Day 1: Selection of nanobodies against a recombinant target antigen
Principle:
The binding of nanobody-expressing phages to a target antigen immobilised onto a
solid support permits the selection of the gene encoding the specific nanobody. The
procedures and protocols are nicely described by Marks et al J Mol Biol. 1991. This
protocol is for the selection of EGFR nanobodies.
The first steps have already been performed to save time:
 Coat Maxisorp wells with 1:2000 rabbit anti-human Fc (# 38) in PBS; 100l
per well, incubate 8 hours at 4oC
 Wash wells three times with PBS
 Block with 200l 2% (w/v) milk in PBS (MPBS) for 30 minutes at RT
shaking. All further incubations are performed in this buffer.
 Capture the EGFR ECD-FC fusion incubating with 100l of a 1:10 dilution
of EGFR ECD-FC (0.1 mg/ml stock) in MPBS overnight at 4oC
START
Before you start, take a good look at the plate. What can you find in the three
wells?
 Wash the wells 3x with MPBS
 Mix phages with MPBS in an eppendorf tube: per well you need 100 μl
phages + 100l of 4% MPBS (for 3 wells, take 300 μl phages and 300 μl
MPBS)  Incubate for 30min rotating
 Add the phages and shake for 1.5hrs at RT
In the meantime, find out how you are going to elute the phages further on in the
procedure. Calculate how much competitor protein you are going to use for
specific elution.
Each group will perform another type of elution. Total elutions will be
performed with TEA (triethylamine). Specific elutions will be performed in
the presence of excess of Erbitux (compared to the antigen, you will use a 10x
molar excess of your elution protein)
What do you do with the control? Elute? If so, how?
 Wash wells extensively with PBS-Tween 0.05% (10 times) and PBS (10
times)
 Elute bound phage as follows:
TEA: add 100 l of TEA. Leave for 20 minutes on the shaker. Add 50 l of
1M Tris/HCl and transfer to a clean eppendorf tube.
Erbitux: add 100 l and leave for 60 minutes on the shaker. Transfer to a
clean eppendorf tube and add 50 l of PBS.
 Make a 10-fold dilution series of the output phages in a 96-wells plate. For
this, take 5 l of phage and add this to 45 l of PBS (this is the 10-1 dilution).
Mix well by carefully pipetting up and down. Refresh the pipet tip and take 5
l of the 10-1 dilution, add it to 45 l PBS to get a 10-2 dilution. Repeat this
until the 10-6 dilution.
 Also make a 10-fold dilution series of the input phages. Pipette as above.
Repeat until you have the 10-12 dilution.
 In new wells: Mix 95 l of exponentially growing TG1 E.coli cells with 5 μl
of each dilution.
Incubate the plate in a 37°C incubator for 30 minutes.
 Spot 5 l of each phage infection on a LB-Agar-Amp plate. Wait until the
spot are dry and grow overnight in the 37C incubator.
 Plate the rest of the 10-2 and 10-3 TG1 infection on other LB-Agar-Amp
plates. Grow overnight in the 37C incubator.
During Incubations:
 Draw the principle of the selection.
 What's the principle of blocking with milk?
 Why do we coat with Ra a-huFc?
 Why do we have a well that is coated with Ra a-huFc, but has no captured
EGFR-ECD-Fc?
 How do we elute and recover phages?
Day 2: ELISA to test Nanobody clones for binding to EGFR and
Her2 ECD
Principle:
The binding of the selected nanobodies on both EGFR-ECD and Her2-ECD is
evaluated with an ELISA.
The first steps have already been performed to save time:
 Pick colonies and make a master plate. Grow O/N at 37oC and store in
glycerol.
 Let phages be produced in cultures started from the master plate, adding the
helper phage. Grow O/N at 37oC. Spin down the plate and collect the
supernatant, where the phages are.
 Coat an ELISA plate with 1:2000 rabbit anti-human Fc in PBS; 100l per
well, overnight at 4°C
 Wash wells three times with PBS (submerge the plate in PBS, discard it and
slap the plate on some tissues; all further wash steps are performed in this
way)
 Block with 200l 2% (w/v) BSA in PBS (PBS/BSA) for 30 minutes at RT
with shaking. All further incubations are performed in this buffer.
START:
 Carefully label your plate; determine which wells are the controls.
Attention: ALL VOLUMES INDICATED ARE PER WELL!!
 Capture EGFR-ECD-FC fusion and Her2-ECD-FC fusion incubating with
100l of a 1:200 dilution of ECD-FC (0.1 mg/ml stock) in PBS/BSA for 1
hour at RT.
 Wash the plate four times with PBS
 Add 20 l phages in a total of 100μl PBS/BSA to the wells. Incubate for 1.5
hours at RT shaking.
Make sure you include controls (no phage added).
 Wash wells five times with PBS.
 Detect bound phage with 100 l anti-M13-HRP (1:10.000 diluted) in
PBS/BSA. Incubate for 1 hour at RT shaking.
 Wash wells three times with PBS.
 Develop with 50l TMB. Stop the reaction with 50l 2M sulphuric acid.
 Discuss the results and the next steps.
During incubations:
 Draw a scheme of the principle of the assay
 Why do we use BSA and not (!) milk?
 Discuss dilutions and results of the day of selections:
 What is the number of phages used in the input?
 What is the number of phages in each output?