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 Table of Contents  
SHORT ARTICLE
Year : 2021  |  Volume : 6  |  Issue : 2  |  Page : 224-227

Comparison of indigenous enzyme-linked immunosorbent assay and chemiluminescent immunoassays for screening for immunoglobulin G antibodies against SARS-CoV-2 in India


1 Department of Transfusion Medicine, S. N. Medical College, Agra, Uttar Pradesh, India
2 Department of Medicine, S. N. Medical College, Agra, Uttar Pradesh, India
3 Department of Transfusion Medicine, Super Speciality Paediatric Hospital and Post Graduate Teaching Institute, Noida, Uttar Pradesh, India
4 Department of Microbiology, S. N. Medical College, Agra, Uttar Pradesh, India

Date of Submission04-Jul-2021
Date of Decision08-Oct-2021
Date of Acceptance08-Oct-2021
Date of Web Publication30-Nov-2021

Correspondence Address:
Dr. Satyam Arora
Department of Transfusion Medicine, Super Speciality Paediatric Hospital and Post Graduate Teaching Institute, Noida
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/gjtm.gjtm_73_21

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  Abstract 


Serological screening for antibody against SARS-CoV-2 has been gaining more attention in view of selection for an appropriate donor for COVID-19 convalescent plasma, documenting the past/ recent infection due to the virus as well as now for evaluating the immune response to the vaccination. In beginning of the pandemic an indigenous ELISA kits were developed in India to screen for IgG type of antibody against the whole-cell virus particle. Our retrospective analysis provides a head-to-head comparison of an indigenous ELISA kits (COVID KAWACH IgG MICROLISA) and CLIA (SARS-CoV-2 IgG by Abbott) platform for screening of IgG type of antibody. Total 380 samples from adults were screened on both the platforms, out of which 250 had a history of a positive RT-PCR report and 130 were RT-PCR negative but with a history of symptoms suggestive of COVID-19 or exposure to COVID-19 individual. The overall concordance of detection IgG type of antibody by both the platforms was 52.2% (higher in individuals with known RT-PCR positivity; 70%). The median S/CO ratio (on CLIA) was 3.34 in concordant positive samples and 2.48 in cases of CLIA yield samples. Our study highlights the variation in detection of the antibody by both the platforms due to inherent difference in the design of these platforms and kits.

Keywords: Coronavirus disease 2019 pandemic, coronavirus disease 2019 serology, immunoglobulin G antibody


How to cite this article:
Chauhan N, Chahar AS, Dua S, Mohan Y, Arora S, Agrawal A, Singh P, Kanaujia G. Comparison of indigenous enzyme-linked immunosorbent assay and chemiluminescent immunoassays for screening for immunoglobulin G antibodies against SARS-CoV-2 in India. Glob J Transfus Med 2021;6:224-7

How to cite this URL:
Chauhan N, Chahar AS, Dua S, Mohan Y, Arora S, Agrawal A, Singh P, Kanaujia G. Comparison of indigenous enzyme-linked immunosorbent assay and chemiluminescent immunoassays for screening for immunoglobulin G antibodies against SARS-CoV-2 in India. Glob J Transfus Med [serial online] 2021 [cited 2022 Aug 10];6:224-7. Available from: https://www.gjtmonline.com/text.asp?2021/6/2/224/331620




  Introduction Top


The World Health Organization declared coronavirus disease 2019 (COVID-19) as a public health emergency in January 2020 and since then SARS-CoV-2 virus have spread all across the globe. Majority of diseases caused by the virus are milder whereas with multiple mutations the virus is becoming more aggressive in terms of transmissibility and severity of presentations of the disease. Currently, viral RNA detection by polymerase chain reaction (PCR) is one of the gold standards tests to confirm the diagnosis of COVID-19. Apart from molecular detection of the virus, serological tests for the immune response generated due to the infection or vaccination are also gaining interest.

Serological screening tests have many roles to play during the different phases of the pandemic. From public health perspective, serological tests may be used to conduct seroprevalence and seroepidemiological studies to understand the extent of the spread of the virus. These tests may also be used as a surrogate marker of the infection with COVID-19 (in cases of reverse transcription–PCR [RT-PCR] negative). Screening for the type of antibody, i.e. immunoglobulin G (IgG) and immunoglobulin M (IgM), also helps in differentiating between acute or chronic infections. These tests have also been used to screen donors for convalescent plasma donation postrecovery from COVID-19. Finally, these tests are also used to screen for immunity from the virus (after natural infection or vaccination) and the risk of re-infection.

