INFE

CTI

OU

S D

ISEA

SE

FOR OUTSIDETHE US

AND CANADA ONLY

LIAISON® SARS-CoV-2 SOLUTIONSThe combined testing solutions for the detection of antibodies to SARS-CoV-2

LIAISON® SARS CoV-2 S1/S2 IgG

Concordance with PRNT90

The comparison to PRNT90 was evaluated by testing 87 samples collected during the outbreak from subjects whose PRNT90 result was available: 69 were PRNT assay-negative, and 18 were PRNT assay-positive (Titer >1:40).

LIAISON® SARS-CoV-2 S1/S2 IgGPRNT titers

Totalnegative positive

Negative (< 12.0 AU/mL) 68 1 69

Equivocal (12.0 – 15.0 AU/mL) 0 0 0

Positive (> 15.0 AU/mL) 1 17 18

Total 69 18 87

High positive concordance (94.4%) at LIAISON cut off for PRNT90 at 1:40 ratio, was observed between IgG assay and PNRT titers, indicating the presence of neutralizing antibodies.

Possible use for Plasma

donors (3):

• Neutralizing antibodies are commonly considered to be protective (*) and DiaSorin assay positive agreement with PRNT 90 is 94.4%

• Although the role of neutralizing antibodies to SARS-CoV-2 is under investigation, information on their presence can be important from an epidemiological point of view

• The DiaSorin assay can be a valuable tool for plasma donation screening• S1 and S2, the antigens that DiaSorin uses in his IgG test, are the most promising

for vaccine (7)

(*) Reminder:It should be noted that lack of scientific data at this time does not allow yet to determine if neutralizing IgG antibodies against SARS-CoV-2 provide long term immunity to the virus or if they protect patients against re-infection.By blocking the virus entry to the cell, Neutralizing antibodies can block viral replication: this does not remove need for PCR testing to confirm patient is not infectiveNOTE: Neutralizing antibodies are produced via a maturation process, can take longer to be produced than non-neutralizing Abs (6)

• Shorten the diagnostic window (combined use with IgM + IgG)• IgM testing could help to stratify patients to evaluate new vs previous infection • IgG testing to identify potential donors of iperimmune plasma, thanks to the

correlation with neutralizing antibodies• Moreover, the Spike protein seems to be most promising target for the vaccines

development

Potential use of Antibodies

testing solutions:

FOCUS on the VALUE of neutralizing

antibodies correlation

• 1:160 titer PRNT90 is a possible cut off to select donor serum for plasmapheresis in Italy and by CDC, i.e. hyperimmune serum donations

• At a LIAISON value of 80 AU/ml a positive agreement of 100% with a 1:160 PRNT90 Titer was observed.

• Our assay can be used as upstream pre-screening tool: LIAISON test first to identify donors >80AU/ml, then proceed with Quality Protocol for evaluating plasma donations (PRNT, screening for blood pathogens, etc)

Envelope Protein

Nucleocapsid

Spike Protein

MembraneProtein

RNA

LIAISON® SARS-CoV-2 The value of DiaSorin assaysDiaSorin provides diagnostic laboratories with 2 antibodies detection kits to SARS-CoV-2: • LIAISON® SARS-CoV-2 IgM• LIAISON® SARS-CoV-2 S1/S2 IgGThe testing solutions are based on:• 2 fully automated assays to be run on LIAISON® XL• Two results from a single sample to determine both antibodies results• The selection of Spike protein antigens for both assays, recognized to be the most

likely target of future COVID-19 vaccines and for IgG neutralizing antibodies• High sensitivity and specificity to ensure accurate results along the infection window

Timing of seroconversion

Timing of seroconversion is a key factor in establishing the appropriate time window to use serology tests. Seroconversion has been observed as early as within 5 days after symptom onset for IgM and within 5-7 days for IgG. (1, 2) 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. (3, 4)

The estimated variation over time in diagnostic tests for detection of SARS-CoV-2 Infection relative to symptom onset can be depicted for illustrative purposes as follows (5)

The selection of the antigens for the assays: the Value of using Spike Protein

Both IgM and IgG assays use Spike protein on the solid phase

IgM antibody IgG antibodyNasopharyngeal swab PCR

Virus isolation from respiratory tract

SARS-CoV-2exposure

Before symptom onset After symptom onset

Week -2 Week -1 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6

Symptom onset

Incr

easi

ng p

roba

bilit

y of

det

ectio

n

Detection unlikely PCR - Likely positive PCR - Likely negative

Antibody detection

Furthermore, since viral RNA can be detected in patients after 20 or more days (6) a positive IgG result should not be interpreted as a sign that a patient has stopped being infective.

