Research code: 28671
Ethics code: IR.IUMS.FMD.REC.1402.526
Clinical trials code: IR.IUMS.FMD.REC.1402.526
Immunology Research Center, Institute of Immunology and Infectious Diseases, Iran University of Medical Sciences, Tehran, Iran, & Department of Immunology, Iran University of Medical Sciences, Tehran, Iran , assarehma@gmail.com
Abstract: (85 Views)
Background: Systemic lupus erythematosus (SLE) is a chronic, systemic autoimmune disorder that can affect various tissues and organs including the skin, joints, kidneys, and the central nervous system. The pathogenesis of SLE involves the production of autoantibodies that target nuclear and cytoplasmic antigens, leading to immune complex formation and widespread tissue inflammation. Among these autoantibodies, anti-double-stranded DNA (anti-dsDNA) antibodies are highly specific for SLE and are considered a key biomarker for diagnosis and disease monitoring, especially in lupus nephritis [1,4,5,6,7].
These antibodies may be detected in the serum up to two years before the onset of clinical symptoms [5], and their titers often correlate with disease activity. Rising levels of anti-dsDNA antibodies can serve as an early predictor of disease flare-ups, particularly within six months. Moreover, their presence is associated with increased risk of renal involvement and progressive kidney damage [6].
Multiple laboratory techniques are currently used to detect anti-dsDNA antibodies, each with distinct strengths and limitations. ELISA (enzyme-linked immunosorbent assay) is widely utilized due to its high sensitivity, ease of use, and capability for high-throughput processing [14]. However, ELISA can produce false-positive results due to nonspecific binding of antibodies or cross-reactivity with other nuclear components [10]. On the other hand, the Crithidia luciliae immunofluorescence test (CLIFT) offers superior specificity by utilizing Crithidia luciliae, a flagellated protozoan that contains a unique organelle known as the kinetoplast a highly concentrated, circular, double-stranded DNA structure devoid of interfering nuclear proteins [9,10]. The kinetoplast’s composition provides a highly specific antigenic target for anti-dsDNA antibodies, minimizing the likelihood of cross-reactivity and nonspecific results [11].
Despite this advantage, CLIFT generally exhibits lower sensitivity compared to ELISA. This limitation is primarily due to the inaccessibility of some antigenic epitopes within the compact kinetoplast DNA and the intracellular location of the dsDNA, which may limit binding by low-affinity antibodies [11,14]. Given the complementary nature of these two methods, a combined approach may improve diagnostic confidence.
The objective of this study was to design and evaluate a diagnostic immunofluorescence slide using Crithidia luciliae as the substrate for the indirect detection of anti-dsDNA antibodies and to compare its diagnostic performance with that of a commercially available ELISA kit.
Materials and methods: Crithidia luciliae cells were cultured aerobically in RPMI 1640 medium (pH 7.4) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin-streptomycin. The culture was maintained at 26°C and harvested at the stationary growth phase after approximately 72 hours [13]. To improve morphology and antigen exposure, the cells were washed with PBS and treated with 40 mM hydrogen peroxide. A 10 µL aliquot of the suspension (2 × 10⁷ cells/mL) was applied to each slide well, air-dried, and fixed using 96% ethanol. Slides were stored at –20°C until use. Microscopic examination confirmed the preservation of cell morphology and the uniform distribution of cells.
A total of 100 human serum samples were analyzed, including 50 samples previously confirmed positive and 50 confirmed negative for anti-dsDNA IgG antibodies using a commercial ELISA kit (Generic Assay, Germany). According to the manufacturer, the kit reports a sensitivity of 94.6% and specificity of 99%.
For the indirect immunofluorescence assay, serum samples were diluted 1:10 in PBS and applied to the prepared slides. Following a 30-minute incubation in a humid chamber, slides were washed with PBST and treated with FITC-conjugated sheep anti-human IgG antibodies. After a second 30-minute incubation, final washes were performed, and slides were mounted with glycerol-PBS before examination under a fluorescence microscope. The presence of specific fluorescence in the kinetoplast region was interpreted as a positive result.
Statistical analysis included calculation of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall accuracy. Agreement between CLIFT and ELISA was evaluated using Cohen’s Kappa coefficient, and the statistical significance of the differences between methods was assessed using Fisher’s exact test.
