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A protocol for determination of anticardiolipin antibodies by ELISA

Nature Protocols volume 3, pages 840–848 (2008)Cite this article

Abstract

The anticardiolipin (aCL) test has been widely used by physicians since the mid-1980s for diagnosing patients with antiphospholipid syndrome (APS). Establishment of this diagnosis has enabled effective management of patients with recurrent thrombosis or recurrent pregnancy losses. The test was first established in 1983 as a radioimmunoassay and soon thereafter converted into ELISA. There have been numerous efforts to standardize the aCL test, but precise reproducible measurement of aCL levels is difficult and the use of semiquantitative measurements (high, medium and low) is recommended as this is probably sufficient for clinical diagnosis. Using validated ELISAs for measuring aCL Abs offers greater reproducibility, would reduce interlaboratory variations and limit discrepancies in results between different laboratories. This article details a procedure that takes ∼2 h and summarizes the information available on the aCL ELISA test.

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Figure 1
Figure 2: Examples for calibration curves for anticardiolipin (aCL) ELISA test.

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References

  1. Harris, E.N. Syndrome of the black swan. Br. J. Rheumatol. 26, 324–326 (1987).

    Article  CAS  PubMed  Google Scholar 

  2. Wilson, W.A. et al. International consensus statement on preliminary classification for definite antiphospholipid syndrome. Arthritis Rheum. 42, 1309–1311 (1999).

    Article  CAS  PubMed  Google Scholar 

  3. Miyakis, S. et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J. Thromb. Haemost. 4, 295–306 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Lockshin, M.D., Sammaritano, L.R. & Schwartzman, S. Validation of the Sapporo criteria for antiphospholipid syndrome. Arthritis Rheum. 43, 440–443 (2000).

    Article  CAS  PubMed  Google Scholar 

  5. Harris, E.N. et al. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis. Lancet 2, 1211–1214 (1983).

    Article  CAS  PubMed  Google Scholar 

  6. Gharavi, A.E., Harris, E.N., Asherson, R.A. & Hughes, G.R. Anticardiolipin antibody isotype distribution and phospholipid specificity. Ann. Rheum. Dis. 46, 1–6 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Harris, E.N., Gharavi, A.E., Patel, S. & Hughes, G.R. Evaluation of the anticardiolipin antibody test: report of an international workshop held 4 April 1986. Clin. Exp. Immunol. 68, 215–222 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Harris, E.N. The second international anticardiolipin standardization workshop: the Kingston Anticardiolipin Antibody Study (KAPS) group. Am. J. Clin. Pathol. 101, 616–624 (1990).

    Article  Google Scholar 

  9. Pierangeli, S.S., Stewart, M., Silva, L.K. & Harris, E.N. Report of an anticardiolipin workshop during the VIIth International Symposium on antiphospholipid antibodies. J. Rheumatol. 25, 156–162 (1998).

    CAS  PubMed  Google Scholar 

  10. Reber, G. et al. Multicenter evaluation of nine commercial kits for the quantitation of anticardiolipin antibodies. Thromb. Haemost. 73, 444–452 (1995).

    Article  CAS  PubMed  Google Scholar 

  11. Harris, E.N., Pierangeli, S. & Birch, D. Anticardiolipin wet workshop report: Vth international symposium on antiphospholipid antibodies. Am. J. Clin. Pathol. 101, 616–624 (1994).

    Article  CAS  PubMed  Google Scholar 

  12. De Moerloose, P., Reber, G. & Vogel, J.J. Anticardiolipin antibody determination: comparison of three ELISA assays. Clin. Exp. Rheumatol. 8, 575–577 (1990).

    CAS  PubMed  Google Scholar 

  13. Reber, G. et al. Multicenter evaluation of nine commercial kits for the quantitation of anticardiolipin antibodies. Thromb. Haemost. 73, 444–452 (1995).

    Article  CAS  PubMed  Google Scholar 

  14. Wong, R., Wilson, R., Pollock, W., Steele, R. & Gillis, D. Anticardiolipin antibody testing and reporting practices among laboratories participating in a large external Quality Assurance Program. Pathology 36, 174–181 (2004).

