a rapid agglutination assay to detect anti-streptokinase antibodies

7/27/2019 A rapid agglutination assay to detect anti-streptokinase antibodies

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A rapid agglutination assay to detect anti-strepokinase antibodies

204 Irish Journal of Medical Science Volume 173 Number 4

A rapid agglutination assay to detectanti-streptokinase antibodies

JP McRedmond1, NT Mulvihill2, M Kane3, B Burke3, B Aloul1, T Forde3, M Walsh2, DJ Fitzgerald1

Department of Clinical Pharmacology1, Royal College of Surgeons in Ireland, Department of Cardiology 2, CResT

Directorate, St Jamess Hospital, National Diagnostics Centre3, BioResearch Ireland, National University of Ireland,

Galway, Ireland

I n t ro d u c t i o nS t reptokinase is a widely used thrombolytic agent. It is ap roduct of -haemolytic streptococcal bacteria and isantigenic. Low, but variable levels of anti-stre p t o k i n a s eantibodies are present in the population due to pre v i o u ss t reptococcal infections and can affect therapy in three ways.Antibodies may neutralise str eptokinase, rendering thed rug inef fective. For this reason it is administered at a dosehigh enough to overcome any antibodies.1A n t i - s t re p t o k i n a s eantibodies may lead to hypersensitivity re a c t i o n s .2-5 This doesnot reduce the effectiveness of the dru g6 and rarely leads tothe cessation of therapy.7 - 9 A n t i - s t reptokinase antibodiesmay be associated with platelet activation,10which may lead toacute re-occlusion of the affected coro n a ry art e ry11 and in-hospital re i n f a rc t i o n .7-9 At the standard dose antibodies donot alter the outcome of streptokinase therapy in navep a t i e n t s .12,13

S t reptokinase remains the most widely used thro m b o l y t i cd rug worldwide. With the increasing use of thro m b o l y t i ctherapy many patients presenting with myocardial infarc t i o n(MI) have received streptokinase pre v i o u s l y. St reptokinase isassociated with the development of high levels of neutralisingantibodies. Antibodies capable of neutralising an entires t a n d a rd dose are detectable five days after administrat ion. 14

Antibody levels peak within two weeks and decrease slowlyt h e re a f t e r. Antibodies may persist for up to seven years.15,16

Readministration within one year is not recommended, but inmany institutions streptokinase is not used if the patient hasreceived it at any stage, or if the patients thrombolytic historyis uncert a i n .17,18Within six months of therapy 44% of patientshave low levels of neutralising antibodies and within twelve

months this figure increases to 76%. 14 N e v e rtheless, somepatients have high levels for up to 7 years after treatment and

possibly longer.14-16,19 If individuals who have low levels ofantibodies could be distinguished from those with highantibody levels, it could be re a d m i n i s t e red to suitablepatients. Several assays have been developed, but none ares u fficiently rapid to be used clinically.19-24

Latex agglutination assays are widely used as a rapid,convenient and inexpensive means of detecting viral orm i c robia l antibodies or antigens , or drugs, such as horm o n e sor substances of abuse.25-27 Smal l antigen-coated latex part i c l e svisibly agglutinate in the presence of bivalent antibodies, dueto cross-linking by antibodies of adjacent beads. Theagglutination of latex particles from a milky suspension is seenwith the naked eye agains t a contras ting backgro u n d .Antigens can be detected by their ability to inhibit theagglutination of beads in the presence of antibody, or dire c t l y,using antibody-coated beads. We describe an assay usings t reptokinase-coated latex beads to detect high levels of

antibodies, sufficient to neutralise a standard dose. The assaywas designed as simple, rapid and inexpensive. Ascomparators, a highly sensitive clot lysis assay was developedto measure the streptokinase-neutralising capacity in seru mand an enzyme-linked immunoassay (EIA) to measures t reptokinase-binding IgG. Al l three assay s were applied tosamples from patients with acute MI prior to and at varioustimes after treatment. This latex bead assay may be used toidentify patients for whom streptokinase re - a d m i n i s t r a t i o nwould be safe. It is also used to screen stre p t o k i n a s e - n a v epatients to identify those in whom stre p t o k i n a s e - t h e r a p ymight fail due to pre-existing neutralising antibodies.

Patients and methods

Patient re c ru i t m e n tThe study protocol was approved by the Joint Institutional

Abstract

Background Streptokinase resistance may cause suboptimal thrombolytic therapy.Aim To develop a rapid latex-bead assay to detect streptokinase antibodies.Methods Sera were obtained from 16 patients presenting with acute myocardial infarction (MI) before treatment withstreptokinase and 1 and 6 months post treatment, and from 100 controls. Sera were assayed for anti-streptokinase

antibodies using a functional streptokinase-neutralising assay.

