 s pulses of 30 mM HCl 

at a flow rate of 10 Al/min. The binding response 

was recorded for each warfarin standard concentration 

(R AG ) and divided by the response measured in 

the presence of zero antigen concentration (R 0 ) to 

give normalised binding responses (i.e., R AG /R 0 ). A 

logarithmic calibration plot was then constructed of 

the normalised binding response versus warfarin 

concentration (ng/ml) using BIAEvaluationk software 

version 3.1 and a four-parameter fit fitted to 

the calibration plot. 

2.14. HPLC studies 

The mobile phase for these studies was prepared by 

mixing 570 ml of acetonitrile with 426 ml of water 

and 4 ml of glacial acetic acid. The mobile phase was 

filtered through a 0.2-Am filter and was sonicated for 

20 min. Samples were analysed using a Supelco 

Discoveryk C18 column (150 4.6 mm, particle 

size 5 Am) with UV detection at 308 nm and a mobile 

phase flow rate of 1.0 ml/min. Postrun sample analysis 

of chromatograms was carried out using Varian 

Star software (version 5.1). 

3. Results 

3.1. Stability of the immobilised ligand and nonspecific 

binding studies 

The binding capacity of directly immobilised 4Vaminowarfarin 

(Fig. 1) and 4V-azowarfarin–BSA (Fig. 

Fig. 1. Ligand-binding capacity of a 4V-aminowarfarin drug surface. One hundred forty consecutive binding–regeneration cycles of an 

antiwarfarin antibody over the directly immobilised 4V-aminowarfarin drug surface demonstrated a coefficient of variation for binding response 

over this cycle of 0.82%, with no change in the surface antibody-binding capacity (i.e., Cycle 1 = 446.2, Cycle 140 = 446.4).


Fig. 2. Ligand-binding capacity of a 4V-azowarfarin–BSA surface following 75 consecutive binding–regeneration cycles of an antiwarfarin 

monoclonal antibody. The binding response can be observed to plateau after approximately 20 regeneration cycles. All assays performed using 

such surfaces were thus exposed to at least 20 cycles prior to sample analysis. The binding response demonstrated a coefficient of variation of 

1.88% following the initial priming cycles, with a decrease of approximately 7.2% in the antibody-binding capacity of the immobilised surface 

over this regeneration cycle range. 

2) conjugate surfaces was compared following a series 

of binding and regeneration sequences to ascertain 

which surface provided the optimal ligand-binding 

characteristics over the course of the regeneration 

studies. The surface regeneration studies conducted 

showed that the directly immobilised 4V-aminowarfarin 

drug surface provided excellent stability over the 

regeneration cycles performed (Fig. 1). The coefficient 

of variation of the measured antibody-binding response 

over the course of the 140 regeneration cycles was 

determined to be 0.82%, underlining the stability and 

reproducibility of the measured binding response using 

the immobilised ligand 4V-aminowarfarin. Surface regeneration 

was achieved using two consecutive pulses 

of 30 mM HCl. Such surfaces were found to be 

essentially ‘inexhaustible’ with respect to antibodybinding 

capacity and were in fact used for greater than 

1000 cycles. 

The immobilised 4V-azowarfarin–BSA conjugate 

surface initially demonstrated a relatively high decrease 

in antibody-binding capacity, which began to 

plateau after approximately 20 regeneration cycles 

(Fig. 2). This initial decrease in antibody-binding 

capacity may be attributed to poorly immobilised 

conjugate, and consequently all assays conducted 

using such surfaces were subjected to at least 20 

binding regeneration cycles prior to sample analysis. 

The reduction in binding capacity of the immobilised 

surface, following these initial ‘priming’ regeneration 

cycles, showed a decrease in antibody-binding capacity 

of the immobilised surface of approximately 

7.2% over the following 50 regeneration cycles, 

which is within the previously recommended guidelines 

(Wong et al., 1997), which suggested that the 

surface-binding capacity should remain within 20% 

of the initial measured binding response over the 

course of the assay procedure. 

