Doc. No. GB-TCF-106-07/ Rev. No · 2021. 1. 14. · Doc. No. GB-TCF-106-07/ Rev. No.2 Technical File 1 Performance Evaluation Plan 1.1 Purpose To confirm the performance and effectiveness

Doc. No. GB-TCF-106-07/ Rev. No.O

Technical File

Doc. No. GB-TCF-106-07/ Rev. No.2

Technical File

1 Performance Evaluation Plan

1.1 Purpose

To confirm the performance and effectiveness of GenBody Influenza/COVID-19 Ag Multi through

the performance evaluation test and clinical trial designed with reference to the CLSI guideline

1.2 Responsibility

- Test specialist name : Seo seul ki at the GenBody Biotech Institute

- Team leader/first reviewer : Jedae Moon at the GenBody Biotech Institute.

1.3 Test guidance / regulation documents

- diagnostic kit

- European harmonised standard EN13612:2002 and EN23640:2015,

- NCCLS (EP17-A2, EP06-A, EP07-A2, MM17-A, EP05-A3, EP12-A2, EP10-A3, EP09A2)

1.4 Information of the test diagnostic kit - Kit name : GenBody Influenza/COVID-19 Ag Multi

- Catalog No. : CVIFG025

- Batch No : 3 Lots (FMFOS25201, FMFOS25202, FMFOS25203, FAFG04161, FAFG11162,

FAFG18163)

1.5 Intended use

GenBody Influenza/COVID-19 Ag Multi is an immunochromatographic assay kit for

qualitatively and simultaneously detecting the antigens of influenza virus (type A and B) and in

nasopharyngeal swab from human.

1.6 Information of instruments

Confiscope G20 (Analyzer) is optional.

1.7 Information of specimen

Human nasopharyngeal swab and oropharyngeal swab


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2 Analytical performance evaluation

2.1 Analytical Sensitivity / Reference Material for SARS-CoV-2

2.1.1 Protocols

- Material: SARS-CoV-2 Iisolate: USA-WA1/2020) Culture Fluid (Heat inactivated)

(Zeptomatrix corp.)

No Serial dilution Titer

P0 (stock) 1/1x 3.55.E+05

P1 1/300x 1.18.E+03

P2 1/400x 8.88.E+02

P3 1/500x 7.10.E+02

P4 1/600x 5.92.E+02

P5 1/700x 5.07.E+02

P6 1/800x 4.44.E+02

- Method: serial dilution of material spiked in matrix

- No. of tests: single per sample

- Test Kit: GenBody Influenza/COVID-19 Ag Multi

- Protocol: followed by the manual of GenBody Influenza/COVID-19 Ag Multi

- Result analysis

Sample P1 P2 P3 P4 P5 P6

Number

of

repeats

1 Pos Pos Pos Pos Pos Pos








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8 Pos Pos Pos Pos Pos Neg













Pos: positive result, Neg: negative result

Sample Serial dliution Agreement to

expected result Titer

P1 1/300x 20/20 100% 1.18.E+03

P2 1/400x 20/20 100% 8.88.E+02

P3 1/500x 20/20 100% 7.10.E+02

P4 1/600x 20/20 100% 5.92.E+02

P5 1/700x 20/20 100% 5.07.E+02

P6 1/800x 18/20 90% 4.44.E+02



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2.1.2 Conclusion

- As shown in the result tables, GenBody COVID-19 Ag concentration was detected

1/3200x and it is 3.59x 103 TCID50/ml

2.2 Anlytical Sensitivity / Reference Material for influenza A/B Ag

- Material: See under below;


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- Method: Each Instructions for Use and serial dilution

- Test Kit: GenBody Influenza A/B Ag(Lot No.: FAFG04161)

- Protocol: Follow GenBody Influenza A/B Ag IFU and each product IFU

- Test guidance : Evaluation of Detection Capability for Clinical Laboratory Measurement

Procedure; Approved Guideline, EP17-A2, NCCLS

- Results


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- *ELISA analysis

- -sample value : >1.1 : positive,


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- -sample value: >1.1 : positive,


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- -sample value : >1.1 : positive,


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- No. of tests: 20 times per sample