Results Streptokinase-neutralising activity was low in controls (545U/ml) and patients prior to treatment (10118),increasing to 2,110823 and 1,017169 at 1 and 6 months (meanSEM). The latex assay had a sensitivity of 94%

and a specificity of 93% for detecting individuals with >350U/ml of streptokinase resistance, which is sufficient to

neutralise the drug clinically.

Conclusions Estimation of streptokinase resistance using an enzyme immunoassay and a latex bead assaycorrelated well with serum neutralising activity. This assay can rapidly identify patients who have a high level of

streptokinase-neutralising activity.


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205Irish Journal of Medical Science Volume 173 Number 4

R e s e a rch and Ethics Committee of St Jamess Hospital,Dublin. Patients presenting with acute MI were astre p t o k i-naseed to participate in the study and gave written inform e dconsent. The patients were treated with streptokinase 1.5MUinfused intravenously over 60 minutes. Clinical details of pre-vious MI and prior administration of a thrombolytic agentw e re re c o rded. Patients who had received streptokinase with-in the previous four years were excluded from the study.

Blood samples were taken before the administration ofs t reptokinase, and at one month and s ix months. Samples

w e re also obtained for estimation of fibrinogen levels beforet reatment and after completion of the infusion ofs t reptokinase. Serum was removed from whole blood samplesand stored at -20oC until assayed. Serum samples were alsoobtained from 100 blood donors attending the BloodTransfusion Service Board to serve as normal controls. Noneof these subjects had a history of MI or had re c e i v e ds t re p t o k i n a s e .

S t reptokinase neutralising activityS t reptokinase neutralising activity (SNA) was determ i n e dusing a clot lysis assay, based on that described by Urano eta l .28 B r i e f l y, diluted sera were mixed with streptokinase (2.5-80U/ml) and added to a 96 well plate together with human

plasminogen (0.1U/ml) and thrombin (5U/ml, Sigma,Poole, Dorset). Fibrinogen (9.6mg/ml, Calbiochem,

Nottingham, UK) was added and a clot formed immediately.The clot lysis time was monitored at 405nm using a micro t i t replate reader (Bio Tek Instruments, Winooski, VT). The timetaken for 50% clot lysis was determined at each stre p t o k i n a s econcentration for each serum sample (see Figure 1A). Clotlysis times were compared to a standard curve in which nos e rum was incubated with streptokinase (see Figure 1B). Theresidual streptokinase activity in serum-incubated samples wasd e t e rmined and from this the SNA of the sample was calculat-ed. Sera were diluted to between 1:5 (for most control seraand patient samples prior to receiving streptokinase) and1:500 (for some patient samples in the first months afterreceiving streptokinase) as re q u i red to give clot lysis timeswithin the standard curv e .

As a control, samples were also analysed using tissueplasminogen activator (t-PA [Genentech, San Francisco, CA])as the thrombolytic agent within the assay. None of thesamples exhibited t-PA neutralising activity (data not shown).Antibodies were removed from some samples using mouseanti-human Ig and Sepharose-linked goat anti-mouse IgG(Sigma), followed by centrifugation. The neutralising activityagainst streptokinase was abolished following this tre a t m e n t(data not shown), demonstrating that it was due to antibodiesin the seru m .

Enzyme immunoassay of anti-streptokinase IgGWells of polystyrene plates were coated overnight with a

20mg/ml solution of streptokinase, then washed four timeswith 300ml wash buffer (150mM NaCl, 0.05% Tween 20)and allowed to dry. Wells were then blocked with 200ml assayb u ffer (10mM NaPO4, 150mM NaCl, 0.1% bovine seru malbumin (BSA), 0.1g/l thimerosal, pH 7.4) containing 1%BSA for 30 minutes at 37 oC, washed as before ands t o red dry at 4oC .

Samples were diluted to 1:100 in assay buffer for analysis.Diluted samples (50ml) were added to duplicate wellstogether with 150ml of anti-human IgG horseradishp e roxidase conjugate (Dako, Cambridge, UK). Plates werewashed three times before addition of 150ml of substratesolution (5mM ortho-phenylenediamine dihydro c h l o r i d e(Sigma)). Plates were incubated in the dark for 30 minutes

b e f o re addition of 50l of stop solution (4M H2S O4) andw e re read immediately at 492nm using a micro t i t re plate

Figure 2.True and false positive rates of assays of streptokinase

antibodies. The ability of the EIA and latex assays to correctly

identify samples with >350U/ml streptokinase neutralising activity

was assessed by comparing true and false positive rates at various

cut-off points. The chosen optical density cut-off of 0.35 for the EIA

is indicated by the solid line.

