1.

Specification of COVID-19
Coronavirus is a group of viruses that belongs to the Family Coronaviridae; a type of RNA virus of 27~32kb commonly
found in birds and mammals including human. Coronavirus is divided into four genera: alpha, beta, gamma and delta.
The virus causes illness ranging from the common cold to more severe diseases such as Middle East Respiratory
Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome (SARS-CoV).
Coronavirus disease 2019 (COVID-19) is a new strain caused by severe acute respiratory syndrome coronavirus 2 (SARS-
CoV-2). The disease originated from Wuhan city of China in December 2019. The World Health Organization (WHO)
publicly named this virus ‘COVID-19’ and declared it a pandemic and a Public Health Emergency of International
Concern. The infection is typically spread from one person to another via direct contact or respiratory droplets from
cough or sneeze. Latent period from exposure to onset of symptoms is between one to fourteen days (four to seven
days on average). Common symptoms and signs of infection include fever, cough, and shortness of breath and
breathing difficulties. In severe cases, infections can cause pneumonia, severe acute respiratory syndrome, kidney failure
and even death.
Due to the wide variety of symptoms, it is difficult to differentiate COVID-19 from other existing respiratory viruses or
bacteria. Diagnosing COVID-19 through isolating the virus or detecting specific genes from the collected respiratory
droplet specimens is a challenge in terms of time and accessibility as it requires long hours, well-equipped laboratory
and advanced technology which are often not available to many public.

2. Analytical sensitivity
2.1. Limit of Detection
2.1.1. Test objective
The study was performed to evaluate analytical sensitivity of the Humasis COVID-19 Ag Home Test.

2.1.2. Test information

a. Materials
(1) Specimen
① Negative standard material (dilution material):
- PBS
- Pooled negative clinical matrix
② Positive standard material:
SARS-CoV-2 virus inactivated by beta-Propiolactone (Conc. 6.3  105 TCID50/mL or 2.5  108 RNA copies/mL,
NMC-nCoV02 #24, sourced from Chungbuk National University)
(2) Reagent
① Humasis COVID-19 Ag Home Test (Lot No. RN-20-611, Exp. 2021.11.05)
② Humasis COVID-19 Ag Home Test (Lot No. RN-20-612, Exp. 2021.11.06)

b. Method
Negative sample was prepared by collecting nasopharyngeal swab samples from 20 healthy donors (negative
clinical matrix) eluted in PBS.
The positive standard materials are prepared with the six different concentrations of SARS-CoV-2 inactivated
virus (Conc. 6.3 x 105 TCID50/mL, NMC-nCoV02 #24, Chungbuk National University) that is serially diluted in
extraction buffer and PBS and negative clinical matrix.
The diluted positive standard materials are applied to the swab tip with 100 μL of approximate absorption
volume. Then the swab was applied to the prepared negative standard material. The swab was moved up and
down inside the tube 10 times and taken out by pressing to remove the extracted liquid. The filter cap was
 equipped onto the test tube, then three drops of extracts (100 μL) was dispensed into the sample inlet. The
result was read 15 minutes after applying the sample.
Serial dilutions of the inactivated SARS-CoV-2 were tested in 3 replicates. The lowest concentration at which all
3 replicates were positive was treated as the tentative LoD for each test. The LoD of each test was then
confirmed by testing 20 replicates with concentrations at the tentative limit of detection. The final LoD of
Humasis COVID-19 Ag Home Test was determined to be the lowest concentration resulting in positive detection
more than 95% of the time, which is at least 19 out of 20 replicates.

1) Positive standard material was spiked into negative standard material and serial dilution was made to obtain
6 level of concentrations as shown in below table. Each concentration level was tested using Humasis COVID-19
Ag Home Test according to instructions in 3 replicates.
COVID-19 Virus 1 2 3 4 5 6
Dilution rate 1/1,000 1/5,000 1/20,000 1/50,000 1/80,000 1/320,000
Concentration 1X102.8 2X101.8 5X100.8 2X100.8 1.25X100.8 0.3X100.8
(TCID50/mL)

2) Concentration near suspected cut-off was diluted to make 3 concentration levels as shown in below table and
each level was tested in 20 replicates.
COVID-19 Virus 1 2 3
Dilution rate 1/20,000 1/50,000 1/80,000
Concentration 5X100.8 2X100.8 1.25X100.8
(TCID50/mL)

2.1.3. Interpretation of results

1) Place the Humasis COVID-19 Ag Home Test on the level surface. Dispense the prepared samples into the device
according to instruction for use. Read the result at 15 minutes after applying the sample to determine limit of
detection.
2) Result interpretation is done according to the internal standard operating procedure (Humasis SOP QA-004)
and the results are recorded.

2.1.4. Acceptance criteria

Limit of detection is defined as the concentration of SARS-CoV-2 virus that produces positive results more than
95% of the time when tested using Humasis COVID-19 Ag Home Test.

2.1.5. Test result

① Serial dilution
COVID-19 Virus 1 2 3 4 5 6
Dilution rate 1/20,000 1/50,000 1/80,000 1/20,000 1/50,000 1/80,000
Concentration (TCID50/mL) 5X100.8 2X100.8 1.25X100.8 5X100.8 2X100.8 1.25X100.8
Test result 1/20,000 1/50,000 1/80,000 1/20,000 1/50,000 1/80,000
(No. of positive/ No. of
replicates)

② Dilution near cut-off

COVID-19 Virus 1 2 3
Dilution rate 1/20,000 1/50,000 1/80,000
Concentration (TCID50/mL) 5X100.8 2X100.8 1.25X100.8
Test result 20/20 18/20 0/20
(No. of positive/ No. of
replicates)
 2.1.6. Conclusion
The limit of detection (LoD) of Humasis COVID-19 Ag Home Test is 32 TCID50/mL (5 x 100.8 TCID50/mL).
1 TCID50/mL is equivalent to approximately 4,000 RNA copies/mL, indicating the LoD of Humasis COVID-19 Ag
Home Test 128,000 RNA copies/mL.

2.2. Reactivity/ Inclusivity

2.2.1. The live virus inclusivity tests of the Alpha and Beta variants (Wet testing)
a. Test objective
The study was performed to evaluate analytical reactivity and inclusivity of the Humasis COVID-19 Ag Home Test on
SARS-CoV-2 variants.

b. Test information
Negative sample was prepared by collecting nasopharyngeal swab samples from healthy donors eluted in extraction
buffer.
Alpha variant (B.1.1.7_hCoV-19/Korea/KDCA51463/2021) and Beta variant (B.1.351_hCoV-
19/Korea/KCDA55905/2021) at a concentration of 1.3 х 105 TCID50/mL sourced from Chungbuk National University
was used for the study. The titer was determined by measuring it cytopathic effect (CPE) using 3-day cell culture
virus.
The positive standard materials of 6 levels of concentrations are prepared with each cell culture virus strains that is
diluted in negative sample. The diluted positive standard materials are applied to the sterile swab, which is included
with the test, and conducted the testing following the instructions for use. Dilutions of each cell culture virus strains
were tested in 3 replicates using the Humasis COVID-19 Ag Home Test.
COVID-19 Virus 1 2 3 4 5 6
Dilution rate 1/100 1/500 1/1,000 1/5,000 1/10,000 1/50,000
Concentration
1.3  103 2.5  102 1.3  102 2.5  101 1.3 x 101 2.5  100
(TCID50/mL)

c. Test results
According to the results, the Alpha variant and Beta variant were detected up to 25 TCID50/mL using Humasis
COVID-19 Ag Home Test.
Alpha Beta
Conc.
(TCID50/mL) GR (B.1.1.7) GH (B.1.351)
(positive/positive) (positive/positive)
1/100
3/3** 3/3**
1.3  103
1/500
3/3** 3/3**
2.5  102
1/1,000
3/3** 3/3**
1.3  102
1/5,000
3/3** 3/3**
2.5  101

