Clinical Chemistry Unit II - Lesson II: Animal Immunology and Serology

Suggested Reading:  pp.264-280, 282-286 Laboratory Procedures for Veterinary Technicians, 5th Ed,

 Hendrix & Sirois; pp. 264-266 Clinical Textbook for Veterinary Technician, 6th Ed, McCurnin and Bassert



Immunology is the study of the immune system.

Serology is commonly defined as the testing of antigen and or antibody levels of the blood, but it is strictly
defined as study of the serum.


Some definitions:

Antigen: A foreign substance, usually a protein. Antigens are part of the cell walls of bacteria,
viruses, protozoans, fungi and endogenous (self) cells that have become “altered”, for example
cancer cells.


Antibody: An immunoglobulin molecule that is produced by specialized B lymphocytes.
Antibodies are produced in response to a specific antigen. Antibodies are unique, specific and
form a “complex” with “their” antigen, with the intent to inactivate the antigen.

Antibodies are also known as immunoglobulins, abbreviated IgG, IgM, IgE, IgA, and IgD.

I) Brief explanation of the functioning of the immune system:
The immune system is a complex group of cells, tissue and organs located throughout the body that
interact to defend the body from all type of “foreign” substances (infectious agents, toxins, chemicals).

The immune system includes
- Cells: lymphocytes and monocytes (white blood cells circulating in the blood), tissue macrophages

 (which leave the bloodstream and migrate in the tissues)
- Tissues: bone marrow and the sub-mucosa of GIT
- Organs: spleen, thymus, liver (fetal), lymph nodes

The immune system is divided into two parts.
 - Innate immunity: Innate immunity is considered generalized, because it protects the body against
    all invaders.

 -  Acquired immunity: Acquired immunity is specific and reacts against specific antigens

A) Innate immunity: Also known as the “passive” immune system, is formed before
exposure to a foreign invader (antigen).

Types of innate immunity include:
- The skin and mucous membranes, which act as physical barriers to invaders.
- Neutrophils and monocytes (both types of white blood cells), and tissue macrophages
  (white blood cells which have migrated to the tissue).These cells engulf all invaders, which
  is termed non-specific phagocytosis.

- Colostrum which contains preformed antibodies from the mother.
- Protective secretions from the body such as mucus
- Non-pathological bacteria that normally live in the body. These populations of friendly
  bacteria often inhibit the growth of pathogens.

B) Acquired Immunity: This part of the immune system is much more complex and
involves the interactions of many different actions and cells. This system “reacts”
after exposure to foreign invaders (antigens) by producing very specific antibodies to
fight the antigen.
When the first line of defense is broken (for example if bacteria gets through the skin
and overwhelms the macrophages) then the acquired immune system begins to work. 

It is the acquired immune system that stimulates vaccination response, and causes immune
deficiency diseases and autoimmune diseases.

1) The acquired system is divided into:  
- Humoral immunity:
Humoral immunity is mediated by B lymphocytes (lymphocytes that mature in the bone marrow)
which are spread primarily to lymph nodes and the spleen.
These “B” cells produce plasma cells in response to antigens.
The plasma cells then produce specific antibodies or immunoglobulins to a huge number
(perhaps millions) of antigens.
Some of the “B” cells also produce memory cells that recognize previous antigens and
with a second exposure quickly clone to make huge numbers of specific antibodies also
known as immunoglobulins.

Immunoglobulins (also known as antibodies) are classified according to their molecular weight,
there are five classes of immunoglobulins.
        a) IgG immunoglobulin is the most common type of antibody and essential in body defense
mechanisms, it “coats” the antigen and enables the destruction of the antigen by macrophages.
        b) IgM immunoglobulin is a “first responder” it is the second most common immuoglobulin
and produces the primary immune response to “new” antigens. IgM antibodies are very efficient
at neutralizing virus.
        c) IgA immunmoglobulins are secreted at mucosal surfaces. These antibodies protect the body
by binding to antigens in the respiratory and gastrointestinal tract and not allowing the antigens to
attach and enter the body.
         d) IgE immunoglobulins are involved with many types of allergic reactions, including skin and
food allergies. IgE antibodies attach to mast cells and cause the release of histamine and other
substances that cause inflammation.
         e) IgD immunoglobulins are not found in all species, they are present in low concentration in
primates, rodents and probably dogs

-  Cellular immunity: Cellular immunity is the other type of acquired immunity and is mediated by
T lymphocytes. T-lymphocytes  become “activated T cells” after exposure to a specific antigen, then
they circulate throughout the body often for months. The activated cells perform much like antibodies,
by binding to specific antigens. There are several types of T cells.   

