Treatment with Monoclonal

antibodies for patients with

SARS-CoV-2infection
[In The plasma]

[08/09/2021]
FACULTY OF PHARMACY
 Monoclonal antibodies

STUDENTS NAME: Nael Kamel Eltewacy 01100728639 nael.eltwacy@gmail.com

Amr Aly Resha 01283257524 amr.resha.student@pua.edu.eg

Huda Anas Gallhoum 01120352766 hudaanas369@gmail.com

Yara Mohamed Arif 01012072019 yara.aref.student@pua.edu.eg

Ahmed Abdel Aleem

Rana Khaled

DEPARTMENT: microbiology
PI:
CO-PI:
DATE OF SUBMISSION: 08/09/2021
 ABSTRACT

Recently, the entire world and the science committee, in particular, have stepped out to confront
Coronavirus COVID-19, which has stopped human lives because no specific vaccine has been
discovered or even successful treatment has helped anyone to confront it, So everybody has
recourse to precautionary steps, and we have worked to develop an antibody vaccine that is
produced in the bodies of patients that have been positively diagnosed with the virus and have been
recovered after the quarantine period.

Some experts and researchers have pointed out the efficacy of blood transfusions containing
antibodies to this virus, However, it remains a question and under experiment, but in our research,
we have relied on more accurate methods to target segments of the virus' spiked glycoproteins using
monoclonal antibody technology, a special type of molecular protein produced in the laboratory, to
find that antibodies act as a frontline defense against disease for the body. As for the monoclonal
antibody solution, it works against a specific antigen and can be manufactured in large quantities,
This bodes well for the scientific work that the science community hopes to discover the vaccine and
combat the epidemic.

In addition to the follow-up to the patient's records in December 2019 to assess the number of deaths
due to cases of pneumonia of uncertain origin, which highlights the probability that this virus has
been present since that time. It also explains the low number of infections in the Arab Republic of
Egypt due to the development of antibodies to this virus in the bodies of the Egyptian people, and
this will also be another strength in the research.
Studies will be extensive in this area and will be retroactive to patients, in addition to our work
extracting vaccine from antibodies, and this is what the science is heading in recent times to discover
any vaccine against any virus.

The role of nanotechnology is very integral in combating this nano-enemy “virus.” Although many
sources are underneath ongoing attention for prevention and care, we would like to start sharing with
readers our vision of the position of inhaled nanomaterials and targeting systems that can play an
important role in the fight against the COVID-19. In this study, we emphasized the genomic structure
of COVID-19, the current mode of virus transmission, infection control measures, pathogenesis, the
scientific manifestation of SARS-CoV-2, and the extent of the virus's impact on the lungs. Even more
than that, Inhaled monoclonal antibodies are mentioned to enhance the target.
Background/Objectives:

Antibody-dependent enhancement (ADE) of viral entry was a major problem in epidemiology,

vaccine development, and antibody-based drug therapy. The molecular mechanism at the return of
ADE is, however, obscure. COVID-19 as virus entry into cells through first binding angiotensin 2
spike glycoprotein converting enzyme to the human cell surface and then fusing viral and host
membranes. This is due to the binding of MAb to the IgG Fc cell surface receptor, which directs viral
entry via canonical viral-receptor-dependent pathways.

The SARS-CoV-2 vaccine is slightly higher than yr away (SN: 2/21/20). In an effort to monitor
COVID-19 in the next few months, the question is, "What kind of interventions should we take that
will break down this pandemic?"Says pathologist John Roback of the Emory University School of
Medicine in Atlanta, who is doing research on transfusion medicine. Pinnacle candidates are drugs
that have already been approved to deal with diseases such as malaria that could potentially be
repurposed for COVID-19 (SN: 3/10/20) and convalescent plasma, he says.

The key to the solution is to take samples from patients who have antibodies to the disease after
making sure they are infected with an IgM or IgG reaction and redirecting it and working to duplicate
and modify Monoclonal Antibodies (mAb or moAb) and then re-injecting it in patients with
Coronavirus through continuous monitoring of cell receptors.