Dilutions of antibody, nominally 1:100, were 

prepared in HBS buffer and passed sequentially 

over both blank CM-dextran chips and immobilised 

BSA surfaces to ascertain the degree of nonspecific 

binding of antibody to the chip surfaces. The 

binding in all cases was negligible, demonstrating 

that the measured binding response was solely due 

to antibody binding to the various warfarin immobilised 

chip surfaces.

18 


Fig. 3. Influence of salt composition on the antibody-binding response. The antibody-binding signal measured over the salt concentration range 

tested (0.120–0.155 M NaCl) spans twice the physiological range of plasma salt concentration normally observed. Results shown are the 

average of duplicate measurements. 

Fig. 4. Binding response curves for equilibrated antibody–warfarin mixtures. Overlaid interaction curves for various equilibrated antibody/ 

warfarin concentrations in plasma ultrafiltrate. The measured binding response (R AG ) was used to calculate the normalised binding response 

values (R AG /R 0 ), which were subsequently used to construct the calibration curves. The bound antibody showed essentially no dissociation from 

the sensor chip. The sensor chip could be easily regenerated using two 30-s pulses of 30 mM HCl.


3.2. Assessment of matrix composition on the 

antibody/antigen-binding reaction 

The composition of plasma within the general 

population is essentially homogenous, and the major 

contributor to the ionic strength of plasma is sodium 

(>90%) and its associated anions, whose ionic 

concentration is normally in the reference range 

0.133–0.145 M (Zilva and Pannell, 1979). To assess 

the potential effect of various salt concentrations on 

the measured antibody-binding response, a titration 

of salt concentration versus antibody-binding response 

was constructed using salt concentrations 

spanning more than twice the normal physiological 

ionic reference range for sodium (i.e., 0.120–0.155 

M NaCl). A 20-Al sample of the respective salt 

solution was allowed to equilibrate with 80 Al of 

antibody solution for 5 min on the BIACore autosampler 

and injected in random fashion over a 4Vaminowarfarin 

sensor chip surface at a flow rate of 

10 Al/min for 4 min, and the binding response was 

recorded. 

Over the range of salt concentrations tested, there 

was statistically no difference between the binding 

responses recorded as tested by analysis of variance 

(ANOVA). The plasma dilution step employed (i.e., 

one part in five) was experimentally found to be 

optimal in serving to minimise the differences in the 

measured binding responses as a result of the 

altered salt composition. The coefficient of variation 

of the measured binding response over the normal 

physiological reference range for sodium was found 

to be 0.38%, demonstrating that the assay was 

sufficiently robust to withstand altered matrix com- 

Fig. 5. Intraassay (reproducibility) calibration and residual plot for the determination of warfarin in plasma ultrafiltrate (n = 3). The mean 

normalised response value (R AG /R 0 ) at each antigen concentration from three independent assays was used to calculate the calibration curve and 

to determine the intraassay variation.

20 


position without any significant effect on the measured 

antibody-binding response (Fig. 3). 

3.3. Application of the assay for the determination of 

warfarin in plasma ultrafiltrate 

Dilutions of antibody were prepared in plasma 

ultrafiltrate as previously described ranging from 

0.48 to 500 ng/ml. Antibody solution (80 Al; i.e., 

nominally 1:100 dilution) was then mixed with 20 

Al of the respective warfarin-spiked plasma ultrafiltrate 

sample of known concentration and allowed to 

equilibrate for 5 min on the BIACore autosampler. 

The equilibrated mixtures were then passed sequentially 

in random order over the chip surface to 

ensure that there was no bias in the recorded 

measurements (typical overlaid antibody/drug equilibrium 

mixture-binding curves are shown in Fig. 

