Tetracore FlexImmArray™ SARS-CoV-2 Human IgG Antibody …

Tetracore

® FlexImmArray™ SARS-CoV-2 Human IgG Antibody Test

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INSTRUCTIONS FOR USE Product Catalog # TC-5040-090

Tetracore FlexImmArray™ SARS-CoV-2 Human IgG Antibody Test

Read this IFU completely before using this product. Follow the instructions carefully while performing the test to

assure accuracy of the results.

Rx ONLY

For in vitro diagnostic use only

For Laboratory Professional use only

Intended Use:

This test is intended for qualitative detection of Human IgG antibody to SARS-CoV-2 in an

unknown human serum or plasma sample.

This test has not been reviewed by the FDA.

Negative results do not rule out SARS-CoV-2 infection, particularly in those who have been

in contact with the virus. Follow-up testing with a molecular diagnostic should be considered

to rule out infection in these individuals.

Results from antibody testing should not be used as the sole basis to diagnose or exclude

SARS-CoV-2 infection or to inform infection status.

Sensitivity of this test early after the infection is currently unknown.

Positive results may be due to past or present infection with non-SARS-CoV-2 coronavirus

strains, such as coronavirus HKU1, NL63, OC43, or 229E.

This is a high complexity test and must be performed by qualified and trained technicians.

Laboratories in USA and its territories are required to report all positive results to the

appropriate public health authorities.

This kit is not for the screening of donated blood.

Introduction: SARS-CoV-2 serology test is a blood-based test to estimate the IgG antibody to SARS-CoV-2 in

people who may have been exposed to the virus. The antibodies specific for SARS-CoV-2 may

also be present in people who were infected but remained asymptomatic or have recovered.

Serological measurement of antibodies can also provide information about the prevalence of

COVID-19 in a population by identifying individuals who have developed antibodies to the

virus.

Principle of the assay:

This assay contains three SARS-CoV-2 specific target proteins for detecting IgG antibodies to

the virus. The test also includes four different internal controls to monitor each step of assay

performance. This multiplex assay uses magnetic microspheres coupled with unique recombinant

proteins specific for SARS-CoV-2 (different spike proteins and nucleoprotein) to capture

antibodies from human serum or plasma. The serum or plasma samples are mixed with the

antigen-coated microspheres in wells of a 96-well plate and incubated for the antigen-antibody

reaction to occur. The antigen-specific antibodies get immobilized on the microspheres, and

unbound material is washed away after the incubation. The CoV-2 antigen captured human IgG

antibodies are then detected using a Fluorescent Anti-human IgG-phycoerythrin (HIgG-PE)

reporter conjugate. After a final wash to remove the unbound conjugate, the microspheres are

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resuspended in the buffer and analyzed using Luminex®

LX 200™ flow analyzer, or MAGPIX®

or FLEXMAP 3D®

instruments. In addition to three different SARS-CoV-2 antigens used in the

multiplex assay, four different controls are also incorporated in every single test. First control

measures the fluorescent intensity measured by Luminex instrument and assures the quality of

fluorescence measurement. The second control measures the non-specific binding that may arise

from the sample. The third control measures the addition of the human serum / plasma sample in

the assay for the measurement of Human IgG antibody. The fourth control monitors for the

addition of fluorescent reporter in the assay thus assuring the assay reagent is added correctly.

Incorporation of these internal controls in every test unmatched quality assurance in the

performance of the test.

In MAGPIX instrument, two spectrally distinct Light Emitting Diodes (LEDs) interrogate the

microspheres and Charge Coupled Device (CCD) images the fluorescence on the beads. One

CCD identifies the microsphere region, and hence the immobilized antigen and the other CCD

measures the amount of PE-derived signal, which is in direct proportion to the bound antibodies.

The bound antibody signal is measured as median fluorescent intensity (MFI).

Table 1. Microsphere regions and the targets

# Microsphere region Target Antigen

1 25 SARS-CoV-2 Protein 1



4 47 IC - Instrument Control

5 54 NC - Non-specific binding Control

6 53 FC - Fluorescent Reporter Control

7 52 ScG - Human IgG Sample Control

Every 96-well test plate can be used for testing up to 90 samples in one test run. Each test run

includes SARS-CoV-2 negative control serum, positive control serum, and calibrator in

duplicates.