First, we want to carry out a retrospective study on chest hospitals in Egypt from November 2019
(period of symptoms such as coronavirus or COVID-19) through March 2020 using a simple
questionnaire and analysis of this data to show the number of patients who have the virus at a later
time and are being treated for themselves How many died without proven reasons.

Second, for these patients, we are instructed to do an IgM or IgG reaction, take plasma samples
from them, then re-adjust and inject them to show the effects.

At this point, we will make sure that we already have the virus and that our bodies have antibodies
and that's why there are fewer cases in Egypt. That also enables the experts to use antibodies as a
way to treat COVID-19.

Key Words: Monoclonal antibodies, COVID-19 infection, SARS-CoV-2, Convalescent plasma.

 INTRODUCTION
The risk of spreading new pathogenic organisms is being a global threat to human beings. Although
the power of vaccination in providing sustained and active protection, the production of new vaccines
is not an easy nor fast step. Behring and Kitasato1 has brought the idea of administering polyclonal
immunoglobulins produced from immunized human or animal origin, and it has been used effectively
as a prophylaxis. Despite this fact, some drawbacks have been associated with using polyclonal
immunoglobulins such as the antigenic reactions that could be produced due to heterologous
proteins, using human blood products is risky and that it's not easy to find immunized donors. A
terrible excessive outbreak of completely new acute respiratory syndrome coronavirus 2 (SARS-
CoV-2) infection has been launched first in Wuhan, China since last December 2019, then it has
speedily extended in 171 countries. From that time till March 24 2020, 379,661 of proven cases and
16,428 deaths worldwide have been recorded because of this virus. And till now, no specific therapy
has been found, only supportive care.
Some treatments are undergoing developments to reinforce surviving from this outbreak. And as
well known that immunotherapy with virus-specific antibodies in convalescent plasma had been used
for serious infectious diseases such as pandemic influenza A, SARS, Ebola virus, middle east
respiratory syndrome coronavirus, and avian-origin influenza A as an only chance for surviving, now
it has shown in some previous reports that treating currently infected patients with convalescent
plasma obtained from recovered patients could reduce the mortality rates resulting from this
outbreak, despite the unclear understanding for the efficacy of convalescent plasma in patients with
SARS-CoV-2 infection with the critically ill condition.
Avian infectious bronchitis virus (IBV), infrequent with other members of the Coronaviridae, contains
three structural proteins: spike (S; peplomer), membrane (M) and nucleocapsid (N) (Siddell et al.,
1983). The spike protein of IBV involves two glycopolypeptides, S1 and S2, of 90K (90 0 mol. wt.).
Parts of each S and the glycosylated M protein (30K) are existing at the virion surface. The N protein
(54K) is related to the viral RNA. Several monoclonal antibodies have been established which react
with the three structural proteins of every other coronavirus, murine hepatitis virus (Collins et al.,
