key: cord-1040605-shwaueev
authors: Steiner, Andrea; Pletz, Mathias; Löffler, Bettina; Baier, Michael
title: A novel SARS-CoV-2 IgG line-blot for evaluating discrepant IgG test results – observations in pre-pandemic and follow-up samples of five patients
date: 2021-03-31
journal: J Microbiol Immunol Infect
DOI: 10.1016/j.jmii.2021.03.004
sha: be7af77090e34e7289811f992ad1d7fade1c248f
doc_id: 1040605
cord_uid: shwaueev

To confirm discrepant SARS-CoV-2-IgG results in four standard assays we applied for the first time a prototype of a coronavirus IgG-line-blot which employs antigens from seasonal coronaviruses, SARS-1 and SARS-CoV-2 combined with avidity testing as a confirmatory tool in a follow-up of five cases including pre-pandemic samples.

Since the beginning of the COVID-19 pandemic a large number newly developed PCR assays detect viral RNA in respiratory samples with high accuracy while several formats of serological assays are available to find SARS-CoV-2 specific antibodies in blood samples with different reliability. [1] [2] [3] Whereas PCR-tests provide a snapshot on the infectiousness, serological tests may predict previous infection and perhaps a certain degree of immune protection in an individual. 4 The latter are also important to reflect and monitor the infection rate in a population and thereby serving as a tool to develop prevention strategies and to implement hygiene measures.

Here we present observations (i) that, depending on the serological assay used, specific SARS-CoV-2 antibodies drop below cut off levels in less than 9 weeks after PCR proven infection, (ii) that false positive results may pretend immune protection in individuals without PCR proven exposure and (iii) that these limitations can be addressed with a SARS-CoV-2 specific line blot to facilitate final and consistent diagnosis.

To prevent hospital outbreaks at the Jena University Hospital with 1.300 beds and 3.500 health care workers (HCW) employed, strict protective measures were introduced, e.g. PCR screening of nasopharyngeal swabs was performed in all HCW returning from risk areas and with occupational or private contacts with confirmed SARS-CoV-2 cases. Members of staff tested positive and with COVID19-typical symptoms were enrolled in a follow up program to monitor viral secretion and specific antibody dynamics. 5 PCR was performed using QIAsymphony DSP Virus/Pathogen Mini Kit (Qiagen, Hilden, Germany) for extraction and LightMix Modular SARS and Wuhan CoV E-gene Germany (T4) and recomLine Coronavirus IgG / avidity (RUO), Mikrogen, Neuwied, Germany (T5)).

The fifth test (T5) is a prototype of a newly developed commercial line blot format containing recombinant nucleocapsid antigen fragments from all four seasonal coronaviruses (CoV), SARS-1 and SARS-CoV-2. Pre-pandemic blood samples from enrolled HCW were included. against seasonal coronaviruses were present. In SARS-CoV-2 positive samples from cases with prepandemic seasonal CoV signals, the corresponding 229E, NL63, OC43 or HKU1-antigens were always co-reacting. Parallel reactivity was more prominent for beta-CoV (OC43, HKU1, SARS-1) than for alpha-CoV (229E, NL63). SARS-1-signals accompany SARS-CoV-2 in all cases, albeit in reduced intensity, but both were never seen in pre pandemic specimen.

We followed up five cases after suspected COVID19-infection with five different SARS-CoV-2 IgG assays including a recently developed line blot prototype for the first time. In all four cases with PCR proven infection we observe IgG sero-conversion in at least three out of four tests at day 20. One case with no positive PCR results turned out to remain seronegative.

With the applied standard tests we note a number of inconsistencies: While early loss of IgG is evident with the ELISA test at day 56 and 58 (T1, case B and C) other assay formats produce delayed (T4, case A), false negative (T3, case B) or false positive results (T2, pre-pandemic samples case B). 3, 7 The capture antigen involved seems to play a role, but other factors may contribute as well, since assay T3, containing both nucleocapsid and spike antigen (the first considered to be more sensitive than the latter) was unable to identify specific antibodies in all samples in case B and one in case C. 1 Avidity testing ad on allows specifying early and late infection phase. This follow up, however, was too short to reach the state of full avidity maturation, but this marker could be a valuable tool in future.

Is there a place for line blots in routine serology diagnostic for SARS-CoV-2? The introduction of a two-step test strategy for e.g. HIV, lyme disease and syphilis using line or westernblot blot systems has largely improved sensitivity and specificity of antibody detection. 9 Now, a similar approach may help to overcome at least in a subset of not conclusive cases some limitation of the available test formats, which do not allow simultaneously assessment of reactivities against other CoV.

In this follow up with PCR proven SARS-CoV-2 infection over nine weeks we observed inconsistencies in SARS-CoV-2 IgG dynamics when different test assays were deployed. Our data demonstrate that a coronavirus line blot format containing specific antigens from different CoV and recruited after a first screening step as a confirmatory tool can help to increase specificity, to distinguish infection with other e.g. seasonal CoV and to define the phase of infection, as IgG avidity can be measured. 

Sensitivity in Detection of Antibodies to Nucleocapsid and Spike Proteins of Severe Acute Respiratory Syndrome Coronavirus 2 in Patients With Coronavirus Disease

Severe Acute Respiratory Syndrome Coronavirus 2-Specific Antibody Responses in Coronavirus Disease

Performance Characteristics of Four High-Throughput Immunoassays for Detection of IgG Antibodies against SARS-CoV-2

Review of Current Advances in Serologic Testing for COVID-19

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Assessment of immune response to SARS-CoV-2 with fully automated MAGLUMI 2019-nCoV IgG and IgM chemiluminescence immunoassays

Kinetics of SARS-CoV-2 specific IgM and IgG responses in COVID-19 patients

The dynamics of humoral immune responses following SARS-CoV-2 infection and the potential for reinfection

Diagnosis, Treatment, and Prevention of Lyme Disease, Human Granulocytic Anaplasmosis, and Babesiosis: A Review

We thank Katrin Dobschal, Kerstin Drexler and Claudia Helgert for performing the line blot assays.J o u r n a l P r e -p r o o f