Choosing an erbb3 antibody, a b7h3 antibody or a trop2 antibody: why an antibody against a tumour surface target has to be matched to the application and the epitope rather than to the gene name, and the expression controls that make a staining result mean something

Specifying the reagent

1. Match the epitope to the application. An antibody against the extracellular domain detects the receptor on intact cells by flow cytometry; one against an intracellular region does not, and will only work on permeabilised or lysed material. This is the most common mismatch on surface targets and it is visible on the datasheet.
2. Ask whether it blocks, if blocking is the point. Detection and function blocking are different products. A reagent claimed to block should show a dose response in a functional assay; binding data alone does not demonstrate it, and the two are frequently presented side by side.
3. Use expression-defined controls, not tissue impressions. A cell line known to express the target strongly and one known not to, run alongside every stain, is what turns an image into evidence. For a surface target these lines are usually available and the control costs one well.
4. Quantify receptor density where you will compare lines. The same antibody gives very different apparent staining on cells expressing different amounts of the target, so comparisons between models need a density measurement rather than an impression. Calibrated beads make this straightforward on a cytometer.
5. Specify the grade for what it will touch. Carrier-free, low endotoxin and azide-free for anything added to living cells; carrier-free also for anything you intend to conjugate. A general purpose preparation with carrier protein cannot be labelled and should not be added to cells.

Authenticate the control lines, or the controls prove nothing

A positive and a negative line are only controls if they are the lines you think they are. Short tandem repeat profiling and a mycoplasma test on both, recorded with the experiment, is what makes the control set defensible.

Expression in a line also drifts with passage, so re-confirm the positive line's expression periodically rather than trusting a figure from a paper or a catalogue.

Tissue staining against flow cytometry

Flow cytometry measures surface expression per cell on a suspension and gives numbers. Tissue staining shows where the target is in an intact architecture and is far harder to quantify. Most questions about these targets need both at some point.

An antibody validated for one is not thereby validated for the other, because fixation and antigen retrieval change the epitope. Ask for evidence in the specific application rather than a list of applications.

Buying for a programme rather than an experiment

Recombinant reagents with a disclosed sequence stay identical indefinitely and can be reformatted, which is what a multi-year target programme needs. Bank one lot for a study and record it with every experiment.

Check the licensing position before a reagent becomes part of anything commercial. Research use only labelling is a licence restriction as well as a safety notice, and on targets with therapeutic interest it matters.

Common questions

Why does my antibody not stain the cell surface?

Most often the epitope is intracellular. Check which region the immunogen came from: an extracellular domain antibody detects intact cells, an intracellular one requires permeabilisation and will look like a failed stain otherwise.

How do I know an antibody blocks rather than just binds?

A functional assay with a dose response and a stated potency in a named system. Binding affinity, however good, is not evidence of blocking, and many catalogue entries present the two together.

What controls does a tumour surface target stain need?

A line known to express the target and one known not to, stained in the same run at the same settings, plus an isotype control. Those three separate a real result from an impression.

Can I compare staining intensity between cell lines?

Only with a receptor density measurement, usually with calibrated beads on a cytometer. Apparent intensity depends on expression level, and comparing lines without quantifying it is how internalisation and efficacy results fail to replicate.

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