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Increased Conjugation Batch Size

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taylorwitte

Participant

Posts: 2

Joined: Sun Aug 15, 2021 7:15 pm

Post Fri Oct 01, 2021 10:27 pm

Increased Conjugation Batch Size

Hi all,

We are looking to increase the amount of antibody we conjugation in one batch. We currently follow a protocol similar to Fluidigms with the X8 polymer kits but would like to conjugate more like 200-400 ug of antibody at a time for some of our antibodies with lower titers. Has anyone had any success increasing their batch size?

Best,
Taylor
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CRStevens

Master

Posts: 60

Joined: Thu Jul 17, 2014 5:07 pm

Post Mon Oct 04, 2021 2:45 pm

Re: Increased Conjugation Batch Size

Hi Taylor,

Hope all is well. We do this routinely. I acutally find bulk conjugations give you better yield. It is very similar to the original directions... see below for help.

Preloading Polymer with Lanthanide:
a. Resuspend the 4 vials of polymer into total of 95 uL L-Buffer (use 95 uL to resuspend one tube then transfer to the next to resuspend, etc) such that the contents of the 4 vials of polymer ends up in one tube in 95uL of L-Buffer.
b. Adjust the amount of metal to match the scale of the antibody (so 400 ug of antibody would need 20 uL of the metal).

Conjugation of Antibody with Metal Loaded Polymer:
a. Resuspend the metal-loaded polymer (4X) in 200 uL of C-Buffer.
b. No change in the centrifugation and incubation steps. Keep those the same as if the quantity of Antibody were only 100 ug.

Recover Metal Conjugated Antibody:
a. Resuspend the recovered antibody in 300 uL of W-Buffer
b. Measure by Nano-drop as usual.

Everything else you do as directed in the directions.

-Chad

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