1982; Fleming et al., 1983).
The majority of monoclonal antibodies neutralized the virus through its spike glycoprotein but not for
the proteins with different structures, though some anti-membrane protein monoclonal antibodies
can neutralize the virus if the complement is present. Their idea was based on creating monoclonal
antibodies specific to the IBV spike glycoprotein to determine if they could neutralize the virus or not.
To recognize if both of the two glycopolypeptides that contain the spike protein, having the concerned
antigenic determinants localized to or not. And lastly to find out if the produced monoclonal
antibodies by the inoculation procedure, could have any other virus-specific properties.
2019-nCoV belongs to lineage B beta coronavirus, with high sequence identity with that of bat or
human extreme acute respiratory syndrome coronavirus-related coronavirus (SARSr-CoV) and bat
SARS-like coronavirus (SL-CoV), based on the phylogenetic analysis.
A wide variety of potent monoclonal antibodies against SARS coronavirus (SARS-CoV) have been
known in previous studies. The target of these antibodies was the spike protein (S) of SARS-CoV
and SL-CoVs which is a type I transmembrane glycoprotein through which the entrance to human
respiratory epithelial cells is mediated via interacting with cell surface receptor angiotensin-
converting enzyme 2 (ACE2). The critical target for neutralizing antibodies specifically is the 193
amino acid size (N318-V510) receptor-binding domain (RBD) inside the S protein. There are different
epitopes on RBD that could be also recognized by some of the antibodies; e.g. the SARS-CoV
neutralizing antibodies CR3014 and CR3022 bound noncompetitively to the SARS-CoV RBD that
synergistically could neutralize the virus. They conceived that the complex structures of 2019-nCoV
RBD with numerous neutralizing antibodies as well as its confirmation and the results they found,
help in the interactions between 2019-nCoV RBD and specific SARS-CoV antibodies. Assuming this
to be as a result of the relatively high identification (73%) of RBD in 2019-nCoV and SARS-CoV. As
indicated by the complex crystal structure that the residues in RBD of SARS-CoV make polar
interactions with a neutralizing antibody m396, are fixedly preserved in 2019-nCoV RBD. R395 in
RBD in the structure of SARS-CoV-RBD-m396 formed a salt bridge with D95 of m396-VL.
Additionally, electrostatic interaction was observed in the model of 2019-nCoV-RBD-m396, forming
via R408 (RBD) and D95 (m396-VL).
So some SARS-CoV-specific monoclonal antibodies might be able in neutralizing 2019-nCoV as
proposed by this evaluation. In contrast, a considerable decrease in the interactions between
antibody F26G19 or 80R and the RBD in 2019-nCoV due to the lack of salt bridges fashioned by
way of R426-D56 in SARS-CoV-RBD-F26G19 or D480-R162 in SARS-CoV-RBD-80R, respectively.
Moreover, as most of the 80R-binding residues on the RBD of SARS-CoV are no longer preserved
on RBD of 2019-nCoV, the effective recognition of 2019-nCoV by antibody 80R is not working.
Subsequently, the need to determine the cross-reactivity of anti-SARS-CoV antibodies with 2019-
nCoV spike protein is very urgent, that may affect the rapid improvement of vaccines and therapeutic
antibodies against 2019-nCoV.

