EUROIMMUN EI 2606-9601 G Government Scientific Source Instruction Manual

Updates with respect to the previous version are marked in grey.
Anti-SARS-CoV-2 ELISA (IgG)
Instructions for use

For in vitro diagnostic use

ORDER NO.| ANTIBODIES AGAINST| IG CLASS| SUBSTRATE| FORMAT
---|---|---|---|---
EI 2606-9601 G| SARS-coronavirus-2
(SARS-CoV-2)| IgG| Ag-coated microplate wells| 96 x 01 (96)

Intended use

The enzyme immunoassay (ELISA) provides semiquantitative in vitro determination of human antibodies  of the immunoglobulin class IgG against SARS-CoV-2 in serum, EDTA, heparin or citrate plasma or dried blood spots (DBS) to support the diagnosis of  SARS-CoV-2 infection and constitutes a supplement to the direct pathogen detection. Moreover, serology can be applied to collect epidemiological data. The product is designed for the use by healthcare professionals and can be processed and evaluated manually  or on automated instruments. The results should always be interpreted together with those of further laboratory diagnostic procedures and based on the clinical picture.

Clinical significance
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously called 2019-nCoV) belongs to the family of coronaviruses and, like SARS-CoV, is classified in the genus Betacoronavirus [1]. At the end of 2019, SARS-CoV-2 was identified as the  causative pathogen of clustered cases of pneumonia of unclear origin. The virus caused an infection wave that has spread rapidly worldwide and was declared a pandemic by the WHO at the beginning of 2020 [2-5].
SARS-CoV-2 is predominantly transmitted by droplet infection via coughing or sneezing and through close contact with infected persons [3, 4, 6]. Health care personnel and family members are especially at risk [6]. The zoonotic reservoir of the virus appears to  be bats [3, 4, 6].
The incubation time of SARS-CoV is three to seven, maximally 14 days [2]. The symptoms of SARS-CoV-2 infection are fever, coughing, breathing difficulties, fatigue and loss of the olfactory and taste sense [2-4, 6, 7]. In most patients the infection manifests with  symptoms of a mild febrile illness with irregular lung infiltrates. Some patients, especially elderly or chronically ill patients, develop acute respiratory distress syndrome (ARDS) [2, 3, 5, 6]. In February 2020, the disease caused by SARS-CoV-2 was named  COVID-19 by the WHO.
Suitable methods for the diagnosis of SARS-CoV-2 infections are the detection of viral RNA by reverse transcriptase polymerase chain reaction (RT-PCR) or of virus protein by means of ELISA or rapid test primarily in sample material from the upper  (nasopharyngeal or oropharyngeal swab) or lower respiratory tract (bronchoalveolar lavage fluid, tracheal secretion, sputum, etc.) [4, 5]. Detection of viral antigens is less sensitive than RT-PCR testing.
The determination of antibodies enables confirmation of SARS-CoV-2 infections in patients with typical symptoms and in suspected cases. It also contributes to monitoring and outbreak control [4, 5]. The spike (S) and nucleocapsid (N) proteins of SARS-CoV-2  are highly immunogenic. More than 90% of the neutralising antibodies in COVID-19 patients are directed against the receptor-binding domain (RBD) of the spike protein. The spike protein is the target protein of almost all vaccines against COVID-19 [8].

