COVID-19 Antibodies

COVID-19 Antibodies 

SARS-CoV-2 (the novel coronavirus that causes coronavirus disease 2019, or COVID-19)

Introduction

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of the coronavirus induced disease 19 (COVID-19) that emerged in China late 2019 and causing a pandemic. As of 19 April 2020, 2,241,778 cases have been reported worldwide, of which 152,551 (6.8%) succumbed to the infection. SARS-CoV-2 belongs to the Sarbecovirus subgenus (genus Betacoronavirus, family Coronaviridae) together with SARS-CoV that emerged in 2002 causing ~8000 infections with a lethality of 10%. Both viruses cross species barriers from an animal reservoir and can cause a life-threatening respiratory illness in humans. Presently, no approved targeted therapeutics are available for COVID-19. Monoclonal antibodies targeting vulnerable sites on viral surface proteins are increasingly recognized as a promising class of drugs against infectious diseases and have shown therapeutic efficacy for a number of viruses.

Coronavirus-neutralizing antibodies primarily target the trimeric spike (S) glycoproteins on the viral surface that mediate entry into host cells. The S protein has two functional subunits that mediate cell attachment (the S1 subunit, existing of four core domains S1_A through S1_D) and fusion of the viral and cellular membrane (the S2 subunit). Potent neutralizing antibodies often target the receptor interaction site in S1, disabling receptor interactions. The spike proteins of SARS-CoV-2 (SARS2-S; 1273 residues, strain Wuhan-Hu-1) and SARS-CoV (SARS-S, 1255 residues, strain Urbani) are 77.5% identical by primary amino acid sequence, are structurally very similar and commonly bind the human angiotensin converting enzyme 2 (ACE2) protein as a host receptor through their S1_B domain. Receptor interaction is known to trigger irreversible conformational changes in coronavirus spike proteins enabling membrane fusion.

COVID-19 Antibodies

Fact Sheet for Patients Elecsys Anti-SARS-CoV-2

You are being given this Fact Sheet because your sample(s) was tested for antibodies to the virus that causes Coronavirus Disease 2019 (COVID-19) using the Elecsys Anti-SARS-CoV-2 immunoassay.

This Fact Sheet contains information to help you understand the risks and benefits of using this test for detecting antibodies to the virus that causes COVID-19. After reading this Fact Sheet, if you have questions or would like to discuss the information provided, please talk to your healthcare provider.

What is COVID-19?

COVID-19 is caused by the SARS-CoV-2 virus. The virus, which can cause mild to severe respiratory illness, was first identified in Wuhan, China, and has now spread globally, including the United States. There is limited information available to characterize the spectrum of clinical illness associated with COVID-19 but it likely spreads to others when a person shows signs or symptoms of being sick (e.g., fever, coughing, sneezing, difficulty breathing, etc.).

What is the Elecsys Anti-SARS-CoV-2 immunoassay?

The test is designed to detect antibodies in a blood sample that would indicate that you may have current or prior COVID-19 infection.

Why was my sample tested?

Testing of your sample(s) will help find out if you have antibodies to the virus that causes COVID-19.

What are the known and potential risks and benefits of the test?

Potential risks include:

·     Possible discomfort or other complications that can happen during sample collection.

·     Possibility of false positive or false negative results.

      Potential benefits include:

·      The results, along with other information, can help your healthcare provider make informed recommendations about your care.

·      The results of this test may help limit the spread of COVID-19 to your family and others in your community.

What does it mean if I have a positive test result?  

you have a positive test result, it is very likely that you MAY have or previously had COVID-19 and that you have developed an antibody response to the virus. Your healthcare provider will work with you to determine how best to care for you based on the test results along with other factors of your medical history, and your symptoms, possible exposures, and geographic location of places you have recently traveled. There is also the small possibility that this test can give a positive result that is wrong (a false positive result).

What does it mean if I have a negative test result?

A negative test result means that antibodies to the virus that causes COVID-19 were not found in your sample. However, it is possible for this test to give a negative result that is incorrect (false negative) in some people with COVID-19. A negative result may occur if you are tested during the early stages of your illness where your body hasn’t had time to produce antibodies to the infection. This means that you could possibly still have COVID-19 even though the test is negative.

If this is the case, your healthcare provider will consider the test result together with all other aspects of your medical history (such as symptoms, possible exposures, and geographical location of places you have recently traveled) in deciding how to care for you. It is important that you work with your healthcare provider to help you understand the next steps you should take. 

Immunoassay for the qualitative detection of 

antibodies against SARS-CoV-2

What is the clinical sensitivity?

The assay exhibited 100% clinical sensitivity for samples collected >14 days after PCR confirmation. 

