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Cover page of the Journal of Health Sciences

 Table of Contents  
Year : 2022  |  Volume : 15  |  Issue : 3  |  Page : 240-243

Correlation of enzyme-linked immunoassay with line immunoassay for the detection of antinuclear antibody in autoimmune diseases

Department of Microbiology, GIPMER, New Delhi, India

Date of Submission06-Dec-2021
Date of Acceptance14-Mar-2022
Date of Web Publication17-Sep-2022

Correspondence Address:
Dr. Abha Sharma
Department of Microbiology, GIPMER, New Delhi
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/kleuhsj.kleuhsj_354_21

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INTRODUCTION: For the diagnosis of autoimmune disorders, detection of antinuclear antibodies (ANAs) is a key step. Indirect immunofluorescence assays are regarded as a gold standard method. Enzyme-linked immunosorbent assay (ELISA) is easily automated and less technical skills are needed and so used for ANA screening. Further testing is done with line immunoassay (LIA) for specific diagnosis of autoimmune disease. However, there is a lack of data comparing ELISA with LIA for ANA detection in the Indian population.
OBJECTIVE: The objective of this study is to find the correlation between ELISA and LIA for ANA detection.
METHODOLOGY: Serum samples received for ANA screen were tested using commercial kits for ELISA and LIA according to manufacturer's instructions.
RESULTS: Total of 108 samples were tested both by ELISA and LIA. The total numbers of samples positive by ELISA were 26 and positive by LIA were 54 of 108. Samples that were both ELISA and LIA positive were 22 and both ELISA and LIA negative were 50. There was a fair level of agreement between the two methods (kappa = 0.333) with 95% confidence interval of 0.259–0.699. There was no significant difference in the distribution of most of the antibody types detected by LIA among ELISA positive and negative groups. However, the correlation of antibody type U1-Sn ribonucleoprotein with ELISA was found to be statistically significant (P = 0.0319).
CONCLUSION: Detection of ANA is an important screening test. This present study shows moderate correlation between ELISA and LIA and hence both methods can be used depending on the workload of laboratories for cost-effectiveness. ELISA may be the preferred method for high-throughput laboratories.

Keywords: Antinuclear antibody, autoimmune disorders, enzyme-linked immunosorbent assay, line immunoassay

How to cite this article:
Bhasin A, Sharma A, Loomba PS, Mishra B, Das M. Correlation of enzyme-linked immunoassay with line immunoassay for the detection of antinuclear antibody in autoimmune diseases. Indian J Health Sci Biomed Res 2022;15:240-3

How to cite this URL:
Bhasin A, Sharma A, Loomba PS, Mishra B, Das M. Correlation of enzyme-linked immunoassay with line immunoassay for the detection of antinuclear antibody in autoimmune diseases. Indian J Health Sci Biomed Res [serial online] 2022 [cited 2022 Sep 25];15:240-3. Available from: https://www.ijournalhs.org/text.asp?2022/15/3/240/356277

  Introduction Top

In patients suffering from autoimmune diseases, wide spectrums of antibodies are produced against nuclear antigens. Detection of antinuclear antibodies (ANAs) can be done by an array of laboratory tests such as enzyme-linked immunosorbent assay (ELISA), line immunoassay (LIA), and indirect immunofluorescence (IIF). IIF is considered the gold standard.[1] However, IIF is labor-intensive and requires technical expertise and expensive equipment such as fluorescent microscopes which may not be available in all laboratories.[2] Two types of ELISAs are used for ANA detection. A generic assay that detects ANA of broad specificity and the other is specific for antigens such as dsDNA, SSA/Ro, SSB/La, Scl-70, and Sm/ribonucleoprotein (RNP).[3] ELISA is simple and does not require much technical expertise as compared to IIF. Hence, it is more commonly used to screen serum samples for ANA. The LIA detects antibodies to individual extractable antinuclear antigens (ENA) and is a development of ELISA only in which the antigens to be tested are adsorbed onto a nylon test strip. LIA is also simple to perform with even less expertise required as compared to ELISA. This study was done with the objective of comparing ELISA with LIA and to find whether any correlation exists between the two methods.

