13.1 Immunodiagnostics

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Questions and Answers

What is the first step in the enzyme-linked immunosorbent assay (ELISA) process?

Create solid phase - coat plates with known antibodies (correct)

Add serum containing antigen

Add substrate for enzyme used to label detecting antibodies

Observe color change or read optical density in a spectrophotometer

What is the purpose of the detecting antibody in the ELISA method?

To bind to antigen in serum

To inhibit enzyme activity

To provide a color change signal (correct)

To create solid phase

In what type of samples is immunohistochemistry mainly utilized?

Body fluids

Frozen, formalin-fixed tissues (correct)

Standard tissue cultures

Blood serum

What type of device modification is a SNAP device classified as?

Modification of ELISA tests (D) Signup and view all the answers

Which component in the ELISA process binds specifically to the antigen present in the serum?

Secondary antibodies (B) Signup and view all the answers

Immunohistochemistry can be used to detect what type of substances in tissues?

Tissue proteins or pathogen antigens (D) Signup and view all the answers

What is the function of the substrate in the ELISA procedure?

To initiate the enzyme reaction resulting in color change (A) Signup and view all the answers

Which of the following is a key feature of immunohistochemistry?

It provides in situ results (B) Signup and view all the answers

What characterizes monoclonal antibodies?

They are specific for a single antigenic epitope. (B) Signup and view all the answers

Which technique is used to detect antibody-antigen reactions with associated color changes?

Enzyme-linked immunosorbent assay (ELISA) (D) Signup and view all the answers

How are polyclonal antibodies produced?

By boosting an initial inoculation with the same antigen. (C) Signup and view all the answers

What distinguishes immunofluorescence from immunohistochemistry?

Use of fluorescent-labeled antibodies (D) Signup and view all the answers

Which type of microscope is necessary for analyzing results in immunofluorescence?

Fluorescent microscope (D) Signup and view all the answers

In the ELISA process, what is the role of secondary antibodies?

To amplify the signal by binding to primary antibodies. (D) Signup and view all the answers

What occurs when the amounts of antigen and antibody are equal in a precipitation assay?

Formation of visible precipitates (A) Signup and view all the answers

Which of the following is NOT a method for detecting antibody-antigen reactions?

Gradient centrifugation (B) Signup and view all the answers

In what situation can antigen-antibody complexes lead to type III hypersensitivity?

Only in vivo due to large complexes (B) Signup and view all the answers

What is the first step in the ELISA procedure?

Coating plates with known antigen. (D) Signup and view all the answers

Which of the following statements regarding polyclonal antibodies is true?

They target multiple epitopes of an antigen. (D) Signup and view all the answers

What is a key characteristic of the gel-based immunoprecipitation method?

Antigens and antibodies diffuse toward each other (C) Signup and view all the answers

Which method uses enzyme-conjugated antibodies to detect the presence of antibodies in a serum sample?

Indirect ELISA (D) Signup and view all the answers

What visual representation might result from an agar gel immunodiffusion assay?

An arc or a straight line (A) Signup and view all the answers

What type of assay is the Coggin’s test considered?

Qualitative gel-based immunoprecipitation assay (B) Signup and view all the answers

What results in no precipitation during a precipitation assay?

Significantly higher amounts of either antigen or antibody (C) Signup and view all the answers

What is the primary blood group involved in blood group incompatibility in dogs?

DEA-1 (B) Signup and view all the answers

Which blood type in cats is associated with a strong transfusion reaction to type A blood?

Type B (B) Signup and view all the answers

What characteristic do untransfused dogs lack concerning DEA-1 antibodies?

They possess naturally occurring anti-DEA-1 antibodies. (A) Signup and view all the answers

What is the main principle utilized in blood typing techniques?

Agglutination (B) Signup and view all the answers

Which of the following blood types in horses is considered clinically important?

Qa (A) Signup and view all the answers

What happens when anti-A antibodies are mixed with type A blood?

The blood coagulates. (D) Signup and view all the answers

How are the blood types of recipient and donor best determined before a transfusion?

