Presentation on theme: "Enzyme Linked Immunosorbent Assay (ELISA)"— Presentation transcript:
1Enzyme Linked Immunosorbent Assay (ELISA) By: Mr. Wael Laithi
2Definition :- Principle :- The enzyme-linked immunosorbent assay (ELISA) is a biochemical technique that uses antibodies and color change to identify a substance. ELISA) is one of the most sensitive and reproducible technologies available. These assays are rapid, simple to perform and easily automated.Principle :-ELISA combine the specificity of antibodies with the sensitivity of simple enzyme assays. using antibodies or antigens coupled to an easily-assayed enzyme. ELISA can provide a useful measurement of antigen or antibody concentration
3Indirect ELISA:Blocking (Competitive) Formatspecific sample antibodies compete with, or block, the enzyme-labeled-specific antibody in the conjugate.The addition of an enzyme substrate-chromogen reagent causes color to develop. This color is inversely proportional to the amount of bound sample antibody.The more antibodies present in the sample, the less color development in the test wells.2 steps of Antibody adding.
5The steps of "indirect" ELISA follows the mechanism below:- Antigens are coated in the wellsThe primary antibody is added, which binds specifically to the test antigen coating the well.A secondary antibody is added, which will bind the primary antibody. This secondary antibody often has an enzyme attached to it, which has a negligible effect on the binding properties of the antibody.A substrate for this enzyme is then addedThe color change shows the secondary antibody has bound to primary antibody.The higher the concentration of the primary antibody present in the serum, the stronger the color change. Often, a spectrometer is used to give quantitative values for color strength.A buffered solution of the antigen to be tested for is added to each well of a microtiter plate, where it is given time to adhere to the plastic through charge interactions.A solution of nonreacting protein, such as bovine serum albumin or casein, is added to well (usually 96-well plates) any plastic surface in the well that remains uncoated by the antigen.
6Sandwich ELISA (Direct) : Antigen-Capture (Direct) Format In the antigen-capture format, the antigen in the sample is sandwiched between antibodies coated on the plate and an enzyme-labeled conjugate.The antibody conjugate can be either monoclonal or polyclonal.The addition of an enzyme substrate-chromogen reagent causes color to develop. This color is directly proportional to the amount of the target antigen present in the samplePlate is coated with a capture antibody. Sample is added, and any antigen present binds to capture antibody.Detecting antibody is added, and binds to antigen.Enzyme-linked secondary antibody is added, and binds to detecting antibody.Substrate is added, and is converted by enzyme to detectable form.
8 Competitive ELISA:A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:Unlabeled antibody is incubated in the presence of its antigen (sample).These bound antibody/antigen complexes are then added to an antigen-coated well.The plate is washed, so that unbound antibody is removed. (The more antigens in a sample, the fewer antibodies will be able to bind to the antigen in the well, hence “competition”).The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme.A substrate is added, and remaining enzymes elicit a chromogenic or florescent signal.For competitive ELISA, the higher the sample antigen concentration, the weaker the eventual signal. The major advantage of competitive ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present.
11ELISA kit :- Micro-plate , coated plates (solid and/or strip plates), Sample diluent,Controls,Wash concentrate,Conjugate,Substrate andStop solution.NOTE: Do not mix or use components from different kit lot numbers.kit lot is optimized and manufactured to work as a unit.
12Coated Well: Is adsorbent and container, so do not react Coated Well: Is adsorbent and container, so do not react. There are lots of materials, usually polystyrene, that can be used in ELISA, polystyrene is strong in adsorbing protein.Its contain 96-well which has been coated with antigen or antibody.Conjugate enzyme: Conjugate is antibody linked with enzyme. The good conjugate possess not only catalytic activity of enzyme but also immunological competence of antibody. The conjugate must be favorable stability.Enzymes used in ELISA should meet requirements such as high purity, high conversion rate, favorable specificity, stable properties, rich resources, and remaining active component and catalytic capacity after becoming conjugate.There are many type of enzymes may be used : Horseradish peroxidase (HRP), widely used, Alkaline phosphatase (AP), β – Galactosidase and Urease.High purified IgG is usually used in conjugate in order to avoid the interference of other proteins when it is linked with enzyme.Conjugate is either ready to use or concentrated. (Need to dilute).
