COVID-19 International Standards & reference reagents

EIA COVID-19

SARS-CoV-2 was first reported in China in December 2019. The virus has spread worldwide, and The World Health Organization (WHO) has declared it a Pandemic in March 2020. To control the pandemic, COVID-19 vaccines are rapidly being developed and administered worldwide. Reliable assays are needed for their evaluation.

A variety of serological assays for the detection of SARS-CoV-2 antibodies (Ab) and antibody response after vaccination are used globally. WHO has highlighted the importance of the availability of International Standards and reference reagents for anti-SARS-CoV-2 antibodies as it will facilitate the development, validation, and assessment of the assays. It will allow for comparability of results from different assays. It will help diagnose the antibodies after infection, determine the antibody levels needed for efficacious vaccines and improve our understanding of virus epidemiology.

The WHO collaborating centre for biological standardization is National Institute for Biological Standards and Control (NIBSC). NIBSC has a portfolio of reference and research reagents for diagnostics, vaccine development, and research on COVID-19 to support the development of accurate and reliable diagnostic tests.

International Standards and panels, which are listed below, were tested on EIA COVID-19 kits in IgA, IgG, and IgM antibody classes. By titrating these Standards, TestLine responded to the requirement to accurately determine the level of antibodies, sensitivity, and dilution of strongly positive results. Thanks to harmonization with International Standards, the kits can be used for monitoring the antibody concentration after vaccination.

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Intended use: Calibration and harmonisation of serological assays detecting antiSARS-CoV-2 neutralisingantibodies. The preparation can also be used as an internal reference reagent for the harmonisation of bindingantibody assays.Material: Antibody, human, convalescent plasma, WHO ISDescription: Pool of convalescent plasma from recovered COVID-19 patients, containing high titer antibodiesagainst SARS-CoV-2. Unitage: The assigned potency is 250 IU/ampoule for neutralising antibody activity. After reconstitution in 0.25 mLof distilled water, the final concentration of the preparation is 1000 IU/ml. For binding antibody assays, an arbitraryunitage of 1000 binding antibody units (BAU)/ml, can be used to assist the comparison of assays detecting thesame class of immunoglobulins with the same specificity.

First WHO International Standard for anti-SARS-CoV-2immunoglobulin, human (NIBSC code: 20/136)

Titration of the First WHO International standard (20/136)

EIA

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TITRE according to WHO U/ml

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1000 500 250 100 3162 15 7

NP IgA NP IgG NP IgM

EIA

U/m

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TITRE according to WHO U/ml

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1000 500 250 100 3162 15 7

RBD IgA RBD IgG RBD IgM

EIA COVID-19

NP IgA NP IgG NP IgM

U/ml VNT U/ml

1000 250 104.2 320 9.33

500 125 37.91 320 7.2

250 63 15.77 320 6.03

125 31 9.73 320 5

62 16 7 96 5

31 8 5.3 24.46 5

15 4 5 11.53 5

7 2 5 7.2 5

EIA COVID-19

WHO titers RBD IgA RBD IgG RBD IgM

U/ml VNT U/ml

1000 250 320 1000 141

500 125 320 1000 69.2

250 63 228.2 1000 36.8

125 31 67.06 258.6 16.67

62 16 28.4 58.23 10.37

31 8 14 16.97 7.4

15 4 9.1 9.43 5.3

7 2 7.27 6.27 5

Interpretation = negative = borderline = positive

Summary: The NIBSC Anti-SARS-Cov-2 standard is designated as Quality Control Reagent and monitors thesensitivity of serological tests. The sensitivity and dilution management of strongly positive antibody levels wereverified by titration of NIBSC 20/162.

Intended use: Monitoring and estimation of the sensitivity of assay for Anti-SARS-CoV-2 by end-point dilution and assessing any variation in the sensitivity of different lots of assay kits by comparing changes in the end-point dilution. Data obtained with this reagent can be used to confirm that the chosen assay is acceptable for clinical use.Material: Antibody, human, convalescent plasmaDescription: High titer Anti-SARS-CoV-2 antibody positive material used to assess and compare the relativesensitivities for Anti-Sars-CoV-2 antibody assays by end-point dilution.Unitage: This reagent has been assigned an arbitrary unitage of 1000U.

Anti-SARS-CoV-2 Antibody Diagnostic Calibrant(NIBSC code: 20/162)

Titration of Diagnostic Calibrant (20/162)

EIA

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TITRE according to WHO U/ml

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1000 500 250 100 3162 15 7

NP IgA NP IgG NP IgM

EIA

U/m

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TITRE according to WHO U/ml

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1000 500 250 100 3162 15 7

RBD IgA RBD IgG RBD IgM

Interpretation = negative = borderline = positive

Summary: The NIBSC Anti-SARS-Cov-2 standard is designated as Quality Control Reagent and monitors thesensitivity of serological tests. The sensitivity and dilution management of strongly positive antibody levels wereverified by titration of NIBSC 20/162.