- Result analysis

Sample P12 P13 P14 P15 P16 P17

Number

of

repeats


















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Pos: positivie result, Neg: negative result

P5 20/20 100%

P6 0/20 0%

2.3.2 Conclusion

- As shown in the result tables, GenBody COVID- is

2.87x 103 TCID50/ml

2.4 Analytical Sensitivity / Limit of Detection for influenza A/B Ag

- Metarial: See under below;


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- Method: each IFU

- Test Kit: GenBody Influenza A/B Ag (Lot No.: FAFG04161)

- Protocol: Follow GenBody Influenza A/B Ag Instruction for use and each product IFU



- Result analysis


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11) Influenza Antigen B/Victoria/2/87(B/Beijing/1/87 -Like) LoD

2.4.1.1 Comparison RDT for influenza A/B Ag

- Material

- Method: Spiked Reference Material in negative sample.

- Protocol: Follow GenBody Influenza A/B Ag manual and each product IFU



- Result analysis


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4) Negative

3. Analytical Specificity

3.1. Analytical Specificity (Interfering substances testing) for SARS-CoV-2

3.1.1. Protocols -

Material:

Sort Product name Concentration Titer

Positive

material

SARS-CoV-2 isolate:

USA-WA1/2020) Culture

Fluid (Heat inactivated),

#0810587CFHI

Low (2.5x

LoD)

7.18*103

TCID50/ml

Negative

material Extraction solution N/A N/A

- Method: Material spiked in matrix

- No. of tests: triplecate gle per sample

- Test Kit: GenBody Influenza/COVID-19 Ag Multi (Lot No.: FMFOS25201)


- Test guidance : Interference Testing in Clinical Chemistry ; Approved GuidelineSecond

Edition, EP07-A2, NCCLS

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Interfering Substance Concentration

(mg/dL)

+SARSCoV-

2

-SARS-CoV-

2

Viral Transport Medium

(VTM) 50% + + + - - -

Whole blood 5% + + + - - -

NasoGEL (NeilMed) 5% v/v + + + - - -

Phenylephrine (Nasal

Drop) 10% v/v + + + - - -

Acetylsalicylic acid 20 mg/ml + + + - - -

Beclomethasone 0.5 mg/ml + + + - - -

Benzocaine (Vicks) 5% + + + - - -

Flunisolide 3 mg/ml + + + - - -

Guaiacol glyceryl ether 20 mg/ml + + + - - -

Menthol 10 mg/ ml + + + - - -

Oxymetazoline (Afrin) 15% v/v + + + - - -

Tobramycin 40 mg/ml + + + - - -

Zanamivir 3.3 mg/ml + + + - - -

Oseltamivir phosphate

(Tamiflu) 12 mg/mL + + + - - -

Cromolyn (Nasal Spray) 40 mg/ ml + + + - - -

Homeopathic (Alkalol) 5% v/v + + + - - -

Zicam Cold Remedy 5% v/v + + + - - -

mucous 35% + + + - - -

+: Positive signal -: Negative signal

3.1.3. Conclusion

No positive, smearing and/or negative interference due to each material test was not observed.

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3.2. Analytical Specificity(Interfering substances testing) for influenza A/B Ag

3.2.1. Protocols

- No. of tests: Triplicate per sample

- Test Kit: GenBody Influenza A/B Ag (Lot No.: FAFG04161, FAFC13184)

- Protocol: Follow GenBody Influenza A/B Ag manual

- Test guidance : Interference Testing in Clinical Chemistry ; Approved Guideline-Second Edition,

EP07-A2, NCCLS

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3.2.2. Results

Microorganism Concentration

+SARSCoV-

2

-

SARSCoV-

2

Adenovirus (e.g. C1 Ad. 71) -

Type 7A

1.41 x 105

TCID50/mL + + + - - -

Enterovirus (e.g. EV68) 5.01 x 105

TCID50/mL + + + - - -

Human Metapneumovirus

(hMPV)

