524 https://oamjms.eu/index.php/mjms/index
Scientific Foundation SPIROSKI, Skopje, Republic of Macedonia
Open Access Macedonian Journal of Medical Sciences. 2023  Apr 14; 11(B):524-528.
https://doi.org/10.3889/oamjms.2023.11611
eISSN: 1857-9655
Category: B - Clinical Sciences
Section: Infective Disease
Quantitative SARS-CoV-2 Spike Receptor-Binding Domain on
Vaccinated Individuals Compared to Natural Infection
Maria Yasintha Lihawa
1
*, Eulis Alwi Datau
2
, Eko Surachmanto
2
, Juwita Soekarno
2
, Budi Dharmono Tulaka
1,3
1
Department of Internal Medicine, Faculty of Medicine, Sam Ratulangi University, Prof. Dr. R. D. Kandou Hospital, Manado,
Indonesia;
2
Division of Allergy and Immunology, Department of Internal Medicine, Faculty of Medicine, Sam Ratulangi
University, Prof. Dr. R. D. Kandou Hospital, Manado, Indonesia;
3
Department of Clinical Pathology, Faculty of Medicine,
Tadulako University, Palu, Indonesia
Abstract
BACKGROUND: Coronavirus disease 2019 (COVID-19) pandemic has been going on for more than 2 years,
with various treatments and diagnostic methods available. One of the most prized structures, the receptor-binding
domain (RBD) of the spike protein in severe acute respiratory syndrome coronavirus 2 has long been thoroughly
researched for its function and becoming the target for various diagnostic methods and treatments, including a
vaccine. The spike-RBD (sRBD) antibody count might be the parameter for antibody response in vaccinated and
infected individuals. However, no direct comparison is made.
AIM: The study aims to compare the sRBD antibody count in the naturally infected individuals to the vaccinated ones.
METHODS: We conducted a cross-sectional study with 49 participants of the infected patients, and vaccinated
individuals were included in this study from Prof. Dr.  R. D. Kandou Hospital, Manado. The participants underwent
a COVID-19 antibody test, using enhanced “Chemiluminescence” Immuno assay to analyze the anti-sRBD IgG
quantitatively. Results were then analyzed and compared using IBM Statistical Package for Social Sciences ver 25.0
with Mann−Whitney non-parametric test.
RESULTS: The study shows a higher median antibody count in the naturally infected group compared to the
vaccinated group (132.70  vs. 11.95 U/mL; p < 0.001). Further studies on the topic should be conducted to determine
the comparison on a larger scale.
CONCLUSION: The s-RBD antibody titer is significantly higher in naturally infected patients than in vaccinated
individuals.
Edited by: Ksenija Bogoeva-Kostovska
Citation: Lihawa MY, Datau EL, Surachmanto E,
Soekarno J, Tulaka BD. Quantitative SARS-CoV-2 Spike
Receptor-Binding Domain on Vaccinated Individuals
Compared to Natural Infection. Open Access Maced J
Med Sci. 2023  Apr 14; 11(B):524-528.
https://doi.org/10.3889/oamjms.2023.11611
Keywords: Coronavirus disease-19; Receptor-binding
domain; IgG; Vaccine; Infection
*Correspondence: Maria Yasintha Lihawa, Department
of Internal Medicine, Faculty of Medicine, Sam Ratulangi
University/Prof. dr. R. D. Kandou Hospital, Manado,
Indonesia. E-mail: [email protected]
Received: 22-Mar-2023
Revised: 01-Apr-2023
Accepted: 04-Apr-2023
Copyright: © 2023 Maria Yasintha Lihawa,
Eulis Alwi Datau, Eko Surachmanto, Juwita Soekarno,
Budi Dharmono Tulaka
Funding: This research did not receive any financial
support
Competing Interests: The authors have declared that no
competing interests exist
Open Access: This is an open-access article distributed
under the terms of the Creative Commons Attribution-
NonCommercial 4.0 International License (CC BY-NC 4.0)
Introduction
The coronavirus disease 2019 (COVID-19)
pandemic has been going on for more than 2  years, with
more than 635 million confirmed cases and more than
6.6 million deaths by November 22, 2022. However, the
case growth has been successfully flattened with various
efforts, particularly COVID-19 vaccine employment [1].
As of November 15, 2022, around 12.9 billion vaccine
doses have been administered, with 68.4% of the world
population receiving at least one vaccine dose, with daily
vaccine administration reaching 2.5 million doses [2].