1/10,000 0/3** 0/3**

 1.3 x 101

1/50,000
0/3** 0/3**
2.5  100
**: positive/positive

2.2.2. The reactivity tests of the mutant NP and RBD using recombinant antigens
a. Test objective
The study was performed to evaluate important mutant NP and RBD proteins.

b. Test information
To evaluate the reactivity of important mutant NP and RBD proteins, the below recombinant antigens were used
for the study.
Nasopharyngeal swab sample from healthy donors were collected and eluted in PBS to be used as a negative
standard material. Antigens were diluted into the negative standard material to obtain 5 levels of concentrations: 1
µg/mL, 100 ng/mL, 10 ng/mL, 1 ng/mL, and 100 pg/mL. Each of the prepared positive samples were added to the
sterile swab included with the test device and conducted the testing according to the instructions for use. Each level
was tested in 3 replicates using the Humasis COVID-19 Ag Home Test.
No Name Spread in Cat. Stock HOST Manufacturer Mutation

1 SARS-CoV-2 Spike protein RBD - - 2.4 mg/mL - Celltrion as control

SARS-CoV-2 (2019-nCoV) Spike 0.25 HEK293

2 Europe (20A.EU2) 40592V08H46 SinoBiological S477N
RBD(S477N)-His Tag mg/mL Cells

SARS-CoV-2 (2019-nCoV) Spike U.K, South Africa, 0.25 HEK293

3 40592-V08H82 SinoBiological N501Y
RBD(N501Y)- His Tag Brazil, mg/mL Cells

SARS-CoV-2(COVID-19) S South Africa,

HEK293
4 Protein RBD(E484K)-His Brazil, U.K, US SRD-C52H3 0.6 mg/mL Acro Biosystems E484K
Tag(MALS verified) Cells
(New York)

SARS-CoV-2(COVID-19) S K417N,
HEK293
5 Protein RBD(K417N, E484K, South Africa SPD-C52Hp 0.6 mg/mL Acro biosystems E484K,
N501Y)-HisTag(MALS verified) Cells
N501Y

SARS-CoV-2 (2019-nCoV) Spike

Europe, South 0.25 HEK293
6 RBD(N439K)-His Recombinant
East Asia
40592-V08H14
mg/mL Cells
SinoBiological N439K
Protein

SARS-CoV-2 (2019-nCoV) Spike

0.25 HEK293
7 RBD(K417N)-His Recombinant - 40592-V08H59 SinoBiological K417N
Protein mg/mL Cells

SARS-CoV-2 (2019-nCoV) Spike

UK, US 0.15 HEK293
8 RBD(L452R)-His Recombinant
(California), India
40592-V08H28
mg/mL Cells
SinoBiological L452R
Protein

SARS-CoV-2 (2019-nCoV) Spike

0.25 HEK293
9 RBD(E484Q)-His Recombinant India 40592-V08E81
mg/mL Cells
SinoBiological E484Q
Protein
 SARS-CoV-2 (2019-nCoV) Spike L452R
0.25 HEK293
10 RBD(L452R, E484Q) Protein India 40592-V08E88 SinoBiological
(His Tag) mg/mL Cells E484Q

SARS-CoV-2 Spike RBD(L452R, 0.25 HEK293 L452R

11 UK, India 40592-V08E90 SinoBiological
T478K) Protein (His Tag) mg/mL Cells T478K
SARS-CoV-2 Nucleocapsid
12 Protein (His tag)
- HC-N001 0.7 mg/mL Insect cells Humasis As control

SARS-CoV-2 (2019-nCoV) R203K

Japan, Brazil, 0.25
13 Nucleocapsid(R203K, G204R)- 40588-V07E1 E. coli SinoBiological
His Recombinant Protein Europe, US mg/mL G204R
SARS-CoV-2 (2019-nCoV) D3L
0.25
14 Nucleocapsid(D3L, S235F)-His Europe 40588-V07E8 E. coli SinoBiological
Recombinant Protein mg/mL S235F
SARS-CoV-2 (2019-nCoV)
0.25
15 Nucleocapsid (T205I) Protein US (California) 40588-V07E9 E. coli SinoBiological T205I
(His Tag) mg/mL

D3L
SARS-CoV-2 (2019-nCoV)
Nucleocapsid(D3L, R203K, Europe (UK), US 0.25 R203K
16 40588-V07E7 E. coli SinoBiological
G204R, S235F)-His (Florida) mg/mL G204R
Recombinant Protein
S235F
SARS-CoV-2 (2019-nCoV)
0.25
17 Nucleocapsid (D377Y) Protein UK, India 40588-V07E16 E. coli SinoBiological D377Y
(His Tag) mg/mL

SARS-CoV-2 Nucleocapsid P199L

0.25
18 (P199L, M234I) Protein (His US (New York) 40588-V07E18 E. coli SinoBiological
Tag) mg/mL M234I

SARS-CoV-2 Nucleocapsid US (Texas, New 0.25

19 40588-V07E12 E. coli SinoBiological P199L
(P199L) Protein (His Tag) York) mg/mL

SARS-CoV-2 Nucleocapsid 0.25

20 US (Texas) 40588-V07E14 E. coli SinoBiological P67S
(P67S) Protein (His Tag) mg/mL

D63G
SARS-CoV-2 Nucleocapsid
21 (D63G, R203M, D377Y) Protein UK, India 40588-V07E29 0.2 mg/mL E. coli SinoBiological R203M
(His Tag)
D377Y
P67S
SARS-CoV-2 Nucleocapsid
0.25
22 (P67S, R203M, D377Y) Protein - 40588-V07E30 E. coli SinoBiological R203M
(His Tag) mg/mL
D377Y

c. Test results
The study using recombinant antigens showed that the Humasis COVID-19 Ag Home Test detected various mutation
of NP and RBD proteins shown in Alpha, Beta, Gamma and Delta variants.
① Test results of recombinant RBD mutant antigen

RBD
RBD mutation
Con. as control

1 2 3 4 5 6

1 μg/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

 100 ng/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

10 ng/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

1 ng/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

100 pg/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

RBD
RBD mutation
Con. as control

1 7 8 9 10 11

1 μg/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

100 ng/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

10 ng/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

1 ng/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

100 pg/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

**: positive/positive
② Test results of recombinant NP mutant antigen

NP
NP mutation
Con. as control

12 13 14 15 16 17

1 μg/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

100 ng/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

10 ng/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

1 ng/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

100 pg/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

NP
NP mutation
Con. as control

12 18 19 20 21 22

1 μg/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

100 ng/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

10 ng/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

1 ng/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

100 pg/mL 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

**: positive/positive

2.2.3. The reactivity tests of recombinant RBD mutant antigens of Pseudo virus
a. Test objective
The study was performed to evaluate important mutant RBD proteins.
 b. Test information
To confirm the reactivity of important mutant RBD (California and New York variant strains) antigens, pseudo virus
which was produced in HEK293T cells using three separate plasmids, encoding mutant RBD (L423R or E484K) spike
protein, a lentiviral gag polyprotein, and a reporter gene were used for this study. The pseudo virus (Lentivirus)
concentration was quantitated by PCR.
Nasopharyngeal swab sample from healthy donors were collected and eluted in sample extraction buffer to be used
as a negative standard material. Each pseudo viruses were diluted into the negative standard material to obtain 6
levels of concentrations as shown in below table. Each of the prepared positive samples were added to the sterile
swab included with the test device and conducted the testing according to the instruction for use. Each level was
tested in 3 replicates using the Humasis COVID-19 Ag Home Test.
Pseudo Virus
(California strain- 1 2 3 4 5 6
L452R)
Dilution rate 1/5 1/10 1/20 1/40 1/80 1/160
Concentration
1.89  108 9.44x107 4.72  107 2.36  107 1.18 x 107 5.9 x 106
(copies/mL)