13Substrate : Contain substrate specific to conjugate enzyme and chromogenic material. Washing solution: PBS-Tween 20 the most used. Must be diluted 1/10 before using.Stop solution: Stop solution used to stop the reaction and to develop the color. H2SO4 is widely used as stop solution of HPR reaction. HCL and NaOH may be used.Occasionally diluent solution
14Equipment Maintenance and Calibration The maintenance and calibration of your laboratory equipment is extremely important in obtaining accurate and reproducible results.The Maintenance and Calibration Schedule should be used as a guideline. Adjust maintenance routines according to the amount of daily testing performed in your laboratory. Always refer to your equipment manufacturer’s guide for their recommendations.
15PipettesSingle-channel, fixed-volume and adjustable-volume (1–20 μL, 10–100 μL, 20–200 μL, etc.)Multichannel, 8- and 12-channelsSemi-automated dispensing units, Fully automated systems that can process multiple platesDilutorsSingle-channelMultichannelAutomated dispensing units
16ELISA Equipment Washer Systems ELISA Plate Readers Other Manual systems that wash one row or column at a timeSemi-automated systems that handle one strip or plate at a timeFully automated systems that can process multiple platesELISA Plate ReadersManual readers that read one row or well at a timeSemi-automated readers that read one plate at a timeFully automated systems that can process multiple plates simultaneouslyOtherH umidity chamber (not required for all ELISA tests)Plate sealers for assays that have long incubation times (to avoid evaporation)ELISA Equipment
18Toxoplasmosis Definition:- Transmission:- Toxoplasmosis is a disease of blood and lymph vessels that caused by a single-celled parasite called Toxoplasma gondii.Transmission:-The infection is most commonly acquired from contact with cats and their feces or with raw or undercooked meat.Cats and dogs are the essential part of the parasite life cycle.The disease is rather mild and undefined illness, associated with fever and lymphoadenopathy.The primary danger is in the congenital infection of the fetus, which is variable, depending upon the time of pregnancy.
19Infection in the first half of pregnancy may lead to:- Intrauterine death ( Abortion ).Microcephaly or hydrocephaly with intracranial calcification.Infection in the second half of pregnancy may lead to:-Fever.Hepatosplenomegaly and lymphoadenopathy.Jaundice at the time of birth.Blindness.Mental retardation.Convulsion.
20Diagnosis:- Serological tests Different serological tests often measure different antibodies that possess unique patterns of rise and fall with time after infection.A combination of serological tests is frequently required to establish whether an individual has been more likely infected in the distant past or has been recently infected.
21IgM Antibodies:An IgM test is used to help determine whether a patient has been infected recently or in the distant past.Because of the significant potential of misinterpreting a positive IgM test result, confirmatory testing should be performed. Despite the wide distribution of commercial test kits to measure IgM antibodies, these kits often have low specificity and the reported results are frequently misinterpreted. In addition, IgM antibodies can persist for months to more than one year.IgM antibodies are measured by the"double-sandwich" or "immuno-capture" IgM-ELISA method. This method avoids false positive results due to the presence of rheumatoid factor and antinuclear antibodies. This test is positive from the beginning of infection.
22 IgG Antibodies:IgG antibodies usually appear within 1 to 2 weeks of the infection, peak within 1 to 2 months, fall at variable rates, and usually persist for life. The titer does not correlate with the severity of illness.A positive test establishes that the patient has been exposed to the parasite. A negative test essentially rules out prior exposure to Toxoplasma gondii (unless the patient is hypogammaglobulinemic). However, in a small number of patients, IgG antibodies might not be detected within 2 to 3 weeks after the initial exposure to the parasite.
23IgA AntibodiesIgA antibodies may be detected in sera of acutely infected adults and congenitally infected infants using ELISA.As is true for IgM antibodies to the parasite, IgA antibodies may persist for many months to more than one year. For this reason they are of little additional assistance for diagnosis of the acute infection in the adult. In contrast, the increased sensitivity of IgA assays over IgM assays for diagnosis of congenital toxoplasmosis represents an advance in diagnosis of the infection in the fetus and newborn. In a number of newborns with congenital toxoplasmosis and negative IgM antibodies, the serological diagnosis has been established by the presence of IgA and IgG antibodies.
24IgE AntibodiesIgE antibodies are detectable by ELISA in sera of acutely infected adults, congenitally infected infants, and children with congenital toxoplasmosis.
25Polymerase Chain Reaction “PCR” Histological tests Immunofluorescence stainSabin field man stainHematoxylene and eosin stainIsolation of T. gondii.Isolation of T. gondii from blood or body fluids establishes that the infection is acute.