Limitations of the Procedure:

The kit should not be used beyond the expiration date on the kit label.

Do not mix or substitute reagents with those from other lots or sources.

The Human IgG antibody response becomes detectable in convalescent samples after about 2

weeks of infection. Acute samples taken within 7 days post onset of symptoms may be

negative for IgG.

At this time research is limited on how long IgG response may persist following infection

and may provide protective immunity. Immunocompromised subjects with COVID 19

disease may have a delayed IgG response. Not much information is available at this time to

understand assay performance in such samples.

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Precautions:

All human samples are presumed hazardous and must be handled according to laboratory safety

guidelines at your institution. Wear appropriate PPE and wash hands thoroughly after handling.

Table 2. Materials Included & Storage Conditions

# PART Description Storage

1 Premixed 7-Plex

Microsphere mix

1 vial of 1.2 mL of ready to use

magnetic microsphere mix

2-8◦C within the expiration

date

2 5X Sample

Dilution Buffer

1 vial of 12 mL 5X Sample dilution

buffer (SDB)


date

3 Fluorescence

Reporter

1 vial of 3 mL ready to use anti-

Human IgG-PE conjugate


date

4 20X Assay Wash

Buffer

2 vials of 15 mL 20X concentrated

assay wash buffer. May develop

crystals when stored at 2- 8 for over

one month.

1 Month after dilution at

room temperature

5 Human IgG

Positive Control

1 vial with 0.2 mL ready to use

control sample for positive IgG

antibodies to the SARS-CoV-2

antigens


date

6 Human IgG

Negative Control

1 vial with 0.2 mL ready to use

control sample for no IgG antibodies

to the SARS-CoV-2 antigens


date

7 Human IgG

assay Calibrator

1 vial with 0.2 mL calibrator ready

to use


date

8 Sample dilution

plates 2 Non-binding 96 well plates for dilution of samples

9 Assay Plate 1 Flat and micro-clear bottom 96-well microplate used for assay

performance

10 Plate sealers 5 Adhesive foils

Material Required but Not Included: Multichannel pipettes and appropriate tips

Deionized or filtered water

Pipettes in common laboratory sizes viz; P10, P20, P100, P1000

Serological pipettes of 10, 15, 25 mL sizes and pipette aid

Equipment required, but not provided:

Automated or manual plate washer for magnetic microspheres

Tabletop centrifuge

Plate shaker

Luminex FLEXMAP 3D, or Luminex 200 system, or MAGPIX instrument

Sample Collection and Storage

1) Serum samples may be collected as per your laboratory guidelines.

2) Plasma samples using EDTA as an anticoagulant in purple top tubes have been validated in

this test.

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3) Aliquot the serum and plasma samples and these aliquots may be stored at 2- 8◦C for up to a

week for testing within that period. If testing is delayed we recommend freezing the sample

per your laboratory guidelines for. Avoid repeated freezing and thawing of samples.

Reagent Preparation

1) Sample Dilution Buffer: Prepare required amount of 1X Sample Dilution Buffer from the

stock 5X Sample Dilution Buffer (SDB) by 5-fold dilution. (e.g. 1mL 5x SDB + 4mL of

distilled water. One mL of 1X buffer is needed per serum sample to be tested.).

2) Wash Buffer – If crystals have formed in the concentrated wash buffer, warm to room

temperature and mix gently until the crystals have completely dissolved. Add 15 mL of 20 X

wash buffer to distilled water to make 300 mL of wash buffer.

3) Positive Control, Negative Control and Calibrator for Human IgG assay are ready to use

and should not be diluted further.

Sample Preparation

We recommend inactivation of serum and plasma samples in a water bath at 56◦C for 30

minutes to 1 hour depending upon the laboratory safety procedures. Inactivation for longer

than an hour and temperature higher than 560C has not been validated and may affect the

assay performance.

1) Following inactivation, bring the samples to room temperature.

2) Centrifuge inactivated human serum samples at room temperature for 2 minutes at 10000

rpm using a tabletop centrifuge to bring the entire sample to the bottom of the tube.

