T Kveder

Anti-β2-Glycoprotein I ELISA : Methodology, determination of cut-off values in 434 healthy caucasians and evaluation of monoclonal antibodies as possible international standards

Abstract Antibodies against β2-glycoprotein I are among the most commonly detected subset of antiphospholipid antibodies. The inter-laboratory comparability of results is hardly possible due to methodological differences, lack of international standards and different cut-off values. We evaluated an ELISA for the detection of anti-β2-glycoprotein I using the analytical goals based on biological variations for similar analytes (immunoglobulins). By our ELISA we fulfilled the optimal (IgA, IgM) or minimal (IgG) analytical goals in longterm imprecision. The determination of cut-off values was based on the frequency distribution of results obtained on 434 healthy Caucasians. To aim at a better inter-laboratory comparability we tested two monoclonal antibodies as possible calibrators: HCAL (IgG) and EY2C9 (IgM). Binding properties determined by dilutional curves showed great similarities with polyclonal sera, used as in-house standards. Cut-off values were expressed by concentrations of IgG and IgM monoclonal antibodies (4.5 and 25.3 μg/l). Our study shows the possibility for a successful application of analytical goals based on biological variation even when data for a particular analyte are not available. The expression of cut-off values, obtained on a large scale Caucasian population, by the concentration of IgG and IgM monoclonal antibodies could make possible a more reliable inter-laboratory comparison of data.

Parasympathetic nervous system dysfunction in primary Sjögren’s syndrome

In the past sicca syndromes were attributed to destruction of glandular tissue. It is now thought that cytokines, autoantibodies, and parasympathetic nervous system dysfunction all have an important role in the xerostomia and xerophthalmia in Sjögren’s syndrome.

Antibodies against annexin A5: Detection pitfalls and clinical associations

Antiphospholipid syndrome (APS) has been defined as a clinical and laboratory entity. Laboratory criteria include the presence of anticardiolipin antibodies (aCL) and/or lupus anticoagulant (LA), collectively termed as antiphospholipid antibodies (aPL). However, there has been a rising interest in antibodies against so-called protein cofactors, particularly in β2-glycoprotein I. In the early 90s, annexins were considered as target antigens for aPL, but at present the exact role of antibodies against annexins (aANX) remains puzzling. This review is concerned with annexin V or annexin A5 (ANXA5), a widespread member of the annexin family, and antibodies directed towards it. We have endeavoured to summarise essential information about the detection of anti-annexin V antibodies (aANXA5) and their clinical relevance. This review has also brought together some relevant published data concerning the structure, physiological role and therapeutic potential of ANXA5.

Concomitant isolation of protein C inhibitor and unnicked β2-glycoprotein I

Abstract β2-Glycoprotein I (β2GPI) is the major target molecule for so-called anticardiolipin antibodies. We evaluated the isolation procedure of β2GPI from human plasma with special emphasis on the time of precipitation, composition of different isolated fractions and their antigenic properties. The isolation was initiated by perchloric acid precipitation for either 3, 18 or 50 min, followed by heparin affinity and cationic exchange chromatography. The properties of isolated proteins were tested by rocket electrophoresis, enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis, immunoblotting and N-terminal sequencing. Each isolation procedure, regardless of the perchloric acid precipitation duration, resulted in three distinct protein peaks, differing in protein composition qualitatively. Comparing sequential peaks between the isolations of different precipitation times, we found that all the three first peaks (set of peaks No. 1), all the three second peaks (set No. 2) as well as the three third peaks (set No. 3) consisted of identical proteins but in different quantities. Set No. 1 was composed of immunoglobulins and a lesser amount of β2GPI. In set No. 2 only unnicked β2GPI was detected. Protein C inhibitor was found in addition to smaller amounts of unnicked β2GPI in set No. 3. Oxidation or degradation of β2GPI during the isolation procedure did not result in a mixture of different forms of β2GPI but rather in a lower yield of wild-type β2GPI. The co-existence of β2GPI and protein C inhibitor in the isolated fractions may suggest their protein-protein interactions in vivo.

Mixed connective tissue disease associated with autoimmune hepatitis and thyroiditis.

