Development of miniaturized immunoassay: influence of surface chemistry and comparison with enzyme-linked immunosorbent assay and Western blot.

Protein microarray technology provides a useful approach for the simultaneous serodetection of various antibodies in low sample volumes. To implement functional protein microarrays, appropriate surface chemistry must be designed so that both the protein structure and the biological activity can be retained. In the current study, two surface chemistries for protein microarrays and immunofluorescent assays were developed. Glass slides were functionalized with N-hydroxysuccinimide (NHS) ester via a monofunctional silane or maleic anhydride-alt-methyl vinyl ether (MAMVE) copolymer to allow covalent grafting of histone proteins. Analytical performance of these microarrays was then evaluated for the detection of anti-histone autoantibodies present in the sera of patients suffering from a systemic autoimmune disease, namely systemic lupus erythematosus (SLE), and the results were compared with those of the classical enzyme-linked immunosorbent assay (ELISA) and Western blot. The detection limit of our MAMVE copolymer microarrays was 50-fold lower than that of the classical ELISA. Furthermore, 100-fold less volume of biological samples was required with these miniaturized immunoassays.

A new target for autoantibodies in patients with rheumatoid arthritis.

Early treatment of rheumatoid arthritis (RA) with disease-modifying antirheumatic drugs can achieve a better disease outcome and reduce the severity of joint damage. The presence of autoantibodies in patient sera can precede onset of the disease and thus be predictive of the development of RA. To date, known autoantibodies in RA are positive in only 50-60% of RA patients at onset of disease and even less before the onset of any RA symptom. The aim of this study was to identify new antibodies that could be useful for the diagnosis of RA using synovial membrane proteins, which represent the best source of candidate RA autoantigens. The humoral reactivity of sera from RA patients was explored using immunoblotting on extracted proteins obtained from synovial membranes from RA after synovectomy or arthroplasty. A new target protein with a molecular weight of 26 kDa was found to be recognized by autoantibodies in RA sera. This protein was identified using MALDI-TOF mass spectrometry and two-dimensional electrophoresis as carbonic anhydrase III with a high level of confidence. In conclusion, this study demonstrates new autoantibodies in RA patients that are directed against carbonic anhydrase III. The sensitivity and specificity of these new autoantibodies for RA have to be further evaluated.

Carbonic anhydrase III: a new target for autoantibodies in autoimmune diseases.

The objective of this study was to identify new autoantibodies that could be useful for the diagnosis of rheumatoid arthritis (RA) using immunoblotting on synovial membrane proteins which represent the best source of candidate RA autoantigens. A new target protein with a molecular weight of 26 kDa was found to be recognized by autoantibodies in RA sera and was identified using MALDI-TOF mass spectrometry and second-dimension electrophoresis as carbonic anhydrase III (CAIII). Three similar protein spots at 26 kDa were recognized by both human sera and monoclonal antibody (mAb) directed against CAIII on immunoblotting using the human recombinant CAIII. Interestingly, CAIII expression within the synovial membrane was not observed in non-RA patients and was differentially expressed among RA patients. The sensitivity of these new autoantibodies for RA, using an immunoenzymatic technique, was 17%. Specificity was high when comparing non-autoimmune diseases (100%), while it was found to be weak (67%) when comparing some other autoimmune diseases, and particularly systemic lupus erythematosus (SLE). In conclusion, this study demonstrates that these new autoantibodies against CAIII are not restricted to RA. However the expression of CAIII in the synovial membrane of RA warrants further investigation of the pathophysiological relevance of this finding.

Calcium-sensing receptor autoantibodies are relevant markers of acquired hypoparathyroidism.