IgM assay uses S1-RBD as antigen: more than 10 antigens were tested and S1-RBD provided the best combination of Specificity and Sensitivity.

IgG assay uses S1 and S2, subunits of the Spike protein.

Proteins are responsible for: binding (S1) & fusion (S2) of virus to cell.

The Spike Protein is the target for neutralizing antibodies and the most promising target for Vaccines. (7)

Internal viral protein, such as Nucleocapsid are not exposed to antibodies directly and unlikely to be neutralizing (8, 9)

NeutralizingAbs

Neutralizing antibodies inhibit SARS-CoV-2 binding

ACE2

Spike Protein SARS-CoV-2

Cell

NeutralizingAbs

ACE2

Spike Protein SARS-CoV-2

SARS-CoV-2 binding to ACE2

Cell

The Value of combining

IgM and IgG assays:

the infection window

The sensitivity was determined by investigating 268 samples collected over the course of time from 233 European patients. Infection with SARS-CoV-2 was confirmed by RT-PCR test at the time of the diagnosis.The LIAISON® SARS-CoV-2 IgM and S1/S2 IgG test were performed on samples collected at the time of admission and the LIAISON® SARS-CoV-2 thereafter up to 30 days. The group included patients hospitalized with moderate symptoms, patients admitted at ICU with severe symptoms and patients not hospitalized without or with mild symptoms.The following table describes combined diagnostic sensitivity in three groups, i.e. the early samples (≤ 7 days after diagnosis), the samples between 8 and 14 days after diagnosis, and the later samples (15-30 days after diagnosis).

Combined detection of IgM and IgG can be used to assess the immune status of patients exposed to and infected by SARS-CoV-2. The use of IgG and IgM in combination can aid to shorten the diagnostic window improving the detection of seroconversion up to day 15 from PCR positivity.

Combined diagnostic sensitivity

Number of LIAISON® IgG and / or IgM Positive results

Sensitivity (Wilson 95% CI)

≤ 7 days 73 / 105 69.5% (60.2% - 77.5%)

8-14 days 43 / 47 91.5% (80.1% - 96.6%)

15-30 days 114 / 116 98.3% (93.9% - 99.5%)

Combined diagnostic specificity

A total of 500 presumed SARS-CoV-2 negative samples were tested with LIAISON® SARS-CoV-2 S1/S2 IgG and LIAISON® SARS-CoV-2 IgM assays resulting in 99.2% combined clinical specificity (496 / 500, 95% CI: 98.0% – 99.7%).

Plaques are generally counted manually and the results, in combination with the dilution factor used to prepare the plate, are used to calculate the number of plaque forming units per sample unit volume. This result represents the number of infective particles within the sample and is based on the assumption that each plaque formed is representative of one infective virus particle.

The concentration of serum to reduce the number of plaques by 90% compared to the serum free virus gives the measure of how much antibody is present or how effective it is. This measurement is denoted as the PRNT90 value.

The science behind PRNT90:The plaque-reduction neutralization test determines relative concentrations of virus-specific (SARS-CoV-2) neutralizing antibodies in a serum sample by measuring the titer at which a serum sample produces 90% or reduction in plaques vs a no serum control. The process has to be performed in a laboratory Biosafety Level 3.

