Question

Q1. You are developing an assay for HTS. Your positive control has a value (all arbitrary units) of 10 and a standard deviation of 1, your negative control has a value of 3 and a standard deviation of 0.5. What is your Z-factor and would you recommend this assay to be run as assay in a HTS campaign?

Q2. a) Describe the key steps of a typical directed evolution experiment

b) Describe/name two techniques to create variant libraries for such an experiment

c) Explain (judge) if the two methods you described can be used to generate a variant library when starting from 1 allele only.

Q3. The Lead identification phase is the drug discovery phase during which the selected hits from the Hit identification are further explored. During this phase, new compounds will be synthesized and tested in a cyclic process often referred to as DMTA cycle. What does DMTA stand for? Explain shortly what is done in each step of this cycle.

Q4. Think about the up-stream process of producing a biopharmaceutical protein. What factors can influence (what can you work with) the yield of our product?

Answer 1

The Z factor is used to measure the assay quality. It shows the separation between the distributions of the positive and negative controls.

Z-factor is calculated as follows:  

Z factor = 1 – [{3* (σp + σn)}/ I μp - μn I]

σp   is the standard deviation of the positive control = 1

σn   is the standard deviation of the negative controls = 0.5

μp is the mean of the positive control = 10

μn is the mean of the negative control = 3

Putting the values in the equation we got,

Z factor = 1 – [{3* (σp + σn)}/ I μp - μn I]

Z factor = 1 – [{3* (1 + 0.5)}/ I 10 - 3 I]

Z factor = 1 – [{3* 1.5}/ I 7 I]

Z factor = 1 – [4.5/ 7]

Z factor = 1 – 0.64

Z factor = 0.36

To interpret the Z-factor, these guidelines are used:

I. Z-factor is 1, ideal.

II. Z-factor between 0.5 and 1.0 is an excellent assay.

III. Z-factor between 0 and 0.5 is marginal, may be acceptable.

IV. Z-factor less than 0 means that the signal from the positive and negative controls could overlap, making the assay not very useful or screening purposes.

The Z score of HTS is 0.36, which is a marginal value. It is not a good assay. The assay needs some modification to makes the Z-factor ranges between 0.5 and 1.0. It is not an ideal assay to be run as assay in a HTS campaign.

Kindly revert for any query and concern.