nAbs against SARS-CoV, MERS-CoV, and SARS-CoV-2

Virus nAbs induced by vaccines or infected viruses play crucial roles in controlling viral infection.
Now the development of SARS-CoV- and MERS-CoV-specific nAbs include monoclonal antibodies
(mAbs), their functional antigen-binding fragment (Fab), the single-chain variable region fragment
(scFv), or single-domain antibodies [nanobodies (Nbs)]. Their inhibition for viral inhibition is by
targeting S1-RBD, S1-NTD, or the S2 region, as also blocking the binding of RBDs to their respective
receptors and interfering with S2-mediated membrane fusion or entry into the host cell. SARS-CoV
and MERS-CoV RBD-specific nAbs are represented but No SARS-CoV-2-specific nAbs have been
reported, Thus we herein introduce SARS-CoV- and MERS-CoV-specific nAbs in the context of their
potential cross-neutralizing activity against SARS-CoV-2 infection.
 LITERATURE REVIEW
Representative SARS-CoV RBD- and MERS-CoV RBD-Targeting nAbs(Jiang, Hillyer, and Du
2020a)

Ab name Source Neutralizing activity Neutralizing mechanism Protective efficacy

S230.1 Human Neutralize human (strains Recognize epitopes Protect mice against challenge
5 m396 GD03, Urbani, Tor2) and (residues 408, 442, 443, of SARS-CoV (strains Urbani,
mAbs palm civet (strains SZ3, 460, 475) on SARS-CoV S1 rGD03, or rSZ16)
SZ16) SARS-CoV infection protein, interfering with
RBD–ACE2 receptor
interaction
S109.8 Human Neutralize human (Urbani, Inhibit the binding of SARS- Protect mice against challenge
S227.14 GZ02, CUHK-W1), palm CoV RBD–ACE2 receptor of SARS-CoV infectious clones
S230.15 civet (HC/SZ/61/03), and (Urbani, GZ02, HC/SZ/61/03)
mAbs raccoon dog or mouse-adapted strain
(A031G) SARS-CoV (MA15)
infectious clones
containing S variants
80R Human Neutralize live SARS-CoV Recognize epitopes on NA
scFv, (strain Urbani) infection SARS-CoV S1 (residues
mAb 261– 672), blocking RBD–
ACE2
binding and inhibiting
syncytium formation
CR3022 Human Neutralize live SARS-CoV Recognize epitopes on CR3014 protects ferrets
CR3014 (strain HKU-39849) SARS-CoV RBD against SARS-CoV (strain
scFv, infection; CR3022 could (residues 318–510); HKU-39849)
mAb neutralize CR3014 escape CR3022 binds infection
variants SARS-CoV-2 RBD with
high affinity
33G4 Mouse Neutralize human (strains Recognize epitopes on NA
35B5 GD03, Tor2) and palm civet SARS-CoV RBD, blocking
30F9 (SZ3) pseudotyped SARS- RBD–ACE2 receptor binding
mAbs CoV infection
MERS- Human Neutralize divergent strains Recognize a number of keyProphylactically and
27 of pseudotyped and live epitopes on MERS-CoVtherapeutically prevent and
m336 (strain EMC2012) MERS- RBD protein, blocking RBD–treat MERS-CoV (strain
MERS- CoV infection DPP4 receptor binding EMC2012) challenge in hDPP4-
GD27 Tg mice, rabbits, or common
MCA1 marmosets
mAbs
, Fabs
4C2 h Humanized Neutralize divergent strains Recognize epitopes Prevent MERS-CoV
hMS- of pseudotyped and live (residues 510, 511, 553) on (strain EMC2012)
1 (strain EMC2012) MERS- MERS-CoV RBD protein, challenge in Ad5/hDPP4-
mAbs CoV infection blocking RBD–DPP4 transduced or hDPP4-Tg
receptor binding mice
Mersmab Mouse Neutralize pseudotyped and Recognize a number of key NA
1 4C2 live (strain EMC2012) epitopes on MERS-CoV
D12 MERS-CoV infection RBD
mAbs protein, blocking RBD–DPP4
receptor binding
HCAb-83 Dromedar Neutralizes live MERS- Recognizes epitope (residue Prophylactically prevents
Nb y camel CoV (strain EMC2012) 539) on MERS-CoV MERS-
infection RBD protein CoV (strain EMC2012)
challenge in hDPP4-Tg mice
NbMS10- Llama Neutralizes multiple strains of Recognizes epitope (residue Prophylactically and
Fc pseudotyped and live (strain 539) on MERS-CoV therapeutically prevents and
Nb EMC2012) MERS-CoV RBD protein treats MERS-CoV (strain
infection EMC2012) challenge in hDPP4-
Tg
mice
Methods:
The mechanism of virus action explains why we resort to antibodies as part of treatment. The virus
enters cells through a glycoprotein bound to the angiotensin 2 converting enzyme on the surface of
the human cell and then fuses the viral membranes and the host, The antibodies produced by the
body attack these spike glycoprotein, preventing the virus from sticking to the angiotensin 2
converting enzyme and entering inside the cell.Therefore, some experiments resorted to the use of
convalescence plasma obtained from convalescence patients with COVID 19 infection proven after
recovery and this plasma was injected in patients with the virus who suffer from symptoms such as
fever and respiratory complications and did not respond to the usual treatment.

Our research steps can be expressed through four axes:

1. How to prove our hypothesis

2. Purification, Identification of the structural sequence and generate Monoclonal for antibodies
3. Amplification
4. Replication

From the first axis, we want to conduct a retrospective study of chest hospitals in Egypt from
November 2019 (period of onset of symptoms such as coronary virus or COVID-19) until March 2020
through a simple questionnaire more likely what have done in China and our questionnaire will be
available Then we will analyze this data to show the number of patients who have the virus later and
recovered and the number of people who died without proven reasons and this gives us a full answer
to the question of why the cases of infections did not explode in Egypt like most countries of the
world despite the delay of the state in closing airports and preventing tourist delegations.