2) Interactions between different parts of the immune system.

- Tissue macrophages after inactivating a foreign invader can carry the antigen via the circulation to a
 lymph node and “present” it to memory cells which reside there, the memory cells will then mount an
immune response (if the antigen is recognized as a previous invader.)
- Helper T cells (of cell mediated immunity) are the primary regulators of the immune system and
produce various substances including, interleukins which are essential in fighting viral infections. 
- Complement (also known as the complement system) is a series of enzymes that are found in the blood
and assist the immune system by combining with the antigen and antibody complex and causing the
phagocytosis of bacteria by white blood cells.  

3) Hypersensitivity reactions: Hypersensitivity is a state of altered reactivity in which the body reacts
with an exaggerated immune response to a foreign agent (antigen).

There are four types of hypersensitivity reactions.
- Type I: This type of hypersensitivity is mediated by IgE antibodies and can range from minor skin
irritation to fatal anaphylactic shock.
- Type II: This type involves the complement system, an example is the blood transfusion reaction.
- Type III: This type is mediated by antigen and antibody complexes (precipitates) that are deposited
in tissues and cause inflammatory reactions, including glomerulonephritis and rheumatoid arthritis.
- Type IV: Type IV is also known as delayed hypersensitivity and is caused by T-lymphocytes,
this inflammatory reaction usually occurs 24 hours or more after exposure. Organ transplant
rejection and tuberculosis testing (TB) are examples.

An in-depth clinical explanation of Type I hypersensitivity example

 “Once contact is made the antigen is processed by tissue macrophages into a form which is presented
with the assistance of T-lymphocytes, to B lymphocytes.  The B cells then produce allergen specific IgE
antibodies and memory cells. The IgE antibodies affix to tissue mast cells and basophils. On re-exposure,
allergens bind the surface IgE molecules. Cross linking of two IgE antibodies results in the degranulation
of mast cells and the releases of inflammatory mediators. This results in erythema, edema and pruritis.”
from Saunders Manual of Small Animal Practice, 2nd Edition, 2000, pg. 339.


                   Testing Methods for Immune Diseases



Agglutination: The aggregation of suspended cells into clumps, often used to describe the antigen
antibody complex formations seen in serology testing.


Titer: The indirect measurement of antibody in the serum. It is the highest dilution of a serum that
will still agglutinate when mixed with a specific antigen.

Titration diagram: In the following diagram a 1 to 8 titer is demonstrated. At the 1 to 16 dilution of
the patient’s blood sample there is no longer enough antibody in the sample to agglutinate with the antigen.



Dilutions of patient’s sample





Dilute by adding 1 ml. of saline to 1 ml sample, to make a 1 to 2 dilution. Successive dilutions are made by adding more saline to the previous dilution







1 :  2


1 : 4


1: 8


1 to 16



Contains nothing














Nothing should happen to the control because it was only saline  begin with
















A titer of 1 to 200 dilution is “stronger” than a titer of 1 to 10 dilution. The 1 to 200 dilution still has some antibodies
capable of reacting even though it has been diluted 20 times more than the 1 to 10 titer.


Sensitivity: The ability of a testing method to detect the presence of infection, when infection IS present.
A test that detects a very small amount of antigens or antibodies is considered sensitive.

Specificity: The probability that a particular test will be positive for only the disease being tested.
The test does not “cross react” (become positive) with other closely related antigens (or antibodies), it is specific
for only one antigen.


Precipitation: The reaction of an antigen and antibody to form a visible deposit of solid particles.


Qualitative test: A test that describes the presence (positive) or absence (negative) of a particular


Quantitative test: A test that describes the amount present of a particular substance in the sample.


Antigen-antibody (Ag-Ab) complex: The reaction between specific antibodies (Ab) and their specific
antigens (Ag).