Around 90% of SARS-CoV-2-infected persons develop specific antibodies until day 10 following symptom onset. IgG, IgA and IgM against the spike protein often occur simultaneously [8]. For significant serological results, two patient samples should be  investigated, one from the acute phase (week 1 of the illness) and one from the convalescent phase (3 to 4 weeks later) [4, 6, 9]. SARS-CoV-2-specific T cells appear a few days after onset of symptoms. A specific T cell response is associated with a milder COVID- 19 course [8].
Neutralising antibodies are associated with protective immunity against reinfection with SARS-CoV-2 or SARS-CoV. Neutralising antibodies against SARS- CoV could be detected 17 years after infection.
SARS-CoV-2 reactive T cells are part of the T cell repertoire from persons who had a SARS-CoV infection in 2003. These cells proliferate following contact with SARS-CoV-2. Cross-reacting T cells were detected in some of the investigated persons without history  of SARS-CoV-2 infection and are supposedly due to prior infections with coronaviruses causing common colds. This may indicate a long- lasting immunity following infection with betacoronaviruses [8, 10, 11].
With regard to COVID-19, the immunological memory is heterogeneous: virus- specific antibodies and memory B and T cells are present in different quantities and their levels change with different dynamics.
Current findings indicate that the T and B cell memory and antibodies in most cases persist over years after SARS-CoV-2 [8].

Antigen
The reagent wells of the ELISA were coated with an S1 domain of the spike protein of SARS-CoV-2, isolate Wuhan-Hu-1, expressed recombinantly in the human cell line HEK 293.

Test principle
The test kit contains microplate strips each with 8 break-off reagent wells coated with recombinant S1 domain of the spike protein of SARS-CoV-2. Information on automated incubation is given in the instructions for use of the respective instruments. In the first  reaction step, diluted patient samples are incubated in the wells. In the case of positive samples, specific IgG (also IgA and IgM) antibodies will bind to the antigens. To detect the bound antibodies, a second incubation is carried out using an enzyme-labelled anti-human IgG (enzyme conjugate) catalysing a colour reaction.

Contents of the test kit

1 . Microplate wells coated with antigens
12 microplate strips each containing 8 individual break-off wells in a frame, ready for use| –| 12 x 8|
2 . Calibrator
(IgG, human), ready for use| dark red| 1 x 2.0 ml|
3 . Positive control
(IgG, human), ready for use| blue| 1 x 2.0 ml|
4 . Negative control
(IgG, human), ready for use| green| 1 x 2.0 ml|
5 . Enzyme conjugate
peroxidase-labelled anti-human IgG, ready for use| green| 1 x 12 ml|
6 . Sample buffer
ready for use| light blue| 1 x 100 ml|
7 . Wash buffer
10x concentrate| colourless| 1 x 100 ml|
8 . Chromogen/substrate solution
TMB/H2O2, ready for use| colourless| 1 x 12 ml|
9 . Stop solution
0.5 M sulphuric acid, ready for use| colourless| 1 x 12 ml|
10.  Protective foil| –| 3 pieces|
11.  Quality control certificate| –| 1 protocol| –
12.  Instructions for use| –| 1 booklet| –

Additional materials and equipment (not supplied in the test kit)

• Automatic microplate washer: recommended. Washing of the microplates can also be carried out manually.
• Microplate reader: wavelength of 450 nm, reference wavelength range from 620 nm to 650 nm
• Calibrated pipettes
• Pipette tips
• Stepper pipette: recommended for the pipetting of enzyme conjugate, substrate, and stop solution
• Distilled or deionised water
• Incubator: for incubation of the microplate at +37 °C
• Incubator or water bath: recommended to warm the wash buffer
• Stop watch

Depending on the instrument, further materials are required for automatic processing. For more information, please refer to the respective instructions for use.

Storage and stability
The test kit has to be stored at +2 °C and +8 °C, do not freeze. Unopened, all test kit components are stable until the indicated expiry date.

In use stability

After initial opening, the reagents are stable until the indicated expiry date when stored at +2°C to +8 °C and protected from contamination, unless stated otherwise below.

Warnings and precautions

• The product must only be used by healthcare professionals in an adequate laboratory environment.

• Do not use the test kit if the packaging of the reagents is damaged.

• Before using the product, read the instructions for use carefully. Only use the valid version, which can be downloaded from the customer portal (https://products.euroimmun.de).

• EUROIMMUN reagents must not be mixed with or replaced by reagents from other manufacturers.

