GU General Microbiology - Lecture 12 PDF

Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 General Microbiology & Immunology Course Code: PMB201 Lecture #12 Immunologically mediated diseases...

Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 General Microbiology & Immunology Course Code: PMB201 Lecture #12 Immunologically mediated diseases Serology Dr. Mariam Hassan Haikal, PhD [email protected] [email protected] @MariamHassanH 12/6/24 PMB201: Microbiology Lecture 12 1 1 Lecture #12 Objectives By the end of this lecture, you should be familiar: v Immunologically mediated diseases Ø Hypersensitivity Ø Graft rejection Ø Autoimmune diseases Ø Immune deficiency v Serological reactions: v Different techniques for measuring Ag/Ab in-vitro 1- Fluorescent-Antibody Techniques 2- Enzyme-Linked Immunosorbent Assay (ELISA) 3- Western Blot 12/6/24 PMB201: Microbiology Lecture 12 2 2 1 Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 Types of grafts Types of grafts A)Permanents B) Rejected Autograft: tissue Allograft: tissue from the same transplanted individual between transferred from genetically one anatomic site different members to another. of the same species. Isograft: tissue Xenograft: tissue from a genetically transplanted identical between different individual. species. 12/6/24 PMB201: Microbiology Lecture 12 3 3 Graft rejection There are two types of graft rejection a) First set response: occur about 10 days. b) Second set response: occur after 7 days. Cellular mechanism, probably delayed hypersensitivity is the main mechanism responsible for the rejection. 12/6/24 PMB201: Microbiology Lecture 12 4 4 2 Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 Autoimmune diseases Natural tolerance develops during embryonic life. Self-reactive clones are eliminated as soon as they arise. Reappearance of these clones will lead to autoimmune diseases 12/6/24 PMB201: Microbiology Lecture 12 5 5 Etiology of autoimmune diseases 1- Failure of tolerance mechanism 2-Release of sequestered (inaccessible, hidden) antigens 3- Alteration of self-Ag to a foreign one. This may occur by: Physical agents as X-rays or UV light. Chemicals or drugs such as penicillin. Infection (specially viral infections) such as mumps 4- Exposure to microbial antigens that cross-react with self-Ag 12/6/24 PMB201: Microbiology Lecture 12 6 6 3 Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 The mechanism of autoimmune diseases Hypersensitivity Type II: autoimmune hemolytic anemia Type III: Rheumatoid arthritis & systemic lupus erythematosus Type IV: Ulcerative colitis 12/6/24 PMB201: Microbiology Lecture 12 7 7 Classification & spectrum of auto-immune diseases I- Organ-specific diseases: II- Non-organ-specific diseases: Auto-immune hemolytic anemia : RBCs Rheumatoid arthritis: IgM cross react with IgG in joints Pernicious anemia: Gastric cells Systemic lupus erythematosus: Abs against nuclear Ags Hashimoto thyroiditis : thyroid proteins 12/6/24 PMB201: Microbiology Lecture 12 8 8 4 Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 Laboratory diagnosis —Elevated level of serum Igs. —Complement level may be decreased. —Auto-antibodies can be detected in serum e.g. rheumatoid factor. —Testing for antibodies specific to the particular Ag involved in organ specific diseases e.g. anti-thyroid antibodies. —Biopsy from organs may show immune complex deposition or lymphocyte infiltration. Management Use of anti-inflammatory drugs & immuno-suppressive drugs e.g. azathioprine. Plasma exchange may be of value in combination with anti-mitotic drugs. 12/6/24 PMB201: Microbiology Lecture 12 9 9 Immune deficiency I-Congenital (Primary): C- Severe combined immunodeficiency A- B-cell deficiency: syndrome (SCID): X-linked Lack of both B and T lymphocytes hypogammaglobulinaemia (fetal within 1 year) Partial defect in immunoglobulins Treated by bone marrow transplant Treated by human pooled gamma globulin D- Phagocytic dysfunction B- T-cell deficiency: Chronic granulomatous disease Digeorge’s syndrome (CGD) -> No oxidase enzyme so no H2O2 Results in severe infections with viral, fungal, and intracellular bacteria E- Complement deficiency Treated by thymic graft or Repeated infection with pyogenic administration of thymic hormones cocci 12/6/24 PMB201: Microbiology Lecture 12 10 10 5 Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 II- Secondary or acquired immunodeficiency: Drugs: immunosuppressive or cytotoxic drugs (in case of organ transplant or cancer) Infections: e.g. HIV (AIDS), or tuberculosis Malignancies: e.g. leukemia Diseases: e.g. diabetes Age: premature birth and elderly. 12/6/24 PMB201: Microbiology Lecture 12 11 11 Serological reactions Serology is the study of the reactions Conversely, by means of known between the antigens and antibodies. antibodies, the various antigens of microorganisms (isolated from patient) Serological reactions can be used to that characterize it, can be identified identify "Ag" or "Ab", if either of these reagents is known. This is particularly valuable in the diagnosis of infections (diseases), or of They can be also used to estimate the certain forms of hypersensitivity. relative quantity of either "Ag" or "Ab", by keeping one reagent constant and N.B.