  Materials and Methods Top

This prospective study was done over 6 months after approval from the ethics committee of the institute. All samples received from patients with clinical symptoms suggestive of autoimmune diseases for ANA screening were tested by both ELISA and LIA. Commercial kits (ANA screen ELISA by CALBIOTECH and IMTEC-ANA-LIA-MAXX by HUMAN, Germany) were used and tests performed according to the manufacturers' instructions. The ELISA kit detected IgG-specific antibody against purified nuclear antigen. LIA kit used is an indirect membrane-based enzyme immunoassay that measures IgG antibodies against dsDNA, nucleosome, histones, SmD1, PCNA, RPP, SSA/Ro60, SSA/Ro52, SSB/La, U1-snRNP, Scl 70, AMA M2, PM-Scl, Mi-2, and Ku. The strip is incubated in diluted patient serum sample and autoantibodies if present in the sample will bind to the antigens on the strip. For the detection of the antibodies bound to the strip, a horseradish peroxidase-labeled secondary antibody (goat) is used that is directed against human IgG. After the addition of a substrate solution comprising precipitating 3, 3, 5, 5-tetramethylbenzidine and addition of stop solution, the bound autoantibodies are visualized as brown lines. Comparison with the evaluation template provided with each kit enables the identification of the patient's autoantibody. Ethical clearance was obtained from Institutional Ethical committee with ref. no. EC/MICRO/249/DEC2020.

Statistical analysis

The level of agreement between both methods was analyzed using the GraphPad software by calculating the kappa value. Association between variables was calculated using Fisher's exact test with significant P < 0.05.

  Results Top

Serum samples of 108 patients were tested for ANA by ELISA and LIA both methods. The total number of samples positive by ELISA were 26 (24.07%) of 108 and positive by LIA were 54 (50%) of 108 [Table 1]. Samples that were both ELISA and LIA positive were 22 and both ELISA and LIA negative were 50. There was a fair level of agreement between the two methods (kappa = 0.333) with 95% confidence interval of 0.21–0404. The types of antibodies detected by LIA in ELISA positive samples and ELISA negative samples are shown in [Table 2]. There was no significant difference in the distribution of most of the antibody types detected by LIA among ELISA positive and negative groups. However, the correlation of antibody type U1-snRNP with ELISA was found to be statistically significant P = 0.0358.
Table 1: Agreement level between two methods

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Table 2: Type of antibodies by line immunoassay correlated with enzyme-linked immunosorbent assay

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  Discussion Top

To the best of our knowledge and literature search, most of the studies have compared ELISA and LIA with the gold standard test IIF. ELISA has not been compared with LIA for the detection of ANA in any of the studies, especially in India. Very few international studies are available. This study has compared ELISA with LIA and has found fair level of agreement between the two methods (kappa = 0.333). Maclachlan et al. observed a good correlation between the two methods in their study,[4] especially for anti- centromere protein B and anti-Jo1 (histidyl-tRNA transferase) antibodies. Conventionally, ANA is detected by IIF, but ELISAs that contain a mixture of known antinuclear antigens are increasingly being used in many laboratories due to easy automation and no requirement of high-level operator skill for performing the test.[5],[6]

LIAs are also simple to use screening assays where up to 17 different autoantibodies can be differentiated with a single assay by spotting antigens in lines on a simple nitrocellulose membrane. LIA is an ideal autoimmune diagnostic tool for differential diagnosis of autoimmune diseases by identifying specific autoantibodies in one assay. LIA was significantly more specific for the detection of antibodies to RNP in the present study with similar findings by Maclachlan et al.[4] In ELISA, ANAs are not detected that are directed against antigens which are not included on the ELISA plate. These may include ribosomal P and PM/Scl and this inability may be the clinical drawback to the use of only ELISA[5] for ANA detection. Furthermore, ELISAs have technical issues of combining different autoantigens in a single assay, and reactivity to some autoantibodies can be missed even if the autoantigens are contained in the mixtures.[7] After the initial screen for ANAs by ELISA, autoantibodies to ENA by LIA should be detected because of their diagnostic and prognostic significance in the diagnosis and management of autoimmune disease.[8],[9] For anti-SSA/Ro, which is the most common antibody type found in this study, there was no significant difference among ELISA positive and negative groups. However, Maclachlan et al.[4] observed that the specificity of the LIA was significantly higher than ELISA if anti-Ro60 was detected. In the case of anti-SS-B/La, anti-Scl70, dsDNA, and PCNA also, no significant difference was noted in correlation to ELISA in this study. Not many studies have correlated types of autoantibodies with ELISA positivity as is presented in this study.

When compared to LIA, ELISA is more economical to perform in high-volume settings [Table 3]. ELISA costs approximately Rs 200 per test while LIA costs apprx Rs 700 per test. Despite that it is easy to use, LIA has one limitation that it is not as feasible to perform in high-volume and high-throughput laboratories, although it can be made automated in such labs but that will add to the cost.[10],[11] Several other new technologies have been developed for testing of sera for autoantibodies in high-throughput laboratories such as fully automated ANA screening assay,[12] a microbead-based ELISA system,[13] and nanobarcode technology.[14]
Table 3: Comparison of enzyme-linked immunosorbent assay versus line immunoassay

