Serology as a Diagnostic Technique

Transcription

1 Serology as a Diagnostic Technique

2 Characteristics of Any Diagnostic Techniques Any useful detection strategy must be: Specific: yield a positive response for only the target organism or molecule. Sensitive: identify very small amount of the target the target organism or molecule, even in the presence of other potentially interfering organisms or substances Simple: to be run efficiently, effectively, and inexpensively on a routine basis Replicable (or Reliability ): to be repeated different times with different technicians under the same experimental conditions and leading for the same

3 Diagnostic Methods Biological assays: Symptomatology Microscope: Observation Serological Assays: Antibodies Molecular Diagnostic Tools: DNA

4 Serological Assays Immunology (sometimes called serology ): is the study of the serum, and the properties and functions of its components. Immuno-technology, is an important arm of biotechnology, constituting the industrial scale application of immunological procedures to produce vaccines, for mass immunisation to prevent prevalent diseases and/or producing immunological therapeutic agents to cure the afflicted. When an infecting organism gains entry into the mammalian system for the first time, the immune system of the mammal reacts, mainly in response to

5 Serological Assays As A Diagnostic Tool Antiserum: Blood serum containing antibodies arising out of immunisation or after an infectious disease. Production of antibodies: Antibodies are produced by the lymphocytes. The process of antibody production and immune response are complex and both the lymphatic and the blood systems are very closely involved: Polyclonal antibodies: antibodies produced by molecules with several different antigenic determinants (epitopes) and/or several different cell populations. Monoclonal antibodies: antibodies produced against a single antigenic determinant (epitope) and/or by a single cell population; hence are very specific.

6 Antigen Recognition by Lymphocytes An antigen is any foreign molecule that is specifically recognized by lymphocytes and elicits a response from them A lymphocyte actually recognizes and binds to just a small, accessible portion of the antigen called an epitope. Antibody A Antigenbinding sites Epitopes (antigenic determinants) Antigen Antibody B Antibody C

7 Antigen Recognition by Lymphocytes Epitope: a part of a protein molecule that acts as an immunogenic/antigenic determinant [and so determines specificities] a macromolecule, such as a protein, may contain many different epitopes, each capable of stimulating the production of specific antibodies, each with a correspondingly specific binding site.

8 B Cell Receptors for Antigens B cell receptors bind to specific, intact antigens, are often called membrane antibodies or membrane immunoglobulins Antigenbinding site Light chain Disulfide bridge C C Antigenbinding site Variable regions Constant regions Transmembrane region Heavy chains Plasma membrane B cell Cytoplasm of B cell (a) A B cell receptor consists of two identical heavy chains and two identical light chains linked by several disulfide bridges.

9 Antibodies: (also known as immunoglobulins, abbreviated Ig) are gamma globulin proteins that are produced by the immune system of an organism in response to exposure to a foreign molecule and characterised by its specific binding to a site, related to an epitope of that molecule; induced response proteins. The antibodies, like all proteins, are formed of chains of amino acids, which undergo very complex packing, giving the proteins a specific and functionally significant final shape (tertiary configuration), which determines most of the characteristics of the protein.

10 Molecular Structure of Antibodies The conventional model of the Ig molecules is a Y shaped configuration, with two heavy chains and two light chains, with two open arms containing the antigen combining sites, which occur on both the light and the heavy chains. The two heavy chains are bound together by disulphide bonds. At any point, the molecule has two chain sections, parallel to each other.

11 Clonal Selection of Lymphocytes In a primary immune response binding of antigen to a mature lymphocyte induces the lymphocyte s proliferation and differentiation, a process called clonal selection Clonal selection of B cells generates a clone of short-lived activated

12 Clonal Selection of Lymphocytes B cells that differ in antigen specificity Antigen molecules Antigen receptor Antigen molecules bind to the antigen receptors of only one of the three B cells shown. The selected B cell proliferates, forming a clone of identical cells bearing receptors for the selecting antigen. Some proliferating cells develop into long-lived memory cells that can respond rapidly upon subsequent exposure to the same antigen. Clone of memory cells Antibody molecules Clone of plasma cells Some proliferating cells develop into short-lived plasma cells that secrete antibodies specific for the antigen.