• Observe Good Laboratory Practice (GLP) and safety guidelines. Some of the reagents contain pre- servatives in non-declarable concentrations. Avoid eye and skin contact with samples and reagents.
  In case of eye or skin contact, rinse thoroughly with water. Remove and wash contaminated clothing.
  In case of ingestion, obtain medical advice.

• The calibrator and controls of human origin have tested negative for HBsAg, anti-HCV, anti-HIV-1 and anti-HIV-2. Nonetheless, all test kit components should be treated as potentially infectious and handled with care.

Preparation and stability of the samples

Samples
Human serum or EDTA, heparin or citrate plasma or dried capillary blood (dried blood spots, DBS), collected with the EUROIMMUN Blood Collection Card (EUROIMMUN order number ZV 9711-01100).

Sample preparation
Patient samples are diluted 1:101 in sample buffer. For example: dilute 10 µl serum in 1.0 ml sample buffer and mix well by vortexing (sample pipettes are not suitable for mixing).

Note

• When using instruments for automated incubation, sample handling is described in the instructions for use.
• When dried blood spots (DBS) are used as the sample material, these must be extracted from the membrane of the blood collection card prior to sample incubation. The instructions for use required for the extraction (EUROIMMUN document number  EI_2606G_A_UK_ZXX) are provided in the customer portal (https://products.euroimmun.de). The quality of the DBS has to be evaluated according to these instructions. Unsuitable DBS must be excluded from the analysis as they can impact the quality  of the test result.

Stability of the patient samples

• stored at +2 °C to +8 °C: up to 14 days
• incubate diluted samples within one working day

Preparation and stability of the reagents

Note
All reagents must be brought to room temperature (+18 °C to +25 °C) before use.
The thermostatically adjustable ELISA incubator must be set to +37 °C ± 1 °C.

Coated wells
Ready for use. Tear open the resealable protective wrapping of the microplate at the recesses above the grip seam. Do not open until the microplate has reached room temperature to prevent the strips from moistening. Immediately replace the remaining wells of  a partly used microplate in the protective wrapping and tightly seal with the integrated grip seam (Do not remove the desiccant bag).
Once the protective wrapping has been opened for the first time, the wells coated with antigens can be stored in a dry place and at a temperature between +2 °C and +8 °C for 4 months.

Calibrator and controls
Ready for use. Mix reagents thoroughly before use.
Enzyme conjugate
Ready for use. Mix the reagent thoroughly before use.
Sample buffer
Ready for use.

Wash buffer
The wash buffer is a 10x concentrate. If crystallisation occurs in the concentrated buffer, warm it to +37 °C and mix well before dilution. Dilute the required volume 1:10 with deionised or distilled water (1 part reagent plus 9 parts water).
For example: For 1 microplate strip, 5 ml concentrate plus 45 ml water. The working-strength wash buffer is stable for 4 weeks if stored at +2 °C to +8 °C and handled properly.

Chromogen/substrate solution
Ready for use. Close the tube immediately after use, as the contents are sensitive to light. The chromogen/substrate solution must be clear on use. Do not use the solution if it is blue coloured.

Stop solution
Ready to use.

Waste disposal

Patient samples, calibrators, controls and incubated microplate strips should be handled as infectious waste. All reagents must be disposed of in accordance with local disposal regulations.

Quality control
For every group of tests performed, the extinction readings of the calibrator and ratios determined for the positive and negative controls must lie within the limits stated for the relevant test kit lot. A quality control certificate containing these reference values is  included. If the values specified for the controls are not achieved, the test results may be inaccurate and the test should be repeated.

Reference material
As no quantified international reference serum exists for antibodies against SARS-CoV-2, the calibration is performed in ratios which are a relative measure for the concentration of antibodies in serum or plasma.