ANA (antinuclear antibody): Specific types of antibodies that are directed at the nucleus of the cell.


Monoclonal antibodies: Types of antibodies specific for only a single antigen. 

Due to monoclonal antibody technology the various “in house” test kits for infectious diseases, parasites,
hormones and blood typing have been developed. Monoclonal antibodies are essential in modern
diagnostic testing! Mice monoclonal antibodies are commonly used.


II) Serological Testing Methods (chart overview)


          Type                              Method                       Interpretation              Examples



Reaction between Ab and large Ag – liquid mixture

Clumping of material demonstrates ag-ab reaction



Coombs, Brucella testing




Large antigens (proteins) bind to antibody and form a precipitate in agar

Precipitate is a white line, if line matches the positive control, then the patient is positive

EIA (equine infectious anemia)

Johne’s disease




Antibody tagged with fluorescent molecule reacts with antigen

Fluorescence is positive

Direct test for tissue- rabies testing


ELISA - enzyme linked immuno-

absorbent assay

 Ag-Ab complex

Detected by enzymatic reaction

Color change is positive

Popular in house kits for FIP, FIV, Lymes disease, HW, etc.

Serum Titers


Serial dilutions of serum tested for

antibody levels

Titer is reported using the denominator of the last dilution to be positive

Used to determine effectiveness of vaccinations or

Paired serum samples, acute and recovery phase

RIM - Rapid Immunomigration or

Lateral flow immunoassay

Gold tagged antibodies detect Ab-Ag complexes

Color change is positive

HW check, Witness kit

Intradermal testing



Antigen injected intrademally to detect specific antibody response

Raised welt at site of injection

Allergy testing

TB testing




Fractions of serum proteins identified using electrical properties

Detection of specific immunoglobins and their levels

Specific diagnosis of many disease referred to reference labs


III) Details of Serological Methods:

A) Agglutination: slide and latex
- Slide agglutination can be seen on a microscope slide as clumping of the red blood cells, depending
on the circumstances agglutination can occur microscopically or grossly (visible with the naked eye).
Agglutination can occur on a slide with only the patient’s own blood (auto-agglutination) or when blood
samples from two animals are mixed (i.e. cross matching blood for transfusions). When agglutination is
noticed on a blood slide it should be differentiated from rouleaux formation. (The stacked “coin-like”
formation of red blood cells common in equine blood samples) If it is true agglutination is should be brought
to the attention of the attending veterinarian.

A simple test to distinguish rouleaux formation from true agglutination is to take a drop of the suspected
blood and mix it with a drop of normal saline and examine it under the microscope for clumping. Rouleaux
formation will be significantly decreased after this treatment, but agglutination will not be decreased.

Usually obvious auto-agglutination is not observed on a blood slide and other diagnostic techniques are needed.

- Latex agglutination: Latex particles are coated with an antigen (or antibody) and mixed with the patient’s blood.
If positive, visible agglutination will be seen. Used primarily for Brucella canis detection. 


B) Immunodiffusion: This is a precipitation test. An agar gel plate with a hole (called a well) in the center and 6
wells located around the central well are
used. The antigen is placed in the central well and patient’s serum is placed in alternating wells surrounding
the central well. A positive control is placed in every other well. The antigen from the central well diffuses
outward towards all the other wells. The positive controls (antibodies against the antigen) also migrate
towards the antigen. When the antigen and antibodies meet they form a visible line of precipitate.
The unknown serum from the patient may or may not form a precipitate line, depending on its antibody status.




Note the positive control precipitation lines .

Are there any positives from patients?















A common immunodiffusion test (the “Coggins test”) is used to detect
the virus causing Equine Infectious Anemia (EIA).*

* EIA is a contagious and usually fatal disease carried by mosquitoes.

Horse people are familiar with EIA because horses that are taken across state lines or going to horse shows etc.
are required to test negative before shipment. The blood is drawn by veterinarians and the test is performed only
at State diagnostic laboratories.

Link to photo of immunodiffusion test


C) Intradermal testing: Intradermal testing is used to detect tuberculosis in cattle and primates and to test for a
variety of environmental and food allergens.

