Serological Tests.pdf

Serological Tests (VETS2007) Dr Noman Naseem [email protected] *Many thanks to Dr. Willy Suen for slide materials * Serological Tests Antigen binding sites • Serological tests take advantage of antigen-antibody interactions to detect either antigen or antibody in fluids. Detection of antigen...

Serological Tests (VETS2007) Dr Noman Naseem [email protected] *Many thanks to Dr. Willy Suen for slide materials * Serological Tests Antigen binding sites • Serological tests take advantage of antigen-antibody interactions to detect either antigen or antibody in fluids. Detection of antigen-specific antibody in serum Detection of antigens in blood/tissues Ex: infectious agents, cancer cell specific components, toxins, cytokines etc. Components of Serological Tests Antigen Epitopes: Each antigen is a complex structure of multiple epitopes, each of which can have a corresponding specific antibody. Components of Serological Tests Antibody • Polyclonal vs Monoclonal (primary ab) • Antiglobulin (secondary ab) Production of Polyclonal Antibodies Antibodies of different classes & subclasses against multiple epitopes on the antigen Production of Monoclonal Antibodies Antibodies against a specific epitope of the antigen • B cells are isolated from spleen, fused with Myeloma cells (neoplastic B cells) = HYBRIDOMA • Multiple clones produced, select the one that produces antibodies against desired epitope • Purify antibodies from the immortal culture = monoclonal antibodies Polyclonal vs monoclonal Production of secondary antibodies (antiglobulins) Animal immunised with purified Ig from serum of another species Anti-globulins • Immunoglobulins purified from the goat’s serum are the antigen -> injected into another species (bunny) -> production of anti-globulin antiserum. These are now antibodies that are specific for immunoglobulin of a different species. (rabbitanti-goat-IgM or IgG) • These are important for Indirect ELISAs and indirect IHC Components of Serological Tests Indicator • Colour change (Enzyme & Substrate) • Fluorescence • Agglutination • Precipitates • RBC lysis/CPE Serological Tests: Types • • • • • • • • Enzyme-linked immunosorbent assay – ELISA, EIA, Luminex Rapid immunoassay devices – Immunofiltration tests – Immunochromatography tests Western blotting Precipitation tests – Agar gel immunodiffusion (AGID) – Immunoelectrophoresis Immunolabelling (“immunostaining”) – Direct or indirect immunofluorescence – Immuno-electron microscopy – Immunohistochemistry & immunocytochemistry Agglutination tests – Bacterial agglutination tests – Passive agglutination – Viral haemagglutination & haemagglutination inhibition tests Complement fixation test Neutralisation test Enzyme-linked Immunosorbent Assay (ELISA) • Used to detect antigen/antibody • Direct ELISA = Indicator enzyme is on primary AB • Indirect ELISA = Indicator enzyme is on secondary AB (antiglobulin) • Requires a pure preparation of a known antigen or antibody (or both) • Rapid, sensitive & relatively cheap • Detection of antibody uses indirect ELISA Enzyme-linked Immunosorbent Assay (ELISA) For detection of antigen (eg., virus) a “sandwich” ELISA (antigen-capture ELISA) is used: 1. Wells are coated with antibody specific for antigen of interest = capture antibody 2. Test sample is added to wells 3. Unbound components are washed away 4. Second antibody (also specific for the antigen) is added 5. 2nd antibody may be tagged with enzyme or a tagged antiglobulin can subsequently be added 6. Enzyme substrate is added and colour development is measured Well coated with antibody. Sample with antigen added, antibody binds Sample Detection antibody added Enzyne labeled antigobulin added Add enzyme substrate, Color changes = Positive In Clinic - Rapid Immunoassay Devices SNAP test Precipitin Reaction-Based Tests • Precipitin-based assays less common now (replaced by ELISA). • Made in a layer of agar that permits radial diffusion, in both of the horizontal dimensions, of one or both reactants • A visible precipitate is formed when sufficient quantities of antibody are mixed with soluble macromolecular antigens • Antigen or antibody excess will hinder immune complex formation – Difficult to interpret • Ex: Equine infectious Anemia, Coggin’s test Immunodiffusion in agar gels Western Blotting 1. Used to identify particular antigens in a complex mixture of proteins (eg., virus antigens in a cell culture lysate). 2. Protein mix is separated on an acrylamide gel by electrophoresis 3. The protein bands are then transferred onto a nitrocellulose membrane by electrophoresis (“blotting”) 4. The protein-containing membrane (“blot”) is immuno-labelled after the same principles as for an ELISA 5. The specific protein bands are visualised with chromogen Western Blotting Immunolabelling of Cells & Tissues • Used for detection of antigens on/incells and tissues • Antigens can be from normal cells, neoplastic cells, pathogens. • Most common approaches: • Immunofluorescence • Immunofluorescence microscopy • Flow cytometry • Immunohistochemistry • Tissue sections • Cell cultures • Cytospin preparations Immunofluorescence Microscopy • Direct immunofluorescence • Fluorochrome bound to 1° antibody • Used to detect antigen in tissue sections or in tissue culture cells (eg., to confirm presence of virus) • Indirect immunofluorescence • Fluorochrome bound