PATIENT SAFETY & EARLY INTERVENTION AND MANAGEMENT

2. • ELISA - Elisa is a laboratory technique method it is an immunological
assay commonly used to measure antibodies, antigens, proteins and
glycoproteins in biological samples .
WHY ELIISA ?
1. Antigen/antibody of interest is absorbed on to plastic surface
(sorbent).
2. Antigen is recognised by specific antibody ( immuno ).
3. This antibody is recognised by second antibody ( immuno ) which
has enzyme attached (enzyme-linked).
4. Substrate reacts with enzyme to produce product, usually
coloured.
BESIC TERM USES OF ELISA-
1. SOLID PHASE.
2. ADSORPTION.
3. WASHING.
4. ANTIGENS.
5. ANTIBODIES.
6. ENZYME CONJUGATE.
7. CHROMOGEN.
8. STOPPING.
9. READING.

3. EQUIPMENTS OF ELISA
1. MULTI CHANEL PIPETTE
2. WASHING DEVICE
3. MICROWELL PLATE
4. MICROPLATE WASHER
5. ELISA READER ( PHOTOMETER )
6. INCUBATOR
REAGENTS OF ELISA
1. HORSERADISH PEROXIDASE (HRP)
2. ALKALINE PHOSPHATASE (AP)

4. ANTIGEN ANTIBODY REACTION
Enzyme-linked immunosorbent assay (ELISA)
ELISAs detect and amplify antigen–antibody reactions by using
covalently bound enzyme–antibody molecules. The presence of the
enzyme (indicating presence of the antigen) is detected by the addition
of the appropriate substrate.
The reactions between Ag and Ab occur in three stages-
• In the first stage reaction involves formation of Ag and Ab complex.
• The second stage leads to visibile events like precipitation,
agglutination etc.
• The third stage includes destruction of Ag or its neutralization.
EACH ANTIBODY BINDS TO A SPECIFIC ANTIGEN; AN INTERACTION
SIMILAR TO A LOCK AND KEY

5. TYPES OF ELISA
• DIRECT ELISA
• INDIRECT ELISA
• SANDWICH ELISA
• COMPETITIVE ELISA

6. WHAT IS DIRECT ELISA ?
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 .
PROCIDURE OF DIRECT ELISA -
1. Apply a sample of known antigen to a surface, often in the well of a microtitre plate .
The antigen is fixed to the surface to render it immobile.
2. The plate wells or other surface are then coated with blocking buffer.
3. Detecting antibody ( labelled by an enzyme ), usually diluted in blocking buffer, is
applied to the plate for binding to the antigen coated on the plate .

7. WHAT IS INDIRECT ELISA ?
Indirect elisa is a two-step binding process involving the use of a primary antibody and a
labeled secondary antibody. In this method, the primary antibody is incubated with the
antigen-coated wells. Next, a labeled secondary antibody that recognizes the primary
antibody is added .
PROCEDURE OF INDIRECT ELISA –
1. Micro-well plates are incubated with antigens, washed up and blocked with BSA.
2. Samples with antibodies are added and washed.
3. Enzyme linked secondary antibody are added and washed.
4. A substrate is added, and enzymes on the antibody elicit a chromogenic or
fluorescent signal.

8. WHAT IS SANDWICH ELISA ?
The Sandwich Enzyme-Linked ImmunoSorbent Assay (ELISA) is a
sensitive and robust method which measures the antigen
concentration in an unknown sample. The antigen of interest is
quantified between two layers of antibodies: the capture and the
detection antibody.
PROCEDURE OF SANDWICH ELISA -
The steps are as follows:
1. Prepare a surface to which a known quantity of capture antibody is
bound.
2. Block any nonspecific binding sites on the surface.
3. Add antigen-containing sample to the plate.
4. Wash the plate, so that unbound antigen is removed.
5. A specific antibody is added, and binds to antigen (hence the
‘sandwich’: the Ag is stuck between two antibodies);
6. Add enzyme-linked secondary antibodies as detection antibodies
that also bind specifically to the antibody’s Fc region (non-specific).
7. Wash the plate, so that the unbound antibody-enzyme conjugates
are removed.
8. Add substrate that is converted by the enzyme into a color or
fluorescent or electrochemical signal.
9. Measure the absorbeance or fluorescence or electrochemical signal
(e.g., current) of the plate wells to determine the presence and
quantity of antigen.

9. WHAT IS COMPETITIVE ELISA ?
Competitive elisa ​​​also known as inhibition elisa or competitive immunoassay, this
assay measures the concentration of an antigen by detection of signal interference.
Each of the previous formats can be adapted to the competitive format.
PROCEDURE OF COMPETITIVE ELISA –
1 . These bound antibody/antigen complexes are then added to an antigen-coated
wall.
2. The plate is washed, so unbound antibodies are removed.
3. The secondary antibody, specific to the primary antibody, is added. This second
antibody is coupled to the enzyme.
4. The substrate is added, and remaining enzymes elicit a chromogenic or
fluorescent signal.
5. The reaction is stopped to prevent eventual saturation of the signal.

10. APPLICATIONS OF ELISA
• Serum Antibody concentrations.
• Detecting potential food allergense ( milk, peanuts,
walnuts, almonds and eggs ).
• Disease outbreaks – tracking the spread of disease e.g.
HIV, birdflu, cholera etc .
• Detection of antigens
e.g. pregnancy hormones, drug
allergen, GMO, mad cow disease.
•Detection of antibodies in blood sample for past exposure
to disease
e.g. Lyme disease, trichinosis, HIV, bird flu .