Blood typing and cross-matching. (D) Signup and view all the answers

What can be inferred about cats with type B blood regarding their acknowledgement of blood type?

They may have a transfusion reaction upon receiving type A blood. (B) Signup and view all the answers

What is the primary purpose of typing gels in blood typing?

To facilitate the agglutination of RBCs with antibodies (B) Signup and view all the answers

Which blood type contains both A and B antigens?

Type AB (D) Signup and view all the answers

What does a major cross-match specifically test for?

The presence of recipient alloantibodies against donor RBCs (C) Signup and view all the answers

In a forked dipstick test, what happens when antigen-positive RBCs are present?

They bind to the monoclonal antibodies at the specified line (A) Signup and view all the answers

What does the presence of agglutination indicate during cross-matching?

There is potential for a transfusion reaction (C) Signup and view all the answers

What is the primary purpose of the Coggin’s test?

To detect antibodies against equine infectious anemia virus (A) Signup and view all the answers

In radial immunodiffusion, what does the area of the precipitate circle indicate?

The concentration of the relevant antigen or antibody (B) Signup and view all the answers

What must occur in the virus neutralization test to confirm the presence of specific antibodies?

No damage to the cell monolayer should be observed (B) Signup and view all the answers

What is the basis of the hemagglutination inhibition assay?

The capability of antibodies to prevent hemagglutination (A) Signup and view all the answers

Which of the following is NOT a factor that can lead to transfusion reactions?

Ongoing hemagglutination reactions (D) Signup and view all the answers

What role do MHC molecules play in blood transfusion?

They present antigens that can prompt an immune response (D) Signup and view all the answers

What are alloantibodies?

Antibodies developed in response to foreign RBC antigens (B) Signup and view all the answers

Which laboratory technique is the most labor-intensive and primarily used in research?

Immunoblotting (Western blotting) (C) Signup and view all the answers

What happens if an animal receives a blood transfusion incompatible with its RBC antigens?

A severe immune transfusion reaction may occur (C) Signup and view all the answers

In what clinical situations is blood transfusion particularly vital?

Under conditions of blood component loss or immune-mediated processes (D) Signup and view all the answers

What is the primary source of antibodies used in laboratory diagnostics?

Serum (C) Signup and view all the answers

Which characteristic of serum is crucial for accurate laboratory testing?

It should be clear to yellowish in color. (C) Signup and view all the answers

How long should blood be left undisturbed at room temperature for clotting to occur?

One hour or longer (D) Signup and view all the answers

What is the maximum volume of pooled sera that can be combined from different animals?

Only two sera from the same species can be pooled. (D) Signup and view all the answers

What is the temperature at which serum samples should be stored for prolonged periods?

-20°C (C) Signup and view all the answers

Which of the following is NOT a requirement for antibody-based techniques?

Non-specific antibodies (B) Signup and view all the answers

During serum preparation, what action should be taken after centrifuging the blood sample?

Pipette the serum into a labeled tube. (C) Signup and view all the answers

What is serology primarily the study of?

Interactions between antibodies in serum and antigens (C) Signup and view all the answers

What process occurs in typing gels when erythrocytes possess the antigen corresponding to the antibody in the gel?

Erythrocytes agglutinate and become suspended in the gel. (D) Signup and view all the answers

In a major cross-match, what is primarily being tested?

If the recipient's serum can contain alloantibodies that destroy donor RBCs. (D) Signup and view all the answers

Which blood type in cats is characterized by the presence of both A and B antigens?

Type AB blood (D) Signup and view all the answers

What occurs during a minor cross-match?

Donor's serum is mixed with recipient's erythrocytes. (C) Signup and view all the answers

What indicates a safe blood transfusion during cross-matching?

No cross-reaction occurring in the test. (A) Signup and view all the answers

What is the main purpose of adding substrate in the ELISA procedure?

To facilitate the detection of enzyme activity (B) Signup and view all the answers

In immunohistochemistry, which type of tissues is primarily used?

