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. 2022 Jul 20;10(7):404.
doi: 10.3390/toxics10070404.

Development and Applications of a Zebrafish (Danio rerio) CYP1A-Targeted Monoclonal Antibody (CRC4) with Reactivity across Vertebrate Taxa: Evidence for a Conserved CYP1A Epitope

Amy L Anderson  1 , Benjamin D Dubanksy  2 , Lindsay B Wilson  3 , Robyn L Tanguay  3 , Charles D Rice  1
Affiliations

Development and Applications of a Zebrafish (Danio rerio) CYP1A-Targeted Monoclonal Antibody (CRC4) with Reactivity across Vertebrate Taxa: Evidence for a Conserved CYP1A Epitope

Amy L Anderson et al. Toxics. .

Abstract

CYP1A is a heme-thiolate enzyme associated with the cytochrome P4501A1 monooxygenase system and is inducible by a wide variety of xenobiotics and endogenous ligands that bind and activate the aryl hydrocarbon receptor (AHR). The AHR-CYP1A axis is important for detoxification of certain xenobiotics and for homeostatic balance of endogenous sex hormones, amine hormones, vitamins, fatty acids, and phospholipids. Herein, we generated and described applications of a zebrafish CYP1A-targeted monoclonal antibody (mAb CRC4) that fortuitously recognizes induced CYP1A across vertebrate taxa, including fish, chicken, mouse, rat, and human. We then demonstrated that mAb CRC4 targets a highly conserved epitope signature of vertebrate CYP1A. The unique complimentary determining region (CDR) sequences of heavy and light chains were determined, and these Ig sequences will allow for the expression of recombinant mAb CRC4, thus superseding the need for long-term hybridoma maintenance. This antibody works well for immunohistochemistry (IHC), as well as whole-mounted IHC in zebrafish embryos. Monoclonal antibody CRC4 may be particularly useful for studying the AHR-CYP1A axis in multiple vertebrate species and within the context of Oceans and Human Health research. By using archived samples, when possible, we actively promoted efforts to reduce, replace, and refine studies involving live animals.

Keywords: conserved CYP1A epitope; monoclonal antibody (mAb) CRC4; vertebrate CYP1A; whole mount IHC; zebrafish CYP1A.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A). ELISA results demonstrating reactivity of three primary hybridoma supernatants against zebrafish CYP1A peptide used for immunizations. (B). ELISA results demonstrating that only mAb CRC4 recognizes a conserved peptide sequence common to all vertebrates. (C). ELISA results demonstrating that the conserved CYP1A sequence blocks the binding of mAb CRC4 to ELISA plate-bound peptide used for immunizations. (D). Cross reactivity of mAb CRC4 with induced CYP1A protein in representative vertebrates. MW, molecular weight markers; C (control); T (treated); ZF, zebrafish liver cells; PLHC-1, Poeciliopsis lucida hepatoma cell line; RBT, β-NF induced rainbow trout microsomes; HEPA1C1C7, mouse hepatoma cell line; Rat, liver S9 fractions; frog, XLK-WG cell line; human CYP1A1, recombinant CYP1A1 expressed in HEK cells. Details are found in Methods and Table 2.
Figure 2
Figure 2
Whole-mount immunolocalization of zebrafish CYP1A protein in a representative DMSO vehicle (control) larva (A,B) and 30 µM retene-exposed (C,D) larvae at 48 hpf using mAb CRC2. Each larva was exposed to their respective treatment from 4 hpf through 48 hpf. BA, brachial vasculature; BV, brain vasculature; H, heart; UP, urogenital pore; PVP, posterior vascular plexus; ISV, intersegmental vessel. Details are found in Methods.
Figure 3
Figure 3
Immunohistochemical detection of CYP1A protein expression using mAb CRC4 in liver tissues from representative vertebrates. (A). Control mouse liver; (B). Liver from a PCB-126 treated mouse; (C). Control chicken embryo liver at 18 days post fertilization (DPF); (D). Liver from a crude oil vapor-exposed chicken embryo at 18 DPF; (E). Liver from CYP1A recalcitrant Gulf killifish; (F). Liver from Gulf killifish collected from a crude oil exposed site; (G). Control Rainbow trout liver; (H). Liver from a β-NF-exposed rainbow trout; (I). Control zebrafish liver; (J). Liver from PCB-126 treated zebrafish. (A,B) are 20× magnification, scale bar = 100 µm (C–J) are 40× magnification, scale bar = 50 µm. Details of tissue sources, experimental conditions, and procedures are found in Methods and Table 2.
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