Antitoxins are antibodies that neutralize toxins and can be used to prevent toxins from causing disease symptoms. Passive immunity can be provided through the administration of antitoxins, immunoglobulins like gamma globulin, and immune sera which provide immediate and temporary protection against various pathogens and toxins. Monoclonal antibodies produced through hybridoma technology allow the isolation of homogeneous antibodies against single antigenic determinants and have various applications in research, diagnosis, and potential treatment.

1. THEME: SPECIFIC PROPHYLAXIS AND THERAPY OF INFECTIOUS
DISEASES. IMMUNE SERA AND IMMUNOGLOBULINS.
I. INDEPENDENT STUDY PROGRAM
1. Immunotherapy (specific therapy) of the infectious diseases. Immediate seroprophylaxis
of the infections.
2. Antibacterial and antiviral immune sera and immunoglobulin.
3. Antitoxic immune sera (antitoxins). The methods of the manufacture, determination of
their activity.
4. Diagnostic antimicrobial and antiviral sera. The methods of obtaining and titration.
Practical use.
5. Monoclones. Hybridomas (myeloma hybrid) technology. The perspectives of usage of
monoclones.
1. Passive immunity can be used to prevent diseases when there is not sufficient time to
develop an acquired immune response through vaccination. The administration of sera, pooled
gamma globulin that contains various antibodies, specific immunoglobulin, or specific antitoxins
provides immediate protection (Table 1).
TABLE 1
Substances Used for Passive Immunization
SUBSTANCE USE
Gamma globulin (human) Prophylaxis against various infections for high-risk individuals,
such as those with immunodeficiencies; lessening intensity of
diseases, such as hepatitis after known exposure
Hepatitis B immune globulin To prevent infection with hepatitis B virus after exposure, such as
via blood contaminated needles
Rabies immune globulin Used in conjunction with rabies vaccine to prevent rabies after a bite
from a rabid animal; used around wound to block entry of virus
Tetanus immune globulin Used in conjunction with tetanus booster vaccine to prevent tetanus
after a serious wound; used around wound to block entry of virus
Rh immune globulin (Rhogam) To prevent an Rh-negative woman from developing an anamnestic
response to the Rh antigen of an Rh-positive fetus; administered
during third trimester or after birth
Antitoxin (various) To block the action of various toxins, such as those in snake venom,
those from spiders, and those produced by microorganisms,
including diphtheria toxin and botulinum toxin
Sera are injected in definite doses intramuscularly, subcutaneously, sometimes intravenously,
with strict observation of all the rules of asepsis. A preliminary desensitization according to Bezredka's
method is necessary. Sera are employed for treatment and for prophylaxis of tetanus, gas gangrene and
botulism. The earlier the serum is injected, the more marked is its therapeutic and prophylactic action.
The length of protective action of sera (passive immunity) is from 8 to 14 days.
At present days sera and immunoglobulin are produced in a purified state. They are treated by
precipitating globulins with ammonium sulphate, by fractionation, by the method of
ultracentrifugation, electrophoresis and enzymatic hydrolysis which allow the removal of up to 80 per
cent of unneeded proteins. These sera have the best therapeutic and prophylactic properties, contain the
least amount of unneeded proteins, and have a less distinct toxic and allergic action.