Summary

SARS‑CoV‑2 is an envelope, single-stranded RNA virus of the family Coronaviridae, genus Betacoronavirus. All coronaviruses share similarities in the organization and expression of their genome, which encodes 16 nonstructural proteins and the 4 structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N). Viruses of this family are of zoonotic origin. 

They cause disease with symptoms ranging from those of a mild common cold to more severe ones such as the Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and Coronavirus Disease 2019 (COVID‑19). Other coronaviruses known to infect people include 229E, NL63, OC43 and HKU1. The latter are ubiquitous and infection typically causes common cold or flu-like  symptoms.1,2 The Elecsys Anti‑SARS‑CoV-2 assay uses a recombinant protein representing the nucleocapsid (N) antigen for the determination of antibodies against SARS‑CoV‑2

Precautions and warnings:

• For in vitro diagnostic use
• Exercise the normal precautions required for handling all laboratory reagents.
• Disposal of all waste material should be in accordance with local guidelines.
• Safety data sheet available for professional user on request. 

     Specimen collection and preparation:

• Only the specimens listed below were tested and found acceptable. Serum collected using standard sampling tubes or tubes containing separating gel.
• Li‑heparin, K2‑EDTA and K3‑EDTA plasma.
• Plasma tubes containing separating gel can be used.
• Criterion: Absolute deviation of negative samples ± 0.3 COI (cutoff index) from serum value; reactive samples: recovery within 70‑130 % of serum value.
• Stable for 3 days at 15‑25 °C, 7 days at 2‑8 °C, 28 days at ‑20 °C (± 5 °C).
• The samples may be frozen once.
• The sample types listed were tested with a selection of sample collection tubes or systems that were commercially available at the time of testing, i.e. not all available tubes of all manufacturers were tested. Sample collection systems from various manufacturers may contain differing materials which could affect the test results in some cases. When processing samples in primary tubes (sample collection systems), follow the instructions of the tube/collection system manufacturer.
• Specimens should not be subsequently altered with additives (e.g. biocides, anti-oxidants or substances that could possibly change the pH or ionic strength of the sample) in order to avoid erroneous findings. Pooled samples and other artificial material may have different effects on different assays and thus may lead to discrepant findings.

Depending on the applied method, seroconversion is observed after a median of 10 - 13 days after  symptom onset for IgM and 12 - 14 days for IgG. Maximum seroconversion occurs at 2 - 3 weeks for IgM, at 3 - 6 weeks for  IgG, and at two weeks for total antibodies. Whereas IgM seems to vanish around week 6 - 7, high IgG seropositivity is seen at that time. Levels and chronological order of IgM and IgG antibody appearance are highly variable, supporting detection of both antibodies simultaneously. Neutralizing antibodies targeting the spike and nucleocapsid proteins are formed as early as day 9 onwards, showing strong neutralizing response, thus seroconversion may ead to protection   at a minimum for a limited for a time.         

Elecsys Anti-SARS-CoV-2 is an immunoassay for the in vitro qualitative detection of antibodies (including IgG) to SARS-CoV-2 in human serum and plasma. The assay users a recombinant protein representing the nucleocapsid (N) antigen for the determination of antibodies against SARS-CoV-2. The test is intended as an aid in the determination of the immune reaction to SARS-CoV-2. 

Electro-chemiluminescence immunoassay (ECLIA)

Test principle: double- antigen sandwich assay (testing time: 18 minutes)

Step - 1 (9 minutes)

20µL/12µL of the patient sample are incubated with a mix of biotinylated and ruthenylated nucleocapsid (N) antigen. Double-antigen sandwich immune complexes are formed in the presence of corresponding antibodies.

Step - 2 (9 minutes)

After addition of streptavidin- coated microparticles, the DAGS complexes bind to the solid phase via interaction of biotin and streptavidin.

Step - 3 (measurement)

The reagent mixture is transferred to the measuring cell, where the micro particles are magnetically captured onto the surface of the electrode.  Unbound substances are subsequently removed. Electrochemilumi­nescence is then induced by applying a voltage and measured with a photomultiplier. The signal yield increases with the antibody titer.

Test process by Cobas 801 analyzer: 

For optimum performance of the assay follow the directions given in this document for the analyzer concerned. Refer to the appropriate operator’s manual for analyzer‑specific assay instructions.

Resuspension of the microparticles takes place automatically prior to use. 

Calibration:  

No international standard is available for Anti‑SARS‑CoV‑2. Calibration frequency: Calibration must be performed once per reagent lot using ACOV2 Cal1, ACOV2 Cal2 and fresh reagent (i.e., not more than 24 hours since the reagent kit / cobas e pack was registered on the analyzer).Calibration interval may be extended based on acceptable verification of calibration by the laboratory.

