The effects of incubation temperature and coating procedure on the measurement of antibodies to cardiolipin

31

Journal of lmmunological Methods, 143 (1991) 31-39

© 1991 Elsevier Science Publishers B.V. All rights reserved 0022-1759/91/$03.50 ADONIS 002217599100287U JIM06062

The effects of incubation temperature and coating procedure on the measurement of antibodies to cardiolipin M . A . F i r e r t, T. Spivak 1, y . S h o e n f e l d 2 a n d H. Slor 3 1Biohytech (Israel) Ltd., 10 Dov Fridman St., Ramat Gan, Israel, 2 Department of Medicine B, Research Unit of Autoimmune Diseases, Sheba Medical Center, Tel-Hashomer 52621, Israel, and 3 Department of Human Genetics, Sackler School of Medicine, Tel-Aviv University, Israel

(Received 7 December 1990, revised received 15 March 1991,accepted 29 May 1991)

Many laboratories have established ELISAs for the the routine detection of anti-cardiolipin antibodies (ACA). Earlier studies had indicated that assay incubation at 37°C may interfere with the antigen binding capacity of these antibodies. We have reexamined this phenomenon by comparing A C A titers obtained when incubations are performed at either 37°C or at room temperature (RT). In addition, the effect of coating antigen in aqueous or organic solution was compared. The sera tested included a set of recognized A C A standards and samples from 19 patients with SLE, two with primary anti-phospholipid syndrome, 71 patients with a variety of autoimmune and non-autoimmune disorders and 210 blood bank controls. The results show that while some sera do perform better under either incubation temperature there was no correlation between A C A titers and incubation temperature on a population basis either for IgG or IgM isotypes. This was seen both for positive standards and patient sera. For IgG ACA a similar p h e n o m e n o n was seen if the microplates were coated with cardiolipin either in sodium carbonate or ethanol. For IgM ACA there was a significant increase in ACA titers at R T when cardiolipin was coated in ethanol. The data suggest that for most sera neither the antigen coating medium nor the assay incubation temperature are important variables in the determination of IgG ACA. Factors contributing to the influence of either variable in individual sera could not be identified. Key words: Anti-cardiolipin;Autoantibody; Autoimmunity; Assay temperature; Coating procedure

Introduction

Antibodies to negatively charged phospholipids, in particular ACA, were originally identiCorrespondence to: M.A. Firer, Advanced Biotechnologies, P.O. Box 1176, Kfar Saba 44111, Israel. Abbreviations: ACA, anti-cardiolipin antibodies; APA, anti-phospholipid antibodies; GPL, IgG anti-phosholipid antibodies; MPL, IgM anti-phospholipid antibodies; PAPS, primary anti-phospholipid syndrome; RT, room temperature; SLE, systemic lupus erythematosus.

fled in the sera of patients with SLE (Harris et al., 1983). These antibodies have been described in association with certain clinical features of the disease such as recurrent thromboembolic phenomena, thrombocytopenia and recurrent fetal loss (Harris et al., 1985a,b; Lockshin et al., 1985). Another group of patients with APA but without typical features of lupus have been identified and the term primary anti-phospholipid syndrome (PAPS) used to describe them (Hughes et al., 1986; Alarcon-Segovia, 1988; Asherson, 1988).

