Choosing antibodies against secreted peptides and growth factors: why a cgrp antibody, a gastrin antibody, a bdnf antibody and an hgf antibody are measured where the molecule goes rather than where it is made, what precursor and mature forms do to a result, and the matrix controls each assay needs

Measuring a secreted factor

1. Measure where the molecule acts, not only where it is made. A lysate tells you a cell contains the peptide; a fluid or a conditioned medium tells you what was released. For most questions about a secreted factor the fluid is the sample, and a tissue stain answers a different question about the source.
2. Establish whether the reagent sees precursor or mature peptide. These molecules are made as precursors and processed, and the biologically active species is usually the processed one. An antibody against a precursor region reports something different, and this is the single largest source of disagreement between assays.
3. Match the assay's matrix and qualify it in yours. An assay validated in serum used on cerebrospinal fluid, conditioned medium or tissue extract is unvalidated until spike recovery and dilution linearity are demonstrated in that matrix.
4. Fix the pre-analytical handling and record it. Peptides are proteolysed rapidly in blood, so tube type, protease inhibitors, time to centrifugation and freeze-thaw all change the number. Fix the protocol before the first sample and apply it to every one.
5. Use a matched pair for immunoassay and buy it as a pair. Capture and detection antibodies must see different epitopes without blocking each other. For small peptides the available epitopes are few, which is exactly why an assembled pair rarely works and a validated one is worth paying for.

Tissue staining for the source

Immunostaining shows which cells contain the peptide and, in nerve fibres, where it is transported, which is a genuinely different result from a concentration. Both are useful and they should not be presented as the same evidence.

For peptides stored in vesicles, fixation and permeabilisation decide whether the granular pattern survives. A diffuse cytoplasmic stain for a stored peptide is usually a preparation artefact.

Blocking experiments with these targets

Neutralising antibodies against secreted factors are used to test necessity, and they need a dose response, an isotype control and a demonstration that the antibody reaches the compartment where the factor acts.

In vivo, half-life and distribution decide whether a neutralising antibody does anything, and a negative result without a pharmacokinetic check is not evidence that the factor is unimportant.

Common questions

Should a secreted peptide be measured in lysate or in fluid?

In fluid for almost every question about signalling, because the molecule acts where it is released. A lysate or a tissue stain tells you about the source cell, which is a different question worth asking separately.

Why do two assays disagree about the concentration?

Usually because they see different forms. These peptides are processed from precursors, and an antibody against a precursor region reports a different species from one against the mature peptide.

What handling do peptide samples need?

Protease inhibitors in the tube where the assay requires them, rapid centrifugation, cold storage and minimal freeze-thaw. Peptides degrade far faster than proteins in blood and the number moves accordingly.

Can I build my own immunoassay for a small peptide?

It is much harder than for a protein, because a short peptide offers few epitopes for a capture and detection pair that do not block each other. Buying a validated pair is usually the cheaper route by the end.

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