1) Humoral Immunity: Skin allergy testing is based on the detecting of IgE humoral antibodies (those antibodies
preformed and found in the blood). A specific antigen is placed intradermally and inflammation results rather
quickly as the Ag-Ab complex forms and the subsequent cascade of events which results in swelling of the tissue.

2) Cell-mediated immunity: The tuberculosis intradermal test, done in the tail fold of cattle or the eyelid of non-
human primates, is based on reaction of the antigen (TB) with T-lymphocytes. This is a delayed reaction because
it takes the tissue lymphocytes longer to get to the site (compared to the antibodies in the skin allergy testing).


D) ELISA: Enzyme Linked ImmunoSorbent Assay

Most of these tests use monoclonal antibodies that are attached to a membrane (in the past often kits containing
“wands” and “wells” were used).
The unknown sample (usually whole blood, plasma, or serum can be used) is added to the membrane.
If the sample is positive for the specific antigen an Ag-Ab complex forms on the membrane.
The membrane is then rinsed to wash off any excess antigen.
A second antibody is added which is specific for the first Ag-Ab complex. This antibody has an enzyme attached
to it. This second antibody attaches to the complex, making an antibody sandwich with the antigen in the middle.
The membrane is washed a second time.
The final step is adding a color indicator that binds with the enzyme linked to the second antibody. A positive
will be observed as a color change. (Remember how enzymes changed color when mixed with reagents in
clinical chemistry? This is the same principle, if the enzyme is present a color change will result)

Many of the tests also have a positive and negative control, to help differentiate between positives and negatives.



E) C-ELISA: The C stands for competitive. This is a variation of the original ELISA test.
In this test a specific monoclonal antibody is attached to the membrane. The patient sample is then added to the
membrane. If there is a positive there will be binding of the patient’s antigen with the membrane antibody. The
next step is the addition of an antigen specific to the antibody attached to the membrane; this specific antigen is
linked to an enzyme.
If the patient sample was positive it contained the antigen specific for the membrane antibody and an
antigen - antibody complex formed on the membrane.  In this case when the enzyme linked antigen was
added it had no place to bind, it was “competitively” inhibited from binding by the patients antigen.

If the patient’s serum was negative there was no binding initially, but when the enzyme linked specific
antigen was added it did bind with the membrane antibody.
The final step is to add a color indicator to the membrane.

If the test shows “color” is this a negative or positive?


D) Electrophoresis:

The principle behind this methodology is that certain substances (immunoglobins, RNA fragments, viral
antigens etc.) have unique molecular properties, i.e. weight and electrical charge. When unknown samples
are placed on a gel membrane (or other suitable medium) and exposed to an electrical current they migrate
upward and have very characteristics “tracks”, based on their molecular weight and electrical charge. These
patterns can be used to positively identify unknowns. 

Electrophoresis is often used to verify test results of other methods because it is very exact.


A type of serological test that uses electrophoresis is the Western Blot Test: In this test specific antigens are
separated by electrophoresis and identified. Often the Western Blot is used for verification of Feline
Immunodeficiency Virus and Lymes Disease

2) Polymerase Chain Reaction (PCR): In this case the unknown virus or bacteria is “broken apart” and
segments of its nucleic acids (RNA, or DNA) are separated and amplified by replication. Again specific
patterns of migration can identify the unknown bacteria or virus. This test is used in diagnosis of Hemobartonella,
Erhlichia, Herpes virus etc.


Polymerase chain reaction diagram


PRC explanation


E) Radioallergosorbent Test (R.A.S.T.) and Radioimmunoassay:

Both of these test are similar to the ELISA test, in that a specific monoclonal antibody is attached to a membrane.
The patient’s serum is added, if the test is positive the serum has antigens that bind to the antibodies.
The next step is to rinse the membrane and add a second monoclonal antibody that is radioactively “tagged”
 and binds with the Ag-Ab complex. The membrane is then measured for radioactivity with a gamma meter.

F) Coombs test: The direct antiglobulin test (DAT) also known as the Coombs test is used after a positive
slide agglutination test (self agglutination). The test is used to determine autoimmune disease.

