Evaluation of diagnostic accuracy of elisa method in the detection of anti-nuclear antibodies compared to immunofluorescence Authors:N.Sudha, P.Balapriya, P.S. Arul Raja Murugan Int J Biol Med Res. 2024; 15(1): 7746-7749 Abstract: Autoimmune connective tissue diseases are group of disorders in which autoantibodies are formed against self-antigen present in the nucleus and other component of the cell. The most common methods to detect antinuclear antibodies are ELISA and indirect Immunofluorescence. This study aims to evaluate the validity of ANA ELISA method as compared to Indirect immunofluorescence (IF) assay which is considered as the gold standard test. This is a descriptive study conducted in the department of Immunology, Madras Medical College Chennai, during the period of October 2021 to December 2021 with 110 samples. ANA ELISA and Indirect Immunofluorescence were done as per the instructions provided by manufacturer of kit. Out of the 110 samples 75 cases were positive, in this 29 were positive by Immunofluorescence alone, 43 cases were positive by both and 3 cases were positive by ELISA alone. The Pearson Correlation coefficient value showed positive correlation between the grading of intensity of the positive immunofluorescence pattern and optical density of antinuclear antibodies by ELISA. So both the methods can be used but in centers with appropriate facilities Immunofluorescence proves to be the gold standard in the diagnosis.

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International Journal of Biological & Medical Research
Int J Biol Med Res. 2025; 15(1): 7746-7749
EVALUATION OF DIAGNOSTIC ACCURACY OF ELISA METHOD IN THE DETECTION OF
ANTI-NUCLEAR ANTIBODIES COMPARED TO IMMUNOFLUORESCENCE
a b c
N.Sudha *, P.Balapriya , P.S. Arul Raja Murugan ,.
The Institute of Microbiology and The Institute of Rheumatology, Madras Medical College and Rajiv Gandhi Government General Hospital, Affiliated to
The Tamil Nadu Dr.M.G.R. Medical University, Chennai.
A R T I C L E I N F O A B S T R A C T
Keywords:
Indirect Immuno-fluorescence
Anti-nuclear antibody
ANA ELISA
Speckled
Homogenous
Anti-cytoplasmic
Anti-mitochondrial.
Original Article
Autoimmune connective tissue diseases are group of disorders in which autoantibodies are formed
against self-antigen present in the nucleus and other components of the cell. The most common
methods to detect antinuclear antibodies are ELISA and indirect Immunofluorescence. This study
aimstoevaluatethevalidityofANAELISAmethodascomparedtoIndirectimmunofluorescence(IF)
assay which is considered as the gold standard test. This is a descriptive study conducted in the
Clinical Immunology Laboratory, Institute of Rheumatology, Madras Medical College Chennai,
during the period of October 2021 to December 2021 with 110 samples. ANA ELISA and Indirect
Immunofluorescence were done as per the instructions provided by manufacturer of kit. Out of the
110 samples 75 cases were positive, in this 29 were positive by Immunofluorescence alone, 43
cases were positive by both and 3 cases were positive by ELISA alone. The Pearson Correlation
coefficient value showed positive correlation between the grading of intensity of the positive
immunofluorescence pattern and optical density of antinuclear antibodies by ELISA. So both the
methods can be used but in centers with appropriate facilities Immunofluorescence proves to be the
goldstandardinthediagnosis.
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1. Introduction
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ISSN: 0976:6685.
