LABtobacillus

LABORATORY
TECHNICAL PROCEDURE

An immunochromatographic assay for rapid, qualitative

detection of novel coronavirus (2019-nCoV) antigen
extracted from the nasopharyngeal swab or
oropharyngeal swab specimen.
 Silliman University Institute of Clinical Laboratory Sciences

LABtobacillus
MT14-CC

2019-nCoV Antigen Test (Lateral Flow Method)

Laboratory Technical Procedure

Overview
The 2019-nCov Antigen Test (Lateral Flow Method), an
immunochromatographic assay that allows for rapid and qualitative
identification of novel coronavirus (2019-nCoV) antigen extracted from the
nasopharyngeal swab or oropharyngeal swab specimen, is described in this
laboratory technical procedure. The test is intended to assist in the diagnosis of
2019-nCoV-caused coronavirus infection illness (COVID-2019). An initial test
result is provided by the test. A negative test does not rule out 2019 COVID-19
infection, nor should it serve as the exclusive foundation for treatment choices or
other management decisions.
For in vitro diagnostic use only. For professional use only.

Description of the Novel Coronavirus Infection (COVID-19)

The novel coronavirus belong to the β genus. COVID-19 is an acute respiratory
infection disease. People are generally susceptible. The primary source of
transmission at this time are patients infected by the novel coronavirus;
nevertheless, infected individuals without symptoms can also spread the
infection. The present epidemiological study indicates that the incubation time
is 1–14 days, with 3–7 days being the most common length. Fever, fatigue and
dry cough are the major symptoms. There are a few instances of nasal
congestion, runny nose, sore throat, myalgia, and diarrhea.

Principle of the Procedure

The 2019-nCoV Antigen Test (Lateral Flow Method) measures the amount of
2019-nCoV antigen that is extracted from nasopharyngeal or oropharyngeal
swab specimens using the immunochromatography sandwich principle. The
specimen is absorbed by capillary action into the test apparatus upon addition,
combines with the 2019-nCoV antibody-dye conjugate, and passes through
the pre-coated membrane. The 2019-nCoV antibody immobilized in the Test
Region (T) of the device combines the antigen bound to the antibody-dye
conjugate with the antigen when the 2019-nCoV antigen level in the specimen is
at or above the target cutoff (the test's detection limit). This results in a colored
test band that signifies a positive test. In the Test Region (T) of the device, no
visible colorful band is seen when the 2019-CoV antigen level in the specimen is
zero or below the specified cutoff. This suggests a negative result.
To serve as a procedure control, a colored lone will appear at the Control region
(C), if the test has been performed properly.

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 Silliman University Institute of Clinical Laboratory Sciences

LABtobacillus
MT14-CC

Precautions in Testing
1. Only in vitro diagnostic use is permitted with this test.
2. Every specimen ought to be handled as though it were able to spread illness.
When gathering, handling, storing, and discarding patient samples and used
kit items, use the necessary safety procedures.
3. When handling the materials of this kit, put on the proper personal protective
equipment (e.g., lab coat, goggles, medical mask, and protective gloves).
4. Without the need for an extraction buffer, the virus can be identified directly if
the specimen processing method uses the virus sampling solution.
5. The success of this test depends on the collection, storage, and
transportation of specimens in an appropriate manner.
6. After usage, discard. There is just one usage allowed for the test equipment,
dropper, and sample extraction tube.
7. A temperature that is too high should not be present during the experiment.
To prevent moisture absorption, test cards and detection buffer that have
been refrigerated must be brought back to room temperature before being
opened.
8. Keep your hands off the test strip's reaction region.
9. Test kits should not be used once they have expired.
10. If the pouch is not tightly sealed or has holes in it, do not use the kit.
11. When a qualified medical expert takes a sample, it should be applied by
properly trained professionals in accredited laboratories or clinics.
12. The doctor should evaluate the test result in conjunction with clinical
observations and results from additional laboratory testing.
13. DISPOSAL OF THE DIAGNOSTIC: There is an infectious risk associated with all
specimens and the used kit. The diagnostic disposal procedure must adhere
to any applicable laboratory regulations or local infectious disposal laws.