Assay procedure

(Partly) manual test performance

Sample incubation:
(1st step)
Transfer 100 µl of the calibrator, positive and negative controls or diluted patient samples into the individual microplate wells according to the pipetting protocol. Incubate for 60 minutes at +37 °C ± 1 °C.
For manual processing of microplate wells, cover the finished test plate with the protective foil. When using an automated microplate processor for incubation follow the recommendations of the instrument manufacturer.

Washing:
Manual: Remove the protective foil. Empty the wells and subsequently wash 3 times using 300 µl of working-strength wash buffer for each wash.
Automatic: Remove the protective foil. Wash the reagent wells 3 times with 450 µl of working-strength wash buffer (program setting: e.g.
TECAN Columbus Washer “Overflow Mode”).
Leave the wash buffer in each well for 30 to 60 seconds per washing cycle, then empty the wells. After washing (manual and automated tests), thor- oughly dispose of all liquid from the microplate by tapping it on absorbent paper with the openings facing  downwards to remove all residual wash buffer.
Note:
Free positions on the microplate strip should be filled with blank wells of the same plate format as that of the parameter to be investigated.

Conjugate incubation:
(2nd step)
Pipette 100 µl of enzyme conjugate (peroxidase-labelled anti-human IgG) into each of the microplate wells. For manual test performance cover the reagent wells with the protective foil.
Incubate 30 minutes at +37 °C ± 1 °C.

Washing:
Remove the protective foil. Empty the wells. Wash as described above.

Substrate incubation:
(3rd step)
Pipette 100 µl of chromogen/substrate solution into each of the microplate wells. Incubate for 30 minutes at room temperature (+18 °C to +25 °C) protected from direct sunlight.

Stopping:
Pipette 100 µl of stop solution into each of the microplate wells in the same order and at the same speed as the chromogen/substrate solution was introduced.

Measurement:
Photometric measurement of the colour intensity should be made at a wavelength of 450 nm and a reference wavelength between 620 nm and 650 nm within 30 minutes of adding the stop solution. Prior to measuring, slightly shake the microplate to ensure a  homogeneous distribution of the solution.

Test performance using fully automated analysis devices
Sample dilution and test performance are carried out fully automatically using an analysis device. The incubation conditions programmed in the respective software authorised by EUROIMMUN may deviate slightly from the specifications given in the ELISA test  instruction, but have been validated in respect of the combination of the EUROIMMUN Analyzer I, the EUROIMMUN Analyzer I-2P, the EUROLabWorkstation ELISA, the Sprinter XL and the DSX from Dynex and this EUROIMMUN ELISA Validation  documents are available on enquiry.
Note: Processing on other fully automated systems is possible but must be validated by the user.

Pipetting protocol

The pipetting protocol for microplate strips 1 to 4 is an example for the semiquantitative analysis of 24 patient sera (P 1 to P 24).
The calibrator (C), the positive (pos.) and negative (neg.) controls, and the patient samples have each been incubated in one well. The reliability of the ELISA test can be improved by duplicate determinations for each sample.
The wells can be broken off individually from the strips. This makes it possible to adjust the number of test substrates used to the number of samples to be examined and minimises reagent wastage.
Both positive and negative controls serve as internal controls for the reliability of the test procedure.
They must be assayed with each test run.

Test evaluation
The extinction of the calibrator defines the upper limit of the reference range of non-infected persons (cut-off) recommended by EUROIMMUN. Values above the indicated cut-off are to be considered as positive, those below as negative.
Semiquantitative
Results can be evaluated semiquantitatively by calculating a ratio of the extinction of the control or patient sample over the extinction of the calibrator. Calculate the ratio according to the following formula:

EUROIMMUN recommends interpreting results as follows:

For duplicate determinations the mean of the two values should be taken. If the two values deviate substantially from one another, EUROIMMUN recommends retesting the samples.

