SARS-CoV-2 Omicron BA.4 and BA.

5 Variant analyses for

Toda Coronadiag Ag® and TODA® Autotest Nasal COVID-19
by TODA PHARMA

1. Purpose of the study

The purpose of this study was to identify if the SARS-CoV-2 variant of concern, lineage BA.4 and BA.5,
which known as the “Omicron” variant could be detected by the Coronadiag Ag® / TODA® Autotest
Nasal COVID-19 and to demonstrate the analytical sensitivity of SARS-CoV-2 variants with recombinant
nucleocapsid proteins.

The purpose of this study was to validate whether the assay performance will be affected by SARS-
CoV-2 variant of concern, lineage BA.4 and BA.5.

2. Reagent
Toda Coronadiag Ag®
Lot 1: I2111176; Lot 2: I2111178; Lot 3: I2111179.

3. Specimen
a. Positive controls used in the study
Recombinant SARS-CoV-2 nucleocapsid protein (NCP)

1) The SARS-CoV-2 variant BA.4(nCOV-PS-Ag159), Lot#20220413, original concentration of 2.9mg/mL;

traceability information: recombinant SARS-CoV-2 NCP.

2) The SARS-CoV-2 variant BA.5 (nCOV-PS-Ag155), Lot#20220105, original concentration of

3.68mg/mL; traceability information: recombinant SARS-CoV-2 NCP.

b. Diluent used in the study

The assay buffer was used as diluent to determine the limit of detection (LOD) for each recombinant
protein.
 Version 1.0: Analytical Sensitivity study for SARS-CoV-2 Omicron Variant

4. Study Design
a. LOD screening
1) 10-fold dilutions of each positive control were made in assay buffer.

2) Apply 100uL of each dilution onto the sample well of test device.

3) Each dilution was tested in triplicate with three kit lots.

4) Result interpretation was performed according to the package insert.

5) The concentration demonstrating 9 of 9 positives was selected for LOD ranging finding.

b. LOD range finding

1) Double dilutions were made of the selected concentration based on LOD screening.

2) Apply 100uL of each dilution onto the sample well of test device.

3) Each dilution was tested in triplicate with three kit lots.

4) The concentration demonstrating 9 of 9 positives was selected for LOD confirmation.

c. LOD confirmation
1) A total of 20 replicates of the selected concentration based on LOD range finding were tested with
three kit lots as described above, to obtain a total of 60 testing results.

2) The LOD was confirmed when 95% of testing results were all positive.

Testing results were documented as + (positive), - (negative) or X (invalid) in the study.

5. Results

Table 1: Result of LOD screening for recombinant SARS-CoV-2 NCP

The SARS-CoV-2 variant BA.4 isolate

Concentration Lot 1 Lot 2 Lot 3

(pg/mL) Number of positives / number of total results
1 2 3 1 2 3 1 2 3
2.9×109 + + + + + + + + + 9/9
2.9×108 + + + + + + + + + 9/9
2.9×107 + + + + + + + + + 9/9
2.9×106 + + + + + + + + + 9/9
2.9×105 + + + + + + + + + 9/9
2.9×104 + + + + + + + + + 9/9
2.9×103 + + + + + + + + + 9/9
2.9×102 + + + + + + + + + 9/9

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 Version 1.0: Analytical Sensitivity study for SARS-CoV-2 Omicron Variant

2.9×101 + + + + + + + + + 9/9
2.9 - - - - - - - - - 0/9
The SARS-CoV-2 variant BA.5 isolate

Concentration Lot 1 Lot 2 Lot 3

(pg/mL) Number of positives / number of total results
1 2 3 1 2 3 1 2 3
3.68×109 + + + + + + + + + 9/9
3.68×108 + + + + + + + + + 9/9
3.68×107 + + + + + + + + + 9/9
3.68×106 + + + + + + + + + 9/9
3.68×105 + + + + + + + + + 9/9
3.68×104 + + + + + + + + + 9/9
3.68×103 + + + + + + + + + 9/9
3.68×102 + + + + + + + + + 9/9
3.68×101 + + + + + + + + + 9/9
3.68 + + + + + + + + + 9/9
0.368 - - - - - - - - - 0/9

Table 2: Result of LOD range finding for recombinant SARS-CoV-2 NCP

The SARS-CoV-2 variant BA.4 isolate

Concentration Lot 1 Lot 2 Lot 3

(pg/mL) Number of positives / number of total results
1 2 3 1 2 3 1 2 3
29 + + + + + + + + + 9/9
14.5 + + - - + + + - + 6/9
7.3 - - - + - - - - + 2/9
3.7 - - - - - - - - - 0/9
The SARS-CoV-2 variant BA.5 isolate

Concentration Lot 1 Lot 2 Lot 3

(pg/mL) Number of positives / number of total results
1 2 3 1 2 3 1 2 3
3.68 + + + + + + + + + 9/9
1.84 + - + - + - + - + 5/9
0.92 - - - - - - + - - 1/9

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 Version 1.0: Analytical Sensitivity study for SARS-CoV-2 Omicron Variant

Table 3: Result of LOD confirmation

The SARS-CoV-2 variant The SARS-CoV-2 variant

BA.4 isolate BA.5 isolate
Recombinant SARS-CoV-2 NCP 29 pg/mL 3.68 pg/mL
Lot
1 2 3 1 2 3
Replicates
1 + + + + + +
2 + + + + + +
3 + + + + + +
4 + + + + + +
5 + + + + + +
6 + + + + + +
7 + + + + + +
8 + + + + + +
9 + + + + + +
10 + + + + + +
11 + + + + + +
12 + + + + + +
13 + + + + + +
14 + + + + + +
15 + + + + + +
16 + + + + + +
17 + + + + + +
18 + + + + + +
19 + + + + + +
20 + + + + + +
Number of positives / number of total 60/60, 100% 60/60, 100%
results

6. Conclusion
The study demonstrated that the assay performance would not be affected by testing SARS-CoV-2
variant BA.4 and BA.5.

Based on the above testing results, the limit of detection of the Toda Coronadiag Ag® for SARS-CoV-2
variant BA.4 was 29pg/ml and for the SARS-CoV-2 variant BA.5 was 3.68pg/ml. And the testing results
were met the criteria. Note that the results are also applicable for the self-test TODA® Autotest Nasal
COVID-19.

Approval:

Approved by Daniel Berros, Head Pharmacist. Date: June 14th, 2022

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