to antiglobulin • Used to detect antigen in tissue sections or antibody in serum Immunofluorescence test Immunohistochemistry HPAI (H5N1) virus antigen in lung infection Double-labelling for virus antigen (red) & microglia cellantigen (black) • Used similarly to immunofluorescence microscopy, but provides a sample with a permanently persevered, visual pigment signal • Direct or indirect labelling – the latter more sensitive • Double-labelling also possible Flow Cytometry • Cells pumped through a narrow tube • Laser beam scatter ➔gives information of cell (e.g. size, internal complexity) • Combined with use of fluorescent ABs ➔ detect antigens (CD markers etc.) Agglutination Assays Agglutination = interaction between antibody and particulate antigen resulting in visible clumping Positive Haemagglutination Assays • Used for typing blood groups and matching compatible donors and recipients for blood transfusion • Individuals will not have antibodies against their own RBC antigen(s). • Haemagglutination occurs when RBCs are mixed with serum that contains the antibodies against the RBCs’ surface antigens. Viral Haemagglutination & Haemagglutination Inhibition • Used for identification of viruses or detection of antibodies to a virus: • Some viruses can agglutinate red blood cells (Ex: from chicken) = virus HA assay • Antibodies to the virus can inhibit this agglutination = HI assay Complement Fixation Test • Used to detect antigen-specific antibody in serum • Based on the principle that when antibody is bound to antigen, complement is activated ➔ membrane attack complexes produced ➔ lyses the antigen • The endogenous complement needs to be inactivated by heating to 56C • Two stages: 1. Antibody (in test serum) + Antigen of interest 2. Anti-RBC antibody + RBCs Interpretation? Interpretation of Serological Tests • Must recognise limitations of testing for antibody • Being positive for antibodies does not always mean that the agent in question is responsible for the disease currently affecting the animal – Other possible interpretations, if animal is antibody positive: • • • • Past exposure Vaccination Maternal antibodies Cross reactivity to related organism Interpretation of Serological Tests • Similarly, being serologically negative for antibodies does not always mean the agent in question was not the cause of disease – Other interpretations, if animal is antibody negative: • Sample taken too early in the course of disease/infection • Immunosuppression or immunodeficiency • Presence of binding/neutralizationinterfering antibodies • Congenital persistent infection (eg., BVD or border disease virus) Disease Course Interpretation of Serological Tests • Therefore a need to take paired blood samples to diagnose infections in individual animals: • First is taken at the acute stage of disease • Second is taken at the convalescent stage (2-3 weeks after the disease period) • If there is a 4-fold or greater increase in titre between the 1st & 2nd sample, we may conclude that infection did occur. Acute sample, titre = 1:2 Convalescent sample, titre = 1:16 Interpretation of Serological Tests However, a single sample may be useful for: – Surveys of infection in populations of animals (eg., determine the proportion of animals in a population that have antibodies to a particular agent) – Confirming vaccination responses (often done in the poultry industry; required for “export” of companion animals) – Determining the infection status of an individual animal to a persistent viral or bacterial pathogen (i.e., an agent the immune response cannot eliminate) • The presence of antibodies against such agents in the face of agent presence suggests persistence (eg., lentiviruses) • Lack of antibodies in the face of agent presence may also suggest persistence, eg., BVD virus Sensitivity & Specificity Sensitivity = measure of the proportion of the true positives (e.g. infected animals) that are scored as positive in the test Sensitivity = infected animals with a positive test result Total # of infected animals A highly sensitive test has very few false negative results, i.e., nearly all infected animals are detected. Total Infected Tested Positive Tested Negative In this test, 6/7 infected animals were detected; i.e. Only 1 false negative Sensitivity & Specificity Specificity = measure of the proportion of uninfected animals that are scored as negative in the test. Specificity = non-infected animals with a negative result Total # of non-infected animals A highly specific test has very few false positive results, i.e., nearly all animals that test positive are truly positive Total Non-Infected Tested Positive Tested Negative In this test, 3/3 non-infected were negative; i.e. No false positive Sensitivity & Specificity: an example Infected animals Non-infected animals +ve test 950 35 -ve test 128 2540 Total 1078 2575 Sensitivity = 950/1078= 0.88 <=> 88% Specificity = 2540/2575 = 0.99 <=> 99% Pulmonary melioidosis Sensitivity & Specificity • • No serological test is absolutely sensitive and specific (i.e., there will always be some infected animals that are missed and some animals that test positive will not actually be infected) To overcome this problem, more than one type of test may be performed, and where discrepancy is discovered, repeat assays are performed or additional samples obtained (in an ideal world). Questions?