Hi,

I find it weird that it uses phosphate buffer since EDC is reactive toward carboxylic, amine and phosphate moiety. That's why it has to be MES (due to its structure and buffering pH range). Anyway I checked the protocol from Luminex and phosphate buffer is used for solubilizing the protein (antibody, etc.). Here is the protocol: http://www.luminexcorp.com/prod/groups/public/documents/lmnxcorp/89-00002-00-319.pdf in case we're talking about the different Luminex protocol.

As for this question of yours:

Isn't amide bond (antibody amine to sulfo-NHS) formation favored in basic conditions?

I have some experience with coupling nanoparticle and antibody, and from what I know we only need to use one buffer (MES buffer pH 5-6), according to the Uptima and Proteochem protocol. The acidic condition is required for the activation of EDC (protonation of imide so the carbodiimide is attacked by the carboxyl moiety).

Even so, as the Luminex protocol mentioned, the microsphere is washed and the protein (which is in phosphate buffer pH 7.4) is added. There is a protocol from Bangs Laboratory that uses phosphate buffer pH 7.2-8.5 for the protein coupling to microbead after being washed with the same phosphate buffer. So yes, I think you are correct.

Hope that help,

Radif

Thanks Radif. I see the protocol you provided uses a single buffer for activation and coupling. Here is the luminex protocol I was referring to which lists 2 different buffers (MES first, mono basic sodium phosphate second) http://www.luminexcorp.com/prod/groups/public/documents/lmnxcorp/protein-coupling-protocol.pdf.

I chose my words poorly in suggesting that the sulfo-nhs bonds to the antibody amine. I understand it is a leaving group, but isn't this much more likely to occur in basic conditions? Thermoscientific tech support told me that the half-life for sulfo-nhs esters at pH 6.5 would be days (as opposed to the 2 hour coupling suggested by luminex at pH 6.0).

Ouch, you caught my unedited answer. Please check my edited answer above.

And I've read the link you provided and I found it weird. And since there are two different protocol with different steps I'd suggest you to contact the Luminex support. But judging from the look, it seems to be an old document compared to the one I provided. I'd rather go with the new one.

Even the newer luminex protocol (pH 6.0 activation/coupling) should result mostly  in adsorption rather than covalent attachment, right?

No, actually I agree with the newer protocol. Check the additional reference from Bangs Laboratory below (Carboxyl-modified microspheres, page 4). It is a bit different though, no sulfo-NHS. But I still have no idea what would make it different with or without the sulfo-NHS, my best guess is to get a controlled reaction so the amount of covalent crosslinking per microsphere is homogenous.

Just found something when I'm doing literature research for my thesis: https://tools.lifetechnologies.com/content/sfs/manuals/MAN0011309_NHS_SulfoNHS_UG.pdf

In the manual you can find a nice explanation about the reason behind the use of NHS or sulfo-NHS too.

Thank you, this is the document that led me here to begin with. I know thatNHS/Sulfo-NHS are used to create amine reactive esters of carboxyl groups, and they increase the efficiency of carbodiimide reactions. As the product insert states:  The activation reaction with EDC and Sulfo-NHS is most efficient at pH 4.5-7.2, Reaction of Sulfo-NHS-activated molecules with primary amines is most efficient at pH 7-8, NHS esters have a half-life of 4-5 hours at pH 7, 1 hour at pH 8 and only 10 minutes at pH 8.6 (and days at pH 6.0 according to tech support). 

For best results in two-step reactions, perform the first reaction in MES buffer (or other non-amine, non-carboxylate buffer) at pH 5-6, then raise the pH to 7.2-7.5 with phosphate buffer (or other non-amine buffer) immediately before reaction to the amine-containing molecule.

This is why I think the majority of any binding that occurs via Luminex labeling procedures are due to adsorption and not covalent coupling. The new protocol has all steps at pH6.0, the old protocol activates at pH 6.2 then couples at pH5.0. If the half-life of NHS or sulfo-nhs esters is days at pH6.0, how much would actually react in the 2hr procedure suggested by luminex (or multiple papers)?

True, BangsLabs does list the reaction correctly (i think). You could simply activate and couple in one step at pH4.5-7.2 with just EDC, but that process is less efficient (i.e. it will crosslink antibodies to each other as well as to the beads). They correctly mention that using NHS would require raising the pH.

Hmmm, yes I get your point. And you are correct, the old protocol is incorrect, and the multiple papers that use such protocol would most likely having inaccurate result. Because as you said, no or very low covalent coupling happens.

I wouldn't go that far. The new and old protocol should result mainly in adsorption of antibody to the beads, rather than covalent bonding, but that doesn't mean the assays would not work satisfactorily. ELISAs use adsorption to attach capture antibody to the plates. I have had good agreement of results between our standard ELISAs and the cytometric bead arrays that I've developed using Luminex' protocol. I imagine stability might become a problem, but only time will tell. I'm in the process of comparing coupling (adsorption) at ph 6.2 to coupling (covalent) at pH 8.

I'm still thinking that the new protocol will give covalent crosslinking, I only disagree with the old one.

OK, good luck :)

Hi

You might find this useful:

www.attana.com/wp-content/uploads/2013/02/TN03-01-Immobilization-of-Antibodies-on-the-Attana-Carboxyl-Sensor-Chip-Surface.pdf

@Evan (" If the half-life of NHS or sulfo-nhs esters is days at pH6.0, how much would actually react in the 2hr procedure suggested by luminex (or multiple papers)?")

The half life refers to the stability of the active ester in aqueous solutions at different pH, without the presence of amines. The NHS esters are hydrolyzed by OH-. -NH2 is a better nucleophile than OH-, but the more OH- you have at increasing pH the worse, as they compete.The actual coupling reaction is completed in very short time.

Thank you, that is helpful. I compared passive adsorption to activation/coupling and found that they performed quite similarly (Il-6 Sandwich), although the passively adsorbed set was a little less sensitive.