Phage display

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F2
F2's picture
Phage display

Dear firends
I 've done phage display and I am testing my clones by ELISA. When I was compairing NSB with specific binding ,NSB was too high ! Then I couldn't seperate any positive clones . Someone said to me that my phage titre is too high,is it truth?
What can I do for having better result in ELISA?

qiang wang
qiang wang's picture
i agree that probably the

i agree that probably the high NSB comes from too high titer.

I have not done ELISA on phage, but I do have done a lot ELISA on other staff. My tips to do good ELISA,

1. the most important thing is to find the proper concentration for both your material and the substrate, to do this, you need to do have serial dilution test

2. for the washing steps, I usually first fill the plate with washing buffer and dump immdiately, then do the washing for a longer time than discribed in protocol.

3. after dumping the liquid in the plate at every step, slam the plate on paper towel real hard to get rid of any trace of liquid

4. when adding anything to the plate, do it as fast as you can, in this assay, good results depends more on the equality than accuracy

5. avoid intercontamination between wells when dumpping the plate

Shubhangi
Shubhangi's picture
Hi

Hi
A very good way to reduce NSB is improving washing step.
Use high concentration of SDS in wash buffer than u r currently using.
i used to pipette updown after adding wash buffer for 5-6 times and then discard this also removes low affinity molecules. It worked well for me.

reza tohidkia
reza tohidkia's picture
Hi. I am intrested in

Hi. I am intrested in immunotherapy of gastric cancer with scFv. I am going to be screening a phage scFv library against a tumor associated antigen of gastric cancer. However i don't know how to earn a naive or synthetic human phage scFv library. Also does anyone know which non-immune human phage scFv library is appropriate for my study?