Protocol | Published:

A protocol for determination of anticardiolipin antibodies by ELISA

Nature Protocols volume 3, pages 840848 (2008) | Download Citation

Subjects

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

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

  2. 2.

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

  3. 3.

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

  4. 4.

    , & Validation of the Sapporo criteria for antiphospholipid syndrome. Arthritis Rheum. 43, 440–443 (2000).

  5. 5.

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

  6. 6.

    , , & Anticardiolipin antibody isotype distribution and phospholipid specificity. Ann. Rheum. Dis. 46, 1–6 (1987).

  7. 7.

    , , & Evaluation of the anticardiolipin antibody test: report of an international workshop held 4 April 1986. Clin. Exp. Immunol. 68, 215–222 (1987).

  8. 8.

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

  9. 9.

    , , & Report of an anticardiolipin workshop during the VIIth International Symposium on antiphospholipid antibodies. J. Rheumatol. 25, 156–162 (1998).

  10. 10.

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

  11. 11.

    , & Anticardiolipin wet workshop report: Vth international symposium on antiphospholipid antibodies. Am. J. Clin. Pathol. 101, 616–624 (1994).

  12. 12.

    , & Anticardiolipin antibody determination: comparison of three ELISA assays. Clin. Exp. Rheumatol. 8, 575–577 (1990).

  13. 13.

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

  14. 14.

    , , , & Anticardiolipin antibody testing and reporting practices among laboratories participating in a large external Quality Assurance Program. Pathology 36, 174–181 (2004).

  15. 15.

    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).

  16. 16.

    & Revisiting the anticardiolipin test and its standardization. Lupus 11, 269–275 (2002).

  17. 17.

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

  18. 18.

    Antiphospholipid antibodies. Arch. Pathol. Lab. Med. 126, 1424–1429 (2002).

  19. 19.

    & 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).

  20. 20.

    , , & Interlaboratory variation in antiphospholipid antibody testing. Am. J. Obstet. Gynecol. 144, 1780–1787 (1992).

  21. 21.

    & 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).

  22. 22.

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

  23. 23.

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

  24. 24.

    , , & Accuracy of anticardiolipin antibodies in identifying a history of thrombosis among patients with systemic lupus erythematosus. Am. J. Med. 98, 559–565 (1995).

  25. 25.

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

  26. 26.

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

  27. 27.

    , , & 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).

  28. 28.

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

  29. 29.

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

  30. 30.

    , & Testing for antiphospholipid antibodies: problems and solutions. Clin. Obstet. Gynecol. 44, 48–57 (2001).

  31. 31.

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

  32. 32.

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

  33. 33.

    , & Anticardiolipin antibodies directed not to cardiolipin but to a plasma protein cofactor. Lancet 336, 1547 (1990).

  34. 34.

    , , & 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).

  35. 35.

    , , , & Anticardiolipin antibodies recognize β2glycoprotein I structure altered by interacting with an oxygen modified solid phase surface. J. Exp. Med. 179, 457–462 (1994).

  36. 36.

    , , & Anticardiolipin autoantibodies recognize β2glycoprotein I in the absence of phospholipid. Importance of Ag density and bivalent binding. J. Immunol. 15, 954–960 (1995).

  37. 37.

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

  38. 38.

    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).

  39. 39.

    , , , & 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).

  40. 40.

    , , & Antiprothrombin antibodies—are they worth assaying? Thromb. Res. 114, L533 (2004).

  41. 41.

    & Induction of antiphospholipid autoantibody during cytomegalovirus infection. Clin. Infect. Dis. 25, 1493–1494 (1997).

  42. 42.

    , , & Autoantibodies in malaria, tuberculosis and hepatitis in a West African population. Clin. Exp. Immunol. 92, 73–76 (1993).

  43. 43.

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

  44. 44.

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

  45. 45.

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

  46. 46.

    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).

  47. 47.

    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).

  48. 48.

    , , , & 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).

  49. 49.

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

  50. 50.

    , , & Anticardiolipin antibodies in infectious diseases. Clin. Rheumatol. 8, 23–28 (1989).

  51. 51.

    , & 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).

  52. 52.

    , , & Presence of antiphospholipid antibodies in Legionnaires' disease. Presse Med. 25, 1649 (1996).

  53. 53.

    , , & Thrombophlebitis in a patient with acute Q fever and anticardiolipin antibodies. Med. Clin. (Barc) 108, 396–397 (1997).

  54. 54.

    , , , & The specificity of anti-cardiolipin antibodies from syphilis patients and from patients with systemic lupus erythematosus. Clin. Exp. Immunol. 76, 178–183 (1989).