Pseudo Virus
(New York strain- 1 2 3 4 5 6
E484K)
Dilution rate 1/5 1/10 1/20 1/40 1/80 1/160
Concentration
1.26  108 6.30x107 3.15  107 1.58  107 7.88 x 106 3.94 x 106
(copies/mL)

c. Test results
According to the results, the California strain (L452R) and New York strain (E484K) were detected up to 5.9 х 106
copies/mL and 3.94 х 106 copies/mL, respectively, using the Humasis COVID-19 Ag Home Test.
Pseudo Virus
(California strain- 1 2 3 4 5 6
L452R)
Concentration
1.89  108 9.44x107 4.72  107 2.36  107 1.18 x 107 5.9 x 106
(copies/mL)
Results 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**

Pseudo Virus
(New York strain- 1 2 3 4 5 6
E484K)
Concentration
1.26  108 6.30x107 3.15  107 1.58  107 7.88 x 106 3.94 x 106
(copies/mL)
Results 3/3** 3/3** 3/3** 3/3** 3/3** 3/3**
**: positive/positive

2.2.4. Conclusion
The Humasis COVID-19 Ag Home Test can detect Alpha and Beta variants up to 25 TCID50/mL.
Reactivity to the following recombinant antigens in which each important amino acid of NP and RBD mutation
observed in various SARS-CoV-2 variants including Alpha, Beta, Gamma and Delta was confirmed up to 100 ng/mL:
S protein RBD mutation N protein mutation
S477N/ N501Y/ E484K/ K417N, E484K, N501Y/ R203K, G204R/ D3L, S235F/ T205I/ D3L, R203K,
N439K/ K417N/ L452R/ E484Q/ L452R, E484Q/ G204R, S235F/ D377Y/ P199L, M234I/ P199L/ P67S/
L452R, T478K D63G, R203M, D377Y/ P67S, R203M, D377Y
3. Precision
3.1. Test objective
The study was performed to evaluate precision of the Humasis COVID-19 Ag Home Test.

3.2. Test information

3.2.1. Materials
1) Standard material
(1) Negative standard material: Specimen extraction buffer
(2) Positive standard material: SARS-CoV-2 inactivated virus (NMC-nCoV02 #24, 1x105.8 TCID50/mL, Chungbuk
National University)
2) Reagent: Humasis COVID-19 Ag Home Test
① Lot# RN-20-610, expiration date: 2022.06.18
② Lot# RN-20-611, expiration date: 2022.06.30
③ Lot# RN-20-612, expiration date: 2022.07.09

3.2.1. Method
Positive standard materials were spiked into negative standard material and were diluted to make high, medium
and low concentration levels as shown in below table. Each concentration level was tested using Humasis COVID-
19 Ag Home Test according to instructions at Humasis Central R&D Center. 4 individual studies were performed:
repeatability (within-laboratory precision), between-operator precision, between-lot precision and between-place
precision.
Level SARS-CoV-2 inactivated virus concentration
(TCID50/mL)
High (approx. 20xLoD) 631
Medium (approx. 4xLoD) 126
Low (approx. 2xLoD) 63
1) Repeatability (Within-laboratory precision)
Prepared negative and positive samples were tested using the Humasis COVID-19 Ag Home Test for 5 consecutive
days, 2 times daily, in 3 replicates per each run.
Lot# RN-20-610
Period 2020.07.27-2020.07.31
Operator S.M.YANG

2) Reproducibility
(1) Between-operator precision
Prepared negative and positive samples were tested by 3 different operators using the Humasis COVID-19 Ag
Home Test for 5 consecutive days, 2 times daily, in 3 replicates per each run.
Lot# RN-20-610
Period 2020.08.03-2020.08.07
Operator S.M.YANG/ S.J.HUH/ D.M.KIM (operator 1, 2, 3)

(2) Between-lot precision

Prepared negative and positive samples were tested using the Humasis COVID-19 Ag Home Test from 3 different
lots for 5 consecutive days, 2 times daily, in 3 replicates per each run.
Lot# RN-20-610/ RN-20-611/ RN-20-612
 Period 2020.08.03-2020.08.07
Operator S.M.YANG

(3) Between-place precision

Prepared negative and positive samples were tested in 3 different places using the Humasis COVID-19 Ag Home
Test for 5 consecutive days, 2 times daily, in 3 replicates per each run.
Lot# RN-20-610
Period 2020.08.03-2020.08.07
Operator S.M.YANG
Place QC laboratory/ Central R&D AC room/ Central R&D laboratory

3.3. Acceptance criteria

Test results of all positive samples should show positive results and all negative samples should show negative results.

3.4. Test result

① Repeatability
Test results
Date
Negative Low pos. Medium pos. High pos.
2020.07.27 6/6* 6/6** 6/6** 6/6**
2020.07.28 6/6* 6/6** 6/6** 6/6**
2020.07.29 6/6* 6/6** 6/6** 6/6**
2020.07.30 6/6* 6/6** 6/6** 6/6**
2020.07.31 6/6* 6/6** 6/6** 6/6**
*: Negative/ **: Positive

② Reproducibility
Test results
Test type
Negative Low pos. Medium pos. High pos.
Operator 1 30/30* 30/30** 30/30** 30/30**
Between-operator Operator 2 30/30* 30/30** 30/30** 30/30**
Operator 3 30/30* 30/30** 30/30** 30/30**
RN-20-610 30/30* 30/30** 30/30** 30/30**
Between-lot RN-20-611 30/30* 30/30** 30/30** 30/30**
RN-20-612 30/30* 30/30** 30/30** 30/30**
R&D AC room 30/30* 30/30** 30/30** 30/30**
Between-place QC Lab 30/30* 30/30** 30/30** 30/30**
R&D Lab 30/30* 30/30** 30/30** 30/30**
*: Negative/ **: Positive

3.5. Conclusion
The Humasis COVID-19 Ag Home Test showed consistent performance within laboratory, between operators, between
lots and between places, and all the results showed 100% agreement with the expected results.
4. Cross-reactivity
4.1. Test objective
The study was performed to evaluate cross-reactivity of the Humasis COVID-19 Ag Home Test.