The continuous research and trial for the diagnosis and
treatment of COVID-19 is based on the novel research
of the severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) vaccine structure.
The SARS-CoV-2 virus or simply called
coronaviruses belongs to the coronaviridae family. The
SARS-Cov-2 is part of the RNA viruses with single-
stranded positive-sense RNA (+ssRNA) with a genome
size of 27–32  kb. The SARS-CoV-2 is constructed with
four structural proteins (S, E, M, and N) and sixteen
non-structural proteins (nsp1–16). The non-structural
proteins are responsible for the reproduction and
virulence of the virus itself. The spike protein (S) of
SARS-CoV-2 is accountable for entering host cells with
spike glycoproteins forming homotrimers that protrude
from the viral surface. The S protein is also composed
of two subunits, the S1, and S2. The S1 subunits consist
of the N-terminal domain and receptor-binding domain
(RBD) to bind the SARS-Cov-2 into the known receptor
on host cells, namely Angiotensin-converting enzyme 2
receptor (ACE2) [3]. The RBD includes two structural
domains, namely the core and external subdomains,
composed of five β antiparallel strands and loops,
stabilized with the di-sulphide bond [3], [5].
The composition of RBD in the spike protein,
making the glycan coat paired with the flexibility of
SARS-CoV-2 spikes, enables them to identify the
host cell surface and bind with ACE2 receptors [6].
The RBD protein also functions through two different
states, the closed “down” and open “up” structure,
to enable human ACE2 receptor identification while
shielding the receptor-binding regions from neutralizing
antibodies [7]. The nature of RBD regions in the S
Since 2002

 Lihawa et al. Quantitative Sars-Cov-2 S-RBD on vaccinated vs natural infection Open Access Maced J Med Sci. 2023  Apr 14; 11(B):524-528.525
protein of SARS-CoV-2 also has been evaluated, with
more sensitive S protein to ACE2 receptor than other
coronaviruses. This might explain the 24% difference in
structure domains in the RBD and S protein of SARS-
CoV-2 compared to different SARS-CoV lineage [8]. The
structure is furtherly evolving throughout the pandemic
as new variants and mutations of the RBD protein,
particularly in the arrangement of the amino acids,
with increasing virulence and binding affinity along with
antibody escape, particularly on the B.1.617.2 (Delta
variant) and B.1.1.529 (Omicron variant) [9], [10].
The S protein, particularly RBD, has become
the target for various diagnostic modalities. The SARS-
CoV-2 RBD IgG test has been implemented as an
antibody test for COVID-19 through the Enzyme-Linked
Immunosorbent Assay (ELISA) method for detecting
SARS-CoV-2 IgG in human serum. The RBD IgG
test is developed for identifying individuals with an
adaptive immune response to SARS-CoV-2 [11]. The
ELISA to detect IgM, IgA, and IgG antibodies against
RBD of SARS-CoV-2 has revealed sensitivity 47%,
80%, and 88%, respectively, with the specificity of
98–100% [12]. The diagnosis using the RBD domain
with chemiluminescent reduction-neutralizing test
also displays the use of diagnostic serology method
for B.1.1.7 and B.1.351 variants using anti-RBD
antibody qualitative assessment, with 99.1% and 100%
sensitivity and specificity, respectively. The presence of
anti-RBD antibodies also correlates with the emergence
of neutralizing antibodies to guide the clinical or public
health decisions during the pandemic [13], [15].
The RBD is also observed to be the
potential target for treatment modalities and vaccine
development using S protein has led to antibody-
blocking therapy and small molecule inhibitors [8]. The
RBD can also be used as an antigen, leading to many
neutralizing antibodies isolated to be used in COVID-19
treatment. The presence of RBD-targeting antibodies,
which have displayed neutralizing characteristics
towards SARS-CoV-2, has been observed to achieve
a cross-neutralizing effect which might lead to the
development of antibodies targeting the SARS-CoV-2
specifically  [16]. The RBD is also used as a target
for vaccine development [17]. Several recombinant
subunit vaccines containing the RBD of SARS-CoV-2
and Fc fragment of human IgG, such as Adimr SC-2f,
S-RBD protein vaccine of China, ZF2001 using a
dimeric fragment of RBD, VIR-7831, AZD7442, or
LY-CoV555  [17]. The more well-known commercial
vaccine such as Moderna mRNA-1273 or BioNTech-
Pfizer BNT162b1 also uses RBD as the vaccine target
by generating the protein through mRNA [18].