Results: Microscopic evaluation confirmed intact morphology and even distribution of Crithidia luciliae cells on the diagnostic slides. The kinetoplast, a distinct organelle rich in dsDNA, was clearly visible under high magnification and provided a consistent substrate for antibody detection.
Among the 50 ELISA-negative samples, all tested negative by CLIFT, yielding a specificity of 100%. Of the 50 ELISA-positive samples, only 23 tested positive by CLIFT, resulting in a diagnostic sensitivity of 46%. The test demonstrated a PPV of 100%, an NPV of approximately 64.9%, and an overall accuracy of 73%. Cohen’s Kappa coefficient was calculated as 0.46, indicating moderate agreement between the two methods. Fisher’s exact test showed a statistically significant difference between CLIFT and ELISA results (p ≈ 0.0001), confirming that the discrepancy is unlikely to be due to random variation.
Conclusion: The designed CLIFT slide demonstrated excellent specificity but moderate sensitivity in detecting anti-dsDNA antibodies. These findings are consistent with prior studies, which reported similarly high specificity for CLIFT but noted its inability to detect antibodies with lower avidity or reduced access to intracellular DNA targets [11,14]. The superior specificity of CLIFT is attributed to the use of kinetoplast dsDNA, which is highly pure and structurally distinct from other nuclear antigens [10].
However, the test’s limited sensitivity may restrict its use as a primary screening tool. Instead, CLIFT is better suited as a confirmatory assay, particularly in cases where ELISA yields equivocal or potentially false-positive results. A combined approach involving ELISA and CLIFT—alongside clinical assessment and measurement of disease activity markers such as serum complement levels (C3, C4, CH50, C1q) and complement split products (C3dg, C4d)—is recommended for improving diagnostic reliability in SLE [15,16,17].
To enhance the performance of CLIFT, further optimization of immunological parameters is warranted. Buffer pH and ionic strength can influence antibody-antigen binding affinity, and strategic adjustment of these conditions may improve test sensitivity. Additionally, the use of permeabilizing agents such as saponin could facilitate antibody access to intracellular dsDNA, though such interventions must be carefully balanced to preserve the structural integrity of Crithidia luciliae cells.
In conclusion, this study supports the use of CLIFT as a valuable adjunct in the serological diagnosis of SLE and provides a foundation for future improvements in immunofluorescence-based detection systems.
Funding: This research was financially supported by Iran University of Medical Sciences (IUMS) and Padyab Teb Company, which provided funding for laboratory materials and experimental procedures. The authors would like to express their gratitude to the company for its contribution to the development and evaluation of the diagnostic immunofluorescence platform presented in this study.
These antibodies may be detected in the serum up to two years before the onset of clinical symptoms [5], and their titers often correlate with disease activity. Rising levels of anti-dsDNA antibodies can serve as an early predictor of disease flare-ups, particularly within six months. Moreover, their presence is associated with increased risk of renal involvement and progressive kidney damage [6].
Multiple laboratory techniques are currently used to detect anti-dsDNA antibodies, each with distinct strengths and limitations. ELISA (enzyme-linked immunosorbent assay) is widely utilized due to its high sensitivity, ease of use, and capability for high-throughput processing [14]. However, ELISA can produce false-positive results due to nonspecific binding of antibodies or cross-reactivity with other nuclear components [10]. On the other hand, the Crithidia luciliae immunofluorescence test (CLIFT) offers superior specificity by utilizing Crithidia luciliae, a flagellated protozoan that contains a unique organelle known as the kinetoplast a highly concentrated, circular, double-stranded DNA structure devoid of interfering nuclear proteins [9,10]. The kinetoplast’s composition provides a highly specific antigenic target for anti-dsDNA antibodies, minimizing the likelihood of cross-reactivity and nonspecific results [11].
Despite this advantage, CLIFT generally exhibits lower sensitivity compared to ELISA. This limitation is primarily due to the inaccessibility of some antigenic epitopes within the compact kinetoplast DNA and the intracellular location of the dsDNA, which may limit binding by low-affinity antibodies [11,14]. Given the complementary nature of these two methods, a combined approach may improve diagnostic confidence.
The objective of this study was to design and evaluate a diagnostic immunofluorescence slide using Crithidia luciliae as the substrate for the indirect detection of anti-dsDNA antibodies and to compare its diagnostic performance with that of a commercially available ELISA kit.
