AGGLUTINATION AND WESTERN BLOT ANALYZES

EXCERCIES NO. 1 V
ELECTROPHORESIS IN THE DETECTION OF IMMUNOGLOBULINS

• Electrophoresis is a process involving the movement of electrified molecules under the influence of an electric field. Different
molecules move at different speeds in an electric field, and the speed depends on their size, shape, and charge. Electrophoretic
methods can be used to separate macromolecules from a mixture. Cross-linked agarose and/or polyacrylamide gels, which can
be placed either horizontally or vertically, are commonly used as separation matrices.

• Proteins are amphoteric (zwitterionic) compounds because they contain both positive and negative side groups. Their charge
originates from the pH-dependent ionization of the side carboxyl and amino groups.
• Therefore, the total charge of a protein is determined by the number and type of amino acids that make up the pH of the medium.
• For each protein, there is a unique pH value at which it has no electric charge, known as the isoelectric point (pI). In a
medium with a pH higher than its pI, it will have a total negative charge and move towards the positive electrode (anode) during
electrophoresis. Conversely, in a medium with a pH lower than its pI, it will have a total positive charge and move towards the
negative electrode (cathode). This highlights the importance of maintaining a constant pH value during electrophoresis, especially
when proteins are separated in their native state.
Cross-linked agarose and polyacrylamide gels, which
can be in the form of horizontal or vertical plates, are
commonly used as separation matrices.

The size of the pores in the gels varies depending on the

concentration of the monomers used, enabling the
preparation of gels with different resolving powers and
the separation of macromolecules with different
molecular masses. From the perspective of protein
separation (with Mr from 5,000 to 200,000 Da),
polyacrylamide gels are more frequently used because
they have smaller pores compared to agarose.
Agarose gels are more commonly used to separate
nucleic acids, very large proteins, and protein complexes
 SEPARATION GELS

• Most often, the separation of proteins is performed in denaturing conditions, during which their secondary and tertiary
structure is lost.
• With this procedure, the shape of the molecule is no longer a factor that limits the movement of proteins during
electrophoresis.

• Protein denaturation is usually achieved using anionic detergents such as sodium dodecyl sulfate (SDS).
• This type of electrophoresis is called SDS-PAGE. Sodium dodecyl sulfate denatures proteins by disrupting hydrophobic
interactions, breaking hydrogen bonds, and forming stable complexes with molecules. As a result, it wraps polypeptide
chains with its hydrophobic tail.

• The amount of SDS that complexes with proteins does not depend on their charge and amino acid sequence, but
only on their size.
•The high negative charge of the SDS complex masks differences in pI (charge) between the different proteins,
affecting electrophoretic mobility.
•Processed in this way, all proteins will have a negative charge, and the magnitude of the negative charge is a function of
their size. Consequently, the only factor on which the electrophoretic mobility of proteins in SDS-PAGE depends is the
molecular mass.
•Based on the number of buffers used in a single protein gel electrophoresis, it can be continuous or discontinuous. In the
continuous system, only one buffer is used both for making the gel and as a medium for electrophoresis, while in the
discontinuous system, several different buffers with different pH are used.
• Although the first is much easier to perform, the discontinuous system is much better because this way many problems
associated with sample precipitation sand aggregation are avoided, and it has much higher resolving power. The gels used
in discontinuous systems are composed of two parts:
• the separation gel, which has smaller pores and in which the proteins are separated. The pH environment in this gel allows
maintaining the negative charge of the proteins, and thus their mobility.
• The application gel, which is non-restrictive and is poured over the separation gel. The pH environment in this gel partially
cancels the negative charge and electrophoretic mobility, allowing the proteins to concentrate in the form of a band at the
interface between the two gels.
• Discontinuous denaturing polyacrylamide electrophoresis, known as Laemmli SDS-PAGE (after its inventor), is today the
most commonly used method for protein separation.
 ANALYSIS OF ELECTROPHOREGRAMS

The analysis of electropherograms includes the detection and quantification of the separated protein fractions after their
visualization using appropriate staining techniques. The detection limit of the Coomassie blue technique is 100-300 ng
protein/lane, while the silver staining technique is about 100 times more sensitive, with a detection limit of 1 ng protein.
The determination of the size of the bands (Mr of the protein) is made possible by comparison with standards of known size,
which are always applied in parallel with the tested samples.

