A rapid agglutination assay to detect anti-strepokinase antibodies

A rapid agglutination assay to detect anti-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 Cardiology2, CResT Directorate, St Jamess Hospital, National Diagnostics Centre3, BioResearch Ireland, National University of Ireland, Galway, Ireland

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 with streptokinase 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 assay correlated well with serum neutralising activity. This assay can rapidly identify patients who have a high level of streptokinase-neutralising activity.

Introduction
Streptokinase is a widely used thrombolytic agent. It is a p roduct of -haemolytic streptococcal bacteria and is antigenic. Low, but variable levels of anti-streptokinase antibodies are present in the population due to previous streptococcal infections and can affect therapy in three ways. Antibodies may neutralise streptokinase, rendering the drug ineffective. For this reason it is administered at a dose high enough to overcome any antibodies.1 Anti-streptokinase antibodies may lead to hypersensitivity reactions.2-5 This does not reduce the effectiveness of the drug6 and rarely leads to the cessation of therapy.7-9 Anti-streptokinase antibodies may be associated with platelet activation, 10 which may lead to acute re-occlusion of the affected coronary artery11 and inhospital re i n f a rction.7-9 At the standard dose antibodies do not alter the outcome of streptokinase therapy in nave patients.12,13 Streptokinase remains the most widely used thro m b o l y t i c drug worldwide. With the increasing use of thro m b o l y t i c therapy many patients presenting with myocardial infarc t i o n (MI) have received streptokinase previously. Streptokinase is associated with the development of high levels of neutralising antibodies. Antibodies capable of neutralising an entire s t a n d a rd dose are detectable five days after administration. 14 Antibody levels peak within two weeks and decrease slowly thereafter. Antibodies may persist for up to seven years. 15,16 Readministration within one year is not recommended, but in many institutions streptokinase is not used if the patient has received it at any stage, or if the patients thrombolytic history is uncertain.17,18 Within six months of therapy 44% of patients have low levels of neutralising antibodies and within twelve months this figure increases to 76%. 14 Nevertheless, some patients have high levels for up to 7 years after treatment and

possibly longer.14-16,19 If individuals who have low levels of antibodies could be distinguished from those with high antibody levels, it could be re a d m i n i s t e red to suitable patients. Several assays have been developed, but none are sufficiently rapid to be used clinically.19-24 Latex agglutination assays are widely used as a rapid, convenient and inexpensive means of detecting viral or microbial antibodies or antigens, or drugs, such as horm o n e s or substances of abuse.25-27 Small antigen-coated latex particles visibly agglutinate in the presence of bivalent antibodies, due to cross-linking by antibodies of adjacent beads. The agglutination of latex particles from a milky suspension is seen with the naked eye against a contrasting backgro u n d . Antigens can be detected by their ability to inhibit the agglutination of beads in the presence of antibody, or dire c t l y, using antibody-coated beads. We describe an assay using streptokinase-coated latex beads to detect high levels of antibodies, sufficient to neutralise a standard dose. The assay was designed as simple, rapid and inexpensive. As comparators, a highly sensitive clot lysis assay was developed to measure the streptokinase-neutralising capacity in serum and an enzyme-linked immunoassay (EIA) to measure streptokinase-binding IgG. All three assays were applied to samples from patients with acute MI prior to and at various times after treatment. This latex bead assay may be used to identify patients for whom streptokinase re-administration would be safe. It is also used to screen stre p t o k i n a s e - n a v e patients to identify those in whom stre p t o k i n a s e - t h e r a p y might fail due to pre-existing neutralising antibodies.

Patients and methods

Patient re c ruitment
The study protocol was approved by the Joint Institutional

204

Irish Journal of Medical Science Volume 173 Number 4

JP McRedmond et al

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 the absence of serum at different streptokinase concentrations. (B) Streptokinase neutralising assay. Clot half-lives versus streptokinase concentration in the absence and presence of serum samples.

