Veterinary diagnostic

techniques and procedures

Faculty of Agricultural Engineering and

Veterinary Medicine- DS
Dr. Ali Mehdy
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview

A- For detection of the agent (infectious organism or toxin), the following kinds
of techniques are possible:
1- Direct Methods:
c) You can detect it through immunologic means, for instance through Agar Gel Immunodiffusion (AGID) or
Fluorescent Antibody (FA) or Enzyme Linked Immunosorbent Assay(ELISA).
d) You can find the toxin through chemical assays.
e) You can detect the nucleic acid of the organism through polymerase chain reaction (PCR).
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview

A- For detection of the agent (infectious organism or toxin) or antibodies

2-Techniques Basics/principle: Immunohistochemistry
 Fluorescent antibody technique: There are two kinds of fluorescent antibody (FA) tests –the Direct
(DFA) and the Indirect FA (IFA).

Fluorescein isothiocyanate (FITC) is a molecule that can easily be attached to an

antibody. FITC appears colorless under normal light, but when ultraviolet light
hits it, it gives off a bright green color that you can see.
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview

A- For detection of the agent (infectious organism or toxin) or antibodies

2-Techniques Basics/principle:Immunohistochemistry
 The Direct (DFA) Test: for the detection antigens in tissues or smears
 Exp, Rabies:
• The brain from the animal is smeared onto a slide
• Fluorescent antibody specific for rabies is put onto the slide, incubated, and then the slide is washed
• Under a fluorescence microscope: bright green spots indicate the presence of antibodies against rabies
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview

A- For detection of the agent (infectious organism or toxin) or antibodies

2-Techniques Basics/principle:Immunohistochemistry
 The IFA Test: for the detection of antibodies in the animal serum
• Antigen is fixed to a surface
• Animal serum is added; if antibodies are present, they bind to the antigen
• Secondary antibody with fluorescent label is added; if the animal antibodies are present, the secondary antibodies
binds to the animal antibodies.
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview

A- For detection of the agent (infectious organism or toxin) or antibodies

2-Techniques Basics/principle: Immunohistochemistry advantages and applications
(a) ease of submitting samples
(b) safe handling of human pathogens
(c) retrospective studies using stored samples
(d) speed of technique.
(e) Detection of abnormal prion protein (PrPSc) in brain in case of
(f) Detection of Brucella antigen in the cytoplasm of inflammatory cells, especially the neutrophils and
macrophages of fixed tissues: Brucellosis in goats
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview
A- For detection of the agent (infectious organism or toxin) or antibodies
2-Techniques Basics/principle: Immunohistochemistry advantages and applications: transmissible spongiform
encephalopathies
o These diseases conversion of normal cellular prion protein (PrPc) into a form that is insoluble and resistant to proteases (PrPSc). The
protease resistant form can polymerize into fibrils which accumulate in infected tissues(lymphoid then nervous) and cause cell death and
tissue damage. Phosphorylation of PrPC at Ser-43 by Cdk5 promotes proteinase K resistance, prion aggregation, and fibril formation.
o The diagnosis of transmissible spongiform encephalopathies (TSE) in ruminants relies on the detection of PrPres. The latter can be
detected in situ by immuno-histochemical methods, or by immunological methods following extraction from tissues.

Western blot analysis Prion protein (PrP)

using an immunohistochemistry
antibody in the frontal cortex
nonphosporylated-
against the of
the brain
Prion Protein shows
extensive deposition
(PrP) and of disease-associated
the
phosphorylated PrP (brown)
(pPrP) Prp
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview

A- For detection of the agent (infectious organism or toxin) or antibodies

2-Techniques Basics/principle:-Indirect Methods
 Hemagglutination inhibition test : for the diagnosis of some enveloped viruses/detection of specific anti-viral
antibodies in the serum of the suspected animal
• A surface Glycoprotein (e.g.hemaaglutinin (HA) of influenza virus) agglutinates red blood cells (RBC) of a variety of species.
• If the serum of an animal infected with the virus is mixed with RBC and the virus, there won’t be any agglutination of RBC. This
phenomenon is known as hemagglutination inhibition.
This arises because antibodies present in the serum of that infected person reacts with the viruses and neutralize them (positive result).
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview

A- For detection of the agent (infectious organism or toxin) or antibodies

2-Techniques Basics/principle:
 Hemagglutination inhibition test : Case of Newcastle Disease
 Serious infectious diseases of poultry , which causes mortalities as high as 100%
 The etiological agent is Newcastle disease virus (avian paramyxovirus 1 (PMV1))
 neurological signs or sever depression are the most obvious clinical signs, and some of non vaccinated birds
may be found dead with no detectable signs
 In the absence of vaccination, the presence of specific antibodies against the ND virus indicates that the bird
has been infected by the virus at some time
 Hemagglutination inhibition (HI) test is the most commonly used test for detection of immune response in
affected birds
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview

