The Detection of IgG and IgM Antibodies in Blood: One of the Many Tools to Help Manage an Epidemic

1. SEROLOGICAL TESTS
The Detection of IgG and IgM Antibodies in Blood:
One of the Many Tools to Help Manage an Epidemic
Antibodies are compelling proteins that are essential to the immune system and extremely powerful in
biotechnology applications; existing as major players in our defence against external agents (viruses,
bacteria, etc.), they are also widely used as tools for research, diagnosis and treatments.
The presence of antibodies in the blood in larger than usual numbers often reflects an infection (viral
or bacterial) or a specific pathology. In addition to be integral components of regulatory-bodies-
approved in vitro diagnostic tests, antibodies can also be used as a biomarker.
Many commercially available tests, known as serological tests, are available to detect antibodies in
blood samples. These tests are particularly useful for finding out if a person has been in contact with
a specific infectious agent. It can also tell whether the contact is recent or older.
We know that most people who have been in contact with the Severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) do not develop symptoms but they are supposed to develop specific
antibodies. If sensitive enough, the serological tests could therefore be a solution during the
coronavirus-2-disease-2019 (Covid-19) epidemic, to identify potential healthy carriers or people who
have encountered the infectious agent and who have developed an immune response against it.

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ANTIBODY OVERVIEW
ntibodies, or immunoglobulins, are complex glycoproteins secreted by B lymphocytes (plasmocytes).
In general, these proteins are produced by the immune system in response to an infectious agent or a
pathogen. Antibodies will specifically recognize this agent, referred to as an antigen, in order to
neutralize and eliminate it. The production of antibodies and the antigen-antibody recognition is
therefore a completely natural defence system enabling the body to act against a pathogen.
The structure of antibodies (Abs) was first described in 1959 by Porter and Edelman. It is a protein of about 150
kDa. With a global structure similar to the letter Y (Figure 1), it consists of:
 2 heavy chains (H for heavy – in blue) each with 1 variable and 3 or 4 constant domains
 2 light chains (L for Light – in green) each with a variable and a constant domain
Heavy and light chains are connected to each
other by disulfide bridges (in yellow).
VH + VL = variable globular domain at the end
of the 2 branches of the « Y ». The antigen is
recognized by the N-terminal ends of the
variable domains.
CH1 + CL = first constant globular domain
between the variable domain and the hinge.
CH2 x 2 = second constant globular domain
under the hinge.
CH3 x 2 = third constant globular domain,
towards the C-terminus of the heavy chains.
FIGURE 1 ANTIBODY MODELLING. MOUSE IgG2a ISOTYPE. THE LIGHT CHAINS ARE GREEN, THE HEAVY CHAINS
ARE CYAN AND BLUE, THE GLYCAN IS IN ORANGE, AND THE INTERCHAIN DISULFIDES ARE YELLOW STICKS.
In humans and in mice, there are different categories of antibodies or isotypes in the blood stream (Figure 2):
 IgG (normal values for IgG are ~70% of the
circulating Abs in humans)
IgG is the most present antibody isotype in normal
human serum. It consists of 4 subclasses (IgG1,
IgG2, IgG3 and IgG4) each containing a different
heavy chain type.
 IgM (10%)
As a soluble globulin, IgM is most often present as a
pentamer, its basic structure held together by a J
chain. It can also exist as a hexameric form in the
serum and as a monomer on the surface of B-cells.
 IgA (<20%)
IgA exists as a monomer or a dimer. In contrast to
the serum where this isotype is in minority, IgA is the
A
FIGURE 2 IMMUNOGLOBULIN ISOTYPES