    Article  PubMed  Google Scholar 

  15. Tincani, A. et al. Anticardiolipin antibody assay: a methodological analysis for a better consensus in routine determinations—a cooperative project of the European Antiphospholipid Forum. Thromb. Haemost. 86, 575–583 (2001).

    Article  CAS  PubMed  Google Scholar 

  16. Harris, E.N. & Pierangeli, S.S. Revisiting the anticardiolipin test and its standardization. Lupus 11, 269–275 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Wong, R.C. Australasian aCL Working Party. Consensus guidelines for anticardiolipin antibody testing. Thromb. Res. 114, 559–571 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Triplett, D.A. Antiphospholipid antibodies. Arch. Pathol. Lab. Med. 126, 1424–1429 (2002).

    CAS  PubMed  Google Scholar 

  19. Favaloro, E.J. & Silvestrini, R. Assessing the uselfulness of anticardiolipin antibody assays: a cautious approach is suggested by high variation and limited consensus in multilaboratory testing. Am. J. Clin. Pathol. 118, 548–557 (2002).

    Article  PubMed  Google Scholar 

  20. Peaceman, A.H., Silver, R.K., MacGRegor, S.N. & Socol, M.L. Interlaboratory variation in antiphospholipid antibody testing. Am. J. Obstet. Gynecol. 144, 1780–1787 (1992).

    Article  Google Scholar 

  21. Favaloro, E.J. & Silvestrini, R. Assessing the usefulness of anticardiolipin antibody assays: a cautious approach is suggested by high variation and limited consensus in multilaboratory testing. Am. J. Clin. Pathol. 118, 548–557 (2002).

    Article  PubMed  Google Scholar 

  22. Harris, E.N. Annotation: antiphospholipid antibodies. Br. J. Haematol. 74, 1–9 (1990).

    Article  CAS  PubMed  Google Scholar 

  23. Harris, E.N. et al. Thrombosis, recurrent fetal loss and thrombocytopenia: predictive value of the anti-cardiolipin test. Arch. Intern. Med. 146, 2153–2156 (1986).

    Article  CAS  PubMed  Google Scholar 

  24. Escalante, A., Brey, R.L., Mitchel, B.D. & Dreiner, U. Accuracy of anticardiolipin antibodies in identifying a history of thrombosis among patients with systemic lupus erythematosus. Am. J. Med. 98, 559–565 (1995).

    Article  CAS  PubMed  Google Scholar 

  25. Levine, S.R. et al. IgG anticardiolipin antibody titer >40 GPL and the risk of subsequent thrombo-occlusive events and death. Stroke 28, 1660–1665 (1997).

    Article  CAS  PubMed  Google Scholar 

  26. Silver, R.M. et al. Anticardiolipin antibodies: clinical consequences of 'low titers'. Obstet. Gynecol. 87, 494–500 (1996).

    Article  CAS  PubMed  Google Scholar 

  27. Galli, M., Luciani, D., Bertolini, G. & Barbui, T. Lupus anticoagulants are stronger risk factors for thrombosis than anticardiolipin antibodies in the antiphospholipid syndrome: a systematic review of the literature. Blood 101, 1827–1832 (2003).

    Article  CAS  PubMed  Google Scholar 

  28. Nash, M.J. et al. The anticardiolipin assay is required for sensitive screening for antiphospholipid antibodies. J. Thromb. Haemost. 2, 1077–1081 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Kaplan, V. et al. Real world experience with antiphospholipid antibodies (APL): how useful is anti-β2glycoprotein (β2GPI) test? Arthritis Rheum. 50, S67 (2004).

    Google Scholar 

  30. Pierangeli, S.S., Gharavi, M.D. & Harris, E.N. Testing for antiphospholipid antibodies: problems and solutions. Clin. Obstet. Gynecol. 44, 48–57 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Kutteh, W.H. & Franklin, R.D. Assessing the variation in antiphospholipid antibody (APA) assays: comparison of results from 10 centers. Am. J. Obstet. Gynecol. 191, 440–448 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. Galli, M. et al. Anticardiolipin (ACA) antibodies directed not to cardiolipin but to a plasma protein cofactor. Lancet 335, 1544–1547 (1990).

    Article  CAS  PubMed  Google Scholar 

  33. Matsuura, E., Igarashi, Y. & Fujimoto, M. Anticardiolipin antibodies directed not to cardiolipin but to a plasma protein cofactor. Lancet 336, 1547 (1990).