4). The measured binding response at each warfarin 

concentration was then divided by the binding 

response measured in the presence of zero antigen 

concentration to give normalised response values 

[i.e., response measured in the presence of antigen 

(R AG )/response measured in the presence of zero 

antigen (R 0 )]. 

BIAEvaluation version 3.1 software was then 

used to construct a standard curve of average 

normalised binding response (R AG /R 0 ) versus warfarin 

concentration (ng/ml) and fitted with a fourparameter 

logistic fit (Fig. 5). To demonstrate the 

application and reproducibility of the inhibition 

immunosensor assay, the assay was conducted over 

3 days at 11 different drug concentrations ranging 

from 0.48 to 500 ng/ml with each standard measured 

in duplicate. Additionally, eight independent 

standards were also prepared in plasma ultrafiltrate 

for each calibration curve constructed. The intraassay 

degrees of accuracy are shown in Table 1 and 

demonstrate the accuracy of the technique as observed 

from the percentage accuracy between the 

interpolated and nominal concentrations. The relative 

precision of the assay was also demonstrated by 

the low percentage difference determined between 

duplicate concentration determinations of each independent 

calibration standard interpolated from the 

four-parameter logistic fit using BIAEvaluation version 

3.1 (Table 2). The interassay variation degree 

of precision and recovery were also calculated 

Table 1 

Intraassay precision for the determination of warfarin plasma 

ultrafiltrate a (n =2) 

Concentration 

(ng/ml) 

BIACore 

interpolated 

concentration 

Percentage 

recovery b 

500 267.89 53.58 

250 208.79 83.52 

125 127.34 101.87 

62.5 66.36 106.18 

31.25 32.012 102.44 

15.63 15.86 101.48 

7.81 7.13 91.27 

3.91 4.50 115.21 

1.95 2.21 113.36 

a Duplicate standards were measured on the same day and the 

back-calculated concentration value interpolated by fitting the 

mean normalised response (R AG /R 0 ) to the standard curve constructed 

using the four-parameter logistic model in BIAEvaluation 

(version 3.1). 

b Percentage accuracy was determined by: (back-calculated 

concentration/nominal concentration) 100%. 

(Table 3), from which, the 95% upper and lower 

confidence limits were calculated (Chapuzet et al., 

1997). In this manner a preliminary estimate of the 

upper limit of quantitation (ULOQ) and lower limit 

of quantitation (LLOQ) were determined for those 

samples whose 95% confidence limits were within 

F 25% of the nominal value (Fig. 6). The calibration 

and residual plot and low v 2 value obtained 

demonstrate the ‘goodness of the fit’ of the fourparameter 

calibration plot applied to the data set 

(Fig. 5). 

The coefficients of variation for the interassay 

curves were typically of the order of 2–7% over 

the assay precision range, which demonstrates the 

reproducibility and corresponding applicability of the 

assay procedure for routine sample analysis. The 

estimated assay precision range was determined to 

be from approximately 4–250 ng/ml in plasma 

ultrafiltrate using the monoclonal antibody preparation 

3–2–19 on a directly immobilised 4V-aminowarfarin 

drug surface (Fig. 6). 

3.4. HPLC analysis of warfarin in plasma ultrafiltrate 

samples 

HPLC analysis of warfarin in the plasma ultrafiltrate 

samples was carried out using a slight


Table 2 

Concentration of back-calculated calibration standards determined by HPLC and BIACore immunoassay 

Warfarin calibration 

standard concentration 

(ng/ml) 

BIACore 

replicate 1 a 

BIACore 

replicate 2 a 

Percentage 

difference of 

replicates 1 and 2 b 

BIACore mean 

concentration 

(ng/ml) 

Percentage 

accuracy 

BIACore c 

HPLC assay 

mean concentration 

(ng/ml) 

