SARS-CoV-2 Rapid Antigen test. As world facing pandemic of coronavirus its rapid detection is of very much important

1. SARS-CoV-2 RAPID
ANTIGEN TEST
VINDHYA.V.V
M.Sc MLT
(MICROBILOGY&IMMUNOLOGY)

2. INTRODUCTION
 Since December 2019,a series of pneumonia cases of unknown causes emerged in
Wuhan,Hubei,China,with clinical presentations greatly resembling viral pneumonia.
 Subsequently ,pathogenic gene sequencing confirmed that the infected pathogen was a
novel Coronavirus, named 2019 novel Corona virus(SARS-CoV-2) and its infection is
called COVID -19,which stands for corona virus disease 2019.
 Its outbreak developed into an epidemic that quickly spread all over China and become
pandemic to more than 120 other countries. The pandemic has resulted in travel
restrictions and nationwide lockdowns in many countries.
 It has been listed as a public health emergency of International concern.
 The COVID 19 virus spreads easily among people, and more continues to be
discovered over time ,data's has shown that it spreads mainly from person to person
among those in close contact(within about 6feet,or 2meters).
 Most people infected with the COVID 19 virus experiences mild to moderate
respiratory illness,some of them with some underlying diseases may develop more
severe illness.

3. Coronavirus
 Coronaviruses constitute the subfamily Orthocoronavirinae,in the Family
Coronaviridae, Order- Nidovirales, and Realm- Riboviria.
 Coronavirus are single stranded positive-sense RNA virus with an envelope of about
80 to 120nm in diameter, often pleomorphic. Its genetic material is the largest of all
RNA viruses.
 They have characteristic club-shaped spikes that project from their surface,which in
electron micrographs create an image reminiscent of the solar corona, from which their
name derives.
 Corona virus are divided into 3 genera α,β,ℽ. α,β are pathogenic to mammals & ℽ
infect birds.SARS-CoV-2 belongs to the genus β corona virus.
 Six species of human coronaviruses are known,with one species subdivided into two
different strains,making seven strains of human coronaviruses together.
 The virus is sensitive to ultraviolet light, heat, and can be effectively inactivated by
56° C for 30 minutes or lipid solvents such as ethyl ether,75%ethanol,chlorine
containing disinfectant, peroxyacetic acid and chloroform etc.
Fig:1;structure
of coronavirus

4. Diagnosis
 Diagnostic testing has played and will continue to play a major role in the
COVID-19 pandemic. The ability to detect the SARS CoV-2 coronavirus in the respiratory
secretions is essential to determine when an individual is infected and potentially infectious
to others.
 Viral detection is used for the identification ,management and isolation of individual
patients.
 Viral detection is also used to determine when the virus has entered a community and
how rapidly it is spreading.

5. DIFFERENT TYPES OF CORONAVIRUS TESTS
Molecular tests Antigen test Antibody test
Also known as/types Diagnostic test,Viral
test,Nucleic acid amplification
test(NAAT),RT-PCR,RT-
LAMP test,Isothermal
amplification assay
Rapid diagnostic tests Serologic test,blood tests/
ELISA,CLIA,Neutralisation
assay
Sample collected Nasopharyngeal ,nasal or
throat swab
Nasal or nasopharyngeal
swab
Blood sample
Turn around time 8 to 10hours 15 to 30minutes
Is another test needed This test is typically highly
accurate and usually does not
need to be repeated
If the test shows positive
result, it should be considered
as true positive, and if
negative confirm with a
molecular test
Sometimes a second Ab test
needed for accurate results.
What it shows…. Diagnoses active corona virus
infection
Diagnoses active corona virus
infection
Shows if infected by
coronavirus in the past.
Timing Most likely to be positive 2
days before the onset of
symptoms,and in early days of
symptomatic
In symptomatic
infection,positive as
symptoms develop and wanes
over time.
Takes at least 7-14 days
after symptom onset to
develop antibodies,and
varies depending on the

6. SARS CoV-2 COVID 19 Ag test
 COVID-19 Ag test is a one-step, rapid, immunochromatographic immunoassay for the
qualitative detection of specific antigens to SARS CoV-2 in human nasal swab
specimens.
 It is a point of care test can be performed outside the conventional lab,and is used to
quickly obtain a diagnostic result.
 Rapid Ag test may be used to detect COVID-19 patients within the first 5days of
illness,leading to quick identification, isolation and treatment.
 This test showed an excellent specificity to rule in COVID-19 patients and had a
moderate sensitivity.
 Patients showing positive results in rapid Ag test need to be immediately triaged and
those with negative results should be reconfirmed by a RT-PCR test.
 RT-PCR is the gold standard test for detecting cases of COVID-19,but it requires
specialised laboratory setup with specific biosafety and biosecurity precautions to be
followed, and it takes more time.

