MDSC 321: Experimental Design

What are the first questions we ask when designing an experiment?

What am I trying to determine? Measure something (antibody - ELISA, gel, antigen - ELISA, Western gel)? Determine disease association or cause?

What are the main assays we use?

ELISA, Western Blot, Agglutination/Precipitation and Gel Diffusion

Walk through and ELISA experimental design.

1. Measure antigen levels (sandwich ELISA)

2. Different antibody - recognizes the same antigen but a different epitope

3. An antibody that recognizes the other antibody (called a secondary antibody) - this antibody is conjugated to a florescent molecule or enzyme that causes a colour change in the media

4. Measure antibody levels (direct ELISA)

5. Coat the dish with antigen

6. Add sample containing antibodies

7. Add detection antibody coupled to fluorescence or enzyme

How do Rapid Antigen Tests (RAT)/Lateral Flow Assays work?

Add analyte to sample pad, capillary flow towards test line and control line.

What can agglutination and precipitation do?

Method can detect very small & soluble antigens very rapidly. Relies on zone of equivalence.

Summary of ELISA to measure antibodies

Coat a plate with antigen, add blood, add secondary antibody (+ enzyme).

Summary of Agglutination to measure antibodies

Attach antigen to a cell, add serum.

Summary of Gel to measure antibodies

Add antigen to well, add plasma to another, look for precipitation.

Summary of ELISA to measure antigens

Coat plate with capture antibody, add sample, add detection antibody that recognizes a different epitope, add secondary (enzyme)

Summary of Western to measure antigen

Run sample on gel, transfer to membrane, stain with specific antibody, add secondary (enzyme)

Summary of Gel to measure antigen

Add sample (antigen) to well, add specific antibody to another, look for precipitation.