Percentage 

accuracy 

HPLC c 

187.50 193.34 176.67 8.62 185.00 98.67 183.93 1.9 

112.50 116.46 109.31 6.14 112.88 100.34 108.55 3.51 

100.00 97.87 95.98 1.94 96.93 96.93 102.35 2.35 

93.75 110.67 106.47 3.80 108.57 115.81 95.12 1.46 

56.25 59.60 59.33 0.46 59.46 105.71 57.1 1.51 

50.00 54.61 53.21 2.57 53.91 107.82 47.35 5.3 

46.88 53.43 53.34 0.17 53.39 113.90 48.9 4.31 

28.13 26.15 24.88 4.85 25.51 90.72 30.55 3.86 

25.00 24.01 23.81 0.83 23.91 95.64 27.98 11.92 

23.44 22.25 21.17 4.82 21.71 92.63 16.09 14.44 

14.06 12.05 11.25 6.63 11.65 82.87 11.04 11.68 

12.50 11.97 11.90 0.58 11.94 95.48 12.87 9.81 

11.72 11.31 10.66 5.77 10.98 93.70 7.22 2.63 

7.03 7.37 7.06 4.27 7.21 102.60 6.28 0.48 

6.25 5.90 5.80 1.73 5.85 93.62 6.16 5.66 

5.86 6.24 5.75 7.77 5.99 102.28 N.D. N/A 

3.52 4.15 3.55 14.38 3.85 109.56 N.D. N/A 

N.D. = nondeterminable; N/A= not applicable. 

a BIACore concentration determinations were made by interpolation of the normalised response into the four-parameter standard curve using 

BIAEvaluation (version 3.1). 

b The percent difference between the replicates was determined by: [(replicate 1 replicate 2)/replicate 1] 100%. 

c Percent accuracy was determined for both analytical techniques using: (mean/nominal) 100%. 

modification of the method described (Ring and 

Bostick, 2000), using a mobile phase flow rate of 

1.0 ml/min with UV detection at 308 nm. Postrun 

analysis of the chromatograms was carried out using 

Varian Star software (version 5.1). The retention 

time of warfarin under the specified experimental 

conditions was found to be 5.7 min. The lower limit 

of detection of the HPLC analytical technique was 

determined to be approximately 5 ng/ml. The peak 

area of the individual chromatograms was integrated 

Table 3 

Interassay variation for warfarin in plasma ultrafiltrate (n =3) 

Warfarin Concentration Calculated Concentration (ng/ml) (n =2) a % Recovery b 

(ng/ml) 

Day 1 Day 2 Day 3 Mean Day 1 Day 2 Day 3 Mean %CV 

500.00 301.29 267.89 262.60 277.26 60.26 53.58 52.52 55.45 4.20 

250.00 198.62 208.79 208.76 205.39 79.45 83.52 83.50 82.16 2.35 

125.00 132.92 127.34 129.41 129.89 106.34 101.87 103.53 103.91 2.26 

62.50 69.88 66.36 72.68 69.64 111.80 106.18 116.29 111.42 5.06 

31.25 30.31 32.01 30.96 31.09 96.98 102.44 99.08 99.50 2.75 

15.63 16.03 15.86 15.15 15.68 102.57 101.44 96.95 100.32 2.97 

7.81 7.42 7.13 7.96 7.50 95.03 91.30 101.92 96.08 5.39 

3.91 4.15 4.50 3.94 4.20 106.10 115.10 100.75 107.32 7.25 

1.95 2.08 2.21 2.01 2.10 106.58 113.54 102.99 107.71 5.36 

a Calculated concentration values were determined from the 4-parameter calibration curve using the mean normalised response value and 

interpolated using BIAEvaluation version 3.1. 

b Percent recovery was calculated as: (calculated concentration/nominal concentration)*100%.

