I am currently trying to measure certain monoclonal antibody levels spiked-in in PBS buffer. Lets say I want to measure anti-X mAb where "X" is the antigen of the mAb of interest. To achieve my goal, I try to modify chip surface with 11-MUA, use EDC NHS to conjugate X protein onto the SAM and expect to see anti-X mAb to bind to X protein on the surface. However, I seem to have little frequency change after chip modification where I conjugate the X protein on surface. Also there is no clear peaks while I try to measure the mAb in PBS buffer between the 1ng/mL to 64ng/mL range.
Is there anything wrong with the protocol I use. Or might there be any other problems causing me to not get good frequency changes? Is it a bad idea to regenerate surface with 0.1 HCl? How can I know that HCl regeneration step doesn't hydrolize the immobilized protein X, or SAM all together from surface instead of just removing the previous sample?
I can't find the problem in here and I am getting short on valuable materials. Please share me your insights. Tomorrow I will update the question with the readings.
The protocol I use as follows:
· Cleaning chip surface
- Wash with ddH2O in an ultra-sonicator.
- Wash with ethanol in an ultra-sonicator
· Coat QCM sensor chip with self assembled monolayer (SAM):
- Immerse the sensors (active and control sensors) in 2mM ethanol solution of mercaptoundecanoic acid overnight.
- Rinse with water and ethanol, dry under nitrogen
- Store at 4 oC till use
· Immobilize protein X (capture) a on the chip using conventional amine coupling chemistry: - Activate sensor surface with 1:1 mixture of 400mM EDC and 100mM NHS by injecting simultaneously across the two sensing spots for 5 min (total volume used 150µL).
- Inject 50 µg/mL protein X (capture protein) (in 1xPBS buffer, pH 5.5) for 5 min (total volume used 150 µL)
- Block surface with 50 µg/mL BSA in PBS (pH 7.4) for 5 min (150µL).
- Non-reacted NHS esters were capped with 1M ethanolamine (pH 8.5) for 5 min (total volume used 150 µL)
- Change running buffer to PBS containing 0.005% Tween (PBS/T) for the binding assay studies.
Detection of anti-X mAb spiked-in in PBS/T
-Dilute antigen of interest at specific concentrations (1-2-4-8-16-32-64 ng/mL) in PBS/T buffer containing 5 µg/mL BSA.
-Inject mAb spiked-in buffer on active sensor and control sensor for 5 minutes (150 µL total volume).
- Record frequency changes 3 min after sample injection.
-Regenerate the surface with 100 mM HCl (1min, 80 uL) between different samples.
-Allow frequency to stabilize under running buffer (PBS/T)
Hello Abbas Güven Akçay ,
In our experience it is important to optimise the spacing of the probe by adjusting the relevant ratios. Of course, maximising the probe density on the surface should maximise the sensitivity. However, in practice if the probe density is too high you will have less successful binding events due to steric hindrance.
Of course, you could look at increasing the surface area of the gold surface by using AuNPs, see our following paper for an example of how the sensitivity can be greatly enhanced vs. a conventional planar gold surface:
Article Self-assembled gold nanoparticles for impedimetric and amper...
A colleauge from a previous lab, Nello Formisano worked on QCM and QCM-D, maybe quickly check his thesis (attached) for protocols as I remember we also used EDC/NHS for a system.
There may be several issues with your protocol:
1. Cleaning: This approach is only sufficient, if your gold surface is very clean. Otherwise, you would need Piranha solution (hazardous!) or similar. https://en.wikipedia.org/wiki/Piranha_solution
2. The activation and conjugation steps are quite short. Keep your protein solution on the chip as long as possible.
3. If your protein X is small, it might be hidden by BSA. Consider to skip the BSA blocking. Use Tris instead of ethanolamine.
4. 100 mM HCl may denature your protein completely. The regeneration conditions need to be optimized very carefully! Do you know, whether your antibody binds to the native protein, to the denatured protein or both?
