E nzyme L inked I mmuno - PowerPoint Presentation

Slide1

E

nzyme

L

inked

I

mmuno

s

orbent

A

ssay

(ELISA

)

Slide2

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

Slide3

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.

Slide4

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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.

Slide6

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

.

Slide7

Slide8

 

 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.

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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.

Slide12

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).

Slide13

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. H

2

SO

4

is widely used as stop solution of HPR

reaction. HCL

and

NaOH

may be used

.

Occasionally diluent solution

Slide14

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.

Slide15

Pipettes

Single-channel, fixed-volume and adjustable-volume (1–20

μL

,

10–100

μL

, 20–200

μL

, etc.)

Multichannel, 8- and 12-channels

Semi-automated dispensing units, Fully automated systems that can process multiple plates

Dilutors

Single-channel

Multichannel

Automated dispensing units

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Washer

Systems

Manual systems that wash one row or column at a time

Semi-automated systems that handle one strip or plate at a time

Fully automated systems that can process multiple plates

ELISA

Plate

Readers

Manual readers that read one row or well at a time

Semi-automated readers that read one plate at a time

Fully automated systems that can process multiple plates simultaneously

Other

H

umidity

chamber (not required for all ELISA tests)

Plate sealers for assays that have long incubation times (to avoid evaporation)

ELISA

Equipment

Slide17

Toxoplasmosis

Slide18

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.

Slide19

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.

Slide20

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

.

Slide21

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:

Slide22

 

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.

Slide23

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

Slide24

IgE

Antibodies

IgE

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

Slide25

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.

Slide26

Thank

you