Neutralization

A Neutralizing antibody, or NAb is an antibody which defends a cell from an antigen or

infectious body by inhibiting or neutralizing any effect it has biologically.[3] An example of a
neutralizing antibody is diphtheria antitoxin, which can neutralize the biological effects of
diphtheria toxin.[4]

Contents

1 Neutralization Method

2 Modern testing
o 2.1 Pharmaceutical Problems

3 References

4 External links

Neutralization Method
Most antibodies work by binding to an antigen, signaling to a white blood cell that this
antigen has been targeted, after which the antigen is processed and consequently destroyed.
The difference between neutralizing antibodies and binding antibodies is that neutralizing
antibodies neutralize the biological effects of the antigen, while binding antibodies flag
antigens.[5] This difference can be shown with IFN-beta;
"Antibodies can simply bind to IFN-beta or glatiramer acetate (binding Ab, or BAb) with no
subsequent effect on function, or they can block or neutralize (neutralizing Ab, or NAb) their
biological activity." --Mark S. Freedman, MD, MSc
This difference is what gives neutralizing antibodies the ability to fight viruses which attack
the immune system, since they can neutralize function without a need for white blood cells
(excluding production)[5]

Modern testing
Neutralizing antibodies have shown potential in the treatment of retroviral infections.
Medical professionals and researchers have shown how the encoding of genes which
influence the production of this particular type of antibody could help in the treatment of
infections which attack the immune system. Professionals in the field have used HIV
treatment as an example of infections these antibodies can treat.[6] Recently, potent and
broadly neutralizing human antibodies against influenza (such as CR6261), HIV and hepatitis
C have been reported, and have suggested possible strategies to generate an improved vaccine
that would confer long-lasting immunity. Another disease which has been linked to the
production of neutralizing antibodies is multiple sclerosis.[2] The use of medicines which
modify diseases is nothing new, used in regulation for multiple sclerosis since 1998 when the
National Multiple Sclerosis Society Consensus Statement recommended their use.[2]

Promise in Medicine
Disease

Promise

Used?

Multiple
Sclerosis

There is some promise but there have been issues with

Pharmaceuticals

Yes

HIV

Research has shown that NAb's may be able to block viral

receptors

No

Pharmaceutical Problems
Although this type of antibody has the ability to fight retroviral infections, in some cases it
attacks pharmaceuticals administered to the body which would otherwise treat multiple
sclerosis. Recombinant protein drugs, especially those derived from animals, are commonly
targeted by neutralizing antibodies. A few examples are Rebif, Betaseron and Avonex.[2]

References

Precipitation and agglutination

A Number of in vitro diagnostic tes are bases on spesificity of interaction
between antibodies and antigens. Whwn soluble antibodies link the
bacterial cells together, forming an agglutinate. Whwn certain viruses
such as the measles virus, are mixed with redd blood cells, the virus
particles react with structure on the surfaces of the red blood cells,
causing hemagglutination. If the patient has measles, the serum will
contain antibodies, againts the measles virus. If the patients serum is
mixed with measles virus and red blood cells are added, hemagglutination
will not occur. Antibodies recting with the virus particles prevent reaction
between the virus and the red blood cells.
Activation of complement
Attraction of phagocytes
Opsonization

The process of coating particles for subsequent phagocytosis is called opsonization, and the
molecules that coat microbes and enhance their phagocytosis are called opsonins. When
several antibody molecules bind to a microbe, an array of Fc regions is formed projecting
away from the microbial surface. If the antibodies belong to certain isotypes (IgG1 and IgG3
in humans), their Fc regions bind to a high-affinity receptor for the Fc regions of heavy
chains, called FcRI (CD64), which is expressed on neutrophils and macrophages. The
phagocyte extends its plasma membrane around the attached microbe and ingests the microbe
into a vesicle called a phagosome, which fuses with lysosomes. The binding of antibody Fc
tails to FcRI also activates the phagocytes, because the FcRI contains a signaling chain that

triggers numerous biochemical pathways in the phagocytes. The activated neutrophil or