13 Antibody concentration (arbitrary units) In the secondary immune response memory cells facilitate a faster, more efficient response 1 Day 1: First 2 Primary 3 Day 28: 4 Secondary response to antigen A produces antibodies exposure to response to Second exposure antigen A antigen A to antigen A; first to A; primary response to antigen B produces antibodies to B produces antibodies to A antigen exposure to B Antibodies to A Antibodies to B Time (days)

14 Antibody Classes IgM (pentamer) First Ig class produced after initial exposure to antigen; then its concentration in the blood declines These are the five major classes of antibodies, or immunoglobulins differ in their distributions and functions within the body IgM: primary response and 10 active site, 5 Y- shape IgG: one single Y- shape 2 active sites, secondary response Secretory component IgG (monomer) IgA (dimer) IgE (monomer) IgD (monomer) Transmembrane region J chain J chain Promotes neutralization and agglutination of antigens; very effective in complement activation Most abundant Ig class in blood; also present in tissue fluids Only Ig class that crosses placenta, thus conferring passive immunity on fetus Promotes opsonization, neutralization, and agglutination of antigens; less effective in complement activation than IgM Present in secretions such as tears, saliva, mucus, and breast milk Provides localized defense of mucous membranes by agglutination and neutralization of antigens Presence in breast milk confers passive immunity on nursing infant Triggers release from mast cells and basophils of histamine and other chemicals that cause allergic reactions Present primarily on surface of naive B cells that have not been exposed to antigens Acts as antigen receptor in antigen-stimulated proliferation and differentiation of B cells (clonal selection)

15 Antibody-mediated mechanisms of antigen disposal Binding of antibodies to antigens inactivates antigens by Viral neutralization (blocks binding to host) and opsonization (increases phagocytosis) Virus Agglutination of antigen-bearing particles, such as microbes Bacteria Precipitation of soluble antigens Activation of complement system and pore formation Complement proteins MAC Bacterium Soluble antigens Foreign cell Pore Enhances Leads to Phagocytosis Cell lysis Macrophage

16 Antiserum Preparation Obtaining PAb for the diagnostic purposes: 1. Choose of the animal: decide wither polyclonal or monoclonal 2. Preparation of antigen 3. Immunization: the antigen is emulsified sometimes with with an equal volume of complete adjuvant (Freund s adjuvant- mannitol monoleate mixed with paraffin oil) 4. Collection of sera: (the supernatant) 5. Titration. 6. Storage: (addition of antiseptic substances (NaN3); lyophilized; at temperature below -20 C)

17 Typical Serological Profile After Acute Infection Note that during reinfection, IgM may be absent or present at a low level transiently

18 IgG Purification with Protein A- spherosis column: 1. Column preparation by washing with Eluting Buffer. [Glycine 0.1 M ph 3] and then with 50 ml PBS 1x. 2. Adding of antiserum [previously preabsorbed with healthy material] to the column 3. Washing the column with 50 ml PBS 1x. 4. Eluting the IgG with Eluting Buffer and collect fraction of 1 ml. Then bring ph to 7.3 with Neutralizing Buffer [Tris 1M ph 9] 5. Titration: Calculate the absorption at the 280 nm for each fraction. Form fractions of 1 ml with concentration of 1 mg/ml (1.445 OD) and store them at -20ºC 6. Wash the column with 50 ml of PBS 1x and conserve them at +4ºC.

19 Monoclonal Antibodies Immunochemical techniques are extremely useful for the rapid and accurate routine detection of host pathogens and ultimately the diagnosis of host disease and their relatedness The introduction of hybridoma technology has provided methods for the production of homologous and biochemically defined immunological reagents of identical specificity Hybridoma are produced by a single cell line and are directed against a unique epitop of the immunizing antigen.

20 Antibodies- Production 1. Spleen cells are removed washed minced and gently agitated to release individual cells, some of which are antibody-producing B cells. 2. Myeloma cells (immortal B-cell Line) are genetically defective for the enzyme: Hypoxanthine-Guanine PhosphoRibosylTransferase (HGPRT-) 3. The combined cells are mixed with 35% PEG (to facilitate fusion between cells) for few minutes to form hybridoma, then transferred to a growth medium containing: Hypoxanthine, Aminopterin, and Thymidine (HAT medium).