    Article  Google Scholar 

  34. McNeil, H.P., Simpson, R.H., Chesterman, C.N. & Krilis, S. Anti-phospholipid antibodies are directed against a complex antigen that includes a lipid binding inhibitor of coagulation: β2glycoprotein I (apolipoprotein H). Proc. Natl. Acad. Sci. USA 87, 4120–4124 (1990).

    Article  CAS  PubMed  Google Scholar 

  35. Matsuura, E., Igarashi, Y., Yasuda, Y., Triplett, D.A. & Koike, T. Anticardiolipin antibodies recognize β2glycoprotein I structure altered by interacting with an oxygen modified solid phase surface. J. Exp. Med. 179, 457–462 (1994).

    Article  CAS  PubMed  Google Scholar 

  36. Roubey, R.A.S., Eisenberg, R.A., Harper, M.F. & Winfield, J.B. Anticardiolipin autoantibodies recognize β2glycoprotein I in the absence of phospholipid. Importance of Ag density and bivalent binding. J. Immunol. 15, 954–960 (1995).

    Google Scholar 

  37. Harris, E.N. et al. Cross-reactivity of antiphospholipid antibodies. J. Clin. Lab. Immunol. 16, 1–6 (1985).

    CAS  PubMed  Google Scholar 

  38. Oosting, J.D. et al. Antiphospholipid antibodies directed against a combination of phospholipids with prothrombin, protein C or protein S: an explanation for their pathogenic mechanism? Blood 81, 2618–2625 (1993).

    CAS  PubMed  Google Scholar 

  39. Bevers, E., Galli, M., Barbui, T., Comfurius, P. & Zwaal, R.D. Lupus anticoagulant IgGs (LA) are not directed to phospholipid only, but to a complex of lipid-bound human prothrombin. Thromb. Haemost. 66, 629–632 (1991).

    Article  CAS  PubMed  Google Scholar 

  40. Tsumi, A.T., Amengual, O., Yasuda, S. & Koike, T. Antiprothrombin antibodies—are they worth assaying? Thromb. Res. 114, L533 (2004).

    Article  CAS  Google Scholar 

  41. Cheng, H.M. & Khairullah, N.S. Induction of antiphospholipid autoantibody during cytomegalovirus infection. Clin. Infect. Dis. 25, 1493–1494 (1997).

    Article  CAS  PubMed  Google Scholar 

  42. Adebajo, A.O., Charles, P., Maini, R.N. & Hazleman, B.L. Autoantibodies in malaria, tuberculosis and hepatitis in a West African population. Clin. Exp. Immunol. 92, 73–76 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Gratacos, E. et al. Prevalence and clinical significance of anticardiolipin antibodies in pregnancies complicated by parvovirus B19 infection. Prenat. Diagn. 15, 1109–1113 (1995).

    Article  CAS  PubMed  Google Scholar 

  44. Santiago, M. et al. Anti-β2glycoprotein I and anticardiolipin antibodies in leptospirosis, syphilis and kala-azar. Clin. Exp. Rheumatol. 19, 425–430 (2001).

    CAS  PubMed  Google Scholar 

  45. Leroy, V. et al. Prevalence and significance of anticardiolipin, anti-β2glycoprotein I and anti-prothrombin antibodies in chronic hepatitis. Br. J. Haematol. 101, 468–474 (1998).

    Article  CAS  PubMed  Google Scholar 

  46. Constans, J. et al. Autoantibodies directed against phospholipids or human β2glycoprotein I in HIV-seropositive patients: relationship with endothelial activation and antimalonic dialdehyde antibodies. Eur. J. Clin. Invest. 28, 115–122 (1998).

    Article  CAS  PubMed  Google Scholar 

  47. Hojnik, M. et al. Anticardiolipin antibodies in infections are heterogeneous in their dependency on β2Glycoprotein I: analysis of anticardiolipin antibodies in leprosy. Lupus 3, 515–521 (1994).

    Article  CAS  PubMed  Google Scholar 

  48. Loizou, S., Cazabon, J.K., Walport, M.J., Tait, D. & So, A.K. Similarities of specificity and cofactor dependence in serum antiphospholipid antibodies from patients with human parvovirus B19 infection and from those with systemic lupus erythematosus. Arthritis Rheum. 40, 103–108 (1997).

