Limited reliability of the indirect immunofluorescence technique for the detection of anti-Rib-P antibodies.

INTRODUCTION: Autoantibodies to the ribosomal P proteins represent a highly specific marker for the diagnosis of systemic lupus erythematosus, where they have been associated with certain clinical manifestations. Historically, autoantibodies against ribosomal P proteins have been detected by indirect immunofluorescence, immunodiffusion, immunoblot, and other immunoassays. More recently, enzyme-linked immunosorbent assays and line and addressable laser bead immunoassays have become more widely used. The primary goal of this study was to determine the sensitivity of indirect immunofluorescence using conventional HEp-2 substrates in the detection of sera with ribosomal P antibodies as detected by other immunoassays. METHODS: Anti-ribosomal P-positive sera (n = 345) as detected by an addressable laser bead immunoassay were collected between 2003 and 2007 and analysed by indirect immunofluorescence. Furthermore, 51 anti-ribosomal P-positive samples from an unselected systemic lupus erythematosus cohort (n = 100) and the Centers for Disease Control and Prevention (CDC) anti-nuclear antibody (ANA) reference sera were tested for anti-ribosomal P reactivity. RESULTS: In the cohort of 345 anti-ribosomal P-positive samples identified by addressable laser bead immunoassay, a low sensitivity (<30%) of indirect immunofluorescence on HEp-2 cell substrates was observed. Although the degree of sensitivity varied among different manufacturers, all immunofluorescence substrates exhibited limited sensitivity and false-negative results were not restricted to samples with low anti-ribosomal P titers. Even the anti-ribosomal P reactivity of CDC ANA reference serum number 12 was not clearly predictable by indirect immunofluorescence. Comparison of five different methods for the detection of anti-ribosomal P found moderate qualitative agreements. CONCLUSIONS: Based on our data, we conclude that indirect immunofluorescence on HEp-2 cells is not a reliable screening test for the prediction of ribosomal P antibodies. As this method is widely used as a first-line screening test for anti-nuclear and other autoantibodies, special considerations for the detection of ribosomal P antibodies are needed. As with many other autoantibodies, further effort is required for the standardisation of ribosomal P immunoassays.

Detection of antibodies against adenovirus protein IX, fiber, and hexon in human sera by immunoblot assay.

The 51 serotypes of human adenoviruses (HAdVs) of the genus Mastadenovirus are classified into the six species HAdV-A to HAdV-F. For the detection of genus- and species-specific antibodies in human sera an immunoblot assay was developed. The recombinant long fiber of HAdV-41[F] (Ad41Fi) and the native hexon of HAdV-5[C] were used as genus-specific antigens. The recombinant capsid protein IX (pIX) of HAdV-2 (Ad2pIX[C]) and HAdV-41 (Ad41pIX[F]), the C-terminal pIX part of HAdV-3 (Ad3pIXC[B]), and the fiber knob of HAdV-8 (Ad8FiKn[D]) were evaluated as representative species-specific antigens. Hence, the pIX amino acid sequences of numerous serotypes of all HAdV species were compared, and the cross-reactivities of pIX antigens with rabbit hyperimmune sera among HAdV-A to -F were analyzed. In an epidemiological study, 667 human patient sera, not selected for viral infection, were screened for adenovirus seroprevalence. The genus-specific antibody prevalences directed against the Ad41Fi and HAdV-5 hexon were 82.8 and 98.8%, respectively. The species-specific antibody prevalence of 44.7% against Ad2pIX[C], 36.6% against Ad41pIX[F], 26.4% against Ad8FiKn[D], and 18% against Ad3pIXC[B] showed an age-dependent distribution and correlated well with the frequency of isolated serotypes of the respective species in earlier studies (except HAdV-D). In conclusion, the immunoblot assay using pIX, fiber, and hexon antigens represents a valuable and new serological tool for refined adenovirus diagnosis as shown in an epidemiological study.