A- For detection of the agent (infectious organism or toxin) or antibodies

2-Techniques Basics/principle:
 Hemagglutination inhibition test : Case of Newcastle Disease
 Collection and processing of blood: Blood is collected from by heart puncture directly, and collecting the blood into universal
bottles, without anticoagulant. Serum is separated from respective clotted blood samples by centrifugation, , then the sera is collected
in the Eppendorf tubes and labeled then stored in deep freeze at - 20˚C until hemagglutination –inhibition (HI) test is performed.
 Preparation of washed chicken red blood cells: Chickens used for the supply of blood for the preparation of red blood cells
should be housed separately from chickens used for other purposes. Usually they are not vaccinated with Newcastle disease vaccine.
Collect blood from more than one chicken.
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview
A- For detection of the agent (infectious organism or toxin) or antibodies
2-Techniques Basics/principle:
 Agar gel immunodiffusion: -Direct/indirect Method
 when antibodies meet antigen in a solid phase, they will bind and form a line of precipitate
 For detection of antigen, the test material is put into one well and the known antibody is put into another well. As they
diffuse toward each other, if there is antigen there, a line of precipitate will form.
 For the detection of antibodies, known antigen is put into one well and the test serum is put into another well. If there is
antibody present, a line of precipitate will form.
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview
A- For detection of the agent (infectious organism or toxin) or antibodies
2-Techniques Basics/principle:
 ELISA : enzyme-linked immunosorbent assay
 ELISAs are typically performed in 96-well (or 384-well) polystyrene plates
 Detecting and quantifying substances such as peptides, proteins, antibodies and hormones
 An antigen must be immobilized on a solid surface and then complexed with an antibody that is linked to an enzyme
 For detection, an enzyme or other tag can be linked directly to the primary antibody or introduced through a secondary antibody
that recognizes the primary antibody
 There are five types of ELISA methods which include:
 Indirect ELISA
 Sandwich ELISA
 Direct ELISA
 Competitive ELISA
 Multiplex ELISA
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview
A- For detection of the agent (infectious organism or toxin) or antibodies
2-Techniques Basics/principle:
 Indirect ELISA Method: To detect sample antibodies
a. Binding Known Antigen - a sample of known antigen being bound to the wells of a microtiter plate.
b. Blocking - The other unoccupied sites in each well are then bound by a concentrated solution of non-interacting protein,
to block or prevent other proteins in the test sample from adhering.
c. Washing – Rinse to remove any unbound antigen and non-interacting protein.
d. Adding Test Sample Primary Antibody - The test sample of serum containing the primary antibodies is added to each
well.
e. Washing – Rinse to remove any antibodies that did not bind to the known antigen.
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview
A- For detection of the agent (infectious organism or toxin) or antibodies
2-Techniques Basics/principle:
 Indirect ELISA Method
e. Washing – Rinse to remove any antibodies that did not bind to the known antigen.
f. Adding Enzyme-linked Secondary Antibody - An enzyme-linked secondary antibody is added next to bind to the test
sample antibodies.
g. Washing – Rinse to remove any secondary antibodies that did not bind to the primary antibody.
h. Adding Substrate - A substrate is then applied which is converted by the enzyme to give a color or fluorescence or
electrochemical signal.
i. Reading Results - By using a spectrophotometer, the results can be read and recorded.
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview

A- For detection of the agent (infectious organism or toxin) or antibodies

2-Techniques Basics/principle:
 Direct vs Indirect ELISA
 Direct and indirect ELISA differ in the detection step strategies
 The direct detection method uses a labeled primary antibody that reacts directly with the
antigen while The indirect detection method uses a labeled secondary antibody for
detection and is the most popular format for ELISA.
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview
A- For detection of the agent (infectious organism or toxin) or antibodies
2-Techniques Basics/principle:
 Comparison of direct and indirect ELISA detection methods
o Direct ELISA detection
 Advantages:
• Quick.
• Cross-reactivity of secondary antibody is eliminated
 Disadvantages
• Immunoreactivity of the primary antibody might be adversely affected.
• Time-consuming and expensive.
• No flexibility in choice of primary antibody label from one experiment to another.
• Minimal signal amplification.
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview
A- For detection of the agent (infectious organism or toxin) or antibodies
2-Techniques Basics/principle:
 Comparison of direct and indirect ELISA detection methods
o Indirect ELISA detection
 Advantages:
• A wide variety of labeled secondary antibodies are available commercially.
• Versatile because many primary antibodies can be made in one species and the same labeled secondary antibody can be
used for detection
• Maximum immunoreactivity of the primary antibody is retained
• Sensitivity is increased because each primary antibody contains several
• Different visualization markers can be used with the same primary antibody.
• Disadvantages
• Cross-reactivity might occur with the secondary antibody
• An extra incubation step is required.
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview
A- For detection of the agent (infectious organism or toxin) or antibodies
2-Techniques Basics/principle: For a 9 minute tutorial overview of Direct, Indirect, and
 ELISA Method Sandwich ELISA, go to https://www.youtube.com/watch?v=nNjlBCnpGZ4