    Article  CAS  PubMed  Google Scholar 

  49. Wilson, W.A. et al. IgA antiphospholipid antibodies in HTLV-1 associated tropical spastic paraparesis. Lupus 4, 138–141 (1995).

    Article  CAS  PubMed  Google Scholar 

  50. Santiago, M.B., Cossemelli, W., Tuma, M.F. & Oliviera, R.M. Anticardiolipin antibodies in infectious diseases. Clin. Rheumatol. 8, 23–28 (1989).

    Article  CAS  PubMed  Google Scholar 

  51. Fiallo, P., Nunzi, E. & Cardo, P.P. β2glycoprotein I-dependent anticardiolipin antibodies as a risk factor for reactions in borderline leprosy patients. Int. J. Lepr. Other Mycobact. Dis. 66, 387–388 (1998).

    CAS  PubMed  Google Scholar 

  52. Durupt, S., Rosselli, S., Manchon, J. & Lopez, M. Presence of antiphospholipid antibodies in Legionnaires' disease. Presse Med. 25, 1649 (1996).

    CAS  PubMed  Google Scholar 

  53. Gálvez, J., Martin, I., Medino, D. & Pujol, E. Thrombophlebitis in a patient with acute Q fever and anticardiolipin antibodies. Med. Clin. (Barc) 108, 396–397 (1997).

    Google Scholar 

  54. Mouritsen, S., Høier-Madson, M., Wiik, A., Orum, O. & Strandberg Pedersen, N. The specificity of anti-cardiolipin antibodies from syphilis patients and from patients with systemic lupus erythematosus. Clin. Exp. Immunol. 76, 178–183 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Harris, E.N., Gharavi, A.E., Wasley, G.D. & Hughes, G.R. Use of an enzyme-linked immunosorbent assay and of inhibition studies to distinguish between antibodies to cardiolipin from patients with syphilis or autoimmune disorders. J. Infect. Dis. 157, 23–31 (1988).

    Article  CAS  PubMed  Google Scholar 

  56. Intrator, L., Oksenhendler, E. & Desforges, L. Anticardiolipin antibodies in HIV infected patients with or without autoimmune thrombocytopenia purpura. Br. J. Haematol. 67, 269–270 (1988).

    Article  Google Scholar 

  57. Canoso, R.T., Zon, L.I. & Groopman, J.E. Anticardiolipin antibodies associated with HTLV-III infection. Br. J. Haematol. 65, 495–498 (1987).

    Article  CAS  PubMed  Google Scholar 

  58. Loizou, S. et al. Measurement of anticardiolipin antibodies and biological false positive serological tests for syphilis in patients with systemic lupus erythematosus. Clin. Exp. Immunol. 56, 193–199 (1994).

    Google Scholar 

  59. Rote, N.S., Dostal-Johnson, D. & Branch, D.W. Antiphospholipid antibodies and recurrent pregnancy loss: correlation between the activated partial thromboplastin and antibodies against phosphatidylserine and cardiolipin. Am. J. Obstet. Gynecol. 163, 575–584 (1990).

    Article  CAS  PubMed  Google Scholar 

  60. Harris, E.N. & Pierangeli, S.S. A more specific ELISA assay for detection of antiphospholipid. Clin. Immunol. News 15, 26–28 (1995).

    Article  Google Scholar 

  61. Merkel, P.A., Chang, Y., Pierangeli, S.S., Harris, E.N. & Polisson, R.P. Comparison between the standard anticardiolipin antibody test and a new phospholipid test in patients with a variety of connective tissue diseases. J. Rheumatol. 26, 591–596 (1999).

    CAS  PubMed  Google Scholar 

  62. Nencini, P. et al. Lupus anticoagulant and anticardiolipin antibodies in young adults with cerebral ischemia. Stroke 23, 189–193 (1992).

    Article  CAS  PubMed  Google Scholar 

  63. Fields, R., Toubbeh, H., Searles, R. & Bankhurst, A. The prevalence of anticardiolipin antibodies in a healthy elderly population and its association with antinuclear antibodies. J. Rheumatol. 16, 623–625 (1989).