Research and Ethics Committee of St Jamess Hospital, Dublin. Patients presenting with acute MI were astre p t o k inaseed to participate in the study and gave written informed consent. The patients were treated with streptokinase 1.5MU infused intravenously over 60 minutes. Clinical details of previous MI and prior administration of a thrombolytic agent were recorded. Patients who had received streptokinase within the previous four years were excluded from the study. Blood samples were taken before the administration of streptokinase, and at one month and six months. Samples were also obtained for estimation of fibrinogen levels before t reatment and after completion of the infusion of streptokinase. Serum was removed from whole blood samples and stored at -20oC until assayed. Serum samples were also obtained from 100 blood donors attending the Blood Transfusion Service Board to serve as normal controls. None of these subjects had a history of MI or had received streptokinase.

Nottingham, UK) was added and a clot formed immediately. The clot lysis time was monitored at 405nm using a microtitre plate reader (Bio Tek Instruments, Winooski, VT). The time taken for 50% clot lysis was determined at each stre p t o k i n a s e concentration for each serum sample (see Figure 1A). Clot lysis times were compared to a standard curve in which no serum was incubated with streptokinase (see Figure 1B). The residual streptokinase activity in serum-incubated samples was determined and from this the SNA of the sample was calculated. Sera were diluted to between 1:5 (for most control sera and patient samples prior to receiving streptokinase) and 1:500 (for some patient samples in the first months after receiving streptokinase) as re q u i red to give clot lysis times within the standard curve. As a control, samples were also analysed using tissue plasminogen activator (t-PA [Genentech, San Francisco, CA]) as the thrombolytic agent within the assay. None of the samples exhibited t-PA neutralising activity (data not shown). Antibodies were removed from some samples using mouse anti-human Ig and Sepharose-linked goat anti-mouse IgG (Sigma), followed by centrifugation. The neutralising activity against streptokinase was abolished following this tre a t m e n t (data not shown), demonstrating that it was due to antibodies in the serum.

Enzyme immunoassay of anti-streptokinase IgG

Wells of polystyrene plates were coated overnight with a 20mg/ml solution of streptokinase, then washed four times with 300ml wash buffer (150mM NaCl, 0.05% Tween 20) and allowed to dry. Wells were then blocked with 200ml assay buffer (10mM NaPO 4, 150mM NaCl, 0.1% bovine serum albumin (BSA), 0.1g/l thimerosal, pH 7.4) containing 1% BSA for 30 minutes at 37 oC, washed as before and stored dry at 4oC. Samples were diluted to 1:100 in assay buffer for analysis. Diluted samples (50ml) were added to duplicate wells together with 150ml of anti-human IgG horseradish peroxidase conjugate (Dako, Cambridge, UK). Plates were washed three times before addition of 150ml of substrate solution (5mM ortho-phenylenediamine dihydro c h l o r i d e (Sigma)). Plates were incubated in the dark for 30 minutes before addition of 50l of stop solution (4M H2SO 4) and were read immediately at 492nm using a micro t i t re plate

S t reptokinase neutralising activity

Streptokinase neutralising activity (SNA) was determined using a clot lysis assay, based on that described by Urano et al.28 Briefly, diluted sera were mixed with streptokinase (2.580U/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,

Irish Journal of Medical Science Volume 173 Number 4

205

A rapid agglutination assay to detect anti-strepokinase antibodies

Table 1. Patient clinical characteristics

Male sex (%) Mean age (SEM) Previous MI (%) Hypertension (%) Smoking (%) Diabetes mellitus (%) Family history (%) Hypercholesterolaemia (%)* Medications: Aspirin (%) Betablocker (%) Calcium blocker (%) ACE inhibitor (%) Statin (%) Nitrate (%) 13 59.5 2 3 12 1 7 6 14 8 7 10 1 6 (81) (2.3) (13) (19) (75) (6) (44) (38) (88) (50) (44) (63) (6) (38)

Table 1. Selected clinical characteristics of the 16 patients treated with streptokinase *(total cholesterol >5.2 mmol/l)

re a d e r. To determine the optimal cut-off between positive and negative samples a graph of true-positive versus false-positive assay results was constructed (see Figure 2). True-positive samples had an SNA >350U/ml and an EIA optical density (OD) greater than the cut-off. False positive samples had an SNA 350U/ml and an OD greater than the cut-off. From F i g u re 2 it can be seen that a cut-off OD of 0.35 gives the optimal balance between a high true-positive rate and a low false-positive rate. Samples with an optical density >0.35 at a dilution of 1:100 were therefore considered positive for antistreptokinase IgG. Positive samples were further diluted from 1:1,000 to 1:107 to obtain an antibody titre. A positive and a negative control were included on each plate.