32 Other associations between anti-phospholipid antibodies and autoimmune or other diseases have also been reported recently (Klemp et al., 1988; Out et al., 1989; Stimmler et al., 1989; Ishii et al., 1990; Maclean et al., 1990). Most of the above studies have used solidphase immunoassays to detect ACA and have exploited variations of the method developed by Harris et al. (1983) and Loizou et al. (1985). In 1985 a study was conducted in which a set of ACA containing sera were distributed to and tested by a number of laboratories. The results were compared and later described (Harris et al., 1987). The report identified common features of the tests and general guidelines for 'acceptable' solid-phase ACA assays were outlined. Features of the assays producing 'incorrect' results included the use of Tween 20 as a detergent in both the assay and wash buffers and incubation of at least the primary antibody with solid phase antigen at 37°C. Despite these recommendations a variety of assays for ACA have been described (Eilat et al., 1986; Smeenk et al., 1987; Cheng, 1988; Shergy et al., 1988; Ishii et al., 1990) and reproducibility between laboratories has been unsatisfactory (Coulam et al., 1990). The effect of assay incubation temperature on anti-cardiolipin binding in a limited number of sera was studied in more detail by Lockshin et al. (1988). These results indicated that the binding of IgG, but not IgM antibodies to microplate bound phospholipid was temperature dependent, being markedly decreased when incubations were performed at 37°C rather than at RT. In these studies the antigen was bound to the solid-phase by evaporation in organic solvent and, interestingly, the binding of IgG ACA to phospholipid micelles was not temperature dependent. In the course of developing a sensitive and specific enzyme immunoassay for ACA, we have re-examined the influence of assay incubation temperature on the ACA binding capacity of a large number of sera under different assay conditions. In the present study, we have also compared two cardiolipin coating procedures and report that on a population basis, incubation temperature (at least up to 37°C) has no influence on the binding capacity of IgG ACA but may influence the binding of IgM ACA.

Materials and methods

Serum samples A total of 323 sera were tested. Of these, 31 were assayed after their routine laboratory evaluation for a variety of antinuclear autoantibodies using a series of ELISAs described elsewhere (Firer et al., 1988). Sera from additional 61 patients were kindly provided by Prof. Y. Naparastek (Hadassah Medical Center, Jerusalem). On retrospective examination of clinical data these 92 patients suffered from a variety of clinical disorders only some of which were of autoimmune etiology. Two patients had been diagnosed as PAPS and 19 were SLE patients. Of these, 14 had been assessed as having active or nonactive disease at the time of sampling. Aliquots of sera from 210 blood bank donors were used as a source of negative sera to determine normal cutoff ACA levels. As recognized positive standards a set of anticardiolipin standards calibrated with respect to G P L and MPL content were obtained from Dr.N. Harris (University of Louisville, U.S.A.).

ELISA for ACA Method 1. Cardiolipin (Sigma) was optimally diluted in 0.05 M sodium carbonate buffer, pH 9.6. ( 3 0 / z g / m l ) and 125/zl added to the wells of Nunc microplates (Nunc, Denmark) for 48 h at 4° C. After coating, plates were either immediately sealed under vacuum and stored at 4°C or first blocked with 1% BSA in 0.01 M PBS, pH 7.4 for 1 h at R T before use. Serum samples were diluted to 1/200 in assay diluent (0.75% gelatin, 0.3% BSA in PBS) (Harris et al., 1987) and 100 /~1 added to duplicate wells. The plates were then incubated either for 30 min at 37 ° C or for 60 min at R T (approximately 22°C). Following incubation, wells were emptied and washed four times with PBS only (Harris et al., 1987; Gharavi and Lockshin, 1988). 100 tzl goat anti-human IgG or IgM alkaline phosphatase conjugates (Sigma), diluted 1/500 in assay diluent, were added to the wells and incubated as described above. Wells were again washed and 100/zl substrate solution (5 mg p-nitrophenylphosphate tablet (Sigma) in 1 ml diethanolamine, MgCI 2 buffer, pH 9.8) added. After incubation for 60 rain at 37°C optical densi-

33

ties were read using a Titertek Multiskan ELISA reader at 405 nm. Optical densities obtained within a single microplate were either compared directly or converted to arbitrary U / m l for interassay comparison by reference to a high titered serum included on each plate. Assay blanks (assay diluent without sample in the first incubation step) as well as known negative and positive sera were also included in each plate run. Method 2. In order to compare the effect of different cardiolipin coating methods on A C A binding, antigen was also coated to microplates by evaporation in ethanol at 50 ~ g / m l overnight at 4°C (Harris et al., 1983). The plates were then used for testing as described for method 1 above.