3) Label the two sample dilutions plate as dilution plate 1 and dilution plate 2 respectively. Each

plate will be used to make 1/20 serial dilution of the serum or plasma sample. Add 190 µL of

1X Sample Dilution Buffer to each well of the two serum dilution plates. Add 10 µL of

serum / plasma sample to assigned wells mix the sample well by pipetting up and down with

the buffer. Take 10 µL from 1st dilution plate and add to the second dilution plate. Mix well.

Samples are now ready for testing at 1 in 400 dilutions in the second dilution plate.

Figure 1. Schema for dilution of a test sample 1/20 followed by 1/20 to achieve 1/400

final dilution.

Instrument Settings

1) Ensure that Luminex analyzer being used is appropriately maintained per Luminex

recommendations in the respective instrument user manual or as per your laboratory

guidelines.

Sample dilution plate 1 Serum / plasma

Sample Sample dilution plate 2

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2) Assign the microsphere region for each of the antigen and control microspheres in the

protocol as per Table 1.

3) Set the protocol parameters as 50 events /region

4) Sample size: 50 µL

5) Data type: Median Fluorescent Intensity (MFI)

Assay Procedure 1) Prepare all reagents and samples as per previous sections.

2) Mix well the 7-plex microsphere mix for about 20 seconds on a vortex.

3) Add 10 µL of well mixed microsphere mix to each well of the plate.

4) Add ready to use controls and calibrators in duplicate to first six wells on the assay plate

as per plate format (Figure 2). The controls should be the first 6 wells for automated

calculation sheet to work properly.

5) Transfer 50 µL of diluted serum samples to assay plate.

6) Cover the plate and incubate at room temperature in dark for 20 minutes while shaking at

800 rpm.

7) Wash the assay plate four times with 200 µL 1X Assay Wash Buffer.

8) Add 25 µL Fluorescence Reporter PE-Anti Human IgG to appropriate wells of assay

plate.

9) Cover the plate with plate seal and incubate at room temperature for 20 minutes while

shaking at 800 rpm.

10) Wash the assay plate four times with 200 µL 1X Assay Wash Buffer.

11) Add 100 µL 1X Assay Wash Buffer to appropriate wells of assay plate.

12) Cover the plate and re-suspend for 10 minutes at 800 rpm shaking.

13) Place the assay plate in correct orientation and read within 60 minutes in the Luminex

Analyzer.

Figure 2. Plate format showing first six wells for controls and calibrator

Results Calculation

All the results are calculated based on a calibrator which is tested in every plate along with the

test samples. The MFI signal ratios of the test sample to the mean of the calibrator on all three

targets are used to qualitatively determine the IgG response in the test sample Table 3.

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𝑀𝐹𝐼 𝑜𝑓 𝑡ℎ𝑒 𝑇𝑒𝑠𝑡 𝑠𝑎𝑚𝑝𝑙𝑒 𝑓𝑜𝑟 𝑒𝑎𝑐ℎ 𝑆𝐴𝑅𝑆 𝐶𝑜𝑉2 𝑃𝑟𝑜𝑡𝑒𝑖𝑛

𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑑𝑢𝑝𝑙𝑖𝑐𝑎𝑡𝑒 𝑣𝑎𝑙𝑢𝑒𝑠 𝑜𝑓 𝑡ℎ𝑒 𝐶𝑎𝑙𝑖𝑏𝑟𝑎𝑡𝑜𝑟 𝑓𝑜𝑟 𝑒𝑎𝑐ℎ 𝑆𝐴𝑅𝑆 𝐶𝑜𝑉2 𝑃𝑟𝑜𝑡𝑒𝑖𝑛

Table 3.Algorithm used for calculating qualitative estimate of human IgG against each of

the three SARS-CoV-2 proteins

𝐓𝐞𝐬𝐭 𝐒𝐚𝐦𝐩𝐥𝐞 𝐌𝐅𝐈

𝐀𝐯𝐞𝐫𝐚𝐠𝐞 𝐂𝐚𝐥𝐢𝐛𝐫𝐚𝐭𝐨𝐫 𝐌𝐅𝐈 Result

> 1.2 SARS-CoV-2 Human IgG Positive

< 0.9 SARS-CoV-2 Human IgG Negative

> 0.9 and < 1.2

SARS-CoV-2 Human IgG Equivocal result. Perform a re-

test using the same sample in duplicate wells. If the

result is same as before sample perform a re-test using

sample collected after 7-10 days.