ELISA Reader Spectrophotometer- A microplate reader Reagents are being loaded into A colored reaction which can be
with a 96-well plate in the sample drawer an ELISA plate read visually
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview
A- For detection of the agent (infectious organism or toxin) or antibodies
2-Techniques Basics/principle:
 ELISA formats-ELISA Sandwish

ELISA sandwich assays

Diagram of common ELISA formats (direct vs. sandwich

 III. Diagnostic Methods
2. LABORATORY TESTING-An overview
A- For detection of the agent (infectious organism or toxin) or antibodies
2-Techniques Basics/principle:
 Blocking ELISA (bELISA)
• Antibodies specific to the targeted antigen are bound to the solid phase of the ELISA plate
• The antigen, prior to adding to the antibody coated plate, is incubated with the samples
• If the samples contain antibodies against that antigen, they will bind (block) to the antigen during this
incubation. When added to the wells, the blocked antigen will be unable to bind to the coating antibodies.
• when the detecting antibody is added to the wells it will not be able to recognize any bound antigen (no color development)
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview
A- For detection of the agent (infectious organism or toxin) or antibodies
2-Techniques Basics/principle:
2 antigens (same type) are competing against the same ab
 Competitive ELISA (cELISA)
• The principle of a competitive assay for the detection of antibodies is competition between the test serum and
the detecting antibody: the sample antigen competes with a reference antigen for binding to a specific
amount of labeled antibody
• The reference antigen is pre-coated on a multi-well plate and the sample is pre-incubated with labeled antibody
and added to the wells.
• Depending on the amount of antigen in the sample, more or less free antibodies will be available to bind the
reference antigen. This means the more antigen there is in the sample, the less reference antigen will be
detected and the weaker the signal or the lower the amount of antigen in the sample, the stronger the signal
due to more labeled antigen in the well.
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview
A- For detection of the agent (infectious organism or toxin) or antibodies
2-Techniques Basics/principle:
 ELISA in Immunohistochemical Staining

NO
• The tissue being studied would be embedded in paraffin and thinly sliced onto a glass microscope slide
• Paraffin is removed, the antigens of the tissues retrieved, and a blocking non-interacting protein would be
added to bind all unoccupied sites on the slide.
• the slide would be washed
• direct or indirect method would be applied:
 an antigen-specific primary antibody is added
 an enzyme-linked secondary antibody is added and then washed
 Add the substrate to be converted by the enzyme and the cell component becomes visible.
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview
A- For detection of the agent (infectious organism or toxin) or antibodies
2-Techniques Basics/principle:
 Proteomics only the definition

• proteomics is the study of proteins, including their expression level, post-translational modification and interaction
with
other proteins, on a large scale
• proteomics can provide an excellent overall view of disease processes at the protein level
• Alterations in the proteome of body tissues or of fluids such as serum, urine or cerebro-spinal fluid can be measured directly
so changes that occur in a disease state can be accurately pinpointed
• Many methods used in proteomics, including two-dimensional gel electrophoresis (2DGE) and mass spectrometry (MS)
• Principle: proteins are separated, usually by 2DGE on polyacrylamide gels, then protein spots are excised, digested with
trypsin, and the resultant peptides analyzed by MS. The masses of these peptides are then compared with the predicted
masses of peptides
• The best-established clinical applications of proteomics so far are in the identification of markers for the early diagnosis of
cancer
 III. Diagnostic Methods
2. LABORATORY TESTING-An overview
A- For detection of the agent (infectious organism or toxin) or antibodies
2-Techniques Basics/principle:
 Proteomics

NO
 The importance of proteomics in the diagnosis of infectious disease Exp: definitive diagnosis of chronic hepatitis B virus
(HBV) infection still relies on liver biopsy, but proteomic analysis of serum samples shows that the expression of at least
seven serum proteins is changed significantly in chronic HBV patients. The ante-mortem differential diagnosis of Creutzfeldt-
Jakob disease (CJD) may be aided by proteomics as preliminary data show that seven proteins in cerebro-spinal fluid (CSF)
are differentially expressed between patients with variant or sporadic CJD (Choe et al., 2002). It Also allows the
identification of novel diagnostic antigens by screening serum from infected and uninfected individuals