During the initial phases of the pandemic, there was nonavailability of an indigenous, approved and cost-effective kit for serological testing; hence, an in-house enzyme-linked immunosorbent assay (ELISA) was developed and validated in India.[1] Gradually, many ELISA and chemiluminescent immunoassays (CLIA) based kits were validated and approved for use as antibody screening kits. This study describes a retrospective experience of a serological screening for COVID-19 and comparison of the in-house prepared ELISA and US Food and Drug Administration (FDA) approved CLIA platforms for COVID-19 antibody testing.


  Material and Methods Top


This is a retrospective analysis of the experience of COVID-19 antibody screening from two tertiary care institutes in Northern India in the state of Uttar Pradesh. The COVID-19 antibody screening service was provided by the department of transfusion medicine at both institutes from July to December 2020. The primary aim for screening/testing for COVID-19 antibodies (IgG type) against N-antigen by CLIA or IgG antibody against any component of viral antigen.

Sample cohort

The screening service was primarily initiated to search for eligible plasma donors with high levels of antibodies (ICMR protocols[2]). The samples were screened from both known RT PCR positive COVID-19 recovered individuals as well as individuals who had a history of contact with a known COVID-19 person or had symptoms suggestive of COVID-19 but tested negative on RT PCR. Each prospective sample (whole blood, EDTA) was screened for IgG type of COVID-19 antibody using two different platforms (both approved by ICMR). Basic history of symptoms/contact or RT PCR positivity was taken.

Enzyme-linked immunosorbent assay platform

IgG antibody for COVID-19 was detected using microtiter plate with whole-cell antigen coating, an in-house validated kit by NIV ICMR.[1](COVID KAWACH IgG MICROLISA) An OD >cutoff value and P/N ratio (positive/negative control ratio) more than 1.5 was considered as positive for IgG antibody.

Chemiluminescent immunoassays platform

SARS-CoV-2 IgG assay using Abbott Architect equipment was used (Abbott Diagnostics Division, Sligo, Ireland). This assay detects IgG type of antibodies against N-antigen of the virus using a two-step chemiluminescent microparticle immunoassay method with n acridinium-labeled anti-human IgG. The assay was used as per the manufacturer's instructions. A signal to cut-off ratio of more than or equal to 1.4 was considered as positive.

Statistical analysis

The concordance of both platforms of antibody detection was analyzed. The signal-to-cut-off ratio of the samples was also analyzed and compared. The results were compared between samples from an individual with known RT-PCR positivity and those without it. Analysis was performed using the Microsoft Excel platform.


  Results Top


Baseline characteristics of the cohort

This retrospective analysis studied the results of 385 individuals screened for the presence of antibody (IgG) for COVID-19. Out of 385 individuals, 5 were pediatric (<18 years) samples and the rest 380 were adult samples. The pediatrics samples were 3 males and 2 females with mean age 10.4 ± 6.1 years (median 12 years). Out of the total 380 adult samples, 315 were males and 65 females with mean age 43 ± 21.2 years (median 43 years) [Table 1].
Table 1: Demographic profile of the adult individuals screened for coronavirus disease 2019 IgG antibody

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Concordance of detection of antibody on both the platform

The IgG antibody was screened on both the platforms (in parallel) and there was overall 52.2% concordance in the detection of antibody. The concordance rate was higher in known RT PCR individuals (70%) as compared to the samples from symptomatic/close contacts of COVID-19, but RT-PCR Negative individuals (20%) [Table 2].
Table 2: Concordance of detection of IgG antibody on both enzyme-linked immunosorbent assay and chemiluminescent immunoassay

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Signal-to-Cutoff ratio (S/CO)