    CAS  PubMed  Google Scholar 

  64. Antiphospholipid Antibodies in Stroke Study Group. Clinical, radiological, and pathological aspects of cerebrovascular disease associated with antiphospholipid antibodies. Stroke 4 (12 suppl.), I120–I123 (1993).

  65. Schved, J.F., Dupuy-Fons, C., Biron, C., Quére, I. & Janbon, C. A prospective epidemiological study on the occurrence of antiphospholipid antibody: the Montpellier Antiphospholipid (MAP) Study. Haemostasis 24, 175–182 (1994).

    CAS  PubMed  Google Scholar 

  66. Ginsburg, K. et al. Anticardiolipin antibodies and the risk of ischemic stroke and venous thrombosis. Ann. Intern. Med. 117, 997–1002 (1992).

    Article  CAS  PubMed  Google Scholar 

  67. Kearon, C. et al. A comparison of three months of anticoagulation with extended anticoagulation for a first episode of idiopathic venous thromboembolism. N. Engl. J. Med. 340, 901–907 (1999).

    Article  CAS  PubMed  Google Scholar 

  68. Schulman, S., Svenungsson, E. & Granqvist, S. Duration of Anticoagulation Study Group. Anticardiolipin antibodies predict early occurrence of thromboembolism and death among patients with venous thromboembolism following anticoagulant therapy. Am. J. Med. 104, 332–338 (1998).

    Article  CAS  PubMed  Google Scholar 

  69. Wahl, D.G. et al. Meta-analysis of the risk of venous thrombosis in individuals with antiphospholipid antibodies without underlying autoimmune disease or previous thrombosis. Lupus 7, 15–22 (1998).

    Article  CAS  PubMed  Google Scholar 

  70. Cervera, R. et al. Morbidity and mortality in systemic lupus erythematosus during a 5 year period. A multicentre prospective study of 1000 patients. Medicine (Baltimore) 78, 167–175 (1999).

    Article  CAS  Google Scholar 

  71. Wahl, D.G. et al. Risk of venous thrombosis related to antiphospholipid antibodies in systemic lupus erythematosus—a meta-analysis. Lupus 6, 467–473 (1997).

    Article  CAS  PubMed  Google Scholar 

  72. Levine, S.R. et al. Antiphospholipid antibodies and subsequent thrombo-occlusive events in patients with ischemic stroke. JAMA 291, 576–584 (2004).

    Article  CAS  PubMed  Google Scholar 

  73. Harris, E.N. & Spinnato, J.A. Should anticardiolipin tests be performed in otherwise healthy pregnant women? Am. J. Obstet. Gynecol. 165, 1272–1277 (1991).

    Article  CAS  Google Scholar 

  74. Budd, R. et al. A re-appraisal of the normal cut-off assignment for anti-cardiolipin IgM tests. J. Thromb. Haemost. 4, 2210–2214 (2006).

    Article  CAS  PubMed  Google Scholar 

  75. Yetman, D.L. & Kutteh, W.H. Antiphospholipid antibody panels and recurrent pregnancy loss: prevalence of anticardiolipin antibodies compared with other antiphospholipid antibodies. Fertil. Steril. 66, 540–546 (1996).

    Article  CAS  PubMed  Google Scholar 

  76. Franklin, R.D. & Kutteh, W.H. Antiphospholipid antibodies (APA) and recurrent pregnancy loss: treating a unique APA positive population. Hum. Reprod. 17, 2981–2985 (2002).

    Article  CAS  PubMed  Google Scholar 

  77. Sugi, T. & McIntyre, J.A. Certain autoantibodies to phosphatidylethanolamine (aPE) recognized factor XI and prekallikrein independently or in addition to the kininogens. J. Autoimmun. 17, 207–214 (2001).

    Article  CAS  PubMed  Google Scholar 

  78. Cabiedes, J. et al. Characterization of anti-phosphatidylcholine polyreactive natural autoantibodies from normal human subjects. J. Autoimmun. 18, 181–190 (2002).

    Article  PubMed  Google Scholar 

  79. Balestrieri, G. et al. Anti-β2glycoprotein I antibodies: a marker of antiphospholipid syndrome? Lupus 4, 122–130 (1995).

