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Practical Immunology Tel:8462530; 8462468(lab) E-mail:[email protected] Weifang Medical University Test Seven Enzyme-linked Immunosorbent Assay Immediate hypersensitivity induced in guinea pig ELISA • Enzyme-linked immunosorbent assay, commonly known as ELISA, is a method which labels antigen or antibody with enzyme depends on an enzyme label. An enzyme conjugated with an antibody reacts with a colorless substrate to generate a colored reaction product. Such a substrate is called a chromogenic substrate. • A number of enzymes have been employed for ELISA including alkaline phosphatase, horseradish peroxidase, -galactosidase. • This method have the advantage of being safe and less costly. ELISA • ELISA allowing qualitative detection or quantitative measurement of either antigen or antibody, each type of ELISA can be used qualitatively to detect the presence of antibody or antigen. • Alternatively, a standard curve based on known concentrations of antibody or antigen is prepared, from which the unknown concentration of a sample can be determined. ELISA: Enzyme-Linked ImmunoSorbent Assay 96-well microtiter plate Run multiple tests on one plate Small volumes Use less of reagents Can use bound Ab to quantitate soluble antigen Can use bound antigen to quantitate serum Ab An ELISA plate • A number of variations of ELISA have been eveloped • Sandwich ELISA • competitive ELISA • ELISPOT assay • indirect ELISA Sandwich ELISA • Antigen can be detected by a sandwich ELISA. • In this method, the antibody is immobilized on a microtiter well. A sample containing antigen is added and allowed to react with the immobilized antibody.After the well is washed, a second enzyme-linked antibody specific for a different epitope on the antigen is added and allowed to react with the bound antigen. After any free second antibody is removed by washing, substrate is added, and the colored reaction product is measured. Competitive ELISA • Antibody is first incubated in solution with a sample containing antigen. The antigen and antibody mixture is then added to an antigencoated microtiter well. The more antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well. • The enzyme conjugated secondary antibody (Ab2) specific for the isotype of the primary antibody can be used to determine the amount of primary antibody bound to the well • In the competitive assay, the higher the concentration of antigen in the original sample, the lower the absorbance. ELISPOT ASSAY • ELISPOT assay allows the quantitative determination of the number of cells in a population that are producing cytokines. • In this method, the plates are coated with the antibody (capture antigen) • A suspension of the cell population under investigation is then added to the coated plates and incubated. The cells settle onto the surface of the plate, and secreted cytokines reactive with the antibodys • The plate is then washed and an enzyme-linked antibody specific for the secreted cytokines is added and allowed to bind, subsequent development of the assay by addition of a suitable chromogenic substrate reveals the position of each antigen-producing cell as a point of color . • • • a well is coated with antibody against the antigen, then a suspension of a cell population secreting the cytokine are layered onto the bottom of the well and incubated. Most of the cytokine secreted by a particular cell react with nearby well-bound antibodies. After the incubation period, the well is washed and an enzymelabeled anti-cytokine antibody is added. After washing away unbound antibody, substrate is added. The colored product precipitates and forms a spot only on the areas of the well where cytokine-secreting cells had been deposited. Indirect ELISA • Humoral as well as tissue constituents can be measured using indirect ELISA. In this method , antibody can be detected or quantitatively determined. Principle: • Serum or some other sample containing primary antibody (Ab1) is added to an antigen-coated microtiter well and allowed to react with the antigen attached to the well. After any free Ab1 is washed away, the presence of antibody bound to the antigen is detected by adding an enzyme-conjugated secondary antibody (Ab2), which binds to the primary antibody. Any free Ab2 then is washed away, and a substrate for the enzyme is added. • The result is measured by specialized spectrophotometric plate readers, which can measure the absorbance of all of the wells of a 96-well plate in seconds. Indirect ELISA Materials • Sheep erythrocytes(SRBC), rabbit antibody (Ab1), anti-rabbit IgG (labeled with enzyme, Ab2). • Washing fluid (PBS), substrate(OPD) for enzyme, 2mol / L H2SO4(stopping fluid). • 96 well assay plate, humidified chamber, icropipetter, tips. Method • • • • • • • To coat the plate with the appropriate antigen. Dump out the fluid into the sink and bang the plate upside down on some paper. Wash the unbound antigen with 200ul (PBS), Wash 3 times. 