Signal-to-cutoff ratio (S/CO) from CLIA is very commonly used to screen high antibody levels plasma donors. The mean S/CO ratio of concomitant positive (ELISA [+] CLIA [+]) samples was 4.3 ± 2.8 (median 3.79) as compared to concomitant negative (ELISA [-] CLIA [-]) samples 0.45 ± 0.41 (median 0.4). The mean S/CO ratio of ELISA yield (ELISA [+] CLIA [-]) samples was 0.7 ± 0.42 (median 0.8) as compared to CLIA yield (ELISA [-] CLIA [+)]) samples 3.2 ± 1.7 (median 2.67) [Figure 1].
Figure 1: Comparison of Signal-to-cutoff ratio (S/CO) on chemiluminescent immunoassays for the detection of antibody

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Days from reverse transcription-polymerase chain reaction positivity

The mean days from RT-PCR positivity and antibody detection were maximum in case of ELISA yield samples 70.8 ± 38.7 days (median 59 days) followed by concomitant negative samples 64.3 ± 34.4 days (median 60 days) then CLIA yield samples 57.3 ± 26.9 (median 50) and concomitant positive samples were 55.2 ± 25.8 days (median 52 days).


  Discussion Top


This study was a retrospective experience of screening for IgG type of antibodies for SARS-CoV-2 at two tertiary care centers in northern India. The study represents a head-to-head comparison of two platforms (ELISA vs. CLIA) for the detection of the IgG type of antibody. ELISA platform is an indigenous platform developed in India and it is compared with an FDA-approved CLIA platform. Out of 380 adults samples analyzed, 250 had a history of RT-PCR positive report whereas the rest 130 were with a history of negative RT-PCR report (with either with history of contact with a known COVID-19 patient or with a history of symptoms suggestive of COVID-19).

The concordance of detection (by both the platforms) of IgG type of antibody was around 52.2% in overall samples (n = 380), whereas it was as high as 70% in individuals with a history of positive RT PCR (n = 250). While Antibody was detected with one or another platform in 23.6%, the remaining 6.4% did not develop detectable antibodies by any of the platforms.

One of the prospective longitudinal serological cohort studies on 3840 COVID-19 recovered individuals from India (using the same platforms: indigenous ELISA and CLIA by Abbott) showed that there was a variation in seroconversion based on the severity of the infection and there was an overall seroconversion of 85.3% at 6–10 weeks of the infection.[3] Seroconversion was 100% in severe cases, 89.6% in mild-to-moderate cases as compared to asymptomatic individuals (77.3%). Overall concordance on both the platform was higher (77.8% [523 out of 672]) at 6–10 weeks of recovery when compared to 7 days (30.1% [202 out of 672]) and 10–28 days (74.9% [503 out of 672]).

Previous studies[4] have also shown that as high as 50% of the recovered individuals do not produce enough neutralizing antibodies of titer of 1:20. Another study from India has shown that individuals with lower neutralizing antibody responses have lower levels of memory B-cells.[5] The reason for variable response may be due to inter-individual differences in human immune response, heterogenicity in the initial viral inoculum,[6] initial viral load, incubation period, and disease severity.[7] In our data, there was a concordance of 70% by both platforms indicating that about 30% of individuals did not produce enough antibodies to be detected by both the platforms.

The use of whole-cell antigen instead of recombinant nucleocapsid antigen or spike protein antigen provides a broader sensitivity[1] to the indigenous ELISA platform when compared to the CLIA platform (Abbott) which is highly sensitive for N-antigen of SARS-CoV-2 virus. ELISA yield as high as 20.4% of individuals with RT-PCR-positive reports indicates detection of antibody other than N-antigen of the virus. One of the reasons may be the days from the RT-PCR positivity as with more days the level of antibodies to N-antigen tends to reduce. The development of a whole-cell antigen-based ELISA platform is a cost-effective and easy-to-use tool for screening for antibodies/seroprevalence studies at a resource-limited setting.

S/CO ratio of IgG against Nucleocapsid protein is frequently being used to provide an indication of the level of neutralizing antibodies in the sample. As per recent FDA recommendation COVID-19, convalescent plasma can be considered high titer if the S/CO ratio is more than or equal to 4.5. The median S/CO ratio was 3.34 in cases of concordant positive and 2.48 in cases of CLIA yield samples.[8] Several researchers have studied IgG antibody of nucleocapsid and neutralizing antibodies and equated approximate S/CO of IgG and concentration of neutralizing antibodies.