22 


Fig. 6. Plot of mean percentage recovery versus log of warfarin concentration for the intraassay curves. A plot of the mean back-calculated 

concentration was constructed against the log of warfarin concentration. The 95% upper and lower confidence limits were then used to assess 

the assay precision range for those samples whose concentrations were within F 25% of the nominal concentration, from which preliminary 

estimates of the upper (ULOQ) and lower (LLOQ) could be made. 

and used to construct a calibration plot of peak area 

versus warfarin concentration (ng/ml; Fig. 7). From 

the equation of the best-fit regression line, it was 

possible to calculate the concentration of warfarin in 

the independent calibration standards as shown in 

Table 2. 

Fig. 7. Standard curve for warfarin (.; 0–500 ng/ml) in plasma ultrafiltrate as determined by HPLC. The peak area determined at each warfarin 

concentration was plotted against the respective warfarin concentration (ng/ml). From the linear calibration plot constructed, the concentration 

of warfarin (ng/ml) in the independently prepared set of calibration standards (o) was back-calculated using the equation of the regression line 

from the constructed standard curve.


Fig. 8. Comparison of analytical techniques. The concentration of warfarin (ng/ml) for the independent set of calibration standards was 

interpolated from each calibration curve using the two independent analytical techniques (i.e., HPLC and BIACore). ANOVA analysis of the 

data sets demonstrated no significant difference between the two analytical techniques for the determination of warfarin in plasma ultrafiltrate 

samples. 

3.5. Comparison of the independent analytical 

techniques 

To assess whether there was any statistical difference 

between the two independent analytical techniques, 

ANOVA analysis was performed on the backcalculated 

concentration of the independent standards 

determined with each assay procedure performed (Table 

2). ANOVA analysis showed there to be no 

statistical difference between the two analytical techniques. 

The constructed X–Y correlation plot of the 

back-calculated concentration values for each analytical 

technique demonstrated excellent correlation between 

the two techniques (R 2 = 0.991; Fig. 8). 

4. Discussion 

The biosensor assay format developed for the analysis 

of warfarin had excellent performance characteristics. 

The directly immobilised 4V-aminowarfarin drug 

surface demonstrated very high stability over the 

course of the regeneration cycle studied, with no loss 

in antibody-binding capacity and high reproducibility 

in the measured binding signal. Any decrease in 

surface-binding capacity was well within the previously 

recommended immunosensor guidelines, which 

suggests that the binding capacity remain within 20% 

of the initial measured binding response over the course 

of the assay study conducted (Wong et al., 1997). 

Directly immobilised drug surfaces could be used for 

greater than 1000 regeneration cycles with no loss in 

antibody-binding capacity. An additional advantage of 

such surfaces is their ability to withstand the use of 

harsh regeneration solutions, such as 3 M guanidine 

hydrochloride, for the dissociation of particularly highaffinity 

antibodies, which can be problematic for conjugate 

surfaces as the harsh regeneration conditions 

required for dissociation of the antibody/antigen complex 

frequently reduce the ligand-binding capacity and 

integrity of the immobilised conjugates (Daly et al., 

2000). In this instance, the bound monoclonal antibodies 

could be routinely dissociated from the drug– 

protein conjugate surface using pulses (i.e., 30 s) of 

mild acid. Protein-A/G may provide specific orientation 

of immobilised antibodies that are capable of 

reproducibly being used for greater than 120 cycles 

(Quinn et al., 1999). However, longterm repeated 

regeneration can result in a very significant reduction 

in antibody-binding capacity. The key benefit arising 

from the use of directly immobilised drug surfaces is 

that the sensitivity of the assay is enhanced when 

detecting the large change in mass, which occurs on 

binding antibody from solution. The degree of nonspe-

24 


cific binding of both antibody and plasma ultrafiltrate 

to the CM-dextran surface and derivatised surfaces was 

negligible. 

