Autoimmune connective tissue disorders (CTDs) are a spectrum of systemic diseases characterized by autoantibody production, chronic inflammation, and immune-mediated tissue injury. These conditions include systemic lupus erythematosus (SLE), systemic sclerosis, Sjögren’s syndrome, mixed connective tissue disease, and others. While the clinical manifestations of CTDs are diverse and often overlapping, the detection of autoantibodies—particularly anti-nuclear antibodies (ANA)—plays a critical role in their diagnosis, classification, and prognostication.

Among the various serological tests, ANA detection is considered the first-line screening tool for suspected autoimmune connective tissue diseases. The presence of ANA is not disease-specific but serves as a useful biomarker for identifying patients who may benefit from further autoimmune workup. The gold standard technique for ANA detection remains indirect immunofluorescence (IIF) on HEp-2 cell substrates, which provides both high sensitivity and the ability to interpret fluorescence patterns that are often disease-specific. For instance, the homogenous pattern is commonly associated with SLE, while the centromere and nucleolar patterns are seen in systemic sclerosis.

Despite its diagnostic value, IIF has notable limitations. It is semi-quantitative, labor-intensive, and subject to observer bias. The interpretation of fluorescence patterns requires trained personnel and can vary between laboratories. These drawbacks have led to increasing use of enzyme-linked immunosorbent assay (ELISA) as an alternative or adjunct ANA detection method. ELISA is a solid-phase assay that is amenable to automation, offers better standardization, and delivers objective results with minimal operator dependence. It is also faster and more practical in high-throughput laboratory settings.

However, ELISA may not detect certain autoantibodies, particularly those directed against complex or conformational antigens not included in the assay’s antigen pool. As a result, false-negative results may occur, particularly in early or atypical CTD presentations. Additionally, ELISA does not provide pattern information, which limits its utility in distinguishing between different autoimmune conditions. Therefore, while ELISA offers convenience, its diagnostic performance compared to IIF remains a subject of ongoing clinical relevance.

Several studies have reported variable degrees of concordance between ELISA and IIF, ranging from moderate to poor agreement, depending on the patient population, disease spectrum, and assay kits used. Regional data, especially from resource-limited settings like India, are still sparse. Understanding the relative strengths and limitations of each method is essential for optimizing diagnostic strategies, particularly in settings where both tests are available.

This study aims to evaluate the diagnostic accuracy of ELISA in comparison with IIF for ANA detection in a cohort of patients clinically suspected of CTDs. Specifically, the study assesses the sensitivity, specificity, concordance rate, and pattern correlation between the two methods. The findings are intended to guide clinicians and laboratory professionals in selecting the most appropriate approach for ANA testing and help establish tiered or combined testing algorithms suited to the local healthcare context.

Study Design and Ethical Consideration

This cross-sectional, comparative study was conducted in the Department of Microbiology, Tirunelveli Medical College, Tamil Nadu, India, over a period of five months from February 2015 to June 2015. Ethical approval was obtained from the Institutional Human Ethics Committee prior to initiation of the study. Informed written consent was obtained from all participants prior to blood sample collection.

Study Population and Sample Selection

A total of 90 serum samples were collected from patients clinically suspected of connective tissue disorders (CTDs), including systemic lupus erythematosus (SLE), systemic sclerosis, Sjögren’s syndrome, mixed connective tissue disease (MCTD), and polymyositis/dermatomyositis. Patients aged 12 years and above of either sex were included based on clinical criteria suggestive of autoimmune disease. Exclusion criteria included pediatric patients under 12 years of age, individuals with known HIV or hepatitis B surface antigen (HBsAg) positivity, and samples that were hemolyzed or insufficient in volume.

Sample Collection and Storage

Venous blood (3 mL) was collected aseptically using plain vacutainer tubes. The samples were allowed to clot at room temperature, and serum was separated by centrifugation. All serum samples were stored at –80°C until tested. Each sample was analyzed by both enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) for the detection of anti-nuclear antibodies (ANA).