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Connective tissue diseases (CTD) are class of autoimmune
disorders identified by the presence of antinuclear autoantibodies
(ANA) in the blood.1 CTD include rheumatoid arthritis, systemic
lupus erythematosus, systemic sclerosis, polymyositis,
dermatomyositis, primary sjogren's syndrome, primary anti-
phospholipid syndrome, undifferentiated connective tissue disease
andmixedconnectivetissuedisease.1ThetermANAisamisnomeras
these antibodies are directed against almost all components of the
cell.2 Detection of ANA is crucial in the diagnosis of SLE according to
the European League against Rheumatology (EULAR) / the American
CollegeofRheumatology(ACR)criteria.3
ANA can be detected by two principal methods namely ANA
indirect immunofluorescence (ANA-IIF) and ANA ELISA.4 Since
1980sANAELISAiswidelyusedasascreeningtestandquantitatively
identifies the specific auto-antibodies against SSA/Ro, SSB/La, Sm,
Sm/RNP, Scl-70/ topoisomerase I, Jo-1, centromere and histones.5
Indirect immunofluorescence using human epidermoid laryngeal
carcinomacells(Hep-2)andHep2000transfectedwithRoantigenas
substrate is a semi-quantitative test.5 IIF test enables to detect
antibodies to more than 30 different nuclear and 50 cytoplasmic
a antigens.6 This is still a gold standard test for ANA antibody
detection but requires skill for interpretation.6 But in some
instances it can be misinterpreted because the antibodies cross
react.AlsoANAcanbepresentin3%ofnormalpopulation.7
ELISA is highly specific and better sensitive with a lesser
turnover time compared to IIF.8Also large number of samples can
be tested hence a best test for screening of patients.8 This study
aims to evaluate the diagnostic accuracy of ELISA in comparison to
ANAImmunofluorescence.
AimsandObjectives:
To detect Anti-nuclear antibodies by Indirect
ImmunofluorescenceandANAELISA
To correlate Anti-nuclear antibody positivity by Indirect
ImmunofluorescenceandANAELISA
MaterialsandMethods
This is a descriptive study conducted in the Clinical
Immunology laboratory, Institute of Rheumatology, Madras
Medical College Chennai, during the period of October 2021 to
December 2021 with 110 samples. Samples were selected by
simple random sampling method including all cases of all age
group attending Rheumatology O.P.D with symptom suggestive of
ANArelatedIllness.
* Corresponding Author : Dr.N.Sudha
The Institute of Microbiology and The Institute of Rheumatology,
Madras Medical College and Rajiv Gandhi Government General Hospital, Chennai
drnsudhamurugan@gmail.com
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2. N.Sudha et al. Int J Biol Med Res. 2025; 15(1): 7746-7749
7747
Detection of ANA by Indirect Immunofluorescence was done as
per the instructions provided by manufacturer of kit with dilution
of 1:80. The diluted sera were pipetted onto respective ANA wells
provided in kit along with the positive and negative controls. The
serum containing anti-nuclear antibodies would bind with the
corresponding antigens present on the slides. The slides were then
incubated for 30 minutes and were then rinsed with phosphate
buffer. Fluorescein labeled antihuman globulin provided in test kit
was added, incubated at moist chamber for 30 minutes. This was
then rinsed and mounted with mounting medium .The slides were
examined under the fluorescent microscope for staining pattern
andintensity.Thepatternswiththeirintensitywerenoted
With the remaining sera ANA ELISA was performed as per
manufacturer's kit instructions. The serum samples along with
positive and negative controls were diluted with freshly prepared
sample diluent in their appropriate wells along with template. This
was incubated at room temperature for 20 minutes and wells were
washed three times with wash buffer. The enzyme conjugate was
added to each well and incubated at room temperature. Then the
wells were washed three times wash buffer. The TMB substrate was
added and incubated at room temperature. To this stop solution
was added. The readings were noted using ELISA reader. Values
between 0.9-1.1 were borderline positive and those more than 1.1
were Positive. The results of ANA IF values and ANA ELISA values
were entered in EXCEL. This was analyzed in SPSS software using
correlationanalysis.
Results:
Thestudyconsistedof110casesofwhich18ofthemweremales
and 92 were female. There was a female predominance of 85% as
showninchart:1,chart:2andtable:1.
Withthe combinationofthetwotest75caseswerepositivebyboth
methodsasshowninChart:3
Out of the total 75 positive cases 29 were positive by
Immunofluorescence alone, 43 cases were positive by both and 3
caseswerepositivebyELISAalone
Chart 4: Out of the total 75 positive cases 29 were positive by
Immunofluorescence alone , 43 cases were positive by both and 3
caseswerepositivebyELISAalone.
The Positive Predictive Value for Immunofluorscence is 96% and
PositivePredictiveValueforANAELISA=61%
Majority of the study population belonged to the age group of
21to50yearsasshowninthechart2.