The use of quantitative spike-RBD (s-RBD)
antibody quantitative assessment has been implemented,
mainly to find antibody titers of infection patients or
vaccinated subjects [19]. However, a direct comparison
between the naturally infected and vaccinated subjects
might not be made. Therefore, this study aims to compare
the s-RBD quantitative antibody titers between naturally
infected and vaccinated individuals.
Materials and Methods
Ethical consideration
All the participants were provided with an
adequate explanation of the reasons for retaking the
study and its procedures. The participants signed
informed consent forms, and their demographic data and
medical history were recorded. The study protocol was
approved by the local ethics committee at each institution
and conducted by the Declaration of Helsinki principles.
Study design and participants
This type of research is analytic observational
with a cross-sectional study design. This research was
conducted at the outpatient department and inpatient
department of the Gastroenterology division of the
Prof. Dr.  RD Kandou Manado and colonoscopy was
carried out at the Gastrointestinal Endoscopy Center,
Prof. Dr.  RD Kandou Hospital Manado. Examination
of fecal SCFA and fecal calprotectin levels was carried
out at the Manado Prodia Laboratory. The study was
conducted from December 2021 to June 2022.
Population and sampling
The study was conducted on Prof. Dr.  R. D.
Kandou Hospital Manado COVID-19  patients. Patients
with positive reverse transcription polymerase chain
reaction results will be considered COVID-19  patients.
COVID-19  patients of adult age (≥18  years old) within
2 weeks after diagnosis, without any autoimmune or
immunodepression diseases or states, is eligible for
the study. The participants must not use any steroid
or immunosuppressant medications before the study.
The team also searched for participants who have been
vaccinated, with any vaccine, through consecutive
sampling methods. Throughout the sampling process,
a total of 49 participants, consisting of 21 infected
participants and 28 vaccinated participants, were
retrieved for the antibody analysis.
The participants were then assessed for
their quantitative antibody count, using s-RBD as the
protein target. The assessment used the Enhanced
“Chemiluminescence” Immunoassay (ECLIA), using
the Elecsys® Anti-SARS-CoV-2 kit [20]. The ECLIA
method uses the binding of specific antibodies on
serum plasma to a specific antigen in the reaction well.
First, the samples will be incubated with recombinant
antigens, namely the ruthenium complex, resulting in
antibody/antigen complexes forming. The complex

B - Clinical Sciences Infective Disease 526 https://oamjms.eu/index.php/mjms/index
was then added with streptavidin-coated microparticles
to bind the complex into a solid phase. Next, the
reaction mixture is aspirated to the measuring cell by
magnetizing the microparticles to the electrode surface.
The application of voltage to the electrode will induce
chemiluminescent emission, which is then measured by
the photomultiplier. The resulting signal will determine
the level of specific antibodies in the patient’s samples.
As per the method above, 20 µL of the patients’ samples
or patients’ serum will be incubated with a mix of
biotinylated and ruthenylated nucleocapsid (n) antigens
forming a double-antigen sandwich complex for 9  min.
The streptavidin-coated microparticles were then
added to create a solid phase of biotin and streptavidin
for another 9  min for incubation. The reagent mixture
was then measured on the measuring cell. A cut-off of
<0.8 U/mL is considered non-reactive, with an antibody
count of ≥0.8 U/mL considered reactive.
Data analysis
The data analysis is conducted using the IBM®
Statistical Package for Social Sciences (SPSS®) ver.
25.0. First, patients’ characteristics on age and gender
are extracted. The outcome of the antibody count was
then classified into a naturally infected group and a
vaccinated group. Data were then assessed for their
normality using the Saphiro−Wilk analysis. Finally,
comparing both groups on the antibody count used the
Mann-Whitney non-parametric test. A p < 0.05 is used
to address the significance of the test result.
Results
A total of 49 participants (21 in the natural
infection group and 28 in the vaccinated group) were
included in the study (Table 1). The age of participants,
based on the median value, is older in the vaccinated
group compared to naturally-infection (38.5  vs. 34  years
old). In addition, the gender distribution for females
is higher in the natural infection group, with the same
number of males and females in the vaccinated group.
All data are not normally distributed; thus, information is
displayed in median (min-max).