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predominant antibody in mucous secretions such as saliva, tears, etc.
 IgE (<1%)
IgE is very rare in the serum but is found on basophils and mast-cells. This class is known for its role in
allergies and to fight infection by parasites.
 IgD (<1%)
IgD is very rare in human serum and more important on the surface of B-cells. Its role is still unknown.
IgM is the predominant antibody in the primary immune response. During the maturation of B lymphocytes, there
is an isotype switching: IgG will be expressed in larger quantities in serum, IgA in the mucosal tissues and IgE
for allergens or parasites.
FIGURE 3 AVERAGE ANTIBODY EXPRESSION AFTER EXPOSURE TO A GIVEN ANTIGEN THAT ELICATES IGGS
SEROLOGICAL TESTS OVERVIEW
erological tests allow to detect soluble proteins, hormones or biomarkers – antibodies in the case that
interests us today – in a blood serum sample. These tests can be qualitative or quantitative and are widely
used for screening, diagnosis, or patient follow-up, but also for epidemiological studies.
As all diagnostics tests these, tools are not 100% reliable. Indeed, due to the time of appearance of
immunoglobulins and also their expression in a wide range of quantities – starting from very low levels – the
tests must be very sensitive to avoid delivering false negatives (when the biomarker is present, but the test fails
to detect it). The question of the limit of detection (LOD) will be discussed below.
On the other hand, the specificity has to be good enough for the test to be meaningful; you do not want to have
too many false alerts that would not come from real infections (false positives), although in the case of Covid-
19, the risk for the patient is relatively low since the consequence will just be a quarantine, as far as no specific
treatment can be given (present situation). A true positive result means that the patient has been in contact with
a pathogen at some time. Unlike virological tests in which viral particles are detected, serological tests allow a
patient to be evaluated even if the virus is no longer present. In addition, long-term follow-up can provide
information, for example if the patient is in the course of its isotype switch.
S

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There are different types of serological tests:
 Precipitation tests based on the precipitation that takes place when antibodies (especially IgMs) and
antigens are mixed together.
 Neutralization tests, which depend on the capacity of antibodies to neutralize the infectious properties
of the infectious organisms.
 Hemagglutinin-inhibition tests based on the agglutination of red blood cells. This agglutination will be
prevented by the presence of antibodies.
 Enzyme Linked Immunosorbent Assay (ELISA) or Lateral Flow Immunoassay (LFIA) based on
antibody binding to the antigen on a solid surface (plate or strip).
We will focus only on these last two techniques which are the most common in routine (see BIOTEM’s
Immunoassay Solutions).
1. ELISA: Enzyme Linked Immunosorbent Assay
Principally used for research and for in vitro diagnostic purposes, ELISA tests are enzymatic immunoassays for
the detection and quantification of analytes in equipped laboratories. Several formats of ELISA are available
(Figure 5):
 Indirect ELISA
This is a simple 2-step ELISA test. The antigen (or pathogen) of interest is coated on the plate. Serum
samples are then deposited and incubated. In the presence of antibodies (IgG / IgM) in the serum, these
will specifically bind to the antigen. Revelation is carried out with an anti-human isotype, secondary
antibody conjugated to a tracer, typically the enzyme HRP. This format is the most commonly used in
routine.
FIGURE 4 SEROLOGICAL TESTS. A) PRECIPITATION TEST (COMPLEMENT FIXATION TEST) ;
B) NEUTRALIZATION TEST ; C) HEMAGGLUTININ-INHIBITION TEST.
Sources: Online Microbiology Notes and Microbe Online

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 Sandwich ELISA
The plate is coated with a capture antibody specific to the antigen / pathogen of interest. This primary
antibody could be commercially available or developed from scratch (see BIOTEM’s Antibody Solutions).
Secondly, the antigen in solution is deposed and incubated. Then serum samples are deposited and
incubated. Specific antibodies (IgG / IgM, but also other isotypes) that are present in the serum will bind
to the antigen. Revelation is carried out with an anti-human IgG or IgM, secondary antibody. The capture
antibody (the one coated on the plate) must be from another species than human in order to avoid non-
specific binding of the anti-human secondary antibody (the one used for revelation).
 Competitive ELISA
The result is negatively proportional to the amount of biomarker to be measured. This format is rarely
used for serological tests.
2. LFIA: Lateral Flow Immunoassay or Rapid tests
Lateral Flow tests are based on the migration of nano or micro particles on strips for analytes detection. These
tests, strips or cassettes (example: the pregnancy test) are widely used as first-line emergency tests performed
at the patient’s bedside or directly in the field, thanks to its user-friendly design.
Several common names are used (although some are less adapted than others):
 Lateral Flow ImmunoAssay (LFIA)
 ImmunoChromatography assays or ImmunoChromatography Test (ICT)
 Rapid Test or Quick Test
 Point of Care (POC) Test
 Strip Test or Dipstick test
As with the ELISA test, the Lateral Flow test can be used in different applications (Human health, Animal health,
Drug presence assessment, Industrial quality control, Agriculture, Biosecurity, Environment, etc.) and with
different types of samples (serum, plasma, urine, saliva, food, environmental media, water, etc.).
Although a Lateral Flow test is easy to use, it is based on a sophisticated device made up of several components
lying on a plastic support strip (Figure 6):
FIGURE 5 SCHEMATIC REPRESENTATION OF TWO TYPES OF ELISA TESTS

