CSDT - Humasis COVID-19 Ag Home Test
CSDT - Humasis COVID-19 Ag Home Test
CSDT - Humasis COVID-19 Ag Home Test
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.
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)
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
1/50,000
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
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
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
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
RBD
RBD mutation
Con. as control
1 7 8 9 10 11
NP
NP mutation
Con. as control
12 13 14 15 16 17
NP
NP mutation
Con. as control
12 18 19 20 21 22
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.
3.2.1. 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 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.
4.4.3. 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.
Sodium cromoglycate
7 15% v/v 3/3* 3/3* 3/3** 3/3**
(CVS nasal spray, Cromolyn)
10 Sore throat Phenol Spray 15% 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**
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.
6.2.2. 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.
6.5. 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.
(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
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
RT-PCR (Ct≤30)
Test result Total
Posi�ve Nega�ve
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.
9.5. 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)