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  Conclusion Top

With the increasing impact of autoimmune diseases, increasing number of ANA detection methods are available. Variation in results of different methods makes reproducibility a critical problem. Tests should be reliable for early diagnosis and timely treatment avoiding unnecessary further investigations. Only the ELISA test may be insufficient to establish the diagnosis of specific autoimmune disease as it lacks sensitivity to detect all ANAs and standardization is an issue. It is recommended that in laboratories with high sample load ELISA can be used for ANA screening as is it less expensive as compared to LIA and then for clinically significant cases, ELISA should be followed by LIA for specific diagnosis. In case sample load is less than LIA alone can be done as it can process samples more quickly with less technical expertise required and detects specific autoantibodies.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Greidinger E, Hoffma RW. Antinuclear antibody testing: Methods, indication and interpretation. Lab Med 2013;34:113-7.  Back to cited text no. 1
Raman S, Adhya AK, Pradhan PK, Dash K, Senapati U. Correlation of indirect immunofluorescence and line immunoassay method in detection of autoimmune diseases. An observational study at a tertiary care hospital. Sch J App Med Sci 2017;5:2520-6.  Back to cited text no. 2
Kumar Y, Bhatia A, Minz RW. Antinuclear antibodies and their detection methods in diagnosis of CTDs: A journey revisited. Diagnostic Pathology 2009;4:1-10.  Back to cited text no. 3
Maclachlan D, Vogt P, Wu X, Rose L, Tyndall A, Hasler P. Comparison between Line Immunoassay (LIA) and Enzyme-Linked Immunosorbent Assay (ELISA) for the determination of antibodies to Extractable Nuclear Antigenes (ENA) with reference to other laboratory results and clinical features. Z Rheumatol 2002;61:534-44.  Back to cited text no. 4
Gerald AM, Amel G, Jeffrey L, Anita YN, Graham W, Sally H, et al. Clinical utility of ANA measured by ELISA compared with ANA measured by immunofluorescence. Rheumatology 2009;48:1014-5.  Back to cited text no. 5
Jeong S, Hwang H, Roh J, Shim JE, Kim J, Kim GT, et al. Evaluation of an automated screening assay, compared to indirect immunofluorescence, an extractable nuclear antigen assay, and a line immunoassay in a large cohort of asian patients with antinuclear antibody-associated Rheumatoid diseases: A multicenter retrospective study. J Immunol Res 2018;2018:9094217.  Back to cited text no. 6
Copple SS, Sawitzke AD, Wilson AM, Tebo AE, Hill HR. Enzyme-linked immunosorbent assay screening then indirect immunofluorescence confirmation of antinuclear antibodies: A statistical analysis. Am J Clin Pathol 2011;135:678-84.  Back to cited text no. 7
Kavanaugh A, Tomar R, Reveille J, Solomon DH, Homburger HA. Guidelines for clinical use of the antinuclear antibody test and tests for specific auto antibodies to nuclear antigens. American College of Pathologists. Arch Pathol Lab Med 2000;124:71-81.  Back to cited text no. 8
Orton SM, Peace-Brewer A, Schmitz JL, Freeman K, Miller WC, Folds JD. Practical evaluation of methods for detection and specificity of auto antibodies to extractable nuclear antigens. Clin Vaccine Immunol 2004;11:297-301.  Back to cited text no. 9
Lee SA, Kahng J, Kim Y, Park YJ, Han K, Kwok SK, et al. Comparative study of immunofluorecent antinuclear antibody test and line immunoassay detecting 15 specific auto antibodies in patients with systemic rheumatic disease. J Clin Lab Anal 2012;26:307-14.  Back to cited text no. 10
Almeida González D, Cabrera de León A, Rodríguez Pérez MC, Brito Díaz B, González Hernández A, García García D, et al. Efficiency of different strategies to detect auto antibodies to extractable nuclear antigens. J Immunol Methods 2010;360:89-95.  Back to cited text no. 11
Hayashi N, Kawamoto T, Mukai M, Morinobu A, Koshiba M, Kondo S, et al. Detection of antinuclear antibodies by use of an enzyme immunoassay with nuclear HEp-2 cell extract and recombinant antigens: Comparison with immunofluorescence assay in 307 patients. Clin Chem 2001;47:1649-59.  Back to cited text no. 12
Grossmann K, Roggenbuck D, Schröder C, Conrad K, Schierack P, Sack U. Multiplex assessment of non-organ-specific auto antibodies with a novel microbead-based immunoassay. Cytometry A 2011;79:118-25.  Back to cited text no. 13
Smith J, Onley D, Garey C, Crowther S, Cahir N, Johanson A, et al. Determination of ANA specificity using the UltraPlex platform. Ann N Y Acad Sci 2005;1050:286-94.  Back to cited text no. 14


  [Table 1], [Table 2], [Table 3]


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