Enzyme-linked immunosorbent assay (ELISA) (lab 6)

ELISA is distinguished among antibody assays because it yields…………………………..

quantitative results.

The principle of ELISA.

It works on measuring the binding of antibodies to antigens. It involves an enzyme-linked antibody for detection of primary antibody binding.

Substrate added—→ becomes colored product through enzyme—→ seen by spectrophotometry.

What’s the difference between primary and secondary detection antibody?

Primary detection antibody only binds to protein (antigen) of interest.

Secondary detection antibody binds to primary antibody and is enzyme-conjugated .

Steps of ELISA immunoassay.

• Coating (with Ab or Ag)

• Sample addition (of either)

• Washing

• Detection

• Final read

Types of ELISA.

1. Direct Elisa

2. Indirect ELISA

3. Sandwich ELISA

4. Competitive ELISA

Describe direct ELISA.

Antigen-coated plates—→Add primary detection antibody with conjugated alkaline phosphate (AP) or horse radish peroxidase (HRP)—→ wash—→ Substrate/chromophore added—→colour change (if AP—→ hydrolysis of phosphate gp. If HRP—→ substrate is oxidized)

Advantages and disadvantages of direct ELISA.

Advantages: eliminating secondary antibody cross reactivity, and due to fewer steps, it is rapid compared to indirect ELISA

Disadvantages: low sensitivity and high costs.

Describe indirect ELISA.

Antigen coating wells—→ sample with primary antibodies is added—→ wash—→another sample with secondary Ab conjugated with enzyme is added—→ bind to primary Ab—→ wash—→ substrate added gives coloured product

Describe sandwich ELISA.

Antibody coated well—→sample with antigen is added—→wash—→ secondary Ab with conjugated enzyme is added—→binds to another part of antigen—→ wash—→substrate added becomes a coloured product.

Describe competitive ELISA for detecting antigen in sample (format 3).

It involves an initial Ag-Ab interaction that leaves some Ab free. This solution is added to an antigen coated plates where the free Ab binds and then washing. A secondary enzyme-conjugated Ab is added and continue as rest.

ELISA applications.

1. Determining presence of Ab or Ag in sample.

2. In food industry to detect allergens.

3. Determine serum concentration of Ab

4. Diagnosis of diseases such as Ebola, pernicious anaemia, AIDS.

5. Evaluate disease outbreak such as COVID-19.

Advantages of ELISA:

• For accurate diagnosis of disease

• Carried out for complex samples

• Rapid test

• Relatively easy and uncomplicated process