Materials (Materials Provided)

W196P000 W196P000 W196P000 W196P000

COMPONENTS
5 6 7 11

Sealed Pouches* (pcs) 1 5 20 20

Drippers (pcs) 1 5 20 20

Extraction Buffer (400μL/tube) 1 5 20 20

Sample Extraction Tube (pcs) 1 5 20 20

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 Silliman University Institute of Clinical Laboratory Sciences

LABtobacillus
MT14-CC

W196P000 W196P000 W196P000 W196P000

COMPONENTS
5 6 7 11

Nasopharyngeal Swab** (pcs) 1 5 20 20

Test Tube Rack (pcs) / / 1 1

Positive Control Swab (pcs) / / / 1

Negative Control Swab (pcs) / / / 1

Procedure Cards (pcs) 1 1 1 1

IFU (pcs) 1 1 1 1

W196P000 W196P000 W196P000 W196P000

COMPONENTS
8 9 10 12

Sealed Pouches* (pcs) 1 5 20 20

Pre-installed Extraction Buffer

1 5 20 20
(400μL/tube)

Nasopharyngeal Swab** (pcs) 1 5 20 20

Test Tube Rack (pcs) / / 1 1

Positive Control Swab (pcs) / / / 1

Negative Control Swab (pcs) / / / 1

Procedure Cards (pcs) 1 1 1 1

IFU (pcs) 1 1 1 1

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LABtobacillus
MT14-CC

Note:
Each sealed pouches containing: 1 Test Cassette and 1 Desiccant Pouch
CE information of nasopharyngeal swab

Materials (Materials Required but Not Provided)

1. Viral Transport Media (VTM)
2. Tongue Depressor
3. Timer
4. Personal protective equipment, such as protective gloves, medical mask,
goggles, and lab coat.
5. Appropriate biohazard waste container and disinfectants.

Storage and Stability

1. Store the sealed pouch at 2–30°C until the date printed on the package; do
not freeze.
2. Use the test cassette within an hour of removing it from the sealed pouch.
Recap the buffer solution promptly after use.
3. Store out of direct sunlight, moisture, and heat.
4. The contents of the kit remain intact until the date of expiration indicated on
the outer box.
5. The exterior box bears an imprint of the production date.

Specimen Collection and Preparation

The test can be done with either a nasopharyngeal or oropharyngeal swab
material.
1. In accordance with the usual protocol for collecting nasopharyngeal or
oropharyngeal swab specimens.
2. To acquire a nasopharyngeal swab specimen, tilt the patient's head back by
70 degrees. Place the swab inside the nostril, making sure it reaches the
same depth as the gap between the nostrils and the outer ear entrance.
Swab should be left in place for a few seconds to collect secretions. Rotate
the swab as you slowly remove it.
3. To collect an oropharyngeal swab specimen, place the swab into the
tonsillar and posterior pharynx. Swabs should be rubbed over the posterior
oropharynx and the tonsillar pillars; do not touch the gums, teeth, or tongue.
4. Testing the samples at the time of specimen collection is advised. The
specimens should be stored in a dry, clean tube that is tightly sealed if they
are not examined right away (swab tip should be placed inside a tube and
the applicator stick should be snapped off or chopped). They can be kept for
up to eight hours at 2–8°C or for an extended period of time at -70°C.

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 Silliman University Institute of Clinical Laboratory Sciences

LABtobacillus
MT14-CC

Note: The sample dilution ratio should be kept to a minimum if the viral transport
medium (VTM) is required for sample transportation. This is because a large
diluent volume may cause a false negative. The diluent volume should, if at all
possible, not exceed 1 mL; nevertheless, the swab tip needs to be completely
submerged in the liquid. Using the influenza virus as a guide, the VT's nasal or
nasopharyngeal swabs can remain steady for a maximum of 72 hours at 2~8°C.

Test Procedure
Please read the instructions for use carefully remove before performing the test.