The Second axis have three phases:

1. Purification
2. sequencing
3. Monoclonal antibodies

1) Purification divided into one of three steps:

 Anion-exchange chromatography
 Polyclonal Antibodies (Serum Purification)
 ELISA
Anion-exchange chromatography
There is a difference between protein A and G chromatography or convergence-based methods
and anion-exchange chromatography, and the second is the most commonly used method for
isolating large amounts of purified IgG. The anion exchange chromatography is much more
affordable and provides the ability to isolate a wide range of IgG sub-layers, and uses milder
conditions.

With the continuous development of therapeutic antibodies to treat a variety of pathological

physiological conditions, the widespread purification of antibodies (for example, mAbs expressed in
viruses) is one of the main applications of chromatography in the pharmaceutical arena. Therefore,
many manufacturers are turning to this field and this device.

If it is achieved on a small scale, it is viable to perform coloration imaging the usage of DEAE-
agarose (or other DEAE-modified resins or membranes) in several ways, this coloration separation
has to be performed based totally on the accessible equipment, the measurement of the
preparations and the purity required. Options include the use of open columns, where buffers go
with the flow through the column the usage of a simple gravity feed; and the use of open columns.
The bulk separation, in which the centrifuge is used for resin pellet with protein binding followed
through washing and elution the usage of a stained glass funnel, multiple centrifuge steps can be
used in more than one way, or the use of small vessels for rapid washing and elution of binding
antibodies. For functions that require large amounts of pretty purified antibodies or these requiring
a lot of constant contact from lot to lot, automated chromatographic systems such as high-protein
liquid chromatography (FPLC) or high-pressure liquid chromatography (HPLC) ought to be used.
These large columns are used, filled with suitable anion change media, membrane-based ceramic
media, or other low-to-high capacity sorts and slow-to-flow-based anion supports, providing large
quantities of high purity.

Polyclonal Antibodies (Serum Purification)

When selecting a polyclonal antibody, either as a most important or secondary antibody in an
immunoassay, the technique consists of some steps, serum purification, and salt precipitation.
Serum Purification
Polyclonal minor antibodies may additionally be
supplied so a serum fraction. toughness serum will
probably include less than 10% unique antibody. The
deficiency of specificity is appropriate according to a
multitude of antibody clones and other proteins,
including albumin, which may also bond non-specifically
within an immunoassay.
Serum will oft be located within individual labs
provided via researchers growing their own
antibodies toughness Serum is the amber-colored
supernatant obtained after blood is allowed
according to clot and is the simplest purification
method stability Salt Precipitation One frequent
method for purifying proteins the solubility concerning proteins is related after the salt concentration
regarding the solution. Increasing the total of salt
among a solution essentially removes water
molecules out of the protein, causing the protein to
precipitate. This is in many instances longevity
known as “salting out.” stability Salt precipitation is an
inexpensive way following consecrate antibodies
beside serum; however, the technique does not remove proteins including a precipitation pattern
comparable to antibodies.
Purification Affinity
There are three sorts of Purification Affinity. Often, the three are used in the mixture when the purest
antibodies are needed.
Immunoglobulin-Specific Affinity Purification
There are three proteins involved in the Immunoglobulin-Specific Affinity Purification used to purify
antibodies, protein A, G protein, and L protein.
Antigen Affinity Purification
Antigen affinity purification is another particular kind of affinity purification and results in the purest
antibodies with the least quantity of cross-reactivity. Affinity-purified antibodies exhibit the best
possible specificity and sensitivity that can be obtained from serum.
Serum Adsorption (Negative Affinity Purification)
Polyclonal antibodies are most used as anti-immunoglobulin secondary antibodies in a variety of
immunoassays Despite the broad use of negative affinity for purification purposes, producers have
traditionally only listed which antibodies have been serum adsorbed.
ELISA (sandwich)
ELISA assays are primarily based upon the principle of antibody/antigen binding. They enable
quantification and characterization of specific analytes and/or molecular interactions.
Antibodies against the target of interest are conjugated to a reporter enzyme. Upon the addition of
its substrate, the enzyme catalyzes the production of a colorimetric molecule. The extent of this
response is measured by the use of a spectrophotometer and is a consultant of antigen
concentration inside a sample.
The most suitable ELISA format for every experiment will depend on many factors, consisting of
desired sensitivity, specificity and assay time. For the identical cost as a standard ELISA, our Simple
Step ELISA™ kits can halve assay time, without compromising sensitivity or reliability.