Specificity of ACA detection by EIA (A) Inhibition studies. To ensure that the EIA method specifically detected ACA, positive and negative sera were incubated at a final dilution of 1/200 with different final concentrations (0-100 /zg/ml) of cardiolipin or a non-specific inhibitor (BSA) prepared in assay diluent. In preliminary experiments the 1/200 dilution was found to lie within the linear portion of the dilution curves of the sera used. The solutions were incubated for 1 h at either 37°C or R T and then added directly to cardiolipin coated microplates. The assay was then developed as described above with incubations at 37°C.

Results

Specificity of ACA detection by ELISA The results of a representative experiment showing the specificity of the ELISA for A C A detection are shown in Fig. 1. The binding of IgG A c A to cardiolipin coated to microplates in aqueous buffer was inhibited by prior incubation of positive sera with cardiolipin but not with a non-relevant protein (BSA). IgM ACA binding could also be similarly inhibited (data not shown). While the ACA activity of one serum was completely inhibited, another serum only reached 55% inhibition over the range of cardiolipin concentrations used. As indicated by the linear shape of the inhibition curve this serum could be further inhibited by higher concentrations of cardiolipin.

ACA levels in control subjects Sera from 210 blood bank donors were tested for IgG and IgM A C A by ELISA with assay incubations performed either at 37°C or RT. OD values were converted to arbitrary U / m l by comparison with the dilution curve of an internal reference serum. Analysis of variance showed there to be no significant difference in the distribution of ACA levels of either isotype measured 100

-

(B) Comparison with an alternative anticardiolipin ELISA. 35 sera were tested for IgG

8(]--!

and IgM A C A with another commercial ELISA (SELISA, Walker Laboratories, U.K.). The test was performed according to the manufacturer's specifications in which incubations are carried out at R T and the results recorded in G P L or M P L / m l notation.

6G~~

4C 2C

I 0

Statistics The Wilcoxon signed rank test, one way analysis of variance and X 2 analyses were used to assess the influence of incubation temperature on the measurement of ACA. When necessary, Yates' corrected X 2 was used. Linear regression analysis was used to compare G P L or M P L versus IgG or IgM arbitrary U.

0.1

0.5 1 #g/rnl inhibitor

10

50

100

Fig. 1. Inhibition of ]gG A C A binding to microplates coated with cardio]ipin in carbonate buffer. Two positive sera ( II, + ) were incubated with solution phase cardiolipin (11, + ) or BSA (Q, *) and then tested for residual antibody activity as described in the materials and methods section. A C A binding of one serum was almost entirely inhibited by incubation with ]00 p.g/ml cardiolipin but not with BSA (11 II). Another serum ( + - + ) required higher concentrations of cardio]ipin to achieve complete inhibition.

34

at the two temperatures (Table I). Normal cutoff levels of mean + 3 standard deviations for each isotype were then determined. The incidence of sera positive for ACA in the blood bank population was similar under both assay conditions (IgG ACA: 0.39% and 0.5% positivity at 37°C and R T respectively; IgM ACA 5.8% and 5% at 37°C and R T respectively).

1,2 1,C 0,8!

0,6 0.4 0.2

Measurement of ACA in standard sera To test the effect of assay incubation temperature on A C A activity in known positive sera, IgG and IgM ACA were measured in a set of recognized, calibrated standards. The results in Fig. 2 show that incubating the sera on the coated microplate at 37°C or at RT resulted in similar

0.0 0.8

1

i

1.0

1.2

1

I

I

1.4 1.6 1.8 Log GPL/ML

Fig. 2. Reactivity of cardiolipin standard assay incubation temperatures at either temperature (It). These standard sera G P L / m l . Each graph point represents separate tests.