21 Explanation of use HAT medium The myeloma (HGPRT-) will not be able to synthesize purines in HAT medium: The (HGPRT-) myeloma alone or as myeloma-myeloma fusion cells cannot use the Hypoxanthine as a precursor for biosynthesis of purines (adenine and guanine), which of course essential for nucleic acid synthesis. They may use the other pathway for purine synthesis by the enzyme dihydrofolate reductase, but the enzyme Aminopterin which included in the media will inhibit the enzyme dihydrofolate reductase activity. Spleen+myeloma fused cells would be the only survivors in HAT medium as they contain HGPRT which allow the use of Hypoxanthine for biosynthesis of purines. Although Thymidine will overcome the blockage of

22 Monoclonal Antibodies Production

23 Serological Techniques In serology Ab acts as a probe for detection of Ag => ppt Direct Methods: it is possible to visualize the Antigen Antibody reaction without the help of other reactions Precipitation or agglutination Immunoelectron microscopy Agar precipitation Indirect Methods: ELISA

24 Comparison among the two Antiserum Preparation Polyclonal Antibodies (Pabs) has two drawbacks: The prepared amount of PAbs produced each time could be varied. PAbs couldn't be used to distinguish two similar target protein. Monoclonal Antibodies (MAbs) are useful for their: Reproducibility Specificity

25 Serological Relationships 1. Serological distinguished Ag2 Ag As 2. Serological related: A. Partial identity reaction B. Non-identity reaction Ag2 Ag A B Ag2 Ag As As 3. Serological identical Ag2 As Ag

26 Serological Relationships Gel preparation 0.8 g agarose 0.2 g NaN ml distilled water Preparation of antiserum: Mix with saline solution (0.85 g NaCl in 100 ml distilled water) The first reading is made after 2 hours and the second reading at 18 hours.

27 Immunological Diagnosis: ELISA ELISA Diagnostic Kits Some Characteristics A number of disease detection kits have been developed for use at the site where a disease is suspected. These kits, which in most cases do not require laboratory equipment, are very useful Some tests only take five minutes to perform. The diagnostic kits are based on a method that uses proteins called antibodies to detect disease causal agents called antigen. One of the technique used is called ELISA (enzyme-linked immunosorbent assay).

28 ELISA Diagnostic Kits This assay is based on the ability of an antibody to recognize and bind to a specific antigen, a substance associated with a host pathogen. The antibodies used in the diagnostic kits are highly purified proteins produced by injecting a warm-blooded animal (like a rabbit) with an antigen associated with one particular host disease. The animal reacts to the antigen and produces antibodies. The antibodies produced recognize and react only with the proteins associated with the causal agent of that host disease. Color changes on the unit s surface indicate a positive (disease present) reaction.

29 ELISA Diagnostic Kits

30 ELISA-test Sample preparations

31 Generalized ELISA-test protocol 1) Coating of the plates (wells): with IgG diluted in coating buffer Incubation at 37 C 2) Adding of the extract : Coating antibody : specific Protein : non specific proteins Incubation at 37 C

32 Generalized ELISA-test protocol 3) Adding of conjugated IgG diluted in conjugating buffer. : Labeled antibody with Alkaline phosphatase 4) Adding of substrate solution Incubation at 37 C p-nitrophenyl-phosphate diluted in substrate buffer. : Substrate Enzyme 5) Observe or measure the amount of colored product

33 ELISA-test Light Filter 405 Healthy O.D * constant (2.5) > Positive control

34 Different Types of ELISA-test Double Antibody Sandwich ELISA (DAS) Triple Antibody Sandwich-Indirect ELISA (TAS) : Coating antibody : Antigen : Labeled antibody : Substrate Enzyme : The intermediate antibody : The labeled anti-mouse IgG Enhanced Double Antibody Sandwich- Indirect ELISA (DAS-I) : Biotinylated anti-body : Streptavidine-enzyme conjugates

35 F(ab') 2 Antibodies Production Papain cleavage: (Fc) Pepsin cleavage [F(ab)2]

36 F(ab') 2 Antibodies Production A system has been devised to carry out DAS-I using a single antiserum The antibodies are treated with the enzyme pepsin to remove the Fc portion of the molecule. The remaining F(ab') 2, fragment still has the antigen binding sites and will bind to the immunosorbent, but will not bind to protein A. The F(ab') 2 fragments are used as trapping antibody and the whole antibody is used as the intermediate antibody. It is possible to use specific substances that recognize Fc portion for increase the reaction sensitivity.