Chart: 1
Table 1 MALE / FEMALE
Table 1 MALE / FEMALE

3. 7748
A correlation analysis was done between the intensity
grading of positive immunofluorescence pattern and optical
Discussion:
An appropriate reporting of ANA antibodies is essential for the
diagnosis of connective tissue disorder. The ELISA method of ANA
detection is a simple technique to perform and can be used in
situation where highly skilled professionals are not available to
recognize microscopic patterns. Hence ELISA is most widely used
methodforroutinescreening4ofANAantibodies.
In our study we included 110 cases with symptoms suggestive
of connective tissue disorder of all age groups and both gender on a
randombasis.Itwasfoundthatthemajorityofthestudypopulation
were females with 85 %(92) and Males were15%(18) as shown in
chart 1.The age group most commonly presenting with symptoms
belongedtotheagegroupof21to50yearsasshowninthechart2.
Out of the total 75 positive cases 29 were positive by
Immunofluorescence alone, 43 cases were positive by both and 3
cases were positive by ELISA alone. Naveen A Kamel et al in their
study included 73 patients and their result showed that ELISA had
higher sensitivity (86.8%) in SLE patients than IIF (84.2%). But in
this study the Positive Predictive Value for Immunofluorescence is
96% and that for ANA ELISA is 61% whereas in the study by Ashish
Tayde et al positive predictive value of ELISA was 72% and by
Immunofluorescencewas98.18%.
The indirect immunofluorescence patterns were observed with
following frequency: out of the 72 positive samples ,35 showed
Speckled pattern (49%),16 were of Homogenous (23%) pattern ,7
showed SSA (10%), 6 cases exhibited Anti-cytoplasmic (8%), 5
wereAnti-mitochondrial(6%),and3wereCentromere (4%).
The Pearson Correlation coefficient value showed positive
correlation between the grading of intensity of the positive
immunofluorescencepatternandopticaldensityofANAbyELISA.
Chart 5
Conclusion
Screening for ANA is essential in early diagnosis of patients
presentingwithsymptomsandsignsofconnectivetissuedisorders.
The commonly used screening techniques either ELISA or indirect
Immunofluorescence are valuable tools. In resource limited places
ELISA technique can be useful in the detection of ANA but in centers
with appropriate facilities Immunofluorescence proves to be the
gold standard in early diagnosis and initiation of treatment to
preventcomplications.
References
[1] Neveen A. Kamel, Ashraf A. Hassaballa, Yomna M. Hasan. Detection of
antinuclear antibody in autoimmune connective tissue diseases: a
comparison between immunofluorescence and solid-phase assay.Al-
AzharAssiutMedicalJournal2018,16:223–228.
[2] Udhayasankar Ranganathan, Gopal Rangasamy1,Mangaiyarkarasi
Thiyagarajan1, Sunil Shivekar Antinuclear antibody patterns by indirect
immunofluorescence test: An experience from a rural tertiary health
centre of Puducherry, South India. Indian Journal of Microbiology
Research2021;8(3):260–262261.
[3] Mohammad J.Khalifah,Omar Almansouri,Syed Sameer Aga . Ammar A.
Aljefri ,Abdulaziz Almalki, Naser Alhmdan ,Wael Al-Mazain , Khalid
A l s a l m i , Ab d u l fa t t a h A l a m r i . C o m p a r i s o n o f I n d i re c t
Immunofluorescence and Enzyme Immunoassay for the Detection of
AntinuclearAntibodies.November03,2022.
[4] O m a r S u h a i l A l s a e d , L a i t h I s h a q A l a m l i h , O m a r
Al-Radideh,Prem.Chandra, Samar Alemadi & Abdul-Wahab
Al-Allaf.Clinical utility of ANA-ELISA vs ANA immunofluorescence in
connectivetissuediseases.ScientificReports|(2021)11:8229.
[5] Olsen, N.J., Choi, M.Y and Fritzler, M.J. Emerging technologies in
autoantibody testing for rheumatic diseases. Arthritis Res Therapy19,
172(2017).