Simple Step ELISA kits

With SimpleStep ELISA kits, an
analyte-capture and detector
antibody sandwich complicated is
formed in solution, which binds to
the microplate by way of an affinity
tag attached to the capture antibody
in the sandwich pair.

Two distinct sites are bound to the

target protein using the traditional
ELISA sandwich using a pair of antibodies. The capture antibody is pre-coated onto the well of a
microplate and selectively binds to the target protein. After a wash step, the detector antibody is
delivered and binds to a 2nd site on the target protein to shape a sandwich complex.

SimpleStep ELISA kits streamline this method by way of the use of a semi-homogeneous layout
wherein the antibody-analyte sandwich complex is shaped in solution in a single step. In simply
one incubation and wash step, the whole sandwich complex forms in the well and is anchored to
the plate with an immunoaffinity tag

Every simple step ELISA® kit is validated the usage of multiple biological samples for assay
specificity. All secreted serum or plasma-based targets are tested and fall within the World Health
Organization blood reference ranges. When available, the SimpleStep ELISA kits are calibrated
against a recognized NIBSC international standards and includes a conversion factor for data
comparison.

Our antibody pairs appear between 2 formats:

Antibody pair kits: include a titrated capture and biotinylated detector antibody pair then a
calibrated protein standard. Available into two sizes, together with enough reagents for either 2 or
ten x 96-well plates the usage of a standard sandwich ELISA.

Carrier-free antibody pairs (BSA, glycerol, and Azide free): include a capture and detector
antibody pair, suitable because of ELISA-based assays. Available as much ten x 96-wells plate.
Antibody pair kits and reagent pair combinations provide consistent, specific and sensitive results.
 batch-to-batch consistency: only recombinant monoclonal antibodies are used in our
antibody pairs.
 Specificity: antibody pairs are screened in plasma and serum to make sure specificity in
complex samples.
 Sensitivity: benchmarked towards commercially reachable antibody pairs to make certain
equivalent or ideal performance compared with the competition.
 Flexibility: Our carrier-free pairs provide even more flexibility in terms of conjugation and
assay set-up.

Sequencing and Monoclonal antibodies

De Novo Antibody Sequencing Workflow.
• Antibody protein is at the start digested into overlapping peptides using more than a few trypsin
• Digestion with LC-MS/MS, then fragmentation optimization
• Overlapping peptides are characterized via de novo peptide sequencing
• Automated antibody sequence assembly the use of characterized peptides
• Data analysis is performed using state-of-the-art computational algorithms that effectively derive the

amino acid sequence of the digested peptide.

Sample Preparation:
We’ll separate the heavy and mild chains of an antibody through SDS polyacrylamide gel
electrophoresis (SDS-PAGE). Briefly, 0.5μg of the antibody will be placed, reduced and denatured
in gel loading buffer. The sample will consequently be loaded into three wells that contained a 10%
precast gel (BioRad). The gel will be subjected to a hundred and eighty constant volts for 50minutes.
Following this, we’ll stain the gel with Coomassie Blue. Gel bands that contained the antibody will be
excised.

