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Troubleshooting Antibody conjugation

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nataliaCW

Participant

Posts: 4

Joined: Mon Jan 31, 2022 4:42 pm

Post Mon Feb 14, 2022 9:50 am

Troubleshooting Antibody conjugation

I have a question regarding self-antibody conjugation. I had such situation twice in my life, that I conjugate 3 antibodies at the same time, and 2 of them had good final concentration, but the concentration of the third one was like 0.06 mg/ml. Could you please give me some advice what should I improve/avoid? I conjugated more or less 50 different antibodies in total, and have no idea why I had such random situation…
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mleipold

Guru

Posts: 5285

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Feb 15, 2022 7:46 pm

Re: Troubleshooting Antibody conjugation

Hi Natalia,

You didn't say whether you've been successful with this particular antibody previously.

Unfortunately, sometimes a given conjugation just doesn't work.

1. Bad lot of antibody from the manufacturer. It's rare, but I've had a bad lot from even big companies like BD and Biolegend: no problem with a product for years, then 2-3 failed conjugations from the same lot, and then no problem once I switched to another lot.
2. Hole in the filter and your antibody is lost in the flow-through. Again, rare, but does happen.

3. Antibody can't survive the reduction process and precipitates. This should be a repeated error (ie, it never works).
4. Didn't fully redissolve if supplied as lyophilized powder. The CCR7 clone I use comes as a powder, and I had bad recovery on it for a long time until I spent extra time letting the powder dissolve (several pipet to mix and wait, pipet to mix and wait steps).


Mike
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Chowduck

Contributor

Posts: 29

Joined: Wed Nov 19, 2014 4:39 pm

Post Wed Feb 16, 2022 1:39 am

Re: Troubleshooting Antibody conjugation

To add to Mike’s comment:

I would recommend that you nano-drop the antibody as provided from the vendor then nano-drop the filtrate after spinning through a 50 kDa filter. Typically, 0-3% of the UV absorbing protein passes through. If it’s more, then it’s likely you’ll have poor recoveries like what you’re describing. (Exception for some BD lots that have UV absorbing components in their buffer)

If you can demonstrate that a large portion of a 150 kDa antibody passes through a 50 kDa filter is pretty clear justification for a refund/replacement. It’s also gives you the chance to abort that reaction and save the cost of Maxpar polymer.

With regards to holes in the filter, it is possible to damage the membrane with a pipette tip.

-Greg Chang

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