1

[

I

I

2.0

2.2

2.4

2.(5

sera in E L I S A with 37°C (4-) or room were calibrated in the m e a n of three

TABLE I P O P U L A T I O N D A T A F O R A C A L E V E L S IN 210 N O R M A L SUBJECTS M E A S U R E D U S I N G D I F F E R E N T ASSAY INCUBATION TEMPERATURES A C A isotype

IgG IgM

Assay

Population

temperature (° C)

Mean

Std.Dev.

Cutoff a

37 22 37 22

170 c 130 130 80

45 50 80 80

AOV b p value

305 282 370 330

0.79 0.68

a Negative cut-off level = m e a n + 3 standard deviations of the mean. b p value for one way analysis of variance. Significance level is p < 0.05. c Arbitrary A C A U / m l determined as descibed in the materials and methods section.

T A B L E II C O M P A R A T I V E P O P U L A T I O N STATISTICS O F P A T I E N T S E R A T E S T E D F O R A C A U S I N G D I F F E R E N T ASSAY CONDITIONS Antigen coating a

ACA isotype

Assay temp.

no. sera

Population Mean

Std.Dev.

Aqueous (carbonate)

IgG

37 22 37 22

113 113 105 105

270 b 280 160 180

IgG

37 22

34 34

IgM

37 22

34 34

IgM Organic solvent (ethanol)

AOV p value

Wilcoxon p value

210 260 140 140

0.76 c

0.866

0.30

0.001

377 470

373 5-10

0.69

0.11

333 410

255 290

0.26

0.001

a For full details of coating procedures see the materials and methods section. b Arbitrary U / m l of either IgG or IgM ACA. c Level of significance was taken as p < 0.05.

35

levels of I g G A C A reactivity at each point of the dilution curve. Similar results were obtained for IgM A C A (data not shown).

TABLE 1II

ACA levels in patient sera The influence of assay incubation t e m p e r a t u r e was further tested by comparing the I g G and IgM A C A levels measured in serum samples from patients with a variety of clinical conditions. Analysis of the population variances (Table II) showed that there was no statistical difference between the two assay conditions either for the m e a s u r e m e n t of I g G A C A ( p = 0.756) or for IgM A C A ( p = 0.298). Paired-tests of the influence of incubation t e m p e r a t u r e on the results obtained with individual sera (Wilcoxon's signed rank test) showed that there was no statistically significant alteration for I g G A C A ( p = 0.8662) although IgM A C A were significantly influenced in favor of R T ( p = 0.001). Wilcoxon's statistical test does not make allowance for recognized interassay variations in tests such as ELISA. Small differences within the acceptable % coefficient of variation (CV) for the assay (in this case + 15%) are then counted as real-time differences resulting in a possibly biased picture of the influence of a particular test variable on the results obtained. Therefore to analyse correctly for a possible influence of assay incubation t e m p e r a t u r e on A C A m e a s u r e m e n t the A C A U / m l measured at 37°C were taken as baseline and those sera giving values at R T outside the acceptable assay % C V were counted. The results are shown in T~ble III. For I g G A C A 16/113 sera gave significan¢ly lower values at R T than at 37°C while 16 o t h e r sera gave significantly higher values at R T than 37 ° C. For IgM A C A 6 / 1 0 5 sera were significantly lower when assayed

ACA

S I G N I F I C A N T A L T E R A T I O N S IN IgG OR IgM ACA IN ELISA P R O C E D U R E S P E R F O R M E D AT 22 ° C Compared to 37 ° C incubation

isotype Unchanged

Increased

Decreased

IgG IgM

16 b 6

16 c 13

81 a 86

A"2

19 value

0 2.15

1.0 0.14

a Sera with ACA U / m l at 22°C within _+ 15% of those at 37 ° C. b S e r a w i t h A C A U / m l a t 2 2 ° C > 15% of those at 37 °C. c S e r a w i t h A C A U / m l a t 2 2 ° C < 15% of those at 37 °C.

at R T while 13 were significantly higher at RT. X 2 analysis showed that these associations were random for both isotypes.