4.2. Test information

4.2.1. Materials
1) Standard material
(1) Negative standard material: Specimen extraction buffer
(2) Positive standard material: SARS-CoV-2 inactivated virus (NMC-nCoV02 #24, 1x105.8 TCID50/mL, Chungbuk
National University)
2) Reagent
Humasis COVID-19 Ag Home Test (Lot# RN-20-610, expiration date: 2022.06.18)
3) Cross-reactive substances
Below 32 viruses (105PFU/mL and above) and bacteria (106CFU/mL and above) were sourced from Korea National
Research Resource Center (KNRRC) for the test.
Virus (≥105PFU/mL)
1 Coronavirus OC43 11 Parainfluenza 1
2 Coronavirus 229E 12 Parainfluenza 2
3 Coronavirus NL63 13 Parainfluenza 3
4 MERS-coronavirus 14 Parainfluenza 4a
5 Human adenovirus 1 15 Rhinovirus 1
6 Human adenovirus 3 16 Metapneumovirus
7 Human adenovirus 5 17 Human Enterovirus
8 Human adenovirus 7 18 Influenza A H1N1
9 Respiratory syncytial virus A 19 Influenza A H3N2
10 Respiratory syncytial virus B 20 Influenza B
Bacteria (≥106CFU/mL)
21 Mycoplasma pneumonia Ag 27 Candida albicans
22 Streptococcus pyogenes 28 Chlamydia pnuemoniae
23 Bordetella pertussis 29 Staphylococcus epidermidis
24 Streptococcus pneumoniae 30 Staphylococcus aureus
25 Legionella pneumophila 31 Enterococcus casseliflavus
26 Haemophilus influenzae
Others (100%)
32 Pooled human nasal wash – to represent diverse microbial flora in the human respiratory tract

4.2.2. Methods for wet testing

Extraction buffer was used as negative sample. Positive standard materials were spiked into negative sample and
were diluted to make low concentration level (63 TCID50/mL, approx. 2xLoD) for testing. Each of the prepared cross-
 reactive substances were added to the negative and positive standard materials and were tested using Humasis
COVID-19 Ag Home Test in 3 replicates following the instructions.

4.2.3. Methods for in-silico

To estimate the likelihood of cross-reactivity with SARS-CoV-2 virus in the presence of organisms that were not
available for wet testing, in silico analysis using the Basic Local Alignment Search Tool (BLAST) managed by the
National Center for Biotechnology Information (NCBI) was used to assess the degree of protein sequence homology.

4.3. Acceptance criteria

Test results of all positive samples should show positive results and all negative samples should show negative results.

4.4. Test result

4.4.3. Wet testing
Test result
No. Cross-reactive substances Positive
Negative
Low positive
1 Coronavirus OC43 3/3* 3/3**

2 Coronavirus 229E 3/3* 3/3**

3 Coronavirus NL63 3/3* 3/3**

4 MERS-coronavirus 3/3* 3/3**

5 Human adenovirus 1 3/3* 3/3**

6 Human adenovirus 3 3/3* 3/3**

7 Human adenovirus 5 3/3* 3/3**

8 Human adenovirus 7 3/3* 3/3**

9 Respiratory syncytial virus A 3/3* 3/3**

10 Respiratory syncytial virus B 3/3* 3/3**

11 Parainfluenza 1 3/3* 3/3**

12 Parainfluenza 2 3/3* 3/3**

13 Parainfluenza 3 3/3* 3/3**

14 Parainfluenza 4a 3/3* 3/3**

15 Rhinovirus 1 3/3* 3/3**

16 Metapneumovirus 3/3* 3/3**

17 Human Enterovirus 3/3* 3/3**

18 Influenza A H1N1 3/3* 3/3**

19 Influenza A H3N2 3/3* 3/3**

20 Influenza B 3/3* 3/3**

21 Mycoplasma pneumonia Ag 3/3* 3/3**

22 Streptococcus pyogenes 3/3* 3/3**

 23 Bordetella pertussis 3/3* 3/3**

24 Streptococcus pneumoniae 3/3* 3/3**

25 Legionella pneumophila 3/3* 3/3**

26 Haemophilus influenzae 3/3* 3/3**

27 Candida albicans 3/3* 3/3**

28 Chlamydia pnuemoniae 3/3* 3/3**

29 Staphylococcus epidermidis 3/3* 3/3**

30 Staphylococcus aureus 3/3* 3/3**

31 Enterococcus casseliflavus 3/3* 3/3**

32 Pooled human nasal wash 3/3* 3/3**

*: Negative/ **: Positive

4.4.3. In-silico
To estimate the likelihood of cross-reactivity with SARS-CoV-2 virus in the presence of organisms that were not
available for wet testing, in silico analysis using the Basic Local Alignment Search Tool (BLAST) managed by the
National Center for Biotechnology Information (NCBI) was used to assess the degree of protein sequence homology.
- Human coronavirus HKU1: 12% homology was found between SARS-CoV-2 Receptor Binding Domain spike
proteins and HKU1 spike protein, and 32% homology was found between SARS-CoV-2 Nucleocapsid protein and
HKU1 Nucleocapsid protein. Therefore, cross-reactivity is highly unlikely but cannot be ruled out.
- Pneumocystis jirovecii: No sequence homology was found between SARS-CoV-2 RBD spike protein/ nucleocapsid
protein and P. jirovecii. Therefore, there is no cross-reactivity.
- Mycobacterium tuberculosis: There was 45.6% homology across 9% of the whole sequence between M.
tuberculosis and SARS-CoV-2 RBD spike protein. No similarity was found between M. tuberculosis and SARS-CoV-2
NP. Therefore, cross-reactivity is highly unlikely but cannot be ruled out.
- SARS-CoV: 72% homology was found between SARS-CoV-2 receptor binding domain spike proteins and SARS-CoV
spike protein, and 96% homology was found between SARS-CoV-2 nucleocapsid protein and SARS-CoV nucleocapsid
protein. Therefore cross-reactivity is highly likely.

4.5. Conclusion
The Humasis COVID-19 Ag Home Test showed no cross-reactivity with above 32 organisms tested.
For organisms not available for wet testing, in silico analysis was conducted and was found that the Humasis COVID-19
Ag Home Test does not differentiate SARS-CoV and SARS-CoV-2. Cross-reactivity with human coronavirus HKU1 and
Mycobacterium tuberculosis is highly unlikely but cannot be ruled out completely.
5. Interference
5.1. Test objective
The study was performed to evaluate interference of the Humasis COVID-19 Ag Home Test.

5.2. Test information

5.2.1. Materials
1) Standard material
(1) Negative standard material: Specimen extraction buffer
(2) Positive standard material: SARS-CoV-2 inactivated virus (NMC-nCoV02 #24, 1x105.8 TCID50/mL, Chungbuk
National University)
2) Reagent
Humasis COVID-19 Ag Home Test (Lot# RN-20-610, expiration date: 2022.06.18)
3) Interfering substances
41 potential interfering substances were sourced and prepared.
5.2.2. Methods
Extraction buffer was used as negative sample. Positive standard materials were spiked into negative sample and
were diluted to make low concentration level (63 TCID50/mL, approx. 2xLoD) for testing. Each of the prepared
interfering substances were added to the negative and positive standard materials and were tested using Humasis
COVID-19 Ag Home Test in 3 replicates following the instructions. Samples without interfering substance added
were also tested in 3 replicates.

5.3. Acceptance criteria

Test results of all positive samples should show positive results and all negative samples should show negative results.