Latex bead agglutination assay

Carboxylated polystyrene micro s p h e res, 0.5 m in diameter (Bangs Laboratories, Fishers, IN) were coated at 1% solids in latex coating buffer (50mM 2-[-N-morpholino-ethanesulfonic acid (MES, Sigma), pH 6.1) with a streptokinase-human serum albumin (100mg/ml streptokinase, 12% total protein) m i x t u re using 250mg/ml 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC, Sigma) as cro s s - l i n k e r. Mixing was carried out on a shaker for one hour at room temperature. The latex was then washed three times by centrifugation at 9000xg for 15 minutes followed by resuspension in latex coating buffer. Latex was blocked overnight in latex buffer (0.1mM glycine, 150mM NaCl, 0.1% BSA, pH 8.2) containing 1% BSA. Latex was finally resuspended at 2% solids in latex buffer and stored at 4oC. A positive control was pre p a red from diluted pooled sera from patients treated with streptokinase for MI. The performance of each batch of latex was assessed by running positive and negative control serum samples in the assay. Batches that did not give the expected response were d i s c a rd e d . For latex agglutination assay, sera were diluted 1:10 in phosphate-buffered saline containing 0.01% BSA. Diluted serum (30 l) and 30l of Streptokinase-coated latex particles were mixed on the surface of a black glass plate (see Figure 3). Up to four samples and the positive and negative contro l s were analysed per plate. Plates were rocked at 35rpm for 5-6 minutes at room temperature, at which time slight agglutination was detectable in the positive control sample. For the purposes of characterising the assay, samples were

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

s c o red from 0 to 6 with re g a rd to the degree of agglutination seen. Samples in which no particles could be seen were scored as 0. The positive control was always score d as 2. The maximum degree of agglutination, in which all the white material was gathered in a small number of clumps, was scored as 6. From Figure 2 it can be seen that deeming samples with a score of 3 as positive gives the assay optimal specificity. However, scoring the assay is somewhat subjective, whereas comparing the degree of agglutination to a positive control on the same plate is more objective. There f o re, samples of score 0 or 1 (<positive control) were deemed to be negative, scores of 2-6 (p ositive control) were considered positive. Scoring was validated by an investigator blinded as to the identity of the samples.

Fibrinogen assay
Plasma fibrinogen concentrations were assayed using the Clauss method.29

Statistical analysis
All data are expressed as meanSEM. Friedmans non-parametric repeated measures test followed by Dunns multiple comparisons post-test were used to investigate differences in SNA, EIA and latex scores at each sampling period. S p e a rmans non-parametric correlation coefficient was calculated between SNA and latex bead score and between SNA

206

Irish Journal of Medical Science Volume 173 Number 4

JP McRedmond et al

and EIA titre. A p-value of 0.05 or less was considered to be statistically significant. Statistical analysis was performed using INSTAT software package (Graphpad software, San Diego, CA).

Results
Clinical characteristics
The clinical profiles of the 16 patients entered into the study a re outlined in Table 1. Two patients had a previous MI; one of these had received Streptokinase for an inferior MI six years previously. The distribution of MIs was: six anteroseptal, two a n t e rolateral, four inferior, 3 inferolateral and one posterior. The mean time from onset of chest pain to thrombolytic therapy was 4.4 hours (range 50 minutes 7 hours 20 minutes). The mean (SEM) peak creatnine kinase was 2,020392U/ml. The mean plasma fibrinogen level pret h rombolysis was 3.370.18g/l. As expected, plasma fibrinogen fell in all patients following the infusion of Stre p t o k i n a s e (to 0.300.05g/l; p<0.01).

administration of streptokinase were negative for anti-streptokinase IgG by EIA (OD<0.35 at 1:100 dilution). All patient samples were positive at one month, with titres ranging from 1:103 to 1:107 (p<0.001). Ti t res fell significantly at six months, ranging from 1:100 (negative) to 1:104 (p<0.01 vs one month) (see Figure 4B).

