L9 Flashcards
1- Complement bending reaction mechanism?
Complement fixation is the classic method for demonstrating the presence of antibodies in serum. This method has two components. The first component is an indicator system consisting of a combination of sheep erythrocytes; a complement-fixing antibody produced against sheep erythrocytes in another animal; and an exogenous source of complement, usually guinea pig serum. When these three components are combined at the optimum concentration, hemolysin, an anti-sheep cell antibody, can bind to the surface of red blood cells. Complement can subsequently bind to this antigen-antibody complex and cause cell lysis.
— The second component consists of a known antigen and patient serum, which are added to a suspension of sheep erythrocytes, hemolysin, and a complement. The two components of the complement fixation procedure are tested in sequence. Patient serum is first added to the known antigen, and complement is added to the solution. If the serum contains antibody to the antigen, the resulting antigen antibody complexes will bind all of the complement. Sheep red cells and hemolysin are then added. If complement has not been bound by an antigen antibody complex formed from the patient serum and known antigen, it is available to bind to the indicator system of indicates both a lack of antibody and a negative complement fixation test.
— If the patient’s serum does contain a complement fixing antibody appositive result will be demonstrated by the lack of haemolysis
- Immunfluorescence: mechanisms of direct and indirect methods
- The fluorescent techniques are extremely specific and sensitive. This technique consists of labeling antibody with fluorescein isothiocyantate, a fluorescent compound with an affinity for proteins to form a complex, conjugate
- Its consistent of 2 method
-direct method :
Direct Immunofluorescent Assay In this technique, Fluorescein- conjugated antibody is used to detect antigen antibody reactions. This method can be applied to the detection of hepatitis B virus & chlamydia. A fluorescent microscope is required to observe the production of color
-result : fluorescein gives a yallow-green light
-indirect method
In the indirect immunofluorescent assay, the antigen source to the specific antibody being tested is fixed to the surface of a microscopic slide. The patent’s serum is diluted and placed on the slide to cover the antigen source. If antibody is present in the serum , it will bind to its specific antigen unbound antibody is then removed by washing the slide, finally antihuman globulin conjugated to a fluorescent substance that will fluoresce when exposed to a fluorescent substance that will fluoresce when exposed to ultraviolet light is placed on the slide. This conjugated marker of human antibody will bind to the antibody already bound to the antigen on the slide and will serve as a marker for the antibody when viewed under a fluorescent microscope
- Enzyme-linked analyses: direct ELISA, indirect ELISA. sandwich ELISA and competitive ELISA mechanisms
-In a direct ELISA, an antigen or sample is immobilized directly on the plate and a conjugated detection antibody binds to the target protein. Substrate is then added, producing a signal that is proportional to the amount of analyte in the sample.
Indirect ELISA is a technique that uses a two-step process for detection, whereby a primary antibody specific for the antigen binds to the target, and a labeled secondary antibody against the host species of the primary antibody binds to the primary antibody for detection.
- Antibody detection by ELISA (competitive ELISA) The antigen-coated reagent wells of the microplate are incubated with diluted patient samples. If a sample contains specific antibodies directed against the antigen, these bind to the antigen-coated reagent wells and inhibit the binding of a biotin-labelled artificial molecular which is added to the reagent well in a subsequent step
- Antigen detection by ELISAS (sandwich ELISA) The reagent wells of the microplate coated with monospecific antibodies are incubated with diluted patient samples. If the sample contains the respective antigens, these bind to the antibody-coated reagent well.
- Radioimmunoassay: classical method
In radioimmunoassay, radioisotopes can be used to measure the concentration of antigen or antibody in serum sample. If antibody concentration is being measured, radioactive labeled antibody competes with patient unlabeled antibody for binding sites on a known amount of antigen. The main advantage of the radioimmunoassay method is the extreme sensitivity and ability to detect trace amounts. Of antigen or antibody. In addition, a large number of tests can be performed in a relatively short time period. The disadvantage is the hazards and instability of isotopes
- Immunoblotting (Western blot)
In the immunoblot, antigens coated on membranes are used as a solid phase in order to detect specific antibodies in patient samples. The test performance is either manual, semi- or fully automated. If a sample contains specific antibodies, these bind to the membrane-bound antigens. In the next step, an alkaline phosphatase (AP) labelled antibody (conjugate) is added, which binds to the specific antibodies. The alkaline phosphatase catalyses a colour reaction with the subsequently added nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP). If specific antibodies are present in the patient sample, a dark line appears at the respective antigen position. The intensity of the resulting staining is proportional to the antibody concentration in the sample