Deglycosylation and Endoprotease Digestion

We will decrease every excised band with
dithiothreitol (DTT). Free cysteine residues will
be then alkylated the usage of iodoacetamide.
Then the heavy chain bands will be
deglycosylated with PNGase F (Roche
Diagnostics). The pH has to be adjusted for
each protease and three enzyme digestions
that would be carried out in a single day according to the manufacturer’s instructions: 1) Asp N, 2)
Chymotrypsin, 3) Trypsin. The peptides will be extracted from the gel bands, desalted the usage of
C18 Zip-Tips® (Millipore) and dried in a speed-vac.
LC-MS/MS Analysis
We will let desalted peptides be suspended in 0.1% formic acid and 1/10 of every of the digests will
be subjected to LC-MS/MS analysis on a Thermo-Fisher Scientific Q-Exactive (Q-E) Orbitrap mass
spectrometer. Then the gradient will be provided the usage of a Thermo-Fisher EASY LLC-1000
UHPLC system and consisted of zero to 40% acetonitrile in 0.1% formic acid over 1hour at 250nL
per minute. We will run the Q-E in a data-dependent mode with 10 MS/MS events per cycle. The
parent ion decision is 70,000 FWHM and the fragment ion resolution is 17,500 FWHM. The 12
ensuing raw data files (6 for every antibody, three for the light chain and three for the heavy chain)
will be used for data analysis.

Amplification and Replication

We will need throughout the next step us Oligonucleotide Primer Design this methodology
for Replication and Amplification of Antibody Genes in PCR. This technique makes it viable to
construct antibody libraries in phage except resort to hybridoma technology. Sensitive research of
B-cell differentiation and ontogeny, B-lymphoma oncogenesis and heterogeneity, and antibody
diversity, as properly as immortalization and manipulation of the antibody repertoire for diagnostic
and therapeutic purposes, are now possible.

The oligonucleotides were designed the usage of the database and taking into account
degeneracies for each amino acid. The 5' primers were constructed from data available on the
conserved sequences of the leader and first framework (FR1) regions. In some instances
subgroups of primers were made earlier than assignment of degeneracy (e.g., human heavy
chain). Generally, organizations of primers have been mixed earlier than PCR. Oligonucleotides
were made on an Applied Biosystems 380B DNA synthesizer. Purification of oligonucleotide
primers was found to be unnecessary. In popular the amplified fragments of DNA are 400-500 bp
in length, corresponding to the dimension of a V region with additional bases in the leader and 5'
portion of the constant regions and restrict enzyme sites.

The rapid direct amplification of antibody variable regions opens many probabilities for future
research. The techniques described above recommend that recombinant DNA technology can
exchange cell fusion as a means of producing monoclonal antibodies. The concept is quite simple:
a mixture of oligomer primers in the 5' leader sequences or framework 1 region mixed with 3'
constant.
Results:
All these acts support the hypothesis, and will be ascertained that most of the Egyptians have already
had the virus 3 months ago and that their bodies have antibodies that's the reason why the number
of cases in Egypt is lower. The efficacy of plasma transfusion and monoclonal antibodies will be
confirmed to covid-19 patients.

Discussion:
The hypothesis is supposed to this year, particularly in November 2019, several people had a
peculiar cold that currently has the same coronavirus symptoms as dry cough and fever (high
temperature) to continue stuck with people for more than a week. Many cases were transferred to
an intensive care unit for the same symptoms, and even all of their bacterial cultures were negative,
referring to first degree viral infection. Many cases have been directed to ARDS (Acute Respiratory
Distress Syndrome).

There are studies on los angeles Between March 12-13 and 15-16, 2020 which have been performed
on One hundred thirty-one tests for SARS-CoV-2 ,and 7 were positive (Spellberg et al. 2020).

Also recorded cases in Wuhan were 640 throat swabs collected from patients with influenza-like
illness from 6 October 2019 to 21 January 2020 and confirmed that 9 of the 640 throat swabs were
positive for SARS-CoV-2 RNA by quantitative PCR (Kong et al. 2020). In Egypt, some Facebook
posts from physicians in chest hospitals and clinics have been observed cases with symptoms like
those of covid-19 and no response for any kind of medical care for more than 40 days.

Conclusion:

Achieved to the perfect treatment of COVID-19, which will be available to all humans through
monoclonal antibodies.
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