Immunodiagnostics (Veterinary Clinical Laboratory) PDF

Immunodiagnostics (Veterinary Clinical Laboratory Immunology) Felix N. Toka Professor, Immunology and Virology Ross University School of Veterinary Medicine  Principles of antibody-based detection techniques Besides protecting the ho...

Immunodiagnostics (Veterinary Clinical Laboratory Immunology) Felix N. Toka Professor, Immunology and Virology Ross University School of Veterinary Medicine  Principles of antibody-based detection techniques Besides protecting the host, antibodies can also be used in laboratory diagnostics and research The most common source of antibodies is serum What is serum? the portion of the blood left after clotting (which removes platelets, white blood cells, RBCs, and clotting factors)  Serum Preparation Collect whole blood using an appropriate technique and transfer to a sterile tube. Approximately Otherwise 1/3 of whole blood volume is serum; therefore collecting 300μl whole blood is required to reach a final volume of 100μl serum. After collection of the whole blood, allow the blood to clot by leaving it undisturbed at room temperature. This may take up to one hour, or longer. Check the blood has clotted by inverting the tube. Remove the clot by centrifuging tubes at 1,000-2,000 x g or 6,000 rpm for 10 minutes. This should preferably be performed in a refrigerated centrifuge but a regular centrifuge will suffice. The resulting supernatant is designated serum, it should be clear to yellowish in colour, not red. Samples which are hemolysed (red in colour) can invalidate some tests. Immediately transfer the serum into a clean, labelled tube using a pipette. If sera are pooled to make a single sample, only TWO sera are to be pooled. Pooled sera should also be from the same species of animals and housed in the same area. The volumes from each animal should be equal. If samples are collected over a prolonged period, freeze, store (at -20°C) and transport frozen. Ensure there is enough packing material or ice to prevent samples from thawing en route. Please label all tubes clearly with a waterproof permanent marker, and have it correspond to the information of the animal it was collected from.  Serology The study of in vitro reactions of antibodies in serum and antigens, usually those of microorganisms that cause infectious diseases Requirements for antibody-based techniques 1. Antigens that can bind to a particular antibody 2. Antibodies that are specific and especially generated to detect a particular antigen - (e.g., antibody against antigen X) 3. Method of “visualizing” the antibody-antigen reactions – (radioisotopes, fluorescent dyes, or enzymes, or the precipitation of antibody–antigen complexes)  Two types of antibodies are used in diagnostics and research Polyclonal antibodies - a mixture of antibodies that are specific for several epitopes of an antigen - products of many different clones of B cells – the source of polyclonal antibodies is serum (antiserum) Monoclonal antibodies - antibodies specific for a single antigenic epitope - products of a single B-cell clone How are polyclonal and monoclonal antibodies produced? Polyclonal antibody production e.g., Inoculate an animal with antigen X Wait 3-4 weeks and inoculate with antigen again (booster) 2 weeks later collect blood and separate serum Purify antibodies on affinity columns or ammonium sulfate precipitation Monoclonal antibody production Detecting Antibody-Antigen Reactions Enzyme-linked immunosorbent assays (ELISA) Immunofiltration technique Immunohistochemistry Immunofluorescent microscopy Precipitation assays Immunoblotting Agglutination Hemagglutination inhibition assays Complement fixation test Virus neutralization test  Enzyme-linked immunosorbent assay (ELISA) 1. Create solid phase - coat plates with known antigen 2. Add serum containing antibodies you are looking for (source of primary antibodies) 3. Add secondary antibodies against species from which the serum originated – these antibodies are labeled with an enzyme 4. Add substrate for the enzyme used to label secondary antibodies 5. Observe colour change or read optical density in a spectrophotometer https://www.biotek.com Indirect ELISA e.g., to detect serum antibodies against B. burgdorferi (Lyme disease) Absorbance Reader  Enzyme-linked immunosorbent assays 1. Create solid phase - coat plates with known antibodies 2. Add serum containing antigen you are looking for 3. Add antibodies against antigen you are looking for – these antibodies are NOT labeled with an enzyme (secondary antibodies) 4. Add detecting antibody against secondary antibodies – these antibodies ARE labeled with enzyme 5. Add substrate for enzyme used to label detecting antibodies 6. Observe colour change or read optical density in a spectrophotometer https://www.biotek.com Sandwich ELISA to detect antigens in the sera Absorbance Reader Pet-side tests, e.g., SNAP devices - are modifications of ELISA tests Immunofiltration