The case is reported of a 27 year old woman who had mixed connective tissue disease (MCTD) associated with chronic active hepatitis and thyroiditis. Although hepatomegaly is sometimes observed in MCTD, only four cases of MCTD and chronic active hepatitis have been described. It is thought that this is the first report of an association between MCTD, chronic active hepatitis and thyroiditis.

Anti-annexin V antibodies in patients with cerebrovascular disease.

Annexin V (ANXV) is a protein with a high affinity for negatively charged phospholipids and shows in vitro a potent anticoagulant activity. It has been suggested that it has a significant role in the prevention of arteriovenous thromboses or fetal loss, or both.1 Increased levels of antibodies against ANXV (aANXV) have been reported in patients with different systemic autoimmune disorders2–4 as well as in women with recurrent fetal loss and pre-eclampsia.5 The presence of aANXV in patients with thromboembolic cerebrovascular disease (CVD), however, has not yet been described. We report on two patients with CVD who had evidently raised levels of IgG aANXV, whereas all the other tested antiphospholipid antibodies (aPL) were negative.nnWe examined 37 young patients with no evident systemic autoimmune …

Counterimmunoelectrophoresis: fast, easy and cost-effective method for the detection of autoantibodies to intracellular antigens.

We read with interest the short communication of Bossuyt et al. about the evaluation of automated enzyme immunoassays for the detection of antibodies to extractable nuclear antigens, published in Clinical Chemistry and Laboratory Medicine 2001; 39(7): 658–659. It was one of many attempts to find an easy and as-fast-as-possible way to detect autoantibodies against intracellular antigens (1). Unfortunately, nobody has yet found the best solution to meet all the needs of rheumatologists and demands of insurance companies. We all seek the best screen at the shortest time possible in the most cost-effective way. Therefore, we believe it is impossible to overlook the ongoing effort of a group of experts constituing the European Consensus Study Group for Autoantibodies who meet annually at the European Workshop for Rheumatology Research (EWRR). The group was formed in 1988 to examine the sensitivity and reproducibility of different methodologies for the detection of autoantibodies to intracellular antigens such as immunofluorescence, gel techniques (radial immunodiffusion (RID) and counterimmunoelectrophoresis (CIE)), ELISA, Western blotting, and then to improve the performance of these tests. Therefore, every year 10 different sera samples are sent to 35–40 leading laboratories from 21 different European countries to test for as many autoantibodies against intracellular antigens as possible, and the results of these exercises show a tremendous improvement in detection rates (2, 3). A significant step in the life of the EWRR has been reached when laboratory experts in the consensus group agreed to submit their protocols, of which the best were chosen and published in the Manual of Biological Markers of Disease (4). Also, participating laboratories are obliged to function as national expert laboratories to disseminate the knowledge from the consensus meetings to other laboratories and clinicians. One of the main conclusions of the consensus group was that different detection techniques could not be directly compared. Immunofluorescence as a good screening method should preceed all the more specific ones. Results obtained by ELISA or immunoblotting tests should be further confirmed by one of the gel techniques (RID or CIE). The Immunology Laboratory at the Department of Rheumatology, University Medical Centre, Ljubljana, Slovenia has been a part of the consensus group since the very beginning, contributing to the CIE protocol for the detection of autoantibodies against different intracellular antigens (5). In the last 10 years, sera from over 30,000 patients have been tested with this version of CIE with most satisfactory results at the lowest possible cost. Because immune complexes formed in CIE gel precipitate as lines, all the reactions between autoantibodies and relevant antigens are actually seen. This includes also those with unknown autoantibodies. Therefore, many rare, but sometimes diagnostically important autoantibodies (PCNA, SL, Ku, PM/Scl, etc.) were discovered by simple routine testing. Considering the results, CIE is neither expensive nor complicated: it can be performed at very low cost in a very reasonable time, 24 hours for the overall positive/negative result and additional 24 hours to test for specific autoantibodies. This is, for this type of analytes, more than acceptable. A trained technician can test over 100 sera in just 2 hours needed for screening for positivity with two antigen substrates and for further characterisation of all positives using relevant standard antisera. The negative CIE test means that the tested serum is negative for all autoantibodies against most common antigens (Ro, La, Sm, U1RNP, Jo-1, Scl-70) and also against some rare, but very important ones (PCNA, PM/Scl, PL-7, PL-4, Ku, SL), as well as against many unknown antigens. Therefore, there is no need to run separate tests for each autoantibody separately as is the case with ELISA technique. Based on over 20 years of experience, we have summarised major practical advantages and disadvantages of the two-step CIE procedure.