The simplest way to quantify the investigated protein is by visually comparing the intensity of the band with the intensity of the
standards, which are of known concentration and run on the same gel. A more accurate method of quantification is
densitometric scanning of stained gels.
WESTERN-BLOTING

•Further analyses involving antibody reactivity to proteins separated by

electrophoresis require the removal of the electrophoretic matrix.

•This is done by protein blotting.

•Protein blotting is the transfer (elution) of the separated protein fractions from
the polyacrylamide gel to a more stable immobilization medium, such as a
membrane filter, which binds the protein molecules at the moment they leave
the gel.

•The immobilization medium is usually made of different materials such as

nitrocellulose, nylon, or polyvinylidene difluoride (PVDF).
 WESTERN BLOTTING

•Capillary blotting involves several sheets of highly absorbent filter paper soaked with transfer buffer and connected to buffer reservoirs.
•The cut from the polyacrylamide gel is placed on the filter paper, and the nitrocellulose membrane is placed on top of it. A few sheets of
filter paper and a sponge are placed over the membrane. The transfer takes 24 to 48 hours.
•ELECTROPHORETIC BLOTTING
•Proteins are transferred from the gel to the membrane by electrophoresis within only 30 minutes to a few hours. The gel and membrane
are placed in a vertical system submerged in transfer buffer. Under the action of an electric field, the separated proteins come out of the gel
and enter the membrane.

•After transfer, the protein fractions, now bound to the membrane, can be detected either through binding with specific antibodies or non-
specifically using various staining techniques. Antibody detection is performed by treating the nitrocellulose membrane with antibodies
specifically directed against a particular protein, and then introducing enzyme-conjugated secondary anti-immunoglobulin antibodies.
•By using a chromogenic substrate specific for the enzyme with which the antibody is marked, staining of the membrane occurs.
WESTERN BLOTTING FOR THE DETECTION OF ANTIBODIES TO HIV
WESTERN BLOT ANTIGENS - ANTIBODIES
 AGGLUTINATION

•Agglutination refers to the sticking together of cells (such as bacteria, erythrocytes) or large particles (such as
polystyrene latex, bentonite, kaolin, cholesterol microcrystals, etc.) on the surface of which antigens are located,
and which interact with specific antibodies.
•When binding to at least two antigens, antibodies establish a kind of bridge between them, leading to the adhesion of the
cells or particles on which those antigens are located.
•Antibodies must overcome the ionic coat of the cell that hosts the antigen to which they bind.

• Agglutination leads to the formation of large aggregates of cells or particles, called agglutinates, which may be visible
microscopically or macroscopically.
•The antibodies that participate in the agglutination reaction are called agglutinins,

•while the antigens with which they interact are termed agglutinogens.
 The agglutination reaction is influenced by the concentration of agglutinins and agglutinogens, the nature of the antigenic structure,

the ionic strength of the medium, temperature, and other factors. Although IgG, IgM, and rarely IgA antibodies are involved in
agglutination reactions, in principle, IgM antibodies are the best agglutinins due to their high valence.

 Based on the principle of the reaction, agglutination tests are divided into:

• Tests of active (direct) agglutination that serve for the detection of particulate antigens;

• Passive (indirect) agglutination tests that serve to detect soluble antigens;

• Agglutination inhibition tests.

PASSIVE (INDIRECT) AGGLUTINATION
TESTS
Active agglutination may be
• rapid agglutination, and is performed on slides,
• spore (classical) agglutination is performed in test tubes.

Rapid agglutination tests are qualitative tests (+/-). T

he determination of blood groups represents a classic example
of rapid direct agglutination.

The presence of agglutinate is examined after incubating

erythrocytes from the subject with anti-A, anti-B, and anti-AB
serum.
•Slow agglutination tests can be used to quantify agglutinins
in serum. The determination is made in test tubes or microtiter
plates using a constant amount of agglutinogens, while serum is
added in different dilutions.
Most often, we work with double dilutions of the serum (1:20, 1:40,
1:80...).
•The greatest dilution of the serum with which agglutination is still
achieved gives the titer of agglutinins.
•A positive reaction is indicated by the agglutinates settling at the
bottom of the test tube, and the remaining liquid being clear, while
the precipitate obtained has uneven edges.
•A negative reacyoon - the content in the test tube does not exhibit
an even turbidity. There is no possibility of the appearance of a
sediment with smooth edges, and it cannot be homogeneously
resuspended by shaking.
 SLOW AGGLUTINATION

Slow agglutination serves for the quantification of agglutinins in the serum.