Formalin-fixed, paraffin-embedded tissues (D) Signup and view all the answers

Which step involves using antibodies that are NOT labeled with an enzyme in the ELISA process?

Add antibodies against the antigen being evaluated (C) Signup and view all the answers

What is a characteristic of SNAP devices in relation to conventional ELISA tests?

They utilize a similar immunofiltration technique (B) Signup and view all the answers

What is primarily analyzed in the immunohistochemistry technique?

Tissue proteins or pathogen antigens (D) Signup and view all the answers

Which of the following steps is NOT part of the enzyme-linked immunosorbent assay (ELISA) process?

Add a fluorescent dye (D) Signup and view all the answers

What type of samples are predominantly analyzed by immunohistochemistry?

Frozen and formalin-fixed tissues (A) Signup and view all the answers

What is the advantage of using enzyme-labeled antibodies in the ELISA method?

They amplify the signal for better detection (C) Signup and view all the answers

What is the primary component used to visualize results in immunofluorescence?

Fluorescent-labeled antibodies (C) Signup and view all the answers

In a precipitation assay, what is the result when there is an excess of either antigen or antibody?

No precipitation occurs (C) Signup and view all the answers

What type of immunoprecipitation assay is characterized as qualitative?

Agar gel immunodiffusion assay (B) Signup and view all the answers

What occurs at the zone of equivalence during a precipitation assay?

Visible precipitate formation (A) Signup and view all the answers

Which of the following describes the role of macrophages in immunofluorescence imaging?

They serve as a source of cytoskeletal proteins. (B) Signup and view all the answers

Which statement best describes the condition under which antigen-antibody complexes lead to type III hypersensitivity in vivo?

Large, insoluble complexes form under specific conditions. (C) Signup and view all the answers

What key feature distinguishes the solution-based immunoprecipitation from the agar gel immunodiffusion assay?

It allows for both qualitative and quantitative analyses. (D) Signup and view all the answers

What type of visual representation indicates a successful agar gel immunodiffusion assay?

An arc or straight line precipitate forms. (C) Signup and view all the answers

What does a negative Coggin’s test indicate for horses?

They can travel across state lines in the USA. (B) Signup and view all the answers

In radial immunodiffusion, what determines the concentration of the antigen or antibody?

The area of the circle formed by the precipitate. (B) Signup and view all the answers

What is the purpose of the virus neutralization test?

To detect virus-specific antibodies in serum. (A) Signup and view all the answers

Which of the following best describes hemagglutination inhibition assay (HAI)?

It assesses the ability of antibodies to inhibit virus-induced clumping of red blood cells. (D) Signup and view all the answers

What is the primary concern associated with transfusion reactions?

Loss of major blood components due to immune-mediated processes. (C) Signup and view all the answers

What is the primary mechanism by which agglutination occurs?

Antibodies cross-link particulate antigens, causing their aggregation. (C) Signup and view all the answers

What happens if an animal with specific alloantibodies receives a mismatched blood transfusion?

A severe immune transfusion reaction may develop. (D) Signup and view all the answers

What is a primary characteristic of immunoblotting (Western blotting)?

It separates proteins based on size before further analysis. (D) Signup and view all the answers

What do alloantibodies represent in an animal's immune system?

Antibodies against blood group antigens from other individuals. (B) Signup and view all the answers

What occurs in dogs that lack DEA 1.1 when exposed to DEA 1.1-positive blood cells?

They produce large amounts of anti-DEA 1.1 antibodies. (B) Signup and view all the answers

Which blood type in cats has the highest risk of transfusion reaction to type A blood?

Type B (C) Signup and view all the answers

What is the primary method used to confirm blood types in cats?

Agglutination tests (D) Signup and view all the answers

What is commonly used to perform blood typing in veterinary practice?

Typing gels and cards (A) Signup and view all the answers

How does agglutination indicate the presence of specific blood group antigens?

By cross-linking and clumping red blood cells. (B) Signup and view all the answers

What is the consequence of mixing anti-A antibodies with type B blood in cats?