Influence of assay temperature on ACA levels in SLE patients 42% of the 19 SLE sera were positive for at least one A C A isotype. Assay t e m p e r a t u r e did not influence the A C A titers in the sera of patients when group variances were compared (IgG A C A p = 0.721; IgM A C A p = 0.537) nor when individual sera were paired-tested ( I g G A C A p = 0.164; IgM A C A p = 0.97). O f these patients ten were known to have active disease at the time of sampling while four were known to be inactive. Disease activity was not associated with differing A C A levels for either of the two incubation conditions. Comparison with an alternative EIA for ACA As an additional test of the influence of assay incubation temperatures on A C A binding in ELISA, a direct comparison was made between the A C A levels measured using method 1 and

TABLE IV Q U A L I T A T I V E D E T E C T I O N OF IgG A N D IgM ACA AT 37°C A N D 22°C ACA

N

isotype IgG IgM

46 45

37°C

22oc

a

Positive

Negative

Positive

Negative

25 c 15

21 30

25 c 16

21 29

a Performed with commercial ACA kit (SELISA, Walker Labs.). b P value of X 2 with Yates' correction. Significance level is p < 0.05. c For each case one sample was positive at one temperature but negative at the other.

X2

p value b

34.7 16.6

0.0001 0.0001

36 220C

700 b

2000 600 1BOC 160C

5 0 0 --

0

140C

400

120C

< L) <[ 100( t9 o3 8 0 ( 60C

40(

< U 0

~ 0 0 0

0 3O0

E

°y°

-

2oo

100

][3

0

2O< I

1

I

I

I

50

100

150

200

250

Selise

O0

t

5

I

10

I 15

i 20 Seliso

GPL/ml

2~5

I

30

I

35

I

40

I 45

I 50

MPL

Fig. 3. Correlation of in-house E L I S A run at 37°C with a commercial E L I S A run at R T for the m e a s u r e m e n t of IgG (a) or IgM (b) ACA. T h e in-house assay was calibrated in IgG or IgM arbitrary U / m l and the commercial assay in G P L or M P L / m l . Linear regression for IgG A C A r = 0.918 and for IgM A C A r = 0.729. ([]) samples; ( ) regression line.

those obtained using another commercial ACA assay (SELISA EIA, Walker Laboratories) which employed R T incubations. For the SELISA test, sera were designated as positive or negative according to the normal ranges indicated by the manufacturer. For our system, samples with ACA levels above the mean + 3 standard deviations for the 210 blood bank control sera tested (Table I) were considered positive. Table IV presents qualitative results obtained by the two methods. The correlation of results was highly significant both for IgG ACA (X 2 = 34.7; p < 0.0001) and for IgM ACA (X z = 16.6; p = 0.0001). Quantitative analysis of the ACA titers measured also showed that results obtained with the two methods were significantly correlated as shown in Fig. 3 (IgG ACA r = 0.918; IgM r = 0.73).

Assay incubation temperature and method of antigen coating T o test whether different methods of antigen coating might indirectly influence any effect of assay temperature on ACA binding, microplates were coated with cardiolipin in organic solvent (ethanol) and used to test the response of 34 patient sera assayed at both 37°C and RT. The results in Table II show that under these coating conditions IgG ACA and IgM ACA population

variations were similar for both assay temperatures (IgG p = 0.407; IgM p = 0.257). In paired tests of each serum the influence of assay temperature was not significant for IgG ACA ( p = 0.07) but was significant for IgM ACA ( p <0.001) measured at RT. This influence on IgM ACA was also significant outside the assay %CV (X 2 = 15.2, p = 0.001).