technique SNAP devices Immunohistochemistry Immunohistochemistry is used principally on frozen, formalin-fixed tissues collected at necropsy or biopsy Thin sections of formalin-fixed, paraffin-embedded tissues; thin sections of frozen tissues; or cells attached to slides In situ results; can detect tissue proteins or pathogen antigens in tissues  Immunohistochemistry Immunohistochemical staining of a hemangiosarcoma using anti–von Willebrand factor antibodies (panel A) and transitional epithelium containing canine distemper virus (panel B) (courtesy of Dr. E. G. Clark) Immunofluorescence Principle: the same as in immunohistochemistry Difference: fluorescent-labeled antibodies https://www.picfair.com are used instead of enzyme-labeled antibodies Samples: tissue or cells on microscope slides Requires a fluorescent microscope to read results  Immunofluorescence http://wendyleesshihtzu.com/Parvovirus.htm A merged image generated by immunofluorescent Canine parvovirus viral antigen in mesenteric microscopy showing the locations of two different lymph node 4 days after oral-nasal infection cytoskeletal proteins (actin and tubulin) within two macrophages. Actin is shown in green, tubulin is shown in red and nuclei are stained blue Immunoprecipitation assays Antigens and antibodies can form complexes Mostly, these are usually small and soluble Under certain conditions these complexes are large and insoluble In vivo can lead to type III hypersensitivity In vitro can form visible precipitates useful for diagnostic analyses Formation of precipitates Precipitation of antigen and antibody complexes in a precipitation assay exploits the concentrations of antibody and antigen (zone of equivalence) to measure their relative quantities (titers) antigen + antibody in equal amounts = precipitation Too much of antigen or antibody = no precipitation Types of immunoprecipitation assays solution based immunoprecipitation – may be quantitative or qualitative  Gel-based immunoprecipitation - also called agar gel immunodiffusion assay - a qualitative assay antigen and antibodies are deposited in separate wells in an agar gel the two substances diffuse in the gel towards each other at the zone of equivalence, the antigen-antibody complexes form a visible precipitate (either an arc or a straight line) A specific example of agar gel immunodiffusion assay is the Coggin’s test. The Coggin’s test is specifically used to detect antibodies against equine infectious anemia virus (a negative Coggin’s test is required for horses to travel into or between most states in the USA)  A modification of the agar gel immunodiffusion assay to measure titers is called radial immunodiffusion – a quantitative assay Either antigen or antibody is incorporated into the gel Only one component diffuses into the gel to form precipitate The precipitate forms in a circle around the well as the sample diffuses out The area of the circle is proportional to the concentration of the relevant antigen or antibody within the sample Radial immunodiffusion to measure serum levels of Igs Immunoblotting (Western blotting) Mostly used in research - it is laborious thus not suitable for routine diagnostics proteins are first separated on the basis of size using polyacrylamide gel electrophoresis separated proteins are then transferred from the gel onto a nitrocellulose membrane membrane is incubated with primary antibody against the protein of interest secondary antibody conjugated to an enzyme is applied followed by substrate substrate may produce color or chemiluminescence Virus neutralization (VN) Test used to detect virus specific antibodies in serum of an animal Test serum is mixed with virus and added to a cell monolayer After appropriate incubation (2-3 days) cell monolayer is inspected for cellular damage If serum contains specific antibodies to virus, they bind the virus thus neutralizing it, and no damage should occur to the cell monolayer However, if serum does not contain specific antibodies against virus, then virus will infect monolayer and cause cellular damage  Agglutination Simple reaction between particulate antigen (e.g., RBCs, bacteria) and antibody OR when soluble antigen is coated onto latex beads Antibody will cross-link particulate antigen leading to agglutination of antigen Clinical application - determination of blood group in dogs and cats No agglutination Agglutination Hemagglutination inhibition assay (HAI) Certain viruses can agglutinate red blood cells – hemagglutination The ability of antibodies to inhibit hemagglutination of red blood cells by certain viruses = HAI If antibodies against that virus are present, they can inhibit hemagglutination of red blood cells Assay is used in diagnostic virology e.g., hemagglutinating viruses, Myxo, Paramyxoviruses Transfusion reactions Loss of major blood components (RBCs, platelets, or clotting factors) results from: direct hemorrhage indirectly in an immune-mediated process (such as