5.4. Test result

Negative + Low pos. +
No. Interfering substances Concentration Negative Interfering Low positive Interfering
substances substances
1 Whole blood 4% 3/3* 3/3* 3/3** 3/3**
2 Mucin 0.5% 3/3* 3/3* 3/3** 3/3**
3 Chloraseptic 1.5 mg/mL 3/3* 3/3* 3/3** 3/3**

4 NeilMed NasoGel 5% v/v 3/3* 3/3* 3/3** 3/3**

5 CVS Nasal drops 15% v/v 3/3* 3/3* 3/3** 3/3**

6 Afrin (Oxymetazoline) 15% v/v 3/3* 3/3* 3/3** 3/3**

Sodium cromoglycate
7 15% v/v 3/3* 3/3* 3/3** 3/3**
(CVS nasal spray, Cromolyn)

8 Zicam 15% v/v 3/3* 3/3* 3/3** 3/3**

9 Homeopathic (Alkalol) 1:10 dilution 3/3* 3/3* 3/3** 3/3**

10 Sore throat Phenol Spray 15% v/v 3/3* 3/3* 3/3** 3/3**

11 Tobramycin 5 μg/mL 3/3* 3/3* 3/3** 3/3**

12 Mupirocin 10 mg/mL 3/3* 3/3* 3/3** 3/3**

13 Fluticasone Propionate 5% v/v 3/3* 3/3* 3/3** 3/3**

Tamiflu (Oseltamivir
14 5 mg/mL 3/3* 3/3* 3/3** 3/3**
Phosphate)
 15 Albumin, human 3000 mg/dL 3/3* 3/3* 3/3** 3/3**

16 Bilirubin 500 μmol/L 3/3* 3/3* 3/3** 3/3**

17 Hemoglobin 500 mg/dL 3/3* 3/3* 3/3** 3/3**

18 Cholesterol 20 μmol/L 3/3* 3/3* 3/3** 3/3**

19 Triglyceride 1000 mg/dL 3/3* 3/3* 3/3** 3/3**

20 Biotin 0.75 mg/mL 3/3* 3/3* 3/3** 3/3**

21 Sodium citrate 25 mg/mL 3/3* 3/3* 3/3** 3/3**

22 Heparin 100 U/mL 3/3* 3/3* 3/3** 3/3**

23 EDTA 5 μmol/L 3/3* 3/3* 3/3** 3/3**

24 K3-EDTA 20 mg/mL 3/3* 3/3* 3/3** 3/3**
Diphenhydramine
25 5 mg/mL 3/3* 3/3* 3/3** 3/3**
hydrochloride

26 Acetaminophen 199 μmol/L 3/3* 3/3* 3/3** 3/3**

27 Acetylsalicylic acid 3.62 mmol/L 3/3* 3/3* 3/3** 3/3**

28 Ibuprofen 2.425 mmol/L 3/3* 3/3* 3/3** 3/3**

29 Olopatadine hydrochloride 5 mg/mL 3/3* 3/3* 3/3** 3/3**

Hanmi Ko-and-Cool Nasal

30 Spray 10%(v/v) 3/3* 3/3* 3/3** 3/3**
(Chlorpheniramine Maleate 250 mg/ 100 mL,
Xylometazoline Hydrochloride 0.1 g/100 mL)

Samchundang Narista-S Nasal

Spray
31 10%(v/v) 3/3* 3/3* 3/3** 3/3**
(Chlorpheniramine Maleate 2.5 mg/mL,
Dipotassium Glycyrrhizinate 3 mg/mL,
Naphazoline Hydrochloride 0.5 mg/mL)

32 Sodium chloride 20 mg/mL 3/3* 3/3* 3/3** 3/3**

33 Zanamivir 5 mg/mL 3/3* 3/3* 3/3** 3/3**

34 Oseltamivir 10 mg/mL 3/3* 3/3* 3/3** 3/3**

35 Artemether-lumefantrine 50 μmol/L 3/3* 3/3* 3/3** 3/3**

36 Doxycycline hyclate 70 μmol/L 3/3* 3/3* 3/3** 3/3**

37 Quinine 150 μmol/L 3/3* 3/3* 3/3** 3/3**

38 Lamivudine 1 mg/mL 3/3* 3/3* 3/3** 3/3**

39 Erythromycin 81.6 μmol/L 3/3* 3/3* 3/3** 3/3**

40 Ciprofloxacin 30.2 μmol/L 3/3* 3/3* 3/3** 3/3**

Rheumatoid factor positive

41 10%(v/v) 3/3* 3/3* 3/3** 3/3**
plasma
*: Negative/ **: Positive

5.5. Conclusion
41 interfering substances tested above did not affect the performance of Humasis COVID-19 Ag Home Test.
6. High-dose hook effect
6.1. Test objective
The study was performed to evaluate high-dose hook effect of the Humasis COVID-19 Ag Home Test.

6.2. Test information

6.2.1. Materials
1) Standard material
(1) Negative standard material: Specimen extraction buffer
(2) Positive standard material: SARS-CoV-2 virus inactivated by beta-Propiolactone(BPL) (NMC-nCoV02 #24, 1x105.8
TCID50/mL, Chungbuk National University)
2) Reagent
Humasis COVID-19 Ag Home Test (Lot# RN-20-610, expiration date: 2022.06.18)

6.2.2. Methods
Positive standard materials were spiked into negative standard material and were diluted to make various high
concentration levels of SARS-CoV-2. Prepared samples of each concentration levels were tested using Humasis COVID-
19 Ag Home Test in 3 replicates following instructions.
6.3. Acceptance criteria
All test results should show positive results.

6.4. Test result

SARS-CoV-2 inactivated virus
(1  105.8 TCID50/mL)
Test results
TCID50/mL
(No. of positives/ No. of replicates)
(concentration)
Lot 1 Lot 2
5 100.8
3/3 3/3
[1xLoD]
2  102.8
3/3 3/3
[4xLoD]
2.5  103.8
3/3 3/3
[500xLoD]
1  105.8
3/3 3/3
[20,000xLoD]

6.5. Conclusion
No high-dose hook effect was observed up to 1x105.8 TCID50/mL, approx. 20,000xLoD.
7. Flex studies
Various flex studies were conducted to evaluate the performance of Humasis COVID-19 Ag Home Test under
possible stressful environment.
Nasopharyngeal swab sample from healthy donors were collected and eluted in extraction buffer to be used as
negative sample. Positive standard materials (NMC-nCoV02 #24, 1  105.8 TCID50/mL, Chungbuk National
University) were spiked into negative sample to make low positive concentration (63 TCID50/mL, approx. 2xLoD)
for testing.
Acceptance criteria for all studies were that the test results of all positive samples should show positive results
and all negative samples should show negative results.