The test is run at 37 degrees C, a sample of “washed” (see description of cross matching for explanation of
washing red blood cells) erythrocytes from the patient are incubated with specific prepared anti-serum.
The antiserum contains antibodies against the Ag-Ab complexes that have formed on the red blood cells.
The sample is examined under the microscope for clumping/agglutination. Because of the expense of the
antiserum this test is done in commercial labs.

Website with various photos and diagrams of serology methods:



I) Case Studies of Some Immune-based Diseases and Testing Methods

Malfunctions of the immune system can take many forms.
Immunological diseases are classified into three major categories:
- Autoimmune

- Immunodeficiency 

- Hypersensitivity-Atopy

Autoimmunity can be caused by genetic defects (inherited) and it can also be triggered after infections,
especially viral infections. Autoimmune diseases maybe organ specific, for example directed at the kidney,
 muscles, blood, skin, nervous system and eye . There are also autoimmune diseases that are systemic and

affect several systems at the same time.
In human medicine these multi-system autoimmune diseases are termed rheumatoid or connective tissue
diseases and include, systemic lupus erythematosus (SLE) and dermatomyositis. Such diseases as
diabetes mellitus (insulin dependant or Type I), hyperthyroidism, polymyositis and
equine recurrent uveitis (moon blindness) are also caused by autoimmunity.

Immunodeficiency diseases can be “primary” (genetic) or secondary (induced by infections, parasites and
toxins). There are many diseases within each category and they affect various parts of the immune system,

for example certain diseases may inhibit the T lymphocytes while other diseases inhibit the B cells, some
diseases target specific immunoglobulins or antibodies. Feline Immunodeficiency Virus depresses the T cells which are essential in
coordinating the entire immune system, resulting in Feline AIDS (Acquired Immunodeficiency Syndrome).

Atopy is a genetically determined predisposition to develop allergies. This may take the form of allergies to
certain foods, vaccines, drugs, insects and inhaled allergens (mold, dust, pollen etc.)  Atopy is usually manifest
in the gastrointestinal and respiratory tracts, and the skin. Type I hypersensitivity is a form of atopy.


Case studied/clinical scenarios:
What does a critically ill 5 month old Arabian filly, a middle aged
terrier that is pruritic and vomiting, a young Cocker Spaniel that is bleeding uncontrollably after
routine surgery, a chronically ill young cat and a jaundiced elderly Pug all have in common?


The Arabian Filly
When this filly was less than 12 hours old the equine practitioner came out to check her for passive transfer of
maternal antibodies using a “mare side” test for IgG antibodies.  (ELISA or RID). The foal had adequate levels
of IgG antibody in her blood.
The antibodies were transferred in the mare’s colostrum within a few hours after birth. Without the antibodies
the foal would need either oral or intravenous supplementation with equine immunoglobulin. Commercial products
containing equine immunoglobulin are now on the market. Without these antibodies she would die.

The owner said the mare was a registered Arabian that she had just picked up at an auction for a very low price.

The veterinarian asked if the owner had ever heard of the syndrome, Severe Combined Immune Deficiency
(SCID or CID).

The owner was not aware of the disease and the veterinarian told her that combined immune deficiency is a genetic
disease found primarily in certain bloodlines of Arabian horses, it is inherited as a recessive gene.

The owner decided not to test for the disease, since it is a fairly expensive genetic test, and she wasn’t even
sure if the foal was a purebred Arabian.

When the foal was four months of age she contracted a severe respiratory infection. After a long course of
antibiotics she recovered but then contracted bacterial diarrhea and a skin infection. The filly did not respond to
the antibiotics and became progressively weaker.

(Presumed) Diagnosis: Severe Combined Immune Deficiency
 Affected foals have hypoplasia of lymphoid tissue and do not produce lymphocytes and consequently do not
produce antibodies. If they are able to nurse and get sufficient amounts of colostrum they have maternal protection
(maternal antibodies) for about 2-5 months. As soon as their maternal antibodies disappear they succumb to
infections. There is no effective treatment, but a genetic test (blood test) is now available to prevent the disease.

Does either parent show signs of immune deficiency with this disease? Why?