Analytical performance

The following data were collected using serum or plasma samples:
Measurement range
Limit of blank (LoB): ratio 0.13
Limit of detection (LoD): ratio 0.14
LoB and LoD were defined according to the requirements defined in guideline EP17-A2 of the CLSI
(Clinical and Laboratory Standards Institute, https://clsi.org/).

Precision
Studies on the intra-lab precision were carried out according to CLSI guideline EP05-A3. Six samples
(reactivity distributed over the entire measurement range) were measured. The precision is given as
standard deviation (SD) and coefficient of variation (CV).

Intra-lab precision

| Sample 1| Sample 2| Sample 3| Sample 4| Sample 5| Sample 6
---|---|---|---|---|---|---
Mean| Ratio 4.74| Ratio 2.74| Ratio 1.09| Ratio 1.25| Ratio 1.14| Ratio 0.22
| SD| % CV| SD| % CV| SD| % CV| SD| % CV| SD| % CV| SD| % CV
Repeatability| 0.125| 2.6| 0.071| 2.6| 0.032| 2.9| 0.064| 5.1| 0.028| 2.5| 0.028| 12.6
Between run| 0.173| 3.6| 0.172| 6.3| 0.067| 6.1| 0.089| 7.1| 0.115| 10.1| 0.023| 10.5
Within day| 0.213| 4.5| 0.186| 6.8| 0.074| 6.8| 0.109| 8.7| 0.119| 10.4| 0.036| 16.4
Between day| 0.139| 2.9| 0.000| 0.0| 0.028| 2.5| 0.047| 3.8| 0.000| 0.0| 0.025| 11.3
Within lab| 0.254| 5.4| 0.186| 6.8| 0.079| 7.3| 0.119| 9.5| 0.119| 10.4| 0.044| 19.9

Cross-reactivity (analytical specificity)
Due to low homologies of the S1 protein within the coronavirus family, cross- reactions to most of the human pathogenic representatives of this virus family are virtually excluded. 163 samples taken prior to the occurrence of SARS- CoV-2 (before January 2020)  that were antibody-positive for at least one human pathogenic coronavirus (HCoV HKU1; HCoV OC43; HCoV NL63; HCoV 229-E) were investigated with the Anti-SARS-CoV-2 ELISA IgG. Positive reactions were only observed in 1.2% of the analyses. Cross  reactions with endemic HCoV are therefore unlikely. However, due to their close relationship, cross-reactions between SARS-CoV(-1) and SARS-CoV-2 are likely.

A ntibodies against| n (positive)/n (total)| P ositive rate [%]
---|---|---
HCoV| 2/163| 1.2

Interference
Haemolytic, lipaemic and icteric samples showed no influence on the result up to concentrations of 10 mg/ml haemoglobin, 20 mg/ml triglycerides and 0.4 mg/ml bilirubin in this ELISA.

The following data were collected using DBS samples:
Precision
Studies on the intra-lab precision were carried out according to CLSI guideline EP05-A3. Six samples (reactivity distributed over the entire measurement range) were measured. The data were determined on 15 days in two runs per day with two replicates each.  Each replicate was yielded by independent extraction from a dried blood spot. The precision is given as standard deviation (SD) and coefficient of variation (CV).

Intra-lab precision

| Sample 1| Sample 2| Sample 3| Sample 4| Sample 5| Sample 6
---|---|---|---|---|---|---
Mean| Ratio 0.26| Ratio 0.90| Ratio 0.99| Ratio 1.38| Ratio 3.73| Ratio 5.95
| SD| % CV| SD| % CV| SD| % CV| SD| % CV| SD| % CV| SD| % CV
Repeatability| 0.045| 17.0| 0.114| 12.6| 0.069| 7.0| 0.140| 10.1| 0.251| 6.7| 0.377| 6.3
Between run| 0.000| 0.0| 0.000| 0.0| 0.115| 11.6| 0.083| 6.1| 0.292| 7.8| 0.000| 0.0
Within day| 0.045| 17.0| 0.114| 12.6| 0.134| 13.5| 0.163| 11.8| 0.385| 10.3| 0.377| 6.3
Between day| 0.025| 9.6| 0.028| 3.1| 0.051| 5.2| 0.053| 3.9| 0.000| 0.0| 0.141| 2.4
Within lab| 0.051| 19.5| 0.117| 12.9| 0.143| 14.5| 0.171| 12.4| 0.385| 10.3| 0.402| 6.8