(1) Temperature and humidity

i. Study objective:
The study was performed to evaluate performance of the opened Humasis COVID-19 Ag Home Test
under extreme temperature and humidity likely to occur in the United States.
ii. Materials used:
Nasopharyngeal swab sample from healthy donors were collected and eluted in extraction buffer to be
used as negative sample. Positive standard materials (NMC-nCoV02 #24, 1  105.8 TCID50/mL, Chungbuk
National University) were spiked into negative sample to make low positive concentration (63 TCID50/mL,
approx. 2xLoD) for testing. Random samples of the Humasis COVID-19 Ag Home Test were selected from
2 different lots for testing.
iii. Test procedure:
Temperature humidity test chamber was used for the study to make two environmental conditions: 40°C
and 95% RH and 5°C and 5% RH.
Each prepared sample were applied to the swab and eluted in the extraction buffer as per the
instructions. Prepared sample extraction tube and opened test devices were stored in the test chamber
for 3 hours and were tested at various time points in 3 replicates following the instructions: immediately,
after 30 minutes, 1, 2, and 3 hours.
iv. Test results:
According to the results, no effect in performance of the Humasis COVID-19 Ag Home Testwas observed
up to 1 hour in both conditions. However it is highly recommended to perform test immediately after
opening of the device for the best results. To minimize the risk of erroneous results under extreme
operating conditions, each test device is individually sealed in aluminum pouch with a desiccant and the
user is advised to release the test device from aluminum pouch only when ready to take test immediately
after opening. Also the QRI includes the statement: “An opened test device is stable up to 1 hour after
released from the aluminum pouch.”
Test results (No. of positives / No. of replicates)
Temperature/
Stored time Lot 1 Lot 2
humidity
Negative Low pos. Negative Low pos.
0 0/3 3/3 0/3 3/3
30 min 0/3 3/3 0/3 3/3
40°C/
1 hr 0/3 3/3 0/3 3/3
95% RH
2 hr 0/3 2/3 0/3 2/3
3 hr 0/3 2/3 0/3 1/3
0 0/3 3/3 0/3 3/3
5°C/
30 min 0/3 3/3 0/3 3/3
5% RH
1 hr 0/3 3/3 0/3 3/3
 2 hr 0/3 1/3 0/3 1/3
3 hr 0/3 0/3 0/3 0/3

(2-1) Delay in sample testing – nasopharyngeal swab sample eluted in extraction buffer
i. Study objective:
Due to workload or staffing limitations, collected specimens may need to be eluted in the extraction
buffer and stored in refrigerator or at room temperature for testing at a later time. The study was
performed to evaluate stability of eluted samples stored at 4°C and 30°C when using the Humasis COVID-
19 Ag Home Test.
ii. Materials used:
Nasopharyngeal swab sample from healthy donors were collected and eluted in extraction buffer to be
used as negative sample. Positive standard materials (NMC-nCoV02 #24, 1  105.8 TCID50/mL, Chungbuk
National University) were spiked into negative sample to make low positive concentration (63 TCID50/mL,
approx. 2xLoD) for testing. Random samples of the Humasis COVID-19 Ag Home Test were selected for
evaluation.
iii. Test procedure:
Each prepared sample were applied to the swab and eluted in the extraction buffer as per the
instructions. Prepared sample extraction tube was stored at 30°C and 4°C and were tested in 3 replicates
at various time points following the instructions: immediately, 2, 3, 4, 6 hours after storage at 30°C, and
immediately, 1, 4, 8, 12, 24, 48, 72 hours after storage at 4°C.
iv. Test results:
According to the results, eluted samples stored at 30°C showed expected results up to 4 hours; eluted
samples stored at 4°C showed expected results up to 48 hours when using the Humasis COVID-19 Ag
Home Test. However, it is highly recommended to perform test immediately after sample collection for
the best results. To minimize the risk of erroneous results due to delay in sample testing, it is clearly
stated in the instructions for use to test the sample immediately after collection at the sample collection
stage.
Temper Test results (No. of positives / No. of replicates)
Stored time
ature Negative Low pos.
0 0/3 3/3
2 0/3 3/3
30°C 3 0/3 3/3
4 0/3 3/3
6 0/3 2/3
0 0/3 3/3
1 0/3 3/3
4 0/3 3/3
8 0/3 3/3
4°C
12 0/3 3/3
24 0/3 3/3
48 0/3 3/3
72 0/3 2/3
* Line data is included in the excel file: flex study

(2-2) Delay in sample testing – nasopharyngeal swab sample in VTM

i. Study objective:
 Due to limitations in capacities, collected specimens may need to be collected in VTM and refrigerated for
testing at a later time or to be transferred to different site for testing. The study was performed to
evaluate stability of nasopharyngeal swab specimen in VTM stored at refrigerated condition (2°C-4°C)
when using the Humasis COVID-19 Ag Home Test.
ii. Materials used:
Nasopharyngeal swab sample from healthy donors were collected in VTM and eluted in extraction buffer
to be used as negative sample. Positive standard materials (NMC-nCoV02 #24, 1  105.8 TCID50/mL,
Chungbuk National University) were spiked into negative sample to make low positive concentration (63
TCID50/mL, approx. 2xLoD) for testing. Random samples of the Humasis COVID-19 Ag Home Test were
selected for evaluation.
iii. Test procedure:
Each prepared sample were applied to the swab and eluted in the extraction buffer as per the
instructions. Prepared sample extraction tube was stored at 30°C and 4°C and were tested in 3 replicates
at various time points following the instructions: immediately, 2, 3, 4, 6 hours after storage at 30°C, and
immediately, 1, 4, 8, 12, 24, 48, 72 hours after storage at 4°C.
iv. Test results:
According to the results, eluted samples stored at 30°C showed expected results up to 4 hours; eluted
samples stored at 4°C showed expected results up to 48 hours when using the Humasis COVID-19 Ag
Home Test. However, it is highly recommended to perform test immediately after sample collection for
the best results. To minimize the risk of erroneous results due to delay in sample testing, it is clearly
stated in the instructions for use to test the sample immediately after collection at the sample collection
stage.
Tempera Test results (No. of positives / No. of replicates)
Stored time
ture Negative Low pos.
0 0/3 3/3
2 0/3 3/3
30°C 3 0/3 3/3
4 0/3 3/3
6 0/3 2/3
0 0/3 3/3
1 0/3 3/3
4 0/3 3/3
8 0/3 3/3
4°C
12 0/3 3/3
24 0/3 3/3
48 0/3 3/3
72 0/3 2/3

(3) Delay in result reading

i. Study objective:
The study was performed to evaluate various reading times of the Humasis COVID-19 Ag Home Test.
ii. Materials used:
Nasopharyngeal swab sample from healthy donors were collected and eluted in extraction buffer to be
used as negative sample. Positive standard materials (NMC-nCoV02 #24, 1  105.8 TCID50/mL, Chungbuk
National University) were spiked into negative sample to make low positive concentration (63 TCID50/mL,
approx. 2xLoD) for testing. Random samples of the Humasis COVID-19 Ag Home Test were selected for
evaluation.
 iii. Test procedure:
Prepared negative and positive samples were tested using the Humasis COVID-19 Ag Home Test in 3
replicates following the instructions and the results were read at various time points: 5, 10, 15, 30 and 60
minutes after sample application.
vi. Test results:
According to the results, there were no problem in reading the results at 15 minutes. To minimize the risk
of erroneous results due to delay in reading time, it is clearly stated in the instructions for use to read
results at 15 minutes after sample application and to not read results beyond 15 minutes.
Test results (No. of positives / No. of replicates)
Reading time
Negative Low pos.
3 min 0/3 1/3
10 min 0/3 3/3
15 min 0/3 3/3
30 min 0/3 3/3
60 min 2/3 3/3