3.80 x 106

TCID50/mL + + + - - -

Influenza A H1N1 (New Cal/20/99) 1.15 x 107

TCID50/mL + + + - - -

Influenza B (Florida/02/06) 1.41 x 105

TCID50/mL + + + - - -

Parainfluenza virus 1 9.12 x 108

TCID50/mL + + + - - -


TCID50/mL + + + - - -


TCID50/mL + + + - - -

Parainfluenza virus 4A 1 x 106.58 TCID50/mL

+ + + - - -

Respiratory syncytial virus -Type A 3.80 x 106

TCID50/mL + + + - - -

Rhinovirus (Type 1A) 1 x 106.58 TCID50/mL

+ + + - - -

Bordetella pertussis 1.13 x 1010 CFU/mL

+ + + - - -

Candida albicans 6.27 x 108 CFU/mL

+ + + - - -

Chlamydia pneumoniae 2.12 x 108

IFU/mL + + + - - -

Haemophilus influenzae 5.43 x 108 CFU/mL

+ + + - - -

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Legionella pneumophila 1.63 x 1010 CFU/mL

+ + + - - -

Mycobacterium tuberculosis 6.86 x 107 CFU/mL

+ + + - - -

Mycoplasma pneumoniae 3.16 x 108 CCU/mL

+ + + - - -

Pneumocystis jirovecii (PJP) -S.

cerevisiae Recombinant

3.45 x 108 CFU/mL + + + - - -

Pseudomonas aeruginosa 3.44 x 109 CFU/mL

+ + + - - -

Staphylococcus epidermis 9.27 x 109 CFU/mL

+ + + - - -

Streptococcus pneumoniae 4.16 x 108 CFU/mL

+ + + - - -

Streptococcus pyogenes 1.64 x 109 CFU/mL

+ + + - - -

Streptococcus salivarius 8.17 x 108 CFU/mL

+ + + - - -

MERS-coronavirus 3.55 x 105

TCID50/mL + + + - - -

Human coronavirus 229E 4.17 x 105

TCID50/mL + + + - - -

Human coronavirus OC43 1.26 x 106

TCID50/mL + + + - - -

Human coronavirus NL63 1.41 x 105

TCID50/mL + + + - - -

SARS-coronavirus (in PBS) 1 x 108

PFU/mL + + + - - -

SARS-coronavirus

(Vero E6 Cell DMEM)

1 x 108

PFU/mL + + + - - -

Pooled human nasal wash 100% + + + - - -

Human coronavirus HKU1 N/A Not Tested Not Tested

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3.3. Cross-reactivity for SARS-CoV-2

3.3.1. Protocols

- Material:

- No. of tests: Triplecate per sample



- Test result: There was cross-reactivity of SARS-coronavirus and no cross reactivity was

observed for any of virus and bacterium tested etc.

3.3.3. Conclusion

Other

pathogens

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cell should not be affected to its reactions excepted SARS-CoV.

3.4. Cross-reactivity (Related virus or bacteria) for influenza A/B Ag

# Substance Concentration Results

1 Adenovirus 1

pfu/ml

Negative Negative Negative

2 Adenovirus 2 Negative Negative Negative

3 Coronavirus Negative Negative Negative

4 Measles virus Negative Negative Negative

5 Parainfluenza virus 1 Negative Negative Negative

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3.4.1. Protocols

- No. of tests: Triplicate per sample


- Protocol: Follow GenBody Influenza A/B Ag Kit manual

- Test result: No cross reactivity was observed for any of virus tested etc.

3.4.2. Result

6 Parainfluenza virus 2 Negative Negative Negative


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7 Respiratory syncytial virus Negative Negative Negative

8 Rhinovirus Negative Negative Negative

9 Escherichia coli

108 cells/ml

Negative Negative Negative

10 Staphylococcus epidermidis Negative Negative Negative

11 Streptococcus pneumoniae Negative Negative Negative

12 Streptococcus pyogenes Negative Negative Negative

13 Streptococcus salivarius Negative Negative Negative

3.5. Whole System Failure for SARS-CoV-2

3.5.1. Protocols

- Test purpose: To determine the variation for multiple results of samples (same concentration)

- Material: each sample spiked in matrix. Samples concentration belew under table.