Figure 1.(A) Streptokinase neutralising assay. Clot lysis times in theabsence of serum at different streptokinase concentrations. (B)

Streptokinase neutralising assay. Clot half-lives versus streptokinase

concentration in the absence and presence of serum samples.


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re a d e r. To determine the optimal cut-off between positive andnegative samples a graph of true-positive versus false-positiveassay results was constructed (see Figure 2). Tru e - p o s i t i v esamples had an SNA >350U/ml and an EIA optical density(OD) greater than the cut-off. False positive samples had anSNA350U/ml and an OD greater than the cut-off. Fro mF i g u re 2 it can be seen that a cut-off OD of 0.35 gives theoptimal balance between a high true-positive rate and a lowfalse-positive rate. Samples with an optical density >0.35 at adilution of 1:100 were there f o re considered positive for anti-s t reptokinase IgG. Positive samples were further diluted fro m1:1,000 to 1:107 to obtain an antibody titre. A positive and anegative control were included on each plate.

Latex bead agglutination assayCarboxylated polystyrene micro s p h e res, 0.5m in diameter(Bangs Laboratories, Fishers, IN) were coated at 1% solids inlatex coating buffer (50mM 2-[-N-morpholino-ethanesulfon-ic acid (MES, Sigma), pH 6.1) with a stre p t o k i n a s e - h u m a ns e rum albumin (100mg/ml streptokinase, 12% total pro t e i n )m i x t u re using 250mg/ml 1-ethyl-3-(3-dimethylaminopro p y l )carbodiimide (EDAC, Sigma) as cro s s - l i n k e r. Mixing was car-ried out on a shaker for one hour at room temperature. Thelatex was then washed three times by centrifugation at 9000xgfor 15 minutes followed by resuspension in latex coatingb u ff e r. Latex was blocked overnight in latex buffer (0.1mMglycine, 150mM NaCl, 0.1% BSA, pH 8.2) containing 1%

BSA. Latex was finally resuspended at 2% solids in latex buff e rand stored at 4oC. A positive control was pre p a red from dilut-ed pooled sera from patients treated with streptokinase forMI. The perf o rmance of each batch of latex was assessed byrunning positive and negative control serum samples in thea s s a y. Batches that did not give the expected response wered i s c a rd e d .

For latex agglutination assay, sera were diluted 1:10 inp h o s p h a t e - b u ff e red sali ne containing 0.01% BSA. Diluteds e rum (30l) and 30l of Streptokinase -coated latex part i c l e sw e re mixed on the surface of a black glass plate (see Figure 3).Up to four samples and the positive and negative contro l sw e re analysed per plate. Plates were rocked at 35rpm for 5-6minutes at room temperature, at which time slight

agglutination was detectable in the positive control sample.For the purposes of characterising the assay, samples were

s c o red from 0 to 6 with re g a rd to the degree ofagglutination seen. Samples in which no particles could beseen were scored as 0. The positive control was always score das 2. The maximum degree of agglutination, in which all thewhite material was gathered in a smal l number of clumps, wass c o red as 6. From Figure 2 it can be seen that deemingsamples with a score of 3 as positive gives the assayoptimal specificity. However, scoring the assay issomewhat subjective, whereas comparing the degree ofagglutination to a positive control on the same plate is more

objective. There f o re, samples of score 0 or 1 (5.2 mmol/l)

Figure 3. Latex bead assay for streptokinase antibodies.

(A) Samples from patient 12 taken before and 1 and 6 months after

MI. (B) Samples from patient 16 (see Table 2). (C) Control sera. To

demonstrate the lack of antibodies in control sera this plate was

incubated for longer than the others, resulting in more agglutination


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and EIA titre. A p-value of 0.05 or less was considered to bestatistically significant. Statist ical analysis was perf o rm e dusing INSTAT software package (Graphpad software, SanDiego, CA).