Hi Michael G. Weller ,
1) My protocol suggests me to use plasma etcher, which we doesn't have. However, luckily last time I used piranha solution to clean my chip. Which I did by dipping the chip into piranha solution together with the teflon coated stand where the chip sits on. I did the dipping into piranha solution for 10 seconds and washed the chip with water 3 times and ethanol 2 times, and followed by placing in 11-MUA solution overnight. However, I don't know if 10 seconds was enough because I don't have a protocol for cleaning via piranha, and I was afraid to lose my gold layer by letting it sit there too long. After that I had
2) I have limited amount of protein solution. Would it work If I decrease the flow speed instead of increasing the flow time. Or can I stop the flow while the protein is flowing through the chip to let it settle on the surface?
3) The protein was bought commercially and the size was around 34 kDa (can be +1 due to tag they used for purification) Do you think BSA blocking might still be an issue. I don't understand how Tris can help? Ethanolamine is shorter than Tris and would cause less steric hinderance, wouldn't it?
4) This protein is commercial. But before we produced and isolated the same protein for ELISA, where it worked quite good although the protein had 10M Urea in it as a remnant of isolation. Which shows that denaturation wasn't a protein in binding to the same antibody in ELISA. However, I don't know how this commercial protein behaves. I realized that 0.1M HCl not only removes protein from surface but more than that. Because when I do around 10 measurements frequency change shows that significant mass loss occurs from surface. But I wasnt able to decide what got torn from surface. So, I removed the regeneration step for now. As I dont have any other solution. Now I measure the proteins in increasing concentration while I try to see the performance.
My results for chip modification and measurements are attached.
In chip modification I wrote on it approx where which solution reached to sensor.
In measurement initial arrow is buffer signal, after that comes first measurement 8ng/mL (235 pM), 2nd measurement =16ng/mL (470 pM), 3rd measurement = 32 ng/mL (970 pM), 4th measurement =64ng/mL (1.88 nM) and lastly 5th measurement= 128ng/mL (3.76 nM).
And an additional question. I have previously produced quite big of a batch of the protein. However, it is in 10M Urea and I don't know how pure the protein is I just pulled the his tagged protein via Ni-NTA. Do you think I can use that protein in chip modification. I may try to buffer exchange to remove Urea without precipitating the protein. However, I am afraid if it will waste my chips with high background or problems due to urea content. I dont have many chips at the moment. Or shall I use HPLC to get rid of residues and urea.
Thanks a lot for your suggestions and time.
Hi Abbas Güven Akçay,
1) The short Piranha treatment might be OK. You are right, a longer incubation might damage your sensor, e.g. by destruction of the layers below the gold.
2) You may stop the flow as long as you like. If your system is first filled with air, you only need a minimum volume of protein solution. If the system contains some buffer, you need more volume to displace the buffer (backmixing).
3) BSA has about double the molecular mass and hence may cause sterical hindrance. I would skip this step. Tris has more OH groups than ethanolamine and hence may lead to a more hydrophilic surface. Tris has only a MW of 0.12 kDa: A sterical hindrance is very unlikely. However, I believe that this step is not necessary too, since after a long protein incubation time, no residual activity of the NHS esters is left. Therefore, these steps may only be performed for the "peace of conscience".
4) I fear that your protein conjugation did not work at all. You perhaps only achieved a noncovalent adsorption of some protein, which is lost during the following regeneration steps. Why the covalent step failed, may have many reasons: One of the most frequent: a) Amino-containing compnents in the protein solution (such as glycine, Tris) -> buffer exchange, b) concentration of protein too low or incubation time to short, c) in the case of proteins, which are only soluble in urea, they may precipitate in your conjugation buffer (check by centrifugation and BCA test of the supernatant).
Additional questions:
Yes, you may use your protein. I never tested a direct immobilization from urea. I would slowly dialyze it in PBS or in your conjugation buffer. Check the effective protein concentration. HPLC is not trivial to purify proteins. We often use PD-10 (or equivalent) columns for such purposes, but in contrast to the general protocols, you should use only 0.5 mL of sample. If you are limited in chips, you also may try to regenerate them with Piranha solution. You end up with the clean gold surface, if the sensor was not damaged by the treatment, but it may by worth a try.
Good luck, Michael
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