Method comparison
Study I: The correlation between extracts of dried blood spots (DBS) from capillary blood and serum from venous blood was determined by analysing 215 patient samples collected in Europe, using the Anti-SARS-CoV-2 ELISA (IgG). For each patient, one  capillary blood sample and one venous blood sample were available.
The agreement between the results of the dried capillary blood spots and the venous blood samples was 100% (positive agreement (PPA): 100%; negative agreement (NPA): 100%). Borderline samples were excluded from the calculation.

n = 215| EUROIMMUN
Anti-SARS-CoV-2 ELISA (IgG) Sera from venous blood
---|---
positive| borderline| negative
EUROIMMUN
Anti-SARS-CoV-2 ELISA (IgG) DBS| positive| 61| 2| 0
borderline| 0| 1| 4
negative| 0| 0| 147

Study II: In another study, 51 patient samples (origin: Brazil) were investigated using the Anti-SARS-CoV-2 ELISA (IgG). Like in the first study, extracts from dried blood spots and serum samples from venous blood were compared.
The agreement between the results of the dried capillary blood spots and the venous blood samples was 100% (positive agreement (PPA): 100%; negative agreement (NPA): 100%). Borderline samples were excluded from the calculation.

n = 51| EUROIMMUN
Anti-SARS-CoV-2 ELISA (IgG) Sera from venous blood
---|---
positive| borderline| negative
EUROIMMUN
Anti-SARS-CoV-2 ELISA (IgG) DBS| positive| 40| 0| 0
borderline| 0| 1| 0
negative| 0| 0| 10

Clinical performance

Diagnostic sensitivity (Prevalence)
To determine the diagnostic sensitivity, samples from patients with confirmed SARS-CoV-2 infection were analysed. The following sensitivity therefore corresponds to the prevalence of antibodies against SARS-CoV-2 in COVID-19 infected persons.
The sensitivity was determined by investigating 166 samples from 152 European patients, using the Anti-SARS-CoV-2 ELISA (IgG). In these patients, infections with SARS-CoV-2 had been confirmed by RT-PCR [10] based on a sample taken at the early phase of  infection. In samples taken prior to day 10 (time point after onset of symptoms or positive direct pathogen detection), the Anti-SARS- CoV-2 ELISA (IgG) showed a sensitivity of 43.7%. The sensitivity of the Anti- SARS-CoV-2 ELISA (IgG) in samples collected  after day 10 was 94.4%. Borderline results (n = 7) were not considered in the calculation.

Days after symptom onset or positive direct pathogen detection| EUROIMMUN
Anti-SARS-CoV-2 ELISA (IgG)
---|---
Positive| Negative| Sensitivity
≤ 10| 38| 49| 43.7%

10| 68| 4| 94.4%

The time course of antibody formation and the antibody activity at specific time points can vary significantly. In most patients, antibodies are detectable after day 10 after symptom onset or positive direct pathogen detection. In individual cases, a significantly  delayed synthesis of IgG (> 4 weeks after onset of symptoms or positive direct pathogen detection) has been reported. The graphs show individual immune responses in COVID-19 patients measured with the EUROIMMUN Anti-SARS-CoV-2 ELISA (IgA) and  the EUROIMMUN Anti-SARS-CoV-2 ELISA (IgG) based on the recombinant S1 domain of the spike protein, and the EUROIMMUN Anti-SARS-CoV-2 NCP ELISA (IgG), for which a modified nucleocapsid protein (NCP) is used as antigen.

