Novel Phospholipid-Protein Conjugates Allow Improved Detection of Antibodies in Patients with Autoimmune Diseases

Abstract

Reliable measurement of clinically relevant autoimmune antibodies toward phospholipid-protein conjugates is highly desirable in research and clinical assays. To date, the development in this field has been limited to the use of natural heterogeneous antigens. However, this approach does not take structural features of biologically active antigens into account and leads to low reliability and poor scientific test value. Here we describe novel phospholipid-protein conjugates for specific detection of human autoimmune antibodies. Our synthetic approach includes mild oxidation of synthetic phospholipid cardiolipin, and as the last step, coupling of the product with azide-containing linker and copper-catalyzed click chemistry with β2-glycoprotein I and prothrombin. To prove utility of the product antigens, we used enzyme-linked immunosorbent assay and three cohorts of samples obtained from patients in Denmark (n = 34) and the USA (n = 27 and n = 14). Afterwards we analyzed correlation of the obtained autoantibody titers with clinical parameters for each patient. Our results prove that using novel antigens clinically relevant autoantibodies can be detected with high repeatability, sensitivity and specificity. Unlike previously used antigens the obtained autoantibody titers strongly correlate with high disease activity and in particular, with arthritis, renal involvement, anti-Smith antibodies and high lymphocyte count. Importantly, chemical composition of antigens has a strong influence on the correlation of detected autoantibodies with disease activity and manifestations. This confirms the crucial importance of antigens’ composition on research and diagnostic assays, and opens up exciting perspectives for synthetic antigens in future studies of autoimmunity.

Assessment of a combination of plasma anti-histone autoantibodies and PLA2/PE ratio as potential biomarkers to clinically predict autism spectrum disorders

“…This phenomenon of phospholipid-autoantibodies complexing was recently ascertained by the work of Nalli et al 69 which demonstrates that anti-phospholipid antibodies analysed by line immunoassay (LIA) can recognize different proteins epitopes depending on to which phospholipids the protein is binding. The obtained higher AUC of combined ROC for anti-histone and PLA2/PE (AUC of 0.991) can find great support in the previous work of Samuelsen et al 70 which indicate high prognostic and scientific potential of cross-linked phospholipid-protein conjugates. Moreover, the finding of the present study can find more support in the previous work of Careaga et al 71 showing an increased anti-phospholipid antibody levels in young children with ASD that is directly related to the severity of the disorder.…”

Citrullinated Peptide Epitope Targets Therapeutic Nanoparticles to Human Neutrophils

“…ELISA procedure was adopted from the paper by Samuelsen, Maity et al 27 Maxisorb 96 well plates (NUNC Thermofisher) were coated with nanoparticle antigens/controls at aconcentration of 8 µg/mL in 1X PBS overnight (room temperature; 100 µl/well). After washing with 1X PT (2 x 300 µl/well, PT: 50 µl Tween-20 in 1 L 1X PBS), the plates were blocked with 1X PTB (1 h, 37 ° C; 100 µl/well, PTB: 20 g BSA, 50 µl Tween-20 in 1 L 1X PBS).…”

“…Having selected the most potent peptide epitope, we included it into nanoparticles loaded with antisense oligonucleotide targeting RA associated microRNA. The structure and properties of the nanoparticles have been evaluated by a series of in vitro assays, ,, and the most potent nanoparticles for specific targeting of human neutrophils in RA have been identified.…”