ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

By: Mr. Wael Laithi

Definition :-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

Indirect ELISA: Blocking (Competitive) Format

• specific 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.

The steps of "indirect" ELISA follows the mechanism below:-• Antigens are coated in the wells• The 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 added• The 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.

Sandwich 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 sample• Plate 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.

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.

ELISA kit :- • Micro-plate , coated plates (solid and/or strip plates),• Sample diluent, • Controls, • Wash concentrate, • Conjugate, • Substrate and • Stop solution.

• NOTE: Do not mix or use components from different kit lot numbers.kit lot is optimized and manufactured to work as a unit.

• 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).

• Substrate : 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

Equipment 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.

• PipettesSingle-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 plates

• DilutorsSingle-channel MultichannelAutomated dispensing units

• Washer SystemsManual 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 plates

• ELISA Plate ReadersManual readers that read one row or well at a timeSemi-automated readers that read one plate at a time Fully automated systems that can process multiple plates simultaneously

• OtherH umidity chamber (not required for all ELISA tests)Plate sealers for assays that have long incubation times (to avoid evaporation)

• ELISA Equipment

TOXOPLASMOSIS

By: Mr. Wael Laithi

Toxoplasmosis• Definition:-

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.

• Infection 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.

Diagnosis:-

• 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.

• 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.

IgM Antibodies:

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.

• IgA 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.

IgA Antibodies

IgE Antibodies

• IgE antibodies are detectable by ELISA in sera of acutely infected adults, congenitally infected infants, and children with congenital toxoplasmosis.

•Polymerase Chain Reaction “PCR”

•Histological tests• Immunofluorescence stain• Sabin field man stain• Hematoxylene and eosin stain

• Isolation of T. gondii.• Isolation of T. gondii from blood or body fluids establishes that

the infection is acute.

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