If one or two of three targets show a

ratio of > 1.2 and one or two show a

ratio of ≥ 0.9 and < 1.2

SARS-CoV-2 Human IgG Equivocal result

Perform a re-test using the same sample in duplicate

wells. If the result is same as before sample is negative.

Performance Characteristics

Analytical sensitivity of the assay

The evaluation of analytical sensitivity of the assay was performed using known SARS-CoV-2

human IgG positive human serum sample. Seven different concentrations (in antibody units,

AU) of the antibody were used for titration. 22 replicates were tested for each concentration and

two different operators performed the testing. Each well measures reactivity of the sample to

three different SARS-CoV-2 antigens. Inter-assay variation was calculated from the testing and

shown in the Table 4. All the three antigens showed good reproducibility and percent co-

efficient of variation of <12.0% for all the concentrations tested during the titration. Each of the

three antigens showed different human IgG antibody reactivity and the intensity of the reactivity

was proportional to the amount of the antibody present in the sample (Figure 3).

Table 4. Analytical sensitivity and precision testing of SARS-CoV-2 Human IgG Assay

SARS-CoV-2

Human IgG

SARS-CoV-2

Protein 1

SARS-CoV-2

Protein 2

SARS-CoV-2

Protein 3

(AU) Mean SD % CV Mean SD % CV Mean SD % CV

2.5 0.41 0.03 7.20 0.39 0.03 7.05 0.32 0.02 5.88

5 0.61 0.05 8.65 0.61 0.05 8.51 0.54 0.04 7.77

10 1.00 0.09 9.12 1.00 0.07 7.23 1.00 0.05 5.42

30 2.91 0.25 8.47 2.85 0.22 7.77 2.84 0.14 4.97

100 7.75 0.63 8.19 7.57 0.56 7.45 6.68 0.29 4.40

300 20.47 2.08 10.15 19.48 1.80 9.25 14.85 0.80 5.40

1000 41.65 4.04 9.69 38.78 3.21 8.28 22.29 1.09 4.89

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Figure 3. SARS-CoV-2 Human IgG Assay reproducibility of 22 individual replicates

Probit analysis was performed to determine the Limit of detection determination. Example of a

probit analysis curve is shown in the following Figure 4. One hundred and fifty-four samples

were used for the LOD determination. 22 replicates of seven concentrations of antibody were

used in this testing.

Table 5. Details about the samples used for LOD determination

Sample size 154

Positive samples 110 (71.43%)

Negative samples 44 (28.57%)

Figure 4. Representative Probit Analysis curve for Limit of detection (LOD) determination for

SARS-CoV-2 Protein 1 antibody.

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All the three SARS-CoV-2 protein antigens gave similar LOD of 7.7 antibody units. The

calibrator that we selected for this test is above the LOD of the test for each of the three SARS-

CoV-2 proteins.

Clinical Performance evaluation of SARS-CoV-2 Human IgG test

Diagnostic sensitivity of assay with respect to RT-PCR positive status

65 de-identified plasma or serum samples from patients tested positive or negative by RT-PCR

were collected and used to evaluate the clinical agreement as a comparator test. SARS-CoV-2

Human IgG test was compared with serum / plasma sampled RT-PCR positive and negative

samples. Positive and negative percent agreements of SARS-CoV-2 Human IgG assay was

determined to be 100 % using results shown in the Table 6.

Table 6. Clinical Agreement between RT-PCR assay and SARS-CoV-2 Human IgG test

Human IgG Positive Human IgG Negative Total

RT-PCR Positive 43 0 43

RT- PCR Negative 0 22 22

Total 43 22 65

Percent Positive Agreement – 100.0% (43/43)

Percent Negative Agreement – 100.0% (22/22)

Clinical Specificity

Three hundred and eight presumed SARS-CoV-2 negative samples from healthy normal subjects

were tested resulting in 100% clinical specificity (CI 99.37% to 100%). Of these samples that

were tested, 26 healthy female and 49 healthy male serum and plasma samples, and 115 gender

unknown samples collected before 2019 were procured from commercial sources. Donor serum

samples from 16 female and 18 male subjects collected in 2020 were also tested. At least 20 /29

samples had known positive 2019 influenza vaccine. They were found to be negative with no

cross reactivity observed in our test. In addition, 84 serum samples from un-identified healthy

samples were also used in this evaluation and were tested negative (Table 7).