Latex bead assay

In the 16 patients followed for at least 6 months, the re s u l t s using the latex agglutination assay mirrored the SNA results. Latex scores were all 0 before administration of streptokinase. Latex scores rose significantly to a median of 5 (range 2-6; p<0.001) at one month. At six months, the latex scores fell to a median of 3.5 (range 0-5; p<0.05 vs one month; p<0.05 vs b e f o re treatment) (see Figure 4C). At six months, four patients had SNA <350U/ml. Of these, three had negative latex agglutination scores. A further two patients had negative latex scores but had SNAs of 1,360 and 643U/ml (see Table 2). In sera from control individuals, only five out of 100 samples showed a latex agglutination score of 2 or above. Of these, two had the highest SNA values in the group (187 and 192U/ml). The remaining three had low SNA values (12, 72 and 91U/ml). Thus, the latex assay appears to be of less value in Streptokinase-nave patients than in patients who previously received the drug.

S t reptokinase neutralising activity

The SNA was measured in U/ml, that is the units of stre ptokinase neutralised per ml of blood in vitro. An SNA of >350 U/ml is significant as this exceeds the peak plasma level of streptokinase (~250100U/ml) following a standard dose of the drug.30 In the samples from the 16 patients, the mean (SEM) SNA was 10119 U/ml (range 0-258 U/ml) before t reatment. The highest level of 258 U/ml was from the patient who had re c e i v e d s t reptokinase six years previously. The SNA levels rose in the whole group to 2,110823U/ml (range 192-14,000U/ml; p<0.001) at one month. At six months, SNA fell to1,020169U/ml (range 298-1,960U/ml; p<0.001 vs before treatment [see Figure 4A]). In 100 samples f rom normal blood bank donors, the mean SNA was 545U/ml (range 0-192U/ml).

Correlation between assays

It was not possible to establish a correlation between the SNA and other assays prior to thrombolysis as all EIA titres were identical and all latex scores were zero. The EIA corre l a t e d with SNA in patients at one month (Spearman corre l a t i o n coefficient r=0.7578; p=0.0007), at six months (r=0.7567; p=0.0007) and in all 146 samples studied (r=0.6688; p<0.0001). Taking all control subjects and patient samples together, the EIA had a sensitivity of 99% and a specificity of 96% for detecting patients with >350U/ml of stre p t o k i n a s e neutralising activity. The latex agglutination assay corre l a t e d with SNA at one month (r=0.645; p=0.007), at six months

EIA for anti-streptokinase IgG

All samples from control subjects and from patients before the

Table 2. Assay results in patients treated with streptokinase

Pre Streptokinase Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 SNA 116 38 19 155 0 72 151 129 105 147 184 158 21 258 11 52 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 EIA 0 Latex 0 1 month SNA 770 1284 2747 373 2135 2225 2385 360 1400 1816 381 1583 464 2470 192 570 EIA 0.74 0.598 0.612 0.334 0.589 0.632 1.048 0.715 0.809 0.317 0.271 0.699 0.638 0.985 0.256 0.639 Latex 5 6 6 4 4 6 5 4 6 6 5 5 6 6 2 5 6 months SNA 1295 1700 1880 339 1360 1881 1930 865 410 288 643 502 590 1960 325 298 EIA 0.485 0.386 0.598 0.181 0.3 0.524 0.96 0.402 0.36 0.139 0.065 0.396 0.354 0.271 0.112 0.173 5 5 4 4 0 0 4 4 3 1 1 Latex 5 3 5 3

Results from all patient samples according to the three assays used. Due to limited sample volumes, assays were performed only once. Preliminary data showed the coefficient of variance of the assays to be less than 15%. With two patients, insufficient serum was obtained immediately prior to thrombolysis to allow EIA and latex assays. SNA: streptokinase neutralising activity (U/ml). EIA: Enzyme immunoassay (Optical Density units)