Patient 1 (86 years old)
Anti-SARS-CoV-2 S1 IgA and anti-SARS-CoV-2 NCP IgG antibodies were detectable as early as 7 days after RT-PCR. The level of anti-SARS-CoV-2 S1 IgG antibodies was still negative 7 days after positive RT-PCR, but was increased in the subsequent sample  taken on day 10.

Patient 2 (47 years old)
The anti-SARS-CoV-2 NCP IgG antibody level was elevated as early as day 8 after the onset of symptoms. Anti-SARS-CoV-2 S1 IgA and IgG antibodies were not yet detectable. A follow-up sample taken on day 11 after the onset of symptoms showed an increase in  the antibody levels for both Ig classes.

Patient 3 (58 years old)
The anti-SARS-CoV-2 S1 IgA antibody level was already highly elevated 12 days after positive RT-PCR.
In contrast, the levels of anti-SARS-CoV-2 S1 and anti-SARS-CoV-2 NCP IgG antibodies increased only slowly until day 43 after positive RT-PCR.
Note: In individual cases, delayed antibody formation may occur, so that antibodies are only detectable after a period of several weeks after the onset of symptoms.

Specificity
The specificity of the Anti-SARS-CoV-2 ELISA (IgG) was determined by analysing 222 patient samples that were positive, for instance, for antibodies against other human pathogenic coronaviruses, other pathogens or for rheumatoid factors. Additionally, 1122  samples from blood donors, children and pregnant women obtained before the occurrence of SARS-CoV-2 (before January 2020) were analysed.
The results in the borderline range (n = 7) were not considered in the calculation. The specificity of the Anti-SARS-CoV-2 ELISA (IgG) amounted to 99.6%.

Specificity
Blood donors| 849| 99.5%
Pregnant women| 199| 99.5%
Children| 74| 100.0%
Elderly people| 97| 100.0%
Infections with other human pathogenic coronaviruses| 23| 100.0%
Influenza (freshly vaccinated, including courses)| 40| 100.0%
Acute EBV infections & heterophile antibodies| 22| 100.0%
Rheumatoid factors| 40| 100.0%
Total| 1344| 99.6%

Limitations of the procedure

• The results should always be interpreted together with those of further laboratory diagnostic procedures and based on the clinical picture.
• A negative serological result does not exclude an infection. Particularly in the early phase of an infection, antibodies may not yet be present or are only present in such small quantities that they are not detectable. In the case of a borderline result, a secure  evaluation is not possible. If there is a clinical suspicion and a negative or borderline serological result, we recommend clarification by means of other diagnostic methods and/or the serological investigation of a follow-up sample. A positive result indicates  that there has been contact with the pathogen. A significant increase in the specific IgG antibody levels (by more than twofold) and/or seroconversion in a follow-up sample taken after at least 7 to 10 days may be interpreted as an indication of acute  infection. The sample and follow-up sample should be incubated in the same test run.
• The specifications in the instructions for use, e.g. pipetting volumes, incubation times, temperatures and preparation steps must be observed to avoid incorrect results
• Correct sample collection and storage are crucial for the reliability of the results.
• The test system is validated for the determination of anti-SARS-CoV-2 IgG in human serum, plasma or dried blood spots only.
• The binding activity of the antibodies and the activity of the enzyme used are temperature- dependent. It is therefore recommended to use a thermostatically controlled ELISA incubator in all incubation steps.
• Insufficient washing (e.g. less than 3 wash cycles, too small wash buffer volumes or too short resi- dence times) can lead to false extinction readings.
• Residual liquid (>10 µl) in the reagent wells after washing can interfere with the substrate conversion and lead to false low extinction readings.
• Partial or complete adaptation of the test system for use with automated sample processors or other liquid handling devices may lead to differences between the results obtained with the automated and manual procedure. It is the responsibility of the user  to validate the automated instruments used for the analysis to ensure that they yield test results within the permissible range.