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● The Sample Pad is necessary for the proper distribution of the
sample towards the conjugate pad. The sample pad can also be
impregnated with different reactants (buffers, detergents, proteins,
etc.) to modify / adapt the chemical / physical characteristics of the
sample or to facilitate its migration and fixation on the test line. It
could also be useful as a filter. Typically, the sample pad is composed
of cellulose fiber filters.
● The function of the Conjugate Pad is to distribute the detector
particles onto the so-called ‘Membrane’ (the real support of the test)
in a consistent volume with respect to the sample. The analyte
present in the sample will therefore come in contact with the detector
particles and migrate together to the test line. The detector particles are typically antibodies conjugated to beads
(gold, coloured latex, etc.) that will allow the appearance of the test and control lines. The conjugate pad is
composed of fibers of cellulose, of glass or plastic, when the ‘membrane’ is usually made of nitrocellulose.
● The Test Line corresponds to the narrow space where the visualization of the target of interest will take place.
● The Control Line is used to check the correct migration of the sample. Whatever the result, this second line
must always be appearing.
● As the sample pad, the Absorbent Pad is a very important component for the good fluidics of the strip. It
facilitates the migration of the sample along the membrane by capillary effect and absorbs the overflow.
Several characteristics must be taken into account for the test and control lines in order to obtain good
performances:
 Thickness
 Weight
 Tensile strength
 Dimensions (length and width)
 Material
As for ELISA tests, several formats are available (Figure 7):
 Indirect or Sandwich LFIA
A positive result is represented by the presence of a coloured line at the test line position
 Competitive LFIA
A positive result is represented by the absence of a coloured line at the test line position. This format is
rarely used for serological tests.
FIGURE 7 COMMON LATERAL FLOW FORMATS
FIGURE 6 LATERAL FLOW COMPONENTS

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3. Test Performances
The performances of a test are highly variable depending on the target, the type of samples (matrix effects) but
also depending on the limit of detection (LOD). Many parameters have to be taken into account in order to have
a test with optimal performance and avoid false negatives and/or false positives (Figure 8).
 Limits of quantification
Limits of quantification are the highest and lowest concentrations of an analyte that have been
demonstrated to be measurable for a given device, with acceptable levels of precision and accuracy.
 Sensitivity (Se)
The sensitivity of a test is the probability that the test will be positive if the person effectively has the
disease. It is the number of true positives divided by the total number of people with the disease (A /
A+C). The more sensitive a test is, the fewer false negatives it delivers.
 Specificity (Sp)
The specificity of a test is the probability that the test will be negative if the person tested is effectively
free of the disease. It is the number of true negatives divided by the total number of people free of the
disease (D / B+D). The more specific a test is, the fewer false positives it delivers.
 Positive Predictive Value (PPV)
The PPV of a test is the probability that the person is really sick if the test is positive. It is the number of
true positives divided by the total number of people with a positive test (A / A+B).
 Negative Predictive Value (NPV)
The NPV of a test is the probability that the person will not have the disease if the test is negative. It is
the number of true negatives divided by the total number of people with a negative test (D / C+D).
 Robustness:
The ability of a method to remain unaffected by small variations in method parameters.
 Precision:
The closeness of agreement between independent test results obtained under specific conditions.
 Reproducibility:
The precision between laboratories (standardization).
 Repeatability:
The precision with the same operating conditions over a short interval of time (intra-assay precision).
 Stability:
The capacity of an analyte (in a matrix) or a test to give the same results under specific conditions
(temperature, humidity, etc.) for given time intervals.
Non exhaustive list
FIGURE 8 DIAGRAM EXPLAINING THE INTRINSIC PERFORMANCE OF A TEST