The presence of IgG type of antibodies in non-RT-PCR positive, concordant positivity of 20%, indicate the presence of infection even in the absence of confirmation by RT PCR for COVID-19. RT PCR is considered a gold standard method for diagnosis and detection of the SARS-CoV-2 virus but a significant amount of false negativity is also being reported recently.[9] Serological tests offer additional test to confirm the history of infection with COVID-19.

Limitation

One of the main limitations of the retrospective analysis was the unavailability of data on disease severity as well as the variable time of infection for the samples. In this study, there was only availability of days from positive RT-PCR. One more limitation was the comparison of platforms which detects two different analytes, i.e. the type of antigen detected (whole virus vs. N-antigen) this may result in the high detection of antibodies by ELISA, which is screening many types of antigens when compared to CLIA. Another limitation was the inability to compare our results based on neutralizing antibody assays which are the gold standard for the detection of antibodies against SARS-CoV-2.


  Conclusion Top


The performance of any assay to detect the antibody depends on many variables such as the principle of the platform (ELISA vs. CLIA vs. LF), type of antibody detected (IgG only, IgM only, and total antibody), target analyte/antigen (N, S1/S2, RBD), the timing of the sample, severity of the disease and individual genetic factors. As per our observation, the concordance for the detection of IgG type of antibody detection by ELISA (COVID KAWACH IgG MICROLISA) and CLIA (SARS-CoV-2 IgG by Abbott) is 70% in individuals with a history of RT-PCR positivity. There is variation in detection of the antibody by both the platforms due to inherent differences in the design of these platforms and kits hence results should be interpreted keeping in view the type of platform used.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Sapkal G, Shete-Aich A, Jain R, Yadav PD, Sarkale P, Lakra R, et al. Development of indigenous IgG ELISA for the detection of anti-SARS-CoV-2 IgG. Indian J Med Res 2020;151:444-9.  Back to cited text no. 1
[PUBMED]  [Full text]  
2.
Agarwal A, Mukherjee A, Kumar G, Chatterjee P, Bhatnagar T, Malhotra P, et al. Convalescent plasma in the management of moderate COVID-19 in adults in India: Open label phase II multicentre randomised controlled trial (PLACID Trial). BMJ 2020;371:m3939.  Back to cited text no. 2
    
3.
Thiruvengadam R, Chattopadhyay S, Mehdi F, Desiraju BK, Chaudhuri S, Singh S, et al. Longitudinal Serology of SARS-CoV-2-Infected Individuals in India: A Prospective Cohort Study. Am J Trop Med Hyg. 2021 May 18;105(1):66–72. doi: 10.4269/ajtmh.21-0164. Epub ahead of print. PMID: 34003792; PMCID: PMC8274753.  Back to cited text no. 3
    
4.
Kalkan Yazıcı M, Koç MM, Çetin NS, Karaaslan E, Okay G, Durdu B, et al. Discordance between serum neutralizing antibody titers and the recovery from COVID-19. J Immunol 2020;205:2719-25.  Back to cited text no. 4
    
5.
Nayak K, Gottimukkala K, Kumar S, Reddy ES, Edara VV, Kauffman R, et al. Characterization of neutralizing versus binding antibodies and memory B cells in COVID-19 recovered individuals from India. Virology 2021;558:13-21.  Back to cited text no. 5
    
6.
Welten SP, Redeker A, Toes RE, Arens R. Viral persistence induces antibody inflation without altering antibody avidity. J Virol 2016;90:4402-11.  Back to cited text no. 6
    
7.
Legros V, Denolly S, Vogrig M, Boson B, Siret E, Rigaill J, et al. A longitudinal study of SARS-CoV-2-infected patients reveals a high correlation between neutralizing antibodies and COVID-19 severity. Cell Mol Immunol 2021;18:318-27.  Back to cited text no. 7
    
8.
Toolkit Updated 04/14/21 COVID-19 Convalescent Plasma (CCP) Under Emergency Use Authorization. Available from: https://www.aabb.org/docs/default-source/default-document-library/regulatory/toolkit-for-ccp-under-eua.pdf?sfvrsn=741be857_18. [Last accessed on 2021 Jun 21].  Back to cited text no. 8
    
9.
Jia X, Xiao L, Liu Y. False negative RT-PCR and false positive antibody tests-Concern and solutions in the diagnosis of COVID-19. J Infect 2021;82:414-51.  Back to cited text no. 9
    


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