Irish Journal of Medical Science Volume 173 Number 4

207

A rapid agglutination assay to detect anti-strepokinase antibodies

Figure 4. Mean results in patients and controls for streptokinase neutralising assay, EIA for anti-streptokinase antibodies and latex score. Assay results in samples from 100 control individuals, and in a cohort of 16 patients before and at 1 and 6 months after receiving streptokinase. (A) Streptokinase neutralising activity. (B) Log10 EIA titres. (C) Latex agglutination assay scores. ***p<0.001 versus pre streptokinase. *p<0.05 versus pre streptokinase. Error bars indicate standard error of the mean. (r=0.505; p=0.046) and in all samples (r=0.5199; p<0.0001). In all samples, the latex assay had a sensitivity of 94% and a specificity of 93% for detecting patients with >350U/ml of SNA (see Figure 2).

Figure 5. Comparison of assays of anti-streptokinase antibodies. Assay results for all 146 samples are plotted. Vertical dotted lines indicate the cut-off level for SNA of 350U/ml. Horizontal lines indicate the cut-off between positive and negative assay results. Most samples fall into the top-right and bottom-left quadrants, indicating agreement between the assays A. EIA and SNA. Samples with an EIA titre >1:100 (log10>2) are positive. B. Latex assay and SNA. Samples with a latex score 2 are positive. neutralisation was measured directly as the SNA. This assay is specific for streptokinase and is due to antibodies in the serum, since neutralising activity is abolished once they are removed and no neutralisiation was found against t-PA. Streptokinase has several distinct antigenic epitopes. Not all patients display the same re p e rt o i re of antibodies to streptokinase, and not all anti-streptokinase antibodies inhibit its plasminogen-activating activity.31 We there f o re conjecture that some of the variance between the latex bead assay and the SNA may be explained by the existence of non-neutralising antibodies 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 had SNA >350U/ml but latex scores <2 (see Figure 5B). The assay could be improved to better detect such individuals by coating latex particles only with those antigenic sequences of s t reptokinase known to be recognised by neutralising antibodies.31 The specificity of the latex assay was lower, as several samples had scores of 2 but with SNA <350U/ml (see Figure 5B). It is more important, however, to avoid administration of streptokinase to patients who have high SNA and these are detected by the latex bead assay. The cutoff level of 350U/ml used by us is more conservative than in

Discussion
P revious studies 14,15,19 have shown that most individuals develop high levels of anti-streptokinase antibodies following tre a tment 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 sing antibodies at one month (when responses tend to be maximal) below the level expected to render a standard dose of streptokinase ineffective. However, levels of antibody suff icient to neutralise a standard dose of streptokinase may persist for 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 and duration of the antibody response to stre p t o k i n a s e . While antibodies to streptokinase have many eff e c t s , including platelet activation10,11 and allergic reactions (2-5), neutralisation of the fibrinolytic capacity of the drug has most implications for repeated use of streptokinase. In this study,

208

Irish Journal of Medical Science Volume 173 Number 4

JP McRedmond et al

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 e achieved following a standard dose of the drug.30 The EIA, while showing better agreement with the SNA than the latex agglutination assay (see Figure 5), is too slow to be useful clinically in deciding a patients suitability to re c e i v e streptokinase. Several previous studies have examined methods of estimating streptokinase resistance due to anti-stre p t o k i n a s e antibodies. Some have looked at clot lysis in streptokinaseserum mixtures15,19,23 in retrospective studies of large groups of patients, charting streptokinase resistance over time. Others have tried to correlate such assays with more rapid assays of a n t i - s t reptokinase antibodies.20,24 None of these assays gave results rapidly enough to be clinically useful. The principal limitation of this study is the small sample size. A larger population would be required to better estimate the specificity and sensitivity of the latex bead assay measure d against a standard such as the SNA. The latex assay is not as specific as the EIA. This could be increased without any loss of sensitivity by increasing the amount of antibody in the positive control, since all samples scored as equal to the positive control were negative (see Figures 2 and 5). H o w e v e r, it is highly sensitive and would pre v e n t administration of streptokinase to individuals with a high level of neutralising antibodies. In conclusion, we have developed an assay based on the agglutination of streptokinase-coated latex beads that detects a n t i - s t reptokinase antibodies. The latex bead assay correlates well with a functional assay measuring the resistance to the t h rombolytic activity of streptokinase. The latex bead assay provides a rapid, bedside tool to detect patients that are likely to be resistant to repeated administration of streptokinase.