    Article  CAS  PubMed  Google Scholar 

  80. Arvieux, J., Pouzol, P., Roussel, B., Jacob, M.C. & Colomb, M.G. Lupus-like anticoagulant properties of murine monoclonal antibodies to β2glycoprotein I. Br. J. Haematol. 81, 568–573 (1992).

    Article  CAS  PubMed  Google Scholar 

  81. Viard, J.P., Armoura, Z. & Bach, J.F. Association of anti-β2glycoprotein I with lupus circulating anticoagulant and thrombosis in SLE. Am. J. Med. 93, 181–186 (1992).

    Article  CAS  PubMed  Google Scholar 

  82. Cabiedes, J., Cabral, A. & Alarcon-Segovia, D. Clinical manifestations of the antiphospholipid syndrome in patients with systemic lupus erythematosus associate more strongly with anti-β2glycoprotein I than with antiphospholipid antibodies. J. Rheumatol. 22, 1899–1906 (1995).

    CAS  PubMed  Google Scholar 

  83. Katano, K. et al. β2glycoprotein I-dependent anticardiolipin antibodies as a predictor of adverse pregnancy outcomes in healthy pregnant women. Hum. Reprod. 11, 509–512 (1996).

    Article  CAS  PubMed  Google Scholar 

  84. Martinuzzo, M.E., Forastiero, R.R. & Carreras, L.O. Anti-β2glycoprotein antibodies: detection and association with thrombosis. Br. J. Haematol. 89, 397–402 (1995).

    Article  CAS  PubMed  Google Scholar 

  85. Ogasawa, M., Aoki, K., Matsuura, E., Sasa, H. & Yagami, Y. Anti-β2glycoprotein I antibodies and lupus anticoagulant in patients with recurrent pregnancy loss: prevalence and clinical significance. Lupus 5, 587–592 (1996).

    Article  Google Scholar 

  86. Tsusumu, A. et al. Antibodies to β2glycoprotein I and clinical manifestations in patients with systemic lupus erythematosus. Arthritis Rheum. 39, 1466–1474 (1996).

    Article  Google Scholar 

  87. Chamley, L.W. Antiphospholipid antibodies or not? The role of β2glycoprotein I in autoantibody-mediated pregnancy loss. J. Reprod. Immunol. 36, 123–142 (1997).

    Article  CAS  PubMed  Google Scholar 

  88. Reber, G., Tincani, A., Sanmarco, M., deMoerloose, P. & Boffa, M.C. Proposals for the measurement of anti-β2glycoprotein I antibodies. Standardization Group of the European Forum on Antiphospholipid Antibodies. J. Thromb. Haemost. 2, 1860–1862 (2004).

    Article  CAS  PubMed  Google Scholar 

  89. Matsuuda, J., Saitoh, N., Gohchi, K., Gotoh, M. & Tuskamoto, M. Anti-annexin V antibody in systemic lupus erythematosus patients with lupus anticoagulants and/or anticardiolipin antibody. Am. J. Hematol. 47, 56–58 (1994).

    Article  Google Scholar 

  90. Reber, G. et al. Inter-laboratory variability of anti-β2glycoprotein I measurement. A collaborative study in the frame of the European Forum on Antiphospholipid Antibodies Standardization Group. Thromb. Haemost. 88, 66–73 (2002).

    Article  CAS  PubMed  Google Scholar 

  91. Pierangeli, S.S. et al. Evaluation of several ELISA tests used in the diagnosis of antiphospholipid syndrome in a large cohort of patient samples. Arthritis Rheum. 54, S553 (2006).

    Google Scholar 

  92. NCCLS. Collection, Transport and Processing of Blood Specimens for Coagulation Testing and General Performance of Coagulation Assays; Approved Guideline—third edition. NCCLS document H21-A3 (NCCLS, Wayne, Pennsylvania, 1998).

  93. Brey, R.L., Cote, S.A., McGlasson, D.L., Triplett, D.A. & Barna, L.K. Effects of freeze-thaw cycles on anticardiolipin antibody immunoreactivity. Am. J. Clin. Pathol. 102, 586–588 (1994).