- The type of "Ag"-"Ab" reaction diluting the other. depends largely on the physical state of the antigen. Therefore, the level or titer of antibodies in the serum can be determined by means of known antigen. 12/6/24 PMB201: Microbiology Lecture 12 12 12 6 Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 1- Precipitation Reactions When a soluble Ag reacts with a specific Ab, they give a precipitate. This reaction is specific, reversible and requires the proper concentration of Ag and Ab for precipitation to occur (equivalence point). Small size lattice, which will not be visible, will be formed in excess Ag or Ab. 12/6/24 PMB201: Microbiology Lecture 12 13 13 Precipitation reactions: o At low Ag o At optimal concentrations, concentration of Ag, extensive cross- there is excess Ab linking occurs and and very little cross- forms a large linking occurs. precipitate little or no § The precipitate (Ag- free Ag or Ab will be Ab complex) is small left in the and the supernatant supernatant fluid. fluid (contain o At high concentration unbound reagents) of Ag, again little has a large cross-linking takes concentration of Ab. place. 12/6/24 PMB201: Microbiology Lecture 12 14 14 7 Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 Methods used in precipitation: — 1- Slide precipitation test. — 2- Tube precipitation test: Simply can be done by slowly layering small volume of soluble Ag over the Ab solution, precipitation will occur as a ring at the interface. 12/6/24 PMB201: Microbiology Lecture 12 15 15 2- Agglutination Tests: Agglutination reactions involve either particulate antigens (particles such as cells that carry antigenic molecules) or soluble antigens adhering to particles. These antigens can be linked together by antibodies to form visible aggregates, a reaction called agglutination They are very sensitive, relatively easy to read They are classified as either direct or indirect. 12/6/24 PMB201: Microbiology Lecture 12 16 16 8 Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 Direct Agglutination Tests ØThey detect antibodies against relatively large cellular antigens, such as those on red blood cells, bacteria, and fungi. Bacterial Agglutination Test: They are used routinely in many laboratories for diagnosis of infectious diseases and identifying bacterial isolates. — When antibodies react with epitopes on antigens carried on neighboring cells, such as bacterial cells, the particulate antigens (cells) agglutinate. — IgM, the most efficient immunoglobulin for agglutination, but IgG also participates in agglutination reactions. 12/6/24 PMB201: Microbiology Lecture 12 17 17 Measuring antibody titer with the direct agglutination test They are usually done in plastic microtiter plates that contain many shallow wells. The amount of particulate antigen in each well is the same, but the amount of serum that contains antibodies is diluted, so that each successive well has half the antibodies of the previous well. The titer is the reciprocal of the dilution in the last positive well 12/6/24 PMB201: Microbiology Lecture 12 18 18 9 Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 Ø Each well in this microtiter plate contains, from left to right, only half the concentration of serum that is contained in the preceding well. Each well contains the same concentration of particulate antigens, Agglutinated (Positive) Non-agglutinated (Negative) Ø The antibody titer is 160 because the well with a 1:160 concentration is the most dilute concentration that produces a positive reaction. 12/6/24 PMB201: Microbiology Lecture 12 19 19 Passive Agglutination Tests Antibodies against soluble antigens or can be detected by agglutination tests In reverse, by using particles coated if the antigens are attached onto with antibodies to detect the antigens particles. The antibody reacts with the against which they are specific attached antigen Reaction in a Reaction in a positive positive test test for antibodies. for antigens. When particles (latex When beads here) are particles are coated with antigens, coated with agglutination antibodies, indicates the agglutination presence of indicates the antibodies, such as presence of the IgM shown here antigens. 12/6/24 PMB201: Microbiology Lecture 12 20 20 10 Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 Hemagglutination Direct Hemagglutination Test Blood typing. Rh Typing oIt is a slide o The Rh (Rhesus) blood gp Ag is agglutination test. present in RBC of 85% of human oIt is based on the beings who are called Rh+ve. fact that RBCs o To detect Rh, put a drop of have one or two person’s blood on slide against Ags (glycolipids) anti-Rh, if agglutination occurs it called A and B indicates that the person is Ags on their Rh+ve. plasma membrane. 12/6/24 PMB201: Microbiology Lecture 12 21 21 Indirect Hemagglutination Test: They are widely used in clinical microbiology to diagnose viral and other infectious diseases. The procedure is based on the fact that many Ags, such as proteins and polysaccharides can be adsorbed (attached) to RBCs. Thus, the RBCs serve as an easily observed indicator system for serological reactions. 