How to buy antibodies for mass cytometry conjugation

So you've designed your mass cytometry panel and realized that Standard Biotools (previously Fluidigm) doesn't offer conjugated antibodies for several of your specific markers. That's where the conjugation service comes in.

We can conjugate almost any antibody to any of the lanthanide metals. We use the MaxPar kits from Standard Biotools and keep all of the metals in stock so we can conjugate your antibodies whenever you need.

How we conjugate your antibodies

To help understand why we recommend different types and formulations of antibody it helps to understand how we conjugate your antibodies.

The antibody is reduced to expose sulphide groups
Specific lanthanides are loaded onto linker molecules.
The linker is joined to the free sulphide groups

BSA-free antibodies, please!

BSA is commonly used as a carrier protein in antibody formulations.

It helps stabilize the antibody making it last longer in the fridge.
It's cheap and compatible with staining media

BUT BSA is a major problem when conjugating antibodies.

BSA has >33 disulphide bridges
IgG has between 3 and 6 disulphide bridges available for conjugation
BSA will conjugate the linker-lanthanide molecule 10x more than IgG
This results in very poor conjugation efficiencies

It's very important to purchase antibodies that are "carrier free"

If BSA-free is unavailable...

Sometimes BSA-free formulations are either

Unavailable
Prohibitively expensive

All is not lost. There are BSA-removal kits available from a number of vendors. At OHRI, we have had success with

Reagent Selection

PRE-CONJUGATED ANTIBODIES

Antibodies already conjugated to lanthanide metals are primarily available from Standard BioTools. They have an extensive catalogue of markers, especially if you are working in:

Immunology
ImmunoOncology
Developmental Biology

The selection of antibodies incresases month by month so it's worth checking to see if your favourite antibodies have become available from Standard BioTools

Reagent Selection

CONJUGATING ANTIBODIES

If you're working in other fields you'll have to source some of your reagents from other sources (your usual antibody vendors will be fine). Purchased antibodies for conjugation they should be:

Carrier-Free
IgG
Monoclonal (preferred)
100ug

We can conjugate these antibodies to any lanthanide for you.

Other Additives

Several different molecules are used as stabilizers in antibody formulations. Let's look at how they might afect your conjugations.

Glycerol

Glycerol is added as a cryopreservative to prevent antibodies freezing easily, so that they don't lose activity from a freeze-thaw. Glycerol under 20% has no deleterious effect on conjugations

Other Additives

Gelatin

Gelatin is added to antibodies as a stabilizing carrier protein

Gelatin contains no disulphide bridges.
It will not hinder the conjugation reaction.
However, we quantify the conjugation process by measuring
A280 absorbance (by NanoDrop). Gelatin will confuse this readout and not allow us to precisely quantify how much antibody we return to you.
The Pierce antibody cleanup kit referred to earlier can remove gelatin from preparations

Other Additives

Trehalose

Trehalose is a cryoprotectant and antibody stabilizer

Trehalose is a carbohydrate with no disulphide bridges.
It will not hinder the conjugation reactions.

Other Additives

Azide or thimerosal

Thimerosal or Sodium Azide are added as preservatives/bacteriostats.

They have no effect on mass cytometry conjugation reactions.

Formulations to look out for

Until recently, getting a carrier-free antibody formulation was relatively difficult. However, many companies are now selling antibodies formulated for mass cytoemtry conjugation reactions. Look for formualtions labelled:

Mass Cytometry Ready
CYTOF Ready
Carrier Free

Alternatively, in-vivo ready formulations like NA/LE or LEAF are perfect for conjugations

Monoclonal Preferred

Monoclonal antibodies are preferred. They are clonal so each molecule will conjugate and stain with the same efficiency.

This is only preference though!

We have had success conjugating polyclonal antibodies so if this is your only option then don't worry - they can and will work!

IgG Preferred

All conjugation protocols are optimized for IgG. Protein reduction can do odd things to IgM and IgA due to their multimeric structures but we (and others) have had success conjugating IgM so if it's your only option it's still worth a shot.

100ug Preferred

The conjugation protocols we use are formulated to use 100ug of antibody as input. We expect to return 60-80% of this to you as conjugated protein. This generally provides enough antibody for validation, testing and completing your project. We can conjugate 50ug of antibody but the efficiency is greatly reduced versus 100ug. We highly recommend providing 100ug if you can.

Supernatants or Ascites

If your antibody is only available cell-line supernatant or ascites all is not lost. You'll need to extract the antibody from media or ascites using a Protein A or G (there are many kits available to do this) and resuspend the antibody in carrier-buffer. After that we can conjugate as normal.