Use of Synthetic Cardiolipin and Lecithin in the Antigen Used by the Venereal Disease Research Laboratory Test for Serodiagnosis of Syphilis

Abstract

The Venereal Disease Research Laboratory (VDRL) test is a microflocculation test for syphilis that uses an antigen containing cardiolipin, lecithin, and cholesterol. For more than 50 years, the preparation of natural cardiolipin and lecithin for this test has been based on the Pangborn method which involves isolating and purifying these components from beef hearts. This process is tedious and time-consuming and results in a variable purity range. In our studies, we found that a VDRL antigen using synthetic tetramyristoyl cardiolipin and synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (lecithin) was as specific in detecting syphilis as a VDRL antigen made with natural components. In 85% of the cases, we obtained an endpoint titer of 1/2 or 1 dilution more than a titer obtained with a VDRL antigen made with natural components. The use of these pure synthetic compounds, with a purity of 99%, would offer advantages in the standardization and stability of the VDRL antigen. Because this antigen is the basic ingredient in the preparation of nontreponemal reagents such as the rapid plasma reagin, toluidine red unheated serum test, and the unheated serum reagin, the use of this synthetic VDRL antigen should also increase the reactivity of these reagents.The Venereal Disease Research Laboratory (VDRL) antigen, suspended in a buffered saline solution, forms a flocculate when combined with antilipoidal antibodies in serum or cerebrospinal fluid (CSF) from persons with syphilis. The VDRL test measures immunoglobulin G (IgG) and IgM antibodies to lipoidal material released from damaged host cells as well as to lipoprotein-like material and possibly cardiolipin released from the treponemes (1, 9).Natural cardiolipin was isolated from extracts of beef hearts by Pangborn in 1941 (10). Natural lecithin is prepared from beef hearts or egg yolks. The process of preparing and purifying cardiolipin and lecithin by the Pangborn method has continued, despite being tedious, time-consuming, and very expensive.Natural cardiolipin is a phospholipid containing approximately 90% unsaturated linoleoyl fatty acid with four 18-carbon chains, each of which has two double bonds. The presence of the double bonds in the fatty acid is conducive to oxidation with reduced reactivity. Cardiolipin possesses a strong antigenic property. The serologic specificity of the antigen reacting with syphilitic sera is attributed to two phosphate groups and the ␤-hydroxyl group of the central glycerol moiety (3). Several investigators have reported on the relationship between the structure and serologic specificity of cardiolipin. The length of the carbon chain of the fatty acids apparently is not of primary importance. Faure and Coulon-Morelec (4) demonstrated that the removal of one or two of the four fatty acids from the phosphatides derived from cardiolipin did not have a detrimental effect, while the esterification of the free hydroxyl group of cardiolipin considerably decreased the activity of the cardiolipin in the VDRL test.Several researchers h...

False‐positive results in ELISA‐based anti FVIII antibody assay may occur with lupus anticoagulant and phospholipid antibodies

“…We observed that 13% of LA‐positive, 10% of anti‐ ß2GP1‐positive and 18% of aCL‐positive samples tested falsely positive in the FVIII IgG ELISA. This finding is not surprising given that FVIII inhibitors and LA/APA antibodies reportedly target similar epitopes in the C2 domain of FVIII [5,27–30]. Antibodies that form in patients with FVIII inhibitors may have multiple epitope targets, whereas LA may bind only to the phospholipid‐binding site (C2) on FVIII.…”

“…This interference from LA and APAs seems to be directed specifically against a phospholipid epitope in the FVIII molecule as other IgG antibodies did not interfere with the FVIII immunoassay. It is not surprising that two of six VDRL‐positive sera tested positive in FVIII IgG ELISA assay, since cardiolipin is the main source of the antigen mixture in the flocculation assays [30]. Of note, there was no interference from LA or APA in FIX IgG immunoassay.…”

Advances in diagnostic tests for bacterial STDs

“…Recently, a synthetic VDRL antigen has been developed using tetramyristoyl cardiolipin and synthetic 1-palmitoyl-2oleoyl-sn-glycerol-3-phosphocholine and was shown to be as specific in detecting syphilis as a VDRL antigen made with natural components. 8 Moreover, this synthetic antigen had a higher level of reactivity with 85% (169/200) of the positive specimens tested than the natural VDRL antigen. The purity of the synthetic VDRL antigen was greater than 99%, which should eliminate this variabile as well as problems with stability that have been encountered with reagents prepared with natural VDRL antigen.…”

An immunofiltration device for the simultaneous detection of non-treponemal and treponemal antibodies in patients with syphilis

“…The non-treponemal antigen was prepared by using synthetic VDRL antigen made from an ethanolic solution of 0.03% tetramyristoyl cardiolipin, 0.14% 1-palmitoyl-2-oleolyl-sn-glycero-3-phosphocholine, lecithin and 0.9% cholesterol (Avanti Polar Lipids, Alabaster, Alabama, USA) 12. A 1:10 emulsion of this antigen in phosphate buffered saline was prepared according to the standard VDRL procedure13 and centrifuged at 4360 g for 45 min and resuspended in 10 mM phosphate buffer pH 6.9 containing 0.25 M EDTA, 0.72 M choline chloride and 0.01% sodium azide.…”