The Adult Terrier-mix
Symptoms: generalized itching, (face, feet, and ears) and also intermittent vomiting and diarrhea.
Diagnosis: food allergy (atopy)

Symptoms related to the disease: Puritis is often the main symptom in food allergies, gastrointestinal signs
may be variable.
There are a number of immune related diseases that are manifest as skin abnormalities, including systemic
lupus erythrematosus (SLE) and pemphigus.


Immune testing: There is no highly accurate test for food allergy in dogs at this time, although skin testing
and serum antibody testing have been used with variable results.

Other immune diseases should be ruled out and the pet can be put on the elimination diet.

A strict diet of only a single type of food is fed for a specified time, then different foods are gradually introduced
and those that cause allergic signs are eliminated from the diet until a safe non-allergenic food is established.



The Young Cocker Spaniel
Symptoms: After routine neuter (castration), the patient bleeds excessively from incision site. 
The diagnosis: Autoimmune thrombocytopenia (primary autoimmune disease).
Certainly many other clotting disorders would also be considered and tested for.

Symptoms related to disease: Antibodies are directed towards platelets and cause the phagocytosis and lysis
of the platelets by macrophages. This leads to weakness, purpura and sometimes generalized bleeding.

Immune testing: There is no easy or inexpensive in-house test for the condition, various methods are used in
reference labs including ELISA and immunofluorscence.
A “presumptive” diagnosis can be tested by treating the patient with corticosteroids and anti-neoplastic medicines.
If improvement is noted then a presumptive diagnosis is made. There is a higher incidence of this abnormality
seen in purebred cockers.



The Young Outdoor Male Tabby

Symptoms: lethargy and weight loss

The diagnosis: an immunosuppressive feline virus

Symptoms related to disease: Feline leukemia (FeLV) and feline immunodeficiency virus (FIV) both are
immunosuppressive viruses that effect T-lymphocyes. This predisposes the cats to a variety of other
infectious diseases.  

Immune testing: In-house ELISA tests are available for both diseases.



The Geriatric Pug

Symptoms: Lethargy, yellow tinged sclera and inner pinnas of ears.

The diagnosis: Autoimmune Hemolytic Anemia (AIHA)

Symptoms related to disease: Symptoms of AIHA are often vague and may include weakness and jaundice.
The primary type of hemolytic anemia is termed autoimmune and is caused by the body directing its’ own
antibodies at the bodies red blood cells. There is a secondary form of hemolytic anemia that is termed immune
mediated hemolytic anemia (IMHA). This type is sometimes seen after infections, neoplasia, vaccinations,
drug toxicity etc.

Immune testing: Coombs Test / DAT



Lesson II on Immunology & Serology - Writing Assignment:    

Learning questions for online lesson: answers are to be completed in a word document downloadable from the college Blackboard site and uploaded into the assignment drop box. All SECTION II assignments are due before taking the final exam.










1) (a) If you see something like this on a microscope slide (blood sample) what is the term for this (see above diagram and use a bit of imagination)?

 (b) What should you check about the above sample or sampling technique?


2) ELISA tests that have antibody as the first layer are testing for ____________ in the patient sample.


3) ELISA tests that have antigen as the first layer are testing for _____________ in the patient sample. 


4) An immunological test for a specific viral antigen came back positive from the lab but on further testing using a more
sophisticated method it was found that the test was positive for a closely related virus. (a) Was this a false-negative or false-positive?

(b) Does this show a lack of sensitivity or specificity?


5) How are monoclonal antibodies are created?

6) Matching Parts of the ELISA Test:

Momoclonal antibody

Membrane (ELISA test)

Patient antigen

Patient blood

Enzyme linked
Monoclonal antibody

Color reagent
Which binds to
Enzyme-linked antibody



                                         Below are the steps in performing an ELISA test
                Match the activity A-G  in the column on the left with the descriptions on the right (1-7) 




1) Color reagent is added


2) Enzyme-linked monoclonal antibodies
are added


3) Patient antigen (blood) is added


4) Specific monoclonal antibodies are
embedded on membrane



5) Antigen binds with membrane bound
monoclonal antibody


6) Color reagent binds with enzyme linked
monoclonal antibody - positive test


7) Enzyme linked monoclonal antibody binds
with antigen forming an "antibody sandwich"