Paratope diversity of anti-prothrombin antibodies

To ascertain the heterogeneity of anti-prothrombin antibodies (aPT), we compared three in-house aPT ELISAs: A) medium binding plates, phosphatidylserine, prothrombin, Tris-buffered saline, calcium; B) high binding plates, prothrombin, Tris-buffered saline; and C) high binding plates, prothrombin, PBS. One serum, exhibiting high positive aPT in all three ELISAs, was selected as the calibrator. Sera from 47 patients (41 with SLE, 4 with pAPS, 2 with arterial thromboses) were tested for IgG and IgM aPT. n nThe results showed six different patterns: 1) similar results were obtained with A, B and C (similar analytical sensitivity); 2) similar results were obtained with A and B while C showed lower analitycal sensitivity; 3) B and C seemed analytically less sensitive than A; 4) A and B were analytically less sensitive than C; 5) A showed very low analytical sensitivity; 6) A and C showed lower analytical sensitivity than B. n nThe analysis of all the presented patterns showed noncomparable results of the three ELISAs. In our experiments, prothrombin was recognized by relevant antibodies when the protein was bound to phosphatidylserine-coated microtiter plates using calcium ions or when it was bound to high binding plates with or without calcium ions. Nevertheless, detected aPT values were not of the same fine specificity. Different binding conditions for prothrombin exposed different epitopes, resulting in the detection of various subgroups of aPT.

Do IgA antiphospholipid antibodies have any clinical importance

Antiphospholipid antibodies (aPL) are strongly associated with clinical manifestations of the antiphospholipid syndrome (APS) such as thromboembolic events and/or pregnancy loss. IgG anticardiolipin (aCL) at moderate/high titre and IgG anti-β2-glycoprotein I (aB2GPI) are closely related to clinical complications, IgM are commonly transient, present during infections, while IgA are not routinely tested. The aim of our study was to evaluate the occurence and possible clinical significance of the IgA isotype of four different aPL: aCL, aB2GPI, anti-prothrombin (aPT) and anti-annexinV (aANXV) antibodies. Sera from 92 patients (87 females and 5 males) with systemic autoimmune disorders (63 with systemic lupus erythematosus (SLE), 19 with secondary APS (SLE with APS) and 10 with primary APS (pAPS)) were assayed with four different in-house ELISA tests. For each of the four antibodies IgG, IgM and IgA isotypes were determined. In evaluating the association of isotypes with particular clinical features (arterial and venous thromboses, CNS disorders, abortuses, thrombocytopenia), IgA isotype did not improve the clinical sensitivity of any measured antibody. The IgA aB2GPI were the only aPL occuring alone in a significant number of patients. Such patients should be followed-up to provide insight into the possible clinical significance of IgA aB2GPI.

Cerebrovascular insult and antiphospholipid antibodies in young adults

Antiphospholipid antibodies (aPL) are strongly associated with arterial and/or venous thrombosis and recurrent foetal loss. The only neurological manifestation satisfying the diagnostic criteria for the antiphospholipid syndrome (APS) is ischemic cerebrovascular insult (CVI). Because of previous contradicting reports on the association between aPL and CVI, we examined the occurrence of different aPL in a group of patients with CVI without an evident systemic autoimmune disease. 39 patients (26 women, 13 men, all under 40 years) were included in study. Blood withdrawal was performed twice after CVI at least 8 weeks apart. Sera were tested by enzyme linked immunosorbent assays (ELISA) for the presence of IgG, IgM and IgA anti-cardiolipin (aCL), anti-β2-glycoproteinI (aβ2GPI), anti-prothrombin (aPT) and anti-annexin V (aANXV) antibodies. Increased levels of IgG aCL were found in 3(8%) patients and for IgA aβ2GPI in 1 (3%). In all aCL and aβ2GPI positive CVI patients only low positive levels of antibodies were detected. aPT were completely absent while aANXV were detected in 4 (10%) patients (3 had IgG and one IgM). aPL directed against serum antigens did not appear with a significant frequency in the studied group of CVI patients. But aANXV, directed against a tissue protein, might be important as one of the possible markers in at least some patients with CVI.