7. PRINCIPLE
 COVID-19 Antigen detection test is an immunoassay for rapid and qualitative
detection of SARS-CoV-2 infection from swab specimens.
 This test is based on a membrane technology with colloidal gold nanoparticles,
uses monoclonal antibodies to detect highly conserved SARS-CoV-2 nucleoprotein
antigen.(N Ag).
 Monoclonal anti-SARS-CoV-2 antibody is coated on the test line region.
 Antigens of SARS-CoV-2 in the specimens react with the anti-SARS-CoV-2
monoclonal antibodies –coupled gold conjugate and form Ag-Ab complex followed
by reaction with anti-SARS-CoV-2 monoclonal antibodies immobilized in the test
line.
 This complex migrates on the membrane, where it will be captured by the
monoclonal anti-SARS-CoV-2antibody.
 A colored test line would be visible in the result window if SARS-CoV-2 antigens
are present in the specimen.
 The intensity of colored test line will vary depending upon the amount of SARS-
CoV-2 Ag present in the specimen.
 If SARS-CoV-2 Ag are not present in the specimen, then no line appears in the test
line.
 The control band is used for procedural control and should always appear if the
test procedure is performed correctly.

8. Fig:2-Nitrocellulose membrane strip

9. Fig:3-Principle of rapid Ag test

10. KIT COMPONENTS
1. Individually packed test devices with desiccant-includes nitrocellulose membrane
striped with Coronavirus N Ag monoclonal Ab as test band and anti-rabbit IgG as
control band.
2. Extraction buffer
3. Extraction tube
4. Disposible dropper cap
5. Sterilized nasopharyngeal swabs for sample collection

11. SAMPLE COLLECTION
NASOPHARYNGEAL SWAB
o Tilt patients head back 70°
o Insert a sterile swab into the nostril of the patient.
o Using gentle rotation ,push the swab till the resistance is met at the level of the turbinate.
o swab over the surface of the posterior nasopharynx for 2to 3 times, hold for few seconds and
withdraw slowly from the nasal cavity.
o Specimen should be tested as soon as possible
o Specimens may be stored at room temperature for up to 1hour or at 2 -8 degree c for up to 48
hours prior to testing.
o Insert the swab into an extraction buffer tube. While squeezing the buffer tube, stir the swab
more than 5times.
o Remove the swab while squeezing the sides of the tube to extract the liquid from the swab.
o Press the nozzle cap tightly onto the tube

12. Procedure
1. Place the device on a clean, flat surface
2. Fill the extraction tube by adding 10-12 drops of buffer
3. Insert the nasopharyngeal swab sample into the extraction solution,then mix the swab
for 8 to 10tmes
4. Remove the swab while pressing against the solution tube in order to extract most of the
specimen.
5. Place dropper cap tightly onto the tube and add 4drops (100micr L) into the sample well
of the test device.
6. Interpret the test results at the end of 20minutes.do not read the result after 30minutes.

13. Fig:4

14. Internal quality control procedure….
 Each Ag test cassette device has a built-in control.
 A red coloured line in the result window at the control line can be considered an
internal positive procedural control.
 The control line will appear if the test procedure has been correctly performed.
 If the control line does not appear ,the test is invalid, and a new test must be performed.
 The product of each lot should be checked with known positive samples for
verification of the positivity.

15. Interpretation of results
 Negative results: If only the control(C )band is developed,the test indicates that the
result is negative.
 Positive result: If the control(C ) and the test (T) bands are developed,the test indicates
for the presence of antigens to SARS-CoV-2in the sample.the result is positive.
 Invalid result:If no control(c )band is developed,then the assay is invalid regardless of
colour development on test (T) band as indicated below.Repeat the assay with a new
device.
Fig:5 Interpretation

16. Precautions
 Handle all the specimens as they contain infectious agents
 The test kit cannot be reused
 Wear personal protective equipments,such as mask, gloves lab coats when handling
reagents and specimens. Wash hands thoroughly after the tests are done.
 Clean up the spills thoroughly using an appropriate disinfectant.
 Follow standard lab procedure and biosafety guidelines for handling and disposal of
potentially infective materials or expired kits or used kits. All remnants of samples,
used materials, expired materials, pipette tips etc. Should be disposed in suitable
biohazard container. Materials should be autoclaved at 121 °C for 30minutes or dipped
in 10% hypochlorite solution for 30minutes prior to disposal.

17. LIMITATIONS
 COVID-19 Ag Detection test is designed for the primary test of SARS-CoV-2 antigen used
as a point-of-care testing .
 The result obtained with this tests should only be interpreted in conjunction with other
diagnostic procedures and clinical findings.
 If the test result is negative and clinical symptoms persists, additional testing using other
clinical methods like viral culture, or molecular assay or ELISA is recommended.
 Positive test result do not rule out co-infection with other pathogens.
 Neither the quantitative value nor the rate of SARS-CoV-2 antigen concentration can be
determined by this qualitative test.
 A negative result may occur if the level of extracted Ag in a specimen is below the sensitivity
of the test or if a poor quality specimen is obtained.
 This test should be done in human nasopharyngeal swab specimens only.
 Failure to follow the test procedure and interpretation of test results may adversely affect the
test performance and produce invalid results.
 Childrens tend to shed virus for longer periods of time than adults, which may result in
differences in sensitivity between adults and childrens.

18. References
1. Gannon CK Mak,Peter KC Cheng,Stephen SY Lau,Kitty KY Wong,CS Lau,Edman
TK Lam,Rickjason CW Chan,Dominic NC Tsang.Evaluation of rapid antigen test
for detection of SARS-CoV-2 virus,Journal of Clinical Virology,June 2020.
2. Clinical management of severe acute respiratory infection when Novel
coronavirus (nCoV)infection is suspected:interim guidance .WHO(2020):2020
3. “Global Progress on COVID-19 Serology –Based testing.Johns Hopkins Center
for Health security.Retrievde 14june2020