Test Procedure I (with Extraction Buffer)

For W196P0005 / W195P0006 / W196P0007 / W196P00011

A. Nasopharyngeal/Oropharyngeal swab or positive/negative control swab

specimen extraction.
1. Transfer all of the extraction buffer into the extraction tube.
2. Insert the swab which has collected secretions into the sample extraction
tube, rotate the swab tip 10 times against the bottom and sides of the
extraction tube to release the specimen from the swab tip. Return the
sample extraction tube to the test tube rack (if applicable) and leave the
swab in the extraction buffer for 1 minute.
3. Take out the swab while squeezing the middle of the extraction tube to
release the fluid from the swab. Discard the used swab in accordance with
the biohazard waste protocol.
4. Cover the dripper.

B. Test Procedure
1. Remove a test cassette from the sealed pouch by tearing at the notch and
place it on a level surface.
2. Invert the extraction sample tube, hold the extraction sample tube vertically
and add 80μL (about 3-4 drops) processed the specimen to the sample well.
Start the timer.
3. As the test begin to work, you will see purple color move across the result
window in the center of the test device.
4. Wait for 15-20 minutes and read the results. Do not read the results after 30
minutes.

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 Silliman University Institute of Clinical Laboratory Sciences

LABtobacillus
MT14-CC

Image source: [Link]

Test Procedure I (with Extraction Buffer)

For W196P0008 / W195P0009 / W196P00010 / W196P00012

A. Nasopharyngeal/Oropharyngeal swab or positive/negative control swab

specimen extraction.
1. Unscrew the lid of the tube.
2. Insert the swab which has collected secretions into the extraction tube (Pre-
installed Extraction Buffer), rotate the swab tip 10 times against the bottom
and sides of the extraction tube to release the specimen from the swab tip.
Return the sample extraction tube to the test tube rack (if applicable) and
leave the swab in the extraction buffer for 1 minute.
3. Take out the swab while squeezing the middle of the extraction tube to
release the fluid from the swab. Discard the used swab in accordance with
the biohazard waste protocol.
4. Cover the dripper.

B. Test Procedure
1. Remove a test cassette from the sealed pouch by tearing at the notch and
place it on a level surface.
2. Invert the extraction sample tube, hold the extraction sample tube vertically
and add 80μL (about 3-4 drops) processed the specimen to the sample well.
Start the timer.
3. As the test begin to work, you will see purple color move across the result
window in the center of the test device.
4. Wait for 15-20 minutes and read the results. Do not read the results after 30
minutes.

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 Silliman University Institute of Clinical Laboratory Sciences

LABtobacillus
MT14-CC

Image source: [Link]

Note: To obtain accurate results, avoid mucoid substances when filling the
micropipette with patient sample in VTM.

Results and Interpretation

Positive: Colored bands appear at both test line (T) and control line (C). It
indicates a positive result for the 2019-nCoV antigen in the specimen.

Negative: Colored bands appear at control line (C) only. It indicates that the
concentration of the 2019-nCoV antigen is zero or below the detection limit of
the test.

Invalid: No visible colored band appears at control line after performing the
test. The directions may have not been followed correctly or the test may
have deteriorated. It is recommended to re-sampling and test.

Image source: [Link]

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 Silliman University Institute of Clinical Laboratory Sciences

LABtobacillus
MT14-CC

Quality Control
The test incorporates a procedural control. An internal procedural control is
indicated by a colored line that appears in the control region (C). It verifies
appropriate membrane wicking, a suitable volume of liquid, and proper
procedural technique. It is recommended by good laboratory practice to
employ the control materials. When it comes to how frequently to assay external
quality control materials, users should abide by the relevant federal, state, and
municipal criteria.

Limitations of Procedure
1. The 2019-nCoV antigen in human nasopharyngeal or oropharyngeal swab
specimens is intended to be detected by this reagent.
2. The procedure used to obtain the sample affects the test's accuracy. The test
result will be impacted by incorrect sample collection, inappropriate sample
storage, or repetitive freezing and thawing of the sample.
3. This is a qualitative test reagent. The quantitative concentration of the 2019-
CoV antigen is not intended to be ascertained by it. Should you require to
assess the quantitative concentration, kindly utilize the appropriate
professional tools.
4. The results of this reagent's tests are intended exclusively for clinical
reference; they shouldn't serve as the exclusive foundation for a clinical
diagnosis or course of therapy. A patient's clinical care should take into
account all relevant factors, including their medical history, symptoms,
indicators, and reaction to treatment.
5. In light of the limitations of antigen test reagents, it is advised to review and
confirm negative test results using nucleic acid detection or virus culture
identification techniques.
6. Co-infections with additional pathogens are not ruled out by positive test
results. The following could result in this reagent working poorly:

1) Inadequate sample handling, transfer, or collection; insufficient viral titer

in the sample;
2) The antigen level of 2019-CoV is below the test's detection limit.
3) Variations in viral genes can lead to modifications in the determinants
of antigens.