ANA Detection by Indirect Immunofluorescence (IIF)

ANA testing by IIF was carried out using the BIOSYSTEMS ANA HEp-2 kit, which uses fixed HEp-2 cells as substrate. Patient serum was diluted at 1:80 using phosphate-buffered saline (PBS), and 25 µL of diluted serum was applied to the slide wells. Slides were incubated in a moist chamber at room temperature for 30 minutes, washed with PBS, and then incubated with FITC-labeled anti-human IgG conjugate. After a second incubation and final wash, slides were mounted with a suitable mounting medium and examined under a fluorescence microscope at magnifications of 250x to 400x. ANA reactivity was recorded as positive or negative, and the pattern of fluorescence was categorized into homogenous, speckled, nucleolar, centromere, peripheral (rim), or cytoplasmic, according to standard IIF interpretation guidelines.

ANA Detection by Enzyme-Linked Immunosorbent Assay (ELISA)

ANA detection via ELISA was performed using the Calbiotech ANA IgG ELISA kit. Serum samples were diluted 1:101 with the provided sample diluent, and 100 µL was added to each microtiter well coated with a mixture of nuclear antigens. After 30 minutes of incubation at room temperature, the wells were washed and incubated with horseradish peroxidase (HRP)-conjugated anti-human IgG for another 30 minutes. Following a second wash, 100 µL of TMB substrate solution was added and incubated for 15 minutes in the dark. The reaction was stopped with stop solution, and absorbance was measured at 450 nm. The results were interpreted based on the manufacturer’s cut-off, classifying samples as positive or negative.

Data Comparison and Evaluation Criteria

Each serum sample underwent parallel testing by ELISA and IIF, and the results were recorded independently. Concordance between the two methods was defined as both tests being either positive or negative, while discordance referred to mismatched results. The diagnostic performance of ELISA was evaluated in terms of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall diagnostic accuracy, using IIF as the reference standard.

Statistical Analysis

All statistical analyses were performed using IBM SPSS software version 20.0. Descriptive statistics were used to summarize demographic data, ANA positivity rates, and fluorescence patterns. The chi-square (χ²) test was applied to assess associations between ANA results and demographic or clinical parameters. The agreement between ELISA and IIF was evaluated using Cohen’s kappa (κ) coefficient, with interpretation as follows: values ≤0.20 indicating slight agreement, 0.21–0.40 fair, 0.41–0.60 moderate, 0.61–0.80 substantial, and ≥0.81 almost perfect agreement. A p-value less than 0.05 was considered statistically significant.

Comparison of ANA Detection by ELISA and IIF

Out of the 90 serum samples analyzed, the indirect immunofluorescence (IIF) assay detected anti-nuclear antibodies (ANA) in 59 cases (65.6%), whereas the enzyme-linked immunosorbent assay (ELISA) identified ANA positivity in 52 cases (57.8%). While both assays showed similar trends in overall positivity, ELISA consistently demonstrated slightly lower sensitivity, missing some cases identified by IIF. Among the 59 IIF-positive cases, 48 were also positive by ELISA, whereas 11 were negative by ELISA, suggesting missed detections. Conversely, of the 31 IIF-negative samples, 4 yielded positive results by ELISA, potentially indicating false positives. Overall, there was agreement in 79 of 90 cases (87.8%) between the two methods, with 11 cases (12.2%) showing discordant results.

Diagnostic Performance of ELISA

Using IIF as the reference standard, the diagnostic characteristics of ELISA were evaluated. The sensitivity of ELISA was found to be 81.4%, and the specificity was 87.1%, indicating strong performance in detecting true positives and true negatives, respectively. The positive predictive value (PPV) was 92.3%, and the negative predictive value (NPV) was 71.0%. The overall diagnostic accuracy of ELISA was calculated at 84.4%, demonstrating a high level of performance, although lower than IIF in certain clinical contexts.

Statistical Agreement between Methods

Agreement between ELISA and IIF was assessed using Cohen’s kappa (κ) statistic, which yielded a κ value of 0.677, indicating substantial agreement between the two diagnostic modalities. This suggests that ELISA can serve as a reasonably reliable alternative to IIF in many routine settings, although not a full replacement due to pattern and antigen limitations.