100ul negative fluid is added into the first well, 100ul rabbit antibody (anti-SRBC,Ab1) is added into the second well, then incubated at 37℃ for 45 minutes. Wash plate as above (3 times). 100ul enzyme conjugated IgG (E-Ab2) is added into two wells, then incubated at 37℃ for 30 minutes. Repeat washing step.100ul substrate for enzyme is added into two wells and then incubated for 20-30 minutes. One drop of stopping fluid is added into each well to cease the reaction, and the color reaction is estimated through naked eyes or spectrophotometer. Steps in the ELISA SRBC antigens pre-coated onto an ELISA plate ELISA plate wells already coated with SRBC antigen Steps in the ELISA Primary antibody (Ab1) serum added which contains antibodies to SRBC antigen Steps in the ELISA Primary antibody Variable domain binds antigen; constant domain free Unbound antibodies washed away Steps in the ELISA e e e e e e e e Secondary antibody Variable domain of 2 antibody binds constant domain of 1 antibody Steps in the ELISA e e e e e Secondary (2) antibody Unbound antibodies washed away Steps in the ELISA Chromagen or substrate added Addition of chromagenic substrate results in color reaction Color intensity increases with concentration of antibodies Darker color = higher titer Steps in the ELISA Primary (1) antibody Negative reaction: There are no antibodies present that bind SRBC antigen Steps in the ELISA Negative reaction : All primary antibodies washed away Steps in the ELISA e e e e e e e e Secondary (2) antibody Negative reaction : There are no primary antibodies for the secondary antibodies to bind Steps in the ELISA Negative reaction : All secondary antibodies washed away Steps in the ELISA Substrate added Negative reaction There is no enzyme to carry out the color reaction NO COLOR CHANGE Negative well Positive well Immediate hypersensitivity induced in guinea pig Principle • I hypersensitive reaction is induced by certain types of antigens referred to as allergens, However, an allergen induces a humoral antibody response by the same mechanisms as other soluble antigens, resulting in the generation of antibody secreting plasma cells and memory cells. • What distinguishes a type I hypersensitive response from a normal humoral response is that the plasma cells secrete IgE. • IgE binds with high affinity to Fc receptors on the surface of mast cells and basophils. Mast cells and basophils coated by IgE are said to be sensitized. A later exposure to the same allergen cross-links the embrane bound IgE on the sensitized mast cells and basophils, causing degranulation of these cells. The pharmacologically active mediators released from these granules act on the surrounding tissues. • These granules dissolve and release histamine serotonin and heparin and other mediators,they act on the smooth muscle and vascular endothelial cells,lead to allergic reaction,even shok and death. • We inject heterogenous protein (allergens ) to guinea pig, after 10-14 days, the IgE level in guinea pig rised and fix to the surface of mast cells and basophils, when the animal is exposed to the same antigen for the second time,the antigen seek out those cell-fixed IgE and react with them at the cell surface, an antigen moleculer combines with two antibody moleculers to form a bridge. • This bridging bring together two IgE receptor moleculers which can trigger an enzymy cascade reaction, this cascade reaction can cause mast cells and basophils release granules and lead to allergic reaction. However, the symptom of some guinea pig are quite slightly for the reason of individual difference, this is called desensitization. Crosslinking of FcεRI on the surface of mast cells by antigen and IgE causes mast-cell activation and degranulation. Materials • Guinea pig:three young healthy guinea pigs each weighs about 150g. • Antigen: horse serum • sterile injection syringes, needles, alcohol tampon. Method • Inject antigen for the first time: inject 0.5ml horse serum to in abdomen(two weeks ago) • Inject antigen for the second time: inject 1ml horse serum into guinea pig’s heart. • Observe the result after 1-5 minutes. Result • guinea pig appears excitement, scratching nose, singultus, spasm, hair standing, urinary and fecal incontinence,even death. Emphysema can be found after been dissected. Announcements • Heart-injection of guinea pig:. Antigen should be injected into guinea pig’s heart on the condition that countercurrent blood appears in the injection syringes. • One grasp and fix , expose the breast , the other one touch the 4,5,6 intercostal space of left hand side.select the site where heartbeat is most obviously. Test report • Principle, Method , Result • Indirect ELISA • Immediate hypersensitivity induced in guinea pig.