[6] Sumanth Kumar GLS, Chaudhury A, Verma A, Kalawat U, Ramana BV,
Siddhartha Kumar B. Comparison of enzyme linked immunosorbant
assaay (ELISA) with indirect immunofluorescence for detection of anti-
n u c l e a r a n t i b o d y. J C l i n S c i R e s 2 0 1 4 ; 3 : 2 3 7 - 4 2 .
DOI:http://dx.doi.org/10.15380/2277-5706.JCSR.14.056.
[7] Kumar Y, Bhatia A, Minz RW. Antinuclear antibodies and their detection
methods in diagnosis of connective tissue diseases: a journey revisited.
Diagn Pathol. 2009 Jan 2;4:1. doi: 10.1186/1746-1596-4-1. PMID:
19121207;PMCID:PMC2628865.
[8] Anupriya Asaithambi, Manjula Gunasekaran, Manivelan S, Palaniappan
Nainar. Antinuclear antibodies in patients with unexplained recurrent
abortions .Asian Journal Pharmaceutical Clinical Research,10 May 2017,
Vol10,Issue8,2017,256-259.
[9] Tesija Kuna A, Đerek L, Drvar V, Kozmar A, Gugo K. Assessment of
antinuclear antibodies (ANA): National recommendations on behalf of
the Croatian society of medical biochemistry and laboratory medicine.
Biochem Med (Zagreb). 2021 Jun 15;31(2):020502. DOI:
10.11613/BM.2021.020502. Epub 2021 Apr 15. Erratum in: Biochem
Med (Zagreb). 2022 Feb 15; 32(1):011201. PMID: 33927550; PMCID:
PMC8047791.
[10] Jacinth Angel, Marina Thomas, Boppe Appalaraju. Evaluation of ELISA
and indirect immunofluorescence in the diagnosis of autoimmune
diseases and their interpretation in the clinical situation.16-Jun-2015.
Volume:17 Issue:1Page:7-11.
[11] Velammal Petchiappan, Adityan Guhan, Sumitra Selvam, V. N. Nagaprabu-
Comparison of immunofluorescence assay with ELISA in detection of
antinuclear antibodies -International Journal of Research in Medical
Sciences-2018Sep;6(9):3140-3146-www.msjonline.org
[12] Yashwant Kumar, Alka Bhatia, and Ranjana Walker Minz Antinuclear
antibodies and their detection methods in diagnosis of connective tissue
diseases: a journey revisited Diagnostic Pathology January 2009 vol. 4 1.
2Jan,DOI:10.1186/1746-1596-41.
N.Sudha et al. Int J Biol Med Res. 2025; 15(1): 7746-7749

4. [13] Ling M, Murali M. Antinuclear Antibody Tests. Clin Lab Med. 2019 Dec;
39(4):513-524. DOI: 10.1016/j.cll.2019.07.001. Epub 2019 Oct 4. PMID:
31668266.
[14] Farha A. El-Chennawi, Youssef M. Mosaad, Hesham M. Habib, and Tamer
El-Degheidi Comparative Study of Antinuclear Antibody Detection by
Indirect Immunofluorescence and Enzyme Immunoassay in Lupus
Patients 2009 -A Journal of Molecular and Cellular Immunology, Volume
38,2009-Issue8.
[15] Susan S. Copple, MS, MT(ASCP) SI, Allen D. Sawitzke, MD, Andrew M.
Wilson, MStat, Anne E. Tebo, PhD, Harry R. Hill, MD Enzyme-Linked
Immunosorbent Assay Screening Then Indirect Immunofluorescence
Confirmation of Antinuclear Antibodies: A Statistical Analysis-American
JournalofClinicalPathology,Volume135,Issue5,January2011.
[16] Ashish Tayde, Chetna Agrawal, A. T. Deshmukh “Comparison of
immunofluorescence assay (IF) with ELISA in detection of antinuclear
antibodies” Indian Journal of Pathology and Oncology, July-September,
2018;5(3):418-420-DOI:10.18231/2394-6792.2018.008.
Copyright 2023 BioMedSciDirect Publications IJBMR -
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