Discussion

Our results demonstrate that alterations in assay incubation temperatures, at least within the range of 22-37°C do not significantly influence the binding of either IgG or IgM ACA in ELISA procedures provided that the cardiolipin is allowed to bind to the microplate in acqueous solution. IgM ACA can be affected by incubation temperature when the antigen is coated in organic solvent (Table II). These results appear to be in contrast to the initial relSort of Harris et al. (1987). Later Lockshin et al. (1988) reported that the binding of IgG but not of IgM ACA to the solid phase bound cardiolipin was inhibited when these were incubated together at 37°C. Those studies reported results on a limited number of sera only and the

37 phenomenon has, to the best of our knowledge, not been re-examined on a more systematic basis using a larger series of samples. If such an influence was to be a general phenomenon its significance could relate not only to the performance reliability of ELISAs now in use by many laboratories for the routine detection of ACA in patient sera, but also to the biological significance of the antibodies measured under different assay conditions. In screening 323 sera by an ELISA in which cardiolipin was attached to the microplate by passive adsorption in aqueous buffer, we found no consistent difference in ACA levels when incubations were performed either at 37°C or RT. This was first demonstrated for 210 control sera where analysis of population statistics showed the distribution of both IgG and IgM ACA titers was similar under both assay conditions (Table I) as was the incidence of positive ACA. This result suggests that the reactivities of normal sera are not influenced by assay incubation temperature as was previously shown by Lockshin et al. (1988). The influence of assay incubation temperature on positive ACA sera was first tested using recognized IgG and IgM ACA standards. The dilution curves of both IgG ACA (Fig. 2) and IgM ACA were similar, indicating that these standards were not adversely affected by incubation at 37°C. Analysis of the population statistics of 113 samples from patients suffering from autoimmune or other disorders also revealed no significant influence of assay temperature on the quantitative detection of either IgG or IgM ACA (Table II). Furthermore, we compared our method (aqueous buffer coating) run at 37°C with that of a commercial ACA test kit (Walker Laboratories) in which the assay is performed at RT. We found a very good correlation between the two systems and the designation by each of the methods of samples as positive or negative was highly concordant (IgG ACA 95.6% and IgM ACA 93.3%, see Table IV). It should be noted that some individual sera are influenced by incubation temperature and that this phenomenon can occur in both directions. For example, with IgG ACA a similar number of sera (16/113) gave significantly higher reactions at 37°C as those that gave higher reac-

tions at RT (Table III). For IgM ACA there were more sera giving higher values at R T than at 37°C but this difference was not statistically significant. We cannot at this time comment in detail on the clinical significance of those individual sera that do react better under one of the two incubation conditions. The 113 patients tested in this study included only 19 well defined SLE patients and two with PAPS. Of the SLE patients, eight (42%) were positive for at least one ACA isotype, a prevalence similar to that found in earlier studies (Harris et al., 1983; Petri et al., 1987). IgG and IgM ACA levels were similar using both assay conditions although 1/10 patients with known active disease showed a significant elevation of IgM ACA at RT. Of the patients with PAPS one showed no change in high levels of both IgG and IgM antibodies while the other showed an elevation in IgM levels at 37°C. These numbers are too small to offer meaningful conclusions as to the influence of incubation temperature on ACA measurements in different disease states. The above results were obtained using microplates coated with cardiolipin in aqueous solution (carbonate buffer). It is possible that this method of coating permits the presentation of antigen in such a way as to overcome any general phenomenon of assay temperature influencing ACA binding. We therefore compared this method of coating with that of evaporation under organic solvent, a procedure often described for other ACA assays (Lockshin et al., 1985; Loizou et al., 1985; Harris et al., 1987). We found that under these coating conditions IgM, but not IgG ACA levels were significantly influenced by assay temperature. These results are in contrast with those of Lockshin et al. (1987) who found that IgG, but not IgM ACA binding was decreased at 37°C. Interestingly, 13/14 of those sera positively influenced by RT with the ethanol coating method were not similarly influenced by RT using the carbonate method suggesting that in the two procedures cardiolipin may assume different three dimensional configurations which might differentially influence the antibody binding capacity of ACA from individual sera. Such a possibility was even more clearly suggested by the IgG data. Of the ten sera significantly influenced by incubation