References
1. Verstraete M, Vermylen J, Amery A, Ve rmylen C. Thrombolytic therapy with streptokinase using a standard dosage scheme. BMJ 1966; 5485: 454-56. McGrath KG, Patterson R. Anaphylactic reactivity to streptokinase. JAMA 1984; 252: 1314-17. McGrath KG, Zeff ren B, Alexander J, Kaplan K, Patterson, R. Allergic reactions to streptokinase consistent with anaphylactic or antigen-antibody complex-mediated damage. J Allergy Clin Immunol 1985; 76: 453-57. Zilliox AP, Domoto DT, Hutchesson PS, Tsai CC, Slavin RG. Henoch-Schloenlein purpura due to streptokinase. J Clin Immunol 1993; 13: 415-23. Taylor BV, Mastaglia FL, Stell R. Guillan-Barre syndrome complicating treatmant with streptokinase. Med J Aust 1995; 162: 214-15. Tsang TS, Califf RM, Stebbins AL et al. Incidence and impact on outcome of streptokinase allergy in the GUSTO-I trial. Am J Cardiol 1997; 79: 1232-35. G ruppo Italiano per lo Studio della Streptochinasi nell'Infart o Miocardico. GISSI-2: A factorial randomised trial of alteplase versus s t reptokinase and heparin versus no heparin among 12 490 patients with acute myocardial infarction. The Lancet 1990; 336: 65-71. ISIS-3 (Third International Study of Infarct Survival) Collaborative G roup. ISIS-3: a randomised comparison of streptokinase vs tissue plasminogen activator vs anistreplase and of aspirin plus heparin vs aspirin alone among 41 299 cases of suspected acute myocardial infarction. The Lancet 1992; 339: 753-70. The GUSTO Investigators. An international randomised trial comparing four thrombolytic strategies for acute myocardial infarction. N Engl J Med 1993; 329: 673-82.

2. 3.

4.

5.

6.

7.

8.

9.

