Establishment of a Direct Competitive ELISA for Camel FGF21 Detection

Abstract

Camels, with the ability to survive under drought and chronic hunger, developed exceptional efficient lipid reserves and energy substance metabolic characteristics. Fibroblast growth factor (FGF) 21 is a hormone that regulates important metabolic pathways and energy homeostasis. However, the absence of a specific detection method for camel FGF21 impacts research on camels' metabolic regulation. This study established a direct competition ELISA assay for detecting camel FGF21. Camel FGF21 antigen was expressed and purified through prokaryotic expression system. Polyclonal antibody was produced and purified via immunizing guinea pigs and affinity chromatography assay. Biotin-labeled FGF21 was synthesized artificially as the competitive antigen. After the determination of optimal conditions, including the working concentrations of the antibody and antigen, blocking solution, dilution buffer, and the competition reaction time, the standard curve with a typical "S" shape was generated using GraphPad Prism. The regression equation was Y = 0.1111 + (X^-0.7894) × (2.162 - 0.1111)/(X^-0.7894 + 15.76^-0.7894), with the IC₅₀ 15.59 ng/mL, the limit of detection (LOD) 0.024 ng/mL, the limit of quantification (LOQ) 1.861 ng/mL, and the linear range IC₂₀~IC₈₀ 2.0~119.22 ng/mL. The verification test showed that the recovery rate ranged from 91.34% to 98.9%, and the coefficients of variation for the intra- and inter-plate both were less than 10%, indicating that the ELISA method had high accuracy, good repeatability, and high stability. In addition, this ELISA method had the potential to detect FGF21 secretion levels in other species such as mouse, human, and pig. This study provided a rapid quantitative tool for conducting research on the FGF21 factor in camels.

Keywords: FGF21; affinity chromatography purification; camel; direct competition ELISA; polyclonal antibody; prokaryotic expression.

Figure 1

Conservation analysis of FGF21 protein. (A) Sequence of camel, human, mouse, and pig FGF21 peptides were aligned with CLUSTALW. (B) Spatial structures of camel, human, mouse, and pig FGF21 protein were predicted with SWISS-MODEL, respectively.

Figure 2

Validation of recombinant FGF21 proteins after purification. (A) SDS-PAGE electrophoresis of purified camel, mouse, human, and pig FGF2 proteins. Protein bands were stained with Caumas Brilliant Blue. (B) Western blot analysis of purified recombinant proteins using anti-His Tag antibody. (C) Western blot analysis of purified recombinant proteins using commercial anti-human FGF21 antibody. M: Protein marker. 1: Recombinant camel FGF21 protein. 2: Recombinant mouse FGF21 protein. 3: Recombinant human FGF21. 4: Recombinant pig FGF21 protein (Supplementary File S1).

Figure 3

Purification and analysis of guinea pig anti-camel FGF21 polyclonal antibody. (A) UV profile during purification of guinea pig IgG using Protein A HP affinity chromatography. (B) SDS-PAGE analysis of purified guinea pig IgG. M: Protein marker. 1: Purified guinea pig IgG. (C) Antibody titer was determined by indirect ELISA mediated by camel FGF21-encapsulated antigen and HRP-labeled goat anti-guinea-pig IgG. (D) Antibody specificity was monitored using western blot. M: Protein marker. 1: Camel FGF21 protein. 2: Mouse FGF21 protein. 3: Human FGF21 protein. 4: Pig FGF21 protein (Supplementary File S1).

Figure 4

Validation of Biotin-labeled antigen (Supplementary File S1).

Figure 5

Optimization of direct competition ELISA reaction conditions. (A) Screening of the optimal blocking solution. (B) Screening of the optimal dilution solution. (C) Determination of the optimal dilution factor of avidin. (D) Determination of the optimal reaction time.

Figure 6

Establishment of camel FGF21 detection standard curve.

Figure 7

Establishment of mouse, human and pig FGF21 detection standard curve.