(4) Extraction buffer volume variability

i. Study objective:
The study was performed to evaluate test results at various sample extraction buffer volumes for the
Humasis COVID-19 Ag Home Test.
ii. Materials used:
Nasopharyngeal swab sample from healthy donors were collected and eluted in extraction buffer to be
used as negative sample. Positive standard materials (NMC-nCoV02 #24, 1  105.8 TCID50/mL, Chungbuk
National University) were spiked into negative sample to make low positive concentration (63 TCID50/mL,
approx. 2xLoD) for testing. Random samples of the Humasis COVID-19 Ag Home Test were selected for
evaluation.
iii. Test procedure:
Prepared negative and positive samples were tested using the Humasis COVID-19 Ag Home Test in 3
replicates following the instructions releasing various sample extraction buffer volumes into the device:
1, 2, 3, 6 drops and whole vial.
iv. Test results:
According to the results, there were no problem in releasing 3 drops of sample extraction buffer into the
device. The risk of erroneous results is mitigated by including words and pictures in the instructions for
use.
Test results (No. of positives / No. of replicates)
Extraction buffer volume
Negative Low pos.
1 drop (3)0/3 (2)1/3
2 drops 3/3 3/3
3 drops 3/3 3/3
6 drops 3/3 3/3
Whole vial (1)0/3 (1)2/3
Note: numbers in () means invalid; e.g. (3)0 = 3 invalids and 0 positives

(5) Swab mixing expression variability

i. Study objective:
 The study was performed to evaluate how mixing the swab in sample extraction buffer affect results of
the Humasis COVID-19 Ag Home Test.
ii. Materials used:
Nasopharyngeal swab sample from healthy donors were collected and eluted in extraction buffer to be
used as negative sample. Positive standard materials (NMC-nCoV02 #24, 1  105.8 TCID50/mL, Chungbuk
National University) were spiked into negative sample to make low positive concentration (63 TCID50/mL,
approx. 2xLoD) for testing. Random samples of the Humasis COVID-19 Ag Home Test were used for
evaluation.
iii. Test procedure:
Prepared negative and positive samples were tested using the Humasis COVID-19 Ag Home Test in 3
replicates following the instructions with various swab mixing actions: none, 3, 5, 10 and 20 times, and
vigorously shaking the test tube until bubbles are formed.
iv. Test results:
According to the results, there were no problem swirling the swab in sample extraction buffer more than
10 times. The risk of erroneous results is mitigated by clearly stating to mix more than 10 times in the
instructions for use.
Test results (No. of positives / No. of replicates)
Sample elution
Negative Low pos.
Non-swirling 0/3 1/3
Swirling 3 times 0/3 3/3
Swirling 5 times 0/3 3/3
Swirling 10 times 0/3 3/3
Swirling 20 times 0/3 3/3
Vigorous shaking until bubbles
0/3 2/3
are formed

(6) Disturbance during testing

i. Study objective:
The study was performed to evaluate how disturbance during testing affects results of the Humasis
COVID-19 Ag Home Test.
ii. Materials used:
Nasopharyngeal swab sample from healthy donors were collected and eluted in extraction buffer to be
used as negative sample. Positive standard materials (NMC-nCoV02 #24, 1  105.8 TCID50/mL, Chungbuk
National University) were spiked into negative sample to make low positive concentration (63 TCID50/mL,
approx. 2xLoD) for testing. Random samples of the Humasis COVID-19 Ag Home Test were used for
evaluation.
iii. Test procedure:
Two types of simulation were conducted for evaluation: a. moving the test device during the test, and b.
dropping the test device during the test.
a. Moving the test device during the test: Prepared negative and positive samples were tested using the
Humasis COVID-19 Ag Home Test in 3 replicates. After applying samples into the test device, the
operator held up the device horizontally and placed it 60cm away from the original spot at: 0, 1, 5, 10
and 15 minutes past from sample application. The results were read at 15 minutes from sample
application.
b. Dropping the test device during the test: Prepared negative and positive samples were tested using the
 Humasis COVID-19 Ag Home Testin 3 replicates. After applying samples into the test device, the
operator dropped it from 80cm height at: 0, 1, 5, 10 and 15 minutes past from sample application. The
results were read at 15 minutes from sample application.
iv. Test results:
According to the results, no problem was observed in performance when the test device was moved from
one place to another. However, when dropping the test device within 1 minute after application, the test
showed erroneous results. This is mitigated by notifying the users through the mobile application training
video to stop the test if the device was dropped after sample application and start new test using new
sample and new test device.
Time of moving the device after Test results (No. of positives / No. of replicates)
sample application Negative Low pos.
0 min 0/3 3/3
1 min 0/3 3/3
5 min 0/3 3/3
10 min 0/3 3/3
15 min 0/3 3/3

Time of dropping of the device after Test results (No. of positives / No. of replicates)
sample application Negative Low pos.
0 min 0/3 2/3
1 min 0/3 3/3
5 min 0/3 3/3
10 min 0/3 3/3
15 min 0/3 3/3

(7) Testing on non-level surface

i. Study objective:
The study was performed to evaluate whether testing on non-level surface affects results of the Humasis
COVID-19 Ag Home Test.
ii. Materials used:
Nasopharyngeal swab sample from healthy donors were collected and eluted in extraction buffer to be
used as negative sample. Positive standard materials (NMC-nCoV02 #24, 1  105.8 TCID50/mL, Chungbuk
National University) were spiked into negative sample to make low positive concentration (63 TCID50/mL,
approx. 2xLoD) for testing. Random samples of the Humasis COVID-19 Ag Home Test selected from 2
different lots were used for evaluation.
iii. Test procedure:
Prepared negative and positive samples were tested using the Humasis COVID-19 Ag Home Test selected
from 2 lots in 3 replicates following the instructions in the following conditions: surface tilted 15˚ towards
the right, left, bottom side and upper side of the device.
iv. Test results:
All test results produced expected results for all inclined positions. However it is highly recommended to
perform test on a level surface for the best results.
Test results (No. of positives / No. of replicates)
Surface tilted 15˚
Lot 1 Lot 2
to
Negative Low pos. Negative Low pos.
Right side 0/3 3/3 0/3 3/3
 Left side 0/3 3/3 0/3 3/3
Bottom side 0/3 3/3 0/3 3/3
Upper side 0/3 3/3 0/3 3/3

(8) Impact of light sources

i. Study objective:
The study was performed to evaluate whether the source of light affects the results of the Humasis
COVID-19 Ag Home Test.
ii. Materials used:
Nasopharyngeal swab sample from healthy donors were collected and eluted in extraction buffer to be
used as negative sample. Positive standard materials (NMC-nCoV02 #24, 1  105.8 TCID50/mL, Chungbuk
National University) were spiked into negative sample to make low positive concentration (63 TCID50/mL,
approx. 2xLoD) for testing. Random samples of the Humasis COVID-19 Ag Home Test selected from 2
different lots were used for evaluation.
iii. Test procedure:
Prepared negative and positive samples were tested using the Humasis COVID-19 Ag Home Test selected
from 2 lots in 3 replicates following the instructions in the following conditions: under direct sunlight
(above 30,000lx, measured by calibrated light meter) and indoor light including fluorescent light and
incandescent light (approx. 500lx).
iv. Test results:
According to the results, the source of light did not affect the performance of the Humasis COVID-19 Ag
Home Test.
Test results (No. of positives / No. of replicates)
Light source Lot 1 Lot 2
Negative Low pos. Negative Low pos.
Direct sunlight 0/3 3/3 0/3 3/3
Fluorescent light 0/3 3/3 0/3 3/3
Incandescent light 0/3 3/3 0/3 3/3