macrophage produces, in its lysosomes, large amounts of reactive oxygen species, nitric
oxide, and proteolytic enzymes, all of which combine to destroy the ingested microbe.
Antibody-mediated phagocytosis is the major mechanism of defense against encapsulated
bacteria, such as pneumococci. The polysaccharide-rich capsules of these bacteria protect the
organisms from phagocytosis in the absence of antibody, but opsonization by antibody
promotes phagocytosis and destruction of the bacteria. The spleen contains large numbers of
phagocytes and is an important site of phagocytic clearance of opsonized bacteria. This is
why patients who have undergone splenectomy for traumatic rupture of the organ are
susceptible to disseminated infections by encapsulated bacteria. Antibody opsonization is
the process by which a pathogen is marked for ingestion and destruction by a phagocyte.
Opsonization involves the binding of an opsonin, e.g., antibody, to a receptor on the
pathogen's cell membrane.[1] After opsonin binds to the membrane, phagocytes are attracted
to the pathogen. The Fab portion of the antibody binds to the antigen, whereas the Fc portion
of the antibody binds to an Fc receptor on the phagocyte, facilitating phagocytosis.[2] The
receptor-opsonin complex can also create byproducts like C3b and C4b which are important
components of the complement system. These components are deposited on the cell surface
of the pathogen and aid in its destruction.[3]
The cell can also be destroyed by a process called antibody-dependent cellular cytotoxicity,
in which the pathogen does not need to be phagocytosed to be destroyed. During this process,
the pathogen is opsonized and bound with the antibody IgG via its Fab domain. Then the
antibody binds an immune effector cell via its Fc domain and this binding triggers a release
of lysis products from the bound immune effector cell (monocytes, neutrophils, eosinophils
and natural killer cells). This process can cause inflammation of surrounding tissues and
damage to healthy cells.

Opsonization is a term that refers to an immune process where particles such as bacteria are
targeted for destruction by an immune cell known as a phagocyte. The process of
opsonization is a means of identifying the invading particle to the phagocyte. Without the
opsonization process the recognition and destruction of invading agents such as bacteria
would be inefficient.
The process of opsonization begins when the immune system recognizes a particle (e.g., a
bacterium) as an invader. The recognition stimulates the production of antibodies that are
specific for the antigenic target. Certain antibody molecules are stimulated to bind to the
surface of the particle. Typically, the binding molecules are a type of antibody classified as
IgG. As well, proteins involved in the complement-mediated clearance of foreign material,
specifically a protein designated C3b, can bind to the surface of the foreign object. Proteins
such as IgG and C3b, which can promote opsonization, are designated as opsonins.
When the IgG antibodies bind to the invading bacterium, the binding is in a specific
orientation. An antibody is somewhat "Y" shaped. The binding of IgG to the bacterium is via
the branching arms of the "Y." The stalk of the molecule, which is termed the Fc region, then
protrudes from the surface. The Fc region is recognized by a receptor on the surface of an

immune cell called a phagocyte. When the Fc region is bound to the phagocytic receptor the
invading particle is taken into the phagocyte and enzymatically digested.
The Cb3 complement protein can bind in a nonspecific manner to an invading particle.
Phagocytes also contain surface receptors that recognize and bind Cb3. As with IgG, the
binding of Cb3 to the phagocytes triggers a process whereby the invading particle is
engulfed, surrounded, and taken inside the phagocytic cell for destruction.
Examples of phagocytic cells that can participate in opsonization are neutrophils and
monocytes.
Bacteria that are associated with the development of infections typically possess a capsule,
which is a layer of carbohydrate material. The capsular material encases the bacterial cell.
The carbohydrate is not recognized as readily by the immune machinery of the body as is
protein. As well, the penetration of antibodies through the capsule network to the surface of
the bacterium is impeded. Thus, possession of a capsule can dampen the opsonization
response.

Stimulation of inflammation
Preventive of bacterial and viral adhesion

Lab 6. Agglutination
When antibodies are mixed with their corresponding antigens on the surface of large,
easily sedimented particles such as animal cells, erythrocytes, or bacteria, the antibodies
cross-link the particles, forming visible clumps. This reaction is termed agglutination.
Agglutination is a serological reaction and is very similar to the precipitation reaction we
learnt last week. Both reactions are highly specific because they depend on the specific
antibody and antigen pair. The main difference between these two reactions is the size of
antigens. For precipitation, antigens are soluble molecules, and for agglutination, antigens
are large, easily sedimented particles. As you will see from this lab exercise, agglutination is
more sensitive than precipitation reaction because it takes a lot of more soluble antigens and
antibody molecules to form a visible precipitation. To make the detection of soluble antigen
and antibody reaction more sensitive, a precipitation reaction can be transformed into an