We investigated the presence of autoantibodies (aAbs) directed against the parathyroid gland in 17 patients with spontaneous isolated acquired hypoparathyroidism. Fourteen patients with acquired hypoparathyroidism (AH) associated with type I or II autoimmune polyendocrinopathy syndrome were also tested in comparison with a control group of 68 subjects without AH, including patients with other autoimmune diseases and healthy blood donors. aAbs against parathyroid tissue were screened using an indirect immunofluorescence technique on primate parathyroid tissue and human parathyroid adenoma. aAbs against the calcium-sensing receptor (CaSR) were analyzed using an immunoblotting assay with the recombinant extracellular domain of the human CaSR as antigen. Seven of the 31 patients with AH were positive for CaSR aAbs. Five of the positive sera were obtained from the group with isolated AH. The two other positive sera were from patients with autoimmune polyendocrinopathy syndrome. The sensitivity of the immunoblotting technique was higher than that of both the radioimmunological test using the extracellular domain of the CaSR and the indirect immunofluorescence technique. There were no positive sera in the control group. In conclusion, using an immunoblotting assay, we demonstrate the presence of CaSR aAbs in about one third of the patients with isolated AH, pointing out the value of detecting such aAbs to assess the autoimmune origin of the disease.

Autoantibodies directed against the UDP-glucuronosyltransferases in human autoimmune hepatitis.

Liver-Kidney Microsomes Type 3 (LKM3) autoantibodies (aAbs) have been described in chronic hepatitis D virus infection in 1983. The detection of such aAbs in autoimmune hepatitis (AIH) Type 2 was thereafter reported. The molecular targets of LKM3 aAbs have been identified as enzymes belonging to the UDP-glucuronosyltransferase family 1. Since 20-30% of suspected AIH are negative for the classical autoimmune serological markers, such as aAbs directed against antinuclear autoantibodies, smooth muscle autoantibodies and Liver-Kidney Microsomes Type 1 aAbs, LKM3 aAbs could be of great interest in the diagnosis of such negative AIH. In this review, we discuss the sensitivity and specificity of these aAbs in AIH in order to stress out their potential clinical use as a marker.

Cross-species reactivity of seven monoclonal antibodies with equine lymphocytes by flow cytometry.

The recognition of equine lymphocyte antigens by monoclonal antibodies (mAbs) directed against human CD11a, CD18, CD21, CD23, CD29 and DR, as well as mouse CD23 was studied by flow cytometry. Unlike anti-CD11a, -CD21, -CD23 and DR mAbs, anti-CD18 and CD29 mAbs labelled the same percentage of horse peripheral blood lymphocytes (PBL) as human PBL. Double-staining with anti-horse immunoglobulin antibodies showed that anti-CD21 and -CD23 mAbs are mainly bound to peripheral blood B lymphocytes. The seven mAbs were also tested on the lymph node and thymus cells. The molecular targets of anti-CD11a, CD18 and CD29 mAbs were confirmed by immunoprecipitation of the membrane proteins. Our results suggest that anti-CD18, -CD29 and -DR mAbs recognise similarly expressed molecular homologues on equine cells, but that anti-CD11a, -CD21 and -CD23 mAbs recognise either different molecules or homologues that are expressed at different levels on horse cells.

Improvement in diagnosis of rheumatoid arthritis using dual indirect immunofluorescence and immunoblotting assays for antifilaggrin autoantibodies: a retrospective 3 year study.

OBJECTIVE: To determine the clinical usefulness of measuring antistratum corneum (ASC) and antifilaggrin autoantibodies (AFA) to discriminate between rheumatoid arthritis (RA) and other rheumatic or autoimmune diseases, using an indirect immunofluorescence (IIF) assay, along with a complementary immunoblotting technique (IB) when IIF detection of ASC was negative. METHODS: Sera from 346 patients were studied: 189 sera from patients with RA seen in the same clinic, 92 from patients with non-RA rheumatic diseases, 24 from nonrheumatic autoimmune diseases, and 41 from healthy blood donors. ASC and AFA were detected using IIF and IB, respectively. RESULTS: ASC detection using IIF showed a specificity of 97.5% for RA with 44.4% sensitivity. When both IIF and IB techniques were used, sensitivity for RA increased significantly (up to 53.4%; p < 0.01) with no decrease in specificity (p < 0.01). CONCLUSION: These data confirm the usefulness of 2 different techniques performed simultaneously for detecting ASC/AFA, and the usefulness of these biological markers for discriminating between RA and other rheumatic diseases in clinical practice.