38

temperature using the ethanol coating method (eight by RT and two by 37°C) three gave the opposite result using the carbonate coating method. We could not correlate these alterations to any particular disease group and additional work is needed to determine whether reactivity to either form of the solid phase coated antigen has relevance to clinical disease a n d / o r other immunological parameters such as antibody affinity or epitope availability. Recent work by Costello et al. (1990) has shown that the phosphate group of the cardiolipin molecule is critical for antibody binding and the availability of this portion of the molecule may be qualitatively a n d / o r quantitatively altered under the different coating conditions compared in this study. We are currently investigating these aspects. The coating conditions used in this study were derived empirically by checkerboard titration and represent optimal conditions for specific antibody detection. Studies by Rauch et al. (1986) have demonstrated that monoclonal anti-lupus coagulant antibodies preferentially bind the hexagonal II rather than the lamellar form of phosphatidylethanolamine (PE) present in normal tissue membrane bilayers. This phospholipid assumes a hexagonal II structure at 37°C in vitro. While antibodies against this structure cross reacted with cardiolipin (Rauch et al., 1990), the clinical significance of antibodies to PE have been recently questioned (Qamar et al., 1990). We do not have data on antibodies to PE in our patients but our results would suggest that any such structural alterations occuring in cardiolipin, at least up to 37°C, do not significantly alter the detection of ACA in ELISA.

Acknowledgements We would like to thank Professor Naparastek for supplying some of the patient sera and to Dr. S. Hirsch, Dr. R. Sa'ag and Dr. N. Zurgil for helpful discussions. References Alaeron-Segovia, D. (1988) Pathogenic potential of antiphospholipid antibodies. J. Rheumatol. 15, 890.

Asherson, R.A. (1988) A primary antiphospholipid syndrome? [editorial]. J. Rheumatol. 15, 1742. Cheng, H.M. (1988) Antigenicity of cardiolipin in Tween 20. J. Immunol. Methods 114, 279. Costello, P.B., Powell, G i . and Green, F.A. (1990) The structural requirements for anti-cardiolipin antibody binding in sera from patients with syphilis and SLE. Clin. Immunol. Immunopathol. 56, 393. Coulam, C.B., McIntyre, J.A., Wagenknecht, D. and Rote, N. (1990) Interlaboratory inconsistencies in detection of anticardiolipin antibodies. Lancet, I, 335. Eilat, D., Zlotnick, A.Y. and Fischel, R. (1986) Evaluation of the cross-reaction between anti-DNA and anti-cardiolipin antibodies in SLE and experimental animals. Clin. Exp. Immunol. 65, 269. Firer, M.A., Shoenfeld, Y., lsenberg, D.A. and Slot, H. (1988) The use of ELISA for the measurement of autoantibodies to nuclear antigens; comparison with other technologies. Isr. J. Med. Sci. 24. 747. Gharavi, A.E. and Lockshin, M.D. (1988) Enhancement of anti-phospholipid antibody activity by Tween 20. J. Immunol. Methods 114, 277. Harris, E.N., Gharavi, A.E., Boey, M.L., Patel, B.M., Mackworth-Young, G.G., Loizou, S. and Hughes, G.R.V. (1983) Anticardiolipin antibodies: detection by radioimmunioassay and association with thrombosis in systemic lupus erythematosus. Lancet II, 1211. Harris, E.N., Asherson, R.A., Gharavi, A.E., Morgan, S.H., Derue, G. and Hughes, G.R.V. (1985a) Thrombocytopenia in SLE and related autoimnmne disorders: association with anticardiolipin antibody. Br. J. Haematol. 59, 227. Harris, E.N., Gharavi, A.E. and Hughes, G.R.V. (1985b) Anti-phospholipid antibodies. Clin. Rheum. Dis. 11,591. Harris, E.N., Gharavi, A.E., Patel, S.P. and Hughes, G.R.V. (1987) Evaluation of the anti-cardiolipin antibody test: report of an international workshop held 4 April 1986. Clin. Exp. Immunol. 68, 215. Hughes, G.R.V., Harris, E.N. and Gharavi, A. (1986) The anticardiolipin Syndrome. J. Rheumatol. 13, 426. Ishii, Y., Nagasawa, T. and Niho, Y. (1990) Clinical importance of persistence of anticardiolipin antibodies in systemic lupus erythematosus. Ann. Rheum. Dis. 49, 387. Klemp, P., Cooper, R.C., Strauss, F.J., Jordaan, E.R., Przybojewski, J.Z. and Nel, N. (1988) Anti-cardiolipin antibodies in ischaemic heart disease. Clin. Exp. Immunol. 74, 254. Lockshin, M.D., Druzin, M.L., Goei, S., Qamar, T., Magid, M.S., Jovanovic, L. and Ferenc, M. (1985) Antibody to cardiolipin as a predictor of fetal distressor death in patients with systemic lupus erythrematosus. Lancet II, 152. Lockshin, M.D., Qamar, T., Levy, R.A. and Best, M.P. (1988) IgG but not IgM anti-phospholipid antibody binding is temperature dependent. J. Clin. Immunol. 8, 188. Loizou, S., McCrea, J.D., Rudge, A.C., Reynolds, R, Boyle, C.C. and Harris, E.N. (1985) Measurement of anticardiolipin antibodies by an enzyme-linked immunosorbant assay (ELISA): standardization and quantitation of results. Clin. Exp. Immunol. 62, 738. Maclean, C., Flegg, P.J. and Kilpatrick, D.C. (1990) Anti-