(9) Test device held at 90˚angle

i. Study objective:
The study was performed to evaluate whether holding the test device at 90˚ vertically and horizontally at
the time of reading results will affect the result reading of the Humasis COVID-19 Ag Home Test.
ii. Materials used:
Nasopharyngeal swab sample from healthy donors were collected and eluted in extraction buffer to be
used as negative sample. Positive standard materials (NMC-nCoV02 #24, 1  105.8 TCID50/mL, Chungbuk
National University) were spiked into negative sample to make low positive concentration (63 TCID50/mL,
approx. 2xLoD) for testing. Random samples of the Humasis COVID-19 Ag Home Test selected from 2
different lots were used for evaluation.
iii. Test procedure:
Prepared negative and positive samples were tested using the Humasis COVID-19 Ag Home Test selected
from 2 lots in 3 replicates following the instructions and were held vertically and horizontally at 90˚ to
read results.
iv. Test results:
According to the results, the positioning angle of the test device did not affect the performance of the
Humasis COVID-19 Ag Home Test. However it is highly recommended to leave the test device on the level
surface for the best results.
Test results (No. of positives / No. of replicates)
Positioning angle Lot 1 Lot 2
Negative Low pos. Negative Low pos.
Vertically 90˚ 0/3 3/3 0/3 3/3
Horizontally 90˚ 0/3 3/3 0/3 3/3
8. Clinical evalua�on
8.1. Study objec�ve
The study was performed to evaluate clinical performance of the Humasis COVID-19 Ag Home Test.

8.2. Study design

The clinical evalua�on of the Humasis COVID-19 Ag Home Test was evaluated by tes�ng a total of 659 clinical
samples prospec�vely collected from individual pa�ents at mul�ple sites (Czech Republic, Brazil, and US. 1
nasopharyngeal swab and 1 nasal swab were collected from single pa�ents consecu�vely. Par�cipants inserted
the en�re absorbent �p of the nasal swab into both nostrils and firmly brushed against the inside of nostril in a
circular mo�on 5 �mes or more for at least 15 seconds each. Collected nasal swab was immediately eluted in
the sample extrac�on buffer provided with the Humasis COVID-19 Ag Home Test kit and tested with the device
immediately. Then the operator collected nasopharyngeal swab from the pa�ent to be used for FDA EUA
approved RT-PCR tes�ng on the same day at each site. Samples that were suspected of contamina�on or
which was not able to obtain any results from RT-PCR was excluded from the test. The results were recorded
and compared.
The total number of evaluable posi�ve samples were 106, which showed ct value of 30 and below with RT-
PCR. The total number of evaluable nega�ve samples were 553.

8.3. Analysis method

The sta�s�cal analysis was carried out as indicated in the CLSI EP12 A2 “User Protocol for Evalua�on of
Qualita�ve Test Performance.” Posi�ve and nega�ve performance agreement between the Humasis
COVID-19 Ag Home Test and the clinical truth confirmed by RT-PCR were calculated as below with 95%
confidence interval:
a. Clinical sensi�vity = True posi�ves / (True posi�ves + False nega�ves) x 100(%)
b. Clinical specificity = True nega�ves / (True nega�ves + False posi�ves) x 100(%)
c. Posi�ve predic�ve value = True posi�ves / (True posi�ves + False posi�ves) x 100(%)
d. Nega�ve predic�ve value = True nega�ves / (True nega�ves + False nega�ves) x 100(%)

8.4. Test result summary

RT-PCR (Ct≤30)
Test result Total
Posi�ve Nega�ve

Posi�ve 101 2 103

Humasis COVID-19 Ag
Home Test
Nega�ve 5 551 556

Total 106 553 659

- Clinical sensi�vity: 95.3% (101/106) (95% CI: 89.4-98.0%) (Ct≤30 with RT-PCR)
- Clinical specificity: 99.6% (551/553) (95% CI: 98.7-99.9%) (Ct≤30 with RT-PCR)
- Posi�ve predic�ve value: 98.1% (101/103) (95% CI: 93.2-99.5%) (Ct≤30 with RT-PCR)
- Nega�ve predic�ve value: 99.1% (551/556) (95% CI: 97.9-99.6%) (Ct≤30 with RT-PCR)
8.5. Posi�ve agreement by ct value
Humasis COVID-19 Ag Posi�ve Percent
Ct value RT-PCR 95% CI (%)
Home Test Agreement (%)
Ct ≤ 24 78 80 97.5 91.3 99.3
Ct ≤ 27 91 93 97.8 92.5 99.4
Ct ≤ 30 101 106 95.3 89.4 98.0

8.6. Posi�ve agreement by days from symptom onset

Days from symptom Humasis COVID-19 Ag Posi�ve Percent
RT-PCR 95% CI
onset Home Test Agreement (%)
0-4 days 73 77 94.8 87.4 98.0
5-7 days 28 29 96.6 82.8 99.4
Total 101 106 95.3 89.4 98.0

8.7. Conclusion
According to test results, clinical sensi�vity and specificity of the Humasis COVID-19 Ag Home Test was as
follows:
- Clinical sensi�vity: 95.3% (101/106) (95% CI: 89.4-98.0%) (Ct≤30 with RT-PCR)
- Clinical specificity: 99.6% (551/553) (95% CI: 98.7-99.9%) (Ct≤30 with RT-PCR)
9. Specimen stability
9.1. Test objective
The study was conducted to evaluate specimen stability of the claimed specimen when testing with the
Humasis COVID-19 Ag Home Test.

9.2. Test method

Nasopharyngeal swab sample from healthy donors were collected and eluted in extraction buffer to be
used as negative sample. Positive standard materials were prepared using the SARS-CoV-2 inactivated
virus (NMC-nCoV02 #24, 1  105.8 TCID50/mL, Chungbuk National University) diluted to low positive
concentration (63 TCID50/mL, approx. 2xLoD) in negative sample, and 20uL of the prepared low positive
materials were coated on the swab to be used as positive sample.
Each prepared negative and positive samples were mixed in extraction buffer and capped as per the
instructions for use and stored in room temperature (30C) for various time periods; immediately, and 1,
2, 3, 4, 6 hours after preparation. Each samples of each conditions were tested in 5 replicates following
the instructions, using randomly selected samples of the Humasis COVID-19 Ag Home Test.

9.3. Acceptance criteria

Test results of all positive samples should show positive results and all negative samples should show
negative results.

9.4. Test results

Test result
Time Negative Low Positive
(No. of negative/ No. of replicates) (No. of positive/ No. of replicates)

Initial 3/3 3/3

1 hour 3/3 3/3

2 hours 3/3 3/3

3 hours 3/3 3/3

4 hours 3/3 3/3

6 hours 3/3 2/3

9.5. Conclusion
According to test results, the sample in extraction buffer can be put in room temperature 4 hours prior to
testing. However, it is recommended to test the sample immediately after collection for best results.
10. Reflected standard
: EN 13612:2002 / Performance evaluation of in vitro diagnostic medical devices
: EP7-A2 / Interference Testing in CLSI document; Approved Guideline

11. References
[1] Zhu N, Zhang D, Wang W, et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med 2020.
[2] Huang C, Wang Y, Li X, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet
2020.
[3] Kang CK, Song KH, Choe PG, et al. Clinical and Epidemiologic Characteristics of Spreaders of Middle East Respiratory
Syndrome Coronavirus during the 2015 Outbreak in Korea. J Korean Med Sci 2017; 32:744-9.
[4] WHO, Novel Coronavirus (2019-nCoV) situation reports. Available at:
https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situationreports/ (Accessed at 2 Feb, 2020)