R e s u l t s

Clinical characteristicsThe clinical profiles of the 16 patients entered into the studya re outlined in Table 1. Two patients had a previous MI; oneof these had received Streptokinase for an inferior MI six yearsp re v i o u s l y. The distri bution of MIs was: six anteroseptal, twoa n t e rolateral, four inferior, 3 inferolateral and one posterior.The mean time from onset of chest pain to thrombolytictherapy was 4.4 hours (range 50 minutes 7 hours 20 min-utes). The mean (SEM) peak creatnine kinase was2,020392U/ml. The mean plasma fibrinogen level pre -t h rombolysis was 3.370.18g/l. As expected, plasma fibrino-gen fell in all patients following the infusion of Stre p t o k i n a s e(to 0.300.05g/l; p350U/ml is significant as this exceeds the peak plasma level ofs t reptokinase (~250100U/ml) following a standard dose ofthe dru g .30 In the samples from the 16 patients, the mean(SEM) SNA was 10119 U/ml (range 0-258 U/ml) b e f o ret reatment. The highest level of 258 U/ml was from the patientwho had re c e i v e d s t reptokinase six years pre v i o u s l y.

The SNA levels rose in the whole group to 2,110823U/ml(range 192-14,000U/ml; p


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(r=0.505; p=0.046) and in all samples (r=0.5199; p350U/ml ofSNA (see Figure 2).

D i s c u s s i o nP revious studies 14,15,19 have shown that most individuals devel-op high levels of anti-streptokinase antibodies following tre a t-ment with streptokinase. However, the response is variable,with some patients showing levels of stre p t o k i n a s e - n e u t r a l i s-ing antibodies at one month (when responses tend to be max-imal) below the level expected to render a standard dose ofs t reptokinase ineffective. However, levels of antibody suff i-cient to neutralise a standard dose of streptokinase may persistfor up to 7.5 years after a single dose of stre p t o k i n a s e .16

These data highlight the variability in the magnitude andduration of the antibody response to str e p t o k i n a s e .While ant ibodies to str eptokinase have many ef f e c t s ,including platelet activation10,11 and allergic reactions (2-5),

neutralisation of the fibrinolytic capacity of the drug has mostimplications for repeated use of streptokinase. In this study,

neutralisation was measured directly as the SNA. This assay isspecific for streptokinase and is due to antibodies in thes e rum, since neutralising activity is abol ished once they areremoved and no neutralisiation was found against t-PA .

S t reptokinase has several distinct antigenic epitopes. Not allpatients display the same re p e rt o i re of antibodies tos t reptokinase, and not all anti-streptokinase antibodies inhibitits plasminogen-activating activity.31 We there f o re conjecture

that some of the variance between the latex bead assay and theSNA may be explained by the existence of non-neutralisingantibodies causing bead agglutination, or conversely,neutralising antibodies that fail to agglutinate the beads.Despite this flaw, the two assays correlated stro n g l y.

The latex assay was highly sensitive, as only two samples hadSNA >350U/ml but latex scores


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other studies of streptokinase resistance (450-625U/ml) (18,19, 24) and is based on the plasma level of stre p t o k i n a s eachieved following a standard dose of the dru g .30 The EIA,while showing better agreement with the SNA than the latexagglutination assay (see Figure 5), is too slow to be usefulclinically in deciding a patien ts suitab ility to re c e i v e

s t reptokinase.Several previous studies have examined methods of

estimating streptokinase resistance due to anti-stre p t o k i n a s eantibodies. Some have looked at clot lysi s in stre p t o k i n a s e -s e rum mixture s15,19,23 in re t rospective studies of large groups ofpatients, charting streptokinase resistance over time. Othershave tried to correlate such assays with more rapid assays ofa n t i - s t reptokinase antibodies. 20,24 None of these assays gaveresults rapidly enough to be clinically useful.

The principal limitation of this study is the small samplesize. A larger population would be re q u i red to better estimatethe specificity and sensitivity of the latex bead assay measure dagainst a standard such as the SNA. The latex assay is not asspecific as the EIA. This could be increased without any loss

of sensitivity by increasing the amount of antibody in thepositive control, since all samples scored as equal to thepositive control were negative (see Figures 2 and 5).H o w e v e r, it is highly sen sitive and would pre v e n tadministration of streptokinase to individuals with a high levelof neutralising antibodies.

In conclusion, we have developed an assay based on theagglutination of streptokinase-coated latex beads that detectsa n t i - s t reptokinase antibodies. The latex bead assay corre l a t e swell with a functional assay measuring the resistance to thet h rombolytic activity of streptokinase. The latex bead assayp rovides a rapid, bedside tool to detect patients that are l ikelyto be resis tant to repeated administration of stre p t o k i n a s e .

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AcknowledgementsS u p p o rted by grants from The Irish Heart Foundation,B i o R e s e a rch Ireland and the Higher Educat ion Authority ofI reland.

Correspondence to: Dr James McRedmond, Conway Institute, University College Dublin, Belfield,Dublin 4.

Email: [email protected]. Tel: 01 7166913. Fax: 01 7166962

a rapid agglutination assay to detect anti-streptokinase antibodies

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