ELISA AND ELISPOT

 ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

·      It is an assay to determine the presence of certain Ag or Ab in a biological assay

·      Primary Ab binds directly to the target Ag. If the primary Ab is conjugated with a reporter enzyme that may alter the specificity of the Ab.

·      Secondary Ab binds the Fc (Fraction crystallizable) region of the primary antibody. This is favored because multiple secondary Ab can bind to the Fc region of a single primary Ab. This increases the sensitivity of the assay and amplifies the results.

·      How to produce secondary Ab in-vivo? – Inject a mouse with certain Ag, and the mouse will produce Abs against that Ag (Primary Ab), now inject the Abs invoked in the mouse into a goat. The goat’s immune system will produce Abs (secondary Ab) against the primary Ab.

·      The visualization of the assay is dependent on the color change of the chromogenic substrate on reaction with enzyme-linked with Abs. Some examples are as follows:

FIGURE 1


 

·      ELISA – DIRECT (SANDWICH ELISA, COMPETITIVE ELISA) & INDIRECT ELISA.

INDIRECT ELISA

·      The antigen from the patient’s serum is immobilized in the microtiter wall, primary Ab against the Ag is added, and the microtiter well is washed to remove unattached primary Ab.

·      Enzyme-linked Secondary Ab is added against primary Ab

·      Substrate added, check for color change

SANDWICH ELISA

·      Capture Ab is immobilized in the microtiter well, Ag from the patient’s serum is added, and washed to remove unattached free Ag.

·       Enzyme- labeled Detection Ab is added to the microtiter well.

·      Substrate added, check for color change

COMPETITIVE ELISA

·      Firstly, the patient’s serum is added with Ab against target Ag and incubated.

·      This Ag-Ab mixture is then added to a microtiter well that has immobilized Ag on its wall.

·      The more the Ag was present in the patient’s sample, the smaller number of Ab would be free to bind to the immobilized Ag on the microtiter well.

·      A secondary enzyme labeled Ab is added.

·      More Ag in the patient’s sample, the lesser intensity of color.

FIGURE 2



BIOTIN- STREPTAVIDIN ELISA

·      Abs are biotinylated. Biotin is a small molecule, that doesn’t affect the specificity of the antibody.

·      Streptavidin is conjugated with an enzyme

·      Streptavidin has the capability of binding 4 molecules of biotin

·      Cross-reactivity of secondary Ab is eliminated.

·      Resistant to pH and temperature change.

FIGURE 3



 

 

ELISPOT

·      ENZYME LINKED IMMUNO SPOT ASSAY

·      Similar to sandwich ELISA

·      Highly sensitive assay to detect Cytokine released from Ag stimulated T cells

·      Following immobilisation of capture Ab, T cells are added to check whether they have encountered Ag and are releasing Cytokines or not.

·      Detection Abs are added hence.

·      Substrate is added, and color change observed.

FIGURE 4

 


 

 

 

 

 

 

 

 

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