No reaction occurs, and the blood remains clear. (A) Signup and view all the answers

Which blood typing technique relies on monoclonal or polyclonal antibodies?

Blood typing (C) Signup and view all the answers

Study Notes

Immunodiagnostics (Veterinary Clinical Laboratory Immunology)

Immunodiagnostics is a field of veterinary clinical laboratory immunology.
Felix N. Toka, Professor of Immunology and Virology at Ross University School of Veterinary Medicine, is the presenter.

Principles of Antibody-Based Detection Techniques

Antibodies are used for laboratory diagnostics and research.
The most common antibody source is serum.
Serum is the portion of blood remaining after clotting, excluding platelets, white blood cells, red blood cells, and clotting factors.

Serum Preparation

Collect whole blood into a sterile tube.
Allow blood to clot at room temperature (approximately 1 hour). (Optional: refrigerate during clotting process)
Centrifuge the blood samples at 1000-2000 x g or 6000 rpm for 10 minutes to remove the clot.
Transfer the serum to a clean, labeled tube.
For pooled samples, only combine two samples from the same species and area. Equally volume each sample.
For samples collected over a prolonged period, freeze, store at −20°C, and transport frozen.

Serology

The study of in vitro reactions of antibodies in serum and antigens, often those of micro-organisms causing infectious diseases.

Requirements for Antibody-Based Techniques

Antigens bind to a specific antibody.
Antibodies are specific to a particular antigen (e.g., an antibody against antigen X).
"Visualization" methods include radioisotopes, fluorescent dyes or enzymes, or precipitation of antibody-antigen complexes.

Two Types of Antibodies Used in Diagnostics and Research

Polyclonal antibodies: A mixture of antibodies that bind to multiple epitopes on an antigen. Serum (antiserum) is the source of these antibodies.
Monoclonal antibodies: Antibodies specific to a single epitope on an antigen. These are generated from a single B-cell clone.

Polyclonal Antibody Production

Animals are inoculated with an antigen.
Booster injections of antigen are given after 3-4 weeks.
Blood is collected 2 weeks after the booster.
Serum is separated, and antibodies are purified using affinity columns or ammonium sulfate precipitation.

Monoclonal Antibody Production

Mice are injected with an antigen.
Spleen cells are collected from the immunized mice.
Myeloma cells are fused with the spleen cells to create hybridomas.
Hybridomas are cultured, and those producing the desired antibody are selected (positive cell selection)
Monoclonal antibodies are harvested from the selected hybridomas.

Detecting Antibody-Antigen Reactions

A variety of techniques (listed below) are used to detect Antibody-Antigen reactions.
Enzyme-linked immunosorbent assays (ELISA)
Immunofiltration technique
Immunohistochemistry
Immunofluorescence microscopy
Precipitation assays
Immunoblotting
Agglutination
Hemagglutination inhibition assays
Complement fixation test
Virus neutralization test

Enzyme-Linked Immunosorbent Assays (ELISA)

ELISA is used to detect serum antibodies.
Different forms of ELISA exist (Direct or Sandwich), e.g., Indirect ELISA and Sandwich ELISA.
Known antigens are coated on solid surfaces (e.g., plates).
Serum containing antibodies is added to the plates.
Secondary antibodies to the species of the serum are added, along with an enzyme label.
A substrate is added, and color changes or optical density in a spectrophotometer is read to determine antibody presence.

Pet-side tests and modifications of ELISA tests

SNAP devices are modifications of ELISA.
Immunofiltration technique is another modification focused on membrane filtration.

Immunofiltration Technique

Modifies ELISA for rapid, on-site diagnoses.
Uses antigen diffusion through a membrane.
Antibodies and colored substrate are added to visualize the result.

Immunohistochemistry

Microscopic technique to detect proteins or pathogen antigens in tissue.
Primarily used on formal-fixed or frozen tissue.
Uses labeled antibodies to visualize proteins within tissues.