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ELISA or LFIA? The choice is essentially based on the practicality of the test and therefore the situation in
which it will be used.
Often used as an emergency test, a rapid test is useful as a first-line test and for large scale testing that
would have to be easy to use. More accurate tests but that would demand scientific instrumentations, can
then be used for positive patient confirmations. ELISA testing is common but requires laboratory equipment
(Table 1).
Pros. Cons.
ELISA Quantitative assay
Optimal limit of detection (LOD)
Sensitivity
Reliable and Robust
Multiplex analysis
Laboratory equipment
Long turnaround time (> 30 min)
Cost
Refrigerated storage
LFIA/ Rapid Test Rapid test and user friendly
Short results (< 10 min)
Emergency test
Economical industrial production
Easy storage (room temperature)
Qualitative or semi-quantitative
Multiplex detection
Sensitivity
SEROLOGICAL TESTS & COVID-19 EPIDEMIC
lready today, more than 140 Covid-19 serological tests are available worldwide. Many of these tests
were available rapidly at the beginning of the epidemic but sometimes with performances that can be
further optimized. Other tests are still under development and should be available in the coming weeks.
Whereas virological tests allow detection of the viral gene (RNA) by PCR, serological tests allow the
detection of antibodies (IgG and IgM) specific to the virus even long time after its clearance from the patient’s
body. Therefore, these second test types are complementary to the first ones and allow the identification of
patients who have been in contact (recently or not) with the virus and who have developed an immune response.
Currently, no specific therapy or vaccine exists to treat or prevent Covid-19 infection. In order to limit the spread
of the virus and to discharge our hospital networks, emergency measures that have not been taken in over a
century have been adopted by many countries. One of the main measures is the confinement of the population.
This is an extremely urgent measure to contain the progression of the epidemic but it raises the question of what
will happen after this period. Several points are currently at steaks, including:
̶ How can we get out of confinement by reducing the number of new contaminations?
̶ Will a vaccine or treatment be available quickly?
Serological tests may be a partial short-term solution to assess the population and their immune response
against the virus, since a first contact might not be enough to fully immunize.
When can these tests be used? Are they effective enough? Do we have enough information about the virus and
its effect on the immune system? These are questions that doctors, specialists and researchers are working on
to identify the best solutions.
One thing is certain, virological testing remains for the moment the solution to identify infected and contagious
people. But serological tests are definitely valuable as part of the arsenal that we need to have against this
epidemic.
A
TABLE 1. COMPARISON ELISA VS. LFIA

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WE WOULD LIKE TO THANK ALL THE PEOPLE MOBILIZED TO FIGHT
THIS EPIDEMIC. THANK YOU TO THE MEDICAL STAFF FOR TAKING
CARE OF OUR PATIENTS. THANK YOU TO THE HOSPITAL STAFF, OFTEN
IN THE SHADOWS, FOR THEIR DAILY INVOLVEMENT. THANK YOU ALSO
TO ALL THE RESEARCHERS WHO ARE WORKING CONSTANTLY ON
FUTURE SCREENING AND THERAPY TOOLS.
THANK YOU TO OUR EMPLOYEES WHO ARE ALSO INVESTED IN
CARRYING OUT PROJECTS WITHRESEARCHERS. FINALLY, THANKYOU
TO OUR CUSTOMERS FOR THEIR TRUST.
TOGETHER WE WILL WIN AGAINST COVID-19!
SAVE LIVES, STAY HOME

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SOURCES
 Antibody Structure and Function: The Basis for Engineering Therapeutics (PMID: 31816964)
 Functional switching (Milestone 8 ; Nature https://www.nature.com/articles/ni.3607)
 Atlas of immunology (Cruse, J.M., and Lewis, R.E. (2010). Boca Raton, FL: CRC Press/Taylor &
Francis).
 Assay Validation Methods (https://www.fws.gov/aah/PDF/QI-Terms%20and%20Defs.pdf)
 A practical guide to immunoassay method validation. (Front. Neurol., 19 August 2015 |
https://doi.org/10.3389/fneur.2015.00179)
 Smart Servier Medical Art

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ABOUT BIOTEM
BIOTEM is a Contract Research Organization providing custom and high added value services in the field of
antibody and immunoassay development.
With 40 years of experience, BIOTEM has developed a large panel of improved strategies enabling it to
complete the most challenging projects. Our staff scientists have extensive experience in antigen development
(peptides, proteins), hybridoma & recombinant antibody generation and production. In addition, the team has
developed an exclusive know-how in antigen design / conjugation, tracer preparation (gold, latex, magnetic
particles, carbon, etc.) and selection, as well as in immunoassay production (ELISA plate, Lateral Flow strip
and cassette).
Our technology platforms allow us to choose among several production processes in order to perfectly match
each project specifications. Thanks to a high success rate (>96%), most of our contracts are proposed with
results commitments and are composed of Go / No-Go phases.
BIOTEM also complies with ISO 9001 and ISO 13485 (IVD) quality standards.
100% fee-for-service: BIOTEM does not claim any intellectual properties or any other rights on the
developed antibodies or immunoassays.
BIOTEM: YOUR PARTNER IN IMMUNOTECHNOLOGIES SINCE 1980!
WWW.BIOTEM-ANTIBODY.COM
Written by Jonathan MAYALI
jonathan.mayali@biotem.fr
+33 476 651 091