this pdf is about different methods of immunoassay that detect antigen or antibody in patient's sample and give us data about his/her condition.

1. comparative review of
immunoassays for
covid_19 detection
seyede Anahid Danesh
supervisor: Dr. sine sepehr

2. rRT_PCR (NAAT)
the sample is taken
from sputum, throat
swab, and secretion of
the lower respiratory
tract.
problems
it is time-consuming (4 - 6 h)
it also needs costly specialist
equipment and skillful
laboratory staff.
FN results due to low viral load
in the upper respiratory may
occur using RT_pcr analysis.
the peak of viral load in upper
respiratory tact secretions is
within the first week of
symptoms and can become
undetectable by 14 days.
reference standard method
detection of unique sequences of
the virus RNA
PCR-based techniques are methods
that rely on the polymerase chain
reaction (PCR) to amplify stretches
of DNA by creating many identical
or near-identical copies.

3. fast
lower cost
high-throughput
less workload
diagnosis of patients more than
diagnosis of patients with negative RT-
PCR test and strong epidemiological and
clinical evidences.
7 days after the onset of symptoms
Strengths + Weaknesses -
serological tests
sero­
conversion usually
occurred at 3–14 d.p.o, so early
diagnosis of COVID-19 using
only serological tests may not
be possible.
the specificity and sensi­
tivity
of serological tests are
affected by infection time.

4. Four major struc­
tural proteins, including membrane (M), envelope (E),
nucleo­capsid (N), and spike (S), as well as non-structural proteins, are
encoded by coronavirus RNA.
N and RBD of S protein are the most
common antigens that can be used as
an immobilized antigen for creating a
serological COVID-19 diagnostic test

5. main methodologies of serological tests
ELISA
LFIA
CLIA
NT assay

7. ELISA
ELISA
To detect patient antibodies, a known
capture antigen is immobi­
lized on the
plate and then the patient’s antibodies in
a sample bind to the immobilized capture
antigen.
an enzyme-labeled detection antibody
specific to any antibody isotypes (i.e., IgG,
IgM, etc.) forms a complex with the
capturedpatientantibodies.
the interaction of enzyme (usually
horseradish peroxidase) and its sub‐­
strate causes a colorimetric change
that can be quantified and
correlated to the presence or
concentration of the antibody.
1.
2.
3.
method
+
ELISA is not a very expensive method.
It can be simply operated and made high
throughput with an automated work
station.
_
this technique is time-consuming and
at risk of contamination. It also needs
infrastructure and qua­
lified personnel

8. CLIAs
In the first step of the
chemiluminescence-based method,
SARS-CoV-2 specific IgA, IgM and
IgG in sera were captured via SARS-
CoV-2 antigen (N or RBD)-coated
magnetic particles.
a second acridinium-conjugated
antibody that detects human IgA,
IgM, or IgG was applied. Reaction of
acridinium with its substrates
caused strong chemiluminescence.
the diagnosed chemiluminescent
signal over the background was
measured as relative light units
The principle of a CLIA is the same
as an ELISA with shorter incubation
steps and no need for a reagent to
stop the enzymatic reaction
1.
2.
3.
Requiring supporting
chemiluminescence
instruments and qualified
personnel are the
disadvantages of CLIA.
It was demonstrated that
the performance of CLIA
for detecting IgM is not
suitable enough.
It was found that CLIA
against multi-antigens (N
+ S proteins) has higher
specificity than single
antigen proteins
Chemiluminescence is a light-emitting process in
which chemical reactions are the energy source
for gener­
ating the electronically excited state.
This laboratory technique benefits from the
specificity of the immune response and also the
sensitivity of the lumi­
nescence reaction.

9. LFIA
+
_
multiple analytes
can be detected
by different test
lines on a device
of LFIA
simple
operation
low cost
high speed
low specificity and sensitivity

10. neutralization assay
virus NT assay is the gold standard for the evaluation
of protective immunity against SARS-CoV-2.
the interaction between RBD and ACE2 recep­
tor is
inhibited by highly specific nAbs in patient or animal
sera in an ELISA plate well.
Advantages of sVNT include its ability to detect total
SARS-CoV-2 nAbs in an isotype-independent and
species-independent manner.
Virus neutralization is a specialized type of
immunoassay because it does not detect all
antigen–antibody reactions. It only detects
antibody that can block virus replication.

11. ELISA CLIA LFIA NT assay
diagnosis time 1.5 - 2.5 h 0.5 h 10 -15 min 1 - 2 h
sensivity 65 - 98 % 77 - 100 % 69 _ 93 % 95 _ 100 %
specifity 71 _ 100 % 90 _ 100 % 80 - 100 % 100 %
disadvantage time consuming
require chemiluescence
equipment
low sensivity during early
infection
generation of neutrilizing AB
has a slower onset and rise in
the primary immune
response.
quantification of the
colorimetric change
diagnosing the
chemiluminescent signal over
the background
visual observation of colored
band
detecting nABs which block
virus replication.
comparison of immunoassay methods

12. conclusion
the fundamental methodology for in vitro diagnosis of SARS-CoV-2 infection is based
on NAAT and It is not possible to properly diagnose acute disease by IgG testing.
serology tests are useful for retrospective identification of COVID-19 patients and for
contact tracing purposes when doubt of false-positive or false-negative PCR results
has existed.
Since serology tests reached their highest positive rates 14 days after symptom onset
and the sensitivity of RT-PCR was decreased at this time, serology testing could be
consid­
ered as a part of the diagnostic panel two-week post-symptom onset.

13. Resource : A comparative
review of immunoassays for
COVID-19 detection (2021)
by Elham Mohit, Zahra
Rostami & Hossein Vahidi
article
link

14. Thank you for your
attention!