The determination is performed in test tubes or microtiter plates using a constant amount of
agglutinogens, while serum is added in different dilutions.
It is usually conducted with double dilutions of the serum (1:20, 1:40, 1:80, etc.). The highest dilution
of the serum with which agglutination is still achieved determines the titer of the agglutinins.

• In the case of a positive reaction, the agglutinates settle at the bottom of the test tube, leaving
the remaining liquid clear, while the sediment exhibits uneven edges.

• In contrast, during the negative reaction, the content in the test tube displays one-dimensional
turbidity, with the possibility of a precipitate forming with smooth edges, which can be
homogeneously suspended by shaking.
 PASSIVE AGGLUTINATION

During passive agglutination, agglutination of dissolved antigens bound to a carrier—either polystyrene latex (latex
agglutination) or sheep erythrocytes (hemagglutination)—occurs. The carrier itself is not immunogenic.
Erythrocytes easily bind to polysaccharide antigens; however, if the antigen is of protein or nucleic acid origin, they must be
pretreated with tannic acid or chromium chloride.
An example of passive hemagglutination is the Waaler-Rose test for determining the rheumatoid factor. It is performed by adding
PATIENT SERUM + HYPERIMMUNIZED RABBIT SERUM WITH SHEEP OR HUMAN ERYTHROCYTES.

If agglutination occurs, it indicates that the patient's serum contains rheumatoid factor, which is anti-IgG.

Additionally, there are non-agglutinating antibodies that can be detected through the Coombs antiglobulin test. An example of the
Coombs test is the determination of antibodies against the erythrocyte antigen Rho or D antigen. This test may involve direct or
indirect agglutination. The direct Coombs test identifies the presence of agglutinins from the Rh-negative mother on the surface of
the erythrocytes of the Rh-positive newborn.
•In passive agglutination, soluble antigens become agglutinated when bound to a carrier

such as polystyrene latex (latex agglutination) or sheep erythrocytes (hemagglutination).

•An example of this is the Waaler-Rose test for determining rheumatoid factors (RF). This

test is conducted by adding patient serum to hyperimmunized rabbit serum containing

sheep or human erythrocytes. If agglutination occurs, it indicates the presence of
rheumatoid factor in the patient's serum, which has an anti-IgG structure.
•Furthermore, there are non-agglutinating antibodies that can be detected through the

Coombs' antiglobulin test. Established by Coombs in 1945, this test, in its direct and
indirect forms, is widely used in immunology.
•In honor of the author, the antiglobulin serum is referred to as Coombs' serum,

obtained through immunization. This involves the introduction of human antibodies-

globulins into the circulation of certain animals, predominantly rabbits, goats, or
rams, which triggers the production of antibodies against the introduced globulins.
•Blood is drawn from these immunized animals, and the serum is separated from it. This Positive
Negative reaction
serum can then be further stabilized, frozen, and stored as a reagent, known as
antiglobulin serum.
 COOMBS TEST

 This serum may be polyspecific, containing antibodies against IgG, IgM, IgA, or monospecific (e.g., against IgG only) if immunization is conducted
with a single class of immunoglobulin.
 An example of such a test is the determination of antibodies against the erythrocyte antigen Rho or D-antigen. This test may also involve direct or
indirect agglutination.
 Performing the direct Coombs test can aid in the diagnosis of:

 Suspected hemolytic anemia

 Hemolytic post-transfusion reactions
 Hemolytic disease of the newborn
 Detection of antibodies when a blood compatibility test is performed
 Detection of antibodies in pregnant women
 Detection of antibodies in transplant patients
 Antibody detection in polytransfused patients
 Detection of antibodies in patients with malignant and autoimmune diseases.
 Procedure

 It can be performed in a test tube or on a microscope slide; A drop of washed red blood cells is added to a drop of

antiglobulin serum, then incubated for 15 minutes at room temperature; After incubation, the test tubes are centrifuged for
30 seconds at 1000 revolutions per minute; The test tube is removed, gently shaken, and the result is read against a light
source, i.e., the appearance of agglutination is observed if antibodies fixed to Er-membrane antigens are present; If the test
is negative, a drop of the test tube is taken for confirmation and observed under a microscope.