- No. of Tests: singal per run, 100 tests

- Test Kit: GenBody Influenza/COVID-19 Ag Multi (Lot No.:FMOS25201)

- Protocol: Followed by GenBody Influenza/COVID-19 Ag Multi manual

3.5.2. Results

- Whole System Failure: Results was determined within intensity

- Whole system Failure rate= 0% (False negative detection number 0/100 tests)

100 samples were detected at Whole system Failure test

3.6. Additional Analytical Sensitivity / Hook effect (prozone effect)


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3.6.1. Protocols

- Material: SARS-CoV-2 isolate: USA-WA1/2020) Culture Fluid (Heat inactivated)

(Zeptomatrix corp.)

No Serial dilution Titer (TCID50/ml)

P1-H (stock) 1/1x 1.15 x 107

P2-H 1/2x 5.75 x 106

P3-H 1/4x 2.86 x 106

P4-H 1/8x 1.43 x 106

P5-H 1/16x 7.15 x 105

P6-H 1/32x 3.59 x 105

P7-H 1/64x 1.80 x 105


- No. of tests: 2 repeats per sample



- Criteria of Hook effect: If intensity of the test was not further increased (= reaching to plateau

point), specify the interval as the hook effect point.

- Result

Sample P1-H P2-H P3-H P4-H P5-H P6-H P7-H

Number

of

repeats

1 Pos Pos Pos Pos Pos Pos Pos

2 Pos Pos Pos Pos Pos Pos Pos

Plateau effect Not

observed Not

observed Not

observed Not

observed Not

observed Not

observed

Not

observed


3.6.2. Conclusion

- As shown in the result tables, plateau effect was not observed at 1.15 x 107 TCID50/ml


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3.7. Hook effect for influenza A/B Ag

3.7.1. Protocols

- Test purpose: To confim the effect of high concentraion treat in samples.

- Materail: See under the table

- No. of Tests: Singular per run

- Test Kit: GenBody Influenza A/B Ag (Lot No.: FAFC13184)


3.7.2. Results


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- No. of Tests: Triplicates per run, 2 run a day; 5 days


3.8.2. Results

- Precision: Results was determined within intensity

Lot No. FMFOS25201

Sample name Day1 Day2 Day3 Day4 Day5

Repeat 1 2 1 2 1 2 1 2 1 2

NC - - - - - - - - - -

P4 + + + + + + + + + +

P5 + + + + + + + + + +

P6 + + + + + + + + + +


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Lot No. FMFOS25202


Repeat 1 2 1 2 1 2 1 2 1 2

NC - - - - - - - - - -

P4 + + + + + + + + + +

P5 + + + + + + + + + +

P6 + + + + + + + + + +

Lot No. FMFOS25203


Repeat 1 2 1 2 1 2 1 2 1 2

NC - - - - - - - - - -

P4 + + + + + + + + + +

P5 + + + + + + + + + +

P6 + + + + + + + + + +

Positive signal: + Negative signal: -

- No. of Tests: Duplicates per run, 2 run a day; 5 days


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3.10. Reproducibility / Inter-site


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- No. of Tests: Duplicates per run, 2 run a day; 5 days



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Positive Signal: + Negative signal: -

3.11. Reproducibility / Total analysis

3.12. Precision assay for influenza A/B Ag

3.12.1. Protocols

- Test purpose: To determine the variation for multiple results of samples (same concentration)

- Material: each sample spiked in matrix. Samples concentration below under table.

- No. of Tests: Triplicates per run, 2 run a day; 5 days



- Test guidance: Evaluation of Precision Performance of Quantitative Measurement Method ;

Approved Guideline-Second Edition, EP05-A3, NCCLS

3.12.2. Results

Lot No. FAG04161


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NC NEG NEG NEG NEG NEG



P1 POS POS POS POS POS























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POS: Positive NEG: Negative

3.13. Reproducibility / Inter-Operator

3.13.1. Protocols

- Test purpose: Experiment was performed by different operators within one laboratory) -

Material: each sample spiked in matrix. Sample concentration below under table.

- No. of Tests: Triplecates per run



- Test guidance: Evaluation of Precision Perfromance of Quantitative Measurement

Method ; Approved Guideline-Second Edition, EP05-A3, NCCLS


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3.14. Reproducibility / Inter-site

3.14.1. Protocols

- Test purpose: Experiment was performed by different sites within three laboratory - Material:

each sample spiked in matrix. Sample concentration below under table.