Table 7. Distribution of normal healthy samples tested to determine specificity of the test

Healthy Controls M F Unknown Influenza Vaccine

status

Human IgG to SARS-

CoV-2

Before 2019 190 49 26 115 Not known Negative

In 2020 118 18 16 84 20 vaccinated Negative

Total 308 67 42 199 Not known Negative

Cross-reactivity Testing

We included 254 other disease state samples to evaluate cross reactivity. No cross reactivity was

observed with any of these samples in our test. 10 Zika IgG positive, 64 Dengue IgG positive, 6

chikungunya IgG positive, 18 HIV positive, 86 HBV positive, 52 HCV positive, 6 EBV positive

and 6 CMV positive serum samples were tested and showed no cross reactivity with our test. We

also included 2 HCoV 229E and 2 HCoV NL63 positive samples. No cross reactivity was

observed in our SARS-CoV-2 Human IgG antibody test (Table 8).

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Table 8. Distribution of serum/plasma samples with different disease states used in cross-

reactivity testing

Other disease states Before 2019 Percent Cross-Reactivity

Zika IgG Positive 10 0 %

Dengue IgG Positive 64 0 %

Chikungunya IgG Positive 6 0 %

HIV positive 18 0 %

HBV positive 86 0 %

HCV positive 52 0 %

EBV positive 6 0 %

CMV positive 6 0 %

HCoV 229 E 2 0 %

HCoV NL 63 2 0 %

HAMA 1 0 %

Rheumatoid Factor 1 0 %

Total 254 0 %

Distribution of 97 PCR positive samples with respect to SARS-CoV-2 specific IgG is given in

Table 9. The assay sensitivity was determined to be 100 % in convalescent samples where the

blood sample was collected more than 14 days after the first PCR positive result. In 24 out of 66

PCR positive samples, the blood sample in 10/24 subjects was collected on the same day as PCR

test, 11/24 samples were collected one day after the PCR test, and one each after 2, 3, and 7

days after PCR test. We are continuing our efforts to collect more convalescent samples,

collected at least 2 weeks after onset of symptoms, for understanding assay sensitivity in samples

where IgG antibody is truly expected to be present.

Table 9. Blood collection with respect to PCR positive sample collection

Days post PCR positive

result

PCR

positive

SARS-CoV-2 human

IgG Positive % Positive

Unknown / possible acute 23 11 47.8%

0 - 7 days 66 24 36.4%

>14 days 8 8 100.0%

Evaluation of assay interferents

Samples positive for HAMA and rheumatoid factors were sourced commercially and tested. We

also tested purified human serum albumin, purified human IgG, conjugated bilirubin,

unconjugated bilirubin, Levofloxacin, Ceftriaxone, Meropenem, Histamine Hydrochloride,

Zanamivir, Ribavirin, Oseltamivir carboxylate, and triglycerides, and cholesterol. We found no

adverse effects or interference by these substances. Table 10 shows the concentration at which

the assay interferents were tested.

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Table 10. Information about the materials and concentrations used for interferents testing

Assay Interferents Tested Concentration

Hemoglobin 10 mg/mL

Bilirubin Conjugated 0.4 mg/mL

Bilirubin Unconjugated 0.4 mg/mL

Triglycerides 15 mg/mL

Cholesterol 4 mg/mL

Human Serum Albumin 60 mg/mL

Histamine hydrochloride 4 mg/L

Zanamivir 1 mg/L

Oseltamivir carboxylate 1 mg/L

Levofloxacin 200 mg/L

Ceftriaxone 400 mg/L

Meropenem 200 mg/L

Ribavirin 40 mg/L

Human IgG 8 mg/mL

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TETRACORE INC.

9901 BELWARD CAMPUS DRIVE SUITE 300

ROCKVILLE, MD 20850

www.tetracore.com

Phone: 240-268-5400

FAX: 240-268-1107

Tetracore® is a registered trademark of Tetracore.

LUMINEX®, LUMINEX® 200TM, MAGPIX®, and FLEXMAP 3D® are registered trademarks of Luminex Corporation.

http://www.tetracore.com/

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