Literature

1. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol. 2020; 5(4): 536-44
2. Borges do Nascimento IJ, von Groote TC, O’Mathúna DP, Abdulazeem HM, Henderson C, et al. International Task Force Network of Coronavirus Disease 2019 (InterNetCOVID-19). Clinical, laboratory and radiological characteristics and outcomes of novel  coronavirus (SARS-CoV-2) infection in humans: A systematic review and series of meta-analyses. PLoS One. 2020; 15(9): e0239235
3. Gralinski LE, Menachery VD. Return of the Coronavirus: 2019-nCoV. Viruses 2020, 12(2), 135
4. Udugama B, Kadhiresan P, Kozlowski HN, Malekjahani A, Osborne M, Li VYC, et al. Diagnosing COVID-19: The Disease and Tools for Detection. ACS Nano. 2020 Apr 9.
5. Cheng MP, Papenburg J, Desjardins M, Kanjilal S, Quach C, Libman M, et al. Diagnostic Testing for Severe Acute Respiratory Syndrome-Related Coronavirus-2: A Narrative Review. Ann Intern Med. 2020; 172(11): 726-34
6. Xiao SY, Wu Y, Liu H. Evolving status of the 2019 novel coronavirus Infection: proposal of conventional serologic assays for disease diagnosis and infection monitoring. J Med Virol. 2020; 92(5): 464-7
7. Lechien JR, Chiesa-Estomba CM, De Siati DR, Horoi M, Le Bon SD, Rodriguez A, et al. Olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (COVID-19): a multicenter European study. Eur  Arch Otorhinolaryngol. 2020; 277(8): 2251-61
8. Sette A, Crotty S. Adaptive immunity to SARS-CoV-2 and COVID-19. Cell. 2021; 184(4): 861-80
9. Okba NMA, Müller MA, Li W, Wang C, Geurts van Kessel CH, Corman VM, et al. Severe Acute Respiratory Syndrome Coronavirus 2-Specific Antibody Responses in Coronavirus Disease 2019 Patients. Emerg Infect Dis. 2020; 26(7)
10. Swadling L, Maini MK. T cells in COVID-19 – united in diversity. Nat Immunol. 2020; 21(11): 1307- 8
11. Ortega N, Ribes M, Vidal M, Rubio R, Aguilar R, Williams S, et al. Seven-month kinetics of SARS- CoV-2 antibodies and role of pre-existing antibodies to human coronaviruses. Nat Commun. 2021; 12(1): 4740

Liability
The test kit, including original accessories, must only be used in accordance with the intended use.
EUROIMMUN accepts no liability for any other use (e.g. non-compliance with the instructions for use and improper use) or for resulting damages

Technical support

In case of technical problems you can obtain assistance via the EUROIMMUN website (https://www.euroimmun.de/en/contact/).

Additional information
Regulatory information for customers in the European Union: Please observe the obligation to report any serious incidents occurring in connection with this product to the competent authorities and to EUROIMMUN.

Meaning of the symbols

Sy m bol| M ean i ng| Sy m bol| M ean i ng
---|---|---|---
| Microplate strips| | Lot description
| Calibrator| | Protect from sunlight
| Positive control| | Storage temperature
| Negative control| | Unopened usable until
(YYYY-MM-DD)
| Conjugate| | CE-marking
| Sample buffer| | Manufacturing date
(YYYY-MM-DD)
| Wash buffer, 10x concentrate| | Manufacturer
| Substrate| | Observe instructions for use
| Stop solution| | Order number
| Protective foil| | Contents suffice for analyses
| Cap| | Biological risks
| In vitro diagnostic medical device| | Unique Device Identifier

EI_2606G_A_UK_C13.docx
Version: 2022-07-01

References

• Clinical & Laboratory Standards Institute: CLSI Guidelines
• EUROIMMUN Customer Portal - Login
• Contact | EUROIMMUN

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