Section8:Assay Validation

From Assay Guidance Wiki

Assay Validation

1. Check all pipettors to ensure that each channel is precise and accurate.
2. Determine signal window. For statistical analysis, it is important to verify that well-to-well variation is minimal and that the signal window (i.e., difference between min and max values) is large enough to yield reproducible analyses. Perform the C-ELISA using the template provided below. Plate 1

   Row	C1	C2	C3	C4	C5	C6	C7	C8	C9	C10	C11	C12
   1	H	M	L	H	M	L	H	M	L	H	M	L
   2	H	M	L	H	M	L	H	M	L	H	M	L
   3	H	M	L	H	M	L	H	M	L	H	M	L
   4	H	M	L	H	M	L	H	M	L	H	M	L
   5	H	M	L	H	M	L	H	M	L	H	M	L
   6	H	M	L	H	M	L	H	M	L	H	M	L
   7	H	M	L	H	M	L	H	M	L	H	M	L
   8	H	M	L	H	M	L	H	M	L	H	M	L

   
   

   Plate 2

   Row	C1	C2	C3	C4	C5	C6	C7	C8	C9	C10	C11	C12
   1	L	H	M	L	H	M	L	H	M	L	H	M
   2	L	H	M	L	H	M	L	H	M	L	H	M
   3	L	H	M	L	H	M	L	H	M	L	H	M
   4	L	H	M	L	H	M	L	H	M	L	H	M
   5	L	H	M	L	H	M	L	H	M	L	H	M
   6	L	H	M	L	H	M	L	H	M	L	H	M
   7	L	H	M	L	H	M	L	H	M	L	H	M
   8	L	H	M	L	H	M	L	H	M	L	H	M

   
   

   Plate 2

   Row	C1	C2	C3	C4	C5	C6	C7	C8	C9	C10	C11	C12
   1	M	L	H	M	L	H	M	L	H	M	L	H
   2	M	L	H	M	L	H	M	L	H	M	L	H
   3	M	L	H	M	L	H	M	L	H	M	L	H
   4	M	L	H	M	L	H	M	L	H	M	L	H
   5	M	L	H	M	L	H	M	L	H	M	L	H
   6	M	L	H	M	L	H	M	L	H	M	L	H
   7	M	L	H	M	L	H	M	L	H	M	L	H
   8	M	L	H	M	L	H	M	L	H	M	L	H

   Where:

   • "H" is the maximum (HI) signal, "M" is the mid-level signal, and "L" is the minimum (LO) signal.
   • See the QB Handbook for definitions of each signal for each assay format.
   
   
   Edge effects, which are not uncommon in this type of assay, will be seen by comparing min and max values throughout the 96 well plate. Consistent deviations in rows or columns will be noted, and should be corrected or accounted for before validation proceeds. The signal window size is also calculated and should be ≥2.
3. Response Surface Design: Example of Experimental Design to Develop and Optimize a C-ELISA
   
   To optimize the assay using experimental design it is best to work directly with a statistician. They will help in setting up the design and with analysis of the data.

Experimental Factors

• HUVEC cells with ERK p42/p44 Antibody
• Serum Starve time (0 - 4 hrs)
• VEGF conc. (originally 10-100. Changed to 30-200)
• Cell Density (2.5 - 10 x103 cells)
• Antibody conc. (1:2000, 1:1000, 1:500)

Results from Response Surface Experiment

• Serum Starvation greater than 4 hours
• VegF conc. higher than 100 ng/ml
• Induction time less than 5 minutes
• Cell Density 20,00 cells per well

Variables Evaluated

• Plating density: Cells at 10 or 20,000 cells per well
• Antibodies: Two antibodies pKDR (996) and pERK p42/44
• Culture conditions: Serum starvation for 4 hours or overnight

Optimal Conditions Results

• HUVEC cells at 20,000 cells per well stimulated for 4 minutes at 150 ng/ml VEGF using 1:500 of pERK p42/44

Assay validation for IC50 determinations - Example from C-ELISA development

After optimal conditions are determined test control compounds to determine if IC50’s can be obtained in the assay.

Follow this with plate to plate and within plate variability using the plate validation templates. If the assay passes plate validation then proceed with analyzing compounds in a test retest assay. See section XII of the QB manual for test retest.