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

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.

Materials and methods Sera Three hundred forty-five serum samples that had a positive anti-Rib-P test as detected by an ALBIA (QuantaPlex ENA8; INOVA, San Diego, CA, USA) between 2003 and 2007 in the Mitogen Advanced Diagnostics Laboratory (Calgary, AB, Canada) were retrospectively analysed for aab by IIF on a HEp-2 substrate kit (HEp-2000; ImmunoConcepts, Sacramento, CA, USA) that included fluorescein-conjugated goat antibodies to human IgG (H+L). IIF patterns were read at serum dilutions of 1:160 and 1:640 on a Zeiss Axioskop 2 plus (Carl Zeiss, Jena, Germany) fitted with a 100-watt USHIO super-high-pressure mercury lamp (Ushio, Steinhöring, Germany) by two experienced technologists with more than 5 years of experience who had no knowledge of the ALBIA results. Of the 345 anti-Rib-P-positive samples, 51 were randomly selected and tested for anti-Rib-P antibodies by ELISA (synthetic peptide; Dr. Fooke Laboratorien GmbH, Neuss, Germany), LIA ( recom Line ENA/ANA IgG with recombinant P0; Mikrogen GmbH, Neuried, Germany), and EliA ® ribosomal P based on recombinant P0, P1, and P2 (Phadia, Freiburg, Germany), retested by IIF on HEp-2 cells, and analysed by IB as described by Mahler and colleagues [ 13 ], with some modifications (see below). Low (20 to 49 median fluorescence units, MFU), medium (50 to 100 MFU), and highly (>100 MFU) reactive samples (15 each) were selected based on the ALBIA results and analysed on HEp-2 slide kits from three different suppliers: INOVA, Euroimmun (Lübeck, Germany), and ImmunoConcepts. All tests were carried out according to the manufacturers' instructions for use. In addition, a clinically defined cohort of SLE patients (n = 100) who met the American College of Rheumatology (ACR) classification criteria [ 19 ] and the international Centers for Disease Control and Prevention (CDC) anti-nuclear antibody (ANA) reference sera [ 20 , 21 ] was assayed for anti-Rib-P antibodies by all methods. This study was approved by the Conjoint Biomedical Ethics Review Board at the University of Calgary.

Immunoblot An in-house

IB with nuclear and cytoplasmic extracts from HeLa cells was employed for determination of Rib-P antibodies. In brief, nuclear and cytoplasmic extracts from HeLa cells were separated by SDS gel electrophoresis on a 13.5% polyacrylamide gel followed by transfer on nitrocellulose. After the nitrocellulose strips were blocked with casein hydrolysate buffer (CHB) (1% casein hydrolysate in phosphate-buffered saline [PBS]/Tween) for 30 minutes at room temperature (RT), they were incubated with serum samples diluted 1:100 in CHB for 1 hour at RT. After washings (3 × 5 minutes) with PBS/Tween, the strips were incubated 1 hour at RT with an alkaline phosphatase (AP)-conjugated anti-human IgG from Sigma-Aldrich (St. Louis, MO, USA) (A-3187), which was diluted 1:10,000 in CHB. The strips were washed again with PBS/Tween (3 × 5 minutes) and then equilibrated with AP buffer (pH 9.5) for 5 minutes and finally developed with the AP enzyme substrate NBT/BCIP (nitro blue tetrazolium/5-bromo-4-chloro-3-indolylphosphate). Development of the blots was stopped after 7 minutes by aspiration of the substrate followed by equilibration with distilled H 2 O. The IB strips were subjected to visual evaluation using a panel of reference strips, among them strips containing nuclear and cytoplasmic extracts that showed the characteristic Rib-P band pattern (37, 19, and 17 kDa). For evaluation of the blots, a subjective semi-quantitative scale was used: -, negative; (+), equivocal; +, weak positive; ++, moderate/strong positive; and +++, very strong positive. A serum was considered to be anti-Rib-P-positive when at least one of the three characteristic Rib-P bands was observed on both strips (nuclear and cytoplasmic).

Statistical evaluation

The data were statistically evaluated using the Analyse-it software (version 1.62; Analyse-it Software, Ltd., Leeds, UK). Chi-square, Spearman correlation, and Cohen kappa agreement tests were carried out to analyse the agreement between portions, and P values of less than 0.05 were considered significant. Differences with P values of less than 0.05 were considered significant.

