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. 2023 Oct 12;24(20):15102.
doi: 10.3390/ijms242015102.

Development of a New Enzyme-Linked Immunosorbent Assay (ELISA) for Measuring the Content of PACAP in Mammalian Tissue and Plasma

Elisabeth Anne Adanma Obara  1 , Birgitte Georg  1 , Jens Hannibal  1
Affiliations

Development of a New Enzyme-Linked Immunosorbent Assay (ELISA) for Measuring the Content of PACAP in Mammalian Tissue and Plasma

Elisabeth Anne Adanma Obara et al. Int J Mol Sci. .

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a naturally occurring neuropeptide found in both the central and peripheral nervous systems of vertebrates. Recent studies have revealed the presence of PACAP and its corresponding receptors, namely, the pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1R), vasoactive intestinal peptide receptor 1 (VIPR1), and vasoactive intestinal peptide receptor 2 (VIPR2), in various structures implicated in migraine pathophysiology, including sensory trigeminal neurons. Human studies have demonstrated that when infused, PACAP can cause dilation of cranial vessels and result in delayed migraine-like attacks. In light of this, we present a novel ELISA assay that has been validated for quantifying PACAP in tissue extracts and human plasma. Using two well characterized antibodies specifically targeting PACAP, we successfully developed a sandwich ELISA assay, capable of detecting and accurately quantifying PACAP without any cross-reactivity to closely related peptides. The quantification range was between 5.2 pmol/L and 400 pmol/L. The recovery in plasma ranged from 98.2% to 100%. The increasing evidence pointing to the crucial role of PACAP in migraine pathophysiology necessitates the availability of tools capable of detecting changes in the circulatory levels of PACAP and its potential application as a reliable biomarker.

Keywords: ELISA; PACAP; assay validation; migraine.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Representative standard curves for PACAP 38 ELISA assay (n = 3). The graph was generated by combining serial dilutions of synthetic PACAP 38 in range 5.12–400 pmol/L and interpolated with GraphPad Prism v. 9.0 software by a four-parameter regression formula. The error bars show the standard deviation between the duplicate samples in three independent runs. y axis: Optical Density (O.D), x axis: PACAP 38 concentration (pmol/L).
Figure 2
Figure 2
Representative distribution of PACAP 38 in the cortex, cerebellum, brainstem and hypothalamus of PACAP wildtype (WT 1, WT2 and WT3) mice.
Figure 3
Figure 3
PACAP expression in the mouse brain: (A) Tissue section showing the (a) cortex and (b) hypothalamus. (B) Tissue section showing the (c) cerebellum and (d) brainstem. Representative immunohistochemical staining for PACAP (green) in the (a) cortex, (b) hypothalamus, (c) cerebellum and (d) brainstem.
Figure 4
Figure 4
Bar graph showing dilution linearity of spiked human plasma samples and mouse tissue extract. Dilution recovery ((%), y-axis) of (A) two spiked human plasma samples (PL1 and PL2), followed by results of serial dilution at 1:2, 1:4 and 1:8 in assay buffer and (B) hypothalamus tissue extracts from wildtype mice (WT1 and WT2), followed by serial dilution at 1:10 and 1:20. The error bars indicate the mean and standard deviation (SD) of each sample after three independent runs. Dilution recovery was deemed acceptable at 100 ± 25%.
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