CGRP antibody and other secreted peptide antibodies: measure where the molecule acts

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 an HGF antibody and the other targets here

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.

A leptin antibody and a hormone measured in plasma

Leptin circulates in proportion to fat mass and is measured by immunoassay far more often than it is stained, so a leptin antibody for a blot and a matched pair for an assay are different purchases. A soluble receptor binds part of the circulating pool, so free and total are separate measurements. Diurnal variation and the fasting state change the number more than the assay does.

A somatostatin antibody and a small peptide's fixation

Somatostatin is a short peptide processed from a precursor into two active forms, so a somatostatin antibody has to state which it detects and the fixation has to crosslink the peptide in place or it washes out. In tissue it marks a neuronal and an islet population, and the granular pattern in processes rather than a filled cytoplasm is what says the stain worked.

A crh antibody and a hypothalamic peptide

CRH is present at very low levels outside the hypothalamus and is rapidly cleared in plasma, so a crh antibody is used in tissue with a validated protocol rather than as a serum measurement, and colchicine treatment to accumulate the peptide is a classical part of that protocol. The precursor and the processed peptide are separate species and the epitope decides which is seen.

An ngf antibody and a factor with a pro form

Nerve growth factor exists as a precursor with its own receptor preference and as the mature dimer, and the two have opposing effects on survival, so an ngf antibody has to say which it detects. A neutralising clone needs functional validation rather than a binding figure. In tissue the factor is scarce, which makes the positive control tissue and the amplification method part of the result.

An epo antibody and glycosylation as identity

Erythropoietin's sugar chains carry much of its activity and distinguish recombinant from endogenous protein, which is the basis of doping analysis, so an epo antibody used for identity has to be read with a separation that resolves the glycoforms. For a plain concentration an immunoassay against a reference standard is the method, and the standard's identity is what makes two figures comparable.

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 using a gastrin antibody or a BDNF antibody disagree?

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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