12/6/24 PMB201: Microbiology Lecture 12 22 22 11 Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 Indirect Hemagglutination Test: When RBCs suspended in a If these RBCs are agglutinated fluid such as physiological by a serological reaction saline are left undisturbed, they between Ab and Ags adsorbed settle to the bottom of the test to the surface of the RBCs, the tube forming a distinct red button does not form. button. Instead, a diffuse layer of RBCs is formed in the bottom of the tube. 12/6/24 PMB201: Microbiology Lecture 12 23 23 Different techniques for measuring Ag/Ab in-vitro 1- Neutralization Reactions Neutralization is an antigen–antibody reaction in which antibodies block the harmful effects of a bacterial exotoxin or a virus. The neutralizing substance, which is called an antitoxin, is a specific antibody produced by a host as it responds to a bacterial exotoxin or its corresponding toxoid (inactivated toxin). The antitoxin combines with the exotoxin to neutralize it 12/6/24 PMB201: Microbiology Lecture 12 24 24 12 Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 A-Toxin neutralization B-Viral neutralization test: Virus + susceptible cell culture = cell destruction (cytopathic effect) Serum antibody + virus + target cell= no CPE. 12/6/24 PMB201: Microbiology Lecture 12 25 25 2- Complement-Fixation Reactions During most antigen–antibody reactions, a complement serum protein binds to the antigen–antibody complex and is used up, or fixed. This process of complement fixation can be used to detect very small amounts of antibody. The complement-fixation test requires great care and good controls. This is one reason why newer, simpler tests are increasingly replacing it. 12/6/24 PMB201: Microbiology Lecture 12 26 26 13 Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 The complement fixation The indicator stage stage Negative test. No Positive test. All available antigen–antibody complement is fixed by the antigen–antibody reaction; reaction occurs. The no hemolysis occurs, so complement remains the test is positive for the free, and the red blood cells are lysed in the presence of antibodies. indicator stage, so the test is negative. 12/6/24 PMB201: Microbiology Lecture 12 27 27 Different techniques for measuring Ag/Ab in-vitro 1- Fluorescent-Antibody Techniques Fluorescent-antibody (FA) techniques can identify microorganisms in clinical specimens and can detect the presence of a specific antibody in serum. These techniques combine fluorescent dyes with antibodies that fluoresce when exposed to ultraviolet light. These procedures are quick, sensitive, and very specific. 12/6/24 PMB201: Microbiology Lecture 12 28 28 14 Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 A- Direct fluorescent antibody (FA) tests The specimen is fixed onto a slide. Fluorescein-labeled antibodies are then added, incubated briefly, and washed to remove any unbound antibodies. Yellow-green fluorescence under the fluorescence microscope from the bound antibody will be visible even if the antigen, such as a virus, is submicroscopic in size. 12/6/24 PMB201: Microbiology Lecture 12 29 29 B- Indirect fluorescent-antibody (FA) tests They are often more sensitive than direct tests. A known antigen is fixed onto a slide, and then the serum added. If antibody that is specific to that microbe is present, it reacts with the antigen to form a bound complex. Fluorescein-labeled anti-human immune serum globulin (anti-HISG), is added to the slide. Anti-HISG will be present only if the specific antibody has reacted with its antigen 12/6/24 PMB201: Microbiology Lecture 12 30 30 15 Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 C- Fluorescence activated cell sorter (FACS) This technique can be used to separate different classes of T cells. For example, a fluorescence- labeled antibody reacts with the CD4 molecule on a T cell. 12/6/24 PMB201: Microbiology Lecture 12 31 31 2- Enzyme-Linked Immunosorbent Assay (ELISA) The most widely used group of tests. They are sensitive and require little interpretive skill to read. Results are highly automated The direct ELISA detects antigens, and the indirect ELISA detects antibodies. 12/6/24 PMB201: Microbiology Lecture 12 32 32 16 Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 Direct ELISA Indirect ELISA 12/6/24 PMB201: Microbiology Lecture 12 33 33 3- Western Blot: This test is typically used to determine whether a positive result in a screening immunologic test is a true- positive or false-positive result. For example, patients who are positive in the screening ELISA for HIV infection should have a western blot performed. 12/6/24 PMB201: Microbiology Lecture 12 34 34 17 Mariam Hassan Haikal, Ph.D. GU General Microbiology – Lecture 12 3- Western Blot: - In this test, HIV proteins are separated - If antibodies are present, they bind to electrophoretically in a gel, resulting the viral proteins. in discrete bands of viral protein. - Bound antibodies can be detected by - These proteins are then transferred adding antibody to human IgG labeled from the gel i.e. blotted, onto a with either radioactivity or an enzyme, membrane and the person’s serum is which produces a visible color added. change when the enzyme substrate is added. 12/6/24 PMB201: Microbiology Lecture 12 35 35 Summary By now, you should be familiar with: v Graft rejection v Autoimmune diseases v Immune deficiency v Serological reactions: v Fluorescent-Antibody Techniques v Enzyme-Linked Immunosorbent Assay (ELISA) v Western Blot 12/6/24 PMB201: Microbiology Lecture 12 36 36 18