IMHA or immune- mediated thrombocytopenia) In such clinical situations, the transfusion of blood or blood products is critical to saving the life of an animal Barriers to blood transfusion several factors, including MHC molecules, present immunological barriers to tissue (including blood) transfer between individuals RBCs do not express either MHC I or MHC II molecules They do express potentially antigenic molecules called erythrocyte or red blood cell (RBC) antigens If an animal with a particular RBC antigen (blood type) is transfused with blood of a different type, a severe immune transfusion reaction can develop  Adverse reactions happen because many animals have naturally occurring antibodies to different RBC antigens called alloantibodies What are alloantibodies? Antibodies of one individual that react against the alloantigens (homologous molecules) of another individual of the same species Dog blood antigens the most important antigens involved in blood group incompatibility are the dog erythrocyte antigen 1 (DEA-1) group Other blood groups exist in dogs but are less commonly implicated in clinical disease Untransfused dogs do not have immunologically significant amounts of naturally occurring alloantibodies to DEA-1 If exposed to DEA 1.1-positive blood cells, dogs lacking DEA 1.1 will produce large amounts of anti-DEA 1.1 antibodies Cat blood antigens Three main blood groups in cats, types A, B, and AB, and a newly recognized group, Mik Some untransfused cats have naturally occurring anti-blood group antibodies Cats that have type B blood produce high titers of anti-A antibodies Therefore, cats with type B blood will have a strong transfusion reaction to type A blood on the first transfusion Horse blood antigens In horses clinically important erythrocyte antigens are Aa, Qa, and to a lesser degree Ca blood transfusion of horses is less common Diagnosis of Blood Type Incompatibilities To prevent or anticipate transfusion reactions due to blood type incompatibilities it is best to know the recipient’s and donor’s respective blood types and their compatibility Two techniques available: Blood typing - patient’s and donor’s blood Cross-matching - patient’s and donor’s blood Blood typing Uses monoclonal or polyclonal antibodies that recognize the common erythrocyte antigens of a species The readout method is based on the principle of agglutination When the antibodies bind to the blood-type antigens on the surface of erythrocytes, they cross-link these cells and cause them to visibly agglutinate Techniques available are, typing cards, typing gels, and membrane dipsticks e.g., to type feline blood A drop of blood is added to a card or gel containing anti-A antibodies The anti-A antibodies bind to any erythrocyte A antigens present and cause the blood to clump or agglutinate This confirms the blood to be type A If type B blood (that does not possess an A antigen) is added, the anti-A antibodies will not bind and thus no agglutination will take place Similarly, blood added to a card with anti-B antibodies will cause agglutination of type B blood but not of type A Blood typing card (available in-clinic for DEA 1.1 or for feline A, B, and AB) Macroscropic agglutination can be observed in the upper box labeled Type A, which is absent from the box labeled Type B, indicating that the A antigen is present on the erythrocytes and B antigen is absent. This cat has type A blood. Typing gels Use monoclonal antibodies against RBC antigens are suspended in gel matrices in test tubes Blood applied to the tube filters through the gel If RBCs possess the antigen to the antibody in the gel, the erythrocytes will agglutinate and become suspended in the gel If RBCs do not posses antigen to the antibody in the gel, they will rapidly pass through the gel The blood from the cat in this image has and settle at the bottom of the tube both A and B antigens and thus has an AB blood type. Membrane dipstick Uses anti-RBC monoclonal antibodies bound to lines on a dipstick The dipstick is dipped into diluted blood, and erythrocytes move up the dipstick by capillary action antigen-positive RBCs, cells will bind to the monoclonal antibodies at the corresponding line http://www.rapid-vet-shop.at/mall/1/files/if68IFU_dog_DEA_1.1_eng.pdf Cross-matching Based on saline-agglutination of RBCs Performed as major and minor cross-matches Sera and RBCs are obtained from both the recipient and donor In a major cross-match, the recipient’s serum is mixed with the donor erythrocytes – to test if recipient has alloantibodies that would destroy donor RBCs In a minor cross-match, the donor’s serum is mixed with the recipient’s erythrocytes- to test if donor has alloantibodies that would destroy recipient’s RBCs Cross-matching of feline A, B, and AB blood types The areas shaded in light purple indicate cross-reactions evidenced by agglutination. In the white boxes, no cross- reaction occurs, indicating that the blood would be safe to transfuse