agglutination reaction by attaching soluble antigens to large, inert carriers, such as

erythrocytes or latex beads.
Agglutination reactions have many applications in clinical medicine. Agglutination
reactions can be used to type blood cells for transfusion, to identify bacterial cultures, and to
detect the presence and relative amount of specific antibody in a patients serum.
Agglutination has been commonly used to determine whether a patient had or has a bacterial
infection. For example, if a patient is suspected of having typhoid fever, the patients serum
is mixed with a culture of Salmonella typhi. If an agglutination reaction occurs, shown as
clumping of the bacteria, the patient either had or has an S. typhi infection. Since certain
antibodies can persist in a patients blood for years after the patent has recovered from the
infection, a positive reaction does not mean that the patient currently has the infection. To
determine whether a patient is currently suffering from typhoid fever, the amount or titer of
the antibody will be determined at the onset of illness and two weeks later. If the titer of
antibody in the patients serum has increased at least four-fold between the two tests, the
patient is currently fighting off the infection, and the pathogen causing the illness is
confirmed.
In this lab exercise, you will learn two different methods of employing agglutination
reactions, rapid slide agglutination and microtiter test. These two tests are valuable methods
commonly used in clinical laboratories. Applications of agglutination include A-B-O blood
typing tests and rapid bacterial identification. The microtiter test is used to quantify the
amount of antibody in patients blood.

Part 1.

Rapid Slide Agglutination

In this lab exercise, you will learn how to use rapid slide agglutination to determine
your blood type. The surface of blood cells of each type expresses a unique oligosaccharide
structure that is called A or B antigens. The A and B antigen are inherited. Each person
carries anti-A and/or anti-B antigen in his or her blood dependent on his or her blood type.
Presence in the blood of anti-A and/or anti-B antibodies is not inherited, but is the result of
(1) prior exposure to A- and B-like antigens in the environment, such as bacterial surface
molecules; or (2) an immunological response to nonself molecules. When a person receives
unmatched blood, these antibodies will either agglutinate blood cells or induce complementmediated cytolysis. Typing blood to match donor and recipient with respect to ABO antigens
is an important and widely used procedure.

SAFETY NOTE: This lab uses your own blood for ABO
blood typing. After obtaining your drops of blood, put on
gloves. Dispose of waste in the biohazard container.
Procedure
1. Label three slides A, B and A+B.
2. Spray your left ring finger with 70% ethanol or wipe it with an alcohol wiper and let it
air dry.
3. Take a sterile lancet and puncture your fingertip. If you have calluses, aim a little to the
side. DO NOT LANCET ANYONE OTHER THAN YOURSELF. When finished with your
lances, place them in the BIOHAZARD CONTAINER.
4. Place a small drop of blood on each of three microscope slides. At this point, put on
GLOVES. DO NOT TOUCH ANYONE ELSES BLOOD.
5. On the A slide, place a 20 l of anti-A antiserum. Place anti-B on the B slide and anti-A +
anti-B on the A+B slide. Mix the antisera in with the blood using a separate toothpick for
each slide. Place toothpicks in the biohazard waste.
6. After several minutes, observe agglutination and determine your blood type.

Part 2.

Microtiter Test

In this lab exercise, you will learn how to use the microtiter test to determine the
amount of anti-sheep red blood cell (RBC) antibody.

Procedure
1. In a round-bottom microtiter plate, add 50 l of PBS to columns 2-9 in rows A-C. Leave
column 1 empty.
2. Add 100 l of rabbit anti-sheep RBC to column 1 in rows A-C.
3. In each row, transfer 50 l from column 1 to column 2 and mix well. Now take 50 l
from column 2 and transfer to column 3. Repeat this process across the columns. Discard
the final 50 ml taken from row 9.
4. Add 50 l of a 2% suspension of sheep RBC to all wells (columns 1-9, rows A-C). Make
sure the RBCs stay adequately resuspended as you are using them. Periodically invert the
capped tube to keep them resuspended evenly.
5. Incubate the plate for 24 hours at room temperature.
6. Come back the next day and observe agglutination. Positive wells will exhibit a diffuse
and confluent settling of the RBC and Ab, while in negative wells, all cells will roll down to
the bottom and it will look more like a dot.