10. McRedmond JP, Harriott P, Walker B, Fitzgerald DJ. Streptokinaseinduced platelet activation involves antistreptokinase antibodies and cleavage of protease-activated receptor-1. Blood 2000; 95: 1301-08. 11. Vaughan DE, Kirshenbaum JM, Loscalzo J. Streptokinase-induced, antibody-mediated platelet aggregation: a potential cause of clot p ropagation in vivo. J Am Coll Cardiol 1988; 11: 1343-48. 12. Shaila G, Chandrashkhar YS, Kumar N, Ganguly NK, Anand IS. A n t i s t re ptokianse antibodies before and after streptokianse therapy in patients with acute myocardial infarction from areas endemic for s t reptococcal infection and influence on reperfusion rates. Am J Cardiol 1994; 74: 187-9. 13. Fears R, Hearn J, Standring R, Anderson JL, Marder VJ. Lack of influence of pre t reatment antistreptokinase antibody on efficacy in a multicenter patency comparison of intravenous streptokinase and a n i s t re plase in acute myocardial infarction. Am Heart J 1992; 124: 305-14. 14. Fears R, Ferres H, Glasgow E et al. Monitoring of streptokinase resistance titre in acute myocardial infarction patients up to 30 months after giving streptokinase or anistreplase and related studies to measure specific antistreptokinase IgG. Br Heart J 1992; 68: 167-70. 15. Lee HS, Cross S, Davidson R, Reid T, Jennings K. Raised levels of a n t i s t re ptokinase antibody and neutralisation titres from 4 days to 54 months after administration of streptokinase or anistreplase. Eur H e a rt J 1993; 14: 84-9. 16. S q u i re IB, Lawley W, Fletcher S et al. Humoral and cellular immune responses up to 7.5 years after administration of streptokinase for acute myocardial infarction. Eur Heart J 1999; 20: 1245-52. 17. Buchalter M.B. Are streptokinase antibodies clinically important? Br H e a rt J 1993; 70: 101-02. 18. C ross D. Repeat thrombolysis. Aust N Z J Med 1993; 23: 749-752. 19. Patel S, Jalihal S, Dutka DP, Morris G.K. Streptokinase neutralisation titres up to 866 days after intravenous streptokinase for acute m y o c a rdial infarction. Br Heart J 1993; 70: 119-21. 20. Bom VJ, Brugemann J, van der Schaaf W, van Wijk RT, van der Meer J. Rapid enzyme immunoassay of anti-streptokinase antibodies in human plasma. Clin Chim Acta 1993; 218: 121-29. 21. Leonardi MS, Gazzara D, Fava C, Foca A, Mastroeni P. EnzymeLinked Immunosorbent Assay (ELISA) for streptokinase antibodies. Diagn Immunol 1983; 1: 64-7. 22. Lynch M, Pentecost BL, Littler WA, Stockley RA. The significance of anti-streptokinase antibodies. Clin Exp Immunol 1994; 96: 427-31. 23. Moran DM, Standring R, Lavender EA, Harris GS. Assessment of a n t i - s t reptokinase antibody levels in human sera using a microradioimmunoassay pro c e d u re. T h romb Haemost 1985; 52: 28187. 24. Buchalter MB, Suntharalingham G, Jennings I et al. Streptokinase resistance: when might streptokinase administration be ineffective? Br H e a rt J 1992; 68: 449-53. 25. Silpapojakul K, Prandutkanchana J, Prandutkanchana S, Kelly DJ. Rapid, simple serodiagnosis of murine typhus. Trans R Soc Trop Med Hyg 1995; 89: 625-28. 26. Fujiyama C, Fujiyoshi T, Matsumoto D, Ta m a s h i ro H, Sonoda S. Evaluation of commercial HTLV-1 test kits by a standard HTLV-1 s e rum panel. Bull World Health Organ 1995; 73: 515-21. 27. Birnbach DJ, Stein DJ, Grunebaum A, Danzer BI, Thys DM. Cocaine screening of parturients without prenatal care: an evaluation of a rapid screening assay. Anest Analg 1997; 84: 76-9. 28. Urano S, Metzger AR, Castellino FJ. Plasmin-mediated fibrinolysis by variant recombinant tissue plasminogen activators. P roc Natl Acad Sci USA 1989; 86: 2568-71. 29. Clauss A. Gerinnungsphysiologische schnellmethode31. Parhami-Seren B, Lynch M, White HD, Reed GL. Mapping the antigenic regions of streptokinase in humans before and after s t re ptokinase therapy. Mol Immunol 1995; 32: 717-24. zur estimmung des fibrinogens. Acta Haematol 1957; 17: 237-46.

Irish Journal of Medical Science Volume 173 Number 4

209

A rapid agglutination assay to detect anti-strepokinase antibodies

30. Gemmill JD, Hogg KJ, Burns JM et al. A comparison of the pharmacokinetic properties of streptokinase and anistreplase in acute myocardial infarction. Br J Clin Pharmacol 1991; 31: 143-47. 31. P a rh a m i - S e ren B, Lynch M, White HD, Reed GL. Mapping the antigenic regions of streptokinase in humans before and after streptokinase therapy. Mol Immunol 1995; 32: 717-24.

Acknowledgements
Supported by grants from The Irish Heart Foundation, BioResearch Ireland and the Higher Education Authority of Ireland. Correspondence to: Dr James McRedmond, Conway Institute, University College Dublin, Belfield, Dublin 4. Email: james.mcredmond@ucd.ie. Tel: 01 7166913. Fax: 01 7166962

210

Irish Journal of Medical Science Volume 173 Number 4