Antiphospholipid antibodies detected by line immunoassay differentiate among patients with antiphospholipid syndrome, with infections and asymptomatic carriers

The persistent presence of aPL is the serological hallmark of APS and represents one of its mandatory classification criteria [2]. It is a well-accepted consensus that aPL interact with PL-binding proteins. While most of the reactivity was against ?2GPI, additional PL-binding proteins were shown to be recognized by aPL [11–13, 16]. The different aPL subpopulations cannot be detected by a single diagnostic assay and this supports the recommendation for performing aCL, a?2GPI, and LA assays in order to identify all the potential aPL.

Between 1 % and 5 % of healthy individuals have circulating aPL that are detectable with the currently recommended aPL assays [4]. This raises the issue of identifying the truly diagnostic aPL and/or those aPL that are really predictive for the clinical manifestations of the syndrome.

The LIA membrane strips provide a unique matrix that allows PL to mimic their natural conformation in tissues as reported for other amphiphatic non-protein antigenic molecules [28, 31–33]. Hence, immobilized PL offer a suitable binding substrate for the main PL-binding proteins. Accordingly, this study investigated the performance of a novel LIA hydrophobic solid phase for the simultaneous detection of multiple aPL in a well-defined cohort of patients with APS and controls including aPL+ asymptomatic carriers.

Of note, the novel LIA solid phase has already proven its usefulness for the specific analysis of auto-antibodies to lipopolysaccharides and glycolipids exhibiting PL-like physicochemical characteristics [28, 33]. In contrast to the planar ELISA solid phase, the porous hydrophobic LIA membrane is assumed to incorporate the hydrophobic PL tail. This shields the by far larger tail of the amphiphatic PL molecule from the reaction environment and, thus, prevents unspecific interactions [11]. Of note, the humoAb RR-7 F interacting with anionic PL only in ELISA also bound readily immobilized anionic PL in the investigated LIA [25]. This reactivity was completely inhibited by CL micelles that expose only hydrophilic CL-heads on their surface in aqueous solutions. Consequently, this confirms the interaction of RR-7 F with the hydrophilic PL-heads on the PVDF membrane. The a?2GPI humoAbs MBB2 and HCAL were able to recognize their target molecule coated on the membrane as well as to react with anionic PL through the bound cationic ?2GPI.

Interestingly, the addition of serum or purified ?2GPI to MBB2 revealed different binding characteristics to the immobilized anionic PL in the novel reaction environment. CL, also referred to as diphosphatidylglycerol, binds ?2GPI far better than its monomeric variant PG. Otherwise, PS bearing only one phosphatic group has a better binding than PI or PG. This supports the assumption that the number, orientation, and accessibility of anionic phosphatic groups in the hydrophilic PL heads determine the binding of ?2GPI and consequently of the ?2GPI-dependent aPL. As MBB2 has been demonstrated to specifically react with D1 of ß2GPI and its conformational epitope [24], its binding to the LIA strips indicates that the immobilized ?2GPI readily exposes D1. This demonstrates the accessibility of this important pathogenic epitope-bearing domain in the LIA reaction environment. As a consequence, these variables may affect the ultimate serum autoantibody binding.

Indeed, favorable assay performance particularly for the specificity of this novel LIA technique for the analysis of aPL has been reported recently and was confirmed in this study [17, 21]. In fact, the prevalence of aCL and aß2GPI IgG was significantly reduced in aPL+ carriers and in VDRL+ individuals compared to patients with APS when analyzed by LIA but not by ELISA. Of note, the significantly qualitative differences of aCL IgG and aß2GPI IgG testing by LIA in APS patients and aPL+ carriers were confirmed by quantitative analyses of the respective median OD levels.

We could not determine the binding of either serum or purified PT with immobilized anionic PL, particularly with PS, in the multiplex LIA environment as shown for ?2GPI. Even in the presence of Ca⁺² ions, which are required for the aPT/PS ELISA reaction environment, no binding was detected. However, it cannot be excluded that the PT in the LIA reaction environment is not able to change its conformation in the presence of Ca²⁺ ions. This point could not be addressed by our experimental setting. Addition of further PT in the presence of the right high Ca++ concentration did not change the binding behavior. Of note, there was a significantly different prevalence of aPT and aPS IgG/IgM (p??0.0001 and p?=?0.0017, respectively) in APS patients by LIA. Altogether, this indicates that aPS reactivity in LIA is mainly based on the reactivity to ?2GPI bound to PS and does not involve PT present in the serum sample. Theoretically in fact, serum PT could bind PS on the PVDF membrane. Accordingly, in our study there was no significant correlation between thrombotic events and either aPT or aPS detected by LIA, which was reported for aPS/PT elsewhere [12]. The latter findings is in line with the hypothesis that aPS/PT abs recognize a peculiar epitope(s) exposed on PT only when complexed with PS-coated polystyrene plates.

A further novelty of this aPL assay is the possibility of detecting antibodies against D1 because of the way the molecule is oriented on the matrix [34, 35]. After binding of ?2GPI to negatively charged surfaces like immobilized anionic PL by D5 (containing the PL-binding site), D1 forms the top of the induced fishhook-like ?2GPI structure that is predisposed to interact with aPL [35]. Assuming a high density of the hydrophilic PL heads on the LIA membrane, ?2GPI D4 and D5 could be indeed engaged in the binding of immobilized PL and no longer available for aPL interaction. Indeed, none out of five patients with APS with D4/D5 reactivity only, had a positive aPL IgG by LIA, whereas all nine patients with D1 reactivity only did have positive aPL by LIA. aPL directed against D1 are significantly more present in sera in APS than in pathological controls such as infectious patients or aPL+ asymptomatic carriers. On the other hand, aPL reacting with ?2GPI D4 and D5 display opposite behavior [36, 37]. Indeed, there is a significant difference in the analysis of aPL binding to anionic PL/?2GPI complexes in LIA compared with ELISA in VDRL+ patients and in particular in asymptomatic aPL+ carriers. Thus, the significantly lower prevalence of aPL detected by LIA in aPL+ carriers and in VDRL+ patients indicates more specific detection of diagnostic aPL by LIA. Remarkably, on aPL analysis by LA testing and ELISA there were no such significant differences, which suggests the detection of aPL to epitopes other than those present on D1. As the aPL reactivity to D4-5 has been reported not to be associated with thromboembolism, our data further support the suggestion that LIA may detect aPL that are more predictive of clinical events in APS [36–38].