The use of a plasma dilution step was employed to 

minimise the variation in antibody-binding response 

due to differing salt concentrations. This proved effective 

in normalising the measured antibody-binding 

response over the salt concentration range assayed, 

which spanned more than twice the normal physiological 

reference range. The coefficient of variation for the 

measured binding response over the normal physiological 

salt concentration reference range was determined 

to be 0.38%, which underlines the robustness of 

the assay procedure to withstand variations in matrix 

composition and its applicability for the analysis of 

samples of plasma ultrafiltrate. The use of ultrafiltration 

techniques has gained widespread use in the 

clinical setting for the preparation of patient plasma 

samples (Menguy et al., 1998; Jortani et al., 1999). It 

offers several advantages over other techniques, such 

as equilibrium dialysis, for the determination of the 

pharmacologically active ‘nonprotein’-bound fraction 

of a drug in plasma. These include rapidity and ease of 

use, with the elimination of the ‘dilution’ effects 

observed using other techniques. Similarly, the essentially 

pure and homogenous nature of plasma ultrafiltrate 

greatly simplifies many of the difficulties 

associated with plasma, particularly the nonspecific 

matrix effects routinely observed. 

The inhibition immunoassay demonstrated excellent 

reproducibility as shown by the interassay variation 

(Table 3). The residual plots constructed and the 

low v 2 values obtained illustrate the ‘goodness of the 

fit’ of the four-parameter equation employed. The 

heteroscedastic nature of the residual plot (Fig. 5) 

illustrates that there is no bias in the constructed plot 

unlike linear calibration models that, when fitted to 

such immunoassay curves, generally exhibit both negative 

and positive deviations from the fitted calibration 

model at both the upper and lower extremities of the 

curve. The preliminary estimated assay precision range 

(4–250 ng/ml) is within the expected physiological 

concentration for warfarin in patient plasma ultrafiltrate 

samples. The percentage difference determined between 

the replicate determinations for the calibration 

standards and closeness of the interpolated mean values 

to the nominal concentration values demonstrates both 

the relative precision and accuracy of the immunoassay 

(Table 2). The constructed regression plot of the backcalculated 

concentration values for the independent 

calibrators demonstrates that excellent correlation 

was achieved between both independent analytical 

techniques (i.e., HPLC assay and BIACore inhibition 

immunoassay, R 2 = 0.991). Indeed, several of the independent 

calibrators quantifiable using the immunosensor 

assay were below the limit of detection of the 

chromatographic technique. 

The work cited in this particular instance highlights 

the applicability of the described immunosensor assay 

format for the detection of any target analyte by simply 

altering the specificity of the antibody and the immobilised 

ligand used, without the need for developing 

individual, chromatographic separation techniques. 

The time required for the analysis of patient samples, 

using the technique described, is typically of the 

order of 30 min from receipt of sample, which is 

considerably less than those of many chromatographic 

techniques which often require extensive sample pretreatment, 

extraction and derivatisation schemes and 

are typically of the order of hours. 

5. Conclusion 

This paper describes the development and application 

of a BIACore-based inhibition immunoassay 

for the detection of the nonprotein-bound fraction of 

warfarin in plasma ultrafiltrate. The use of directly 

immobilised drug surfaces provide exceptional assay 

performance characteristics, stability and reproducibility 

with respect to antibody-binding capacity and, 

consequently, should be employed whenever direct 

attachment of the target analyte to the chip surface is 

feasible. Use of such immunosensor-based assay 

procedures provides a viable alternative to conventional 

chromatographic procedures for the quantitation 

of low molecular weight analytes and may have 

additional advantages with respect to assay performance, 

speed and detection limits attainable. 

Acknowledgements 

The support of PRTLI (Ireland) and the National 

Centre for Sensor Research (Ireland) is gratefully 

acknowledged. We wish to thank Dr. R. O’Donnell,


Beaumont Hospital, Dublin, for his encouragement 

and support. We also wish to thank Enterprise Ireland 

for their support for this research. 

This paper is dedicated to Prof. R.D. Thornes 

whose unfailing enthusiasm, support and advice have 

been of great benefit to our research and who died 

during the preparation of this manuscript. 

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