Reading
Immunobiology A-7

Study Questions
1. What is the difference between precipitation and agglutination? Which one of these two
methods is more sensitive and why?
2. What type of structure on your red blood cells decides your blood type?
3. You used the slide agglutination test to determine your blood type. You got the following
result:
Anti-serum A B AB
Agglutination - +
+
What is your blood type?
If you receive type AB blood for blood transfusion, what is going to happen and why?

4. Describe how to determine whether a patient has ever had a Salmonella typhi infection, the
name of method, reagents used, possible results, conclusions deduced from results and why.
5. In preparing her immunology lab, an instructor purified IgG antibodies that are specific to
sheep red blood cells (SRBCs) and digested some of the antibodies into Fab, Fc, and F(ab)2
fragments. She placed each preparation in a separate tube, labeled the tubes with a watersoluble marker, and left them in an ice bucket. When the instructor returned, she discovered
that the labels had smeared and were unreadable. Determined to salvage the antibodies, she
relabeled the tubes 1, 2, 3, and 4 and proceeded. Based on the test results described below,
indicate which preparation was contained in each tube and explain why you so identified the
contents.

a. The preparation in tube 1 agglutinated SRBCs but did not lyse them in the presence of
complement.
b. The preparation in tube 2 did not agglutinate SRBCs or lyse them in the presence of
complement. However, when this preparation was added to SRBCs before the addition of
whole anti-SRBC, it prevented agglutination of the cells by the whole anti-SRBC antiserum.
c. The preparation in tube 3 agglutinated SRBCs and also lysed the cells in the presence of
complement.
d. The preparation in tube 4 did not agglutinate or lyse SRBCs and did not inhibit
agglutination of SRBCs by whole anti-SRBC antiserum.

umoral Immune Response

The term humoral immune response refers to immunological action of antigen-specific

antibodies, which are secreted by activated B cells. Antibodies produce an immune response
through three main mechanisms: neutralization, opsonization, and activation of the
complement cascade. Neutralization simply refers to the process by which antibodies prevent
virues and intracellular bacteria from entering host cells by binding to the proteins on the
pathogen surface that are necessary for entry into host cells. Opsonization occurs when
antibodies coat the surface of the pathogen, usually a bacteria, and enhance uptake of the
pathogen by phagocytic cells. The complement cascade itself has two possible outcomes:
opsonization by certain complement proteins, usually C3b, and direct killing of the pathogen
by the formation of a pore in the plasma membrane by 10-16 molecules of the complement
protein C9. There are five different isotypes of antibodies with different distributions and
effector functions: IgM, IgD, IgG, IgA, and IgE. IgM is best at complement activation and
transport across epithelium. It is also the first antibody produced when the host is infected
with a pathogen for the first time. The function of IgD is not completely understood at this
time, and it is never secreted. There are many different subtypes of IgG, and their effector
functions vary to include, among others, opsonization and complement activation, and they
often diffuse into extravascular sites. IgA is best at neutralization and is also is often
transported across epithelium as a dimer. IgE is best at sensitization of mast cells and is often
found in extravascular sites. (Janeway et al. 2005).
For most inidividuals, the humoral immune response to S. pyogenes is primarily based on IgG
antibodies' binding to M protein on the bacterial surface. This binding causes opsonization,
resulting in the phagocytosis and the eventual killing of the bacteria. Because the bacteria is
often found on mucosal epithelial surfaces, S. pyogenes-specific IgA antibodies are also
produced in the respiratory tract as protection against another streptococci infection.
However, the protective properties of these antibodies are often negated by the extreme
variability of the M protein (further discussed in the Evasion of the Immune System page).

Figure 4. Transmission electron micrograph of two S. pyogenes bacteria (20,000x). The outer
filamentous material is mostly M proteins, and the clear region between the M proteins and
the darker inside of the bacteria is the cell wall. . Electron micrograph by Maria Fazio and
Vincent A. Fischetti, Ph.D. The Laboratory of Bacterial Pathogenesis and Immunology,
Rockefeller University. Copyright 1995. Used with permission.