    Article  CAS  PubMed  Google Scholar 

  94. Hasselaar, P. et al. Heat treatment of serum or plasma induce false-positive results in the antiphospholipid ELISA. J. Rheumatol. 17, 186–191 (1990).

    CAS  PubMed  Google Scholar 

  95. Agopian, M.S., Boctor, F.N. & Peter, J.B. False-positive test results for IgM anticardiolipin antibody due to IgM rheumatoid factor. Arthritis Rheum. 31, 1212–1213 (1988).

    Article  CAS  PubMed  Google Scholar 

  96. Spadaro, A. et al. Class specific rheumatoid factors and antiphospholipid syndrome in systemic lupus erythematosus. J. Rheumatol. 15, 74–79 (1988).

    Google Scholar 

  97. Cabral, A.R., Cabiedes, J. & Alarcon-Segovia, D. Tween 20 detaches cardiolipin from ELISA plates and makes anticardiolipin antibodies undetectable regardless of the presence of β2glycoprotein I. J. Immunol. Methods 175, 107–114 (1994).

    Article  CAS  PubMed  Google Scholar 

  98. Sammaritano, L.R., Lockshin, M.D. & Gharavi, A.E. Antiphospholipid antibodies differ in cofactor requirement. Lupus 1, 83–90 (1992).

    Article  CAS  PubMed  Google Scholar 

  99. Chamley, L.W., Pattison, N.S. & McKay, E.J. Anomalous anticardiolipin antibody results may be due to cofactor variability. Am. J. Hematol. 37, 289 (1991).

    Article  CAS  PubMed  Google Scholar 

  100. Pierangeli, S.S. & Harris, E.N. Clinical laboratory testing for the antiphospholipid syndrome. Clin. Chim. Acta 357, 17–33 (2005).

    Article  CAS  PubMed  Google Scholar 

  101. Pierangeli, S.S., Gharavi, A.E. & Harris, E.N. Testing for antiphospholipid antibodies: problems and solutions. Clin. Obstet. Gynecol. 44, 48–57 (2001).

    Article  CAS  PubMed  Google Scholar 

  102. Ichikawa, K. et al. A chimeric antibody with human γ1 constant region as a putative standard for assays that detect IgG β2glycoprotein I antibodies. Arthritis Rheum. 42, 2461–2470 (1999).

    Article  CAS  PubMed  Google Scholar 

  103. Schlame, M., Haller, I., Sammaritano, L.R. & Blanck, T.J. Effect of cardiolipin oxidation on solid-phase immunoassay for antiphospholipid antibodies. Thromb. Haemost. 86, 1475–1482 (2001).

    Article  CAS  PubMed  Google Scholar 

  104. Horkko, S. et al. Antiphospholipid antibodies are directed against epitopes of oxidized phospholipids. J. Clin. Invest. 98, 815–825 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Lockshin, M.D., Wamar, T., Levy, R.A. & Best, M.P. IgG but not IgM anti-phospholipid antibody binding is temperature dependent. J. Clin. Immunol. 8, 188–192 (1988).

    Article  CAS  PubMed  Google Scholar 

  106. Pierangeli, S., Greenberg, R. & Harris, E.N. Determination of the calibration equation which achieved best accuracy and precision of the anti-cardiolipin assay. Arthritis Rheum. 32, S123 (1989).

    Google Scholar 

  107. Branch, D.W., Silver, R.M., Pierangeli, S.S., Van Leeuwen, I. & Harris, E.N. Antiphospholipid antibodies in women with recurrent pregnancy loss, fertile controls and antiphospholipid syndrome. Obstet. Gynecol. 89, 549–555 (1997).

    Article  CAS  PubMed  Google Scholar 

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  1. Division of Rheumatology, Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas, USA

    Silvia S Pierangeli

  2. Office of the Vice Chancellor, University of the West Indies, Kingston, Jamaica, West Indies

    Eon Nigel Harris

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Correspondence to Silvia S Pierangeli.

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Competing interests

S.S.P. and E.N.H. are owners of Louisville APL Diagnostics, Inc that distributes aCL Calibrators and the APhL ELISA kit mentioned in the article.

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Pierangeli, S., Harris, E. A protocol for determination of anticardiolipin antibodies by ELISA. Nat Protoc 3, 840–848 (2008). https://doi.org/10.1038/nprot.2008.48

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