Patients Serum

Diluted to different titers(e.g from1:10 to 1:640)

Viral solution

Known viral load

Incubate

1-2hs 37C

cells used are specific for the virus

SERUM DILUTIONS

DISPENSATION ON CELL MONOLAYER

Incubate

1-7 days 37C

PRNT90END-POINT TITERS

CALCULATION

The serum specimen being tested is usually

subjected to serial dilutions prior to mixing with a standardized amount

of virus

The concentration of

virus is held constant such that, when added to

susceptible cells and overlaid with semi-solid media,

individual plaques can be discerned and counted

PRNT90 end-point titers can be calculated for each

serum specimen at any selected percent reduction

of virus activity

The Value of detecting Neutralizing Antibodies:

• Neutralizing Antibodies are defined as an antibody that defends a cell from a pathogen or infectious particle by neutralizing any effect it has biologically

• The presence of NAbs is commonly considered as a sign of protection against a pathogen, even if it should be noted that lack of scientific data at this time does not allow yet to determine if neutralizing IgG antibodies against SARS-CoV-2 provide long term immunity to the virus or if they protect patients against re-infection

The LIAISON® IgG assay correlates with Plaque Reduction Neutralization Test (PNRT90)

A PRNT measures how effective are patient antibodies to stop the virus from infecting cells, that is the neutralizing activity.

Ordering information & Specs - Kit

LIAISON® Control SARS-CoV-2 S1/S2 IgG311451

SARS-CoV-2 IgM 311471

KIT FORMAT 2 levels x 2 vials (up to 40 runs)Open/on board controls stability Up to 2 weeks

LIAISON® SARS-CoV-2 S1/S2 IgG 311450

LIAISON® SARS-CoV-2 IgM 311470

KIT FORMAT 110 tests/ integral

TIME TO FIRST RESULT 35 min

THROUGHPUT Up to 170 test/hour Up to 84 tests/hour

OPEN/ON BOARD KIT STABILITY and CALIBRATION

Up to 2 weeks Up to 1 week

No Biotin potential inference observed Up to 3500 ng/mL 

Ordering information & Specs - Controls

M08

7004

369

07/2

0

Please visit: www.diasorin.com/covid19CEfor more information and updates

LIAISON® is a registered trademark of DiaSorinAvailable only on Product availability subject to required regulatory approval

Diasorin S.p.AVia Crescentino SNC, 13040 Saluggia (VC) - Italy www.diasorin.com

References:1. Liu W, Liu L, Kou G, et al. Evaluation of Nucleocapsid and Spike Protein-Based Enzyme-Linked Immunosorbent Assays for

Detecting Antibodies against SARS-CoV-2. J Clin Microbiol. 2020;58(6):e00461-20. Published 2020 May 26. doi:10.1128/JCM.00461-20

2. To KK, Tsang OT, Leung WS, et al. Temporal profiles of viral load in posterior oropharyngeal saliva samples and serumantibody responses during infection by SARS-CoV-2: an observational cohort study. Lancet Infect Dis. 2020;20(5):565‐574.doi:10.1016/S1473-3099(20)30196-1

3. 3Long Q, Deng H, Chen J, et al. Antibody responses to SARS-CoV-2 in COVID-19 patients: the perspective application ofserological tests in clinical practice. medRxiv. 2020. doi: https://doi.org/10.1101/2020.03.18.20038018

4. Zhao J, Yuan Q, Wang H, et al. Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019 [publishedonline ahead of print, 2020 Mar 28]. Clin Infect Dis. 2020;ciaa344. doi:10.1093/cid/ciaa3444

5. Nandini S., Sundararaj S.  J.; Akihide R. Interpreting Diagnostic Tests for SARS-CoV-2 AuJAMA. Published online May 6,2020. doi:10.1001/jama.2020.8259

6. Derdeyn C., Moore P. et al. Development of Broadly Neutralizing Antibodies from Autologous Neutralizing AntibodyResponses Curr Opin HIV AIDS. 2014 May ; 9(3): 210–216. doi:10.1097/COH.0000000000000057.

7. Shang, W., Yang, Y., Rao, Y. et al. The outbreak of SARS-CoV-2 pneumonia calls for viral vaccines. npj Vaccines 5, 18 (2020).https://doi.org/10.1038/s41541-020-0170-0

8. Wang C., Li W. et al. A human monoclonal antibody blocking SARS-CoV-2 infection. Nature communications (2020)11:2251 | https://doi.org/10.1038/s41467-020-16256-y |ww.nature.com/naturecommunications

9. Ni, L., Ye, F., Cheng, M.-L. Detection of SARS-CoV-2-specific humoral and cellular immunity in COVID-19 convalescent individuals, et al. Immunity (2020) doi: https://doi.org/10.1016/j.immuni.2020.04.023.