Immunofluorescence

Fluorescent-labeled antibodies are used instead of enzymatic labels.
Tissue or cell specimens are prepared on slides.
Fluorescent microscopy is required to read results. This is based on the same principle as immunohistochemistry.

Immunoprecipitation Assays

Antibodies and antigens form complexes.
Complexes are large and insoluble under certain conditions.
These reactions are used in diagnostic analyses.

Formation of Precipitates

Precipitation assays measure antigen-antibody equivalence (and thus relative quantities) using precipitate formation.
Equivalence is achieved when antigen and antibody concentrations are equal to maximum precipitate formation.

Gel-based immunoprecipitations

A qualitative assay that deposits antigens and antibodies in separate wells in an agar gel.
The chemicals diffuse towards each other to form a precipitate.
The Coggin's test is an example of a qualitative agar gel immunodiffusion assay frequently used in equine diagnostics.

Radial Immunodiffusion

A quantitative immunodiffusion assay related to the Coggin's test.
Either the antigen or antibody is introduced in the agar gel.
A circle is formed around the sample well as the single component diffuses through the gel, proportionally proportional to the serum component concentration.

Immunoblotting

Proteins separated by gel electrophoresis and transferred to a nitrocellulose membrane.
Primary antibody is incubated with the membrane to detect the protein of interest(s). A secondary antibody with an enzyme label is applied and substrate reveals the protein(s).

Virus Neutralization (VN)

Detects virus-specific antibodies.
Serum is mixed with a virus, and the mixture is incubated on cell monolayer.
Antibody binding to the virus neutralizes the virus and prevents cellular damage.
The absence/presence of cellular damage in the monolayer allows determination of neutralization.

Agglutination

A simple reaction between a particulate antigen (e.g., RBCs or bacteria) and an antibody.
The antibody cross-links the antigen, resulting in clumping/agglutination.
Agglutination is used for diagnostics, including blood typing in dogs and cats.

Hemagglutination Inhibition Assay (HAI)

Measures the ability of antibodies to inhibit hemagglutination (clumping) of red blood cells by a virus.
If antibodies are present, the virus is neutralized.

Transfusion Reactions

Can be caused by a loss of blood components (RBCs, platelets, clotting factors).
Reactions can be direct (hemorrhage) or indirect (immune-mediated process such as IMHA or ITP).

Barriers to Blood Transfusion

Multiple factors create immunological barriers (e.g., MHC molecules).
Red blood cells (RBCs) express specific antigens which may lead to incompatibility. If an animal is transfused with a different type, the reaction may result in immune transfusion reaction.

Adverse Blood Reactions (Alloantibodies)

Alloantibodies occur naturally in many animals in response to different RBC antigens (types).
These can cause transfusion reactions.

Dog Blood Antigens

DEA-1 blood group is the primary group for blood incompatibility diagnoses in dogs.

Cat Blood Antigens

Cats have three primary blood groups (A, B, AB, and Mik).
The B blood type often produces high titers of anti-A antibodies.

Horse Blood Antigens

Important erythrocyte antigens are Aa, Qa, and Ca in horses.

Diagnosis of Blood Type Incompatibilities

Techniques include blood typing and cross-matching.

Blood Typing

Uses monoclonal or polyclonal antibodies to identify common erythrocyte antigens for species.
The technique's readout depends on the principle of agglutination (clumping of RBCs).

Cross-Matching

Based on agglutination (clumping) of RBCs when the recipient's serum is mixed with the donor's RBC's (major crossmatch). Donor serum is then mixed with the recipient's RBC's in a minor crossmatch.
Used to anticipate potential transfusion reactions due to blood type incompatibility.

Typing Gels

Gel-based technique to determine blood types.
RBCs are added and allowed to filter through gels with antibodies.
Results are determined based on how the RBCs behave in the gel's matrix.

Additional Methods

Membrane dipsticks can be used for rapid diagnosis, using a similar technique to typing gels/cards.

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Test your knowledge on the fundamentals of enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. This quiz covers essential concepts such as antibody functions, sample types, and detection techniques. Perfect for students in immunology or related fields.