Materials and methods: Crithidia luciliae cells were cultured aerobically in RPMI 1640 medium (pH 7.4) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin-streptomycin. The culture was maintained at 26°C and harvested at the stationary growth phase after approximately 72 hours [13]. To improve morphology and antigen exposure, the cells were washed with PBS and treated with 40 mM hydrogen peroxide. A 10 µL aliquot of the suspension (2 × 10⁷ cells/mL) was applied to each slide well, air-dried, and fixed using 96% ethanol. Slides were stored at –20°C until use. Microscopic examination confirmed the preservation of cell morphology and the uniform distribution of cells.
A total of 100 human serum samples were analyzed, including 50 samples previously confirmed positive and 50 confirmed negative for anti-dsDNA IgG antibodies using a commercial ELISA kit (Generic Assay, Germany). According to the manufacturer, the kit reports a sensitivity of 94.6% and specificity of 99%.
For the indirect immunofluorescence assay, serum samples were diluted 1:10 in PBS and applied to the prepared slides. Following a 30-minute incubation in a humid chamber, slides were washed with PBST and treated with FITC-conjugated sheep anti-human IgG antibodies. After a second 30-minute incubation, final washes were performed, and slides were mounted with glycerol-PBS before examination under a fluorescence microscope. The presence of specific fluorescence in the kinetoplast region was interpreted as a positive result.
Statistical analysis included calculation of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall accuracy. Agreement between CLIFT and ELISA was evaluated using Cohen’s Kappa coefficient, and the statistical significance of the differences between methods was assessed using Fisher’s exact test.
Results: Microscopic evaluation confirmed intact morphology and even distribution of Crithidia luciliae cells on the diagnostic slides. The kinetoplast, a distinct organelle rich in dsDNA, was clearly visible under high magnification and provided a consistent substrate for antibody detection.
Among the 50 ELISA-negative samples, all tested negative by CLIFT, yielding a specificity of 100%. Of the 50 ELISA-positive samples, only 23 tested positive by CLIFT, resulting in a diagnostic sensitivity of 46%. The test demonstrated a PPV of 100%, an NPV of approximately 64.9%, and an overall accuracy of 73%. Cohen’s Kappa coefficient was calculated as 0.46, indicating moderate agreement between the two methods. Fisher’s exact test showed a statistically significant difference between CLIFT and ELISA results (p ≈ 0.0001), confirming that the discrepancy is unlikely to be due to random variation.
Conclusion: The designed CLIFT slide demonstrated excellent specificity but moderate sensitivity in detecting anti-dsDNA antibodies. These findings are consistent with prior studies, which reported similarly high specificity for CLIFT but noted its inability to detect antibodies with lower avidity or reduced access to intracellular DNA targets [11,14]. The superior specificity of CLIFT is attributed to the use of kinetoplast dsDNA, which is highly pure and structurally distinct from other nuclear antigens [10].
However, the test’s limited sensitivity may restrict its use as a primary screening tool. Instead, CLIFT is better suited as a confirmatory assay, particularly in cases where ELISA yields equivocal or potentially false-positive results. A combined approach involving ELISA and CLIFT—alongside clinical assessment and measurement of disease activity markers such as serum complement levels (C3, C4, CH50, C1q) and complement split products (C3dg, C4d)—is recommended for improving diagnostic reliability in SLE [15,16,17].
To enhance the performance of CLIFT, further optimization of immunological parameters is warranted. Buffer pH and ionic strength can influence antibody-antigen binding affinity, and strategic adjustment of these conditions may improve test sensitivity. Additionally, the use of permeabilizing agents such as saponin could facilitate antibody access to intracellular dsDNA, though such interventions must be carefully balanced to preserve the structural integrity of Crithidia luciliae cells.
In conclusion, this study supports the use of CLIFT as a valuable adjunct in the serological diagnosis of SLE and provides a foundation for future improvements in immunofluorescence-based detection systems.
Funding: This research was financially supported by Iran University of Medical Sciences (IUMS) and Padyab Teb Company, which provided funding for laboratory materials and experimental procedures. The authors would like to express their gratitude to the company for its contribution to the development and evaluation of the diagnostic immunofluorescence platform presented in this study.
Keywords: Systemic Lupus Erythematosus (SLE), Anti-dsDNA Antibody, Crithidia luciliae, Indirect Immunofluorescence, ELISA, CLIFT
Type of Study: Research |
Subject:
Immunology
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