Table 1: Patients’ characteristics and antibody count outcome
on natural infection and vaccinated group
Variable (s) Natural infection
(n = 21)
Vaccinated
(n = 28)
p-value
(If available)
Total (n = 49)
Age (years old) 34 (25–67) 38.5 (25–78) 38 (25–78)
Gender
Male 6 14 20
Female 15 14 29
Antibody count (U/mL)132.70
(0.00–250.00)
11.95
(0.88–250.00)
<0.001
1)
24.26
(0.00–250.00)
1)
Mann–Whitney non-parametric test.The antibody count based on the ECLIA
antibody test for IgG on SARS-CoV-2 has displayed a
significantly higher count in the naturally infected group
than in the vaccinated group (132.70  vs. 11.95 U/mL)
Figure 1. Also shows the considerably lower antibody
count in most vaccinated participants than in the
naturally infected group.
Figure 1: Antibody count using enhanced “chemiluminescence”
immunoassay analysis on both the natural infection group and the
vaccinated group. A higher value of antibody count in the natural
infection group is observed
Discussion
Our study has compared the antibody count
of the naturally infected individuals with the vaccinated
individuals, with a significantly higher antibody count
in the infected group than in the vaccinated group. Up
to the writing of this manuscript, the authors believe
this study is the first to compare the IgG antibodies
toward s-RBD level between the naturally infected
and vaccinated individuals. In addition, the increase in
antibody levels and seroconversion rate after the use
of the COVID-19 vaccine have been proven by various
trials on COVID-19 vaccine efficacy.
However, other studies on a similar topic
might not support our study. The COVID-19 vaccine
achieves almost 98% seroconversion rate as compared
to the antibody seroconversion rate of non-vaccinated
individuals with prior infection [21]. The results are also
in line with a study by Alatab et al., showing lower anti-
s-RBD IgG levels in the previously infected subjects
compared to the vaccinated subjects (2,110  vs. 1,341
BAU/mL), with higher IgG levels found in subjects with
the third dose of vaccine (booster) [19], [22]. Roltgen et al.
also stated the higher IgG levels observed in patients
vaccinated with the BioNTech/Pfizer mRNA vaccine
BNT162b2, as IgG is more dominant in the vaccinated
patients as compared to increased IgM or IgA in the
infected patients. The report is still suitable even with
the new variants such as B.1.1.7 or B.1.351 [23]. Most

 Lihawa et al. Quantitative Sars-Cov-2 S-RBD on vaccinated vs natural infection Open Access Maced J Med Sci. 2023  Apr 14; 11(B):524-528. 527
studies evaluated the comparison of antibody count from
the vaccinated subjects with prior infection compared
to vaccinated subjects without previous infection. Ali et
al., report higher IgG, IgA, and neutralizing antibodies
in the vaccinated with prior infection [24]. Similar
results were also reported by Demonbreun et al. and
Pantelidou et al., with higher values of median IgG and
neutralization rate as compared to the non-infected
patients [25], [26].
The result of this study, however, is quite in
line with one of the results in a study by Jaworska et
al., Despite the initial results of higher IgG value in the
vaccinated group, the IgG decay curve was found steeper
in the vaccinated group, particularly the BNT162b2 Thus,
after 6  months of post-vaccination, a lower IgG level is
observed in patients as compared to hospitalized COVID-
19 patients 6  months earlier [27]. This also might explain
the phenomenon found in this study, considering the non-
defined vaccination period of the included participants.
Our study also factored in the age and gender
of the participants. Sasso et al. reported higher s-RBD
IgG levels in the female group compared to the male,
despite a similar decay rate of the IgG [22]. Ikezaki et al.
also reported that the mean titer of anti-spike IgG was
lower in the older age group after adjustment for sex,
body mass index smoking habits, and alcohol drinking
habits. Other variables are found not significant on the
IgG antibody count [28]. However, age is considered a
significant positive negative correlation toward (s-RBD),
with lower antibodies found between 30 and 39  years
old [29]. Lower length of stay in the hospital and lower
oxygen need are also associated with the increased
s-RBD antibody count [30].
This study is not without limitations. Without
defining the time frame, the diverse definition of
COVID-19  cases and vaccination status might lead to
non-conclusive results due to IgG decay status.
Conclusion
The s-RBD antibody titer is significantly
higher in naturally infected patients than in vaccinated
individuals. Further analysis and study on the topic,
particularly in defining a suitable time frame, is needed
to properly understand the effect of the COVID-19
vaccine compared to infection in terms of antibodies
with a larger sample size.
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