- No. of Tests: Triplicates per run



- Test guidance: Evaluation of Precision Perfromance of Quantitative Measurement



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3.15.1. Protocols

- Test purpose: Experiment was performed by different sites within three lot

- Material: each sample spiked in matrix. Sample concentration below under table.


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- Test Kit: GenBody Influenza A/B Ag (Lot No.: FAFG04161, FAFG11162, FAFG18163)


- Test guidance: Evaluation of Precision Performance of Quantitative Measurement



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3.16.1. Protocols

- Material: each sample spiked in matrix. Sample concentration below under table.




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- Test guidance: Evaluation of Precision Performance of Quantitative Measurement Method ; Approved Guideline-Second Edition, EP05-A3, NCCLS

3.16.2. Results

Speci

men Rep

eat 1

D 2

D 3

D 4

D 5

D 6

D 7

D 8

D 9

D 10

D 11

D 12

D 13

D 14

D 15

D 16

D 17

D 18

D 19

D 20

D

N 1 N N N N N N N N N N N N N N N N N N N N

2 N N N N N N N N N N N N N N N N N N N N

3 N N N N N N N N N N N N N N N N N N N N

P1

1 P P P P P P P P P P P P P P P P P P P P



P2




P3




P4




P5




P6




P7




P8




P9




P: positive, N: negative, D: days


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4. Clinical Evaluation/Diagnostic sensitivity & specificity for SAR-CoV-2

4.1. Test Protocol

- Material : 130 clinical samples(30 positive specimens, 100 negative specimens/ see under

each result)

- No. of Tests : Single test per sample

- Test Kit : GenBody Influenza/COVID-19 Ag Multi

- Protocol: Follow GenBody Influenza/COVID-19 Ag Multi manual

- Test guidance : User Protocol for Evalution Qualitative Test Performance ;

Approved Guideline, EP12-A2, NCCLS

- Result analysis : Each target in positive samples were tested Real-Time PCR kit

- * User Protocol for Evalution Qualitative Test Performance ; Approved Guideline, EP12-

A2, NCCLS

4.2. Test Result

CT ≤ 30

Real-time RTPCR

Total Positive Negative

GenBody

INFLUENZA/COVID-

19 Ag Multi Test

Positive 122 3 125

Negative 4 366 370

Total 126 369 495


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-Positive Percent Agreement:

1) CT ≤ 30: 96.83% (95% CI: 92.07% to 99.13%)

2) CT > 30: Less than 50%

-Negative Percent Agreement: 99.18% (95% CI: 97.25% to 99.70%)

-Positive Predictive Value: 96.83% (95% CI: 92.00% to 98.78)

-Negative Predictive Value: 98.92% (95% CI: 97.21% to 99.59%)

5. Clinical Evaluation/Diagnostic sensitivity & specificity for influenza A/B Ag

5.1. Test Protocols

- Material : clinical specimen(see under each result)

- No. of Tests : 53 Influenza A, 62 Influenza B, Single test per sample

- Test Kit : GenBody Influenza A/B Ag


- Clinical evaluation site: Korea University Guro Hospital in Korea

- Test guidance : User Protocol for Evalution Qualitative Test Performance ; Approved

Guideline, EP12-A2, NCCLS

- Result analysis : Each target in positive samples were tested between RT-PCR

confirmed.

Comparison of GenBody Influenza A/B Ag and Asan Easy Test Influenza A/B for

Influenza Type A and Type B result.

- * User Protocol for Evalution Qualitative Test Performance ; Approved Guideline,

EP12-A2, NCCLS


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5.2. Test Results

5.2.1. Sensitivity and specificity

6. Performance claim in IFU Sample type: Nasopharyngeal swab and oropharyngeal swab from human

Documents

Doc. No. GB-TCF-106-07/ Rev. No · 2021. 1. 14. · Doc. No. GB-TCF-106-07/ Rev. No.2 Technical File 1 Performance Evaluation Plan 1.1 Purpose To confirm the performance and effectiveness