39 cardiolipin antibodies and HIV infection. Clin. Exp. Immunol. 81,263. Out, H.J., De Groot, P.G., Hasselaar, P., Vliet, M.V. and Derksen, R.H.W.M. (1989) Fluctuations of anticardiolipin antibody levels in patients with systemic lupus erythematosus:a prospective study. Ann. Rheum. Dis. 48, 1023. Petri, M., Rheinschmidt, M., Whiting-O'Keefe, Q., Hellmann, D. and Corash, L. (1987) The frequency of lupus anticoagulant in systemic lupus erythematosus. Ann. Int. Med. 106, 524. Qamar, T., Levy, R.A., Sammaritano, L., Gharavi, A.E. and Lockshin, M.D. (1990) Characteristics of high-titer IgG antiphospholipid antibody in systemic lupus erythematosus patients with and without fetal death. Arthritis Rheum. 33, 501. Rauch, J. and Janoff, A.S. (1990) Phospholipid in the hexagonal II phase is immunogenic: Evidence for immunorecognition of nonbilayer lipid phases in vivo. Proc. Natl. Acad. Sci. U.S.A. 87, 4112.

Rauch, J., Tannenbaum, M., Tannenbaum, H., Ramelson, H., Cullis, P.R., Tilcock, C.P.S., Hope, M.J. and Janoff, A.S. (1986) Human hybridoma lupus anticoagulants distinguish between lamellar and hexagonal phase lipid systems. J. Biol. Chem. 261, 9672. Shergy, W.J., Kredich, D.W. and Pisetsky, D.S. (1988) The relationship of anticardiolipin antibodies to disease manifestations in pediatric systemic lupus erythematosus. J. Rheumatol. 15, 1389. Smeenk, R.D.T., Lucassen, W.A.M. and Swaak, T.J.G. (1987) Is anticardiolipin activity a cross-reaction of anti-DNA or a separate entity? Arthritis Rheum. 30, 607. Stimmler, M.M., Quismorio, F.P., McGehee, W.G., Boylen, T. and Sharma, O.P. (1989) Anticardiolipin antibodies in acquired immunodeficiency syndrome. Arch. Intern. Med. 149, 1833.