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 Silliman University Institute of Clinical Laboratory Sciences

LABtobacillus
MT14-CC

Performance Characteristics
A. Sensitivity and Specificity
574 clinical case samples which include 212 confirmed as COVID-19 positive and
362 confirmed as COVID-19 negative by PCR assay, were obtained for testing,
and then compared the test results between 2019-nCoV Antigen Test (Lateral
Flow Method) and the PCR results. The results are shown below.

PCR

Reagents Total

Positive Negative

Positive 208 1 209

2019-nCoV
Antigen Test
(Lateral Flow
Method)
Negative 4 361 365

Total 212 362 574

Sensitivity: 98.11% (95% Cl: 95.24%-99.48%)

Specificity: 99.72% (95%Cl: 98.47%-99.99%)
Total Agreement: 99.13% (95% Cl: 97.98%-99.72%)

B. Cross-Reactivity
Cross-reactivity of the 2019-nCoV Antigen Test (Lateral Flow Method) was
evaluated using specimens containing the antigens listed below. The results
showed no cross-reactivity of the following:

Common Coronavirus (NL63, 229E, OC43)

Enterovirus A/B/C/D antigen
antigen

Coronavirus (MERS) antigen EB Virus antigen

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LABtobacillus
MT14-CC

Influenza A H1N1 abtigen Measles Virus antigen

Influenza A H3N2 antigen Human Cytomegalovirus antigen

Influenza B Yamagata antigen Rotavirus antigen

Influenza B Victoria antigen Norovirus antigen

Respiratory Syncytial Virus A/B antigen Mumps Virus antigen

Rhinovirus-A/B antigen Varicella-zoster Virus Positive antigen

Adenovirus-1/-2/-3/-4/-5/-7/55 antigen Mycoplasma Pneumoniae antigen

C. Interference
The test result of 2019-nCoV Antigen Test (Lateral Flow Method) do not be
interfered with the following substance:

TYPE SUBSTANCE

Allergic Symptoms Histamine Dihydrochloride

Systemic Antibacterial Drugs Tobramycin

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LABtobacillus
MT14-CC

TYPE SUBSTANCE

Interferon Alpha

Zanamivir

Ribavirin

Oseltamivir

Antiviral Drugs

Palamivir

Lopenavir

Ritonavir

Abidor

Levofloxacin

Azithromycin

Antibiotics

Ceftriaxone

Meropenem

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LABtobacillus
MT14-CC

D. Hook Effect
Within the titer range of clinically positive samples of 2019-nCoV antigens, there
is no hook effect in the test results of this product.

E. Precision
1. Within run precision was determined by testing positive specimens ten times.
The agreement rate was 100%.
2. Between run precision was determined by testing three different specimens
including positive and negative in three different slots of test devices. The
negative agreement rate and the positive agreement rate were 100%

F. Limit of Detection
The LoD of this test is 1.1 x 10^2 TCID50/mL

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 Silliman University Institute of Clinical Laboratory Sciences

LABtobacillus
MT14-CC

References
[Link]. (n.d.). Wondfo 2019-nCoV Antigen Test. Retrieved from
[Link]

JOYSBIO. (2024, May 24). COVID-19 Antigen Rapid Test Kit - JOYSBIO
Biotechnology. Retrieved from [Link]
rapid-test-kit/

[Link]. (n.d.-a). COVID-19 Diagnostic Solution: RT-PCR, Antigen

and Antibody Test - Wondfo. Retrieved from
[Link]

Prepared by:

JOMIL EDWARD S. OMANDAM

Microbiology Supervisor

Approved by:

MARISTELLA URIELLE E. CORTEZ

Laboratory Director

EFFECTIVE IMMEDIATELY UPON APPROVAL

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