 With the direct Coombs test, the presence of agglutinins from the Rh-negative mother on the surface of the erythrocytes of

the Rh-positive newborn is determined. The test is performed by adding antiglobulin antiserum that binds to the Fc
fragment of the agglutinins, leading to agglutination of the erythrocytes. A positive reaction indicates the presence of
agglutinins in the mother's serum. These agglutinins surround the erythrocytes of the newborn, but without the involvement
of anti-immunoglobulin antibodies, they cannot cause agglutination of the erythrocytes of the newborn in vitro.
The INDIRECT COOMBS TEST is used for the detection of free antibodies circulating in
the plasma. It can be performed in a test tube or on a microscope slide.
Procedure: Three drops of washed red blood cells from blood group O are added to a test
tube, followed by three drops of the serum being tested to determine the presence of
antibodies. The test tube is then incubated for 45 minutes at 37°C, during which time the
antibodies (if present) will be fixed to the antigens on the erythrocyte membrane. After
incubation, the contents of the test tube are washed with physiological saline solution,
three times by centrifugation at 1500-2000 revolutions per minute for 1 minute each time.
After the final wash, two drops of antiglobulin serum are added to the sediment. After each
centrifugation, the supernatant is completely removed using a pipette through aspiration.
After the final wash, two drops of antiglobulin serum are added to the sediment. The tube
is then centrifuged again at 1000 revolutions per minute for 1 minute. The result is
macroscopically read against a light source. Presence of agglutination indicates a positive
test. If the test is negative, a drop of the test tube is taken for confirmation and observed
under a microscope.
The INDIRECT COOMBS TEST is used to determine the presence of antibodies directed
against the Rho antigen on fetal erythrocytes in the serum of pregnant women.
The test is performed by adding serum from a woman to serum from an individual with
blood group O Rh-positive, followed by anti-immunoglobulin antiserum that will enable in
vitro agglutination of sensitized erythrocytes with Rh blood group specificity. A positive
reaction confirms the presence of agglutinins in the woman's serum.
Tests for agglutination inhibition can be used to determine whether an individual is using certain types of illegal
substances and narcotics, such as cocaine or heroin.

A urine or blood sample is first incubated with specific antibodies for the suspected substance.

Then, red blood cells (or other particles) coated with the substance are added. If the red blood cells are not agglutinated
by the antibody, it indicates that the sample contains an antigen recognized by the antibody and that the individual has
been exposed to the presence of the tested substance. One problem with these tests is that some drugs have chemical
structures similar to those of abused substances, and these drugs may cross-react with the antibodies, yielding false-
positive reactions. For this reason, any positive result obtained must be confirmed with another non-immunological
method.

Some viruses and mycoplasmas have the ability to bind to the surface receptors of erythrocytes from certain animal
species and agglutinate them. This phenomenon allows for the detection of the presence of antiviral antibodies in the
serum of patients using agglutination inhibition tests. If, in the mixture of virus and erythrocytes, the patient's serum is
added and there is no agglutination, it means that antiviral antibodies are present in the serum, blocking the virus from
agglutinating the erythrocytes. This is a positive diagnosis that the patient is infected with the tested virus.
 • Agglutination inhibition is used to determine the presence of an antigen even in very small quantities. This
test is based on the competition between a complex and soluble antigen that appropriately binds to an
antibody.

• The antibodies and the test sample react mutually. If a soluble antigen is present in the sample, the antibody
reacts with it and is not free to react further after the additional addition of indicator particles or cells. Thus,
the absence of agglutination in the sample indicates a positive result.

• An example of this type of analysis is the detection of human chorionic gonadotropin (hCG), which is used in
the pregnancy confirmation test, as well as in other pathological conditions where hCG levels are
important.

Agglutination Reactions (FL-Immuno/60) – YouTube Direct Vs Indirect Coombs Test - YouT

ube
Indirect Coombs Test – YouTube Direct Coombs Test - YouTube