2. Sera thus produced are subdivided into antitoxic and antimicrobial sera. Antitoxic sera include
antidiphtheritic, antitetanic sera and sera effective against botulism, anaerobic infections, and snake
bites.
Antimicrobial sera are used against anthrax, encephalitis and influenza in the form of globulins
and gamma globulins.
Before the development of antibiotics, passive immunization – often using horse sera – was
widely practiced. Unfortunately, precipitation from extensive antigen-antibody complex formation
caused kidney damage when horse serum was routinely administered. Today the use of passive
immunity to treat disease is limited to cases of immunodeficiency and to specific reactions to block the
adverse effects of pathogens and toxins.
Various antitoxins (antibodies that neutralize toxins) can be used to prevent toxins of microbial
or other origin from causing disease symptoms. The administration of antitoxins establishes passive
artificial immunity. Antitoxins are used to neutralize the toxins in snake venom, saving the victims of
snake bites. The toxins in poisonous mushrooms can also be neutralized by administration of
appropriate antitoxins. The administration of antitoxins and immunoglobulin to prevent disease occurs
after exposure to a toxin and/or an infectious microorganism.
Antitoxins are antibodies that neutralize toxins and can be used to prevent toxins from
causing disease symptoms.
Flocculation test is used for determination of antitoxic serum activity (table 7). This test is
similar to precipitation test. Flocculation phenomenon is formation of turbidity in the tube with toxin
(toxoid) and antitoxic serum mixture. It is specific reaction which is used for determination of activity
of toxin (toxoid) or antitoxic serum.
The activity of antitoxic serum is determined in international unit (IU). This is amount of serum
which can neutralize certain amount of Dlm of bacterial toxins.
Table 2
Schematic representation of the flocculation test
Ingredients (ml)
Tubes
1 2 3 4 5 6
Diphtherial anatoxin (30 IU per ml) 2.0 2.0 2.0 2.0 2.0 2.0
Tested serum 0,1 0,2 0,3 0,4 0,5 0,6
Incubate the tubes in water bath at 40 °C until initial flocculation will appear.
For example, initial flocculation has been appeared in the 3 tubes. It means that 0.3 ml of tested
serum connects 60 IU of anatoxins, and 1 ml of serum can connect 200 IU (1ml x 60 IU : 0.3 ml
= 200 IU). Thus, antitoxic serum activity is 200 IU.
It is also possible to establish passive immunity by the administration of gamma globulin, which
contains mainly IgG and some IgM and IgA . It is important that the gamma globulin used for
establishing passive immunity is pooled in order to combine the immune functions from many
people. Passive immunity lasts for a limited period of time because IgG molecules have a finite
lifetime in the body. The administration of IgG does not establish an anamnesis response
capability. The administration of IgG is also particularly useful therapeutically in preventing
disease in persons with immunodeficiency and other high-risk individuals.
Monoclonal Antibodies. Most proteins possess many different antigenic determinants. As
a result, serum from an animal or human producing Abs to a protein or cellular constituent
contains a complex mixture of Abs. This mixture contains Abs to all determinants as well as Abs
that are heterogeneous with respect to heavy chain isotype, light chain type, allotype, variable
region sequence, and idiotype. A long held dream of biomedical scientists was to isolate a single

3. Ab producing cell and grow it in vitro to provide a source of homogenous Abs that would bind
to only a single antigenic determinant.
Normal cells producing the desired Ab from an immunized animal are fused with myeloma
cells (malignant lymphocytes that can be propagated easily in vitro) in the presence of a
chemical that promotes cell fusion (polyethylene glycol or Sendai virus). Such fused cells, called
hybridomas, have the Ab producing capability of the normal cell parent and the in vitro growth
properties of the malignant myeloma parent. The normal, nonfused spleen cells cannot survive in
culture, whereas the unfused myeloma cells, which can grow in vitro, carry a mutant gene in a
critical biosynthetic pathway (ie, a drug marker). The presence of this mutant gene allows the
unfused myeloma cell to be killed by adding the appropriate drug in culture. The fused cell is
protected from this drug, because the normal spleen cell provides the normal biosynthetic gene.
The procedure used to produce hybridoma cell lines secreting monoclonal Abs is shown in
Figure 3.
Production or hybridoma cell lines secreting monoclonal antibodies .The procedure for
producing monoclonal antibodies is shown. Activated B cells from an immunized individual (eg,
spleen cells from an immunized mouse) are fused with malignant plasma cells isolated from
plasmacytomas and adapted to tissue culture. The myeloma cell has a mutant gene that renders
it sensitive to the drug aminopterin. The activated B cells, although resistant to aminopterin,
have a limited lifetime in culture and die naturally. The B cell—myeloma cell hybrid is resistant
to aminopterin because the B cell provides the missing genes. Therefore, the B cell-myeloma cell
hybrid (the hybridoma) is the only fusion product that can survive in the hypoxanthine,
aminopterin, thymidine (HAT) selective culture medium used. The hybrids are distributed into
many culture wells in the multiwell culture plates and are allowed to grow for a short period.
The culture supernatant or these wells then is tested for the desired antibody. Those cultures that
are positive are cloned, and the hybridoma cell producing the desired antibody is propagated
and used as a source of the monoclonal antibody.
Monoclonal Abs are available for thousands of different determinants and are being used
widely as research tools to study protein structure and virus and toxin neutralization, and to
isolate specific proteins from complex mixtures. Moreover, many commercially available
monoclonal Abs are being used in extremely sensitive and specific techniques for the
diagnosis of various diseases and, as mentioned earlier, for the experimental treatment of
several human diseases.
Summary:
Artificial Passive Immunity
• Passively acquired immunity comes about when antibodies produced in another organism
are introduced into the body. Artificially acquired passive immunity is derived from the injection
of antibodies into an individual to provide immediate protection against a pathogen or toxin.
II. Student practical activities
1. Finish the preparing of the inactivated vaccine (from 5 to 7 stage).
2. Acquaint with immune sera and immunoglobulun and diagnostic serum preparations.