Renewed calibration is recommended as follows:

• After 3 days when using the same reagent lot
• After 3 days when using the same reagent kit / cobas e pack on the analyzer
• As required: e.g. quality control findings outside the defined limits

Quality control:  

For quality control, use controls prepared as follows:

Negative control: Determine the COI of ACOV2 Cal1 by measuring it as a routine sample. Pool serum samples with a COI result of ≤ 150 % compared to the COI result of ACOV2 Cal1 (pooling of ≥ 5 non-reactive samples in this range is recommended). 

Mix carefully, avoiding foam formation. Prepare aliquots of at least 250 μL from this sample pool and store frozen at ‑20 °C (± 5 °C) or colder. Use these aliquots to perform regular quality control.

This negative control has a target value range of COI < 0.8 (qualitative assay result “non-reactive”).

Positive control: Determine the COI of ACOV2 Cal2 by measuring it as a routine sample. Pool serum samples with a COI result that is higher than the COI result of ACOV2 Cal2 (pooling of ≥ 3 reactive samples in this range is recommended). 

Dilute the sample pool by adding pooled negative serum (for pooling criterion see negative control) or Diluent Multi Assay to obtain a COI between 3 and 15. Mix carefully, avoiding foam formation. It is recommended to confirm calculated reactivity after dilution by a measurement. 

Prepare aliquots of at least 250 μL from this sample pool and store frozen at ‑20 °C (± 5 °C) or colder. Use these aliquots to perform regular quality control. Upon first use of this control, determine the COI of the control by measurement of the control in triplicate and using a freshly opened and calibrated reagent rack pack / cobas e pack.

The obtained median of these measurements serves as target value for this positive control. Subsequent measurements of all aliquots of this control material must match this target value ± 45 % (3SD = 45 %, 1SD = 15 %; qualitative assay result "reactive").

In rare cases, interference due to extremely high titers of antibodies to analyte‑specific antibodies, streptavidin or ruthenium can occur. These effects are minimized by suitable test design. The results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.

A negative test result does not rule out the possibility of an infection with SARS‑CoV‑2. Serum or plasma samples from the early (pre‑seroconversion) phase of illness can yield negative findings. Therefore, this test cannot be used to diagnose an acute infection. Also, over time, titers may decline and eventually become negative. 

Testing with a molecular diagnostic should be performed to evaluate for active infection in symptomatic individuals. 

It is not known at this time if the presence of antibodies to SARS‑CoV‑2 confers immunity to reinfection.

Clinical Sensitivity: 

A total of 204 samples from 69 symptomatic patients with a PCR confirmed SARS-CoV-2 infection were tested with the Elecsys Anti-SARS-CoV-2 assay. One or more consecutive specimens from these patients were collected after PCR confirmation at various time points.

S.No.	Days post PCR confirmation	N	Sensitivity (95% CI)
1	0 – 6 days	116	65.5% (56.1 – 74.2%)
2	7 – 13 days	59	88.1% (77.1 – 95.1%)
3	≥14 days	29	100% (88.1 – 100%)

Interpretation of the results:

Results obtained with the Elecsys Anti‑SARS‑CoV‑2 assay can be interpreted as follows:

S.No.	Numeric result	Result message	Interpretation
1	COI < 1.0	Non-reactive	Negative for anti-SARS-CoV-2 Antibodies
2	COI ≥ 1.0	0 Reactive	Positive for anti-SARS-CoV-2 Antibodies

The magnitude of the measured result above the cutoff is not indicative of the total amount of antibody present in the sample.

The individual immune response following SARS‑CoV‑2 infection varies considerably and might give different results with assays from different manufacturers. Results of assays from different manufacturers should not be used interchangeably.

Limitations - interference

The effect of the following pharmaceutical compound on assay performance was tested. Interference was tested up to the listed concentration and no impact on results was observed. 

S.No.	Compound	Concentration
1	Biotin	≤ 4912 nmol/L or ≤ 1200 ng/mL

This assay has no biotin interference in serum concentrations up to 1200 ng/mL. Some studies have shown that serum concentrations of biotin can reach up to 355 ng/mL within the first hour after biotin ingestion for subjects consuming supplements of 20 mg biotin per day and up to 1160 ng/mL for subjects after a single dose of 300 mg biotin.

Potential endogenous interferences e.g. hemolysis, bilirubin, rheumatoid factors and pharmaceutical compounds other than biotin have not been tested and interference cannot be excluded.

All sample results were non‑reactive.

In addition, due to prevalence of immunization and/or exposure to common viral agents in the general population, the Specificity section below indicates no major cross reactivity to antibodies for Hepatitis B, Influenza A/B, Haemophilus, common colds (i.e. Rhinovirus), Respiratory Syncytial Virus, or the Coronavirus strains listed.

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