Analysis of Discordant Cases

The 11 discordant cases were examined to explore reasons for disagreement. The 11 ELISA-negative/IIF-positive samples were mostly low-titer ANA reactions with less common patterns such as nucleolar or peripheral, which are typically associated with systemic sclerosis and mixed CTD—conditions where IIF tends to outperform ELISA due to broader antigen presentation. The 4 ELISA-positive/IIF-negative cases could reflect nonspecific reactivity, cross-reactivity with other autoantibodies, or early subclinical autoimmunity.

Operational Considerations

From an operational standpoint, ELISA offered advantages in terms of reduced hands-on time, automated processing, and objective output, making it suitable for high-throughput environments. However, its limitations in detecting conformational epitopes and lack of pattern recognition support the continued use of IIF, especially for initial screening and in diagnostically ambiguous cases.

Table 1: ELISA vs IIF Result Comparison

Table 2: Diagnostic Performance Metrics of ELISA (Using IIF as Reference)

The accurate detection of anti-nuclear antibodies (ANA) is crucial in the diagnostic evaluation of connective tissue disorders (CTDs), where early identification can significantly influence treatment outcomes. In this study, we evaluated the diagnostic performance of enzyme-linked immunosorbent assay (ELISA) in comparison to the gold standard indirect immunofluorescence (IIF) for ANA detection in clinically suspected CTD patients.

Our results revealed that IIF detected ANA in 65.6% of samples, while ELISA detected ANA in 57.8%, demonstrating a modest reduction in sensitivity with ELISA. While both assays showed overall agreement in 87.8% of cases, 11 samples showed discordant results—specifically, ELISA failed to detect ANA in 11 IIF-positive samples. This reduced sensitivity may be attributed to the narrower antigen range used in ELISA kits, which often lack certain conformational or rare nuclear antigens. These findings are consistent with previous studies, including Ghosh et al. and Wiik et al., which reported that ELISA tends to underperform in detecting non-standard ANA patterns or low-titer antibodies.

The diagnostic sensitivity and specificity of ELISA, as observed in this study, were 81.4% and 87.1%, respectively. The positive predictive value (PPV) was high at 92.3%, indicating that a positive ELISA result is strongly predictive of ANA positivity confirmed by IIF. However, the negative predictive value (NPV) was lower at 71.0%, highlighting that a negative ELISA result does not reliably exclude autoimmune disease. These figures align with global studies, such as those by Von Mühlen and Mahler, who similarly observed lower NPV and sensitivity for ELISA-based ANA detection.

Cohen’s kappa coefficient for agreement between the two methods was 0.677, reflecting substantial agreement. This statistical relationship supports the use of ELISA as a supplementary or screening test but not a replacement for IIF in diagnostic workflows. The discordant ELISA-negative/IIF-positive cases were predominantly associated with nucleolar or peripheral patterns, often linked to systemic sclerosis and mixed connective tissue disease. These patterns are known to be underrepresented in ELISA panels, reaffirming the importance of IIF in detecting broader autoantibody profiles.

Operationally, ELISA offers advantages such as automation, objective readout, faster processing, and minimal observer bias. These features make it attractive for high-throughput diagnostic settings. However, it lacks the qualitative insight offered by IIF, particularly the fluorescence pattern, which can provide disease-specific clues and aid in clinical differentiation.

Given these findings, a tiered diagnostic approach may be most appropriate. ELISA could be employed for initial high-volume screening, with reflex testing by IIF in ELISA-negative patients with strong clinical suspicion or when specific pattern information is necessary. Alternatively, laboratories with sufficient expertise and resources should continue using IIF as the primary ANA testing modality, especially in tertiary care centers handling complex autoimmune cases.

ELISA demonstrates good specificity and agreement with IIF for ANA detection but may miss low-titer or rare pattern cases. While suitable for high-throughput screening, ELISA should be supplemented with IIF, especially in diagnostically challenging CTD cases. A tiered testing strategy incorporating both methods is recommended to enhance diagnostic accuracy.

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