EIA COVID-19

NIBSC titers NP IgA NP IgG NP IgM

U/ml U/ml

1000 25.06 320 9.9

500 13.23 320 6.97

250 8.7 320 5.5

125 6.23 191.6 5

62 5.07 42.8 5

31 5 13.83 5

15 5 8.07 5

7 5 5.93 5

EIA COVID-19

NIBSC titers RBD IgA RBD IgG RBD IgM

U/ml U/ml

1000 255,2 1000 17,57

500 69,11 1000 12,67

250 25,91 1000 9,2

125 12,1 1000 6,73

62 9,4 868,6 5,4

31 7,1 123,2 5

15 5,87 25,49 5

7 5,4 12,83 5

Intended use: For use as a verification panel to detect claims by manufacturers on the detection of SARS-CoV-2 antibodies. It can be used as part of a laboratory‘s verification process when adopting a new AntiSARS-CoV-2 assay.Material: Antibody, human, convalescent plasma Anti-SARS-CoV-2 negative plasmaDescription: This product is CE marked for use as an IVD within the EU member states and EEA countries. Inall other territories, it can be used for research purposes only. The Verification Panel consists of 37 samples (23 positive and 14 negative).Unitage: Tables below summarise results obtained for the Anti-SARS-CoV-2 Verification panel for Serology Assays on 13 commercially available assays and one in-house assay. These results are intended only as a guide to the approximate levels of reactivity to be expected and may not be exactly reproduced in other laboratories.

Anti-SARS-CoV-2 Verification Panel for Serology Assays(NIBSC code: 20/B770)

Titration of Verification Panel IgA, IgG, and IgM (20/B770) on EIA COVID-19 kits and comparison with othercommercially available assays and declared samples.

Interpretation = negative = borderline = positive

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declared

14

23

Abbot Siemens Fortress TestLineRoche

Total Antibodies

14

23

14

23

14

23

13

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15

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12

25

Euroimmnun S1

14

23

TestLine RBD+S2

IgA

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35

Liaison S1/S2

14

23

EuroimmnunS1

Siemens RBD EuroimmunNP

TestLine NPDiaPro NP+Spike

TestLine RBD

IgG

14

23

14

23

15

22

16

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14

22

1

12

24

1

0

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35

19

18

Fortress

17

6

14

DiaPro NP+Spike

23

1

13

TestLine RBD

13

24

DiaSorin RBD

IgM

Interpretation = negative = borderline = positive

Summary: The verification Panel was tested on EIA COVID-19. Results from testing show that EIA has comparable results with other 13 commercially available assays and one in-house assay. In some cases, the results are even better. These results are intended only as a guide to the approximate levels of reactivity to be expected.

Intended use: Detection and quantification of anti-SARS-CoV-2 antibodies in an assessment and development of assays.Material: Antibody, human, convalescent plasma, WHO reference panelDescription: It comprises of 5 panel members; four pools of convalescent plasma from recovered COVID-19patients, containing high, medium, low anti-S but relatively high anti-N, low antibodies against SARS-CoV-2,and negative control (healthy donors collected before 2019).Unitage: No unitage has been assigned.

First WHO International Reference Panel for anti-SARS-CoV-2immunoglobulin, human (NIBSC code: 20/268)

Reactivity of anti-RBD antibodies – correlation with Ab 20/268 WHO Reference Panel

High 20/150 Mid 20/148 Low S, High N 20/144 Low 20/140 Neg 20/142

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900Anti-RBD IgG (BAU/ml) EIA RBD IgG

anti-RBD IgG (BAU/ml) EIA COVID-19 RBD IgG

High 20/150 817 914.90

Mid 20/148 205 605.20

Low S/High N 20/144 66 25.29

Low 20/140 45 28.45

Neg 20/142 0 5.69

Reactivity of anti-Nucleocapsid antibodies – correlation with 20/268 WHO Reference Panel

anti-N IgG (BAU/ml) EIA COVID-19 NP IgG

High 20/150 713 728.9

Mid 20/148 146 267.2

Low S/ High N 20/144 146 45.27

Low 20/140 12 5.25

Neg 20/142 0 5.79

High 20/150 Mid 20/148 Low S, High N 20/144 Low 20/140 Neg 20/142

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Anti-N IgG (BAU/ml) EIA NP IgG

Interpretation = negative = borderline = positive

Summary: Testing Reference Panel for anti-SARS-CoV-2 immunoglobulin on EIA COVID-19 is used forassessment and quantitation between antibodies from WHO reference panel and TestLine EIA COVID-19 kits.

TestLine Clinical Diagnostics Ltd.Krizikova 68, 612 00 Brno, Czech Republic

+420 549 121 203sales@testlinecd.comwww.testlinecd.com

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Ordering information

Cat. No. Product No. of wells

CoNA96 EIA COVID-19 NP IgA 96

CoNG96 EIA COVID-19 NP IgG 96

CoNM96 EIA COVID-19 NP IgM 96

CoRA96 EIA COVID-19 RBD IgA 96

CoRG96 EIA COVID-19 RBD IgG 96

CoRM96 EIA COVID-19 RBD IgM 96

SK-CoNA96 SmartEIA COVID-19 NP IgA 96

SK-CoNG96 SmartEIA COVID-19 NP IgG 96

SK-CoNM96 SmartEIA COVID-19 NP IgM 96

SK-CoRA96 SmartEIA COVID-19 RBD IgA 96

SK-CoRG96 SmartEIA COVID-19 RBD IgG 96

SK-CoRM96 SmartEIA COVID-19 RBD IgM 96

SmartEIA kits are designed for automated processing using the Agility® analyser.