  55. 55.

    , , & 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).

  56. 56.

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

  57. 57.

    , & Anticardiolipin antibodies associated with HTLV-III infection. Br. J. Haematol. 65, 495–498 (1987).

  58. 58.

    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).

  59. 59.

    , & 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).

  60. 60.

    & A more specific ELISA assay for detection of antiphospholipid. Clin. Immunol. News 15, 26–28 (1995).

  61. 61.

    , , , & 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).

  62. 62.

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

  63. 63.

    , , & The prevalence of anticardiolipin antibodies in a healthy elderly population and its association with antinuclear antibodies. J. Rheumatol. 16, 623–625 (1989).

  64. 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. 65.

    , , , & A prospective epidemiological study on the occurrence of antiphospholipid antibody: the Montpellier Antiphospholipid (MAP) Study. Haemostasis 24, 175–182 (1994).

  66. 66.

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

  67. 67.

    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).

  68. 68.

    , & 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).

  69. 69.

    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).

  70. 70.

    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).

  71. 71.

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

  72. 72.

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

  73. 73.

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

  74. 74.

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

  75. 75.

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

  76. 76.

    & Antiphospholipid antibodies (APA) and recurrent pregnancy loss: treating a unique APA positive population. Hum. Reprod. 17, 2981–2985 (2002).

  77. 77.

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

  78. 78.

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

  79. 79.

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

  80. 80.

    , , , & Lupus-like anticoagulant properties of murine monoclonal antibodies to β2glycoprotein I. Br. J. Haematol. 81, 568–573 (1992).

  81. 81.

    , & Association of anti-β2glycoprotein I with lupus circulating anticoagulant and thrombosis in SLE. Am. J. Med. 93, 181–186 (1992).

  82. 82.

    , & 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).

  83. 83.

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

  84. 84.

    , & Anti-β2glycoprotein antibodies: detection and association with thrombosis. Br. J. Haematol. 89, 397–402 (1995).

  85. 85.

    , , , & Anti-β2glycoprotein I antibodies and lupus anticoagulant in patients with recurrent pregnancy loss: prevalence and clinical significance. Lupus 5, 587–592 (1996).

  86. 86.

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

  87. 87.

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

  88. 88.

    , , , & 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).

  89. 89.

    , , , & Anti-annexin V antibody in systemic lupus erythematosus patients with lupus anticoagulants and/or anticardiolipin antibody. Am. J. Hematol. 47, 56–58 (1994).

  90. 90.

    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).

  91. 91.

    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).

  92. 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. 93.

    , , , & Effects of freeze-thaw cycles on anticardiolipin antibody immunoreactivity. Am. J. Clin. Pathol. 102, 586–588 (1994).

  94. 94.

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

  95. 95.

    , & False-positive test results for IgM anticardiolipin antibody due to IgM rheumatoid factor. Arthritis Rheum. 31, 1212–1213 (1988).

  96. 96.

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

  97. 97.

    , & 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).

  98. 98.

    , & Antiphospholipid antibodies differ in cofactor requirement. Lupus 1, 83–90 (1992).

  99. 99.

    , & Anomalous anticardiolipin antibody results may be due to cofactor variability. Am. J. Hematol. 37, 289 (1991).

  100. 100.

    & Clinical laboratory testing for the antiphospholipid syndrome. Clin. Chim. Acta 357, 17–33 (2005).

  101. 101.

    , & Testing for antiphospholipid antibodies: problems and solutions. Clin. Obstet. Gynecol. 44, 48–57 (2001).

  102. 102.

    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).

  103. 103.

    , , & Effect of cardiolipin oxidation on solid-phase immunoassay for antiphospholipid antibodies. Thromb. Haemost. 86, 1475–1482 (2001).

  104. 104.

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

  105. 105.

    , , & IgG but not IgM anti-phospholipid antibody binding is temperature dependent. J. Clin. Immunol. 8, 188–192 (1988).

  106. 106.

    , & Determination of the calibration equation which achieved best accuracy and precision of the anti-cardiolipin assay. Arthritis Rheum. 32, S123 (1989).

  107. 107.

    , , , & Antiphospholipid antibodies in women with recurrent pregnancy loss, fertile controls and antiphospholipid syndrome. Obstet. Gynecol. 89, 549–555 (1997).

Download references

Author information

Affiliations

  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

Authors

  1. Search for Silvia S Pierangeli in:

  2. Search for Eon Nigel Harris in:

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.

Corresponding author

Correspondence to Silvia S Pierangeli.

About this article

Publication history

Published

DOI

https://doi.org/10.1038/nprot.2008.48

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.