Confirmation of anti-Rib-P reactivity in 51 samples by other methods When 51 samples with a positive anti-Rib-P test result by ALBIA were tested for anti-Rib-P reactivity by other assays, 13 of 51 (25.5%) samples were confirmed by LIA (recombinant P0), 27 of 51 (52.9%) by ELISA (synthetic peptide), 21 of 51 (39.6%) by EliA ® Rib-P, and 20 of 51 (39.2%) by IB. Of 20 samples with anti-Rib-P reactivity by IB, only 8 (40.0%) showed the IIF CSP that is considered to indicate the presence of anti-Rib-P antibodies (kappa = 0.19, P = 0.1514). The results of all methods showed no statistically significant agreement with an IIF CSP. When only medium and highly reactive (>1,000 MFU) samples as detected by ALBIA were considered, the percentage of confirmed results significantly increased (Table 1 ). The agreement between the individual methods and the IB was found at 0.57 ( P < 0.0001) (ELISA), 0.71 ( P < 0.0001), and 0.96 ( P < 0.0001) according to the kappa method. Repeat analysis of these 51 sera by IIF on HEp-2 cells revealed CSP in 14 (27.5%) of the sera. The percentage of confirmed results did not increase significantly in medium- and/or high-titer samples for LIA and ELISA but did increase slightly for IIF. Table 1 Sensitivity of LIA, ELISA, EliA ® Rib-P, IB, and IIF versus ALBIA All Medium a + high MFU Only high b MFU n = 51 n = 27 n = 20 LIA Positive + borderline, number (percentage) 19 (37.3) 9 (33.3) 7 (35.0) Positive, number (percentage) 13 (25.5) 8 (29.6) 7 (35.0) ELISA Positive + borderline, number (percentage) 36 (70.6) 21 (77.8) 14 (70.0) Positive, number (percentage) 27 (52.9) 17 (63.0) 12 (60.0) EliA ® Positive + borderline, number (percentage) 22 (43.1) 12 (44.4) 8 (40.0) Positive, number (percentage) 21 (41.2) 11 (40.7) 8 (40.0) IIF (CSP) Positive, number (percentage) 14 (27.5) 11 (40.7) 8 (40.0) IB At least one Rib-P band, number (percentage) 20 (39.2) 10 (37.0) 7 (35.0) a Medium 50 to 100 median fluorescence units (MFU); b high ≥ 100 MFU. ALBIA, addressable laser bead immunoassay; CSP, cytoplasmic staining pattern; ELISA, enzyme-linked immunosorbent assay; IB, immunoblot; IIF, indirect immunofluorescence; LIA, line immunoassay; Rib-P, ribosomal P protein. When a serum sample that was 'monospecific' for anti-Rib-P antibodies (no antibodies to dsDNA, Sm, U1-RNP, SS-A/Ro, and so on) was tested by IIF, it was noted that there was variation in the staining pattern produced on the HEp-2 substrates from three different manufacturers (Figure 1 ). We expanded this study to ensure that the limited sensitivity of IIF for the detection of anti-Rib-P antibodies is not restricted to slides of a certain manufacturer. Fifteen sera having low, medium, or high titers of anti-Rib-P as determined by ALBIA were analysed on HEp-2 slides from the three different suppliers (Table 2 ) and this analysis indicated two main observations. First, there is inter-manufacturer difference in the display of the typical CSP IIF patterns when medium or highly reactive sera were analysed and none of the manufacturers' HEp-2 substrates had a typical CSP pattern in sera with low levels of anti-Rib-P. Second, anti-Rib-P sera characterised by higher titer (MFU) tended to have a higher frequency of the typical CSP pattern, irrespective of manufacturer. Nevertheless, even on HEp-2 substrates from the manufacturer with the apparently highest frequency of CSP, less than 50% had the typical CSP Rib-P staining pattern. Figure 1 Indirect immunofluorescence staining pattern of anti-Rib-P-positive samples. One anti-Rib-P-positive serum that did not have autoantibodies to other known antigens (a-c) and the Centers for Disease Control and Prevention (CDC) anti-nuclear antibody reference serum number 12 (d-f) were tested at dilutions of 1:500 and 1:100, respectively, on slides from three different suppliers. Significant differences were observed in patterns of staining for the monospecific anti-Rib-P sera (a-c) on HEp-2 substrates from INOVA (San Diego, CA, USA) (a), ImmunoConcepts (Sacramento, CA, USA) (b), and Euroimmun (Lübeck, Germany) (c). Furthermore, the indirect immunofluorescence of the high-titer CDC anti-Rib-P reference serum produced only weak cytoplasmic staining on HEp-2 substrates from the same manufacturers (d-f). Rib-P, ribosomal P protein. Table 2 Presence of Rib-P-like cytoplasmic indirect immunofluorescence pattern on HEp-2 substrates from different manufacturers Manufacturer Low a MFU, number (percentage) Medium b MFU, number (percentage) High c MFU, number (percentage) All, number (percentage) n = 15 n = 15 n = 15 n = 45 INOVA (San Diego, CA, USA) 0 3 (20.0) 7 (46.6) 10 (22.2) ImmunoConcepts (Sacramento, CA, USA) 0 1 (6.7) 5 (33.0) 6 (13.3) Euroimmun (Lübeck, Germany) 0 0 3 (20.0) 3 (6.7) a Low = 20 to 49 median fluorescence units (MFU); b medium = 50 to 100 MFU; c high ≥ 100 MFU. Rib-P, ribosomal P protein.